• A kinase anchor protein 200
• calcineurin A and calcineurin B (calmodulin stimulated protein phosphatase)
• Calmodulin (CaM)
• CrebB-17A - Cyclic AMP response element binding protein
• dunce
• Exchange protein directly activated by cAMP
• Fasciclin II
• G protein-coupled receptor kinase 2
• G protein salpha
• radish
• rutabaga
• Dfos
• Djun
• Protein kinase A (PKA) - cyclic AMP dependent

• Amnesiac
• Calcium/calmodulin dependent protein kinase 1
• ether à go-go (See Calcium/calmodulin dependent protein kinase 1)
• Dopamine receptor
• G protein salpha
• latheo
• Neurofibromin 1
• Serotonin receptor 1A and Serotonin receptor 1B

• Accessory gland peptide 70A
• Adh transcription factor 1
• Adipokinetic hormone-like
• Ataxin-2
• Ataxin-2 binding protein 1 (common alternative name: Rbfox)
• blistered
• Crustacean cardioactive peptide (Cardioacceleratory peptide)
• debra
• discs lost
• Dopamine/Ecdysteroid receptor
• ebony
• Ecdysis triggering hormone
• Excitatory amino acid transporter 1
• foraging
• genderblind
• Gld2
• homer
• hugin
• inactive
• kurtz
• leonardo
• Lnk
• lush
• Neprilysin 4
• neuralized
• neuropeptide F
• NMDA receptor 1 and NMDA receptor 2
• Octopamine receptor in mushroom bodies
• Odorant receptor 22a and Odorant receptor 22b
• Odorant receptor 65a
• Odorant receptor 67d
• Ora transientless
• orb2
• oskar
• painless: pain response defective, nociceptor
• pale
• Protein kinase C
• pumilio
• pumpless
• Resistant to dieldrin
• Rfx
• Rad, Gem/Kir family member 1
• Ribosomal protein S6 kinase II
• sarah
• scab
• scribbled
• Sensory neuron membrane protein 1
• Serotonin receptor 7
• sex-peptide
• sex peptide receptor
• sickie
• slowpoke
• small conductance calcium-activated potassium channel
• staufen
• Synapsin
• takeout
• Tomosyn
• Tyramine β hydroxylase
• Tyrosine decarboxylase 2
• unextended

The cyclic AMP second messenger system is often called the learning pathway. But what is the first messenger system? How does cAMP function, and what are the consequences to the cell of cAMP signaling?

In synaptic transmission the chemical signal sent from cell to cell is a neurotransmitter, a small molecule constituting the first signal (first messenger) received by target cells. Neurotransmitters can act directly on ion channels to modify the permiability of cells to ions. One type of channel allows extracellular Ca2+ ions to enter the cell. Alternatively, mutation in K+ channels, ether à go-go (eag) and Shaker, each affecting different K+ channels, display greatly enhanced nerve activity as a result of reduction in K+ currents. Changes in Ca++ ion levels can result in activation of the cyclic AMP pathway, or, in the case of K+ channels, can result in increased neuronal activity and increase in synaptic structure and branching. K+ channel mutation is thought to increase motoneuron activity and synaptic transmission by increasing the cAMP second messenger signaling.

Rutabaga is an adenyl cyclase that creates cAMP in response to neurotransmitter signaling. Rutabaga is activated by the protein Calmodulin, an excellent sensor of the level of intracellular Ca2+. When Ca2+ levels are high, Rutabaga converts ATP into cAMP, which in turn activates PKA, the cyclic AMP dependent protein kinase. PKA in turn transduces the cAMP signals to downstream targets by phosphorylating (modifying with a phosphate residue) target proteins capable of effecting biological activity, such as transcription factors.

Where does Amnesiac fit in this system? Amnesiac is a neuropeptide, the kind of protein that interacts with non-channel receptors called G-protein coupled receptors. Instead of serving as ion channels, G-protein coupled receptors signal through G-proteins. G-proteins are biochemical marvels. Upon receptor activation, heterotrimeric G-proteins bind GTP and dissociate into constituent parts which in turn initiate further signal transduction. In vertebrates, one target of G-proteins is the enzyme adenyl cyclase which converts ATP, the prevelent store of energy of the cell, into the second messenger, cyclic AMP.

We are thus brought by a second route to Rutabaga, an adenyl cyclase of the fly, and its potential role in transmitting G-protein coupled receptor signaling to activate cell responses. How does all this lead to learning? This is discussed in the dunce and rutabaga sites and at sites for other genes involved in the cAMP second messenger pathway.

Just as the cyclic AMP pathway is involved in learning, it is also involved in determining the strength of the neuromuscular junction. In fact, the neuromuscular junction, because of its accessability and the ease with which is observed, is used as a model system for studying the synaptic changes that take place on learning. For more information about the neuromuscular junction, see the Dunce, Fasciclin II, Discs large, MEF2 and Rutabaga sites.

The fruit fly Drosophila melanogaster has been a popular model to study cAMP signaling and resultant behaviors due to its powerful genetic approaches. All molecular components (AC, PDE, PKA, CREB, etc) essential for cAMP signaling have been identified in the fly. Among them, adenylyl cyclase (AC) gene rutabaga and phosphodiesterase (PDE) gene dunce have been intensively studied to understand the role of cAMP signaling. Interestingly, these two mutant genes were originally identified on the basis of associative learning deficits. Findings on the role of cAMP in Drosophila neuronal excitability, synaptic plasticity and memory are summarized in this review. It mainly focuses on two distinct mechanisms (global versus local) regulating excitatory and inhibitory synaptic plasticity related to cAMP homeostasis. This dual regulatory role of cAMP is to increase the strength of excitatory neural circuits on one hand, but to act locally on postsynaptic GABA receptors to decrease inhibitory synaptic plasticity on the other. Thus the action of cAMP could result in a global increase in the neural circuit excitability and memory. Implications of this cAMP signaling related to drug discovery for neural diseases are also described (Lee, 2015; Full text online).

The balance among different subtypes of glutamate receptors (GluRs) is crucial for synaptic function and plasticity at excitatory synapses. This study shows that the two subtypes of GluRs (A and B) expressed at Drosophila neuromuscular junction synapses mutually antagonize each other in terms of their relative synaptic levels and affect subsynaptic localization of each other. Upon temperature shift-induced neuromuscular junction plasticity, GluR subtype A increased but subtypeB decreased with a timecourse of hours. Inhibition of the activity of GluR subtype A led to imbalance of GluR subtypes towards more GluRIIA. To gain a better understanding of the signalling pathways underlying the balance of GluR subtypes, an RNA interference screen of candidate genes was performed and postsynaptic-specific knockdown of dunce, which encodes cAMP phosphodiesterase, was found to increase levels of GluR subtype A but decreased subtype B. Furthermore, bidirectional alterations of postsynaptic cAMP signalling resulted in the same antagonistic regulation of the two GluR subtypes. These findings thus identify a direct role of postsynaptic cAMP signalling in control of the plasticity-related balance of GluRs (Zhao, 2020).

A negative correlation between subtype A and B receptors has been reported previously at Drosophila NMJs. However, the mechanism by which the antagonistic balance of different subtypes of GluRs is regulated remains unclear. The present study revealed that bidirectional alterations of cAMP levels in the postsynaptic muscle cells alter the balance of GluR subtypes in a cell-autonomous manner. This study thus provides new insights into the mechanism underlying synaptic plasticity by altering the balance of GluR subtypes (Zhao, 2020).

Most previous conventional microscopy studies have reported substantial colocalization or differential localization of GluRIIA and GluRIIB. This study reports an apparently non-overlapping localization of GluRIIA and GluRIIB at the postsynaptic densities (PSDs) of NMJ synapses (see The subtype B forms a doughnut-shaped ring, with a smaller subtype A ring in the centre). Although clear interpretations are lacking for the distinct localization of GluRIIA and GluRIIB at PSDs, there could be two possibilities: either different classes of receptors might be associated with specific interacting proteins that could mediate, directly or indirectly, the concentric localization of GluR subtypes at PSDs or concentric rings of GluR subtypes A and B in wild-type larvae might associate with their specific biophysical properties. Desensitization is the process by which receptors are inactivated in the prolonged presence of an agonist; it occurs faster in response to a lower concentration of agonist. On the postsynaptic side, GluR subtype A exhibits slower desensitization kinetics than GluR subtype B. It is therefore speculated that the slower desensitization of subtype A receptors might be caused, in part, by a higher concentration of glutamate released on the presynaptic side, because subtype A rings are more closely juxtaposed to presynaptic Cacophony calcium channels than subtype B rings (Zhao, 2020).

This study showed that subtype A rings become enlarged (both the inner and outer ring diameters are increased) when the synaptic levels of GluRIIA are increased, whereas subtype B rings are enlarged in a specific manner, i.e., the inner diameter decreases, but the outer diameter of the ring increases when the level of GluRIIB is increased. A simple explanation for the enlarged GluR rings might therefore be increased synaptic levels of GluRIIA or GluRIIB. Given that GluR-enriched PSDs are confined to specific spatial domains by cell adhesion molecules and the spectrin-actin network, it is possible that an increase in the level of one subtype of GluR might take up the space left by a reduced level of the other (Zhao, 2020).

Synaptic plasticity is the ability of synapses to strengthen or weaken over time, in response to increases or decreases in their activity. It is well established that GluRs are involved in synaptic plasticity at excitatory synapses. However, it is not entirely known how different types or subtypes of GluRs are involved in synaptic plasticity. Drosophila glutamatergic NMJs with two subtypes of GluRs, rather than mammalian NMJs with multiple subtypes of GluRs, are an effective model for studying synaptic plasticity. Hyperexcitable double mutants of eag sh show persistent strengthening of larval NMJs, which represents long-term plasticity. This study found that increased presynaptic release by warm-activated TrpA1 led to increased GluRIIA but normal GluRIIB, which was consistent with increased GluRIIA in eag sh double mutants. In addition, increased GluRIIA but decreased GluRIIB were observed in high temperature-induced synaptic plasticity of long-term strengthening of neurotransmission. Thus, it is speculated that increased presynaptic release might result in long-term plasticity by enhancing postsynaptic responses through increased GluRIIA (Zhao, 2020).

Increased GluRIIA is consistently observed in different models of synaptic plasticity. However, this study observed normal and reduced GluRIIB in TrpA1- and high temperature-induced synaptic plasticity, respectively. The discrepant changes in GluRIIB levels in different models of synaptic plasticity could be caused by different timescales, such as a limited time (8 h) for elevating presynaptic release through activating TRPA1 versus 4 days for raising larvae at 27°C, or the change in the level of GluRIIB might be too low to be detected by overexpressing TRPA1, or both (Zhao, 2020).

Whether the antagonistic balance of GluRs is actively (as a functional requirement) or passively (as a physical competition) regulated depends on specific conditions. It appeared that GluRIIA and GluRIIB competed with each other for the essential subunits when the expression levels of either GluRIIA or GluRIIB were changed, consistent with previous reports. These results support a passive competition between GluRIIA and GluRIIB. However, an actively regulated antagonistic balance of GluRs also occurs. When the essential subunit GluRIIC, GluRIID or GluRIIE was limited, both GluRIIA and GluRIIB decreased. If only passive regulation of GluRIIA and GluRIIB occurs, GluR subtype A and B decreased at similar levels would be expected. However, the ratio of GluRIIA to GluRIIB increased, indicating that the GluRIIA subtype is maintained preferentially when the total GluRs are limited and supporting an active regulation of the balance between GluRIIA and GluRIIB. Given that GluRIIA is mainly responsible for the postsynaptic responses, the relative increase in GluRIIA when an essential subunit of GluRs was knocked down might be a functional compensation for the decrease of synaptic strength (Zhao, 2020).

In addition to the antagonism of GluRIIA and GluRIIB reported in this study, there are a few reports on the regulation of synaptic levels of single GluR subunits. For example, GluRIIA but not GluRIIB receptors are anchored at the PSD by the actin-associated Coracle (Chen, 2005) and are regulated by a signalling pathway involving the Rho-type GEF (Pix) and its effector, Pak kinase (Albin, 2004). Recent studies also showed specific upregulation of GluRIIA but not GluRIIB when the calcium-dependent proteinase calpains were mutated (Zhao, 2020).

A previous study showed that the numbers of terminal varicosities and branches were increased in dnc but not rut mutants. Given that elevated cAMP levels induced an antagonistic balance of GluRs at the postsynaptic side, it was important to test whether the antagonistic balance of GluRs was associated with NMJ overgrowth. The results showed that the number of varicosities remained normal when dnc or rut was knocked down by RNAi in the postsynaptic muscles, suggesting that an alteration in the cAMP pathway at the postsynaptic side did not affect NMJ development (Zhao, 2020).

The importance of the GluR subtype balance in synaptic plasticity has been documented in mammals. The major forms of AMPA receptors in the hippocampus include GluA1/2 and GluA2/3 heteromers, in addition to GluA1 homomers. The relative abundance of GluA1- and GluA2-containing receptors is a well-established determinant of synaptic plasticity in diverse brain circuits; GluA1-containing receptors are recruited to synapses after long-term potentiation, whereas GluA2-containing receptors are required for long-term depression. Together with the mammalian findings, the results support the notion that the GluR subtype balance contributes to synaptic plasticity at excitatory synapses (Zhao, 2020).

It is widely known that cAMP signalling plays an important role in regulating synaptic plasticity by increasing presynaptic neurotransmitter release. However, it is not known whether the cAMP pathway acts postsynaptically in regulating the ratio of GluRs, which plays a crucial role in synaptic plasticity. In the present study, we showed, for the first time, that the cAMP pathway regulates the balance of different GluR subtypes on the postsynaptic side; either increased or reduced cAMP leads to an altered ratio of GluR subtypes at Drosophila NMJ synapses. Thus, an optimal level of cAMP in postsynaptic muscles might be required for the normal ratio of synaptic GluR subtypes (Zhao, 2020).

When cAMP levels are elevated, cAMP binds to the regulatory subunits of PKA and liberates catalytic subunits that then become active. Active PKA in muscles decreases the activity of GluRIIA in Drosophila (Davis, 1998). Thus, an increase in the level of synaptic GluRIIA might compensate for the reduced activity of GluRIIA caused by overexpression of wild-type or constitutively active PKA. Conversely, inhibition of PKA activity in muscles causes a significant increase in the average amplitude of miniature excitatory junctional currents, consistent with the notion that PKA negatively regulates the activity of GluRIIA. Surprisingly, an increase was observed in synaptic GluRIIA when the cAMP level was downregulated in rut mutants or when PKA was knocked down by RNAi. It appears that the negative regulation of GluRIIA activity by PKA is not sufficient to account for the increase of GluRIIA at NMJ synapses (Zhao, 2020).

Analysis of western blots showed that the protein level of GluRIIA increased significantly, regardless of whether postsynaptic cAMP pathway was up- (dnc RNAi and PKAOE) or downregulated (rut RNAi and PKA RNAi), suggesting that the similar antagonistic balance of GluR subtypes induced by both up- and downregulation of cAMP might be caused by an elevated protein level of GluRIIA the cAMP pathway regulates the antagonism between GluRIIA and GluRIIB at two distinct steps, GluRIIA activity and protein level. Exactly how bidirectional changes of cAMP lead to a similar alteration of GluR subtypes remains to be investigated (Zhao, 2020).

Although Dnc and Rut regulate cAMP levels in opposite directions, physiological studies in Drosophila have shown that activity-dependent short-term plasticity is altered in a similar manner at larval NMJs in both dnc and rut mutants. Specifically, synaptic facilitation and post-tetanic potentiation are both weakened, indicating that the bidirectional change of cAMP signalling might result in similar abnormalities in synapse plasticityn. The mechanisms underlying synaptic facilitation and post-tetanic potentiation are exclusively presynaptic. Synaptic facilitation and post-tetanic potentiation both result from increased presynaptic calcium concentrations, leading to an enhanced release of neurotransmitters. A bell-shaped model was proposed to explain this mode of regulation, i.e. mutations in dnc and rut, which regulate cAMP levels in opposite directions, result in a similar plasticity phenotype. It is proposed that the bell-shaped model might also explain a similar increase in GluRIIA at NMJ synapses caused by bidirectional changes in cAMP levels in postsynaptic muscles (Zhao, 2020).

The antagonistic balance of GluRIIA and GluRIIB is induced by the postsynaptic cAMP/PKA pathway. However, whether the antagonism between GluRIIA and GluRIIB requires the cAMP/PKA pathway is unclear. GluRIIA or GluRIIB nulls were recombined with postsynaptic RNAi knockdown of PKA (i.e. inhibition of the cAMP pathway). It is noted that PKA null mutants are lethal at the first larval stage and thus cannot be used for the genetic interaction assay. Compared with simple null mutants of GluRIIA (or GluRIIB), PKA RNAi in the mutant background of GluRIIA (or GluRIIB) did not change the synaptic levels of GluRIIB (or GluRIIA), suggesting that the antagonistic balance of GluRIIA and GluRIIB does not require the cAMP pathway at the postsynaptic side. Thus, an altered cAMP pathway leads to the antagonistic balance of GluRIIA and GluRIIB, but the antagonistic balance of GluRIIA and GluRIIB appears not to be dependent on the cAMP pathway, at least for the antagonism induced by null mutations of GluRIIA or GluRIIB, or the remaining PKA upon RNAi knockdown is sufficient to support the antagonistic balance of GluRIIA and GluRIIB (Zhao, 2020).

It will be of great interest to determine how the cAMP-PKA-GluR signalling pathway acts on the postsynaptic side to contribute to synaptic plasticity and whether this pathway is also effective and conserved in mammalian central synapses (Zhao, 2020).

The fruit fly can discriminate and remember visual landmarks. It analyses selected parts of its visual environment according to a small number of pattern parameters such as size, colour or contour orientation, and stores particular parameter values. Like humans, flies recognize patterns independently of the retinal position during acquisition of the pattern (translation invariance). The central-most part of the fly brain, the fan-shaped body, contains parts of a network mediating visual pattern recognition. Short-term memory traces have been identified of two pattern parameters -- elevation in the panorama and contour orientation. These can be localized to two groups of neurons extending branches as parallel, horizontal strata in the fan-shaped body. The central location of this memory store is well suited to mediate translational invariance (Liu, 2006).

A fly tethered to a torque meter, with its head (and hence its eyes) fixed in space, can control its orientation with respect to the artificial scenery in a flight simulator. In this set up, the fly is conditioned to avoid certain flight directions relative to virtual landmarks and recognizes these visual patterns for up to at least 48 h. Visual pattern recognition in Drosophila has been studied in some detail. Flies store values of at least five pattern parameters: size, colour, elevation in the panorama, vertical compactness, and contour orientation. Moreover, they memorize spatial relations between parameter values. The neuronal substrate underlying visual pattern recognition is little understood in any organism (Liu, 2006).

In Drosophila, memory traces can be localized to groups of neurons in the brain. Using the enhancer GAL4/UAS expression system, short-term memory traces of aversive and appetitive olfactory conditioning have been assigned to output synapses of subsets of intrinsic neurons of the mushroom bodies (MBs). The Rutabaga protein -- a type 1 adenylyl cyclase that is regulated by Ca²⁺/Calmodulin and G protein, and is considered a putative convergence site of the unconditioned and conditioned stimulus in olfactory associative learning, selectively restores olfactory learning if expressed in these cells in an otherwise rutabaga (rut)-mutant animal. Moreover, expressing a mutated constitutively activating Galpha_s protein (Galpha_s*) in the MBs interferes with olfactory learning. Blocking the output from these neurons during memory retrieval has the same effect, while blocking it during acquisition has no effect. Interestingly, memory traces for other learning tasks seem to reside in other parts of the brain: for remembering its location in a dark space, the fly seems to rely on a rut-dependent memory trace (Zars, 2000) in neurons of the median bundle and/or the ventral ganglion (Liu, 2006).

The present study localizes short-term memory traces for visual pattern recognition to the fan-shaped body (FB), the largest component of the central complex (CX; also called the central body in other species). The CX is a hallmark of the arthropod brain. It has been characterized functionally as a pre-motor centre with prominent, but not exclusive, visual input. In the locust, large-field neurons sensitive to the e-vector orientation of polarized light have been described in the CX. Because of its repetitive structure and the precisely ordered overlay of fiber projections from the two hemispheres in the FB, neighbourhood relations of visual space might still be partially preserved at this level (retinotopy). Using the genetic approach, this study shows that a small group of characteristic stratified neurons in the FB house a memory trace for the pattern parameter 'elevation', and a different set of neurons forming a parallel stratum contain a memory trace for 'contour orientation' (Liu, 2006).

Of ten mutants with structural abnormalities in the CX, all were impaired in visual pattern recognition. They were able to fly straight and to avoid heat, yet they failed to remember the patterns. Did they really lack the memory or had they lost their ability to discriminate between patterns? Fortunately, individual flies often display spontaneous preferences for one of the patterns. In three lines, these preferences were consistent enough to reveal intact pattern discrimination, suggesting that aberrant circuitry of the central complex can affect visual learning independent of visual pattern discrimination (Liu, 2006).

Since the developmental and structural defects in these mutants are not well characterized, the GAL4/UAS system was used to acutely interfere with CX function. A GAL4 driver line (c205-GAL4) was used with expression in parts of the CX and, the gene for tetanus toxin light chain (CntE) was used as the effector. CntE blocks neurons by cleaving neuronal Synaptobrevin, a protein controlling transmitter release. For temporal control, the temperature-sensitive GAL4-specific silencer GAL80 was added under the control of a tubulin promoter (tub-GAL80^ts). Flies (UAS-CntE/+; tub-GAL80^ts/c205-GAL4) were raised at 19 °C, and were transferred for 14 h to the restrictive temperature (30 °C) just before the behavioural experiment to induce GAL4-driven toxin expression. Flies kept at the low temperature showed normal memory scores, while after inactivation of GAL80^ts no pattern memory was observed. Again, flight control and heat avoidance were normal, and Fourier analysis confirmed that flies at the high temperature had retained their ability to tell the patterns apart. As with the structural mutants, interrupting the circuitry of the CX by tetanus toxin expression seemed to specifically interfere with visual pattern memory. In addition, the use of tub-GAL80^ts excluded the possibility that toxin expression in unknown tissues during development might cause the memory impairment in the adult. These results do not, as yet, address the question of memory localization (Liu, 2006).

Visual pattern memory in the flight simulator requires an intact rut gene. Mutant rut flies (rut²⁰⁸⁰) showed normal visual flight control, heat avoidance and pattern discrimination. To confirm that the defect was indeed due to the mutation in the rut gene rather than an unidentified second-site mutation, rut was rescued by the expression of the wild-type rut cDNA (UAS-rut⁺) using the pan-neuronally expressing driver line elav-GAL4. Indeed, flies of the genotype rut²⁰⁸⁰/Y;elav-GAL4/UAS-rut⁺ have normal memory (Liu, 2006).

Visual pattern memory in the flight simulator has been shown to depend upon at least two kinds of behavioural plasticity: (1) an associative classical (pavlovian) memory trace is formed linking a particular set of values of pattern parameters to heat; (2) the fly's control of the panorama operantly facilitates the formation of this memory trace (Brembs, 2000). Either of the two processes might depend upon the Rut cyclase (Liu, 2006).

To address this issue, rut mutant flies were tested in a purely classical variant of the learning paradigm. During training, panorama motion was uncoupled from the fly's yaw torque and the panorama was slowly rotated around the fly. Heat was made contingent with the appearance of the 'punished' pattern in the frontal quadrant of the fly's visual field. All other parameters were kept as described. For testing memory, panorama motion was coupled again to yaw torque and the fly's pattern preference was recorded as usual. Even in the absence of operant facilitation, visual pattern memory required the intact rut gene. Therefore, the rut-dependent memory trace investigated in this study represents the association of a property of a visual pattern with the reinforcer (Liu, 2006).

As a first step in localizing the memory trace, it was asked in which neurons of the rut mutant expression of the wild-type rut gene would be sufficient to restore learning. To this end, a total of 27 driver lines expressing GAL4 in different neuropil regions of the brain was used to drive the UAS-rut⁺ effector gene in the rut mutant background. The parameter 'elevation' was measured. With seven of the driver lines, pattern memory was restored (104y, 121y, 154y, 210y, c5, c205 and c271) (Liu, 2006).

Comparison of the expression patterns of the 27 lines allowed the putative site of the memory trace to be narrowed down to a small group of neurons in the brain. The seven rescuing lines all showed transgene expression in a stratum in the upper part of the FB. In three of them staining is rather selective. It comprises, in addition to the FB, only a layer in the medulla, several cell clusters in the subesophageal ganglion and a few other scattered neurons (Liu, 2006).

Evidently, rut⁺ expression in the MBs is neither necessary (104y, c5, c205, 154y) nor sufficient for rescue. This result is in line with the earlier observation that elimination of more than 90% of the MBs by hydroxyurea treatment of first-instar larvae has no deleterious effect on visual pattern memory. The MBs were ablated in one group of rescue flies (rut²⁰⁸⁰/Y;UAS-rut⁺/ +;c271/+). They showed full visual pattern memory (Liu, 2006).

Although GAL4 expression in the optic lobes is prominent in all seven rescuing lines, it occurs in distinctly different layers that do not overlap. For instance, in 104y expression is restricted to layer 2, whereas in 210y it is found only in the serpentine layer (layer 7). A similar situation is found for the s ganglion, although there the staining patterns are more difficult to evaluate. Finally, expression in the ellipsoid body is again not necessary (104y, c5, c205, 154y) or sufficient (c232, 78y, 7y, and so on) for rescue. Thus, the expression patterns favour the conclusion that the neurons of the upper stratum of the FB might be the site of the memory trace for the parameter 'elevation' in visual pattern memory (Liu, 2006).

Neurons in this stratum, labelled in all seven rescuing lines, have a very characteristic shape. Their cell bodies are located just lateral to the calyces. Their neurites run slightly upward in an antero-medial direction, forming an upward-directed tufted arborization just behind the alpha/alpha'-lobe of the MB. From there, the fiber turns sharply down and backward towards the midline just in front of the FB. Finally, it turns horizontally backward, spreading as a sharp stratum through all of the FB across the midline. These neurons have been described in Golgi preparations. They belong to a larger group of tangential FB neurons called F neurons. Besides the stratum in FB, most of them have an arborization in a particular part of the unstructured neuropil. The layer stained in 104y, and the other six rescuing lines, is tentatively classify as layer 5 (from bottom upward), and hence provisionally the neurons are called F5, although, without further markers, it is difficult to reliably number the layers. In summary, expression of Rut cyclase in F5 neurons rescues the rut-dependent memory defect for pattern elevation, whereas no rescue effect is observed in any of 20 strains without expression of Rut cyclase in F5 neurons (though Rut cyclase was expressed in other regions of the brain). Hence, a rut-dependent memory trace for pattern elevation may reside in F5 neurons (Liu, 2006).

This finding does not exclude the possibility that memory is redundant, and that other rut-dependent memory traces for pattern elevation might be found elsewhere. Therefore, it was asked whether plasticity in the F5 neurons is necessary for visual pattern memory. The Rut cyclase is regulated by G protein signaling, and olfactory learning/memory can be blocked by a constitutively active form of the Galpha_s protein subunit (Galpha_s*). The Galpha_s* mutant protein was expressed in the FB using the driver line c205, and the flies were tested for their memory of 'elevation'. Memory was fully suppressed. Since in olfactory learning, overexpression of the wild-type protein does not interfere with learning, these results support the hypothesis that continuous upregulation of Rut cyclase in the F5-neurons interferes with visual short-term memory, implying that F5 neurons are the only site of a rut-dependent memory trace for pattern elevation (Liu, 2006).

The patterns used in the experiments so far exclusively addressed the parameter 'elevation' (upright and inverted Ts or horizontal bars at different elevations). It was of interest to discover whether the mutant defect in rut and the Rut rescue in the F5 neurons affects only this parameter, or whether it applies to other pattern parameters as well. Therefore, the study looked at to two further parameters: 'size' and 'contour orientation'. Three driver lines -- c205, NP6510 and NP2320 -- were chosen showing different expression patterns in the FB. In the line NP6510, as in c205, a group of F neurons is marked. They are putatively classified as F1, since their horizontal stratum lies near the lower margin of the FB. Their cell bodies form a cluster in the dorso-frontal cellular cortex above the antennal lobes. Like the F5 neurons, they have large arborizations in the dorsal unstructured neuropil. The line NP2320 expresses the driver in columnar neurons running perpendicular to the strata of F neurons, with their cell bodies scattered singly or in small groups between the calyces. Since they seem to have no arborizations outside the FB, they are tentatively classified as pontine neurons (Liu, 2006).

Initially, it was shown that pattern memory requires the rut gene for each of the three parameters. Next, the Rut rescue flies were studied (for example, rut²⁰⁸⁰/Y;c205/UAS-rut⁺). In the line c205, memory was restored only for 'elevation', not for 'size' or 'contour orientation'. Correspondingly, the memory impairment by expression of dominant-negative Galpha_s* in this driver line should be specific for 'elevation', as is indeed the case. With the driver line NP6510, memory was not restored for either 'elevation' or for 'size,' but memory was restored for 'contour orientation'. The third driver line, NP2320, labelling columnar neurons of the FB, did not restore the memory for any of the three pattern parameters. Among the 27 GAL4 lines, a second was found with a very similar expression pattern as NP6510 (NP6561). The P-element insertions in the two lines are only 124 nucleotides apart from each other. Like NP6510, NP6561 restores the memory for 'contour orientation' but not for 'size' or 'elevation'. These results strongly suggest that memory traces for distinct visual pattern parameters are located in different parts of the FB, and that, in addition to the memory trace in F5 neurons, a memory trace for the parameter 'contour orientation' is located in F1 neurons (Liu, 2006).

A pertinent question in rescue experiments is whether the rescue is due to the provision of an acute function in the adult or to the avoidance of a developmental defect. Therefore, the tub-GAL80^ts transposon was added to the system. The driver lines c205 and NP6510 were chosen. Groups of adult males (for example, rut²⁰⁸⁰/Y;+/tub-GAL80^ts;NP6510/UAS-rut⁺), raised at 19°C, were kept as adults for 14 h at 19°C or 30°C. Afterwards, pattern memory for the corresponding pattern parameter was tested. In both cases, flies that had been kept at 30°C showed normal memory, indicating that Rut cyclase induced just a few hours before the experiment had restored an immediate neuronal function rather than preventing a developmental defect. This conclusion was further supported by the finding that Galpha_s* expression in the adult (using tub-GAL80^ts) was sufficient to disrupt memory (Liu, 2006).

Several conclusions can be drawn from the above results. Memory traces in Drosophila are associated with specific neuronal structures: odor memories with the MBs, visual memories with the CX, and place memory (tentatively) with the median bundle. Memory traces are not stored in a common all-purpose memory centre. Even within the visual domain, memories for distinct pattern parameters are localized within distinct structures: a rut-dependent short-term memory trace for the pattern parameter 'elevation' to F5 neurons, and a corresponding memory trace for 'contour orientation' to F1 neurons. Moreover, if the constitutively activating Galpha_s* protein indeed interferes with the regulation of Rut cyclase, it follows that the brain contains no other redundant rut-dependent memory traces for these pattern parameters. The Rut-mediated plasticity is necessary and sufficient, at least in F5 neurons. As in the earlier examples, the memory traces are confined to relatively small numbers of neurons. At least in flies, and probably in insects in general, memory traces appear to be part of the circuitry serving the respective behaviour (Liu, 2006).

This study provides a first glimpse of the circuitry within a neural system for visual pattern recognition. Though the picture is far from complete, it invites (and may guide) speculation. The FB is a fiber matrix of layers, sectors and shells. The F1- and F5-neurons form two sharp parallel horizontal strata in this matrix. If the width of the FB represents the azimuth of visual space as has been proposed, the horizontal strata of the F neurons would be well suited to mediate translation invariance. In any case, it is satisfying to find a translation invariant memory trace in the CX where visual information from both brain hemispheres converges. These first components of the circuitry may encourage modelling efforts for pattern recognition in small visual systems (Liu, 2006).

Memories are formed, stabilized in a time-dependent manner, and stored in neural networks. In Drosophila, retrieval of punitive and rewarded odor memories depends on output from mushroom body (MB) neurons, consistent with the idea that both types of memory are represented there. Dorsal Paired Medial (DPM) neurons innervate the mushroom bodies, and DPM neuron output is required for the stability of punished odor memory. Stable reward-odor memory is also DPM neuron dependent. DPM neuron expression of amnesiac (amn) in amn mutant flies restores wild-type memory. In addition, disrupting DPM neurotransmission between training and testing abolishes reward-odor memory, just as it does with punished memory. DPM-MB connectivity was examined by overexpressing a DScam variant that reduces DPM neuron projections to the MB α, β, and γ lobes. DPM neurons that primarily project to MB α′ and β′ lobes are capable of stabilizing punitive- and reward-odor memory, implying that both forms of memory have similar circuit requirements. Therefore, these results suggest that the fly employs the local DPM-MB circuit to stabilize punitive- and reward-odor memories and that stable aspects of both forms of memory may reside in mushroom body α′ and β′ lobe neurons (Keene, 2006).

It is widely believed that memory is encoded as changes in synaptic efficacy between neurons in a network. This concept of synaptic plasticity predicts that it will be possible to localize memory to discrete synapses in neural networks in the brain. The relatively small brains of insects are well suited to this endeavor, and genetic manipulation in the fruit fly Drosophila has greatly aided neural circuit mapping of odor memory. Flies can be taught to associate an odor conditioned stimulus (CS) with either a punitive electric shock or a rewarding sugar unconditioned stimulus (US). Strikingly, learning and memory with these opposing unconditioned stimuli requires differential transmitter involvement: sugar-rewarded odor memory is dependent on intact octopamine signaling (see Tyramine β hydroxylase), while shock-punished (punitive) odor memory is dependent on dopamine signaling. However, despite the differential requirement for these monoamine transmitters, blocking MB output during retrieval impairs both punitive- and reward-odor memories, implying that these memories rely on overlapping brain regions. Stability of reward-odor memory is reliant on the same MB extrinsic neurons that are required for stability of punitive-odor memory (Keene, 2006).

amnesiac mutant flies can associate odors with a punitive or a rewarding US, but they quickly forget this information, which suggests that amn might be generally involved in memory. The amn gene is expressed throughout the brain and strongly in Dorsal Paired Medial neurons—two large modulatory neurons that appear to ramify throughout the approximately 5000 neurons of the MBs. Prolonged DPM neuron output is required for the stability of punitive-odor memories. Since DPM neurons heavily ramify in the MBs, these data support the importance of the MB as a crucial locus for memory and also suggested that the neural network involving MB and DPM neurons could be critical for all MB-dependent memory. Therefore whether the circuitry involving DPM neurons was involved in the stability of rewarded olfactory memory was tested (Keene, 2006).

It was first confirmed that amn mutant flies have a memory defect when conditioned with odors and sugar reward. A modified protocol was used that more closely resembles the odor-shock conditioning protocol and that produces robust memory that lasts for more than 6 hr. In brief, approximately 100 starved flies were exposed to an odor for 2 min in the absence of sugar, followed by a clean air stream for 30 s and a second odor with sugar reward for 2 min. Olfactory memory was tested 3, 60, 180, and 360 min after training. Flies homozygous for the strong amn alleles—amn¹ or amn^X8—learn to associate the appropriate odor with sugar reward (they have a small but significant initial performance defect), but they forget this association within 60 min of training. These data are consistent with the earlier report that amn¹ flies have defective reward-odor memory (Keene, 2006).

Since amn mutant flies forget quickly when trained with either a punitive or a rewarding US, it was of interest to see whether similar neural circuitry was involved in both types of memory. Although the amn gene is expressed throughout the brain, expressing the amn gene in DPM neurons restores punitive odor memory performance to amn mutant flies. Therefore whether restoring amn expression in DPM neurons of amn mutant flies would rescue the reward-odor memory defect was tested. The c316 {GAL4} line was used to transgenically express the amn gene in DPM neurons of amn mutant flies. 3 hr memory of amn^X8/amn¹;c316/uas-amn and amn¹;c316/uas-amn flies was similar to wild-type flies and was statistically different from the memory of amn^X8 and amn¹; uas-amn mutant flies. These data demonstrate that amn expression in DPM neurons is sufficient to restore reward-odor memory to amn mutant flies and suggest that DPM neurons are generally critical for olfactory memories (Keene, 2006).

Next an acute role of DPM neurons in reward-odor memory was directly tested by temporally blocking their output during the course of the experiment. The dominant temperature-sensitive shibire^ts1 transgene was tested in DPM neurons by using the c316{GAL4} and Mz717{GAL4} drivers and a sugar reward conditioning experiment was performed at either the permissive (25°C) or the restrictive (31°C) temperature. At the restrictive temperature, shibire^ts1 blocks vesicle recycling and thereby blocks synaptic vesicle release. At 25°C, reward-odor memory of c316; uas-shi^ts1 and Mz717; uas-shi^ts1 flies was comparable to memory of wild-type and uas-shi^ts1 flies. However, at 31°C, memory of c316; uas-shi^ts1 and Mz717; uas-shi^ts1 flies was statistically different from wild-type and uas-shi^ts1 flies. Therefore, DPM synaptic release is necessary for stable reward-odor memory as it is with punitive-odor memory (Keene, 2006).

Stable punitive-odor memory requires prolonged DPM output between acquisition and retrieval, and DPM output is dispensable during training and testing. Therefore whether DPM neurons were similarly required for reward-odor memory was tested. Again DPM output was blocked by expressing uas-shi^ts1 with c316{GAL4}, but this time the inactivation was restricted to either the training, testing, or storage period. Blocking DPM neurons during acquisition did not produce memory loss; memory of c316; uas-shi^ts1 flies was comparable to wild-type and uas-shi^ts1 flies. Similarly, DPM neuron output was not required during memory retrieval; memory of c316; uas-shi^ts1 flies was comparable to wild-type and uas-shi^ts1 flies. However, blocking DPM output for 30 min after training significantly reduced reward-odor memory; memory of c316; uas-shi^ts1 flies is statistically different from wild-type and uas-shi^ts1 flies. These data parallel results with punitive-odor memory and suggest that there is a similar requirement for DPM neuron output to stabilize both punitive- and reward-odor memory. DPM block from 30 to 60 min after training decreased punitive-odor memory similar to a 0–30 min block. However, disrupting DPM neuron output from 30 to 60 min had an insignificant effect on reward-odor memory. These data imply that the role of DPM neurons is diminished at 30–60 min for reward-odor memory. The Mz717 driver was used to increase the confidence that the temporal uas-shi^ts1 disruptive effect can be ascribed to blocking DPM neurons. Blocking DPM output for 60 min after training with Mz717 significantly reduced reward-odor memory. Memory of Mz717; uas-shi^ts1 flies is statistically different from wild-type and uas-shi^ts1 flies (Keene, 2006).

DPM neurons innervate all the lobes of the MBs, and previous imaging studies suggest that the DPM projections there may be both transmissive and receptive. In addition, expression of n-syb::GFP in DPM neurons has been reported to label DPM projections to the MB lobes. In an attempt to gain further insight into DPM neuron organization, a collection of pre- and postsynaptic compartment markers was overexpressed in DPM neurons. However, no clear evidence was seen for asymmetry within DPM neurons or between projections to individual MB lobes. Therefore, understanding DPM polarity and organization will require further work (Keene, 2006).

During this analysis, it was found that expression of the DScam17-2::GFP fusion protein (which has been described to label the presynaptic compartment when overexpressed in certain neurons, in DPM neurons, with c316{GAL4}, affected DPM neuron development and resulted in DPM neurons that predominantly project to the MB α′ and β′ lobe subsets. Coexpressing uas-DScam17-2::GFP and uas-CD2 or uas-lacZ in DPM neurons reveals that DScam17-2::GFP labels the remaining projections rather than a subset of existing projections. To identify projections to MB α/β neurons versus α′/β′ neurons, brains were costained with anti-FASII, which labels α/β and γ neurons , and anti-TRIO, which labels α′/β′ and γ neurons (Keene, 2006).

A functional role for the MB α′ and β′ lobes in memory has not been reported. Therefore, uas-DScam17-2::GFP; c316 flies were used to assess the role of DPM neuron projections to the MB α′ and β′ lobe subset in punitive- and reward-odor memory. Heterozygous uas-DScam17-2::GFP flies were included as a control as well as wild-type and amn^X8 flies for comparison. The presence of the uas-DScam17-2::GFP transgene did not significantly affect punitive-odor memory. Remarkably, DPM neurons that primarily project to the α′ and β′ lobe subsets retain punitive-memory function. Memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amn^X8 flies. Therefore, DPM neuron projections to the α′ and β′ lobes of the MB are apparently sufficient for punitive-odor memory. Next the function of uas-DScam17-2::GFP; c316 flies in reward-odor memory was tested. Again, memory of uas-DScam17-2::GFP; c316 flies was similar to memory of uas-DScam17-2::GFP flies and was significantly greater than that of amn^X8 flies. These data indicate that the DPM neuron projections to the α′ and β′ MB lobe subsets are also apparently sufficient for reward-odor memory and imply that the circuit requirements for the stability of rewarded and punished odor memory are very similar. Although redundancy of DPM projections or retention of a few critical projections to the α, β, and γ lobes cannot currently be ruled out, these data are consistent with the notion that DPM projections to the α′ and β′ MB lobes are sufficient for stabilizing memory. The data also suggest that DScam may play a role in wiring the DPM-MB circuit (Keene, 2006).

In Drosophila, there is a striking dissociation of monoamine transmitters for reward and punishment. Dopamine is required for aversive-odor memory formation, whereas octopamine is necessary for appetitive-odor memory. Octopaminergic and dopaminergic neurons project throughout the brain including to the MBs. Although it is not known whether the MB arborization of these monoaminergic neurons is required for odor memories, blocking MB output is required to retrieve both aversive- and appetitive-odor memory (Keene, 2006).

DPM neurons ramify throughout the MB lobes and provide a general stabilizing mechanism for both punitive- and reward-odor memory. This DPM neuron analysis enhances resolution of memory processing and provides further weight to the idea that components of both punitive- and reward-odor memory reside at synapses within MB neurons. Imaging studies suggest that DPM neurons are both receptive and transmissive to MB neurons, and a model is favored where DPM neurons represent recurrent feedback neurons that consolidate conditioned changes in synaptic weight in MB neurons. However, if MB neurons provide drive to DPM neurons, one would expect MB neuron output and DPM neuron output to have similar temporal requirements. Current published data conclude that MB neuron output is not required during memory storage (but is exclusively required for retrieval) when DPM neuron output is required. However, the work described here suggests a role for MB α′ and β′ lobe neurons in memory stability, and it is noteworthy that MB studies did not employ GAL4 drivers with extensive expression in MB α′/β′ neurons. Further detailed analysis of the role of MB α′/β′ neurons in memory should resolve this conundrum (Keene, 2006).

Memory consolidation is a time-dependent process occurring over hours, days, or longer in different species and requires protein synthesis. An apparent exception is a memory type in Drosophila elicited by a single olfactory conditioning episode, which ostensibly consolidates quickly, rendering it resistant to disruption by cold anesthesia a few hours post-training. This anesthesia-resistant memory (ARM), is independent of protein synthesis. Protein synthesis independent memory can also be elicited in Drosophila by multiple massed cycles of olfactory conditioning, and this led to the prevailing notion that both of these operationally distinct training regimes yield ARM. Significantly, this study shows that, unlike bona fide ARM, massed conditioning-elicited memory remains sensitive to the amnestic treatment two hours post-training and hence it is not ARM. Therefore, there are two protein synthesis-independent memory types in Drosophila (Bourouliti, 2022).

This study identified dopaminergic neurons whose activity inhibits ARM and therefore, as in mammals, positively affects LTM formation. That regulation of memory consolidation seems to involve precisely cadenced oscillations in MV1 and MP1 neurons is of particular conceptual interest. Indeed, it was recently suggested that a subset of hypothalamic dopaminergic neurons in rats, robustly oscillating at 0.05 Hz, may be responsible for lactation inhibition. Thus, inhibition through rhythmic oscillations appears to be a widespread functional feature of dopaminergic networks. In addition, a slow oscillatory firing mode, in the 0.5-1.5-Hz frequency range, has been identified in the dopaminergic neurons in the ventral tegmental area of rats. This oscillatory firing pattern would underlie subsecond synchronization between ventral tegmental area and prefrontal cortex, an area of major importance in learning and memory. Thus, and although such an assumption remains quite speculative, LTM regulation by dopaminergic neurons in mammals might involve mechanisms similar to those described in this study in Drosophila (Plaçais, 2012).

Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). This study sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, this study provides the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrates that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories (Schwartz, 2023).

The signal pathway of actin remodeling, including LIM-kinase 1 (LIMK1) and its substrate cofilin, regulates multiple processes in neurons of vertebrates and invertebrates. Drosophila melanogaster is widely used as a model object for studying mechanisms of memory formation, storage, retrieval and forgetting. Previously, active forgetting in Drosophila was investigated in the standard Pavlovian olfactory conditioning paradigm. The role of specific dopaminergic neurons (DAN) and components of the actin remodeling pathway in different forms of forgetting was shown. This research investigated the role of LIMK1 in Drosophila memory and forgetting in the conditioned courtship suppression paradigm (CCSP). In the Drosophila brain, LIMK1 and p-cofilin levels appeared to be low in specific neuropil structures, including the mushroom body (MB) lobes and the central complex. At the same time, LIMK1 was observed in cell bodies, such as DAN clusters regulating memory formation in CCSP. GAL4 x UAS binary system was applied to induce limk1 RNA interference in different types of neurons. The hybrid strain with limk1 interference in MB lobes and glia showed an increase in 3-h short-term memory (STM), without significant effects on long-term memory. limk1 interference in cholinergic neurons (CHN) impaired STM, while its interference in DAN and serotoninergic neurons (SRN) also dramatically impaired the flies' learning ability. By contrast, limk1 interference in fruitless neurons (FRN) resulted in increased 15-60 min STM, indicating a possible LIMK1 role in active forgetting. Males with limk1 interference in CHN and FRN also showed the opposite trends of courtship song parameters changes. Thus, LIMK1 effects on the Drosophila male memory and courtship song appeared to depend on the neuronal type or brain structure (Zhuravlev, 2023).

Flexible goal-driven orientation requires that the position of a target be stored, especially in case the target moves out of sight. The capability to retain, recall and integrate such positional information into guiding behaviour has been summarized under the term spatial working memory. This kind of memory contains specific details of the presence that are not necessarily part of a long-term memory. Neurophysiological studies in primates indicate that sustained activity of neurons encodes the sensory information even though the object is no longer present. Furthermore they suggest that dopamine transmits the respective input to the prefrontal cortex, and simultaneous suppression by GABA spatially restricts this neuronal activity. Fruit flies possess a similar spatial memory during locomotion. Using a new detour setup, flies are shown to be able to remember the position of an object for several seconds after it has been removed from their environment. In this setup, flies are temporarily lured away from the direction towards their hidden target, yet they are thereafter able to aim for their former target. Furthermore, it was found that the GABAergic (stainable with antibodies against GABA) ring neurons (Hanesch, 1998) of the ellipsoid body in the central brain are necessary and their plasticity is sufficient for a functional spatial orientation memory in flies. The protein kinase S6KII (ignorant; Putz, 2004) is required in a distinct subset of ring neurons to display this memory. Conditional expression of S6KII in these neurons only in adults can restore the loss of the orientation memory of the ignorant mutant. The S6KII signalling pathway therefore seems to be acutely required in the ring neurons for spatial orientation memory in flies (Neuser, 2008).

Previous studies have shown that walking flies heading for an object maintain their direction even when the target disappears. This persistence of orientation can last for several seconds, indicating that flies store the position of, or the path towards, the hidden object for further targeting. It is therefore proposed that flies form a spatial memory for objects that is similar to the working memory in vertebrates. To investigate this putative memory in Drosophila a detour paradigm for walking flies was established. Single flies were put into a cylindrical virtual-reality arena, in which two dark vertical stripes were presented at opposite sides. Normally, flies patrol between the two visual objects for a considerable length of time. In the new paradigm, the stripes disappeared when the fly crossed the invisible midline of the circular walking platform, and a new target appeared laterally at a 90° angle to the fly. In most cases wild-type flies turned towards this new target if it was presented for more than 500 ms. After the fly had oriented itself towards the new object (deviation of the fly's longitudinal body axis from the ideal course to the stripe below +/-15°), this target also disappeared within 1 s and no objects were visible to the fly. It was then determined whether the fly turned back to continue its approach to its initial, but still invisible, target. The walking traces reveal a direct course towards the former location of the first target. The flies therefore retained positional information on the former object, although it was no longer present in the environment (Neuser, 2008).

Wild-type (Canton-S) flies recall the old target and integrate it into a guided behaviour with a median frequency of 80% as measured in ten consecutive trials for each fly. Longer presentation of the distracter stripe did not significantly change the percentage of positive choices. These data strongly suggest that flies stored the relative position of the first target in a spatial orientation memory for at least 4 s. To exclude the possibility that flies used chemical traces of former runs for their orientation the absolute positions of the stripes were randomly changed after each trial. As a result of this randomization, flies had to update their memory continuously. Moreover, no training effect could be observed, because the frequency of positive turns did not change during the ten consecutive trials. Similar performances were observed when two opposing distracters were presented to the fly. This orientation memory for vanished objects is considered to be to be idiothetic. Because no visible landmarks were presented to the fly after the distracter disappeared, the fly could not use a stored reference picture of the environment for its guidance. It is therefore suggested that the fly uses online stored information of its own angle towards the former target, a strategy known as path integration. Path integration has been shown to be used by other insects, such as ants and bees, to navigate through a familiar landscape (Neuser, 2008).

In an attempt to localize this type of memory to discrete parts of the insect brain, several mutant lines with structural central-complex defects of Drosophila were analysed. The central complex is composed of four different neuropils and has been implicated in supervising motor output during locomotion. First tests showed that the persistence of orientation towards a removed target is reduced or lost whenever the ellipsoid body of the central complex was defective. Therefore the ellipsoid body open mutant (ebo^KS263) was tested in the detour paradigm; these flies did not show a preference for the first target after the detour, suggesting that an intact ellipsoid body is required for establishing a spatial orientation memory. In contrast, the use of hydroxyurea to ablate the mushroom bodies, which are important in olfactory memory, did not disturb the orientation memory (Neuser, 2008).

One prominent type of neuronal cells of the ellipsoid body is the group of GABAergic ring neurons. The fibres of these neurons run in a prominent tract, the RF tract (ring-neuron and tangential fan-shaped-body neuron tract), and form bushy thin endings in the ipsilateral lateral triangle and bleb-like endings in the ellipsoid body. Four different kinds of ring neuron (R1-R4) can be distinguished by their arborization pattern around the ellipsoid body canal. R1-R3 neurons project outwards from the ellipsoid body canal, whereas the arborization of R1 is restricted to the inner zone, that of R2 to the outer zone, and that of R3 to both zones. R4 neurons project from the periphery inwards and arborize in the outermost zone. It was next proposed that the ring neurons might be necessary for the orientation memory. The GAL4/UAS system was used to silence distinct subsets of ring neurons through the expression of tetanus toxin (TNT) by using the GAL4 driver lines c232, c481 and c105. For temporal control, TNT was induced conditionally by using the temperature-sensitive GAL4 repressor GAL80^ts under the control of the ubiquitous Tubulin promoter (Tub-GAL80^ts). Experimental and control flies were raised at 18°C, tested within the detour paradigm, and retested after the induction of TNT. Pairwise comparison revealed that the preference for the original target was lost whenever the toxin was expressed in ring neurons of the ellipsoid body. This finding confirms the hypothesis that the ellipsoid-body ring neurons are necessary components of the orientation memory (Neuser, 2008).

To investigate which molecular pathways are involved in this kind of memory, focus was placed on the cyclic-AMP signalling pathway. Variable levels of cAMP have been shown to have a crucial function in memory formation during associative learning in Drosophila. cAMP levels are modulated by the opposing actions of adenylyl cyclases and cAMP phosphodiesterases. Mutants for the adenylyl cyclase gene rutabaga (rut¹ and rut²⁰⁸⁰) were unable to target visual objects and could not be tested in the paradigm. Therefore mutants of the dunce gene (dnc), which encodes a cAMP phosphodiesterase, were tested in the detour paradigm. The dnc¹ mutant is a hypomorph and displays about half of the enzyme activity in the wild type. dnc¹ mutant flies show deficits in several paradigms of associative classical learning and operant conditioning. In contrast, dnc¹ mutants showed no defects in the detour paradigm, indicating that a tight modulation of cAMP levels might not be critically required for spatial orientation memory (Neuser, 2008).

Another molecule involved in memory formation in Drosophila is a member of the ribosomal serine kinase family. ignorant (ign) encodes the S6 kinase II (S6KII), which interacts with mitogen-activated protein (MAP) kinase signalling in Drosophila. S6KII does not seem to be involved in cAMP signalling pathways. The null allele ign^58/1 has been shown to be defective in classical aversive conditioning and operant learning (Putz, 2004). Therefore ign^58/1 flies were tested in the detour paradigm. Although the mutants readily targeted visible objects, they showed no directional preference for the position of the original target after it disappeared, suggesting that they had lost their memory. In contrast, walking speed, walking activity and orientation towards visual objects were similar to those of the wild type. Next whether ign is required in the ring neurons targeted by c232-GAL4 was tested with the use of a UAS-ign RNA-mediated interference (RNAi) effector line. RNAi silencing in these ring neurons decreased the performance by half. This decrease in memory constitutes only a partial phenocopy of the null mutant, because the performance was not significantly different from that of the wild type or ign^58/1. Nevertheless, this result is interpreted to suggest that ign is required in the ring neurons for spatial orientation memory (Neuser, 2008).

To address the question of whether restoring S6KII levels is sufficient for regaining memory, neuron-specific rescue experiments were performed in the ign^58/1 mutant background. S6KII was expressed pan-neuronally with Appl-GAL4 and elav-GAL4, and also specifically in the R3 and R4 ring neurons with c232-GAL4. In all three cases a complete rescue was observed. Next, whether ign function in the R3 and R4 ring neurons is acutely required for orientation memory was examined. Therefore, again use was made of the GAL80^ts transgene to rescue the ign phenotype only in the adult. Conditional expression of S6KII only in the R3 and R4 ring neurons resulted in a perfect rescue of the ign mutant. This result -- that acute S6KII expression in the R3 and R4 ring neurons accomplished a complete rescue -- reveals that this very narrow subset of cells is sufficient for regaining a functional orientation memory. It has been reported that Drosophila S6KII negatively regulates extracellular signal-regulated kinases (ERKs) by acting as a cytoplasmic anchor of the MAP kinase. Further studies will determine whether the MAP kinase signalling pathway is required for this kind of memory task (Neuser, 2008).

It is concluded that the relevant ring neurons use the inhibitory neurotransmitter GABA. Their circuitry and interconnections within the ellipsoid body are not yet known. Expression of the dDA1 dopamine receptor in the ellipsoid body has recently been shown. It is therefore possible that the same neurotransmitter systems as those used for visual-spatial memory in the monkey prefrontal cortex are used to establish orientation memory in the central complex of flies (Neuser, 2008).

Memory guides behavior across widely varying environments and must therefore be both sufficiently specific and general. A memory too specific will be useless in even a slightly different environment, while an overly general memory may lead to suboptimal choices. Animals successfully learn to both distinguish between very similar stimuli and generalize across cues. Rather than forming memories that strike a balance between specificity and generality, Drosophila can flexibly categorize a given stimulus into different groups depending on the options available. This study asked how this flexibility manifests itself in the well-characterized learning and memory pathways of the fruit fly. It was shown that flexible categorization in neuronal activity as well as behavior depends on the order and identity of the perceived stimuli. These results identify the neural correlates of flexible stimulus-categorization in the fruit fly (Modi, 2023).

Given the relationship between sleep and plasticity, this study examined the role of Extracellular signal-regulated kinase (ERK, Rolled in Drosophila) in regulating baseline sleep, and modulating the response to waking experience. Both sleep deprivation and social enrichment increase ERK phosphorylation in wild-type flies. The effects of both sleep deprivation and social enrichment on structural plasticity in the within the Pigment Dispersing Factor (PDF)-expressing ventral lateral neurons (LNvs) can be recapitulated by expressing an active version of ERK (UAS-ERK^SEM) pan-neuronally in the adult fly using GeneSwitch (Gsw) Gsw-elav-GAL4. Conversely, disrupting ERK reduces sleep and prevents both the behavioral and structural plasticity normally induced by social enrichment. Finally, using transgenic flies carrying a cAMP response Element (CRE)-luciferase reporter it was shown that activating ERK enhances CRE-Luc activity while disrupting ERK reduces it. These data suggest that ERK phosphorylation is an important mediator in transducing waking experience into sleep (Vanderheyden, 2013).

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. This study identified the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, this study demonstrated that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. It is proposed that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories (Poe, 2023).

The brain as a central regulator of stress integration determines what is threatening, stores memories, and regulates physiological adaptations across the aging trajectory. While sleep homeostasis seems to be linked to brain resilience, how age-associated changes intersect to adapt brain resilience to life history remains enigmatic. This study provides evidence that a brain-wide form of presynaptic active zone plasticity ("PreScale"), characterized by increases of active zone scaffold proteins and synaptic vesicle release factors, integrates resilience by coupling sleep, longevity, and memory during early aging of Drosophila. PreScale increased over the brain until mid-age, to then decreased again, and promoted the age-typical adaption of sleep patterns as well as extended longevity, while at the same time it reduced the ability of forming new memories. Genetic induction of PreScale also mimicked early aging-associated adaption of sleep patterns and the neuronal activity/excitability of sleep control neurons. Spermidine supplementation, previously shown to suppress early aging-associated PreScale, also attenuated the age-typical sleep pattern changes. Pharmacological induction of sleep for 2 days in mid-age flies also reset PreScale, restored memory formation, and rejuvenated sleep patterns. The data suggest that early along the aging trajectory, PreScale acts as an acute, brain-wide form of presynaptic plasticity to steer trade-offs between longevity, sleep, and memory formation in a still plastic phase of early brain aging (Huang, 2022).

Elucidating how the distinct components of synaptic plasticity dynamically orchestrate the distinct stages of memory acquisition and maintenance within neuronal networks remains a major challenge. Specifically, plasticity processes tuning the functional and also structural state of presynaptic active zone (AZ) release sites are widely observed in vertebrates and invertebrates, but their behavioral relevance remains mostly unclear. This study provides evidence that a transient upregulation of presynaptic AZ release site proteins supports aversive olfactory mid-term memory in the Drosophila mushroom body (MB). Upon paired aversive olfactory conditioning, AZ protein levels (ELKS-family BRP/(m)unc13-family release factor Unc13A) increased for a few hours with MB-lobe-specific dynamics. Kenyon cell (KC, intrinsic MB neurons)-specific knockdown (KD) of BRP did not affect aversive olfactory short-term memory (STM) but strongly suppressed aversive mid-term memory (MTM). Different proteins crucial for the transport of AZ biosynthetic precursors (transport adaptor Aplip1/Jip-1; kinesin motor IMAC/Unc104; small GTPase Arl8) were also specifically required for the formation of aversive olfactory MTM. Consistent with the merely transitory increase of AZ proteins, BRP KD did not interfere with the formation of aversive olfactory long-term memory (LTM; i.e., 1 day). These data suggest that the remodeling of presynaptic AZ refines the MB circuitry after paired aversive conditioning, over a time window of a few hours, to display aversive olfactory memories (Turrel, 2022).

Synapses are key sites of information processing and storage in the brain. Notably, synaptic transmission is not hardwired but adapts through synaptic plasticity to provide appropriate input-output relationships as well as to process and store information on a circuit level. Still, there are fundamental gaps in understanding of exactly how the dynamic changes of synapse performance intersect with circuit operation and consequently define behavioral states. This is partly due to the inherent complexity of synaptic plasticity mechanisms, which operate across a large range of timescales (sub-second to days) and use a rich spectrum of both pre- and post-synaptic molecular and cellular mechanisms. Lately, refinement processes following the immediate engram formation have been described, which might promote specific neuronal activity patterns to select neurons for longer-term information display and storage (Turrel, 2022).

Synaptic transmission across chemical synapses is evoked by action potentials that activate presynaptic Ca2+ influx through voltage-gated Ca2+ channels to trigger the fusion of synaptic vesicles (SVs) containing neurotransmitter at sites called active zones (AZs). AZs assemble from conserved scaffold proteins, including ELKS (Drosophila ortholog: BRP), RIM, and the RIM-binding protein (RBP) family. Recent work in Drosophila showed that discrete SV release sites form at AZ. In the AZ, the ELKS-family BRP master scaffold protein localizes the critical Munc13 family release factor Unc13A in defined nanoscopic clusters around Ca2+ channels (BRP/Unc13A nanomodules). This AZ architecture of the nanoscale organization between BRP/Unc13 release machinery and the AZ-centric Ca2+ channels is present across all Drosophila synapses, including Kenyon cell (KC) derived AZs, and munc13-clusters also define release sites at central mammalian synapses. Importantly, AZ structure and function is dynamic and can remodel within 10 min, as shown at Drosophila neuromuscular junction (NMJ) synapses (Turrel, 2022).

The Drosophila mushroom body (MB) forms and subsequently stores olfactory memories. Importantly, a depression of SV release from the AZ of intrinsic KCs within specific compartments of the MB lobes was found to promote the formation of olfactory memories within a few minutes of paired conditioning. Indeed, Ca2+ in vivo imaging experiments indicate that dopamine bidirectionally tunes the strength of KC synapses to output neurons, with forward conditioning driving depression of those synapses and backward conditioning generally driving potentiation. How this tuning is executed at AZ level is not yet known (Turrel, 2022).

This study present evidence for AZ remodeling (BRP, Syd1, and Unc13A) to take place within MB lobes after paired conditioning for a few hours and provide genetic evidence that this AZ remodeling within the MB-intrinsic KCs is crucial for mid-term aversive olfactory memories. To identify candidate mechanisms of presynaptic remodeling to then be tested in MB-dependent olfactory memory, the role of AZ remodeling was studied during extended larval NMJ plasticity and relevant transport factors were identified. These data suggest that broad but transient changes of presynaptic AZs depending on the transport of new biosynthetic material support refinement processes within KC and MB circuitry and are specifically needed for stable formation of mid-term olfactory memories (Turrel, 2022).

Historically, postsynaptic plasticity mechanisms have been analyzed extensively, and molecular and cellular processes targeting postsynaptic neurotransmitter receptors have been convincingly connected to learning and memory. At the same time, the necessity of using postsynaptic neurons as reporters of presynaptic activity (and, thus, setup paired recordings) has imposed an additional obstacle specific to the functional study of presynaptic forms of mid- and long-term plasticity. Furthermore, the cellular and molecular processes remodeling presynaptic AZs are not characterized as extensively as those at the postsynapse. Consequently, although widely expressed by excitatory and inhibitory synapses of mammalian brains, the behavioral relevance of longer-term presynaptic plasticity remains largely obscure (Turrel, 2022).

This study combined the possibility of genetically analyzing memory formation and stabilization within discrete neuron populations of the Drosophila MB with the identification of molecular machinery remodeling presynaptic AZs in vivo. Evidence is provided for an extended but temporally restricted (a few hours post training) upregulation of presynaptic AZ proteins across the MB lobes, a process seemingly needed in MB intrinsic neurons to display olfactory MTM (Turrel, 2022).

Notably, the acute formation of aversive STM was previously shown to trigger synaptic depression at the KC::MBON synapse in the respective MB compartments. It is emphasized that the exact relation of the AZ remodeling described in this study to this STM-controlling short-term depression is presently unknown. Particularly, it is not possible to tell whether the conditioning-associated presynaptic remodeling described in this study is indeed potentiating KCs and MB AZs or whether overlapping sets of synapses are involved in STM and MTM formation and display. What can be concluded, however, is that molecular machinery that executes structural remodeling at NMJ AZs is critically needed for MTM within the MB intrinsic neurons. Establishing the degree to which synaptic weight changes are associated with the mechanism of MB presynaptic remodeling will have to await the development of protocols to directly follow synapses in vivo for hours after conditioning. Different from presynaptic remodeling being part of the memory trace or engram itself, the idea is favored that synaptic upregulation might instead execute a refinement function extending over larger parts of the MB AZ populations. Refinement is an emerging concept stating that stable propagation and maintenance of memory traces might depend on homeostatic regulations of neuronal circuitry. Sleep-dependent synaptic plasticity is suggested to similarly play an important role in neuronal circuit refinement after learning (Turrel, 2022).

Notably, it has been recently shown that a similar upregulation of AZ proteins (BRP/Unc13A) is indeed a functional part of Drosophila sleep homeostasis, where it suffices to trigger rebound sleep patterns. It thus appears conceivable that the AZ changes associated with conditioning reported in this study might promote specific MB activity patterns instrumental for MTM. An alternative, not mutually exclusive, option is that the initial synaptic depression associated with aversive conditioning must, on a longer term, be compensated by the MB AZ changes (and potential potentiation) described in this study (Turrel, 2022).

Notably, compartment-specific synaptic changes occur in the MB in response to sheer odor presentation or DAN activity although AZ remodeling in this study behaved strictly conditioning dependent, meaning it was not observed after unpaired conditioning, and appeared broadly distributed. It cannot be excluded, however, that smaller size, compartment-specific AZ changes, have been missed, given the limited resolution of the staining assays (Turrel, 2022).

Cell biological processes remodeling presynaptic AZs at larval NMJ synapses can also be of relevance for memory formation in the adult fly KCs. Concretely, this study found that the MB KC-specific KD of transport factors, which at the NMJ level provoked plasticity profiles similar to BRP, also specifically affected MTM but spared STM. Given that several molecular factors, including transport proteins not directly physically associated with the AZ, fulfilled this relation, it indeed appears likely that retrieving axon-transported biosynthetic AZ precursor material is what is critical here (Turrel, 2022).

Speaking of the specificity of rthe MTM phenotypes in relation to AZ remodeling, this study found STM formation undisturbed, but at the same time, MTM to be severely affected after BRP and transport factor KD. This is strong evidence against the possibility of baseline synaptic defects being responsible for the observed MTM deficits. It is also emphasized that this study achieved behavioral phenotypes by comparatively mild and strictly post-developmental KD and that odor Ca2+ responses in MBON neurons postsynaptic to KC appeared normal in BRP KD flies (Turrel, 2022).

When analyzing in a MB-lobe-specific manner, α/&betal and α'/β' neurons showed stronger and more sustained upregulation of BRP/Unc13A than the γ lobes. This might indicate that the extent and role of refinement across the MB lobes is adapted to their specific roles in memory acquisition and retrieval. This is also in accordance with previous observations showing heterogeneity in the exact AZ protein composition across synapses of the Drosophila brain (Turrel, 2022).

Interestingly, Syd-1 levels are significantly increased 1 h after conditioning in the α/β and α'/β' lobes, whereas it has been shown that Syd-1 levels are not increased 10 min after PhTx treatment at the NMJ. This finding indicates that some of the AZ proteins may be affected differently in those two plasticity processes (Turrel, 2022).

Given the generally observed sparse representation of odors within the MB KCs, one might expect initial synaptic changes to be specific to only a few odor-response KCs. Still, this analysis apparently reveals more extended changes of synaptic AZs across the lobes. Potentially, upon successful conditioning, the initial, more restricted, synaptic changes might be followed by an extended communication between the neurons involved in the memory circuit, potentially including KC::KC communication. Indeed, there is ample evidence for a transfer of requirement between different subsets of KCs in the temporal evolution of olfactory memory. This communication seemingly involves gap junctions between KCs but might in parallel also use chemical synapses and their AZs. Concerning the broad distribution of the AZ changes across compartments, it is interesting to mention that KC-global, conditioning-dependent metabolic changes have been observed, being critical for LTM but also MTM (Turrel, 2022).

It is tempting to speculate that the initial, compartment-specific changes, confined to a few odor-responding KCs, might overcome a threshold to also trigger more global synaptic changes. Also interesting in this context, dorsal paired medial (DPM) neurons' odor response increase following spaced conditioning, also indicating that opposite synaptic strength changes might counterbalance the initial synaptic changes occurring in the memory-relevant compartment or depending on post-synaptic partner neurons provoke either potentiation or depression (Turrel, 2022).

As mentioned above, this study found that KD of BRP in the adult MB lobes did not affect LTM, whereas MTM was decreased both at 1 and 3 h. Such a phenotype, a deficit of MTM but subsequent memory phases being intact, was only rarely observed before (Nep2-RNAi in adult DPM neurons, synapsin mutants with memory deficits up to 1 h but normal memory later on). On one hand, this reinforces the idea that MTM and LTM might form using separate circuits, and on the other hand, that cell types other than KCs might contribute to aversive olfactory LTM formation. Different sets of proteins in the same lobes might operate in parallel circuits similar to what has been observed in the honeybee. However, it might also well be that the presynaptic AZ remodeling observed in this study is indeed specific for the display of MTM and that the synaptic memory traces orchestrating the later recall of LTM are mediated by independent parallel molecular/synaptic mechanisms or distinct circuit (Turrel, 2022).

The formation of long-term memories requires changes in the transcriptional program and de novo protein synthesis. One of the critical regulators for long-term memory (LTM) formation and maintenance is the transcription factor CREB. Genetic studies have dissected the requirement of CREB activity within memory circuits, however less is known about the genetic mechanisms acting downstream of CREB and how they may contribute defining LTM phases. To better understand the downstream mechanisms, a targeted DamID approach (TaDa) was used. A CREB-Dam fusion protein was generated using the fruit fly Drosophila melanogaster as model. Expressing CREB-Dam in the mushroom bodies (MBs), a brain center implicated in olfactory memory formation, identified genes that are differentially expressed between paired and unpaired appetitive training paradigm. Of those genes we selected candidates for an RNAi screen in which genes were identified causing increased or decreased LTM (Sgammeglia, 2023).

Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odor-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. A system was developed to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrate that they are indeed an exception where activation of the cAMP/protein kinase A pathway induces LTD. Contrary to the prevailing model, cAMP imaging finds no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, it was found that forskolin-induced cAMP increase alone is insufficient for plasticity induction; it additionally requires simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induces slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioral study. Finally, KC subtype-specific interrogation of synapses reveals that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, this work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model (Yamada, 2024).

In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. This study provides evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). The nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons (MBONs) for appetitive memory induction, but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein Dlg. This study shows that ostsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla (Pribbenow, 2023).

Long term memory (LTM) requires learning-induced synthesis of new proteins allocated to specific neurons and synapses in a neural circuit. Not all learned information, however, becomes permanent memory. How the brain gates relevant information into LTM remains unclear. In Drosophila adults, weak learning after a single training session in an olfactory aversive task typically does not induce protein-synthesis-dependent LTM. Instead, strong learning after multiple spaced training sessions is required. This study reports that pre-synaptic active-zone protein synthesis and cholinergic signaling from the early α/β subset of mushroom body (MB) neurons produce a downstream inhibitory effect on LTM formation. When inhibitory signaling was eliminated from these neurons, weak learning was then sufficient to form LTM. This bidirectional circuit mechanism modulates the transition between distinct memory phase functions in different subpopulations of MB neurons in the olfactory memory circuit (Chen, 2023).

Maladaptive operant conditioning contributes to development of neuropsychiatric disorders. Candidate genes have been identified that contribute to this maladaptive plasticity, but the neural basis of operant conditioning in genetic model organisms remains poorly understood. The fruit fly Drosophila melanogaster is a versatile genetic model organism that readily forms operant associations with punishment stimuli. However, operant conditioning with a food reward has not been demonstrated in flies, limiting the types of neural circuits that can be studied. This study presents the first sucrose-reinforced operant conditioning paradigm for flies. In the paradigm, flies walk along a Y-shaped track with reward locations at the terminus of each hallway. When flies turn in the reinforced direction at the center of the track, they receive a sucrose reward at the end of the hallway. Only flies that rest early in training learn the reward contingency normally. Flies rewarded independently of their behavior do not form a learned association but have the same amount of rest as trained flies, showing that rest is not driven by learning. Optogenetically-induced sleep does not promote learning, indicating that sleep itself is not sufficient for learning the operant task. The sensitivity of this assay to detect the effect of genetic manipulations was validated by testing the classic learning mutant dunce. Dunce flies are learning-impaired in the Y-Track task, indicating a likely role for cAMP in the operant coincidence detector. This novel training paradigm will provide valuable insight into the molecular mechanisms of disease and the link between sleep and learning (Wiggin, 2021).

Navigation through the environment requires a working memory for the chosen target and path integration facilitating an approach when the target becomes temporarily hidden. Previous studies have shown that this visual orientation memory resides in the ellipsoid body, which is part of the central complex in the Drosophila brain (see Neuronal architecture of the central complex in Drosophila melanogaster in Niven's Visuomotor control: Drosophila bridges the gap). Former analysis of foraging and ignorant (ign) mutants have revealed that a hierarchical PKG and RSKII kinase signaling cascade in a subset of the ellipsoid-body ring neurons is required for this type of working memory in flies. This study shows that mutants in the ellipsoid body open (ebo) gene, which encodes the actin-binding protein Exportin 6, exhibit excessive nuclear accumulation of actin during development and in the adult brain. ebo mutants lack the orientation memory independent of the structural defect in the ellipsoid-body neuropil, and EBO activity in any type of adult ring neurons is sufficient for orientation-memory function. Moreover, genetic interaction studies revealed that nuclear actin accumulation in ebo mutants inhibits the Drosophila coactivator myocardin-related transcription factor A (dMRTF) and therefore the transcriptional activator serum response factor (dSRF). dSRF also functions in different ring neurons, suggesting that it regulates abundance of a diffusible factor that enables a working memory in ellipsoid-body ring neurons. To date, SRF has only been implicated in longer forms of memory formation like synaptic long-term potentiation and depression. This study provides the first evidence that SRF-mediated gene regulation is also required for a working memory that lasts only for a few seconds (Thran, 2013).

The central complex (CX) of the adult fly brain consists of four compartments that interconnect the protocerebral hemispheres: the protocerebral bridge, the fan-shaped body, the ellipsoid body (EB), and the ventrally located paired noduli. The analysis of several Drosophila mutant strains with structural defects in one or more neuropils of the CX has suggested that the CX represents a higher control center for locomotion and orientation behavior. These mutants walk slower than wild-type flies, have a delayed reaction to changing stimuli during flight, and show deficits in the orientation behavior toward landmarks. A major sensory input region for external stimuli, especially visual information, appears to be the ellipsoid body and the fan-shaped body, and in larger insects this includes information on the orientation of polarized light, which is used for navigation. In Drosophila, it has been shown that the protocerebral bridge is required for step-length control in walking flies and visual targeting during climbing events. Visual input is also processed in the fan-shaped body, which mediates visual pattern recognition to memorize different objects during flight control], a function that has also been attributed to the EB. The EB also holds a memory trace for the position of landmarks, as revealed in a Morris water maze-like paradigm for visual place learning in flies. In addition, the analysis of the ellipsoid body open (ebo) mutant has shown that the EB is necessary to establish a visual orientation memory for a vanishing object in walking Drosophila (Thran, 2013).

In this so-called detour paradigm, a fly approaching an attractive target is lured out of the way by a dark stripe on one side while simultaneously the original object vanishes. After the distracting stripe has also been removed, the fly is left without any visual cue. However, in over 80% of the cases, wild-type flies use idiothetic information on their past movements with respect to the original target to resume their originally intended approach. This type of working memory lasts about 4 s and must be updated during every turn the fly takes (Neuser, 2008). Analysis of two Drosophila memory mutants in ignorant (ign) and foraging (for), which encode the ribosomal-S6 kinase II (RSKII) and cGMP-dependent protein kinase (PKG), respectively, revealed that both kinases share one signaling pathway that is required in a specific type of EB ring neurons to display an orientation memory (Kuntz, 2012; Thran, 2013).

To further elucidate the structural and biochemical components that enable the EB to hold an orientation memory, the ebo mutants were genetically and molecularly analyzed. This study reports that the ebo gene encodes the actin-binding protein Exportin 6, which is required for the export of globular actin (G-actin) from the nucleus, thus preventing actin-filament formation there. This analysis of the ebo mutant confirms these findings because elevated levels of an Actin-GFP fusion protein can be found in nuclei of the mutant. Interestingly, excessive G-actin has been shown to inhibit the myocardin- related transcription factor A in vertebrates (MRTF-A), a coactivator of the transcriptional regulator serum response factor (SRF). This genetic interaction analysis of ebo with the Drosophila ortholog of MRTF, as well as rescue experiments of the dSRF mutant blistered (bs), revealed that elevated levels of nuclear actin in EB neurons and the subsequent malfunction of the dMRTF/dSRF transcription regulator complex prevent the visual orientation memory in flies (Thran, 2013).

In vertebrates, more than 200 putative target genes of SRF have been postulated, most of them involved in cytoskeleton dynamics and cell motility, but immediate early genes like c-fos have also been identified. Unfortunately, downstream targets of Drosophila dSRF in neuronal cells are yet unknown, complicating the identification of further gene products that are involved in development of the EB and a functional visual orientation memory in adult flies. Based on the current results, it is hypothesized that the effect of EBO on the transcriptional activity of the dMRTF/dSRF complex is responsible for both the developmental and behavioral phenotype (Thran, 2013).

Although transheterozygous mutants for hypomorphic bs alleles displayed no structural EB defect and the morphological defects of homozygous MrtfD7 mutants are less prominent than those of ebo mutants, it nevertheless is possible that dMRTF/dSRF-mediated gene regulation is also required in EB development. For instance, in vertebrates, transcription of profilin, an F-actin promoting factor, is activated by SRF, and the structural ebo phenotype could indicate that dSFR is also promoting transcription of the profilin encoding gene chickadee in Drosophila. Alternatively, nuclear actin in the ebo mutant could sequester profilin in the nucleus, thus reducing levels at the growth cone required in axonal outgrowth (Thran, 2013).

However, it is obvious that a memory that lasts only a few seconds cannot depend on changes in transcriptional regulation. The rescue experiments described in this study have established that EBO and dSRF can restore memory function of the respective mutant independent of the specific ring-neuron subtype. This is surprising, considering the biochemical activity of EBO and dSRF, which are definitively cell autonomous. Therefore, it is proposed that dSRF promotes gene expression that ultimately results in the production of a diffusible factor that has to be delivered to the ring neurons. Presumably, this factor feeds into pathways that enable rapid changes in synaptic transmission of the ring neurons necessary to encode an orientation memory. For instance, ring neurons might need a higher density of synaptic release sites, a highly efficient synaptic vesicle reserve pool, or elevated expression of ion channels for prolonged excitation. Similarly, a high density of dendritic neurotransmitter receptors and elevated levels of second messenger molecules at the postsynaptic site of the ring neurons could be necessary to exert their specific function in working memory formation and/or retrieval. Finding the dSRF target genes that mediate the orientation memory in flies might lead to new insights how working memories are orchestrated in general (Thran, 2013).

Drosophila mushroom bodies (MB) are bilaterally symmetric multilobed brain structures required for olfactory memory. Previous studies suggested that neurotransmission from MB neurons is required only for memory retrieval. An unexpected observation that Dorsal Paired Medial (DPM) neurons, which project only to MB neurons, are required during memory storage but not during acquisition or retrieval, led a revisiting og the role of MB neurons in memory processing. This study shows that neurotransmission from the α'β' subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory, but is dispensable during memory retrieval. In contrast, neurotransmission from MB αβ neurons is required only for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in an MB α'β' neuron-DPM neuron loop is required to stabilize memories formed in the MB αβ neurons (Krashes, 2007).

It is often said that form follows function. While this postulate would argue the striking multi-lobed arrangement of the insect mushroom bodies implies functional differences between the different types of MB neurons: αβ, α'β' and γ, very limited data exists describing the individual function of these anatomical subdivisions. Although several complex behaviors in insects appear to require the MBs and a differential role for distinct MB neuron groups has been suggested, most conceptual models of memory treat the MBs as a single unit (Krashes, 2007).

One of the most detailed examinations of MB function has been in the context of Drosophila aversive olfactory memory, where flies are trained to associate specific odors with the negative reinforcement of electric shock. Genetic studies over the last thirty years have suggested that the MBs play an essential role in fly olfactory memory, but the role of the MBs in memory acquisition, storage, and retrieval has been examined only recently. Taking advantage of a dominant, temperature-sensitive dynamin transgene, uas-shi^ts1, a number of laboratories concluded that MB output was required only for recall, but not acquisition or storage. These and other findings have led to a simple model whereinDrosophila olfactory memory is formed and stored at MB output synapses (Krashes, 2007).

Functional studies of DPM neurons, MB extrinsic neurons that ramify throughout the MB lobes, demonstrated they were specifically required during consolidation, but not acquisition or storage. Furthermore, genetically-modified DPM neurons that primarily innervate the MB α'β' lobes retain function implying that MB α'β' neurons might also have a similar function in memory consolidation (Keene, 2006; Krashes, 2007 and references therein).

Examination of the GAL4 enhancer trap lines used to express the uas-shi^ts1 transgene in the earlier MB studies revealed that c309, c747, and MB247 only express in a few MB α'β' neurons compared to αβ and γ neurons, while c739 expresses exclusively in αβ neurons. Thus it seems likely that prior studies utilizing these drivers did not observe requirements for MB activity during olfactory memory acquisition and storage because of insufficient expression in α'β' neurons (Krashes, 2007).

Subsequently two GAL4 enhancer traps that strongly express in MB α'β' neurons were identified in order to test this hypothesis. The expression of c305a appears to be entirely restricted to α'β' neurons within the MBs whereas c320 expresses in α'β', αβ, and a few γ neurons. Both of these lines also express in additional non-MB neurons so a MB{GAL80} tool was used to more rigorously test the requirement for MB activity, when the neurons labeled with either {GAL4} line were manipulated. With these new reagents the role of MB α'β' neurons in memory was investigated and it was found that MB α'β' neuron output during and after training is critical to form, and consolidate, both appetitive and aversive odor memory from a labile to a more stable state. For comparison the requirements for MB αβ neurons were also examined using c739, confirming the results of McGuire (2001). Thus, output from the MB α'β' neuron subset is required for memory acquisition and stabilization whereas, as previously described (McGuire, 2001), output from αβ neurons is apparently dispensable during training and consolidation but is required for memory retrieval (Krashes, 2007).

Based on c305 and c739 data, it was recognized that c320 flies, which express in both α'β' and αβ neurons, might be expected to exhibit memory loss if MB neuron output was blocked during both the consolidation and recall time windows. However, it is possible that a retrieval effect was not observed with c320 because it expresses GAL4 in fewer αβ neurons, or is in a different subset of αβ neurons, relative to the c739 driver (Krashes, 2007).

Despite this caveat, it is believed that the data suggest that different lobes of the MB have different roles in memory and therefore provide a significant shift in understanding of the role of the MB in memory. Older models implied that MB αβ, α'β' and γ neurons are largely interchangeable and that each of the MB neurons that respond to a particular odor receive coincident CS and US input and modify their presynaptic terminals to encode the memory. The present data suggest that MB αβ and α'β' neurons are functionally distinct (Krashes, 2007).

This study did not investigate the role of the unbranched γ lobe neurons. Previous work with c309, c747 and MB247 suggests that neurotransmission from γ neurons is likely dispensable for acquisition and consolidation. In addition, another study indicated that γ neurons are minimally involved in MTM and ARM. However, it is possible that experiments to date have not employed odors that require γ neuron activation. The response of γ neurons may be tailored to ethologically relevant odors such as pheromones. It is notable that fruitless, a transcription factor required for male courtship behavior, is expressed in MB γ neurons and blocking expression of the male-specific fru^M transcript in the MB γ neurons impairs courtship conditioning. If the relevant odors can be identified, it will be interesting to determine if MB α'β' neurons and DPM neurons are required to stabilize these odor memories in the γ neurons. Recent work by Akalal (2006) is supportive of the idea that odor identity is an important factor in determining the requirement for the function of distinct subsets of MB neurons in olfactory learning (Krashes, 2007).

Stable aversive and appetitive odor memory requires prolonged DPM neuron output during the first hour after training and DPM neuron output is dispensable during training and retrieval. DPM neurons ramify throughout the MB lobes but DPM neurons that have been engineered to project mostly to the MB α'β' lobes retain wild-type capacity to consolidate both aversive and appetitive odor memory (Keene, 2006). This study has demonstrated that similar to wild-type DPM neurons, blocking output from these modified DPM neurons for one hour after training abolishes memory. Thus finding a specific role for both DPM neuron output to MB α'β' lobes and MB α'β' neuron output during the first hour after training is consistent with the notion that a direct DPM-MB α'β' neuron synaptic connection is important for memory stability. It should be reiterated that the focus of this paper has been on protein-synthesis-independent memory and whether or not a similar processing circuit is utilized for protein synthesis-dependent LTM remains an open question (Krashes, 2007).

Beyond simply attributing an additional function to the MBs, when taken in conjunction with work on the role of DPM neurons in memory, the data presented in this study suggest a new model for how olfactory memories are processed within the MBs. It is proposed that olfactory information received from the second-order projection neurons is first processed in parallel by the MB αβ and α'β' neurons during acquisition. Activity in α'β' neurons establishes a recurrent α'β' neuron-DPM neuron loop that is necessary for consolidation of memory in αβ neurons and subsequently memories are 'stored' in αβ neurons, whose activity is required during recall. It is plausible that MB α'β' neurons are directly connected to MB αβ neurons and/or that DPM neurons provide the conduit between MB neurons. However, the finding that DPM neurons that project primarily to MB α'β' neurons are functional implies that only a few connections from DPM neurons to MB αβ neurons are necessary (Krashes, 2007).

The requirement for α'β' neuron output during training also potentially provides a source for the activity that drives DPM neurons. DPM neuron activity is not required during training and the current data are consistent with the idea that olfactory conditioning triggers activity in MB α'β' neurons that in turn elicits DPM neuron-dependent activity. It is proposed that after training recurrent MB α'β' neuron-DPM neuron activity is self-sustaining for 60-90 minutes (Yu, 2005). This recurrent network mechanism is similar to models for working memory in mammals (Durstewitz, 2000). It is also conceivable that MB α'β' neurons receive prolonged input after training from the antennal lobes (AL) via the projection neurons (PN). Olfactory conditioning has been reported to alter the odor response of Drosophila PNs in the AL but the observed effects are short-lived. Nevertheless, AL plasticity for a few minutes after training could contribute to the required MB α'β' neuron activity. If continued activity from the AL is required for consolidation, blocking PN transmission with shibire^ts1 for one hour after training should abolish memory. The bee AL and MB are clearly involved in olfactory memory and may function somewhat independently in learning and memory consolidation respectively. However, biochemical manipulation of the bee AL can also induce LTM and therefore it is possible that either plasticity in the AL alone can support LTM, or that the AL and MB interact during acquisition and consolidation. A differential role for the AL and MBs has also been suggested from neuronal ablation studies of courtship conditioning in Drosophila. Short-term courtship memory can be supported by the AL but memory lasting longer than 30 min requires the MBs (Krashes, 2007 and references therein).

This work also has significant implications for the organization of aversive and appetitive odor memories in the fly brain. Stability of both appetitive and aversive memory is dependent on DPM neurons (Keene, 2006) and MB α'β' neurons. It therefore appears that processing of aversive and appetitive odor memories may bottleneck in the MBs. Schwaerzel (2003) demonstrated that aversive memory formation requires dopaminergic neurons whereas appetitive memory relies on octopamine providing a possible mechanism to distinguish valence. However, Schwaerzel also found that MB output is required to retrieve aversive and appetitive odor memory suggesting that both forms of memory involve MB neurons and that both US pathways may converge on MB neurons. It will be important to understand how the common circuitry is organized to independently process the different types of memory and to establish if, and how, such memories co-exist (Krashes, 2007).

These data imply that stable memory may reside in MB αβ neurons because blocking output from MB αβ neurons impairs retrieval of MTM and ARM (both components of 3-hour memory). It has been previously proposed that AMN peptide(s) released from DPM neurons cause prolonged cAMP synthesis in MB neurons that is required to stabilize memory. The finding that genetically-engineered DPM neurons mostly projecting to the MB α'β' lobes, are functional (Keene, 2006) taken with the idea that stable memory resides in MB αβ neurons is somewhat inconsistent with the notion that crucial AMN-dependent memory processes occur in MB αβ neurons. However, it is plausible that AMN, or another DPM product that is released in a shibire-dependent manner, could diffuse locally from the aberrant DPM neurons to MB αβ neurons (Krashes, 2007).

This work demonstrates that MB αβ neurons and α'β' neurons have different roles in memory. Beyond gross structural and gene expression differences, it will be essential to establish the precise connectivity, relative excitability and odor responses of the different MB neurons. Future study may also reveal further functional subdivision within the MB lobes and it should be possible to refine current MB α'β' neuron GAL4 lines with appropriate GAL80 transgenes and FLP-out technology (Krashes, 2007).

In the mammalian brain, memories that initially depend on the function of the hippocampus lose this dependence when they are consolidated. This transient involvement of the hippocampus has led to the idea that consolidation of memory results in the transfer of memory from the hippocampal circuits to the cortex. An alternate view is that aspects of the memory are always in the cortex but they are dependent on the hippocampus because recurrent activity from cortex to hippocampus to cortex is required for consolidation. Hence, disrupting hippocampal activity during consolidation leads to memory loss (Krashes, 2007).

The current data suggest the simpler fruit fly brain similarly employs parallel and sequential use of different regions to process memory. MB α'β' neuron activity is required to form memory, MB α'β' neurons and DPM neurons are transiently required to consolidate memory and output from αβ neurons is exclusively required to retrieve memory. It is therefore proposed that aversive and appetitive odor memories are formed in MB αβ neurons and are stabilized there by recurrent activity involving MB α'β', DPM neurons and the MB αβ neurons themselves (Krashes, 2007).

It is becoming increasingly apparent that neural circuit analysis will play an important role in understanding how the brain encodes memory. The ease and sophistication with which one can manipulate circuit function in Drosophila, combined with the relative simplicity of insect brain anatomy should ensure that the fruit fly will make significant contributions to this emerging discipline (Krashes, 2007).

This study distinguishes the memory response of flies to appetitive vs. aversive long-term memory. In Drosophila, formation of aversive olfactory long-term memory (LTM) requires multiple training sessions pairing odor and electric shock punishment with rest intervals. In contrast, this study shows that a single 2 min training session pairing odor with a more ethologically relevant sugar reinforcement forms long-term appetitive memory that lasts for days. Appetitive LTM has some mechanistic similarity to aversive LTM in that it can be disrupted by cycloheximide, the dCreb2-b transcriptional repressor, and the crammer and tequila LTM-specific mutations. However, appetitive LTM is completely disrupted by the radish mutation that apparently represents a distinct mechanistic phase of consolidated aversive memory. Furthermore, appetitive LTM requires activity in the dorsal paired medial neuron and mushroom body α'β' neuron circuit during the first hour after training and mushroom body αβ neuron output during retrieval, suggesting that appetitive middle-term memory and LTM are mechanistically linked. Finally, this study describes experiments in which feeding and/or starving flies after training reveal a critical motivational drive that enables appetitive LTM retrieval (Krashes, 2008).

A single 2 min training session pairing odor with sucrose forms appetitive memory that lasts for days. The term 'session' rather than 'trial' is used cautiously because, although the conditioned odor stimulus is continuously presented for 2 min, it is not known how often the flies sample the sugar unconditioned stimulus. One session of the established aversive training paradigm presents 12 shocks at 5 s intervals overlapping with 1-min-long odor exposure, and therefore neither protocol is strictly 'single-trial' learning. Nevertheless, the results present a profound difference between the training protocol requirements to form aversive and appetitive LTM in flies. Formation of aversive LTM requires 5-10 training sessions with rest intervals, whereas a single 2 min session is sufficient to form robust protein synthesis-dependent appetitive LTM. Appetitive LTM is disrupted by cycloheximide (CXM) feeding, inhibition of CREB-dependent transcription, and the crammer (Comas, 2004) and tequila (Didelot, 2006) genes, which suggests that it is bona fide LTM. Furthermore, these data indicate some mechanistic parallel between aversive and appetitive LTM. Appetitive conditioning forms more distributed memory traces in the brain and more efficiently forms LTM than aversive conditioning. It is speculated that these properties of appetitive memory result from the ethological relevance of feeding and the salience of sucrose reinforcement. Furthermore, the salience is likely to be enhanced in hungry flies because they are motivated to seek food. There are a few other reports of single-trial training forming LTM. With the notable exception of fear conditioning in rodents, most involve feeding behavior and the gustatory pathway. In conditioned taste aversion experiments, rodents develop a long-lasting avoidance of a novel tastant after a single exposure of the tastant and delayed drug-induced malaise. Similarly, pond snails develop long-lasting conditioned taste aversion if carrot juice is paired with salt exposure, and 1-d-old chicks develop LTM to avoid pecking a colored bead if that bead was tainted with a bitter tasting compound when first presented. There are also examples in which single-trial conditioning forms appetitive LTM. Rats deficient in thiamine can be trained to prefer non-nutritious saccharin-flavored water by pairing it with delayed an intramuscular thiamine injection. Pond snails form appetitive LTM for the odorant/tastant amylacetate after a single trial of appetitive conditioning pairing it with sucrose. Last, a single trial of appetitive conditioning in honeybees forms robust day-long memory that, surprisingly, does not require new protein synthesis after training. Therefore, it is possible that the innate importance of food-seeking behavior and memory makes it particularly prone to fast consolidation to LTM (Krashes, 2008).

The single training session appetitive LTM assay provides a unique advantage for the study of memory consolidation because one can manipulate the brain immediately after training during the initial period of memory formation. In contrast, 10 cycles of aversive spaced training takes 150 min to complete, and therefore one cannot perturb neural processing during this period without also interfering with acquisition. Using cold-shock anesthesia, it was found that appetitive memory is quickly, and perhaps entirely, consolidated to anesthesia-resistant forms within 2 h after training (Krashes, 2008).

Previous work in flies suggests that cold shock-resistant memory can be broken into two independent components, ARM that depends on the radish (rsh) gene and is resistant to CXM and LTM that is unaffected by rsh and is sensitive to CXM. Feeding flies CXM disrupted appetitive LTM and produced a statistically significant defect 6 h after training, suggesting that protein synthesis-dependent LTM guides behavior at that time. Although the effect of CXM feeding is estimated to inhibit only 50% of global protein synthesis and has to be partial, these data are consistent with the notion that consolidated memory before 6 h might be ARM. However, whereas aversive LTM requires protein synthesis and is not affected by rsh, appetitive LTM requires new protein synthesis and rsh, suggesting appetitive LTM and rsh-dependent appetitive memory do not represent separable memory phases. This result highlights a potentially major mechanistic difference between aversive and appetitive LTM, and that the relationship between ARM and LTM is worth revisiting. Unfortunately, the cloning of rsh does not provide any mechanistic insight because its primary sequence does not contain any known functional domains (Krashes, 2008).

These data reveal a slight discrepancy in the notion that rsh, dCreb-dependent transcription and new protein synthesis are all necessary components of appetitive LTM. Cold-shock anesthesia indicates that appetitive memory consolidation is nearly complete 2 h after training and rsh mutant flies display defective performance 3 h after training, but neither dCreb2-b repressor transgene nor CXM feeding produced a significant difference in memory performance 3 h after training. It is speculated that expression of early forms of appetitive LTM (E-LTM) depend on rsh and that because Radish protein immunolocalizes to neuropil, Radish might function in a synaptic tagging process that marks the relevant synapses for capture of dCreb2-dependent transcripts. This idea provides a plausible reason why radish is required both for E-LTM and for later appetitive LTM (L-LTM), whereas dCreb2-b (Cyclic-AMP response element binding protein B) only interferes with L-LTM. Similarly, it is posited that CXM feeding blocks the translation of mRNAs that are direct and indirect targets of CREB and that are necessary for L-LTM. Similar models have been proposed based on work in rodents and Aplysia (Krashes, 2008).

Previous work has determined that stable olfactory memory (MTM) observed 3 h after aversive and appetitive training requires the sequential involvement of different MB neuron subsets. MB α' β' neurons are required during and after training to acquire and stabilize memory (Krashes, 2007), whereas MB αβ neuron output is required only to retrieve the memory. Stable aversive and appetitive MTM also requires the action of MB-innervating dorsal paired medial (DPM) neurons during the first hour after training. Similarly timed manipulation of these distinct neural circuit elements strongly impairs appetitive LTM, suggesting a tight mechanistic link between appetitive MTM and LTM (Krashes, 2008).

Finding that consolidation of appetitive memory to a protein synthesis-dependent form requires the DPM-MB neural circuitry and that retrieval requires MB αβ neuron output is consistent with the idea that consolidated memory is represented in MB αβ neurons themselves. Several studies have now reported that MB neuron output is required to retrieve olfactory memory, and a few have indicated that MB αβ neurons are particularly important to retrieve aversive and appetitive MTM. A recent live-imaging study provided additional evidence that consolidated aversive LTM is represented in MB αβ neurons (Yu, 2006). Flies that had been space trained with odor and shock exhibited enhanced odor-evoked Ca²⁺ signals in the vertical α branch of MB αβ neurons 9-24 h after conditioning. The development of this memory 'trace' was disrupted by CXM administration, by mutations in the amnesiac gene, and by expressing a transgenic dCreb2-b in MB αβ neurons. Furthermore, expression of the dCreb2-b transgene in MB αβ neurons also impaired aversive LTM behavior. These data are highly consistent with the current findings for appetitive LTM after a single training session and therefore indicate that there are common mechanistic components to aversive and appetitive LTM. It is also worth noting that radish is strongly expressed in MB αβ neurons. Therefore, this collection of findings provides strong evidence that consolidated aversive and appetitive LTM involves MB αβ neurons (Krashes, 2008).

These results do not support the recently proposed idea that LTM consolidation involves transfer from MB to EB (Wu, 2007). Although an appetitive memory assay was used, it was found that Feb170;uas-shi^ts1 flies have a pronounced locomotor defect and therefore these flies are not suitable for memory analysis. Furthermore, Ruslan GAL4 (Wu, 2007) and c305a (Krashes, 2007) express in EB ring neurons, but blocking these neurons does not affect appetitive LTM retrieval. These data are instead consistent with the notion that the transfer of the MB lobe requirement within the first few hours after training may be the fly equivalent of systems consolidation (Krashes, 2008).

These data clearly demonstrate that flies have to be hungry to effectively retrieve appetitive memory. Feeding them ad libitum after training suppressed memory performance, but restarving them restored memory performance. It is proposed that this apparent context dependence of appetitive memory retrieval reflects a motivational state to seek food and therefore it is predicted to be regulated by neuromodulatory systems that signal hunger (Krashes, 2008).

Dopaminergic neurons with distinct projection patterns and physiological properties compose memory subsystems in a brain. However, it is poorly understood whether or how they interact during complex learning. This study identified a feedforward circuit formed between dopamine subsystems and showed that it is essential for second-order conditioning, an ethologically important form of higher-order associative learning. The Drosophila mushroom body comprises a series of dopaminergic compartments, each of which exhibits distinct memory dynamics. A slow and stable memory compartment can serve as an effective 'teacher' by instructing other faster and transient memory compartments via a single key interneuron, which was identified by connectome analysis and neurotransmitter prediction. This excitatory interneuron acquires enhanced response to reward-predicting odor after first-order conditioning and, upon activation, evokes dopamine release in the 'student' compartments. These hierarchical connections between dopamine subsystems explain distinct properties of first- and second-order memory long known by behavioral psychologists (Yamada, 2023).

A long-standing question in the study of long-term memory is how a memory trace persists for years when the proteins that initiated the process turn over and disappear within days. Previously, it was postulated that self-sustaining amyloidogenic oligomers of cytoplasmic polyadenylation element-binding protein (CPEB) provide a mechanism for the maintenance of activity-dependent synaptic changes and, thus, the persistence of memory. This study found that the Drosophila CPEB Orb2 forms amyloid-like oligomers, and oligomers are enriched in the synaptic membrane fraction. Of the two protein isoforms of Orb2, the amyloid-like oligomer formation is dependent on the Orb2A form. A point mutation in the prion-like domain of Orb2A, which reduced amyloid-like oligomerization of Orb2, did not interfere with learning or memory persisting up to 24 hr. However the mutant flies failed to stabilize memory beyond 48 hr. These results support the idea that amyloid-like oligomers of neuronal CPEB are critical for the persistence of long-term memory (Majundar, 2012).

Learning changes the efficacy and number of synaptic connections. Memory is the maintenance of that altered state over time. Synaptic modification is likely to include both quantitative and qualitative changes in local protein composition. However, this model of memory raises a fundamental question that remains unanswered: how does the altered protein composition of a synapse persist for years when the molecules that initiated the process should disappear within days (Majundar, 2012)?

The protein composition of a synapse can be altered in several ways including synthesis of new proteins. Local synthesis of new proteins at the synapse has been shown to be essential for stabilizing the functional changes and physical growth of the activated synapse. Previously, a family of RNA-binding proteins, known as cytoplasmic polyadenylation element-binding proteins (CPEBs), were identified as regulators of activity-dependent protein synthesis at the synapse. In the sea snail Aplysia, a neuron-specific variant of CPEB, ApCPEB, is required not for the initial changes in synaptic efficacy or growth following serotonin stimulation but for the maintenance of these changes beyond 24 hr). In Drosophila, reduction in Orb2, a member of the CPEB protein family, does not affect short-term memory (< 3 hr) but does prevent the memories from persisting beyond 12 hr. In mice, deletion of the CPEB-1 gene reduces long-term potentiation evoked by theta-burst stimulation and growth-hormone applicatio. Together, these observations suggest that CPEBs play a role in stabilizing activity-dependent changes in synaptic efficacy. However, how CPEB-dependent changes in molecular composition of the synapse persist over time is unknown (Majundar, 2012 and references therein).

Previously, based on the self-sustaining amyloidogenic (prion-like) properties of Aplysia CPEB, it was hypothesized that the activity-dependent conversion of CPEB to the amyloidogenic state provides a self-sustaining mechanism for the persistent change in molecular composition of the synapse and thereby persistence of memory. Consistent with this idea, in Aplysia sensory neurons ApCPEB forms amyloidogenic aggregates when overexpressed, and the number of aggregates increases following stimulation with serotonin. Moreover, an antibody that recognizes oligomeric ApCPEB selectively blocks the persistence of long-term facilitation of the sensory-motor neuron synapse beyond 24 hr. However, the central question of whether such conversion of neuronal CPEB to the amyloid-like state is necessary for the persistence of memory remains unanswered. To address the behavioral significance of the amyloid-like state of CPEB, Drosophila Orb2 was studied. Drosophila Orb2 protein carry a prion-like domain and target synaptic growthrelated proteins, suggesting that Orb2 is not only structurally but also functionally similar to ApCPEB (Majundar, 2012 and references therein).

This paper has asked two specific questions. First, does the Orb2 protein form amyloid-like oligomers in the adult Drosophila brain in an activity-dependent manner? Second, is this oligomerization necessary for long-term memory? It was found that Drosophila Orb2 forms stable SDS-resistant, amyloid-like oligomers upon neuronal stimulation, and Orb2 mutant defective in activity-dependent oligomerization is specifically impaired in forming stable long-term memories. These observations support the hypothesis that self-sustaining amyloid-like conversion of neuronal CPEB is involved in the persistence of memory (Majundar, 2012).

Previously, based on studies primarily with Aplysia CPEB, it was postulated that self-sustaining amyloidogenic oligomers, similar to yeast prion-like proteins, might be the basis of the persistence of activity-dependent increase in synaptic efficacy and the persistence of memory. However some of the earlier analysis was performed under overexpression or in heterologous conditions. This study found that like other amyloids, in physiological concentration, in the adult Drosophila brain, Orb2 forms tetramers or hexamers that are resistant to heat, SDS, and chaotropic reagents. Stimulation of behaviorally relevant neurons increases the level of oligomeric Orb2, which is enriched in the synaptic membrane fraction. These observations suggest that the unusual amyloidogenic oligomerization of Orb2/CPEB is conserved across species, and the oligomer may indeed act to stabilize activity-dependent increase in synaptic efficacy (Majundar, 2012).

A mutation in the rare Orb2A isoform that results in reduced oligomerization, without lowering the amount of Orb2B protein, produces a selective deficit in the stabilization of memory beyond 24 hr. This is different than loss of both isoforms, which leads to earlier memory deficit. One interpretation of these data is that Orb2B activity is required for the formation of long-term memory, whereas Orb2A activity is required for the persistence of memory beyond 24 hr. However, both Orb2A and Orb2B form amyloidlike oligomers and when overexpressed in Dorb2/+ background can rescue the male courtship suppression memory as well as olfactory-reward memory at 24 hr, suggesting functional similarity. How can these observations be reconciled (Majundar, 2012)?

When expressed in S2 cells, both Orb2A and Orb2B act as translation repressor (Mastushita-Sakai, 2010). In the adult brain, the Orb2B protein is constitutively expressed in a large number of neurons, but the Orb2A protein is 100-fold less abundant, expressed in fewer neurons, and deletion of Orb2A reduces overall Orb2 oligomerization in vivo. Together, these results suggest the following model. Orb2B-mediated translation repression is critical for the formation and consolidation of memory up to 24 hr, and when ectopically expressed, Orb2A rescues this repressive function of Orb2B. However, regulated conversion of Orb2 proteins to the oligomeric state is necessary for long-term stabilization of memory beyond 24 hr, and the Orb2A protein regulates this conversion. This model implies that Orb2A and Orb2B have nonredundant functions in long-term memory, and neurons in which Orb2 oligomerizes are the site for long-term memory storage in Drosophila (Majundar, 2012).

The recent modENCODE project has reported four new protein isoforms in the Orb2 locus that would be affected in the Orb2 deletion mutants. However, the Orb2 oligomers or the behavioral phenotypes observed in this study are not dependent on these isoforms. The Orb2 antibodies used in this study do not recognize the common region between Orb2B and the new isoforms. Moreover, Orb2A and Orb2B cDNA as well as a genomic construct that does not code for any of these new proteins isoforms can rescue behavioral deficit. The function of these new isoforms has yet to be determined (Majundar, 2012).

Although the amyloidogenic forms of PrP and other proteins are pathogenic, it is now evident that amyloids can underlie epigenetic heritable phenotypes in yeast and can serve normal physiological functions in other organisms. However, in most cases it is unclear how amyloid formation is regulated, if at all. The low level of Orb2 oligomers in the adult brain and their virtual absence from the body raise the possibility that although Orb2B protein is widely expressed, Orb2 oligomerization per se is limited, perhaps only in neurons in which Orb2A is expressed. The Orb2A protein, due to its propensity to oligomerize, may form the seed that recruits Orb2B protein, resulting in the temporally and spatially restricted conversion of Orb2A/Orb2B into self-sustaining oligomers. In this regard, Orb2 oligomerization may resemble seeded formation of curli amyloid on the surface of bacteria, in which oligomerization of the major curli subunit CsgA is seeded by the membrane-bound minor subunit CsgB (Majundar, 2012 and references therein).

Curiously, this study found that Orb2A mRNA in the adult brain retains an intronic sequence with stop codons. Among age-matched pCasper-Orb2AEGFP flies, heterogeneity in the Orb2A protein level was observed, particularly in the synaptic-neuropil region. The low abundance, presence of unprocessed mRNA, and immense propensity to oligomerize imply that in the adult head, expression of Orb2A is regulated and may constitute the rate-limiting step in Orb2 oligomerization and thereby long-lasting memory formation. Moreover, although Orb2A mRNA is present in the body, the Orb2A protein was undetectable, suggesting that it is present either in very low levels or only in certain cell types. What function Orb2A serves outside the nervous system is not known (Majundar, 2012).

It is now evident that a number of proteins with very different primary amino acid sequences can form self-templating amyloids. What sequence and structural features distinguish a regulated functional amyloid from unregulated inactive or pathogenic amyloids? Although these studies were initiated with the observation that Aplysia CPEB contains a Q-rich unstructured domain, this study found that the Q-rich region of Orb2 is important for the stability but not formation of oligomer. Similarly, a coiled-coil domain outside the amyloid-forming domain of Aplysia CPEB regulates its oligomerization. These observations suggest that identification of functional amyloid based on primary amino acid sequence is challenging. Highlighting this point, it was found that a single nonpolar to polar amino acid change in the N-terminal 8 amino acids of Orb2A affects not only the efficiency of oligomerization but also the nature of the amyloid oligomer. Structural analysis of wild-type and mutant Orb2 proteins may help us to understand what features distinguish functional amyloid from nonfunctional amyloid (Majundar, 2012).

Extensive molecular, genetic, and anatomical analyses have suggested that olfactory memory is stored in the mushroom body (MB), a higher-order olfactory center in the insect brain. The MB comprises three subtypes of neurons with axons that extend into different lobes. A recent functional imaging study has revealed a long-term memory trace manifested as an increase in the Ca²⁺ activity in an axonal branch of a subtype of MB neurons. However, early memory traces in the MB remain elusive. This paper reports learning-induced changes in Ca²⁺ activities during early memory formation in a different subtype of MB neurons. Three independent in vivo and in vitro preparations were used, and all of them showed that Ca²⁺ activities in the axonal branches of α'/β' neurons in response to a conditioned olfactory stimulus became larger compared with one that was not conditioned. The changes were dependent on proper G-protein signaling in the MB. The importance of these changes in the Ca²⁺ activity of α'/β' neurons during early memory formation was further tested behaviorally by disrupting G-protein signaling in these neurons or blocking their synaptic outputs during the learning and memory process. The results suggest that increased Ca²⁺ activity in response to a conditioned olfactory stimulus may be a neural correlate of early memory in the MB (Wang, 2008).

Functional Ca²⁺ imaging was used in three different preparations to search for memory traces in the MB after a single-cycle training that produces short-term memory (STM), middle-term memory (MTM), anesthesia-resistant memory (ARM). Flies were trained in groups in the standard T-maze or individually while held immobilized. These two different settings in training both led to enhanced Ca²⁺ activity in response to trained odors in the axonal branches of the α'/β' neurons. The enhancement was specific to the odor that was paired with electric shock and was detected at ~1 hour after conditioning. The semi-in vivo preparation that mimicked the single-cycle olfactory training with paired stimulation of the antennal nerves (ANs) and the ventral nerve cord (VNC) led to a similar observation. In addition, an immediate enhancement in Ca²⁺ response in α'/β' lobes after the paired event was observed in this preparation. Targeted disruption of the G-protein signaling in the MB blocked the enhancement of Ca²⁺ response after paired stimulation of AN and VNC. These results indicate that the enhanced Ca²⁺ response in α'/β' lobes after conditioning may represent a memory trace associated with early forms of memory. This was further confirmed in a behavioral experiment in which the 3 min and 1 h memory was impaired by disrupting the G-protein signaling in the α'/β' neurons or blocking synaptic output from these neurons (Wang, 2008).

This finding does not preclude the possible existence of memory traces in other output branches of the MB neurons. There are a number of possibilities why the enhanced Ca²⁺ response to trained odors was only observed in axonal branches of the α'/β' neurons. Memory traces in the α/β and γ neurons may appear in forms other than alteration of Ca²⁺ response. It is known that a Ca²⁺-independent PKC signaling can potentiate synaptic transmission presynaptically. Another possibility is that for α/β and γ neurons, memory traces may occur postsynaptically in downstream neurons. Postsynaptic mechanisms play a key role in long-term potentiation and long-term depression. A third possibility is that the low G-CaMP responses in α/β and γ lobes are making it difficult to detect changes in these lobes. Activities in MB neurons are sparse, and the γ neurons tend to show a lower probability of response. Changes in a small amount of activity may be hard to detect with the imaging approach that was used. Finally, learning may lead to changes in the fine temporal pattern of neuronal activities in the MB that cannot be detected by Ca²⁺ imaging (Wang, 2008).

Olfactory learning and memory in Drosophila seem to involve multiple brain structures in parallel and sequential pathways. Blocking the output from the MB impairs retrieval of memory at multiple stages that encompass STM, MTM, ARM, and LTM. This requirement of the MB output has been further dissected and assigned to different subtypes of MB neurons for different stages of memory processing. The initial study with the Shi^ts has indicated that retrieval of 3 min and 3 h memory may be exclusively through α/β neurons. The most recent study on 3 h memory confirmed the role for α/β neurons in retrieval and further revealed that output of the α'/β' neurons is required for acquisition and consolidation. Several places outside the MB are also involved in olfactory learning and memory. Output from a pair of neurons called dorsal paired medial (DPM) neurons that innervate the entire MB lobes are required for 3 h memory in a time window of 30-150 min after conditioning. One of the NMDA receptor subunits, NR1, which is preferentially expressed in a small number of neurons that innervate the MB dendritic region, affects learning and LTM consolidation. Several genes expressed in a small number of neurons outside the MB were identified in a large-scale mutagenesis to affect 1 d memory. Dopaminergic and octopaminergic neurons, which both innervate the MB lobes, are believed to carry the aversive and appetitive unconditioned stimulus reinforcement, respectively (Wang, 2008).

Correspondingly, learning and memory-associated cellular changes, collectively called memory traces, have been found in some of these structures at different stages of memory processing. In dorsal paired medial (DPM) neurons, odor-evoked Ca²⁺ response in the branch that innervates the MB vertical lobes (α and α') is enhanced 30 min after conditioning. In the MB, enhanced odor-evoked Ca²⁺ response is observed in the α lobe 24 h after spaced training (multiple training sessions with a rest between sessions), which produces LTM. In addition, a memory trace appearing as recruitment of new projection neurons (PNs) in the AL occurs immediately after conditioning but lasts only a few minutes, disappearing at 7 min after conditioning (Wang, 2008).

How these memory traces interact with each other is not yet clear. The memory trace in the AL PNs and that was observed in the MB α'/β' neurons may appear in parallel after conditioning so STM may be formed in two independent places. The following observations support this possibility. (1) The memory trace in PNs is observed in the presynaptic specializations of PNs localized in the AL glomeruli. (2) No change was observed in the amount of odor-evoked Ca²⁺ response after olfactory training in the calyx, where synaptic connections are made between MB neurons and PNs. The spatial distribution of activity in the calyx was also not changed after olfactory training. Therefore, the recruited new synaptic activities in the AL may not be transmitted to the MB to drive activity changes there at time points studied (Wang, 2008).

The involvement of DPM neurons in memory consolidation relies on their projections to the axonal branches of the MB α'/β' neurons. This suggests that memory consolidation may be performed by local interactions between DPM neurons and the MB α'/β' neurons, and the memory may reside in the α'/β' neurons during this process. This idea is also supported by the finding that the α'/β' neurons are required for memory acquisition and consolidation. The observation of a memory trace in the axonal branches of the MB α'/β' neurons also provides a strong support to the idea that the memory initially resides in the α'/β' lobes. From this perspective, it is interesting that the memory trace in the DPM neurons is only observed in branches that innervate the MB vertical lobes. It is not known whether this memory trace is restricted further to branches that innervate the α' lobe or is expressed in all branches that innervate vertical lobes. The memory trace observed in the current study in α'/β' neurons seems to appear before the memory trace in the DPM neurons, which was detected ~30 min after conditioning. Currently, it is not known whether there is any causal link between the two memory traces or whether they may exist independently. It should be interesting to see how the two memory traces are affected by manipulating activities of the DPM neurons and α'/β' neurons (Wang, 2008).

The LTM trace in the α lobe of the α/β neurons start to appear at 9 h after spaced training. It will be interesting to investigate whether the early memory trace observed in the α'/β' neurons also exists after spaced training and how the STM trace is converted into the LTM trace. The LTM trace is blocked by mutation of the amnesiac gene, which is expressed in the DPM neurons. Given the facts that the amnesiac mutation also blocks the intermediate memory trace in DPM neurons and that DPM projections to the axonal branches of the α'/β' neurons are sufficient for its role in memory consolidation, there is a very good possibility that the amnesiac mutation will disrupt the memory trace in the α'/β' neurons and this disruption results in the elimination of the LTM trace in the α/β neurons. Future studies addressing these issues should help derive a better understand how memory is formed and maintained in the brain (Wang, 2008).

Memories are not created equally strong or persistent for different experiences. In Drosophila, induction of long-term memory (LTM) for aversive olfactory conditioning requires ten spaced repetitive training trials, whereas a single trial is sufficient for LTM generation in appetitive olfactory conditioning. Although, with the ease of genetic manipulation, many genes and brain structures have been related to LTM formation, it is still an important task to identify new components and reveal the mechanisms underlying LTM regulation. This study shows that single-trial induction of LTM can also be achieved for aversive olfactory conditioning through inhibition of highwire (hiw)-encoded E3 ubiquitin ligase activity or activation of its targeted proteins in a cluster of neurons, localized within the α/β core region of the mushroom body. Moreover, the synaptic output of these neurons is critical within a limited posttraining interval for permitting consolidation of both aversive and appetitive LTM. It is proposed that these α/β core neurons serve as a 'gate' to keep LTM from being formed, whereas any experience capable of 'opening' the gate is given permit to be consolidated into LTM (Huang, 2012).

The current study began with the finding that 24 hr memory resulting from single session was enhanced in two hiw mutant alleles. This enhanced memory component was identified as facilitated LTM, given that it was sensitive to protein synthesis inhibition. The behavioral effect of hiwδRING and the presence of a hiw-GAL4 line allowed mapping the neural circuitry to a cluster of MB α/β core neurons, within which Hiw and its downstream targets regulate LTM. Furthermore, it was shown that the MB α/β core neurons are involved in the consolidation of both aversive and appetitive LTM. In conclusion, the observations that the MB α/β core neurons are capable of both facilitating and limiting LTM suggests a working model in which the ability to form LTM is gated through these neurons. The significance of these results is further elaborated below (Huang, 2012).

Not only synthesis but also degradation of proteins plays a critical role in the remodeling of synapses, learning, and memory. Altered memory formation in ubiquitin ligase mutants has been reported in mice and Drosophila. This study reports that Hiw, an evolutionarily conserved E3 ubiquitin ligase, negatively regulated LTM formation through restraining its downstream target Wallenda (Wnd). The results indicated Hiw function as an inhibitory constraint, as the memory suppressor gene, on LTM formation. Removal of this suppressor or direct activation of its downstream signals could lead to the facilitated LTM induction without the repetitive training that is normally required. So far, the physiological consequence of Hiw or Wnd in core neurons still remains an open question. Because of the extensively shared components with hiw’s function in synaptic growth and transmission, the attenuated Hiw activity may elevate Wnd level and then lead to the excessive synaptogenesis or abnormal synaptic activity in core neurons. It will also be interesting to test whether the hiw-mediated LTM facilitation shares some common molecular components with the corkscrew-regulated spacing effect in LTM induction, in which the MB α/β lobes also play an important role (Huang, 2012).

In a recent report, Hiw was shown to regulate the axon guidance in MB (Shin, 2011). The morphological defect was also observed in MB α/β lobes in a portion of hiw mutant flies. It is striking that hiw mutants with MB defect can form LTM even more efficiently, given the observation of LTM impairment in another MB structural mutant, ala. However, comparing to the total loss of vertical lobes (including α and α') in ala mutant, most hiw mutants had the abnormal thickness of α/β lobes caused by the unequal distribution of the MB axonal projections between the α and β lobes, and about 39% of hiwDN mutant had the shortened α lobe. One of the possible explanations is that the remaining function of α/β lobe in hiw mutants is sufficient to support the LTM. Moreover, expression of Hiw dominant-negative protein or acutely increasing Wnd protein level in MB was sufficient to promote LTM but did not give rise to any observable gross morphological change in MB. Thus, it is suggested that Hiw mediates memory phenotype through a different mechanism from the one that led to the structure change in the MB (Huang, 2012).

The involvement of MB in the hiw-mediated LTM facilitation led an examination of the function of this structure in LTM regulation. It has been well documented that the MB, a bilateral brain structure that consists of approximately 2,500 neurons in each hemisphere, plays the central role in olfactory memories, both aversive and appetitive. Intrinsic MB neurons are organized into physically distinct α, β, α', β' and γ lobes. All three lobes exhibit different functions in memory processing, such that the output of α/β lobes is required for retrieval of memory, α' β' lobes are transiently required to stabilize memory or to retrieve immediate memory, and γ lobe mediate a rutabaga-dependent mechanism and dopaminergic signal to support short-term memory (STM) and LTM formation. Moreover, memory traces mapped to different lobes exhibit different temporal features (Huang, 2012).

Through gene expression patterns and enhancer trap lines, each lobe of MB can be classified into more specific subgroups such as the posterior, surface, and core regions in the α/β lobes. The current work shows that α/β core neurons play a distinct role in LTM induction. The synaptic outputs of these neurons are critical during consolidation of LTM for both aversive and appetitive conditioning, but these neurons are not involved in LTM cellular consolidation per se because a landmark of LTM cellular consolidation, CREB-mediated protein synthesis, occurs in non-MB neurons. Thus, one of the roles for this cluster of neurons can be viewed as simply providing connections to channel learning information to the downstream neurons in which LTM is formed. However, the remarkable feature of enabling single-trial induction of aversive LTM through targeted genetic manipulation of this cluster of neurons suggests that they play a unique permissive role in determining whether an experience should be consolidated (Huang, 2012).

This newly identified function for permitting an experience to be consolidated leads to proposal of a gating theory. This theory proposes that the α/β core neurons serve as a 'gate,' and activation of this gating mechanism functions as a checkpoint that keeps LTM from being formed for general experiences, whereas only specific experiences, capable of 'opening' this gate, can and are bound to trigger LTM consolidation and to form LTM ultimately. There is little survival advantage in committing never-to-be-repeated episodes to memory, particularly because the very act of LTM formation may be deleterious to the fly. In contrast, repetitively occurring experience, such as spaced repetitive aversive conditioning, and events critical for survival, such as finding food or single-trial appetitive conditioning, would be able to 'open' the gate, and therefore, LTM is formed for such experiences (Huang, 2012).

Behavioral expression of food-associated memory in fruit flies is constrained by satiety and promoted by hunger, suggesting an influence of motivational state. This study identified a neural mechanism that integrates the internal state of hunger and appetitive memory. Stimulation of neurons that express neuropeptide F (dNPF), an ortholog of mammalian NPY, mimics food deprivation and promotes memory performance in satiated flies. Robust appetitive memory performance requires the dNPF receptor in six dopaminergic neurons that innervate a distinct region of the mushroom bodies. Blocking these dopaminergic neurons releases memory performance in satiated flies, whereas stimulation suppresses memory performance in hungry flies. Therefore, dNPF and dopamine provide a motivational switch in the mushroom body that controls the output of appetitive memory (Krashes, 2009).

Drosophila can be efficiently trained to associate odorants with sucrose reward. Importantly, fruit flies have to be hungry to effectively express appetitive memory performance (Krashes, 2008). This apparent state dependence implies that signals for hunger and satiety may interact with memory circuitry to regulate the behavioral expression of learned food-seeking behavior. The mushroom body (MB) in the fly brain is a critical site for appetitive memory. Synaptic output from the MB α′β′ neurons is required to consolidate appetitive memory whereas output from the αβ subset is specifically required for memory retrieval (Krashes, 2007; Krashes, 2008). This anatomy provides a foundation for understanding neural circuit integration between systems representing a motivational state and those for memory (Krashes, 2009).

Neuropeptide Y (NPY) is a highly conserved 36 amino acid neuromodulator that stimulates food-seeking behavior in mammals. NPY messenger RNA (mRNA) levels are elevated in neurons in the arcuate nucleus of the hypothalamus of food-deprived mice. Most impressively, ablation of NPY-expressing neurons from adult mice leads to starvation. NPY exerts its effects through a family of NPY receptors and appears to have inhibitory function. NPY therefore must repress the action of inhibitory pathways in order to promote feeding behavior. Drosophila neuropeptide F (dNPF) is an ortholog of NPY, which has a C-terminal amidated phenylalanine instead of the amidated tyrosine in vertebrates. Evidence suggests that dNPF plays a similar role in appetitive behavior in flies. dNPF overexpression prolongs feeding in larvae and delays the developmental transition from foraging to pupariation. Furthermore, overexpression of a dNPF receptor gene, npfr1, causes well-fed larvae to eat bitter-tasting food that wild-type larvae will only consume if they are hungry (Krashes, 2009 and references therein).

This study exploited dNPF to identify a neural circuit that participates in motivational control of appetitive memory behavior in adult fruit flies. Stimulation of dNPF neurons promotes appetitive memory performance in fed flies, mimicking the hungry state. npfr1 is required in dopaminergic (DA) neurons that innervate the MB for satiety to suppress appetitive memory performance. Directly blocking the DA neurons during memory testing reveals performance in fed flies, whereas stimulating them suppresses performance in hungry flies. These data suggest that six DA neurons are a key module of dNPF-regulated circuitry, through which the internal motivational states of hunger and satiety are represented in the MB (Krashes, 2009).

It is critical to an animal's survival that behaviors are expressed at the appropriate time. Motivational systems provide some of this behavioral control. Apart from the observation that motivational states are often regulated by hormones or neuromodulatory factors, little is known about how motivational states modulate specific neural circuitry. Hungry fruit flies form appetitive long-term memory, after a 2 min pairing of odorant and sucrose, and memory performance is only robust if the flies remain hungry (Krashes, 2008). Therefore, this paradigm includes key features of models for motivational systems: the conditioned odor provides the incentive cue predictive of food, there is a learned representation of the goal object (odorant/sucrose), and the expression of learned behavior depends on the internal physiological state (hunger and not satiety). This study identified a neural circuit mechanism that integrates hunger/satiety and appetitive memory (Krashes, 2009).

The signals that ordinarily control dNPF-releasing neurons is unknown. In mammals, NPY-expressing neurons are a critical part of a complex hypothalamic network that regulates food intake and metabolism. In times of adequate nutrition, NPY-expressing neurons are inhibited by high levels of leptin and insulin that are transported into the brain after release from adipose tissue and the pancreas (Figlewicz, 2009). In hungry mice, leptin and insulin levels fall, leading to loss of inhibition of NPY neurons. Flies do not have leptin, but they have several insulin-like peptides, that may regulate dNPF neurons. Some NPY-expressing neurons are directly inhibited by glucose (Levin, 2006). Fly neurons could sense glucose with the Bride of Sevenless receptor (Kohyama-Koganeya, 2008). In blowflies, satiety involves mechanical tension of the gut and abdomen. Lastly, it will be interesting to test the role of other extracellular signals implicated in fruit fly feeding behavior, including the Hugin and Take-out neuropeptides (Krashes, 2009).

NPY inhibits synaptic function in mammals, and the data from this study suggest that dNPF promotes appetitive memory performance by suppressing inhibitory MB-MP neurons [named according to the regions of the MB that they innervate: medial lobe and pedunculus (MP)]. A model is proposed in which MB-MP neurons gate MB output. Appetitive memory performance is low in fed flies because the MB αβ and γ neurons are inhibited by tonic dopamine release from MB-MP neurons. Hence, when the fly encounters the conditioned odorant during memory testing, the MB neurons encoding that olfactory memory respond, but the signal is not propagated beyond the MB because of the inhibitory influence of MB-MP neurons. However, when the flies are food deprived, dNPF levels rise, and dNPF disinhibits MB-MP neurons, and other circuits, through the action of NPFR1. dNPF disinhibition of the MB-MP neurons opens the gate on the MB. Therefore, when hungry flies encounter the conditioned odorant during memory testing, the relevant MB neurons are activated and the signal propagates to downstream neurons, leading to expression of the conditioned behavior (Krashes, 2009).

Satiety and hunger are not absolute states. Sometimes above-chance performance scores are observed in fed flies, and shorter periods of feeding after training suggest that inhibition of performance is graded. This could be accounted for by a competitive push-pull inhibitory mechanism between dNPF and MB-MP neurons (Krashes, 2009).

By gating the MB through the MB-MP neurons, hunger and satiety are likely affecting the relative salience of learned odor cues in the fly brain. However, MB-MP neurons are unlikely to change the sensory representation of odor in the MB because flies trained with stimulated MB-MP neurons perform normally when tested for memory without stimulation. Therefore, odors are likely perceived the same irrespective of MB-MP neuron activity. Furthermore, the MB-MP neurons did not affect naive responses to the specific odorants used. It will be interesting to test whether MB-MP neurons change responses to other odorants and/or modulate arousal, visual stimulus salience, and attention-like phenomena (Krashes, 2009).

There are eight different morphological classes of DA neurons that innervate the MB (Mao, 2009), and the current data imply functional subdivision. Previous studies concluded that DA neurons convey aversive reinforcement (Krashes, 2009).

This study specifically manipulated the MB-MP DA neurons. MB-MP neurons are not required for acquisition of aversive olfactory memory, consistent with a distinct function in controlling the expression of appetitive memory. Since several studies have implicated the MB α lobe in memory, other DA neurons in protocerebral posterior lateral 1 (PPL1) that innervate the α lobe (like those labeled in MBGAL80;krasavietz) may provide reinforcement. The MB-MP neurons may also be functionally divisible and independently regulated to gate MB function. The idea that a specific DA circuit restricts stimulus-evoked behavior is reminiscent of literature tying dopamine to impulse control in mammals. Previous studies of DA neurons in Drosophila have simultaneously manipulated all, or large numbers of DA neurons. The current data suggest that the DA neurons should be considered as individuals or small groups (Krashes, 2009).

Flies have to be hungry to efficiently acquire appetitive memory, but whether this reflects a state-dependent neural mechanism or results from the failure to ingest enough sugar is unclear. Stimulation of MB-MP neurons in hungry flies did not impair appetitive memory formation, and therefore MB-MP neurons are unlikely to constrain learning in fed flies. Other dNPF-regulated neurons may provide this control since NPY has been implicated in learning (Krashes, 2009).

The dNPF-expressing neurons innervate broad regions of the brain and may simultaneously modulate distinct neural circuits to promote food seeking. MB-MP neurons represent a circuit through which the salience of learned food-relevant odorant cues is regulated by relative nutritional state. Given the apparent role of the MB as a locomotor regulator, MB-MP neurons may also generally promote exploratory behavior. There are likely to be independent circuits for other elements of food-seeking behavior including those that potentiate gustatory pathway sensitivity and promote ingestion (Krashes, 2009).

NPY stimulates feeding but inhibits sexual behavior in rats. Modulators exerting differential effects could provide a neural mechanism to establish a hierarchy of motivated states and coordinate behavioral control. dNPF may potentiate activity in food seeking-related circuits while suppressing circuits required for other potentially competing behaviors, e.g., sexual pursuit (Krashes, 2009).

This study has provided the first multilevel neural circuit perspective for a learned motivated behavior in fruit flies. The work demonstrates a clear state-dependence for the expression of appetitive memory. Odorants that evoke conditioned appetitive behavior in hungry flies are ineffective at evoking appetitive behavior in satiated flies. Therefore, the fly brain is not simply a collection of input-output reflex units and includes neural circuits through which the internal physiological state of the animal establishes the appropriate context for behavioral expression (Krashes, 2009).

It has been proposed that a satiated fly receives maximum inhibitory feedback so that sensory input is behaviorally ineffective. As deprivation increases inhibition wanes and sensory input becomes increasingly effective in initiating feeding. The current data provide experimental evidence that this prediction is also likely to be accurate for expression of appetitive memory in the fruit fly where the mechanism involves neuromodulation in the central brain. The DA MB-MP neurons inhibit the expression of appetitive memory performance in satiated flies, whereas dNPF disinhibits the MB-MP neurons in food-deprived flies. The likelihood that appetitive behavior is triggered by the conditioned odorant is therefore determined by the competition between inhibitory systems in the brain. The concept that continuously active inhibitory forces in the insect brain control behavioral expression has also be proposed many years ago. This study provides evidence that these neurons exist and that their hierarchical arrangement is a key determinant of behavioral control (Krashes, 2009).

It was also shown that subsets of neurons in the fly brain (ring neurons of the ellipsoid body) are critical for visual place learning, probably by implementing, storing, or reading spatial information. Strikingly, flies in which ellipsoid body neurons were silenced have a basic 'circling' search routine that is reminiscent of the behaviour displayed by rats with hippocampal lesions. Imaging of neuronal activity in the fly brain while the animal is executing a navigation task should help further define the role of the central complex, and ellipsoid body neurons in particular, in spatial memory (for example in a head-fixed preparation with a virtual-reality arena. Ultimately, elucidating the cellular basis for place learning in Drosophila will help uncover fundamental principles in the organization and implementation of spatial memories in general (Ofstad, 2011).

The β-amyloid precursor protein (APP) plays a central role in the etiology of Alzheimer's disease (AD). APP is cleaved by various secretases whereby sequential processing by the β- and γ-secretases produces the β-amyloid peptide that is accumulating in plaques that typify AD. In addition, this produces secreted N-terminal sAPPβ fragments and the APP intracellular domain (AICD). Alternative cleavage by α-secretase results in slightly longer secreted sAPPalpha fragments and the identical AICD. Whereas the AICD has been connected with transcriptional regulation, sAPPalpha fragments have been suggested to have a neurotrophic and neuroprotective role.Loss of the Drosophila APP-like (APPL) protein impairs associative olfactory memory formation and middle-term memory that can be rescued with a secreted APPL fragment. This study show that APPL is also essential for visual working memory. Interestingly, this short-term memory declines rapidly with age, and this is accompanied by enhanced processing of APPL in aged flies. Furthermore, reducing secretase-mediated proteolytic processing of APPL can prevent the age-related memory loss, whereas overexpression of the secretases aggravates the aging effect. Rescue experiments confirmed that this memory requires signaling of full-length APPL and that APPL negatively regulates the neuronal-adhesion molecule Fasciclin 2. Overexpression of APPL or one of its secreted N termini results in a dominant-negative interaction with the FASII receptor. Therefore, these results show that specific memory processes require distinct APPL products (Rieche, 2018).

Age-related memory impairment (AMI) affects all animals, and cognitive decline is one of the devastating features of Alzheimer's disease (AD). Although APP, and more specifically the β-amyloid peptide, has been connected with memory deficits in AD, the role of full-length APP and its various other fragments in AMI is unknown. Wild-type Drosophila flies display AMI at middle age (30-40 days) when tested for middle-term or long-term olfactory memory. Furthermore, Drosophila not only encodes an ortholog for APP, called amyloid precursor protein-like (APPL), but also homologs for all three types of secretases; kuzbanian (kuz) corresponds to ADAM10 considered to be an α-secretase, dBace, the fly β-secretase, and Presenilin (Psn), the catalytic subunit of γ-secretase. APPL is processed in a similar way as human APP; however, the cleavage sites of the α- and β-secretase are reversed. Therefore, cleavage by KUZ produces a shorter secreted N-terminal fragment (NTF) than processing by dBACE. Nevertheless, subsequent γ-processing of the β-cleaved C-terminal fragment (βCTF) results in a neurotoxic dAβ peptide, whereas cleavage by KUZ does not. This study asked whether the very short-term (~4 s) visual working memory in flies is also affected by AMI and whether it requires APPL or one of its proteolytic fragments. Therefore, wild-type flies and heterozygous mutants for the three secretases were aged, and their visual orientation memory was assessed (Rieche, 2018).

This working memory is tested in the detour paradigm where walking flies navigate between two inaccessible landmarks. During an approach, the targeted landmark disappears and the fly is lured toward a novel distracting landmark. This distracter disappears one second after reorientation so that the fly is now left without any landmarks. Nevertheless, wild-type Canton-S (CS) flies can recall the position of the initial landmark and try to approach it although still invisible ('positive choices'). Whereas young CS males make about 80% positive choices, aged flies showed a reduced memory when tested at 4 weeks of age and a complete memory loss when 6 weeks old. Interestingly, heterozygosity for any of the three secretases prevented AMI, with 4- and 6-week-old Psn¹⁴³/+ and kuz^e29-4/+ flies being indistinguishable from young CS flies. When using heterozygous dBace⁵²⁴³ flies, the improvement compared to age-matched CS controls did not reach significance; however, they made significantly more positive choices than chance level at 6 weeks, whereas CS did not. These findings show that visual working memory is deteriorating with age, and they suggest that reducing APPL processing can suppress AMI (Rieche, 2018).

To address whether increased processing of APPL disrupts this memory, the secretases were overexpressed in the R3 ring neurons of the ellipsoid body (using 189Y-GAL4) the seat of visual working memory. Expression of any of the secretases reduced the performance already in 3-day-old flies compared to controls, supporting a requirement of full-length flAPPL for this type of memory. On the other hand, western blot analyses using an antiserum directed against the NTFs of APPL (Ab952M) established that heterozygous secretase mutants have an overall increase especially in flAPPL which supported the hypothesis on the role of secretases and APPL processing in AMI. Furthermore, a quantitative analysis of the levels of APPL in head extracts from different ages revealed that flAPPL declines with age, whereas the NTF/flAPPL ratio increases, also suggesting that reduced levels of flAPPL are involved in AMI. To verify that APPL is indeed required for visual working memory, homozygous Appld-null mutants and transheterozygous combinations of hypomorphic Appl alleles were tested. All these mutants performed at chance level already when young, confirming that APPL is necessary for this short-termed memory. This function is dose sensitive because even young heterozygous Appld/+ showed a reduced memory that declined faster with age than in CS females (Rieche, 2018).

Next, an RNAi-mediated knockdown of Appl was introduced in the R3 neurons, which resulted in severe memory deficits already in 3- to 5-day-old flies, showing that APPL is required in these neurons for visual working memory. To identify domains in APPL that mediate this function, rescue experiments were performed expressing different APPL constructs via 189Y-GAL4 in young Appld mutant flies. This included full-length flAPPL, secretion-defective sdAPPL, specific deletion constructs, and secreted fragments. Because the exact cleavage sites in APPL are unknown, the secreted fragments are referred to as sAPPLLong (L), which comprises the N-terminal 788 amino acids and should represent the β-cleaved fragment, whereas the 758-amino-acid (aa)-long sAPPLShort (S) should represent the α-cleaved form of Drosophila APPL. In contrast to full-length wild-type APPL, neither of the secreted forms could rescue the memory deficit of Appld when induced in R3 neurons. Notably, sdAPPL very effectively rescued the memory phenotype, confirming that unprocessed flAPPL is crucial for visual working memory (Rieche, 2018).

Next, whether APPL functions as a receptor or ligand in R3 neurons was investigaged. Expression of sdAPPL that in addition lacks the intracellular C terminus (sdAPPL-ΔC) did rescue the Appld memory phenotype, which suggests that intracellular signaling is not required and that APPL does not act as a receptor. Rescue experiments with sdAPPL forms that, in addition, lack one of the two ectodomains (sdAPPL-ΔE1 and sdAPPL-ΔE2) revealed a requirement for E2 for the rescue but not for E1. To confirm this, the rescue experiments were repeated with the hypomorphic Appl4460 allele and APPL, sdAPPL, and sdAPPL-ΔE1 rescued to full extent, whereas sdAPPL-ΔE2 did not. These results suggest that membrane-bound APPL functions as a ligand in the ring neurons. This function was conserved in human APP because expression of APP695 via 189Y-GAL4 in Appld also resulted in a rescue. Using conditional expression of APPL (by combining 189Y-GAL4 with the temperature-sensitive GAL4 repressor Tub>GAL80ts) resulted in the same rescue as constitutive expression, revealing that expression in adult R3 neurons is sufficient to restore the memory in 3- to 5-day-old flies. Notably, using the same expression system to induce moderate overexpression of sdAPPL (at 25°C), it was possible to rescue the AMI in a wild-type background, demonstrating that the secretion-deficient unprocessed APPL can prevent the decline of visual working memory of aged flies. Together with the finding that the levels of endogenous flAPPL decrease with age, this suggests that a loss of flAPPL underlies the visual working memory deficits that occur during normal aging. Interestingly, an age-related increase in BACE1 activity has been described in vertebrates that could reduce levels of full-length APP (Rieche, 2018).

Most of APPL functions in synaptogenesis, neurite outgrowth, and guidance described so far required signaling via the C-terminal domain. Analyzing heterozygous Appld/+ mutant flies or inducing an adult-specific knockdown of Appl in the relevant mushroom body neurons, Preat and Goguel (2016) showed that APPL function is not required for olfactory learning, but for a 2-hr associative memory and long-term memory formation. Similar to findings in mice, overexpression of secreted sAPPLL (as well as APPL and sdAPPL) could restore the 2-hr memory in heterozygous Appld/+ flies, whereas only wild-type APPL could rescue the long-term memory deficit. Because endogenous APPL was still expressed in this rescue experiments, sAPPLL and sdAPPL might act as ligands that bind to flAPPL in mushroom body neurons. The authors therefore suggested that distinct memory phases require different forms of APPL and maybe different intracellular signaling pathways. Notably, overexpression of KUZ in the mushroom body did not affect the 2-hr olfactory memory, whereas KUZ in this study significantly reduced visual working memory. It should also be noted that unprocessed APPL can be deleterious, because expression of sdAPPL in photoreceptor cells caused cell death of lamina glia via an unknown receptor, further emphasizing that individual neuronal networks may require different APPL fragments and signaling pathways (Rieche, 2018).

Due to recent studies suggesting that output from the R3 neurons into the ellipsoid body is instrumental for visual working memory, it was hypothesized that full-length APPL is present at the axonal terminals of R3 neurons in the ellipsoid body. To analyze the sub-cellular localization of APPL and its fragments, a double-tagged version of APPL (dtAPPL) was used that carries an EGFP tag near the N terminus and RFP tag at the C terminus, resulting in yellow fluorescence of full-length dtAPPL (or co-localized fragments that have not been separated yet). Using 189Y-GAL4 to induce dtAPPL, it was observed that dtAPPL is processed differently in individual R3 neurons, whereby full-length as well as fragments of dtAPPL are found in the cell bodies and axonal/dendritic projections. That significant amounts of unprocessed APPL can be found in the R3 axons in the ellipsoid body supports the model, that full-length APPL is needed at the R3 output sites. Note that dtAPPL was able to rescue the memory deficit, as did a dtAPP695, which showed a similar distribution pattern as dtAPPL (Rieche, 2018).

Having established that full-length APPL can be found at the relevant output sides, it was asked whether proteolytic processing of APPL also changes in the R3 neurons with age. Comparing the pattern of dtAPP695 expressed with the 189Y-GAL4 driver in 3-day-old and 6-week-old flies indicated less full-length dtAPP695 in aged flies, but, when quantifying this, it did not reach significance. Therefore, another R3-neuron-specific GAL4 line (VT42759) was used that results in reduced levels of dtAPP695 with increasing age but could nevertheless be used to rescue the memory phenotype of Appld. Compared to 3-day-old flies, there was little co-localization of GFP-tagged N termini and RFP-tagged C termini in 6-week-old VT42759>dtAPP flies, revealing enhanced proteolytic processing of APP. This suggests that AMI of the visual orientation memory is caused by increased ectodomain shedding of APPL and western blot analysis of aged flies supports this notion because during aging the ratio of NTFs to flAPPL increases (Rieche, 2018).

To identify a possible receptor for APPL in visual working memory, focus was placed on the neural cell adhesion molecule Fasciclin 2 (FASII). FASII is enriched in most, if not all types of ring neurons in the ellipsoid body, and it has been shown to interact with APPL in synaptic bouton formation at the neuromuscular junction (NMJ) (Ashley, 2005). Moreover, FASII is the insect homolog of neural cell adhesion molecule (NCAM)-140, which has been demonstrated to bind APP in an E2-depending fashion. When young hemizygous mutants were tested for the strong hypomorphic FasII^e76 allele in the detour paradigm, they showed no working memory, and the same phenotype was observed when an RNAi against FasII was introduced in R3 neurons of 3- to 5-day-old flies. This phenotype could be rescued by expression of FASII in R3 neurons, which reveals that FASII is required in the same ring neuron subtype as APPL, providing a possible binding partner for APPL. At the NMJ, loss of APPL suppressed the increase in bouton number in heterozygous i>FasII^e76/+ larvae. This study therefore investigated whether removing one copy of Appl could rescue the memory deficits of homozygous i>FasII^e76 mutants. This resulted in a significant improvement in performance, as did one copy of the i>FasII^e76 mutant allele in homozygous Appld-null mutant flies. Moreover, reducing FASII expression in R3 neurons by RNAi also ameliorated the memory deficit of Appld-null mutants. Together, this suggests that APPL negatively regulates FASII in R3 neurons and that FASII acts downstream of APPL. That this negative interaction is essential to prevent AMI is demonstrated by the observation that heterozygosity for v suppresses the memory loss of 4-week-old heterozygous Appld/+ flies (Rieche, 2018).

To further investigate interactions between APPL and FASII, overexpression studies were performed using young flies, and elevated levels of flAPPL or sdAPPL were found to ameliorate the memory deficit induced by FASII overexpression. However, only wild-type APPL induced memory deficits when overexpressed without FASII. This suggested that a secreted form of APPL can induce a gain-of-function phenotype, and this study therefore overexpressed sAPPLL and sAPPLS in R3 neurons. Whereas sAPPLL had no effect, sAPPLS caused a severe impairment of visual working memory. Moreover, overexpression of sAPPLS suppressed the effects of elevated FASII levels, whereas sAPPLL did not. This shows that sAPPLS, which corresponds to the α-cleaved (KUZ) fragment, has deleterious effects when overexpressed and that these are also mediated by an interaction with FASII. Whether the lack of an effect of sAPPLL overexpression is due to a less efficient interaction with FASII or an inability to induce downstream pathways causing this gain of function remains to be determined (Rieche, 2018).

Interestingly, overexpression of the dAICD in R3 neurons also disrupted visual working memory of young flies, suggesting that α- and γ-cleavage of APPL has deleterious effects on visual working memory. This is in agreement with the finding that heterozygosity for kuz and Psn ameliorated the age-related decline in visual working memory, whereas heterozygosity for dBACE had only a modest effect. This could be explained by assuming that most of the ectodomain shedding in flies is done by KUZ activity. Therefore, reducing dBACE levels might result in a small increase of flAPPL. In addition, competitive KUZ cleavage in dBace/+ flies could result in more detrimental sAPPLS. Whereas in the case of kuz/+, the effects on memory are mediated by the interaction with FASII, in the case of Psn/+ this may be mediated by a transcriptional function of the AICD, affecting a so far unknown target (Rieche, 2018).

In summary, the results show that full-length APPL acts as a membrane-bound ligand that inhibits the FASII receptor (both acting in R3 neurons), thereby promoting visual working memory. Increased proteolytic APPL processing and therefore reduced suppression of FASII signaling then seems to cause AMI in flies. A similar interaction might also be required for working memories in vertebrates. Aging mice show reduced expression of NCAM-140 in the medial prefrontal cortex and a conditional knockout of NCAM in the forebrain promotes AMI in a delayed matching-to-place test in the Morris water maze and in a delayed reinforced alternation test in the T-maze (Rieche, 2018).

Memory stability across learning contexts in Drosophila has some common genetic mechanisms, but the timing for gene action depends on the type of learning. That this study has added several genes here, including lat, pst, and rac as regulators of memory stability in operant place memory suggests that there are at least some common molecular processes in memory stability across different learning types. However, the timing of these genetically-defined phases depends on what is learnt. It is speculated that an ideal system to regulate memory stability would be one that activates its own decline. That is, a given memory type should activate the process of decreasing memory expression. This might work with the recruitment of a reinforcing pathway, like the dopaminergic signal that is important for both the acquisition of an associative olfactory memory and the active process of forgetting that association. In this case an odor associated with shock gives rise to a memory trace in mushroom body neurons that depends on a set of dopamine neurons that is important for both memory acquisition and decline. Whether this type of aminergic-based system applies to other forms of memory is not yet known. However, if an aminergic-based signal is common in memory decline, as appears to be the case with the Rac-based mechanism, differences in the types of aminergic neurons or innervation targets could give rise to the altered stabilities of behaviorally expressed memories (Ostrowski, 2014).

These data suggest the existence of a reward circuit in which the PAM neurons integrate gustatory reward and other relevant regulatory inputs, and then convey the summed positive value signal to specific subdomains of the MBs. The MB lobes can be anatomically divided into 35 subdomains that are defined by specific combinations of intrinsic and extrinsic neurons. Distinct sets of dopamine neurons may provide functionally independent local circuits within the MBs, potentially allowing appetitive and aversive modulation of the same odour. The PAM neurons may drive positive associative modulation of concomitant olfactory signals of the Kenyon cells. The dual processing of appetitive and aversive stimuli may be a conserved function of dopamine, highlighting the physiological pleiotropy of a neurotransmitter (Liu, 2012).

Multiple spaced trials of aversive differential conditioning can produce two independent long-term memories (LTMs) of opposite valence. One is an aversive memory for avoiding the conditioned stimulus (CS+), and the other is a safety memory for approaching the non-conditioned stimulus (CS-). This study shows that a single trial of aversive differential conditioning yields one merged LTM (mLTM) for avoiding both CS+ and CS-. Such mLTM can be detected after sequential exposures to the shock-paired CS+ and unpaired CS-, and be retrieved by either CS+ or CS-. The formation of mLTM relies on triggering aversive-reinforcing dopaminergic neurons and subsequent new protein synthesis. Expressing mLTM involves αβ Kenyon cells and corresponding approach-directing mushroom body output neurons (MBONs), in which similar-amplitude long-term depression of responses to CS+ and CS- seems to signal the mLTM. These results suggest that animals can develop distinct strategies for occasional and repeated threatening experiences (Zhao, 2021).

To survive in a complex environment, animals need to learn from threatening experiences to avoid potential dangers. From invertebrates to humans, aversive differential conditioning is widely used to study memories produced by threatening experiences. After repetitive spaced trials of conditioning, animals form two complementary long-term memories (LTMs) of opposite valence, including the aversive memory to the conditioned stimulus (CS+) and the rewarding memory to the non-conditioned stimulus (CS-). Such complementary LTMs result in enhanced long-lasting discrimination between CS+ and CS- through guiding avoidance to CS+ and approach to CS-. However, it remains unclear whether and how occasional threatening experiences, such as single-trial conditionings, would induce long-lasting changes in future escape behavior (Zhao, 2021).

From invertebrates to humans, experience-dependent long-lasting behavioral modifications mainly rely on the formation of LTMs. In Drosophila, there are at least two categories of aversive olfactory LTMs that last for more than 7 days. One is the spaced training-induced LTM that can be observed after repetitive spaced training with inter-trial rests (multiple trials with a 15 min rest interval between each), but not after either single-trial training or repetitive massed training without interval. Forming such aversive LTM requires new protein synthesis and the paired posterior lateral 1 (PPL1) cluster of dopaminergic neurons (DANs) to depress the connection between odor-activated Kenyon cells (KCs) in the mushroom body (MB) αβ lobe and downstream α2sc (MB-V2) MB output neurons (MBONs). The other is a recently reported context-dependent LTM that forms after single-trial training, which does not require protein synthesis-dependent consolidation. The expression of context-dependent LTM relies on multisensory integration in the lateral horn and is not affected by blocking KCs. However, all these observations derived from the same design principle that evaluates memory performance through testing the discrimination between CS+ and CS-. Thus, direct responses to CS+ and CS- have been largely overlooked (Zhao, 2021).

The current study introduced a third-odor test, in which flies were given a choice between either CS+ and a novel odor, or CS- and a novel odor. It was therefore identified that the single-trial differential conditioning produces a merged LTM (mLTM) guiding avoidances of both CS+ and CS- for several days after training. The encoding and expression of such mLTM involve new protein synthesis, PPL1 DANs, αβ KCs, and α2sc MBONs. These findings suggest that animals utilize distinct escape strategies for facing occasional and repeated dangers (Zhao, 2021).

In the current study, the use of third-odor test leads to a conclusion that single-trial training produces an mLTM for guiding flies to avoid both CS+ and CS- for more than 7 days. Three categories of evidence in support of this conclusion are outlined below (Zhao, 2021)

First, throughout this study, the amplitudes of long-term avoidances of CS+ and CS- are always at a similar level under various conditions, including pharmacological treatment, cold-shock treatment, odor re-exposure, paradigm alteration, and neural circuitry manipulations. Second, re-exposure to either one of CS+ and CS- alone can extinguish both CS+ avoidance and CS- avoidance. Third, the long-term avoidances of CS+ and CS- can be recorded as the depression of odor-evoked responses in the same α2sc MBONs, meanwhile, CS+ avoidance and CS- avoidance both involve the same PPL1 DANs, αβ KCs, and α2sc MBONs. Thus, CS+ avoidance and CS- avoidance derive from the same aversive mLTM, instead of two parallel LTMs of the same valence. The significance of these findings is further discussed below (Zhao, 2021).

Combining with a recent report that uses a similar third-odor test to dissect LTMs induced by multi-trial spaced training, it is concluded that spaced multi-trial aversive differential conditioning produces two independent LTMs of opposite valence for avoiding CS+ and approaching CS-, whereas single-trial aversive differential conditioning yields one mLTM that guides avoidances of both CS+ and CS-. Thus, animals can develop distinct escape strategies for different categories of dangers. When the same dangerous situation has been experienced repeatedly, animals would remember the detailed information to guide behavior in the next similar situation. However, when the dangerous event has only been experienced occasionally, animals would choose to avoid all potentially dangerous cues as a more reserved survival strategy (Zhao, 2021).

Moreover, the differences between single-trial training-induced mLTM and multi-trial training-induced complementary LTMs lead to the question of how these differences are induced by different training sessions. Jacob (2020) reported that multi-trial spaced training induces depressed responses to CS+ in α2sc MBONs and α3 MBONs are required for aversive LTM to CS+, whereas the modulated responses to CS- in β'2mp MBONs and γ3, γ3β'1 MBONs appears to be responsible for the safety memory to CS-. In contrast, this study found that single-trial training is sufficient to induce the depressed responses to both CS+ and CS- in α2sc MBONs. Therefore, the results suggest a lower threshold and specificity of the plasticity between KCs-α2sc MBONs, compared to KCs-α3 MBONs, KCs-β'2mp MBONs, and KCs-γ3, γ3β'1 MBONs connections. Consequently, changing these synaptic connections requires involving more training sessions (Zhao, 2021).

In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. This work finds that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, these findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit (Zhou, 2019).

This report has shown that a 28 kDa protein initiates from the internal ATG2 codon, that it functions as a CRE-dependent transcriptional activator both in vitro and in vivo, and is responsible for the original report of memory enhancement. Although ATG2 is infrequently used, and the resulting protein is expressed at low levels, its existence has been shown using multiple antibodies and different two-step enrichments (EMSA supershifts and Western identification of proteins on EMSA complexes) (Tubon, 2013).

he ATG2 codon is also used on endogenous dCREB2-encoded mRNAs, since all of the sequenced dCREB2 cDNAs contain ATG1 and ATG2 on the same molecule. Interestingly, internal translation initiation is also used on both of the mammalian CREM and CREB genes. 'Intronic' or internal ATGs can become positioned to be the first start codons through alternative promoter usage and alternative splicing. The mammalian CREB β isoform is a minority species that becomes upregulated upon deletion of the α and Δ isoforms. This study has not caracterized the transcriptional regulation of the dCREB2 gene, so it is possible that ATG2 is the first initiation codon on a minor, currently uncharacterized, dCREB2 transcript (Tubon, 2013).

A number of related issues have complicated molecular analysis of dCREB2-encoded protein isoforms, and are likely to be relevant in the characterization of these proteins in all species. First, the number and variety of posttranslational modifications that occur on dCREB2-encoded proteins is large. The KID region contains up to 7 phosphorylation sites, and other modifications, including O-GlcNac glycosylation, SUMOylation, ubiquitylation, and cysteine oxidation and/or nitrosylation, occur elsewhere on CREB proteins. These posttranslational modifications can dramatically affect the apparent mobility of protein species, and make it difficult to determine whether Western blots that contain many bands are due to a nonspecific or specific recognition of dCREB2-encoded proteins. A related observation is that these modifications can alter the binding affinity of many of the antibodie, suggesting that any given antibody reports a specialized subpool of protein. Finally, the blocker (40 kDa doublet) and activator (22-35 kDa cluster) species seem to be differentially modified, further complicating detailed analysis. It is likely that combinations of modifications are used to regulate the complex subcellular localization and activity of dCREB2 protein isoforms (Tubon, 2013).

Various parameters contribute to the inconsistency of memory enhancement. The expression of the 28 kDa protein off of the original 572 transgene is low but detectable. However, this modest level of expression is not responsible for inconsistent enhancement of olfactory avoidance memory, since the 807 transgenic fly (which has consistently higher levels of expression) also sporadically enhances memory formation. Instead, the limited duration of dCREB2-mediated transcriptional activation can place serious timing constraints on the requisite interval between transgene induction and behavioral training (the temporal window). A second temporal parameter is the time-of-day when induction and behavioral training occur. There is a growing awareness that the time-of-day-of training can affect memory formation, and this literature highlights the importance of circadian/sleep-related physiological processes and their relationship with the neuroanatomy and molecular machinery of memory formation. Careful control of these different timing issues greatly increases the reproducibility of behavioral enhancement using the olfactory avoidance assay. The consistent enhancement of the courtship behavior reinforces the original observation that the 28 kDa protein can enhance memory formation (Tubon, 2013).

Why does 807 enhance memory of courtship suppression reliably, but affects memory of olfactory avoidance less consistently? Comparing two diverse behavioral paradigms is difficult, since there are many parameters that differ. However, this type of approach is necessary, and will be useful. Another behavioral paradigm was developed using conditioned place preference. In the place preference behavioral assay, the 807 transgene enhances memory formation consistently, reinforcing the conclusion that the 28 kDa protein can have important effects on memory formation. Current experiments are directed at determining what behavioral factor(s) differ between courtship suppression and place preference (where consistent enhancement is seen) and olfactory avoidance (where enhancement is less consistent). One possibility is that enhancement in flies specifically requires a 'behavioral state' that is difficult to control experimentally, and which can be epistatic to the other parameters such as expression levels, activity windows, and the time-of-day of training. This behavioral state appears to be an 'all-or-nothing' group effect, with all of the flies in a given experiment affected similarly (Tubon, 2013).

Recent work using acute interventions in mice and other systems has shown that increasing CREB activity increases the intrinsic excitability of neurons, while interfering with CREB activity has the opposite effect (see for example Liu, 2011 and Suzuki, 2011). The CREB-dependent increase in excitability is correlated with memory enhancement, and vice versa. If dCREB2 enhances memory formation partially through affecting the excitability of relevant neurons, then the 'excitable state' of those neurons at the time of training might determine whether additional dCREB2 protein has enhancing potential or not (Benito, 2010). Since excitability is saturable, there are two simple outcomes, depending upon the state of the neurons at the time of training. If neurons are more quiescent, dCREB2 induction can increase excitability, and enhancement will occur in response to training (relative to equally quiescent neurons that just receive training). However, if the neurons are already excitable at the time of training, then extra dCREB2 will not have any effect, since excitability is saturable. The pretraining handling and housing of flies differs between various behaviors, and is somewhat variable even with the same behavior. These parameters could affect the baseline excitability of the flies, and indirectly affect enhancement. The behavioral data are consistent with this view, since enhancement usually becomes significant when the control fly population has lower memory scores, rather than the experimental population having higher memory scores. The effect of the time-of-day on enhancement also is consistent with this general hypothesis, since excitability is known to vary across the circadian cycle, at least for certain neurons. This possibility and its relevance has important implications for the role that dCREB2 plays in memory formation are currently being tested in a non-transgenic fly (Tubon, 2013).

In Drosophila, long-term memory (LTM) requires the cAMP-dependent transcription factor CREBB, expressed in the mushroom bodies (MB) and phosphorylated by PKA. To identify other kinases required for memory formation, Trojan exons encoding T2A-GAL4 were integrated into genes encoding putative kinases and genes expressed in MB were selected for. These lines were screened for learning/memory deficits using UAS-RNAi knockdown based on an olfactory aversive conditioning assay. A novel, conserved kinase, Meng-Po (MP, CG11221, SBK1 in human) was identified; loss severely affects 3 hr memory and 24 hr LTM, but not learning. Remarkably, memory is lost upon removal of the MP protein in adult MB but restored upon its reintroduction. Overexpression of MP in MB significantly increases LTM in wild-type flies showing that MP is a limiting factor for LTM. PKA phosphorylates MP and both proteins synergize in a feedforward loop to control CREBB levels and LTM (Lee, 2018).

Using MiMIC technology, 27 genes encoding putative protein kinases were converted with the Trojan T2A-GAL4 exon, and an image screen was performed for genes expressed in MBs. This tagging approach is especially useful for genes that are expressed at low levels in the CNS. By tagging the proteins with GFP, a conditional and reversible knockdown can be achieved in almost any tissue or cell. This allowed identification of a novel serine/threonine protein kinase, Meng-Po (MP), that is a critical player in LTM formation in Drosophila. MP is a homologue of SBK1 in mammals, a gene that is expressed in the hippocampus and the cortex. Loss of this gene in mice is associated with embryonic lethality, whereas in flies, loss of MP leads to a reduction in viability as well as sterility (Lee, 2018).

The data show that CREBB stability is highly susceptible to loss of MP. CREBB activity is modulated by phosphorylation via PKA and CamKII in Drosophila. Although the findings indicate that MP kinase activity is critical for maintaining CREBB levels and that MP kinase activity acts in synergy with PKA, it has not been possible to demonstrate that CREBB is a direct target of MP. However, some kinases require a previously phosphorylated residue as part of their recognition sequence, and various kinases were not mixed with MP in in vitro assays. Hence, it remains to be established how CREBB is degraded in the absence of MP (Lee, 2018).

A reduction in CREB levels has been shown to be associated with an age-dependent memory loss in rodents. Interestingly, delivery of CREB protein in the hippocampus using somatic cell transfer attenuated LTM impairement. However, no gene has so far been shown to affect CREBB stability in vivo and the current findings that MP, together with PKA, synergize to dramatically affect CREBB levels via a feedforward loop, reveal another mechanism to control CREBB levels during memory formation. This model is supported by the observation that overexpression of MP increases CREBB activity and promotes memory formation, suggesting that it is a central player in LTM (Lee, 2018).

Insects adapt their response to stimuli, such as odours, according to their pairing with positive or negative reinforcements, such as sugar or shock. Recent electrophysiological and imaging findings in Drosophila melanogaster allow detailed examination of the neural mechanisms supporting the acquisition, forgetting, and assimilation of memories. It is proposed that this data can be explained by the combination of a dopaminergic plasticity rule that supports a variety of synaptic strength change phenomena, and a circuit structure (derived from neuroanatomy) between dopaminergic and output neurons that creates different roles for specific neurons. Computational modelling shows that this circuit allows for rapid memory acquisition, transfer from short term to long term, and exploration/exploitation trade-off. The model can reproduce the observed changes in the activity of each of the identified neurons in conditioning paradigms and can be used for flexible behavioural control (Gkanias, 2022).

Antimicrobial peptides act as a host defense mechanism and regulate the commensal microbiome. To obtain a comprehensive view of genes contributing to long-term memory. mRNA sequencing from single Drosophila heads was performed following behavioral training that produces long-lasting memory. Surprisingly, it was found that Diptericin B, an immune peptide with antimicrobial activity, is upregulated following behavioral training. Deletion and knock down experiments revealed that Diptericin B and another immune peptide, Gram-Negative Bacteria Binding Protein like 3, regulate long-term but not short-term memory or instinctive behavior in Drosophila. Interestingly, removal of DptB in the head fat body and GNBP-like3 in neurons results in memory deficit. That putative antimicrobial peptides influence memory provides an example of how some immune peptides may have been repurposed to influence the function of nervous system (Barajas-Azpeleta, 2018).

In most animals modifying behavior based on past experiences is important for survival and reproductive success. To achieve these experience-dependent behavioral modifications, organisms must form memories of specific situations and maintain them to guide future behavior. Given that animals encounter different types of experiences, the resulting memories also vary in nature and duration. Moreover, not only the types of event, but also the internal state of the organism, influences whether an animal will form memory of a given experience, or, if memory is formed, how long it will persist. At molecular level it remains unclear how an animal forms various types of memories with different durations in different context (Barajas-Azpeleta, 2018).

The immune system and nervous system rely on their ability to detect and discriminate many cues from the external environment and produce appropriate responses. Similarly, once a cue is encountered, both systems possess the ability to modify their response to the same cue in subsequent encounters. Given the similarity in functional logic, therefore, it is perhaps not surprising that several immune genes also function in the nervous system. One of the earliest examples of this is the major histocompatibility complex 1, which is expressed both in the developing and mature nervous system of mice. The MHC1 genes are important for synaptic pruning as well as synaptic plasticity. Likewise, the complement system has been shown to be important for synapse formation, and immune receptors, such as Toll receptors, peptidoglycan pattern recognition receptor (PGRP), or interleukin receptors, are important for synaptic plasticity. In Drosophila immune peptides have been implicated in sleep regulation and nonassociative learning (Barajas-Azpeleta, 2018).

In the course of exploring how animals form long-lasting memories, this study discovered, surprisingly, that peptides that are known to be induced in the body upon bacterial infection, such as Diptericin B (DptB), are induced in the adult fly head following behavioral training that produces long-term memory. DptB activity is required to modulate long-term memory. In the course of these experiments it was also found that Gram-Negative Bacteria Binding Protein like 3 (GNBP-like3), although it is not induced by behavioral training at mRNA level, is nonetheless required for efficient long-term memory formation. These peptides attenuate bacterial growth consistent with their posited antimicrobial activity. Antimicrobial peptides modulating specific aspects of memory provides a novel example of the emerging link between the immune and nervous systems and leads to the proposal that some immune peptides might have been repurposed in the nervous system to 'moonlight' as neuromodulators over the course of evolution. It is unclear at this stage how these immune peptides modulate long-term memory (Barajas-Azpeleta, 2018).

This study observed that some of the known memory related genes that were changed in the trained groups, were also changed upon starvation or exposure to sorbose that does not produce robust long-term memory. This suggests that genes that are changed in some of the control conditions may also be important for memory, although they fail to satisfy the criteria set up in this study. Moreover, this analysis would miss genes that are involved in memory, but not transcriptionally induced, as illustrated by GNBP-like3 and other memory related genes. Also, our analysis would miss transcripts that are up- or down- regulated in small number of neurons or specific substructure, since isolation of mRNA from whole head would dilute the differences. Nonetheless, despite these inherent limitations of tis analysis, a few immune peptides were found to be consistently upregulated in adult head in groups that are trained to form long-term memory (Barajas-Azpeleta, 2018).

For most animals, including insects such as Drosophila melanogaster, the ability to remember a potential food source or modulate reproductive behavior based on prior experiences is a valuable trait. Both feeding, and copulation expose the inside of the animal to the external environment. Therefore, these events are likely to engage the immune system in preparation for the exposure to external agents, including pathogens. It is postulated that DptB, GNBP-like3, and other AMPs are upregulated in the body to deal with immune challenges. Subsequently, over evolutionary time, in addition to their protective roles in immunity, some immune related genes were repurposed to act as modulators of nervous system function. The nervous system perhaps co-opted these immune genes to convey and store information about specific aspects of experiences. The co-option would be appropriate given that the AMPs and memory are both immediately downstream of stress of starvation or rejection, and AMP proteins would be uniquely available after acute stress. However, what exact information represented by these peptide signals in the brain remains unclear at this stage (Barajas-Azpeleta, 2018).

There is increasing evidence that components of the immune system also function in the nervous system. In Drosophila, AMPs, such as Metchnikowin (Mtk), Drosocin and Attacin, are implicated in regulation of sleep; moreover, the innate immune receptor PGRP-LC is involved in homeostatic plasticity of neuromuscular junction synapse. More recently, Dpt, a different antimicrobial peptide, has been shown to be important for a form of nonassociative learning, where ethanol preference is modified upon exposure to predatory wasp. However, this is the first time that AMPs made in different tissues in adult head have been found to be involved in modulating long-term associative memories (Barajas-Azpeleta, 2018).

Interestingly, this study found that while both DptB and GNBP-like3 have similar requirement for long-term courtship suppression memory, their requirement in associative appetitive memory is different. What accounts for this differential dependency on a set of molecules? It is possible that the animals prioritize survival over reproductive success, and therefore remembering a food source involves several molecules that can compensate for the absence of each other. Indeed appetitive memory is quite robust and requires only one training for 5 minutes, while to elicit long-term courtship suppression memory requires multiple training lasting for 6 hours. In any event, these observations raise the possibility that in addition to common molecular processes, different types of memories may have unique molecular requirements. Indeed, a different group of immune peptides are up-regulated when Drosophila forms memory of a predator, such as wasp (Barajas-Azpeleta, 2018).

A key unanswered question of considerable interest is how and where DptB, and GNBP-like3, act to influence memory. In innate immunity of Drosophila, it is well characterized how invading pathogen induces the expression of AMPs via the PGRP-IMD and Toll- myD88 pathway. However, in spite of decades of work, with few exceptions, it remains unclear how majority of the AMPs function. Nonetheless, in addition to directly disrupting the bacterial membrane, there are other proposed activities of AMPs that can provide some clues to how they can influence cellular functions. For example, the AMPs are known to modulate the host inflammatory responses by acting as chemoattractant, inducing cytokines expression or stimulating cellular migration or proliferation. These actions of AMPs are mediated by directly or indirectly acting on some host cell surface receptors and engaging downstream signalling pathways. It is envisioned that in the nervous system, DptB and GNBP-like3 may similarly directly or indirectly influence neuronal activity by activating specific signalling pathway that may be similar to or distinct from immune cells. Among proposed mechanisms of AMP-mediated activation of signalling pathways that may be relevant in this context is indirect activation of receptor by displacing ligands, altering membrane microdomain, or directly acting as an alternate ligand. Indeed, the possibility of AMPs acting as an alternate ligand is not unprecedented. For example, mammalian β-defensin acts as a ligand for the melanocortin receptor 1 (Mc1r) to control melanin synthesis. Therefore, to understand how these AMPs act at molecular and cellular level it would be important to identify the 'receptors' of these AMPs in the adult brain. Identification of interacting molecules would uncover in which cell population these AMPs act, how they change cellular function and when the AMP-mediated modulation of the cellular function is important for memory (Barajas-Azpeleta, 2018).

Is there additional significance to the observation that AMPs modulate nervous system functions? Curiously, some neuropeptides, like NPY, possess antimicrobial activity, and innate immunity-related peptides are expressed in the mammalian brain. However, the expression of AMPs in the brain is often associated with dysfunction. For example, overexpression of antimicrobial peptides in Drosophila brain accelerates neurodegeneration. Recently, Aβ-42, the truncated product of amyloid-precursor-protein (APP) and a causative agent for Alzheimer's disease, has been postulated to be an AMP. In this view, although the central nervous system is isolated by the blood-brain-barrier, these AMPs are present in the brain to fight invading pathogens, or the AMPs are produced in the brain in response to inflammation or other stress. It is speculated that AMPs are made in the brain, not necessarily exclusively for immune related functions, but also to regulate nervous system functions. Indeed, the requirement of GNBP-like3 in neurons and its presence in synaptosomes are consistent with such a possibility. That some AMP expression eventually leads to dysfunction is perhaps an unintended consequence of a normal process (Barajas-Azpeleta, 2018).

The mechanisms that constrain memory formation are of special interest because they provide insights into the brain's memory management systems and potential avenues for correcting cognitive disorders. RNAi knockdown in the Drosophila mushroom body neurons (MBn) of a newly discovered memory suppressor gene, Solute Carrier DmSLC22A, a member of the organic cation transporter family, enhances olfactory memory expression, while overexpression inhibits it. The protein localizes to the dendrites of the MBn, surrounding the presynaptic terminals of cholinergic afferent fibers from projection neurons (Pn). Cell-based expression assays show that this plasma membrane protein transports cholinergic compounds with the highest affinity among several in vitro substrates. Feeding flies choline or inhibiting acetylcholinesterase in Pn enhances memory, an effect blocked by overexpression of the transporter in the MBn. The data argue that DmSLC22A is a memory suppressor protein that limits memory formation by helping to terminate cholinergic neurotransmission at the Pn:MBn synapse (Gai, 2016).

Genetic studies have now identified hundreds of genes required for normal memory formation. Some of these genes regulate the development of the cells and circuits required for learning; some mediate the physiological changes that occur with acquisition and storage. Of particular interest are gene functions that suppress normal memory formation and, by analogy with tumor suppressor genes, are referred to as memory suppressor genes. These genes and their products can, in principle, suppress memory formation by antagonizing the process of acquisition, limiting memory consolidation, promoting active forgetting, or inhibiting retrieval. Recently, a large RNAi screen of ∼3,500 Drosophila genes has bee carried out, and several dozen new memory suppressor genes were identified (Walkinshaw, 2015), identified as such, because RNAi knockdown produces an enhancement in memory performance after olfactory conditioning (Gai, 2016).

Aversive olfactory classical conditioning is a well-studied type of learning in Drosophila and consists of learning a contingency between an odor conditioned stimulus (CS) and most often an unconditioned stimulus (US) of electric shock. Many cell types in the olfactory nervous system are engaged in this type of learning, including antennal lobe projection neurons (Pn), several different types of mushroom body neurons (MBn), dopamine neurons (DAn), and others, but a focused model of olfactory memory formation holds that MBn are integrators of CS and US information with the CS being conveyed to the MBn dendrites by the axons of cholinergic, excitatory Pn of the antennal lobe, and the US conveyed to the MBn by DAn Gai, 2016).

A memory suppressor gene identified and describe in this report encodes a member of the SLC22A transporter family. The Solute Carrier (SLC) family of transporters in humans consists of 395 different, membrane-spanning transporters that have been organized into 52 different families. Some of these are localized pre-synaptically and involved in neurotransmitter recycling, others localize to glia for clearance of neurotransmitter from the synapse. In addition, glutamate transporters can be localized post-synaptically to regulate neurotransmission strength via clearance mechanisms. Some of these SLC transporters have prominent roles in neurological and psychiatric disorders and in drug design, including SLC1A family members that are responsible for glutamate uptake and clearance of this neurotransmitter from the synaptic cleft and SLC6A2-4 proteins that transport monoamines into cells. Inhibitors of these proteins, which include the serotonin-specific reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs), increase monoamine dwell time at the synapse and are used to treat depression and several other neuropsychiatric disorders (Gai, 2016).

The SLC22A family of transporters is distinguished into two major classes that carry either organic cations (SLC22A1-5, 15, 16, and 21) or anions (SLC22A6-13 and 20) across the plasma membrane, with generally low substrate binding affinity and high capacity. They transport numerous molecules with diverse structures, including drugs, acetylcholine, dopamine, histamine, serotonin, and glycine among others. Ergothioneine has been identified as a high-affinity substrate for SLC22A4 and spermidine for SLC22A16. Mice mutant in the two organic cation transporters, SLC22A2 and SLC22A3, exhibit behavioral phenotypes suggestive of functions in anxiety, stress, and depression. These observations point out the importance of the SLC22A family for brain function and cognition. Recently, a Drosophila SLC22A family member, CarT (CG9317) was identified and found to transport carcinine into photoreceptor neurons for the recovery of essential visual neurotransmitter histamine (Gai, 2016).

This study shows that the Drosophila gene, CG7442, functions as a memory suppressor gene and is a member of the SLC22A family. This transporter is expressed most abundantly in the dendrites of the MBn, at the synapses with the cholinergic antennal lobe Pn. Cell-based expression assays show that Drosophila SLC22A transports choline and acetylcholine with the highest affinity among several substrates. Pharmacological and genetic data support the model that Drosophila SLC22A functions at the Pn:MBn synapse to terminate cholinergic neurotransmission, differing from well-characterized presynaptic choline transporters for neurotransmitter recycling, and mechanistically explaining its role in behavioral memory suppression (Gai, 2016).

These data connect the SLC22A family of transporters and memory suppression. DmSLC22A, located on the dendrites of the adult α/β and α'/β' MBn, removes ACh from the Pn:MBn synapses in the calyx. The normal expression level of this plasma membrane transporter limits the transference of olfactory information to the MBn by removing neurotransmitter from the synapse. Overexpression of DmSLC22A hardens this limit, weakening the CS representation and weakening memory formation. Reducing DmSLC22A expression has the opposite effect of softening the limit, producing a stronger CS representation and stronger memory formation. Thus, the data indicate that acetylcholinesterase and postsynaptic SLC22A transporter function jointly to regulate neurotransmitter persistence at the synapse. This conclusion is notable, given the longstanding emphasis on ACh degradation as the primary route for termination of the cholinergic synaptic signal. Although the evidence is strong for the proposed mechanism shown in Figure 8A, the transporter exhibits broad substrate specificity and expression outside of the Pn:MBn synapse. Alternative or additional mechanisms of action in memory suppression thus remain a possibility (Gai, 2016).

The current data are consistent with the model that ACh persistence at the Pn:MBn synapse is a surrogate for the strength of the CS and therefore a primary effector of olfactory memory strength. Other data similarly point to the strength of stimulation of MBn as an important variable for regulating memory strength. The MBn also receive inhibitory input through GABAA receptors expressed on the MBn. Overexpressing the MBn-expressed GABAA receptor Rdl impairs learning, while RNAi knockdown of this receptor in the MBn enhances memory formation. This regulation of memory strength is independent of the US pathway involved in classical conditioning, functioning similarly for both aversive and appetitive USs. However, it is noted that the in vivo functions for the SLC22A class of transporters must be broader than the focused model presented above. For instance, the data indicate that the Drosophila SLC22A protein transports both acetylcholine and dopamine in ex vivo preparations. Moreover, the protein's memory suppressor function maps to both MBn and the DAn. How DmSLC22A might function in DAn to suppress memory formation has not been explored, but one reasonable hypothesis is that DmSLC22A transports acetylcholine at the synapse between upstream and putative cholinergic neurons that provide input to the DAn that convey the US in classical condition. Testing this hypothesis requires identifying the presynaptic neurons to the DAn that carry the US information (Gai, 2016).

One unexplained observation is that although DmSLC22A knockdown enhances the duration of memory produced from stronger memory traces instilled at acquisition, it slows the rate of acquisition as measured by acquisition curves. However, this observation has been made with another memory suppressor gene as well. A knockdown of the pre- and post-synaptic scaffolding protein, Scribble, has the same effect of producing more enduring memories but slowing acquisition. In addition, similar observations have been made in mouse: injection of muscarinic acetylcholine receptor antagonists impairs memory acquisition but enhances retention (Easton, 2012; Gai, 2016 and references therein).

These studies bring a focus on the SLC22A family of plasma membrane transporters as potential targets for neurotherapeutics. Of the 24 members of this family, only a few have been studied in some detail in the nervous system. RNA expression experiments have shown that SLC22A1-5 are all expressed in the brain, with SLC22A3 and A4 being the most abundant, and immunohistochemistry experiments have revealed that SLC22A4-5 are localized at dendrites within the hippocampus. Mammalian members of this family of transporters and, by extension, probably DmSLC22A, are subject to regulation by multiple signaling molecules including protein kinase A, calcium/calmodulin-dependent protein kinase II, and the mitogen-activated protein kinases. Knockout mice for SLC22A2 and A3 show reduced basal level of several neurotransmitters in a region-dependent manner and decreased anxiety-related behaviors, although the effects of SLC22A3 on anxiety-related behaviors is debated. In addition, the knockouts or antisense insults reveal behavioral changes in depression-related tasks, with SLC22A2 knockouts exhibiting increased behavioral despair, and SLC22A3 antisense-treated animals exhibiting decreased behavioral despair. Little is known about the biological or behavioral functions of the other members of the SLC22A family. The current results show that the SLC22A family of transporters is also involved in memory suppression (Gai, 2016).

DmSLC22A is a unique and new type of memory suppressor gene. There are, to date, about two dozen memory suppressor genes identified in the mouse and about three dozen such genes in Drosophila. The mechanisms by which all of these genes suppress memory formation are not yet known, but a few themes have emerged. For instance, several of the genes suppress memory formation by limiting excitatory neurotransmitter release and function, or the expression and function of post-synaptic receptors. DmSLC22A appears to fall into this category. Another example is Cdk5, which negatively influences the expression of NR2B and limits memory formation. Knockouts of some GABA receptors reduce inhibitory tone of learning circuitry so as to facilitate memory formation. Several of the known memory suppressor genes are known to function in active forgetting processes. These include damb, a dopamine receptor involved in forgetting mechanisms; scribble, a pre- and post-synaptic scaffolding gene; and rac, a small G protein involved in the biochemistry of active forgetting. Memory suppressor genes can also encode signaling molecules that negatively regulate transcription factors required for long-term memory and the transcription factors themselves, such as repressing isoforms of Aplysia Creb; ATF4, a transcription factor homolgous to ApCreb-2; and protein phosphatase I. Elucidating all of the genetic constraints on memory formation and their mechanisms will have profound consequences for understanding of how the brain forms and stores memories and for the development of cognitive therapeutics (Gai, 2016).

A major bottleneck to understanding of the genetic and molecular foundation of life lies in the ability to assign function to a gene and, subsequently, a protein. Traditional molecular and genetic experiments can provide the most reliable forms of identification, but are generally low-throughput, making such discovery and assignment a daunting task. The bottleneck has led to an increasing role for computational approaches. The Critical Assessment of Functional Annotation (CAFA) effort seeks to measure the performance of computational methods. In CAFA3, selected screens were performed, including an effort focused on long-term memory.Homology and previous CAFA predictions were used to identify 29 key Drosophila genes, which were tested via a long-term memory screen. 11 novel genes were found that are involved in long-term memory formation and show a high level of connectivity with previously identified learning and memory genes. This study provides first higher-order behavioral assay and organism screen used for CAFA assessments and revealed previously uncharacterized roles of multiple genes as possible regulators of neuronal plasticity at the boundary of information acquisition and memory formation (Kacsoh, 2018).

Long-lasting, consolidated memories require not only positive biological processes that facilitate long-term memories (LTM) but also the suppression of inhibitory processes that prevent them. The mushroom body neurons (MBn) in Drosophila melanogaster store protein synthesis-dependent LTM (PSD-LTM) as well as protein synthesis-independent, anesthesia-resistant memory (ARM). The formation of ARM inhibits PSD-LTM but the underlying molecular processes that mediate this interaction remain unknown. This study demonstrates that the Ras-->Raf-->rho kinase (ROCK) pathway in MBn suppresses ARM consolidation, allowing the formation of PSD-LTM. The initial results revealed that the effects of Ras on memory are due to postacquisition processes. Ras knockdown enhanced memory expression but had no effect on acquisition. Additionally, increasing Ras activity optogenetically after, but not before, acquisition impaired memory performance. The elevated memory produced by Ras knockdown is a result of increased ARM. While Ras knockdown enhanced the consolidation of ARM, it eliminated PSD-LTM. These effects are mediated by the downstream kinase Raf. Similar to Ras, knockdown of Raf enhanced ARM consolidation and impaired PSD-LTM. Surprisingly, knockdown of the canonical downstream extracellular signal-regulated kinase did not reproduce the phenotypes observed with Ras and Raf knockdown. Rather, Ras/Raf inhibition of ROCK was found to be responsible for suppressing ARM. Constitutively active ROCK enhanced ARM and impaired PSD-LTM, while decreasing ROCK activity rescued the enhanced ARM produced by Ras knockdown. It is concluded that MBn Ras/Raf inhibition of ROCK suppresses the consolidation of ARM, which permits the formation of PSD-LTM (Noyes, 2020).

Consolidation is a process required for the formation of long-lasting memories. This process of converting memories that are initially sensitive to disruption from a variety of insults to more resilient ones is well conserved and many of its characteristics are shared across species. For example, memory in invertebrates and vertebrates lasts longer following multiple spaced training sessions, undergoes both molecular/cellular and systems consolidation, and can be disrupted by inhibition of protein synthesis (Noyes, 2020).

The fruit fly Drosophila melanogaster forms two distinguishable types of consolidated aversive olfactory memory: 1) anesthesia-resistant memory (ARM), which reportedly decays to negligible levels by 4 d after conditioning, can be generated by a single training session; 2) protein synthesis-dependent long-term memory (PSD-LTM), which shows limited decay, requires spaced training. These two types of consolidated memory are not independent from one another. The formation of ARM impairs either the formation or expression of PSD-LTM. Although circuit mechanisms possibly responsible for this relationship are beginning to be dissected, the molecular requirements in the mushroom body (MB), a brain region critical for the storage and retrieval of PSD-LTM and ARM remain unknown (Noyes, 2020).

The small GTPase Ras85D (Ras) is a Drosophila homolog of the mammalian Ras family genes KRAS, NRAS, and HRAS. Activated Ras proteins act as signaling switches, initiating signaling cascades through multiple downstream effector proteins. Precise induction and regulation of Ras activity is essential for mammalian synaptic plasticity and memory. Although upstream regulators of Ras, like NF1 and DRK, have been explored for their roles in Drosophila learning and memory Ras itself has not been thoroughly examined. A large RNA interference (RNAi) screen identified Ras85D as a memory suppressor gene but did not detail its specific role in memory suppression (Noyes, 2020).

This study reports that Ras activity in the MB acts as a switch between the two forms of consolidated memory, required both for PSD-LTM and inhibition of ARM. Increasing Ras activity dramatically reduced memory expression. This effect was determined to be due to Ras regulation of ARM. Knockdown of Ras enhanced the consolidation of ARM, leading to an overall increase in memory, while Ras knockdown eliminated PSD-LTM following spaced training. Although the effect of Ras on both ARM and PSD-LTM was found to be mediated by Raf, it is independent from the canonical downstream extracellular signal-regulated kinase (ERK). Instead, Ras/Raf-mediated inhibition of rho kinase (ROCK) suppresses ARM and is required for PSD-LTM (Noyes, 2020).

Based on the results, a model in which ARM consolidation is suppressed by a training-induced increase in Ras activity. Raf activity is increased in γ MBn following training, presumably through Ras, but the receptor(s) initiating this signaling are not known. Ras can be regulated through G-coupled protein receptors. It is possible that dopamine or an unknown coneurotransmitter released from dopaminergic neurons (DAn) during training initiates Ras signaling. This would provide a link between MP1 DAn, which are proposed to gate LTM, and Ras. The participation of ROCK in consolidation suggests that PSD-LTM and ARM are modulated by changes in the actin cytoskeleton (36) but does not directly indicate whether these changes occur in the pre- or post-synaptic compartments. Of the several genes known to be required for ARM, Bruchpilot (Brp) is the only one with a well-established, specific subcellular compartmentalization. Brp is localized to presynaptic active zones and is required for normal presynaptic morphology and synaptic transmission, indicating that ARM may result from a form of presynaptic plasticity in the MB. Additionally, the DAn that are required for memory formation innervate MB axons and modulate synaptic strength between MBn and downstream MB output neurons. The results demonstrating that artificial activation of Ras increases axonal pERK in γ MBn is evidence that Ras/Raf signaling participates in axonal signal transduction and is consistent with a previous report highlighting a role for presynaptic Raf activity in γ MBn. ROCK activity in mammalian axons is critical for a number of processes; however, it has not been tested whether ROCK signaling occurs in γ MBn axons (Noyes, 2020).

The hypothesis that ARM inhibits the formation PSD-LTM was based on the observation that spaced training, which generates PSD-LTM, eliminates or precludes ARM. Subsequent research at the systems neuroscience level revealed that two sets of neurons, MP1 DAn and serotonergic projection neurons (SPn), appear to be responsible for the promotion of PSD-LTM through the suppression of ARM. The activity of these neurons is increased during spaced training. This activity reduces ARM, while inhibiting their activity enhances ARM. Blocking the activity of either set of neurons during spaced training does not prevent memory formation but prevents the formation of PSD-LTM. This suggests that without SPn and MP1 DAn activity, ARM occurs by default and is preferentially expressed at the expense of PSD-LTM. Ras fulfills the requirements as the intracellular and molecular switch regulating the inverse relationship between ARM and PSD-LTM. The suppression of ARM and formation of PSD-LTM both require Ras in γ MBn, which are downstream in the circuit from the ARM/PSD-LTM-gating MP1 DAn that synapse directly on to γ MBn (Noyes, 2020).

The mammalian counterpart for ARM, if one exists, is unknown. Protein synthesis-independent ARM has been reported to be measurable up to 4 d after conditioning, while mammalian protein synthesis-independent memory lasts only hours. Despite the lack of a clear and direct mammalian counterpart to ARM, it is becoming apparent that many of the same genes that are involved in ARM also play a role in mammalian memory and plasticity. Ras, Raf, and CDC42 negatively regulate ARM but in mammals are positive regulators of LTM. Conversely, reduced ROCK or dunce, the latter purported to function through the SPn, impair ARM. In mammals, inhibition of ROCK or a mammalian ortholog of dnc (48), PDE4, enhances memory. It seems likely that discovering more genetic regulators of ARM will reveal previously unknown genetic regulators of mammalian memory. Based on the genes and their functions discussed in this paper, it is possible that factors that promote ARM in Drosophila function in memory suppression in mammals (Noyes, 2020).

The effect of ROCK on memory is not restricted to γ MBn. ROCK is also required in α/β MBn for ARM. In this neuron type, the effects of ROCK are not mediated by Ras but through Drk, the Drosophila homolog of Grb2. It is interesting to consider whether the ROCK substrate(s) mediating enhanced ARM in α/β and γ MBn are the same even though the upstream signaling components are distinct. Several ROCK targets have been established as important for normal memory, including cofilin and nonmuscle myosin II (Noyes, 2020).

A recent report indicates that ERK activity in γ MBn slows forgetting. The current results revealing that ERK knockdown reduces memory support this conclusion. However, the former report finds that Raf RNAi expression in γ MBn reduces memory, which is at odds with the finding that Raf RNAi enhances memory. The most likely explanation for this discrepancy is the use of different gal4/UAS-RNAi combinations that produce different levels of gene knockdown. It is interesting to consider that Raf signaling in γ MBn might regulate three forms of memory: consolidated ARM and PSD-LTM through ROCK and labile memory through ERK (Noyes, 2020).

Consolidated memory can be preserved or updated depending on the environmental change. Although such conflicting regulation may happen during memory updating, the flexibility of memory updating may have already been determined in the initial memory consolidation process. This study explored the gating mechanism for activity-dependent transcription in memory consolidation, which is unexpectedly linked to the later memory updating in Drosophila. Through proteomic analysis, it was discovered that the compositional change in the transcriptional repressor, which contains the histone deacetylase Rpd3 and CoRest, acts as the gating mechanism that opens and closes the time window for activity-dependent transcription. Opening the gate through the compositional change in Rpd3/CoRest is required for memory consolidation, but closing the gate through Rpd3/CoRest is significant to limit future memory updating. These data indicate that the flexibility of memory updating is determined through the initial activity-dependent transcription, providing a mechanism involved in defining memory state (Takakura, 2021).

In mammals, learning circuits play an essential role in energy balance by creating associations between sensory cues and the rewarding qualities of food. This process is altered by diet-induced obesity, but the causes and mechanisms are poorly understood. This study exploited the relative simplicity and wealth of knowledge about the D. melanogaster reinforcement learning network, the mushroom body, in order to study the relationship between the dietary environment, dopamine-induced plasticity, and food associations. Flies that are fed a high-sugar diet were shown to be unable make associations between sensory cues and the rewarding properties of sugar. This deficit was caused by diet exposure, not fat accumulation, and specifically by lower dopamine-induced plasticity onto mushroom body output neurons (MBONs) during learning. Importantly, food memories dynamically tune the output of MBONs during eating, which instead remains fixed in sugar-diet animals. Interestingly, manipulating the activity of MBONs influenced eating and fat mass, depending on the diet. Altogether, this work advances fundamental understanding of the mechanisms, causes, and consequences of the dietary environment on reinforcement learning and ingestive behavior (Pardo-Garcia, 2023).

This study took advantage of the simplicity of the associative learning circuits in D. melanogaster to investigate how the dietary environment affects food learning. Consumption of a sugar diet (SD) disrupts associative learning by decreasing the reinforcing power of Dopamine (DA) signals onto the MBONs (mushroom body output neurons). This decrease in DA transmission results from changes in sweetness sensation that develop in the taste neurons with exposure to dietary sugar, even in the absence of fat accumulation. Thus, these impairments in food memories result from diet, not obesity. These findings establish the critical role of nutrients in brain processes and support accumulating evidence from mammalian studies on the effects of diet exposure on reinforcement learning (Pardo-Garcia, 2023).

DA transmission is essential to the neuromodulation of MBON activity that underlies the learning process; this study demonstrates that the decrease in DA signal with high dietary sugar is insufficient to drive the neurophysiological changes that link a sensory cue with reward, preventing the formation of the food memory. To understand how this was linked to the output of MBONs, the presynaptic activity of this circuit in response to sensory cues before and after learning. Yhe formation of food associations was found to shift MBON output; interestingly, responses to cues change dynamically during eating (fasted versus 30 min re-fed). In the absence of learning in the SD animals, however, the output of MBONs remains static during eating. When the effects of MBON activity on eating and energy balance was examimed, no effect was found of activating MBONs when flies were fed a control diet (CD). However, activation while the animals were on an SD corrected eating and fat accumulation. Importantly, inhibiting MBON activity promoted higher eating and fat accumulation. A model is proposed where components of processed food contribute to deficits in food associations independently of weight gain by decreasing the reinforcing power of DA signals. It was also shown that the activity of associative learning circuits affects eating in diet-dependent contexts. These results are consistent with the known impairments in reinforcement learning that occur with obesogenic diets in mammals and provide causes and mechanisms for these effects. Beyond this, the data also support the satiety cascade's theoretical framework, where cognitive circuits and processes are postulated to affect intake.How does a high-sucrose diet change DA-induced plasticity during learning (Pardo-Garcia, 2023)?

The etiology of alterations in DA-neuron activity with diet-induced obesity in mammals remains unresolved. This work demonstrates that changes in β' 2 PAM DANs arise from changes in the peripheral sensory processing of sweetness. In previous studies, it was found that the responses of the sweet gustatory neurons to sucrose and the transmission of the sweetness signal were reduced by exposure to a high SD. Correcting these sensory deficits with opto- and neurogenetic tools or pharmacological interventions restored normal DAN responses to sucrose as well as feeding behavior and fat mass in flies fed a high-sucrose diet. Here, the same pharmacological manipulation corrected the neural signatures of learning, suggesting that sensory changes in response to the dietary environment play an essential role in deregulating food associations and eating. Of note, similar sensory alterations have been observed in mammals and humans exposed to high-fat and high-sugar diets or with a high body mass index, raising the possibility that these chemosensory alterations may contribute to changes in DA-induced plasticity and higher food intake observed with some diets. This result is interesting because how sensory processing promotes satiety is still not understood, although sensory components of food play an important role. Thus, diet-induced chemosensory plasticity could provide a new lens to explain how some food environments or COVID-1974 affect food intake (Pardo-Garcia, 2023).

Perturbations in DA plasticity, however, could also arise from changes in the expression or function of DA receptors or transporters or even in DA synthesis, all of which have been described with diet-induced obesity in mammals. In flies, the DA receptor 1 (D1R) and DA receptor 2 (D2R) play important roles in associative learning and food seeking. The effects of diet on the expression or activity of these receptors have not been investigated in flies, but if they occur, these could also underlie some of the phenotypes observed in this study. Finally, plasticity at the MBON synapse could also reflect changes in mushroom body network activity, especially the contributions of antagonistic MBON and DAN circuits, some of which may also be important for energy homeostasis and foraging (Pardo-Garcia, 2023).

A growing body of data in mammals supports the model that obesity arises from changes in food learning rather than innate 'food pleasure or liking' because manipulations of circuits necessary for reinforcement learning and memory influence eating and fat mass. In the current work, a strong effect of MBON activity on feeding behavior and fat accumulation was foun. In flies, the activity of MBONs affects the innate animal's preference (avoidance versus approach) for cues, depending on experience. Because of this, the feeding and obesity phenotypes observed with manipulations of MBON activity could be due to their effect on innate or learned preferences. Although these experiments, like those in rodents, do not provide a direct causal link between food associations and eating, the second interpretation is favore. First, if MBONs affected feeding solely through their innate regulation of avoidance/approach, activation of these neurons to decrease eating and fat mass would not be expected under CD conditions. However, this is not what was observed: closed-loop activation of MBONs while flies were on a CD did not affect feeding or fat mass. Only when MBONs were activated in animals eating an SD did was a reduction in eating and protection from diet-induced obesity observed. The most parsimonious explanation for these results is that on an SD, the activity of the neurons is lower, and activating them corrects this deficit. This interpretation is also consistent with the observation that inhibiting the activity of MBONs recapitulated the higher eating and obesity found in SD flies. Thus, although the possibility that MBONs affect eating exclusively through learning cannot be ruled out, it cannot be proven that learning deficits drive eating, it is believed that the weight of the evidence better aligns with the idea that diet-driven impairments in associative learning contribute to the escalation of food intake (Pardo-Garcia, 2023).

MBONs project to sensory-motor integration areas in the fly central complex involved in motor aspects of the eating program, such as foraging, proboscis extension, and eating rate. This part of the fly brain is genetically, functionally, and anatomically related to the mammalian basal ganglia, which receive input from the limbic circuitry involved in reinforcement learning. This connection between reinforcements, associative learning, and pre-motor areas provides a neural pathway to turn food associations into actions. In flies, the dynamic changes in the responses of MBONs to cues observed during eating could control the activity of downstream pre-motor circuits and the animal's interaction with food. In the absence of these, as seen in the SD condition, the animal may stay 'locked' in its interaction with food until pre- and post-absorptive signals disengage it from eating. Another possibility is that a mismatch between the different rewards contributing to food associations creates incentives to eat more. In humans, rodents, and flies, a mismatch created by giving animals non-caloric sweeteners along with sugars (or other carbohydrate-containing foods) changes food associations and central responses to sugar. In a similar way, a high SD may uncouple taste and nutrient rewards by degrading the sweetness-reinforced CS+, or generalizing the reward to the CS−, which may result in a higher-than-expected reward from nutrients and drive intake (Pardo-Garcia, 2023).

In summary, this work sheds light on the causes and mechanisms through which processed food components impair the formation of food memories. Future functional dissections of the circuits in this network in flies and pre-clinical models, as well as investigations of the molecular and cellular mechanisms involved, will provide new insights into understanding the connection between food memories and eating (Pardo-Garcia, 2023).

Dietary magnesium (Mg(2+)) supplementation can enhance memory in young and aged rats. Memory-enhancing capacity was largely ascribed to increases in hippocampal synaptic density and elevated expression of the NR2B subunit of the NMDA-type glutamate receptor. This study shows that Mg(2+) feeding also enhances long-term memory in Drosophila. Normal and Mg(2+) enhanced fly memory appears independent of NMDA receptors in the mushroom body and instead requires expression of a conserved CNNM-type Mg(2+)-efflux transporter encoded by the unextended (uex) gene. UEX contains a putative cyclic nucleotide-binding homology domain and its mutation separates a vital role for uex from a function in memory. Moreover, UEX localization in mushroom body Kenyon Cells is altered in memory defective flies harboring mutations in cAMP-related genes. Functional imaging suggests that UEX-dependent efflux is required for slow rhythmic maintenance of Kenyon Cell Mg(2+). It is proposed that regulated neuronal Mg(2+) efflux is critical for normal and Mg(2+) enhanced memory (Wu, 2020).

Magnesium (Mg2+) plays a critical role in cellular metabolism and is considered to be an essential co-factor for more than 350 enzymes. As a result, alterations of Mg2+ homeostasis are associated with a broad range of clinical conditions, including those affecting the nervous system, such as glaucoma, Parkinson's disease, Alzheimer's disease, anxiety, depression, and intellectual disability (Wu, 2020).

Perhaps surprisingly, increasing brain Mg2+ through diet can enhance neuronal plasticity and memory performance of young and aged rodents, measured in a variety of behavioral tasks. In addition, elevated Mg2+ reduced cognitive deficits in a mouse model of Alzheimer's disease and enhanced the extinction of fear memories. These apparently beneficial effects have led to the proposal that dietary Mg2+ may have therapeutic value for patients with a variety of memory-related (Wu, 2020).

Despite the large number of potential sites of Mg2+ action in the brain, the memory-enhancing property in rodents has largely been attributed to increases in hippocampal synaptic density and the activity of N-methyl-D-aspartate glutamate receptors (NMDARs). Extracellular Mg2+ blocks the channel pore of the NMDAR (see Drosophila NMDA receptors) and thereby inhibits the passage of other ions. Importantly, prior neuronal depolarization, driven by other transmitter receptors, is required to release the Mg2+ block on the NMDAR and permit glutamate-gated Ca2+ influx. The NMDAR therefore plays an important role in neuronal plasticity as a potential Hebbian coincidence detector. Acute elevation of extracellular Mg2+ concentration ([Mg2+]e) within the physiological range (0.8-1.2 mM) can antagonize induction of NMDAR-dependent long-term potentiation. In contrast, increasing [Mg2+]e for several hours in neuronal cultures leads to enhancement of NMDAR mediated currents and facilitation of the expression of LTP. The enhancing effects of increased [Mg2+]e were also observed in vivo in the brain of rats fed with Mg2+-L-threonate. Hippocampal neuronal circuits undergo homeostatic plasticity to accommodate the increased [Mg2+]e by upregulating expression of NR2B subunit containing NMDARs. The higher density of hippocampal synapses with NR2B containing NMDARs are believed to compensate for the chronic increase in [Mg2+]e by enhancing NMDAR currents during burst firing. In support of this model, mice that are genetically engineered to overexpress NR2B exhibit enhanced hippocampal LTP and behavioral memory (Wu, 2020).

Olfactory memory in Drosophila involves a heterosynaptic mechanism driven by reinforcing dopaminergic neurons, which results in presynaptic depression of cholinergic connections between odor-activated mushroom body (MB) Kenyon cells (KCs) and downstream mushroom body output neurons (MBONs). In addition, olfactory information is conveyed to KCs by cholinergic transmission from olfactory projection neurons. Although it is conceivable that glutamate is delivered to the MB network via an as yet to be identified route, there is currently no obvious location for NMDAR-dependent plasticity in the known architecture of the cholinergic input or output layers. The fly therefore provides a potential model to investigate other mechanisms through which dietary Mg2+ might enhance memory (Wu, 2020).

The reinforcing effects of dopamine depend on the Dop1R D1-type dopamine receptor, which is positively coupled with cAMP production. Moreover, early studies in Drosophila identified the dunce and rutabaga encoded cAMP phosphodiesterase and type I Ca2+-stimulated adenylate cyclase, respectively, to be essential for olfactory memory. Studies in mammalian cells have shown that hormones or agents that increase cellular cAMP level often elicit a significant Na+-dependent extrusion of Mg2+ into the extracellular space. However, it is unclear whether Mg2+ extrusion plays any role in memory processing (Wu, 2020).

This study demonstrates that Drosophila long-term memory (LTM) can be enhanced with dietary Mg2+ supplementation. The unextended (uex) gene, which encodes a functional fly ortholog of the mammalian Cyclin M2 Mg2+-efflux transporter (CNNM) proteins, is critical for the memory enhancing property of Mg2+. UEX function in MB KCs is required for LTM and functional restoration of uex reveals the MB to be the key site of Mg2+-dependent memory enhancement. Chronically changing cAMP metabolism by introducing mutations in the dnc or rut genes alters the cellular localization of UEX. Moreover, mutating the conserved cyclic nucleotide-binding homology (CNBH) domain in UEX uncouples an essential role for uex from its function in memory. UEX-driven Mg2+ efflux is required for slow rhythmic maintenance of KC Mg2+ levels suggesting a potential role for Mg2+ flux in memory processing (Wu, 2020).

This study observed an enhancement of olfactory LTM performance when flies were fed for 4 days before training with food supplemented with 80 mM [Mg2+]. This result resembles that reported in rats, although longer periods of feeding were required to raise brain [Mg2+] to memory-enhancing levels. A difference in optimal feeding time may reflect the size of the animal and perhaps the greater bioavailability of dietary Mg2+ in Drosophila. Whereas Mg2+-L-threonate (MgT) was a more effective means of delivering Mg2+ than magnesium chloride in rats, a similar enhancement of memory performance was observed when flies were fed with magnesium chloride, magnesium sulfate, or MgT (Wu, 2020).

Elevating [Mg2+]e in the rat brain leads to a compensatory upregulation of expression of the NR2B subunit of the NMDAR and therefore an increase in the proportion of postsynaptic NR2B-containing NMDARs. This class of NMDARs have a longer opening time suggesting that this switch in subunit composition represents a homeostatic plasticity mechanism to accommodate for the increased NMDAR block imposed by increasing [Mg2+]e. Moreover, overexpression of NR2B in the mouse forebrain can enhance synaptic facilitation and learning and memory performance, supporting an increase in NR2B being an important factor in Mg2+-enhanced memory. However, even in the original in vitro study of Mg2+-enhanced synaptic plasticity, it was noted that NMDAR currents were insufficient to fully explain the observed changes (Wu, 2020).

NMDAR subunit loss-of-function studies in the Drosophila KCs did not impair regular or Mg2+-enhanced memory. Furthermore, no obvious change was detected in the levels of brain-wide expression of glutamate receptor subunits in Mg2+-fed flies. Although NMDAR activity has previously been implicated in Drosophila olfactory memory, the effects were mostly ascribed to function outside the MB. In addition, overexpressing Nmdar1 in all neurons, or specifically in all KCs, did not alter STM or LTM. Ectopic overexpression in the MB of an NMDARN631Q version, which cannot be blocked by Mg2+, impaired LTM. However, this mutation permits ligand-gated Ca2+ entry, without the need for correlated neuronal depolarization, which may perturb KC function in unexpected ways. It is perhaps most noteworthy that learning-relevant synaptic depression in the MB can be driven by dopaminergic teaching signals delivered to cholinergic output synapses from odor-responsive KCs to specific MBONs. It is conceivable that KCs receive glutamate, from a source yet to be identified, but there is currently no obvious place in the MB network for NMDAR-dependent plasticity. Evidence therefore suggests that normal and Mg2+-enhanced Drosophila LTM is independent of NMDAR signaling in KCs. In addition, MagFRET measurements indicate that Mg2+ feeding also increases the [Mg2+]i of αβ KCs by approximately 50 μM (Wu, 2020).

This study identified a role for uex, the single fly ortholog of the evolutionarily conserved family of CNNM-type Mg2+ efflux transporters. There are four distinct CNNM genes in mice and humans, five in C. elegans, and two in zebrafish. The uex locus produces four alternatively spliced mRNA transcripts, but all encode the same 834 aa protein. The precise role of CNNM proteins in Mg2+ transport is somewhat contentious. Some propose that CNNM proteins are direct Mg2+ transporters, whereas others favor that they function as sensors of intracellular Mg2+ concentration [Mg2+]i and/or regulators of other Mg2+ transporters. This study found that ectopic expression of Drosophila UEX enhances Mg2+ efflux in HEK293 cells and that endogenous UEX limits [Mg2+]i in αβ KCs in the fly brain. Therefore, if UEX is not itself a Mg2+ transporter, it must be able to interact effectively with human Mg2+ efflux transporters and to influence Mg2+ extrusion in Drosophila. Since UEX is the only CNNM protein in the fly, it may serve all the roles of the four individual mammalian CNNMs. However, the ability of mouse CNNM2 to restore memory capacity to uex mutant flies suggests that the memory-relevant UEX function can be substituted by that of CNNM2 (Wu, 2020).

Interestingly, none of the disease-relevant variants of CNNM2 were able to complement the memory defect of uex mutant flies. The CNNM2 T568I variant substitutes a single amino acid in the second CBS domain. The oncogenic protein tyrosine phosphatases of the PRL (phosphatase of regenerating liver) family bind to the CBS domains of CNNM2 and CNNM3 and can inhibit their Mg2+ transport function. It will therefore be of interest to test the role of the UEX CBS domains and whether fly PRL-1 regulates UEX activity (Wu, 2020).

RNA-seq analysis reveals that uex is strongly expressed in the larval and adult fly digestive tract and nervous systems, as well as the ovaries suggesting that many uex mutations will be pleiotropic. The uex^Δ allele, which deletes 272 amino acids (including part of the second CBS and the entire CNBH domain) from the UEX C-terminus, results in developmental lethality when homozygous, demonstrating that uex is an essential gene. Mammalian CNNM4 is localized to the basolateral membrane of intestinal epithelial cells. There it is believed to function in transcellular Mg2+ transport by exchanging intracellular Mg2+ for extracellular Na+ following apical entry through TRPM7 channels. Lethality in Drosophila could therefore arise from an inability to absorb sufficient Mg2+ through the larval gut. However, neuronally restricted expression of uex^RNAi with elav-GAL4 also results in larval lethality, suggesting UEX has an additional role in early development of the nervous system, like CNNM2 in humans and zebrafish. Perhaps surprisingly, flies carrying homozygous or trans-heterozygous combinations of several hypomorphic uex alleles have defective appetitive and aversive memory performance, yet they seem otherwise unaffected (Wu, 2020).

Genetically engineering the uex locus to add a C-terminal HA tag to the UEX protein allowed localization of its expression in the brain. Labeling is particularly prominent in all major classes of KCs. Restricting knockdown of uex expression to all αβ KCs of adult flies, or even just the αβc subset reproduced the LTM defect. The LTM impairment was evident if uex^RNAi expression in αβ neurons was restricted to adult flies, suggesting UEX has a more sustained role in neuronal physiology. In contrast, knocking down uex expression in either the αβs or α'β' neurons did not impair LTM. Activity of α'β' neurons is required after training to consolidate appetitive LTM, whereas αβc and αβs KC output, together and separately, is required for its expression. Therefore, observing normal LTM performance in flies with uex loss-of-function in αβs and α'β' neurons argues against a general deficiency of αβ neuronal function when manipulating uex (Wu, 2020).

Dietary Mg2+ could not enhance the defective LTM performance of flies that were constitutively uex mutant, or harbored αβ KC-restricted uex loss-of-function. However, expressing uex in the αβ KCs of uex mutant flies restored the ability of Mg2+ to enhance performance. Therefore, the αβ KCs are the cellular locus for Mg2+-enhanced memory in the fly (Wu, 2020).

It perhaps seems counterintuitive that UEX-directed magnesium efflux is required in KCs to support the memory-enhancing effects of Mg2+ feeding, when dietary Mg2+ elevates KC [Mg2+]i. At this stage, it can only be speculated as to why this is the case. It is assumed that the brain and αβ KCs, in particular, have to adapt in a balanced way to the higher levels of intracellular and extracellular Mg2+ that result from dietary supplementation. Live-imaging of KC [Mg2+]i in wild-type and uex mutant brains suggests that UEX-directed efflux is likely to be an essential factor in the active, and perhaps stimulus-evoked, homeostatic maintenance of these elevated levels (Wu, 2020).

A number of mammalian cell-types extrude Mg2+ in a cAMP-dependent manner, a few minutes after being exposed to β-adrenergic stimulation. The presence of a CNBH domain suggests that UEX and CNNMs could be directly regulated by cAMP. The importance of the CNBH was tested by introducing an R622K amino acid substitution that should block cAMP binding in the UEX CNBH. This subtle mutation abolished the ability of the uex^R622K transgene to restore LTM performance to uex mutant flies. CRISPR was used to mutate the CNBH in the native uex locus. Although deleting the CNBH from CNNM4 abolished Mg2+ efflux activity, flies homozygous for the uex^T626NRR lesion were viable, demonstrating that they retain a sufficient level of UEX function. However, these flies exhibited impaired immediate and long-term memory. In addition, the performance of uex^T626NRR flies could not be enhanced by Mg2+ feeding. These data demonstrate that an intact CNBH is a critical element of memory-relevant UEX function. Binding of clathrin adaptor proteins to the CNNM4 CNBH has been implicated in basolateral targeting, suggesting that uex^T626NRR might be inappropriately localized in KCs. Furthermore, KC expression of the CNNM2 E122K mutant variant, which retains residual function but has a trafficking defect, did not restore the uex LTM defect (Wu, 2020).

Although it has been questioned whether the CNNM2/3 CNBH domains bind cyclic nucleotides, this study found that FSK evoked an increase in αβ KC [Mg2+]i that was sensitive to uex mutation, and that UEX::HA was mislocalized in rut²⁰⁸⁰ adenylate cyclase and dnc¹ phosphodiesterase learning defective mutant flies. Whereas UEX::HA label was evenly distributed in γ, αβc, and αβs KCs in wild-type flies, UEX::HA label was diminished in the γ and αβs KCs and was stronger in αβc neurons in rut²⁰⁸⁰ and dnc¹ mutants. The chronic manipulations of cAMP in the mutants are therefore consistent with cAMP impacting UEX localization, perhaps by interacting with the CNBH. In addition, altered UEX localization may contribute to the memory defects of rut²⁰⁸⁰ and dnc¹ flies (Wu, 2020).

The physiological data using Magnesium Green in mammalian cell culture and the genetically encoded MagIC reporter in αβ KCs demonstrate that fly UEX facilitates Mg2+ efflux. Stimulating the fly brain with FSK evoked a greater increase in αβ KC [Mg2+]i in uex mutant brains than in wild-type controls which provides the first evidence that UEX limits a rise in [Mg2+]i in Drosophila KCs. MagIC recordings also revealed a slow oscillation (centered around 0.015 Hz, approximately once a minute) of αβ KC [Mg2+]i that was dependent on UEX. The physiological function of this [Mg2+]i fluctuation is not yet understood although it likely reflects a homeostatic systems-level property of the cells. Biochemical oscillatory activity plays a crucial role in many aspects of cellular physiology. Most notably, circadian timed fluctuation of [Mg2+]i links dynamic cellular energy metabolism to clock-controlled translation through the Mg2+ sensitive mTOR (mechanistic target of rapamycin) pathway. It is therefore possible that slow Mg2+ oscillations could unite roles for cAMP, UEX, energy flux, and mTOR-dependent translation underlying LTM-relevant synaptic plasticity (Wu, 2020).

Episodic events are frequently consolidated into labile memory but are not necessarily transferred to persistent long-term memory (LTM). Regulatory mechanisms leading to LTM formation are poorly understood, however, especially at the resolution of identified neurons. This study demonstrates enhanced LTM following aversive olfactory conditioning in Drosophila when the transcription factor cyclic AMP response element binding protein A (CREBA) is induced in just two dorsal-anterior-lateral (DAL) neurons. These experiments show that this process is regulated by protein-gene interactions in DAL neurons: (1) crebA transcription is induced by training and repressed by crebB overexpression, (2) CREBA bidirectionally modulates LTM formation, (3) crebA overexpression enhances training-induced gene transcription, and (4) increasing membrane excitability enhances LTM formation and gene expression. These findings suggest that activity-dependent gene expression in DAL neurons during LTM formation is regulated by CREB proteins (Lin, 2021).

CREBA and CREBB both are expressed in DAL neurons, and transgenic manipulations of CREBB have shown an impairment of 1-d memory after 10xS training. These studies, however, did not investigate a functional role for CREBA in DAL neurons and, in particular, did not query whether LTM might be enhanced. This study focused on a role for CREBA in LTM formation. It was first established in vitro that CREBA induced expression of a CRE-luciferase reporter gene in a PKA-dependent manner and was blocked by CREBB. Then, CREBA antibody, a DAL specific Gal4 driver and a crebA-driven KAEDE reporter were used to confirm that CREBA not only was expressed in DAL neurons but also responded transcriptionally to 10xS (but not 3xS or 1x) training. In this in vivo context, it was also shown that 10xS training-induced expression of crebA in DAL neurons was antagonized by overexpression of a crebB (repressor) transgene (Lin, 2021).

These observations suggested that CREBA in DAL neurons might serve as a positive regulator of protein synthesis-dependent LTM. Indeed, inducible transgenic manipulations of crebA only in DAL neurons were sufficient to impair 1-d memory after 10xS training (similar to inhibition of protein synthesis) using crebARNAi, or to enhance 1-d memory after 1x or 3xS training by overexpressing wild-type crebA. Importantly, LTM remained enhanced 4 d after 1x or 3xS training even when induction of transgenic crebA ceased 3 d earlier. Together, these results suggest that CREBA in DAL neurons is involved in learning and/or memory consolidation but not necessarily in memory retrieval (Lin, 2021).

CaMKII and per are two 'downstream' genes that are CREB responsive, are expressed in DAL neurons and impair LTM when disrupted. Using CaMKII and per-driven KAEDE reporter transgenes, it was shown that expression of both genes is induced normally after 10xS training, is blocked after such training by induced expression of a crebARNAi and is enhanced after 1x or 3xS training when a crebA transgene is inducibly expressed. These transgenic manipulations are not required for CaMKII or per expression and LTM to persist for 4 d after training and 3 d after transgenic manipulations are blocked (Lin, 2021).

This role for CREBA in DAL neurons during learning and memory consolidation suggested that the transcriptional response might be activity dependent. This possibility was explored by expressing a NaChBac (a bacterial sodium channel) transgene in DAL neurons, which served to increase membrane excitability and presumably neural activity in response to training. It was found that induced expression of NaChBac in DAL neurons was sufficient to enhance 1-d memory and to enhance expression of crebA, CaMKII, and per after 1x or 3xS training. Together, these observations have suggested a model that illustrates how CREBA and CREBB interact to regulate transcription in DAL neurons and the activity-dependent transcriptional response to gate LTM formation (Lin, 2021).

CREB-dependent long-term memory formation first was shown in Drosophila using inducible transgenes, which were expressed throughout the fly. Acute expression of a transgenic crebB repressor blocked LTM after 10xS training, whereas similar manipulations of a synthetic crebB activator transgene enhanced LTM. An early attempt to identify specific neurons underlying LTM implicated MBs, wherein MB-specific transgenic expression of a crebB repressor was reported to impair LTM after 10xS training. A subsequent study revealed, however, that this behavioral impairment derived from developmental defects in MB structure due to chronic expression of the crebB transgene. In contrast, induced expression of a crebB transgene only in adult-stage MBs did not impair LTM and did not produce any developmental defects. In neither study was a positive (CREB) regulator identified nor was enhanced LTM evaluated (Lin, 2021).

One-trial learning is usually insufficient to produce protein synthesis-dependent LTM, except for those experiences important for survival. This study has demonstrated that LTM can form after a single training session when 'memory genes' in DAL neurons are genetically manipulated. Learning-related and CREB-dependent changes in membrane excitability are well known and explain aspects of neuronal plasticity underlying memory consolidation. Regulation of ion channel genes by CREBA and CREBB transcription factors, for example, modulate plasticity in alcohol tolerance in Drosophila. CREB-dependent regulation of gene expression in DAL neurons appears sufficient to promote systems memory consolidation by modulating neural excitability. Further studies may elucidate whether neural circuits involved in motivation and attention also modulate DAL neurons during LTM formation and whether such prolonged neural activity also produces synaptic plasticity in DAL neurons (Lin, 2021).

CCHamide-2 (CCHa2) is a protostome excitatory peptide ortholog known for various arthropod species. In fruit flies, CCHa2 plays a crucial role in the endocrine system, allowing peripheral tissue to communicate with the central nervous system to ensure proper development and the maintenance of energy homeostasis. Since the formation of odor-sugar associative long-term memory (LTM) depends on the nutrient status in an animal, CCHa2 may play an essential role in linking memory and metabolic systems. This study shows that CCHa2 signals are important for consolidating appetitive memory by acting on the rewarding dopamine neurons. Genetic disruption of CCHa2 using mutant strains abolished appetitive LTM but not short-term memory (STM). A post-learning thermal suppression of CCHa2 expressing cells impaired LTM. In contrast, a post-learning thermal activation of CCHa2 cells stabilized STM induced by non-nutritious sugar into LTM. The receptor of CCHa2, CCHa2-R, was expressed in a subset of dopamine neurons that mediate reward for LTM. In accordance, the receptor expression in these dopamine neurons was required for LTM specifically. It is thus concluded that CCHa2 conveys a sugar nutrient signal to the dopamine neurons for memory consolidation. This finding establishes a direct interplay between brain reward and the putative endocrine system for long-term energy homeostasis (Yamagata, 2022).

Being involved in development of Huntington's, Parkinson's and Alzheimer's diseases, kynurenine pathway (KP) of tryptophan metabolism plays a significant role in modulation of neuropathology. Accumulation of a prooxidant 3-hydroxykynurenine (3-HOK) leads to oxidative stress and neuronal cell apoptosis. Drosophila mutant cardinal (cd¹ with 3-HOK excess shows age-dependent neurodegeneration and short-term memory impairments, thereby presenting a model for senile dementia. Although cd gene for phenoxazinone synthase (PHS) catalyzing 3-HOK dimerization has been presumed to harbor the cd¹ mutation, its molecular nature remained obscure. Using next generation sequencing, this study has shown that the cd gene in cd¹ carries a long deletion leading to PHS active site destruction. Contrary to the wild type Canton-S (CS), cd¹ males showed defective long-term memory (LTM) in conditioned courtship suppression paradigm (CCSP) at days 5-29 after eclosion. The number of dopaminergic neurons (DAN) regulating fly locomotor activity showed an age-dependent tendency to decrease in cd¹ relative to CS. Thus, in accordance with the concept "from the gene to behavior" proclaimed by S. Benzer, this study has shown that the aberrant PHS sequence in cd¹ provokes drastic LTM impairments and DAN alterations (Zhuravlev, 2022).

The gasotransmitter hydrogen sulfide (H(2)S) produced by the transsulfuration pathway (TSP) is an important biological mediator, involved in many physiological and pathological processes in multiple higher organisms, including humans. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) enzymes play a central role in H(2)S production and metabolism. This study investigated the role of H(2)S in learning and memory processes by exploring several Drosophila melanogaster strains with single and double deletions of CBS and CSE developed by the CRISPR/Cas9 technique. The learning and memory parameters of these strains using the mating rejection courtship paradigm and demonstrated that the deletion of the CBS gene, which is expressed predominantly in the central nervous system, and double deletions completely block short- and long-term memory formation in fruit flies. On the other hand, the flies with CSE deletion preserve short- and long-term memory but fail to exhibit long-term memory retention. Transcriptome profiling of the heads of the males from the strains with deletions in Gene Ontology terms revealed a strong down-regulation of many genes involved in learning and memory, reproductive behavior, cognition, and the oxidation-reduction process in all strains with CBS deletion, indicating an important role of the hydrogen sulfide production in these vital processes (Zatsepina, 2022).

The Drosophila Bicra (CG11873) gene encodes the sole ortholog of mammalian GLTSCR1 and GLTSCR1L, which are components of a chromatin remodeling complex involved in neoplasia and metastasis of cancer cells. Bicra is highly expressed in Drosophila larval CNS and adult brain, yet its physiological functions in the nervous system remain elusive. This study reports that Bicra is expressed in both neurons and glia of adult brains, and is required for courtship learning and choice ability of male flies. The function of Bicra in the mushroom body, and in particular, Bicra expression in neurons but not glia, is responsible for the male courtship learning and choice performance. This study unravels a novel function of Bicra in cognition-related courtship behaviors in Drosophila, and may provide insight into the neuronal functions of its mammalian orthologs (Sun, 2022).

The astrocyte-neuron lactate shuttle hypothesis posits that glial-generated lactate is transported to neurons to fuel metabolic processes required for long-term memory. Although studies in vertebrates have revealed that lactate shuttling is important for cognitive function, it is uncertain if this form of metabolic coupling is conserved in invertebrates or is influenced by age. Lactate dehydrogenase (Ldh) is a rate limiting enzyme that interconverts lactate and pyruvate. This study genetically manipulated expression of Drosophila melanogaster Lactate dehydrogenase (dLdh) in neurons or glia to assess the impact of altered lactate metabolism on invertebrate aging and long-term courtship memory at different ages. Survival, negative geotaxis, brain neutral lipids (the core component of lipid droplets) and brain metabolites were also examined. Both upregulation and downregulation of dLdh in neurons resulted in decreased survival and memory impairment with age. Glial downregulation of dLdh expression caused age-related memory impairment without altering survival, while upregulated glial dLdh expression lowered survival without disrupting memory. Both neuronal and glial dLdh upregulation increased neutral lipid accumulation. Evidence is provided that altered lactate metabolism with age affects the tricarboxylic acid (TCA) cycle, 2-hydroxyglutarate (2HG), and neutral lipid accumulation. Collectively, these findings indicate that the direct alteration of lactate metabolism in either glia or neurons affects memory and survival but only in an age-dependent manner (Frame, 2023).

Tauopathies including Alzheimer's disease, are characterized by progressive cognitive decline, neurodegeneration, and intraneuronal aggregates comprised largely of the axonal protein Tau. It has been unclear whether cognitive deficits are a consequence of aggregate accumulation thought to compromise neuronal health and eventually lead to neurodegeneration. This study use the Drosophila tauopathy model and mixed-sex populations to reveal an adult onset pan-neuronal Tau accumulation-dependent decline in learning efficacy and a specific defect in protein synthesis-dependent memory (PSD-M), but not in its protein synthesis-independent variant. It was demonstrated that these neuroplasticity defects are reversible on suppression of new transgenic human Tau expression and surprisingly correlate with an increase in Tau aggregates. Inhibition of aggregate formation via acute oral administration of methylene blue results in re-emergence of deficient memory in animals with suppressed human Tau (hTau)(0N4R) expression. Significantly, aggregate inhibition results in PSD-M deficits in hTau(0N3R)-expressing animals, which present elevated aggregates and normal memory if untreated with methylene blue. Moreover, methylene blue-dependent hTau(0N4R) aggregate suppression within adult mushroom body neurons also resulted in emergence of memory deficits. Therefore, deficient PSD-M on human Tau expression in the Drosophila CNS is not a consequence of toxicity and neuronal loss because it is reversible. Furthermore, PSD-M deficits do not result from aggregate accumulation, which appears permissive, if not protective of processes underlying this memory variant (Yourkou, 2023).

Like many other animals, insects are capable of returning to previously visited locations using path integration, which is a memory of travelled direction and distance. Recent studies suggest that Drosophila can also use path integration to return to a food reward. However, the existing experimental evidence for path integration in Drosophila has a potential confound: pheromones deposited at the site of reward might enable flies to find previously rewarding locations even without memory. Here, we show that pheromones can indeed cause naive flies to accumulate where previous flies had been rewarded in a navigation task. Therefore, an experiment was designed to determine if flies can use path integration memory despite potential pheromonal cues by displacing the flies shortly after an optogenetic reward. It was found that rewarded flies returned to the location predicted by a memory-based model. Several analyses are consistent with path integration as the mechanism by which flies returned to the reward. It is concluded that although pheromones are often important in fly navigation and must be carefully controlled for in future experiments, Drosophila may indeed be capable of performing path integration (Titova, 2023).

The ability to associate neutral stimuli with valence information and to store these associations as memories forms the basis for decision making. To determine the underlying computational principles, this study built a realistic computational model of a central decision module within the Drosophila mushroom body (MB), the fly's center for learning and memory. The model combines the electron microscopy-based architecture of one MB output neuron (MBON-α3), the synaptic connectivity of its 948 presynaptic Kenyon cells (KCs), and its membrane properties obtained from patch-clamp recordings. This neuron is electrotonicly compact and that synaptic input corresponding to simulated odor input robustly drives its spiking behavior. Therefore, sparse innervation by KCs can efficiently control and modulate MBON activity in response to learning with minimal requirements on the specificity of synaptic localization. This architecture allows efficient storage of large numbers of memories using the flexible stochastic connectivity of the circuit (Hafez, 2023).

For understanding of the computational principles underlying learning and memory, it is essential to determine the intrinsic contributions of neuronal circuit architecture. Associative olfactory memory formation in Drosophila provides an excellent model system to investigate such circuit motifs, as a large number of different odors can be associated with either approach or rejection behavior through the formation of both short- and long-term memory within the MB circuitry. In contrast to most axon guidance processes in Drosophila that are essentially identical in all wild-type individuals, processing of odor information in the MB largely, but not exclusively, depends on the stochastic connectivity of projection neurons to KCs that relay odor information from the olfactory glomeruli to the MBONs. However, the role of MBONs within the circuit is largely fixed between animals and many MBONs can be classified as either approach or avoidance neurons in specific behavioral paradigms. Furthermore, individual KCs are not biased in their MBON connectivity but innervate both kinds of output modules, for both approach and avoidance. Learning and memory, the modulation of odor response behavior through pairing of an individual odor with either positive or negative valence, is incorporated via the local activity of valence-encoding DANs that can either depress or potentiate KC>MBON synaptic connections. As KC odor specificity and connectivity differ significantly between flies with respect to the number and position of KC>MBON synapses, this circuit module must be based on architectural features supporting robust formation of multiple memories regardless of specific individual KC connectivity (Hafez, 2023).

Prior computational work addressing properties of central nervous system neurons in Drosophila relied on synthetic (randomly generated) data or partial neuronal reconstructions. This study goes beyond these previous studies by combining precise structural data from the electron-microscopy based synaptic connectome with functional (electrophysiological) data. The structural data consists of the neuroanatomical structure of MBON-α3, including the 12,770 synaptic inputs of all its 948 innervating KCs. While this EM reconstruction may contain some mistakes in synaptic connectivity as e.g. up to 10% of synaptic sites remained unassigned within the dataset, it currently represents the best possible template for an in silico reconstruction. The neuron’s functional properties were determined from ex vivo patch clamp recordings. Near-perfect agreement between experimentally observed and simulated voltage traces recorded in the soma shows that linear cable theory is an excellent model for information integration in this system (Hafez, 2023).

Together, this study obtained a realistic in silico model of a central computational module of memory-modulated animal behavior, that is a mushroom body output neuron (Hafez, 2023).

The data show that the dendritic tree of the MBON is electrotonically compact, despite the complex architecture that includes a high degree of branching. This data is in agreement with a prior electrophysiological characterization of an MBON in locust, and a similar feature has been reported for neurons in the stomatogastric ganglion of crayfish. Here the electrotonic compactness supports linear integration of synaptic inputs across extensive arborizations and likely serves to functionally compensate for inter-individual variability. The location of an individual synaptic input within the dendritic tree has therefore only a minor effect on the amplitude of the neuron’s output, despite large variations of local dendritic potentials. This effect was particularly striking when the analysis was restricted to a population of KCs with identical numbers of synapses that all elicited highly stereotypical responses. The compactification of the neuron is likely related to the architectural structure of its dendritic tree. Together with the relatively small size of many central neurons in Drosophila, this indicates that in contrast to large vertebrate neurons, local active amplification or other compensatory mechanisms may not be necessary to support input normalization in the dendritic tree. In contrast, for axons it has been recently reported that voltage-gated Na+ channels are localized in putative spike initiation zones in a subset of central neurons of Drosophila. In case in vivo physiological data quantitatively describing local active currents become available, they can be incorporated into the model to further increase the agreement between model and biological system (Hafez, 2023).

Encoding of odor information and incorporation of memory traces is not performed by individual KCs but by ensembles of KCs and MBONs. Calcium imaging in vivo demonstrated that individual odors evoke activity reliably in approximately 3–9% of KCs. Simulations of 1000 independent trials with random sets of 50 KCs, each representing one distinct odor that activates approximately 5% of the KCs innervating the target MBON, demonstrate that such activation patterns robustly elicit MBON activity in agreement with in vivo observations of odor-induced activity that elicited robust increases in action potential frequency in MBONs. The low variability of depolarizations observed in these simulations indicates that information coding by such activation patterns is highly robust. As a consequence, labeled line representations of odor identity are likely not necessary at the level of KCs since relaying information via any set of &asymp:50 KCs is of approximately equal efficiency. Such a model is supported by a recent computational study demonstrating that variability in parameters controlling neuronal excitability of individual KCs negatively affects associative memory performance. The authors provide evidence that compensatory variation mechanisms exist that ensure similar activity levels between all odor-encoding KC sets to maintain efficient memory performance (Hafez, 2023).

Optical recordings of in vivo activity of MBONs revealed selective reductions in MBON activity in response to aversive odor training or to optogenetic activation of selective DANs. More generally, both depression and potentiation of MBON activity have been previously observed in different MBON modules in vivo. In addition, recent studies have observed changes in KC stimulus representations after conditioning that may be due to learning-dependent modulations of synaptic PN input to KCs. The computational model allowed implementation and comparison of these two mechanisms changing MBON output: One is a change in the strength of the KC>MBON synapses (over a range of ±25%); the other is a change in the number of activated KCs (over the same range). Interestingly, almost linear relationships were found between the number of active KCs and the resulting depolarizations, and the same between the strength of synapses and MBON depolarization. Decreasing or increasing either of these variables by 25% significantly altered the level of MBON activation with only minor differences in the extent to which these modifications contributed to MBON depolarization. The two different mechanisms of altering MBON output are potentially utilized for the establishment of different types of memory with fast and local alterations of synaptic transmission likely essential for short-term memory while structural changes may ensure maintenance of memory over long periods of time. In addition, differential modes of plasticity may be required at potentially more static parts of the MB circuitry like the food-related part that is not entirely based on stochastic connectivity (Hafez, 2023).

The simulation data thus shows that the KC>MBON architecture represents a biophysical module that is well-suited to simultaneously process changes based on either synaptic and/or network modulation. Together with the electrotonic nature of the MBONs, the interplay between KCs and MBONs thus ensures reliable information processing and memory storage despite the stochastic connectivity of the memory circuitry. While this study focuses on the detailed activity patterns within a single neuron, the availability of large parts of the fly connectome at the synaptic level, in combination with realistic models for synaptic dynamics, should make it possible to extend this work to circuit models to gain a network understanding of the computational basis of decision making (Hafez, 2023).

Learned behavior can be suppressed by the extinction procedure. Such extinguished memory often returns spontaneously over time, making it difficult to treat diseases such as addiction. However, the biological mechanisms underlying such spontaneous recovery remain unclear. This study reports that the extinguished reward memory in Drosophila recovers spontaneously because extinction training forms an aversive memory that can be actively forgotten via the Dia pathway. Manipulating Rac1 activity does not affect sugar-reward memory and its immediate extinction effect but bidirectionally regulates spontaneous recovery-the decay process of extinction. Experiments using thermogenetic inhibition and functional imaging support that such extinction appears to be coded as an aversive experience. Genetic and pharmacological inhibition of formin Dia, a downstream effector of Rac1, specifically prevents spontaneous recovery after extinction in both behavioral performance and corresponding physiological traces. Together, the data suggest that spontaneous recovery is caused by active forgetting of the opposing extinction memory (Yang, 2023).

Multiple lines of evidence from the current study support that the spontaneous recovery of extinguished reward memory is caused by active forgetting of the opposing extinction memory. First, the small G protein Rac1, which is reported to mediate active forgetting of aversive labile memory, is activated in a delayed manner after appetitive memory extinction. It specifically and bidirectionally regulates the decay process of extinction, also known as the process of spontaneous recovery, through its downstream effector Dia. However, it does not affect the acquisition and retention of appetitive memory at multiple time points as well as the early extinction effect within 3 h. Second, the extinction of appetitive memory requires dopaminergic neurons mediating aversive but not rewarding reinforcement. Third, the extinction of appetitive memory produces a physiological trace of aversive memory in a pair of MBON-γ1pedc>α/β neurons. Such an aversive memory trace correlates with the extinction effect at the behavioral level and can be prolonged by inhibiting the Rac1/Dia pathway, which prevents spontaneous recovery. Thus, the extinction of appetitive memory appears to be a kind of aversive labile memory that decays with time due to Rac1/Dia-mediated forgetting. Such time-based forgetting of extinction memory results in spontaneous recovery (Yang, 2023).

Spontaneous recovery, which was first discovered by Pavlov's experiments, has been proposed to occur because extinction is susceptible to forgetting. In recent years, several biological mechanisms of forgetting have been revealed; however, the link between these forgetting mechanisms and spontaneous recovery has never been established. Rac1-mediated forgetting was first discovered in Drosophila, and it regulates the decay of labile aversive memory through its downstream molecule Dia. In the current study, the data support that spontaneous recovery of the original reward memory happens readily because the extinction memory is actively forgotten via the Rac1/Dia pathway over time. Such finding is consistent with Rac1's role in mediating forgetting of aversive memory, given that appetitive memory extinction appears to be a kind of aversive learning. They share similar punishment-teaching DANs and aversive memory traces (Yang, 2023).

Together with previous studies, the current data support that Rac1-mediated time-based forgetting is a delayed-start, slow-completion process. Overexpressing Rac1-CA significantly accelerated the forgetting process of extinction memory at 6 h but not at 3 h, indicating that the upstream forgetting signal of Rac1 might emerge after 3 h and already start to function at 6 h. Consistently, endogenous Rac1 activity was observed to increase 8 h but not 3 h after extinction. Considering that the extinction effect in control flies was still significant at the behavioral level after 12 h, Rac1-mediated forgetting is a slow-completion process. Similarly, there was a significant increase in endogenous Rac1 activity 1 h after aversive learning, when forgetting was not significant at the behavioral level (Yang, 2023).

Rac1 also plays a conserved role in regulating forgetting of different forms of memory in vertebrates and is associated with autism and Alzheimer's disease. In mice, a single-trial contextual fear conditioning induces delayed Rac1 activation. Decreasing or increasing such Rac1 activation suppresses or accelerates fear memory forgetting, respectively. Thus, it is of interest to test whether reward memory extinction also produces a punishment memory and Rac1 also specifically regulates the spontaneous recovery in vertebrates (Yang, 2023).

Rac1 bidirectionally regulates the forgetting of aversive olfactory memory. The current data show that genetic manipulations of Rac1 in MB neurons do not affect the acquisition and retention of the sugar reward memory. These findings together raise an interesting question: is Rac1 specifically responsible for the forgetting of punishment memory but not reward memory? Presynaptic plasticity at KC-MBON synapses plays a key role in aversive learning and memory,while postsynaptic plasticity at KC-MBON synapses was recently found to underlie appetitive learning and memory. Based on these findings, it became easy to understand that the forgetting of punishment memory and reward memory may be achieved through different molecular mechanisms (Yang, 2023).

Spontaneous recovery of aversive memory after extinction is also reported in Drosophila. According to previous studies and the current findings, it is possible that aversive memory spontaneously recovers through forgetting of extinction memory with a positive valence. Up to now, no forgetting molecule of reward memory has been reported. Significant natural decay has been reported in non-nutritious sugar reward memory and water reward memory. It is interesting to find forgetting molecules underlying such short-lived reward memory and to determine whether there are forgetting molecules that could explain the spontaneous recovery of aversive memory (Yang, 2023).

Extinction is vulnerable to relapse. This relapse is a major obstacle to the successful treatment of clinical disorders including addiction and PTSD. Two strategies have been proposed to prevent relapse: enhancing the consolidation of extinction learning and disrupting the reconsolidation of the original memory. The current findings suggest that blocking specific forgetting mechanisms may contribute to the success of relapse prevention, indicating a new strategy for relapse prevention (Yang, 2023).

Rac1 is proposed to be an attractive therapeutic target in treating human diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders. Genetic inhibition of Rac1 prevents spontaneous recovery of extinguished reward memory—one kind of relapse—suggesting that Rac1 may also be a potential target in treating diseases such as addiction. Rac1 can regulate multiple signaling pathways that involve diverse cellular functions such as actin remodeling, microtubule stability, gene transcription, and superoxide production. Formin Dia, a nucleator of linear actin polymerization, is now found to be a downstream effector of Rac1 in regulating the spontaneous recovery of extinguished reward memory. It might be a more specific drug target than Rac1 in preventing relapse in diseases like addiction. Together, these findings suggest a new strategy to prevent relapse and provide potential drug targets (Yang, 2023).

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, the goal of this study was to validate a Drosophila model relevant to CRCI. The chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin were administered to adult Drosophila. Neurologic deficits were observed with all tested chemotherapies, with doxorubicin and in particular cisplatin also resulting in memory deficits. Histologic and immunohistochemical analysis was performed of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, the Drosophila model relevant to CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. This new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI (and chemotherapy-induced neurotoxicity more generally) and pharmacologic screens to identify disease-modifying therapies (Torre, 2023).

Tastes typically evoke innate behavioral responses that can be broadly categorized as acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses also exhibit plasticity through experience-dependent changes in mushroom body circuits. This study developed a novel taste learning paradigm using closed-loop optogenetics. Appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory neurons or dopaminergic neurons encoding reward or punishment. As with olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. This paradigm affords new opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses depend on experience (Jelen, 2023).

Obesity is associated with cognitive decline. Recent observations in mice propose an adipose tissue (AT)-brain axis. This study identified 188 genes from RNA sequencing of AT in three cohorts that were associated with performance in different cognitive domains. These genes were mostly involved in synaptic function, phosphatidylinositol metabolism, the complement cascade, anti-inflammatory signaling, and vitamin metabolism. These findings were translated into the plasma metabolome. The circulating blood expression levels of most of these genes were also associated with several cognitive domains in a cohort of 816 participants. Targeted misexpression of candidate gene ortholog in the Drosophila fat body significantly altered flies memory and learning. Among them, down-regulation of the neurotransmitter release cycle-associated gene SLC18A2 improved cognitive abilities in Drosophila and in mice. Up-regulation of RIMS1 in Drosophila fat body enhanced cognitive abilities. Current results show previously unidentified connections between AT transcriptome and brain function in humans, providing unprecedented diagnostic/therapeutic targets in AT (Oliveras-Canellas, 2023).

Animals can continuously learn different tasks to adapt to changing environments and, therefore, have strategies to effectively cope with inter-task interference, including both proactive interference (Pro-I) and retroactive interference (Retro-I). Many biological mechanisms are known to contribute to learning, memory, and forgetting for a single task, however, mechanisms involved only when learning sequential different tasks are relatively poorly understood. This study dissected the respective molecular mechanisms of Pro-I and Retro-I between two consecutive associative learning tasks in Drosophila. Pro-I is more sensitive to an inter-task interval (ITI) than Retro-I. They occur together at short ITI (<0 min), while only Retro-I remains significant at ITI beyond 20 min. Acutely overexpressing Corkscrew (CSW), an evolutionarily conserved protein tyrosine phosphatase SHP2, in mushroom body (MB) neurons reduces Pro-I, whereas acute knockdown of CSW exacerbates Pro-I. Such function of CSW is further found to rely on the γ subset of MB neurons and the downstream Raf/MAPK pathway. In contrast, manipulating CSW does not affect Retro-I as well as a single learning task. Interestingly, manipulation of Rac1, a molecule that regulates Retro-I, does not affect Pro-I. Thus, these findings suggest that learning different tasks consecutively triggers distinct molecular mechanisms to tune proactive and retroactive interference (Zhao, 2023).

Activation of local translation in neurites in response to stimulation is an important step in the formation of long-term memory (LTM). CPEB proteins are a family of translation factors involved in LTM formation. The Drosophila CPEB protein Orb2 plays an important role in the development and function of the nervous system. Mutations of the coding region of the orb2 gene have previously been shown to impair LTM formation. This study found that a deletion of the 3'UTR of the orb2 gene similarly results in loss of LTM in Drosophila. As a result of the deletion, the content of the Orb2 protein remained the same in the neuron soma, but significantly decreased in synapses. Using RNA immunoprecipitation followed by high-throughput sequencing, more than 6000 potential Orb2 mRNA targets expressed in the Drosophila brain were detected. Importantly, deletion of the 3'UTR of orb2 mRNA also affected the localization of the Csp, Pyd, and Eya proteins, which are encoded by putative mRNA targets of Orb2. Therefore, the 3'UTR of the orb2 mRNA is important for the proper localization of Orb2 and other proteins in synapses of neurons and the brain as a whole, providing a molecular basis for LTM formation (Kozlov, 2023).

During starvation, mammalian brains can adapt their metabolism, switching from glucose to alternative peripheral fuel sources. In the Drosophila starved brain, memory formation is subject to adaptative plasticity, but whether this adaptive plasticity relies on metabolic adaptation remains unclear. This study shows that during starvation, neurons of the fly olfactory memory centre import and use ketone bodies (KBs) as an energy substrate to sustain aversive memory formation. Local providers within the brain, the cortex glia, were identified that use their own lipid store to synthesize KBs before exporting them to neurons via monocarboxylate transporters. Finally, it was show that the master energy sensor AMP-activated protein kinase regulates both lipid mobilization and KB export in cortex glia. These data provide a general schema of the metabolic interactions within the brain to support memory when glucose is scarce (Silva, 2022).

The main energy source for the brain is glucose1. Metabolic communication between neurons and glia is crucial to sustain brain functions such as memory. The main model of this metabolic communication is the astrocyte-neuron lactate shuttle (ANLS), wherein glia take up glucose from blood and provide lactate via glycolysis to neurons as an energy substrate; this lactate production is stimulated by neuronal activity. But how is the brain's energy requirement met during starvation when glucose is scarce? It has been known since the 1960s that, under starvation, the two principal KBs, acetoacetate and β-hydroxybutyrate, are used by the brain to support its energy demand. Nevertheless, the ability of KBs to replace glucose during neuronal oxidative metabolism was fully demonstrated only recently, and no evidence of direct KB oxidation by neurons to sustain memory formation has been reported yet. In mammals, the body's main KB provider is the liver, in which acetyl-CoA used for ketogenesis is produced by β-oxidation of fatty acids (FAs) imported into the mitochondria. Although there is no evidence of ketogenesis in neurons, several in vitro studies in mammals have shown that astrocytes can synthesize KBs due to their ability to oxidize FAs, suggesting that a system for local production and delivery of KBs could exist inside the brain. However, it is unknown whether glia provide KBs to neurons in vivo to sustain higher brain functions (Silva, 2022).

Using Drosophila melanogaster and an associative olfactory memory paradigm, in vivo the metabolic communication between neurons and glia during starvation was investigated. Flies can form long-lasting olfactory aversive memories as a result of several presentations of an odorant paired with electric shocks, the negative stimuli. This association is stored as a memory trace in the mushroom body (MB), the major integrative brain centre for learning and memory in insects. In flies fed ad libitum this study showed that the formation of protein synthesis-dependent long-term memory (LTM) after multiple spaced olfactory trainings crucially relies on the regulation of both pyruvate (a glucose derivative) metabolism in MB neurons and glucose metabolism in glial cells. When flies are starved, LTM formation is blocked, which is beneficial for surviving food restriction. This adaptive plasticity is specific to LTM, as starved flies maintain their ability to form consolidated—but protein synthesis-independent—memory after multiple massed trainings. Because the starved brain cannot rely on glucose as it does in the fed state, this prompted an investigation of the specific metabolic pathways at play during starvation in the MB. The results establish that, during starvation, MB neurons import and use KBs as an energy substrate to sustain associative memory formation, a memory that was named KB-dependent associative memory (K-AM). Additionally, a local provider of KBs in the brain, the cortex glia, was identified, and it was show that cortex glia mobilize FAs from their own lipid droplets (LDs) to synthesize KBs. Key actors were characterized in KB metabolic pathways and transport between cortex glia and MB neurons. Finally, this study showed that KB production and delivery in cortex glia are regulated by AMP-activated protein kinase (AMPK), the cellular master energy sensor, thus allowing cortex glia to adapt their support to neurons depending on the brain's energy status (Silva, 2022).

This study investigated in vivo the metabolic communication between neurons and glia that are used to sustain brain functions during starvation. KBs were shown to be imported and oxidized by neurons to sustain associative memory formation during starvation. Interestingly, these KBs are provided by a local glia source. By using cell-specific knockdown of enzymes involved in each of the key steps of KB production (that is, FA mobilization, FA mitochondrial import and ketogenesis), it was established that cortex glia produce KBs from their own FA internal store and transfer them to neurons. This metabolic communication is critical for K-AM formation in the MB. A combination of behavioural and imaging experiments using the trans-acceleration properties of MCTs allowed identified Sln and Chk (Chaski) as the specific MCTs involved in KB transport during starvation in neurons and cortex glia, respectively. Finally, it was shown that AMPK, the master energy sensor of the cell, regulates this metabolic communication during starvation by activating KB production and its export by cortex glia (Silva, 2022).

These results indicate that the cortex glia mobilize their own internal store of FAs to produce KBs and provide them to neurons. But could this be a more general feature of glial cell types during starvation? Neither astrocyte-like glia nor ensheathing glia, the two other glial cell types in the Drosophila brain that are in close contact with neurons, contribute to KB production to sustain memory formation in neurons during starvation. Thus, the role of LDs as an energy reservoir to sustain neuronal function during starvation seems to be specific to cortex glia. In contrast, its function in other glial cells in which they have been observed should be more related to neuroprotection from damage by reactive oxygen species, as proposed in several Drosophila and mammalian studies. If the cortex glia in the Drosophila brain are the main local provider of KBs, this raises the question of a shared function by glial cells across species, and more specifically in mammals. Even if astrocyte-like glia are the Drosophila glial cell type most commonly referred to as the equivalent of mammalian astrocytes, the cortex glia also share some essential morphological features with mammalian astrocytes such as the encompassing of neuronal cell bodies, as well as functions including the modulation of neuronal excitability. Interestingly, mammalian astrocytes present three key points that this study has shown to be critical for cortex glia in providing KBs to neurons for sustaining K-AM: (1) they contain LDs; (2) they have (at least in vitro) the metabolic capacity to produce KBs; and (3) they express the KB transporter MCT1. Altogether, these arguments suggest that astrocytes in the mammalian brain could provide an additional source of KBs for neurons to sustain neuronal function during starvation. However, at the molecular level, the results show that ketogenesis in Drosophila cortex glia depends on a two-step reaction from acetoacetyl-CoA to acetoacetate that relies on HMGS and HMG-lyase, as in the classical path described in the mammalian liver. This pathway is different from the one described to occur in vitro in mammalian astrocytes for ketogenesis, which is a one-step reaction catalysed by the reversible enzyme SCOT. Even if succinyl-CoA, the by-product of acetoacetate production by SCOT, is an allosteric inhibitor of HMGS, it is not known if these two pathways used to produce acetoacetate from acetyl-CoA are exclusive, or if they can occur in the same cell in parallel. Further in vivo investigations of the mammalian glia role as a local provider of KBs to neurons, as well as other possible pathways of KB production in Drosophila cortex glia, will make it possible to discriminate between experimental set-up bias (in vitro experiments in which only glial cells are present with no neuronal environment), or even differences between mammals and insects (Silva, 2022).

In insects, the KB concentration increases in the haemolymph during starvation. However, it has still not been clearly established in Drosophila if the fat body (functionally equivalent to the liver) synthesizes and delivers KBs to the haemolymph during starvation. A local provider within the brain such as the cortex glia would be advantageous due to its proximity to neurons, as compared to peripheral organs such as the fat body. This would also circumvent the need to transport KBs across the blood-brain barrier for their uptake by the brain. In addition, a local provider within the brain ensures that the brain will have a KB source with limited competition from other organs, as compared to when KBs are taken up from the haemolymph (Silva, 2022).

The results show that AMPK is required in cortex glia to sustain K-AM, suggesting a basal mechanism of KB production and delivery that is activated during starvation. This study identified two well-known downstream effectors of AMPK, namely Bmm, the homologue of ATGL, and CPT1 as essential actors of FA mobilization and the subsequent mitochondrial import necessary to sustain K-AM during starvation. Bmm and CPT1 expression are upregulated during starvation in fly heads, and AMPK in glial cells is required to mediate this transcriptional regulation. The regulation of Bmm and CPT1 by AMPK at the transcriptional level revealed in this study does not rule out additional post-transcriptional regulations such as phosphorylation of Bmm, as described for ATGL in the activation of its TAG hydrolase activity, and the indirect activation of CPT1 through inhibitory phosphorylation of ACC by AMPK, a mechanism described in various mammalian tissues including the brain that is also conserved across species. Finally, the results demonstrate that, in starved flies, Chk-dependent KB transport is not directly coupled to KB production, whereas it requires AMPK in cortex glia. Further investigation is required to determine whether the regulation of KB transport via Chk is achieved by regulating Chk activity or Chk trafficking and expression at the membrane, and how AMPK regulates this process (Silva, 2022).

In mammals, it seems that the brain relies on KB metabolism at two particular times of life: during the postnatal development period; and during ageing, when glucose metabolism becomes impaired. The model proposed in this study of the metabolic coupling between glia and neurons during memory formation based on KB metabolism can provide a framework for further investigations into what occurs during ageing when glucose metabolism is impaired, and how a ketogenic diet might be beneficial in the treatment of neurodegenerative diseases (Silva, 2022).

Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. This study shows that the time-of-day (TOD) of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation. The results demonstrate notable correlations between the TOD behavioral effects and the circadian profile of dCREB2 proteins. At ZT = 20, there is a significant depression in memory formation, an event which coincides with apparent increases in blocker-related species clearly visible on the Western blots. At ZT = 16, a significant increase was measured in performance. This time point correlates with the end of a window (ZT = 13-15) when nuclear levels of the activator are elevated. Based on these relationships, it is hypothesized that the dynamics of dCREB2 protein levels contribute to the TOD effects on memory formation (Fropf, 2014).

Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. This study reports that 1) spaced training induces crebB expression in MB α-lobe neurons and 2) elevating specific CREBB isoform levels in the early α/β subpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training 3) induces 5-HT1A serotonin receptor synthesis, 4) activates 5-HT1A in early α/β neurons, and 5) inhibits LTM formation. 6) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. These findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/β MB neurons to modulate LTM consolidation (Lin, 2022).

Recurrent spaced learning has been shown to relieve inhibition and gate LTM formation in animal models. However, gene regulatory mechanisms that act to filter relevant signals of repeated events and override inhibitory constraints in identified circuit elements remain unknown. The current data suggest that MB neurons in Drosophila provide a compelling cellular gating mechanism for LTM formation. Weak learning is sufficient to increase 5-HT1A synthesis in early α/β neurons, and these neurons produce a downstream inhibitory effect on LTM formation. After spaced training, CREBB expression represses further 5-HT1A synthesis, thereby relieving the inhibitory effect on LTM formation. These conclusions are supported by several lines of evidence: i) CREBB transcription increased after 5xS or 10xS but not after 1x (Fig. 1); and ii) RNAi-mediated knockdown of CREBB in α/β impaired LTM (Fig. 1), while overexpression of a crebB-a or crebB-c transgene enhanced LTM. iii) Conversely, RNAi-mediated knockdown of 5-HT1A in early α/β neurons enhanced LTM, while overexpression of a 5-HT1A transgene impaired LTM; and iv) 1x was sufficient to activate 5-HT1A, and this activation was inhibited by expression of CREBB proteins. v) Furthermore, overexpression of 5-HT1A-mediated LTM impairment was fully rescued by CREBB overexpression. Together, these findings suggest that synthesis of 5-HT1A and CREBB proteins in response to training operate like an opposing molecular switch to inhibit or disinhibit downstream LTM formation, respectively (Lin, 2022).

Previous reports suggested that expression of a chimeric CREBB-a transcriptional activator and a CREBB-b transcriptional repressor throughout whole fly enhanced and impaired LTM formation, respectively. Subsequently, CREBB-a-dependent enhancement of LTM was not observed using a hs-Gal4 driver that has low expression in MB. Chronic expression of a CREBB-b in all α/β neurons was shown to impair 1-d memory after spaced training. It has been documented, however, that these chronic disruptions of CREBB-b produced developmental abnormalities in MB structure. In contrast, acute induced expression of CREBB-b only in adult α/β neurons did not impair 1-d memory after spaced training (and did not produce structural defects). Using a different inducible system (MB247-Switch) to acutely expresses CREBB-b in γ and α/β neurons showed a mild impairment of 1-d memory after spaced training. More interestingly, various molecular genetic tools were used to show that interactions among CREBB, CREB-binding protein, and CREB-regulated transcription coactivator in MB were clearly involved in LTM formation or maintenance, respectively. Using the same inducible gene switch tool, a positive regulatory loop has been shown between Fos and CREBB in MB during LTM formation - but that study did not show behavioral data pertaining to manipulation of CREBB per se - nor did that study restrict experiments to early α/β neurons (Lin, 2022).

Zhang (2015) expressed a CRE-luciferase transgene in different subpopulations of MB neurons and then monitored luciferase activity in live flies at various times after spaced training. Immediately after spaced training, some patterns of luciferase expression decreased (OK107 expressing in all MB neurons; c739 expressing in all α/β neurons; 1471 expressing in γ neurons), or increased (c747 and c772 expressing variably in all MB neurons), or showed no detectable change (c320 expressing variably in γ, α'/β' and α/β subpopulation, 17d expressing primarily in late α/β and in early α/β neurons). Indeed, the Zhang paper pointed out that, because the CRE-reporter was expressed in more than one subpopulation of MB neurons, only net effects of CREB function could be quantified. Furthermore, this study did not elucidate which CREBB isoforms might increase or decrease after spaced training. Obviously, this information would be critical if different isoforms have opposing activator and repressor functions in specific MB neuron subpopulations. The current study provides a dramatic example of this point. By restricting manipulation to early α/β neurons in adult stage animals, this study showed that enhanced LTM formation after acute CREBB-c overexpression is comparable to the net effect of chimeric CREBB-a overexpression in whole flies, and that spaced training serves to increase the expression of CREBB in these early α/β neurons (Lin, 2022).

Yin (1995) reported that the CREBB-a isoform functions as a PKA-responsive transcriptional activator and the CREBB-b isoform functions as a repressor of CREBB-a-induced gene activation. Using new KAEDA synthesis as a reporter for temporal gene activation, it has been previously shown that CREBB-b in DAL neurons represses CREBA-mediated gene activation to inhibit LTM formation. In early α/β MB neurons, KAEDA experiments indicate that CREBB-a and CREBB-c, but not CREBB-b, both repress 5-HT1A-mediated inhibition to gate LTM formation. These findings demonstrate a neuron- and training-specific CREBA activation and CREBB repression of effecter genes involved in modulating LTM formation. Although crebB promoter-driven Gal4 expression, crebBRNAi downregulation, and cell-type specific transcriptomes show CREBB expression in early α/β neurons, it remains unclear whether specific naturally occurring CREBB isoforms in these neurons serve to modulate LTM formation (Lin, 2022).

How is the learning-induced LTM gating mechanism differentially regulated by different [1x, 10xM (ten massed cycles of training without rest intervals) or 10xS (spaced trials)] training protocols? Expression of both 5-HT1A and crebB in early α/β MB neurons was elevated 24 h after 10xS, whereas only 5-HT1A was induced after 1x, and neither gene was induced after 10xM. Why is elevated 5-HT1A seen after 10xS, when constitutive expression of CREBB proteins suppresses 5-HT1A expression? A possible explanation is that 5-HT1A may be normally activated as an early response to 1x, whereas crebB induction by 10xS is not evident for about 3 h. Gradual cessation of 5-HT1A transcription by the delayed 10xS-induced CREBB expression may account for lower KAEDE levels observed in one odor/shock pairing experiment. Interestingly, the data showed that even with elevated 5-HT1A, CREBB proteins can still enhance 1-d memory, suggesting that CREBB-mediated inhibition is rather complex (Lin, 2022).

Massed training appears not to activate or suppress learning-induced transcriptional activity in early α/β neurons, and 5-HT1A nor crebB is activated after 10xM. Nevertheless, massed training may antagonize LTM formation. For instance, in MB neurons, spaced training induces repetitive waves of Ras/mitogen-activated protein kinase (MAPK) activity, activates MAPK translocation to the nucleus mediated by importin-7 (29), increases CREBB expression and, in dorsal-anterior-lateral (DAL) neurons, training induces activity-dependent crebA, CamKII, and per gene expression - all of which are not activated after massed training. These notions above suggest that massed training produces a more upstream general suppression of these 1x- and 10xS-induced genes required for inhibitory/gating mechanisms allocated in MB and DAL neurons, respectively (Lin, 2022).

An LTM enhancing role associated with CREBB expression and protein synthesis inhibition is a novel aspect of this gating mechanism. A previous study showed that inhibition of protein synthesis in MB after strong spaced training did not reduce LTM. Since it would not be possible to detect enhanced performance in these experiments, the possibility cannot be excluded that this inhibition might eliminate downregulation of LTM effector genes, with a net effect of promoting the formation of LTM rather than impairing it. This study estalished that synthesis of new 5-HT1A proteins in early α/β neurons after weak learning provides negative regulation and produces a downstream inhibitory effect on LTM formation. Surprisingly, CREBB protein synthesis in early α/β neurons after strong spaced training provides positive regulation by antagonizing this negative effect of 5-HT1A on LTM . Thus, CREBB-mediated repression is equivalent to the net effect of blocking protein synthesis in MB. Both relieve downstream inhibition and enhance rather than impair LTM formation. It is proposed that CREBB-mediated inhibition operates both directly by repressing gene transcription and indirectly through activating their downstream translational suppression (Lin, 2022).

Together, these experiments uncover a biochemical LTM gating mechanism that requires delicate regulation of protein synthesis and repression after training within identified neurons. More broadly, these observations also highlight the need to confirm the regulatory functions of specific CREB isoforms in identified neuronal subtypes before making conclusions about their roles in LTM formation (Lin, 2022).

The discovery that molecules in early α/β neurons inhibit LTM formation is relevant to future studies. Another persistent anesthesia-resistant form of memory (ARM) is also mediated by α/β neurons and has been shown to inhibit LTM formation. 5-HT1A appears to be a key protein involved in both ARM and LTM. Furthermore, the interaction of serotonin released from dorsal paired medial neurons and 5-HT1A in α/β neurons is necessary for sleep. CREBB expression in MB is also under circadian regulation, which together suggests mechanistic links between ARM, LTM, sleep, and circadian timing in early α/β neurons (Lin, 2022).

The ability to integrate experiential information and recall it in the form of memory is observed in a wide range of taxa, and is a hallmark of highly derived nervous systems. Storage of past experiences is critical for adaptive behaviors that anticipate both adverse and positive environmental factors. The process of memory formation and consolidation involve many synchronized biological events including gene transcription, protein modification, and intracellular trafficking: However, many of these molecular mechanisms remain illusive. With Drosophila as a model system this study used a nonassociative memory paradigm and a systems level approach to uncover novel transcriptional patterns. RNA sequencing of Drosophila heads during and after memory formation identified a number of novel memory genes. Tracking the dynamic expression of these genes over time revealed complex gene networks involved in long term memory. In particular, this study focuses on two functional gene clusters of signal peptides and proteases. Bioinformatics network analysis and prediction in combination with high-throughput RNA sequencing identified previously unknown memory genes, which when genetically knocked down resulted in behaviorally validated memory defects (Bozler, 2017).

A key function of the brain is to filter essential information and store it in the form of stable, long-term memory (LTM). The Dunce (Dnc) phosphodiesterase, an important enzyme that degrades cAMP, acts as a molecular switch that controls LTM formation in Drosophila. During LTM formation, Dnc is inhibited in the SPN, a pair of newly characterized serotonergic projection neurons, which stimulates the cAMP/PKA pathway. As a consequence, the SPN activates downstream dopaminergic neurons, opening the gate for LTM formation in the olfactory memory center, the mushroom body. Strikingly, transient inhibition of Dnc in the SPN by RNAi was sufficient to induce LTM formation with a training protocol that normally generates only short-lived memory. Thus, Dnc activity in the SPN acts as a memory checkpoint to guarantee that only the most relevant learned experiences are consolidated into stable memory (Scheunemann, 2018).

The brain filters the most important experiences and encodes them into LTM. Deficient filtering underlies many psychological disorders and may lead to indiscriminate remembering or even to the perturbation of the memory process itself. Selective filtering is thought to be executed by the default inhibition of selective neuronal activity, although the precise mechanisms remain obscure. This study deciphered that the Dnc PDE controls neuronal activity and represents a limiting step for LTM within a single pair of Drosophila serotonergic neurons (Scheunemann, 2018).

Not only is LTM formation, the ability to evaluate an experience and retain the information over time, involved in forming an individual's identity over the course of a lifetime, but it is also crucial for the fitness and survival of any organism. However, which mechanisms does the brain utilize to evaluate the relevance of information that will be consolidated into a long-lasting memory? Molecular memory checkpoints, e.g., a default inhibition of neuronal activity that is released only in a relevant context, could effectuate this selected memory consolidation. PDE-4 activity and cAMP degradation have previously been proposed as promising candidates for such a checkpoint. Nevertheless, it was still necessary to determine in vivo that PDE-4-mediated restriction of cAMP and PKA activity are, indeed, released upon the early steps of LTM formation. Thus, several previous studies focused primarily on memory pathways downstream of associative processes. However, to address the issue of context evaluation and modulation of memory storage, it is crucial to identify memory checkpoints that are upstream of brain structures involved in the association between stimuli. This study found that Dnc represents such a memory checkpoint in a serotonergic circuit that controls memory consolidation via modulation of dopaminergic input, upstream of the olfactory memory center in Drosophila (Scheunemann, 2018).

At the circuit level, Dnc was found to play a major role as a modulator of network properties by controlling serotonergic release from the SPN, aside from its potential role in memory processes via regulation of cAMP in the MB (Scheunemann, 2012). An integrated mechanism of LTM control is proposed in which a salient (alerting) experience leads to inhibition of Dnc in the SPN. The resulting PKA activation leads to serotonin release by SPN terminals, which, in turn, triggers MP1 oscillations and allows LTM formation downstream in the MB (Scheunemann, 2018).

Notably, the SPN has wide arborizations within the GNG, a region that is relevant for the processing of nutrient stimuli and feeding behavior (Gordon, 2009). MP1 signaling has been demonstrated to convey energy-related signals that trigger downstream memory processes in the MB for appetitive memories but, strikingly, also for aversive memories. The SPN-MP1 axis, therefore, represents a potential link that connects metabolic state with memory processing (Scheunemann, 2018).

Is there an equivalent serotonin-dopamine axis involved in aversive LTM in the mammalian brain? While many studies in mammals support the critical role of dopamine signals in reward and positive motivation involving mainly the ventral tegmental area (VTA) and nucleus acumbens (NA), it is increasingly acknowledged that the VTA also transmits signals related to salient but non-rewarding experiences, such as aversive and alerting events. These dopaminergic pathways -- one promoting motivation value and the other encoding alert salience -- have been hypothesized to cooperate in order to support adaptive behavior (Bromberg-Martin, 2010). Serotonin and dopamine interactions play a key role in neuropsychiatric diseases with symptoms of cognitive decline; and, interestingly, the implication of serotonin in dopamine-dependent cognitive dysfunction has been suggested. Dopamine is released after artificial serotonin microinfusion in the VTA; additionally, a 5HT-2A receptor antagonist has been shown to play a role in changes of oscillatory dopamine release by VTA neurons rather than changing baseline dopamine activity. Likewise, this study demonstrated that knockdown of the 5HT-2A receptor in MP1 abolishes dopamine oscillation but not spontaneous activity. Serotonin is well known to act as a behavioral switch that controls alternative emotional and physiological states across all phyla. A serotonin-dopamine axis as described here in Drosophila could, therefore, represent a generic design principle that coordinates how metabolic states integrate into behavior control (Scheunemann, 2018).

Historically, the dnc¹ mutant has been shown to display a strong memory defect that can be detected immediately after a single training cycle; furthermore, this phenotype has been regularly observed. Strikingly, this study reveals that the dnc¹ mutation, as well as Dnc knockdown by RNAi in the SPN, leads to a facilitation of LTM formation. Initially, it was reported that dnc¹ performs poorly in the short term as well as at 24 hr after a single training cycle. Notably, at the time of the initial report, the conditions had not yet been established to generate protein-synthesis-dependent LTM in wild-type flies, which may explain why the authors did not observe any increased dnc¹ performance at 24 hr. However, the possibility cannot be excluded that other factors, such as genetic background effects, could account for these differences in memory scores at 24 hr for the dnc¹ mutant used in this study (Scheunemann, 2018).

According to the current findings, Dnc loss of function is not deleterious for memory formation in general. Instead, Dnc-deficient flies exhibit selective facilitation of consolidated LTM. In fact, contradictory results can be found within studies investigating the consequences of reduced PDE activity. In addition to memory deficits, studies on improved memory are found in other insects and, remarkably, in mammals. Thus, several studies have revealed an improvement of memory after PDE-4-specific inhibitor treatment; moreover, PDE inhibitors are known targets for anti-depressive drugs. Defective Dnc PDE activity may, therefore, link symptoms of psychological disorders with impaired cognitive functions. However, the mechanisms involved have remained obscure. Indeed, significant gain in understanding PDE action in memory formation has been hampered by both the complexity of the mammalian brain and the existence of about 100 different types and isoforms of PDEs (Scheunemann, 2018).

One open question is how learning and 3-hr memory are impaired in the dnc¹ mutant, while LTM is facilitated at 24 hr. Interestingly, prevyious studies have shown that Dnc loss of function is specifically linked to defects in ARM forms of Drosophila memory that are measurable immediately after training and at 3 hr (Scheunemann, 2012, Bouzaiane, 2015). In addition, this study demonstrated that Dnc inhibition in the SPN as well as artificial SPN stimulation impairs ARM. Based on previous findings, which established that ARM and LTM are exclusive memory phases, it was hypothesize that ARM and LTM can be oppositely tuned by the activity of Dnc in the SPN-MP1 axis. Nevertheless, this study did not identify how Dnc could be inhibited in wild-type flies upon intensive LTM training. Interestingly, biochemical data indicate that ERK2 MAP kinases are able to inhibit Dnc activity. Furthermore, ERK2 mitogen-activated protein (MAP) kinases have been demonstrated to play a crucial role in LTM, making them likely candidates for the inhibition of Dnc upon LTM formation (Scheunemann, 2018).

In conclusion, contrary to most studies that have addressed suppressor mechanisms primarily by pharmacological inhibition that can artificially elevate PKA, this study has demonstrated that inhibition of Dnc in the SPN is a physiological state that gates LTM after intensive training. In addition to the increasing attention given to PDE inhibitors in recent years, due to their memory facilitation role, there is ongoing research on the specific role of PDEs in symptoms of Alzheimer's disease. These findings therefore offer great potential for revealing the complex action of PDEs in the brain (Scheunemann, 2018).

Sparsening of odor representation through GABAergic inhibition in the mushroom body (MB) neurons is thought to be a possible mechanism for information storage in locusts (Perez-Orive, 2002). GABAergic local neurons are known to be involved in olfactory information processing in Drosophila (Wilson, 2005; Olsen, 2008) indicating that GABAergic transmission plays a crucial role in shaping odor response. The MB shows extensive GABAergic innervation in both locusts (Perez-Orive., 2002) and Drosophila (Yasuyama, 2002). This, along with the observation that cAMP pathway genes like dunce and rutabaga are preferentially expressed in the MB (Davis, 2011), indicates that cAMP mediated GABAergic plasticity may be important for learning in Drosophila. Consistent with this hypothesis, this study observed altered cAMP mediated GABAergic IPSCs in the cAMP mutants dnc1 and rut1. The effect of cAMP on suppression of GABAergic currents was less pronounced in the mutants. This suggests that the altered inhibition contributes to their observed learning defects. In fact, recent studies have shown that GABAA RDL receptors expressed in the MB and GABAergic neurons projecting to the MB are essential for olfactory learning (Liu, 2007; Liu, 2009). It is thus possible that altered cAMP mediated GABAergic plasticity at the MB neurons may account for some forms of the learning defects in Drosophila (Ganguly, 2013).

GABAergic IPSCs are known to act through picrotoxin-sensitive postsynaptic GABA receptors in both Drosophila embryonic and pupal neuronal cultures. This study observed that the suppression of GABAergic IPSCs by FSK is completely abolished in the presence of a membrane impermeable PKA inhibitor restricted to the postsynaptic neuron. This indicates that PKA may modulate GABAergic IPSCs by regulating GABA receptor sensitivity by phosphorylation, similar to what has been suggested in the mammalian hippocampus (Ganguly, 2013).

There are three known ionotrophic GABA receptor gene homologs in Drosophila - RDL, LCCH3 and GRD. Amongst them, the GABA RDL subunit is widely expressed in several regions of the Drosophila brain and its expression in the MB is inversely correlated to olfactory learning. Therefore, RDL-containing GABA receptors may play an important role in cAMP-dependent synaptic plasticity and thus be involved in learning and memory. The data suggests that the majority of synaptic GABA receptors contain the RDL subunit while a small fraction of synaptic GABA receptors lack RDL, providing evidence of heterogeneous synaptic GABA receptors in Drosophila for the first time. However, it is still not known what particular subunit of GABA receptors is involved in regulation of cAMP-dependent GABAergic plasticity. Based on the observation that RDL containing GABA receptors mediate the majority of GABAergic IPSCs in Drosophila primary neuronal cultures, the action of FSK on GABAergic IPSCs is probably through the GABA RDL subunit. While the detailed molecular mechanism remains to be explored, it is proposed that PKA-mediated phosphorylation of RDL subunits and subsequent GABA receptor internalization may occur in the postsynaptic region. In this scenario, the only functional synaptic GABA receptors will be those lacking the RDL subunit at the postsynaptic regions. This will account for a decrease in mIPSC frequency with response to FSK, while leaving mIPSC amplitude almost unchanged (Ganguly, 2013).

Although GABA receptor subunits would be the target of cAMP-PKA signaling the possibility that other molecules can be phosphorylated and then indirectly regulate GABA receptor subunits can still not be rule out. Future work using heterologous expression systems for GABA subunits will help to determine whether GABA receptors are directly phosphorylated (Ganguly, 2013).

Recordings from embryonic and pupal MB neurons of both dunce and rutabaga mutants show a defect in ionotropic GABA receptor response in the presence of FSK. Interestingly, this response to FSK is similar in both the mutants despite their contrasting levels of cellular cAMP. Recent imaging studies in the rutabaga mutant have shown that AC is required for co-incidence detection in the MB neurons. FSK application also fails to increase PKA to wild-type levels in the MB neurons of rutabaga. Thus in the current experiments, the changes in receptor response in rutabaga can be explained by a lack of increase in cAMP/PKA levels due to defects in FSK-mediated AC activation (Ganguly, 2013).

The dunce mutants with high levels of cAMP also show defects in short-term memory due to alterations in the spatiotemporal restriction of dunce phosphodiesterase to the Drosophila MB. Further, the dunce MB neurons show an increase in PKA levels on FSK application similar to the wild-type strains. These findings suggest that FSK-mediated inhibition of GABA receptor should be greater in dunce neurons. However, in the current results the dunce and rutabaga mutants, despite having opposing effects on cellular cAMP levels, showed very similar FSK mediated effects on GABAergic IPSCs. Several other studies have also shown that dunce and rutabaga have similar defects in growth cone motility, excitatory synaptic plasticity and more importantly, short-term memory. Even though the effect of FSK on GABAergic IPSCs in dunce and rutabaga mutants is similar, it is very likely that the molecular mechanisms underlying these responses differ in the two mutants. It has been shown that elevated cAMP signaling reduces phosphorylation in rat kidney cells through activation of protein phosphatase 2A. In addition, increased PKA activity in mouse hippocampus hyper-phosphorylates several downstream molecular targets including a tyrosine phosphatase (STEP), correlates with decreased phosphodiesterase protein (PDE4) levels and results in memory defects. Therefore, it is tempting to speculate that high levels of cAMP due to the dunce mutation leads to the activation of phosphatase(s) and thus reduces the effects of FSK as seen in the current study. Taken together, all these findings strongly suggest that the disruption of cellular cAMP homeostasis can alter inhibitory GABAergic synaptic plasticity and hence cause defects in olfactory learning, although the underlying mechanisms leading to this effect can be different (e.g. reduced PKA activity in rut1 versus increased phosphatase activity in dnc1) (Ganguly, 2013).

Strengthening in the efficacy of excitatory transmission underlies enhanced synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia. It is thus possible that the suppression of inhibitory transmission by a common second messenger like cAMP, which can enhance excitatory synaptic transmission, may lead to synaptic strengthening. Previous work has shown that the cAMP activator FSK increases excitability at the cholinergic synapses in Drosophila primary neuronal cultures. However the effect of FSK on other synapses like the GABAergic synapses has not been explored. This study shows that FSK elevates overall cellular excitability at GABAergic synapses as demonstrated by the increase in spontaneous AP frequency. Moreover, when PKA in the postsynaptic neuron is completely blocked by an inhibitor, an increase is seen in the frequency of GABAergic IPSCs. Together with previous studies on cholinergic synapses (Yuan, 2007), the current results indicate that FSK/cAMP act as common molecules regulating globally presynaptic excitability at both the cholinergic as well as GABAergic synapses. It is also noted that FSK inhibits the response of postsynaptic GABA receptors in a specific manner leading to a decrease in GABAergic synaptic strength. These studies demonstrate a novel dual regulatory role of cAMP by showing that it increases overall presynaptic function on one hand; and, acts specifically on postsynaptic GABA receptors to decrease GABAergic plasticity on the other. This action of cAMP could result in global increases in excitability and learning (Ganguly, 2013).

The key finding in this study is the identification of two parallel neural pathways that additively express 3-h aversive ARM through Drosophila MB αβ and α'β' neurons. After training, Radish in MB αβ neurons and octopamine signaling in α'β' neurons independently consolidate ARM, which is additively retrieved by αβ-MBON-β2β'2a and α'β'm-MBON-β'2mp circuits for memory expression. Five lines of evidence support this scenario. First, the output from αβ or α'β' neurons is required for ARM retrieval, and the effect of blocking αβ output and that of blocking α'β' output during retrieval are additive. Second, knockdown of radish in αβ neurons, but not in α'β' neurons, impaired ARM, while knockdown of octβ2R in α'β' neurons further impaired the residual ARM in rsh¹ mutant flies. Third, blocking output from α'β' neurons, but not from αβ neurons, during retrieval further impaired the residual ARM in rsh¹ mutant flies. Forth, glutamatergic output from neurons downstream of the αβ or α'β' neurons, i.e., MBON-β2β'2a or MBON-β'2mp neurons, is required for ARM retrieval, and the effects of knockdown of VGlut are additive. Finally, output from α'β'm neurons, but not α'β'ap neurons, is required for ARM retrieval, consistent with the dendritic distribution of MBON-β'2mp neurons (Yang, 2016).

The parallel pathways for 3-h ARM expression were spatially defined by the requirements of neurotransmission from two sets of circuits during retrieval, the αβ-MBON-β2β'2a neurons and the α'β'm-MBON-β'2mp neurons. In addition, blocking neurotransmission from αβ or α'β' neurons during retrieval reduced ARM expression by about 50% whereas simultaneous blockade produced an additive effect that completely abolished ARM expression. Similar additive effects were repeatedly observed in experiments that utilize manipulations in both pathways: an rsh¹ mutant background plus octβ2R RNAi knockdown or plus retrieval blockade in α'β' neurons and knockdown of VGlut in MBON-β2β'2a plus MBON-β'2mp neurons. Thus, total four lines of evidence support the additive expression of 3-h ARM (Yang, 2016).

The parallel pathways for 3-h ARM expression shown in this study differ from the degenerate parallel pathways for the stomatogastric ganglion of the crab or CO₂ avoidance in the fly, as the latter enable mechanisms by which the network output can be switched between states. In the current study, the two parallel neural pathways additively contribute to the expression of 3-h ARM. The nature of the ARM parallel pathways may be similar to that for cold avoidance behavior in the fly, where parallel pathways in the β' and β circuits additively contribute but only the β circuit allows age-dependent alterations for potential benefits against aging (Shih, 2015). Considering the robustness of ARM through the course of senescence, it's unlikely to be age-dependent alterations in ARM system (Yang, 2016).

In studies of Drosophila neurobiology, C305a-GAL4 is a common GAL4 line for α'β' neurons. In this study, by examining three different zoom-in sections of the MB lobes and counting the cells, the following GAL4 lines expressing in α'β' neurons were extensively characterized: VT30604-GAL4 and VT57244-GAL4, which cover most α'β'ap and α'β'm neurons; VT37861-GAL4 and VT50658-GAL4, which cover α'β'ap neurons; and R42D07-GAL4 and R26E01-GAL4, which cover most α'β'm neurons. In contrast, C305a-GAL4 sporadically expresses in about half as many MB neurons as VT30604-GAL4 or VT57244-GAL4 does. Although covering both subsets of α'β' neurons, the expression pattern of C305a-GAL4 in α'β'm neurons is too few and/or weak to lead to a perturbation of synaptic transmission. This is shown by the data that retrieval of 3-h ARM was disrupted by shibire manipulation using all-α'β' neurons driver or α'β'm-specific driver, but neither α'β'ap-specific driver nor C305a-GAL4 for 3-h memory. Note that the GFP signals were acquired from flies carrying two copies of 5XUAS-mCD8::GFP reporter and without any immunostaining-mediated amplification. With the assistance of immunostaining and/or advanced reporter such as increasing copy number of UAS or incorporating a small intron to boost expression, some studies have shown appreciable GFP signal in most α'β' neurons. Given that shibire-mediated neurotransmission blockade and RNAi-mediated knockdown require high enough expression level, the imaging method adopted in this study can faithfully reflect the regions that were effectively manipulated in these behavioral assays. Regarding the pervasive use of C305a-GAL4 for shibire or RNAi manipulation, some functional studies of α'β' neurons might need to be carefully revisited. This study showed, by close examination and cell counting, that VT30604-GAL4, VT37861-GAL4, and R42D07-GAL4 are useful GAL4 lines to study α'β', α'β'ap, and α'β'm neurons, respectively, especially when split-GAL4 lines that span the second and third chromosomes are not genetically feasible (Shih, 2015).

ARM was thought to be diminished in radish mutant flies, in which a truncated RADISH is expressed. It's noteworthy that radish mutants still show a residual 3-h ARM with a PI of roughly 10, which is equal to the 3-h ARM score in wild-type flies fed with an inhibitor of serotonin synthesis to hinder the serotonergic DPM neurotransmission. Interestingly, feeding radish mutant flies with the drug didn't make the 3-h memory score worse, which has already implied that RADISH mediates the consolidation of ARM in the serotonergic DPM-αβ neurons circuit. Indeed, in this study advantage was taken of RNAi-mediated knockdown to identify αβ neurons with RADISH-mediated ARM consolidation. However, only the output from αβs neurons among three subsets of αβ neurons is required for aversive memory retrieval. Whether the αβs neurons are the only aversive ARM substrate of RADISH remains to be identified (Yang, 2016).

APL and DPM neurons are two pairs of modulatory neurons broadly innervating the ipsilateral MB, although the DPM neuron's fiber is lacking in the posterior part of pedunculus and the calyx. Broad, extensive fiber and non-spiking feature allow these two pairs of neurons to have multiple functional roles through different types of neurotransmission. The APL neuron has been shown to receive odor information from the MB neurons and provide GABAergic feedback inhibition as the Drosophila equivalent of a group of the honeybee GABAergic feedback neurons. This feedback inhibition has been proposed to maintain sparse, decorrelated odor coding by suppressing the neuronal activity of MB neurons, which can be somewhat linked to the mutual suppression relation with conditioned odor and the facilitation of reversal learning. Interestingly, Pitman (2011) proposed that the feedback inhibition from APL neurons sustains the labile appetitive ASM based on shibire manipulation. Since shibire manipulation can impact small vesicle release, and APL neurons have been demonstrated to co-release at least GABA and octopamine, it might worth conducting GABA-specific manipulation in APL neurons to confirm the role in appetitive ASM. For aversive olfactory memory, acute RNAi-mediated knockdown of Glutamic acid decarboxylase in APL neurons had no effect on 3-h memory. Instead, the octopamine synthesis enzyme mutant, Tβh^nM18, knockdown of Tβh in APL neurons, the octopamine receptor mutant, PBac{WH}octβ2R^f05679, and knockdown of octβ2R in α'β' neurons all phenocopied the 3-h ARM impairment caused by shibire-mediated neurotransmission blockade in APL neurons. Together with the serotonergic DPM-αβ neurons circuit , a model that is favored that two sets of triple-layered parallel circuits, octopaminergic APL-α'β'-MBON-β'2mp and serotonergic DPM-αβ-MBON-β2β'2a, additively contribute to 3-h aversive ARM (Yang, 2016).

Although the data showed that 3-h ARM consolidation requires recurrent output from α'β'ap neurons but not from α'β'm neurons, RNAi-mediated knockdown of octβ2R in α'β'ap or α'β'm neurons impaired ARM, suggesting that Octβ2R functions for normal ARM expression in the entire population of α'β' neurons. On the other hand, neuronal activity during memory consolidation is naturally more quiescent than that during memory retrieval, and the shibire-mediated neurotransmission blockade requires an exhaustion of already-docked vesicles. Together with the unfavorable performance for experiments blocking the output from α'β'm neurons during consolidation, the possibility cannot be excluded that output from α'β'm neurons is also required for ARM during consolidation. Alternatively, octopamine signaling may also be involved in ARM retrieval (Yang, 2016).

Accumulating evidence suggests that transcriptional regulation is required for maintenance of long-term memories (LTMs). This study characterized global transcriptional and epigenetic changes that occur during LTM storage in the Drosophila mushroom bodies (MBs), structures important for memory. Although LTM formation requires the CREB transcription factor and its coactivator, CBP, subsequent early maintenance requires CREB and a different coactivator, CRTC. Late maintenance becomes CREB independent and instead requires the transcription factor Beadex, also know as LIM-only. Bx expression initially depends on CREB/CRTC activity, but later becomes CREB/CRTC independent. The timing of the CREB/CRTC early maintenance phase correlates with the time window for LTM extinction and this study identified different subsets of CREB/CRTC target genes that are required for memory maintenance and extinction. Furthermore, it was found that prolonging CREB/CRTC-dependent transcription extends the time window for LTM extinction. These results demonstrate the dynamic nature of stored memory and its regulation by shifting transcription systems in the MBs (Hirano, 2016).

This study has identified Bx and Smr as LTM maintenance genes and has characterize a shift in transcription between CREB/CRTC-dependent maintenance (1-4 days) to Bx-dependent maintenance (4-7 days). In addition, a biological consequence of this shift was identified in defining a time window during which LTM can be modified, β-Spec was identified as being required for memory extinction (Hirano, 2016).

LTM maintenance mechanisms change dynamically during storage. In particular, CRTC, which is not required during memory formation, becomes necessary during 4-day LTM maintenance and then becomes dispensable again. Consistent with this, CRTC translocates from the cytoplasm to the nucleus of MB neurons during 4-day LTM maintenance and returns to the cytoplasm within 7 days. On the other hand, Bx expression is increased at both phases, suggesting that transcriptional regulation of memory maintenance genes may change between these two phases. Supporting this idea, it was found that Bx expression requires CRTC during 4-day LTM maintenance but becomes independent of CRTC 7 days after training. It is proposed that CREB/CRTC activity induces Bx expression, which subsequently activates a feedback loop where Bx maintains its own expression and that of other memory maintenance genes (Hirano, 2016).

Although it is proposed that the shifts in transcriptional regulation that were observed occur temporally in the same cells, the possibility cannot be discounted that LTM lasting 7 days is maintained in different cells from LTM lasting 4 days. MB Kenyon cells can be separated into different cell types, which exert differential effects on learning, short-term memory and LTM. Thus, it is possible that LTM itself consists of different types of memory that can be separated anatomically. In this case, CRTC in one cell type may exert non-direct effects on another cell type to activate downstream genes including Bx and Smr. However, as that CRTC binds to the Bx gene locus to promote Bx expression and both CRTC and Bx are required in the same α/β subtype of Kenyon cells, it is likely that the shift from CRTC-dependent to Bx-dependent transcription occurs within the α/β neurons (Hirano, 2016).

Currently, it is proposed that the alterations in histone acetylation and transcription that were uncovered are required for memory maintenance. However, it is noted that decreases in memory after formation could be caused by defects in retrieval and maintenance. Thus, it remains formally possible that the epigenetic and transcriptional changes reported in this study are required for recall, but not maintenance. However, this is unlikely, as inhibition of CRTC from 4 to 7 days after memory formation does not affect 7 day memory, whereas inhibition from 1 to 4 days does. This suggests that at least one function of CRTC is to maintain memory for later recall (Hirano, 2016).

Consistent with a previous study in mice, which suggests distinct transcriptional regulations in LTM formation and maintenance (Halder, 2016), the data indicate that memory formation and maintenance are distinct processes. Although the HAT, CBP, is required for formation but dispensable for maintenance, other HATs, GCN5 and Tip60, are required for maintenance but dispensable for formation. Through ChIP-seq analyses, those downstream genes, Smr and Bx, were identified as LTM maintenance genes and these are not required for LTM formation. Collectively, these results suggest differential requirements of histone modifications between LTM formation and maintenance. Although other histone modifiers besides GCN5 and Tip60 were identified in the screen, knockdown of these histone modifiers did not affect LTM maintenance. There are ~50 histone modifiers encoded in the fly genome, raising the possibility that the lack of phenotype in some knockdown lines is due to compensation by other modifiers (Hirano, 2016).

The results indicate some correlation of increase in CRTC binding with histone acetylation and gene expression. Interestingly, DNA methylation shows higher correlation to gene expression in comparison with histone acetylation in mice. Notably, flies lack several key DNA methylases and lack detectable DNA methylation patterns. Hence, histone acetylation rather than DNA methylation may have a higher correlation with transcription in flies. Reduction in histone acetylation was detected, overlapping with increase in CRTC binding. Those reductions could be due to CRTC interacting with a repressor isoform of CREB, CREB2b or other transcriptional repressor that binds near CREB/CRTC sites. These interactions would decrease histone acetylation and gene expression, and may be related to LTM maintenance. Although this study focused on the upregulation of gene expression through CREB/CRTC, downregulation of gene expression by transcriptional repressors may also be important in understanding the transcriptional regulation in LTM maintenance. The results demonstrate the importance of HATs for LTM maintenance; however, the data do not conclude that histone acetylation is a determinant for gene expression, but rather it might be a passive mark of gene expression. HATs also target non-histone proteins and also interact with various proteins, both of which could support gene expression in LTM maintenance (Hirano, 2016).

Similar to traumatic fear memory in rodents, this study found that aversive LTM in flies can be extinguished by exposing them to an extinction protocol specifically during 4-day LTM maintenance. These observations suggest the time-limited activation of molecules that allows LTM extinction only during the early storage. Supporting this concept, it was found that CRTC is activated during the extinguishable phase of LTM maintenance and prolonging CRTC activity extends the time window for extinction. Thus, CRTC is the time-limited activated factor determining the time window for LTM extinction in flies. In cultured rodent hippocampal neurons, CRTC nuclear translocation is not sustained, suggesting that other transcription factors may function in mammals to restrict LTM extinction (Hirano, 2016).

This work demonstrates that LTM formation and maintenance are distinct, and involve a shifting array of transcription factors, coactivators and HATs. A key factor in this shift is CRTC, which shows a sustained but time-limited translocation to the nucleus after spaced training. Thus, MB neurons recruit different transcriptional programmes that enable LTM to be formed, maintained and extinguished (Hirano, 2016).

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. This study deconstructed the larval mnemonic organization after aversive olfactory conditioning. After odor-high salt conditioning (establishing an aversive olfactory memory) larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, this study shows for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C (PKC) activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution (Widmann, 2016).

Memory formation and consolidation usually describes a chronological order, parallel existence or completion of distinct short-, intermediate- and/or long-lasting memory phases. For example, in honeybees, in Aplysia, and also in mammals two longer-lasting memory phases can be distinguished based on their dependence on de novo protein synthesis. In adult Drosophila classical odor-electric shock conditioning establishes two co-existing and interacting forms of memory--ARM and LTM--that are encoded by separate molecular pathways (Widmann, 2016).

Seen in this light, memory formation in Drosophila larvae established via classical odor-high salt conditioning seems to follow a similar logic. It consist of LSTM (larval short lasting component) and LARM (anesthesia resistant memory). Aversive olfactory LSTM was already described in two larval studies using different negative reinforcers (electric shock and quinine) and different training protocols (differential and absolute conditioning). The current results introduce for the first time LARM that was also evident directly after conditioning but lasts longer than LSTM. LARM was established following different training protocols that varied in the number of applied training cycles and the type of negative or appetitive reinforcer. Thus, LSTM and LARM likely constitute general aspects of memory formation in Drosophila larvae that are separated on the molecular level (Widmann, 2016).

Memory formation depends on the action of distinct molecular pathways that strengthen or weaken synaptic contacts of defined sets of neurons. The cAMP/PKA pathway is conserved throughout the animal kingdom and plays a key role in regulating synaptic plasticity. Amongst other examples it was shown to be crucial for sensitization and synaptic facilitation in Aplysia, associative olfactory learning in adult Drosophila and honeybees, long-term associative memory and long-term potentiation in mammals (Widmann, 2016).

For Drosophila larvae two studies by Honjo (2005) and Khurana (2009) suggest that aversive LSTM depends on intact cAMP signaling. In detail, they showed an impaired memory for rut and dnc mutants following absolute odor-bitter quinine conditioning and following differential odor-electric shock conditioning. Thus, both studies support the interpretation of the current results. It is argued that odor-high salt training established a cAMP dependent LSTM due to the observed phenotypes of rut, dnc and syn mutant larvae. The current molecular model is summarized in A molecular working hypothesis for LARM formation. Yet, it has to be mentioned that all studies on aversive LSTM in Drosophila larvae did not clearly distinguish between the acquisition, consolidation and retrieval of memory. Thus, future work has to relate the observed genetic functions to these specific processes (Widmann, 2016).

In contrast, LARM formation utilizes a different molecular pathway. Based on different experiments, it was ascertained, that LARM formation, consolidation and retrieval is independent of cAMP signaling itself, PKA function, upstream and downstream targets of PKA, and de-novo protein synthesis. Instead it was found that LARM formation, consolidation and/or retrieval depends on radish (rsh) gene function, brp gene function, dopaminergic signaling and requires presynaptic signaling of MB KCs (Widmann, 2016).

Interestingly, studies on adult Drosophila show that rsh and brp gene function, as well as dopaminergic signaling and presynaptic MB KC output are also necessary for adult ARM formation. Thus, although a direct comparison of larval and adult ARM is somehow limited due to several variables (differences in CS, US, training protocols, test intervals, developmental stages, and coexisting memories), both forms share some genetic aspects. This is remarkable as adult ARM and LARM use different neuronal substrates. The larval MB is completely reconstructed during metamorphosis and the initial formation of adult ARM requires a set of MB α/β KCs that is born after larval life during puparium formation (Widmann, 2016).

In addition, this study has demonstrated the necessity of PKC signaling for LARM formation in MB KCs. The involvement of the PKC pathway for memory formation is also conserved throughout the animal kingdom. For example, it has been shown that PKC signaling is an integral component in memory formation in Aplysia, long-term potentiation and contextual fear conditioning in mammals and associative learning in honeybees. In Drosophila it was shown that PKC induced phosphorylation cascade is involved in LTM as well as in ARM formation. Although the exact signaling cascade involved in ARM formation in Drosophila still remains unclear, this study has established a working hypothesis for the underlying genetic pathway forming LARM based on the current findings and on prior studies in different model organisms. Thereby this study does not take into account findings in adult Drosophila. These studies showed that PKA mutants have increased ARM and that dnc sensitive cAMP signaling supports ARM. Thus both studies directly link PKA signaling with ARM formation. (Widmann, 2016).

KCs have been shown to act on MB output neurons to trigger a conditioned response after training. Work from different insects suggests that the presynaptic output of an odor activated KCs is strengthened if it receives at the same time a dopaminergic, punishment representing signal. The current results support these models as they show that LARM formation requires accurate dopaminergic signaling and presynaptic output of MB KCs. Yet, for LARM formation dopamine receptor function seems to be linked with PKC pathway activation. Indeed, in honeybees, adult Drosophila and vertebrates it was shown that dopamine receptors can be coupled to Gαq proteins and activate the PKC pathway via PLC and IP3/DAG signaling. As potential downstream targets of PKC radish and bruchpilot are suggested. Interference with the function of both genes impairs LARM. The radish gene encodes a functionally unknown protein that has many potential phosphorylation sites for PKA and PKC. Thus considerable intersection between the proteins Rsh and PKC signaling pathway can be forecasted. Whether this is also the case for the bruchpilot gene that encodes for a member of the active zone complex remains unknown. The detailed analysis of the molecular interactions has to be a focus of future approaches. Therefore, the current working hypothesis can be used to define educated guesses. For instance, it is not clear how the coincidence of the odor stimulus and the punishing stimulus are encoded molecularly. The same is true for ARM formation in adult Drosophila. Based on the working hypothesis it can be speculated that PKC may directly serve as a coincidence detector via a US dependent DAG signal and CS dependent Ca²⁺ activation (Widmann, 2016).

Do the current findings in general apply to learning and memory in Drosophila larvae? To this the most comprehensive set of data can be found on sugar reward learning. Drosophila larva are able to form positive associations between an odor and a number of sugars that differ in their nutritional value. Using high concentrations of fructose as a reinforcer in a three cycle differential training paradigm (comparable to the one used in this study for high salt learning and fructose learning) other studies found that learning and/or memory in syn⁹⁷ mutant larvae is reduced to ~50% of wild type levels. Thus, half of the memory seen directly after conditioning seems to depend on the cAMP-PKA-synapsin pathway. The current results in turn suggest that the residual memory seen in syn⁹⁷ mutant larvae is likely LARM. Thus, aversive and appetitive olfactory learning and memory share general molecular aspects. Yet, the precise ratio of the cAMP-dependent and independent components rely on the specificities of the used odor-reinforcer pairings. Two additional findings support this conclusion. First, a recent study has shown that memory scores in syn⁹⁷ mutant larvae are only lower than in wild type animals when more salient, higher concentrations of odor or fructose reward are used. Usage of low odor or sugar concentrations does not give rise to a cAMP-PKA-synapsin dependent learning and memory phenotype. Second, another study showed that learning and/or memory following absolute one cycle conditioning using sucrose sugar reward is completely impaired in rut¹, rut²⁰⁸⁰ and dnc¹ mutants. Thus, for this particular odor-reinforcer pairing only the cAMP pathway seems to be important. Therefore, a basic understanding of the molecular pathways involved in larval memory formation is emerging. Further studies, however, will be necessary in order to understand how Drosophila larvae make use of the different molecular pathways with respect to a specific CS/US pairing (Widmann, 2016).

Effective and stimulus-specific learning is essential for animals' survival. Two major mechanisms are known to aid stimulus specificity of associative learning. One is accurate stimulus-specific representations in neurons. The second is a limited effective temporal window for the reinforcing signals to induce neuromodulation after sensory stimuli. However, these mechanisms are often imperfect in preventing unspecific associations; different sensory stimuli can be represented by overlapping populations of neurons, and more importantly, the reinforcing signals alone can induce neuromodulation even without coincident sensory-evoked neuronal activity. This paper reports a crucial neuromodulatory mechanism that counteracts both limitations and is thereby essential for stimulus specificity of learning. In Drosophila, olfactory signals are sparsely represented by cholinergic Kenyon cells (KCs), which receive dopaminergic reinforcing input. KCs were found to have numerous axo-axonic connections mediated by the muscarinic type-B receptor (mAChR-B). By using functional imaging and optogenetic approaches, it was shown that these axo-axonic connections suppress both odor-evoked calcium responses and dopamine-evoked cAMP signals in neighboring KCs. Strikingly, behavior experiments demonstrate that mAChR-B knockdown in KCs impairs olfactory learning by inducing undesired changes to the valence of an odor that was not associated with the reinforcer. Thus, this local neuromodulation acts in concert with sparse sensory representations and global dopaminergic modulation to achieve effective and accurate memory formation (Manoim, 2022).

This study showed that KC-KC axonal interaction is mediated by mAChR-B. This mAChR-B-mediated neuromodulation has dual roles: it decreases both odor-evoked Ca2+ elevation and DA-induced cAMP elevation. Thus, this neuromodulation suppresses both signals that are required for KC-MBON synaptic plasticity. In behavior experiments, it was demonstrated that mAChR-B knock-down (KD) in KCs impairs stimulus specificity of learning. This study reveals a novel form of local neuromodulation, which improves sensory discrimination during learning (Manoim, 2022).

This study identified the first biological functions of axo-axonic synapses between KCs. Olfactory coding in the insect MB is a well-established model system to study the circuit mechanisms and benefits of sparse sensory representations. The abundance of KC-KC synapses at the axons discovered by the EM connectome surprised the field at first because excitatory cholinergic interactions may ruin the very benefit of the sparse coding in olfactory learning. However, Ca2+ imaging demonstrated that the net effect of those cholinergic transmissions is, in fact, inhibitory. The lateral inhibition mediated by mAChR-B should further enhance, rather than ruin, the benefit of sparse coding and thereby improve the stimulus specificity of learning. Although the population of KCs that show reliable responses to a given odor is sparse (~5%), many more KCs are activated in a given odor presentation. This is because there is a larger population of unreliable responders, making up to ~15% of total KCs active in a given trial (Manoim, 2022).

Since those unreliable responders tend to show weaker Ca2+ responses than the reliable ones, it is reasonable to speculate that mAChR-B-mediated mutual inhibition would preferentially suppress unreliable responders, letting reliable responders win the lateral competition. Since even a single, 1-s odor-DAN activation pairing can induce robust KC-MBON synaptic plasticity, presence of unreliable responders can significantly compromise the synapse specificity of plasticity. Restricting Ca2+ responses to reliable responders should therefore greatly enhance the stimulus specificity of learning (Manoim, 2022).

To support thos finding, selective inhibition of Go signaling in KCs by expressing pertussis toxin (PTX) impairs aversive learning, and this effect was mapped to αβ and γ KCs, which were found to express mAChR-B most abundantly. Furthermore, expression of PTX disinhibits odor-evoked vesicular release in γ KCs, and PTX-induced learning defect was ameliorated by hyperpolarization or blocking synaptic output of γ KCs (Manoim, 2022).

It is argued that mAChR-B-mediated inhibitory communication between γ KCs contributes at least in part to those previous observations. Lateral communication through mAChR-B also suppresses cAMP signals in KCs, which counteracts Dop1R1-mediated DA action during associative conditioning. Since DA release in the MB likely takes a form of volume transmission, it cannot provide target specificity of modulation. Furthermore, although induction of LTD depends on coincident activity of KCs and DANs, elevation of cAMP can be triggered by DA application alone, although DA input followed by KC activity could induce opposite plasticity (i.e., potentiation) via another type of DA receptor (Manoim, 2022).

Thus, lateral inhibition of cAMP signals by Gi/o-coupled mAChR-B plays an essential role in the maintenance of target specificity of modulation. Taken together, dual actions of mAChR-B on local Ca2+ and cAMP signals at KC axons, where plasticity is supposed to take place, should directly contribute to synapse specificity of plasticity. If animals lack mAChR-B in KCs, axons of unreliable responders to CS+ would stay mildly active during conditioning. Furthermore, DA release on KCs causes some unchecked increase in cAMP in inactive and mildly active KCs. Consequently, some plasticity occurs in these KCs, even if to a lesser extent than in the KCs that are reliably and strongly activated by the CS+. Thus, absence of mAChR-B would minimally affect plasticity of KCs that are reliably activated by the CS+, assuming that those KCs are nearly maximally depressed by learning-related plasticity in the presence of mAChR-B. However, other KCs, which may include reliable responders to the CS−, will also undergo plasticity. This should result in unspecific association and that is exactly the type of learning defect observed in mAChR-B KD flies (Manoim, 2022).

The above model suggests that mAChR-B is required during memory acquisition. However, previous studies suggested that blocking KC synaptic output during memory acquisition does not affect aversive memory. How can one reconcile these two seemingly contradictory results? The experimental approach (i.e., RNAi KD of mAChR-B) precluded the ability to control the receptor function with high temporal specificity, and therefore, it was not possible to directly test whether mAChR-B is required during memory acquisition. Nevertheless, it is plausible that KC output affects memory acquisition via mAChR-B. Previous literature relied on temperature-sensitive Shibirets1 (shits1), which blocks synaptic release at the restrictive temperature, to demonstrate that KC output is not required during memory acquisition. However, it has been shown that substantial release is still maintained with shits1 even at the restrictive temperature (Manoim, 2022).

GPCRs are known to be activated at extremely low concentrations, ranging in the nM. On the other hand, nicotinic receptors operate at higher concentrations, often in the range of μM. Thus, it is possible that in the presence of shits1, there is some residual release from KCs at the restrictive temperature that is sufficient to activate mAChR-B but not the nicotinic receptors on downstream neurons. Thus, these results shed light on the role of KC output during memory acquisition, which may have been overlooked in previous studies (Manoim, 2022).

What may be the cellular mechanisms underlying the effects of mAChR-B on cAMP and Ca2+ level? mAChR-B was shown to be coupled to Gi/o, which is known to inhibit the cAMP synthase, adenylate cyclase, which is widely expressed in KCs (Manoim, 2022).

In addition, the Gβγ subunits have been demonstrated to be able to directly block voltage-gated Ca2+ channels. Gβγ can also directly open inward rectifying potassium channels that would oppose the changes in membrane potential required for the gating of voltage-gated Ca2+ channels, although these potassium channels are not broadly expressed in KCs. In this regard, it would be interesting to note that behavioral and physiological effects of mAChR-B KD were observed only whend KD was performed in γ KCs, although the results indicate that those receptors are also expressed in αβ KCs. This could be due to potential diversity in the intracellular signaling molecules among KC subtypes. Another possibility is that the efficiency of RNAi KD is somehow different between those KCs. It is also possible that the relatively lower number of KC-KC connections between αβ KCs may be insufficient to activate mAChR-B in the experimental contexts. Nevertheless, it is noted that a number of studies have demonstrated that γ KCs have a dominant role at the stage of acquisition of short-term memory, which is consistent with the model that proposes the critical role of mAChR-B during memory acquisition (Manoim, 2022).

Although the majority of studies on population-level sensory coding has focused on somatic Ca2+ or extracellular electrophysiological recordings, this study sheds light on the importance of local regulation of Ca2+ and other intracellular signals at the axons when it comes to stimulus specificity of learning. Are there other mechanisms that may be involved in reducing unspecific conditioning? One potential source of such mechanisms is the APL neuron, a single GABAergic neuron in the MB that is excited by KCs and provides feedback inhibition to KCs (Manoim, 2022).

Since activity of APL neuron contributes to sparse and decorrelated olfactory representations in KCs, it is possible that GABAergic input to KC axons also serves to prevent unspecific learning. Release of GABA onto KC axons is expected to have similar effects as the activation of mAChR-B. Specifically, the activation of the Gi/o-coupled GABA-B receptors that are widely expressed in KCs should have similar effects as activation of mAChR-B. However, in the current experiments, lateral inhibition induced by optogenetic activation of a subset of KCs was completely suppressed by mAChR-B KD, suggesting that APL neuron did not contribute to lateral suppression of Ca2+ response at least in the current experimental condition. This result is consistent with the prediction that individual KCs inhibit themselves via APL neuron more strongly than they inhibit the others due to the localized nature of the activity of APL neuron's neurites and the geometric arrangement of the ultrastructurally identified synapses (Manoim, 2022).

Nonetheless, whether APL neuron contributes to sparsening of axonal activity to prevent unspecific conditioning remains to be examined. In summary, the current study identifies functional roles of axo-axonic cholinergic interactions by uncovering previously unknown local neuromodulation that can enhance the stimulus specificity of learning and refines the DA-centric view of MB plasticity (Manoim, 2022).

Animals, including humans, form oppositely valenced memories for stimuli that predict the occurrence versus the termination of a reward: appetitive 'reward' memory for stimuli associated with the occurrence of a reward and aversive 'frustration' memory for stimuli that are associated with its termination. This study characterized these memories in larval Drosophila melanogaster using a combination of Pavlovian conditioning, optogenetic activation of the dopaminergic central-brain DAN-i1864 neuron, and high-resolution video-tracking. This reveals their dependency on the number of training trials and the duration of DAN-i1864 activation, their temporal stability, and the parameters of locomotion that are modulated during memory expression. Together with previous results on 'punishment' versus 'relief' learning by DAN-f1 neuron activation, this reveals a 2×2 matrix of timing-dependent memory valence for the occurrence/termination of reward/punishment. These findings should aid the understanding and modelling of how brains decipher the predictive, causal structure of events around a target reinforcing occurrence (Thoener, 2022).

The most basic models of learning are reinforcement learning models (for instance, classical and operant conditioning) that posit a constant learning rate; however many animals change their learning rates with experience. This process is sometimes studied by reversing an existing association between cues and rewards, and measuring the rate of relearning. Augmented reversal-learning, where learning rates increase with practice, can be an important component of behavioral flexibility; and may provide insight into higher cognition. Previous studies of reversal-learning in Drosophila have not measured learning rates, but have tended to focus on measuring gross deficits in reversal-learning, as the ratio of two timepoints. These studies have uncovered a diversity of mechanisms underlying reversal-learning, but natural genetic variation in this trait has yet to be assessed. A reversal-learning regime was conducted on a diverse panel of Drosophila melanogaster genotypes. Highly significant genetic variation was found in their baseline ability to learn. It was also found that they have a consistent, and strong (1.3x), increase in their learning speed with reversal. No evidence was found, however, that there was genetic variation in their ability to increase their learning rates with experience. This may suggest that Drosophila have a hitherto unrecognized ability to integrate acquired information, and improve their decision making; but that their mechanisms for doing so are under strong constraints (Foley, 2017).

Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, this study examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca(2+) responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, this plasticity was found to be dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli (Louis, 2018).

Learning generates plasticity in neuronal responses to input stimuli, which is distributed across multiple cells and synapses in the brain. Molecularly, dopamine and downstream cAMP signaling are involved in multiple forms of memory, including olfactory learning. For instance, dopamine is required in the amygdala for olfactory classical conditioning in mammals. Similarly, dopamine and downstream cAMP signaling molecules play a central role in olfactory classical conditioning in Drosophila. This pathway is particularly critical in the mushroom body (MB), a brain region that receives olfactory information and is required for olfactory learning. Dopaminergic neurons are postulated to convey a reinforcement signal to the MB-stimulating certain subsets of MB-innervating dopaminergic neurons drives aversive or appetitive reinforcement in lieu of a physical reinforcer. The dopamine released from these neurons acts directly on intrinsic MB neurons, and possibly other neurons in the area as well. The D1-like receptor DopR, type I adenylyl cyclase Rutabaga (Rut), catalytic domain of protein kinase A, and Dunce phosphodiesterase (Dnc) are all required for olfactory classical conditioning. Importantly, rescuing the expression of DopR or Rut -- specifically in intrinsic MB neurons of otherwise mutant animals -- restores normal olfactory learning and memory. Further downstream, both Epac and PKA, as well as phosphorylation targets such as synapsin, have been shown to regulate learning and memory via effects in MB neurons. Thus, dopamine and cAMP are critical in intrinsic MB neurons for normal memory. Furthermore, broadly elevating cAMP generates plasticity in MB neurons, demonstrating that this pathway influences the responsivity of MB neurons. However, the role of this pathway in driving coherent patterns of plasticity that encode memory is unknown (Louis, 2018).

Recent advances have opened up the possibility of understanding how olfactory memory is encoded in exquisite detail. Recent studies of memory encoding in the Drosophila MB have suggested that mushroom body neurons are highly plastic, exhibiting learning-related changes in odor responses. This is supported by observations of memory traces using in vivo Ca2+ imaging of neurons innervating the MB. However, the neuronal changes associated with cAMP-dependent, short-term memory are unclear. Conditioning generates plasticity in α'/β'-neurons within a few minutes of training, a time point at which the animals exhibit robust short-term memory. However, the Rut cyclase is not required in α'/β'-neurons for learning, leaving the functional role of cAMP-dependent plasticity in the MB unclear. MB γ-neurons exhibit depression in response to an aversive conditioned odor that is sensitive to manipulations of G αo-signaling, though it is not clear how this relates to dopaminergic modulation via G αs. Finally, blocking the synaptic output of MB neurons during conditioning does not impair aversive learning, suggesting that a significant proportion of the engram resides in the MB neurons and/or upstream connections (Louis, 2018).

In contrast, other studies have described a major role for plasticity in downstream MB output neurons (MBONs), which may arise via pre- and/or postsynaptic plasticity. Robust, dopamine-dependent plasticity has been observed in MBONs, but not at the cellular level in MB neurons. This emphasizes the role of the MB in encoding sparse, relatively invariant olfactory representations. Learning-induced plasticity is then layered in at the MB-MBON synapses, possibly via synaptic depression. This leaves the requirement of cAMP signaling molecules in the MB, and the dispensability of MB output during memory acquisition, unresolved. Thus, there is a paradoxical dissociation of anatomical loci between where cAMP signaling is required and where robust, short-term, learning-induced plasticity has been reported. This study has examined the role of cAMP-dependent plasticity in the MB using in vivo imaging, combined with pharmacological and optogenetic manipulation of cAMP levels. Results suggest that cAMP-dependent plasticity localizes to intrinsic MB neurons and mirrors the plasticity induced during olfactory classical conditioning, with a bias toward appetitive conditioning (Louis, 2018).

The present data support several major conclusions about the role of cAMP-dependent plasticity in the memory-encoding MB: (1) Intrinsic MB neurons exhibit robust cAMP-dependent plasticity; (2) cAMP-dependent plasticity is heterogeneous, both across and within anatomical classes of MB neurons; (3) the directionality and magnitude of plasticity parallel Rut-dependent associative changes in MB responsivity following appetitive classical conditioning; and (4) appetitive conditioning produces changes in cAMP of a magnitude that generates plasticity in odor-evoked responses. Thus, cAMP-dependent plasticity plays a major role in modulating intrinsic MB neurons, directly linking the physiology of MB neurons to the behavioral roles for cAMP signaling molecules in learning and memory. One caveat to the interpretation of imaging studies is that the preparations require tethering the animal under a microscope. Future developments enabling recording of brain activity in freely behaving animals will be necessary to test how responses in the MB neurons facilitate behavioral output in real time (Louis, 2018).

In the context of olfactory learning, the MB encodes a sparse representation of olfactory space, which is computationally advantageous for learning and potentially modulated by learned valence. If neurons responded homogeneously to input stimuli, coincidence detection would result in uniform plasticity across the sparse set of neurons that encode the odor and receive a reinforcement signal. However, the heterogeneity observed in cAMP-dependent plasticity in this study suggests that olfactory memory traces may be driven to specific subsets of 'eligible' neurons in the MB. This could play an analogous role to memory allocation, which drives memory traces to subsets of eligible neurons in the mammalian amygdala during fear conditioning. Molecularly, heterogeneity may be driven by differential expression of genes that function downstream of cAMP/Epac/PKA to regulate neuronal excitability or presynaptic function. Such differences in expression could be set up via developmental or epigenetic mechanisms (Louis, 2018).

It is proposed that the cAMP-dependent plasticity in the MB plays two roles during olfactory learning: filtering MB responses based on salience and encoding valence. A role for MB plasticity in salience filtering is suggested by the observation that appetitive conditioning produced enhancement of MB responses across spatial compartments. These compartments have been suggested to route olfactory signals to valence-encoding output neurons, driving learned approach or avoidance via heterosynaptic plasticity. Therefore, the broad pattern of plasticity observed in this study would affect multiple downstream output pathways of opposing valence, suggesting that it does not encode valence per se. Rather, it may function to heighten relative MB sensitivity to salient stimuli. Across multiple sensory systems, ascending information is filtered according to salience, typically enhancing responses to stimuli that are biologically important. Alternatively, appetitive conditioning may modulate MB neurons in a fundamentally different way from aversive conditioning. While these opposing forms of memory require many overlapping MB-associated neurons, there are some differences in the circuits recruited during these forms of learning, and plasticity across MB neurons may be one difference. Regardless of the interpretation, the data reveal cAMP-dependent plasticity at the cellular level in intrinsic MB neurons. This may be layered on top of synaptic plasticity at MB output synapses, which have been proposed to encode valence by altering how olfactory signals flow through the neuronal networks that mediate behavioral approach or avoidance (Louis, 2018).

Several additional lines of evidence support the idea that cAMP-dependent plasticity serves as an overall gain control, regulating MB responses based on stimulus salience. First, the MB and MB-innervating dopaminergic neurons modulate salience-based decision making in a visual flight simulator paradigm. Second, dopaminergic neurons innervating the MB respond broadly to sensory stimuli that do not have an acquired valence, and exhibit activity that is correlated with locomotion. Activation of these neurons elevates cAMP in the downstream MB neurons in a compartmentalized manner, which in turn modulates their sensitivity and neurotransmission at the MB-MBON synapses. Thus, the MB neurons receive dynamically regulated dopaminergic inputs that alter the function of both the MB and downstream network components as a function of behavioral state. This may facilitate learning in situations in which the animal is likely to experience biologically important events (e.g., during foraging). Similar modulatory mechanisms modulate plasticity and memory in other animals as well. For instance, in honeybees, appetitive conditioning prolongs odor responses in MB neurons. Likewise, in the mammalian amygdala, coactivation of neuromodulatory and Hebbian plasticity is necessary for plasticity and memory (Louis, 2018).

Aversive conditioning produced no significant plasticity in the current study, consistent with results from some optogenetic reinforcement substitution imaging experiments. However, since Rut is required in MB neurons for normal aversive memory, cAMP-dependent plasticity is likely present in some form. Indeed, a previous study detected plasticity in the γ-lobe following aversive conditioning, which could be tightly localized to specific output synapses or neuronal subsets. Pairing odor with stimulation of tyrosine hydroxylase Gal4-labeled dopaminergic neurons produces aversive memory and detectable plasticity in MB γ-neurons. In this study, robust plasticity differentially following appetitive conditioning. This may be due to a bias toward learning about stimuli that guide motivationally relevant behaviors, such as approaching food-associated odors. Consistent with such an interpretation, appetitive conditioning produces memory that is more stable over time than aversive memory. A single trial of appetitive conditioning leads to the formation of long-term memory, while aversive conditioning requires multiple-spaced trials. The cAMP-dependent plasticity during appetitive conditioning could trigger downstream molecular pathways necessary to engage long-term memory formation. This presumably interacts with Ca2+ levels in neurons to regulate short- and long-term memory. In honeybees, elevating intracellular Ca2+ during a single-trial conditioning, which normally only triggers short-term memory, can induce long-term memory, whereas decreasing intracellular Ca2+ during multiple-spaced training impaired long-term memory formation. In addition, appetitive memory retrieval is motivationally gated by hunger state, suggesting a tie-in with motivational state. Integrating these observations, this suggests that motivationally relevant stimuli may enhance the sensitivity of MB neurons via cAMP-dependent plasticity, modulating the overall gain of the system in a salience-dependent manner (Louis, 2018).

The MB is involved in multiple distinct yet potentially interrelated behaviors, including several forms of learning and memory, regulating sleep and activity, context generalization, habituation, temperature preference, context dependence of olfactory behaviors, and salience-based decision making. The common thread among these behaviors is that they revolve around selection of an appropriate action based on context. Thus, a primary function of the MB and its modulatory input may to be alter the probability of action based on integrating environmental cues and internal state. In such a scenario, modulating the overall gain of the circuit could function in concert with fine-scale synapse-specific plasticity to alter the flow of information to downstream motor areas. Thus, these data support a model in which dopaminergic neurons and downstream cAMP-dependent plasticity modulate MB responses to stimuli based on their salience, priming the animal to engage in appropriate goal-oriented behaviors (Louis, 2018).

How compartment-specific local proteomes are generated and maintained is inadequately understood, particularly in neurons, which display extreme asymmetries. This study shows that local enrichment of Ca(2+)/calmodulin-dependent protein kinase II CaMKII) in axons of Drosophila mushroom body neurons is necessary for cellular plasticity and associative memory formation. Enrichment is achieved via enhanced axoplasmic translation of CaMKII mRNA, through a mechanism requiring the RNA-binding protein Mub and a 23-base Mub-recognition element in the CaMKII 3' UTR. Perturbation of either dramatically reduces axonal, but not somatic, CaMKII protein without altering the distribution or amount of mRNA in vivo, and both are necessary and sufficient to enhance axonal translation of reporter mRNA. Together, these data identify elevated levels of translation of an evenly distributed mRNA as a novel strategy for generating subcellular biochemical asymmetries. They further demonstrate the importance of distributional asymmetry in the computational and biological functions of neurons (Chen, 2022).

Local protein synthesis at synapses has been studied extensively in the context of specialized processes like activity-dependent plasticity and axon guidance. Recent theory and experimental work, however, suggests that local translation occurs much more generally and may be used to establish differential proteomes in functionally-specialized subcellular regions. This study resolves two long-standing questions about CaMKII: how and why it achieves extraordinary levels in axons. It was demonstrated that resting adult levels of CaMKII protein are translationally accrued, and that the high levels in this compartment form a computational scaffold critical for formation of associative memory and the cellular memory trace. While previous studies using mutants and RNAi have shown a role for CaMKII in plasticity, the current manipulations of the 3'UTR, which do not affect somatic kinase levels, establish the necessity of synaptic enrichment. This enrichment requires cis-elements present only in the long form of the 3'UTR and Mub, the Drosophila poly-C-binding-protein homolog demonstrating a new, activity-independent function for the CaMKII 3'UTR (Chen, 2022).

Activity-dependent translation and differential polyadenylation are ancient conserved features of CaMKII mRNAs. For mammalian CAMK2A, early work in which the 3'UTR was deleted demonstrated its requirement for mRNA stability and dendritic localization, and also for protein accumulation and activity-dependent synthesis (Chen, 2022).

A handful of studies attempted to identify cis-elements regulating dendritic CAMK2A mRNA localization and transport, but there is as yet no information on 3'UTR cis-elements controlling translation, though in silico prediction suggests that the CAMK2A 3'UTR may have polyC-binding protein motifs (Chen, 2022).

At the Drosophila larval neuromuscular junction, it has been shown that the CaMKII 3'UTR controls activity-dependent synthesis of CaMKII. The fact that the rodent CAMK2A 3'UTR can support activity-dependent protein synthesis in the fly suggests that there will be shared mechanisms for this aspect of CaMKII regulation. But while there are many similarities between mammals and flies, there are also differences. In Drosophila, the 3'UTR appears to have little effect on mRNA localization, and only a small effect on stability that is ascribable to a proximal cis-element. How CaMKII mRNA reaches synapses in Drosophila is yet to be determined, but the differences in localization mechanism may reflect the ca. 100-fold difference in distances that mRNAs need to travel to reach synapses (Chen, 2022).

The ability of Mub, which is present at low levels in MB axons and at high levels in MB and other cell bodies, to specifically regulate axonal accumulation of CaMKII protein without affecting somatic protein levels suggests several models. One possibility is that MB axons have either compartment-specific translational machinery or a distinct set of auxiliary proteins that allow Mub to regulate axonal ribosomes. The presence of Mub protein in MB axons, but not in other neuropils, may indicate the existence of unique translational complexes in that compartment. Another possibility is that Mub is a general translation enhancer, but MB soma contain repressor proteins that locally inhibit its actions. This would be consistent with the finding that there are cis elements that appear to act as general repressors in the CaMKII 3'UTR. While these ideas remain speculative, the robust interaction of Mub with CaMKII provides an opportunity to deepen understanding of how local protein synthesis can shape neuronal function and build the synaptic proteome (Chen, 2022).

In addition to mechanisms promoting protein-synthesis dependent long-term memory (PSD-LTM), the process appears to also be specifically constrained. This study presents evidence that the highly conserved Receptor Tyrosine Kinase dAlk is a novel PSD-LTM attenuator in Drosophila. Reduction of dAlk levels in adult α/β mushroom body (MB) neurons during conditioning elevates LTM, whereas its overexpression impairs it. Unlike other memory suppressor proteins and miRNAs, dAlk within the MBs constrains PSD-LTM specifically, but constrains learning outside the MBs as previously shown. Dendritic dAlk levels rise rapidly in MB neurons upon conditioning, a process apparently controlled by the 3'UTR of its mRNA and interruption of the 3'UTR leads to enhanced LTM. Because its activating ligand Jeb is dispensable for LTM attenuation, it is proposed that post-conditioning elevation of dAlk within α/β dendrites results in its auto-activation and constrains formation of the energy costly PSD-LTM, acting as a novel memory filter (Gouzi, 2018).

The findings reveal a novel role for dAlk in regulation of PSD-LTM formation in addition to its established role in learning. dAlk constrains both processes, but whereas learning attenuation requires its activity outside the MBs, suppression of PSD-LTM formation requires its elevation within the dendrites of α/β MB neurons. Moreover, while its activating ligand Jeb is required for learning attenuation, it is dispensable for PSD-LTM constraint. Global pharmacological inhibition of dAlk activity resulted in both STM and LTM enhancement as expected, because it addressed all neurons expressing this RTK. dAlk activity outside the MBs is known to be required for learning/3-minute memory suppression and these neurons are clearly affected by the inhibitor TAE684, as also are the MBs yielding enhanced PSD-LTM. In contrast, dAlk levels were specifically abrogated within the MBs where converging studies established that STM and LTM are engage different MB neuron types. dAlk attenuation in γ MB neurons did not affect 3 minute memory, or PSD-LTM strongly suggesting that dAlk in not expressed therein. The notion of memory suppression almost invariably refers to forgetting, broadly defined as a decay of memory that either actively dissipates in time or undergoes interference by additional learning of unrelated or irrelevant information (Gouzi, 2018).

Forgetting an odor/shock association in Drosophila requires the small G protein Rac, or Dopamine (DA) signalling predominantly through the DAMB receptor, and its suppression appears as an enhancement of all types of 3-24-hour memories. However, 24-hour memory enhancement resulting from Rac attenuation appears distinct from PSD-LTM and inhibition of DA signalling in the MB-afferent DAN neurons does not enhance 16 to 24-hour memories. Hence, Rac and/or DA signalling inhibit recently acquired labile memories rather than consolidated forms (Gouzi, 2018).

dAlk also acts during the labile stage of memory formation, but not its dissipation and is specific to PSD-LTM, not 3-hour memory or ARM. Furthermore, dAlk is not required within the essential for forgetting dopaminergic neurons. Therefore dAlk-mediated LTM inhibition is distinct from dissipation of labile memories (Gouzi, 2018).

A number of memory suppressor genes have been recently described in Drosophila and mice indicating that although its exact role is unclear, memory restraint is evolutionarily conserved. Constraining memory may limit the conditioned associations processed towards the energetically demanding PSD-LTM, ensure the fidelity of associations that progress towards consolidation, or inhibit proactive or retroactive interference (Gouzi, 2018).

The role of all apparent memory suppressor proteins and miRNAs identified to date in Drosophila has not been fully delineated, but some mechanistic aspects emerge. Drosophila memory suppressor miRNAs ostensibly regulate translation of postsynaptic proteins involved in MB excitability, hence attenuation of their levels and the resultant hyper-excitability could underlie enhanced memory. Accordingly, loss of the apparent signal-tempering acetylcholine transporter DmSLC22A from MB neurons enhances their excitability and elevates memory. Interestingly, like dAlk, DmSLC22A is found in mushroom body calycal microglomeruli. Therefore, in conjunction with the current results, it appears that memory constraining mechanisms depend on the level of postsynaptic proteins that limit the amplitude or duration of neuronal excitation. In contrast to other memory suppressor proteins and miRNAs, dAlk elevation in the MBs is not required for 3-hour memory, but appears specific to PSD-LTM. The temporal specificity of dAlk suggests that its activity may not constrain MB excitability, but rather LTM consolidation mechanisms, a hypothesis under investigation (Gouzi, 2018).

Conditioning-dependent dAlk elevation in MB dendrites appears to result via local translation regulated by the 3'UTR of its mRNA. This 3'UTR-conferred property is shared with multiple dendritic proteins, including another RTK involved in memory formation, the BDNF receptor trkB. Similarly, 3'UTR sequences direct the mRNA of Drosophila CaMKII, a kinase also implicated in memory, to be translated in the postsynaptic zones of MB calyces (Gouzi, 2018).

Significantly, the 3'UTR of dAlk mRNA contains more numerous regulatory elements, than those on CaMKII transcripts, including several stabilizing AREs. The putative miRNA binding sites include those for miR-305 and miR-932, both implicated in memory formation and possibly in dAlk local translation. Translational regulation may also involve identified putative RBP binding sequences at the dAlk 3'UTR. Some like Pumilio (Pum) and the cytoplasmic polyadenylation element binding protein (CPEB) Orb2, are translation suppressors with known function in memory. Whether others, like Rox8, the ortholog of the stress granule-associated vertebrate protein TIA1, play a role in memory formation is currently unknown. Whether miRNAs and RBPs interact with dAlk mRNA upon conditioning to regulate its dendritic levels, will be the focus of forthcoming work (Gouzi, 2018).

The PSD-LTM constraint depends on dAlk activity and the increased levels per se as demonstrated by the elevated memory upon treatment with the inhibitor TAE684. How is calycal dAlk activated to constrain LTM formation since Jeb is dispensable for LTM attenuation? Presently, the possibility cannot be excluded that a yet unidentified ligand may activate dAlk upon spaced conditioning. However, another explanation that is currently favored is that upon spaced training, dAlk can auto-activate in response to its local elevation in the calyx. Level-dependent auto-activation has been reported for human ALK-positive cancers, or neurons transfected with ALK, a feature shared by almost all RTKs. Local elevation-dependent autoactivation of dAlk is in agreement with the current experimental data that acute dAlk elevation attenuates LTM and that conditioning elevates the endogenous protein in MB dendrites. Moreover, dAlk autoactivation is consistent with the independence of dAlk-dependent PSD-LTM attenuation from Jeb. This conditioning dependent dAlk elevation and auto-activation in MB dendrites is likely considerably slower than acute activation by Jeb of extant dAlk outside the MBs required to constrain learning/3-minute memory formation. Furthermore, paneuronal elevation of Jeb left PSD-LTM unaffected, consistent with the notion that the two methods of dAlk activation, Jeb-dependent and autoactivation are operant spatially distinct neurons (outside and inside the MBs and of distinct functional consequences (Gouzi, 2018).

Hence, it is proposed that conditioning results in local elevation of unliganded dAlk monomers in MB dendrites, raising the probability of encounter, lateral dimerization, auto-phosphorylation and activation of the kinase domain at the postsynaptic plasma membrane (Gouzi, 2018).

Unfortunately, an antibody specific to phosphorylated, hence activated dAlk is not currently available and therefore it is not possible to test this prediction in situ. Downstream mechanisms engaged by dAlk to restrain LTM are still unknown. In a previous study, dAlk outside the MBs was described as an upstream activator of a dNf1-regulated Ras/ERK signaling pathway responsible for learning/STM attenuation. Intrestingly, dAlk and dNf1 co-localize extensively in MBs calyces, suggesting that they could also interact to mediate PSD-LTM attenuation. However, unlike for dAlk abrogation, dNf1 loss results in PSD-LTM deficits restored by re-expression of the protein MB neurons under c739-Gal4. Therefore, although possible that dAlk and dNf1 interact within these neurons they are likely antagonistic with respect to PSD-LTM formation, a process potentially engaging and requiring suppression of Ras signaling, a hypothesis currently under investigation (Gouzi, 2018).

In conclusion, this study has identified dAlk as a specific negative regulator of PSD-LTM formation. Thus far, dAlk appears unique among RTKs in that it constrains LTM formation, possibly acting as a memory filter. The nature of the specific signals engaged by dAlk and the downstream PSD-LTM constraining mechanisms remain yet to be elucidated in future work (Gouzi, 2018).

For aversive olfactory memory in Drosophila, multiple components have been identified that exhibit different stabilities. Intermediate-term memory generated after single cycle conditioning is divided into anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM), with the latter being more stable. This study determined that the ASM and ARM pathways converged on the Rgk1 small GTPase and that the N-terminal domain-deleted Rgk1 was sufficient for ASM formation, whereas the full-length form was required for ARM formation. Rgk1 is specifically accumulated at the synaptic site of the Kenyon cells (KCs), the intrinsic neurons of the mushroom bodies (MBs), which play a pivotal role in olfactory memory formation. A higher than normal Rgk1 level enhanced memory retention, which is consistent with the result that Rgk1 suppressed Rac-dependent memory decay; these findings suggest that rgk1 bolsters ASM via the suppression of forgetting. It is proposed that Rgk1 plays a pivotal role in the regulation of memory stabilization by serving as a molecular node that resides at KC synapses, where the ASM and ARM pathway may interact (Murakami, 2017).

Drosophila olfactory learning and memory, in which an odor is associated with stimuli that induce innate responses such as aversion, has served as a useful model with which to elucidate the molecular basis of memory. Olfactory memory is divided into several temporal components and the intermediate-term memory (ITM) generated after single cycle conditioning is further classified into two distinct phases, anesthesia-sensitive memory (ASM) and anesthesia-resistant memory (ARM). Evidence has suggested that ASM and ARM are distinctly regulated at the neuronal level and at the molecular level (Murakami, 2017).

Mushroom bodies (MBs) represent the principal mediator of olfactory memory. Kenyon cells (KCs) are the intrinsic neurons of MBs, which are bilaterally located clusters of neurons that project anteriorly to form characteristic lobe structures and are a platform of MB-extrinsic neurons that project onto or out of the MBs. To elucidate the molecular mechanisms that underlie olfactory memory, screenings for MB-expressing genes have been a useful strategy. A technique used to examine gene expression in a small amount of tissue samples has enabled the investigation of the expression profile in MBs with a substantial dynamic range of expression levels and high sensitivity, thereby representing a promising approach with which to identify novel genes responsible for memory. This study deep sequenced RNA isolated from adult MBs and identified rgk1 as a KC-specific gene (Murakami, 2017).

The RGK protein family, for which Drosophila Rgk1 exhibits significant protein homology, belongs to the Ras-related small GTPase subfamily, which is composed of Kir/Gem, Rad, Rem, and Rem2. Their roles include the regulation of Ca2+ channel activity and the reorganization of cytoskeleton. Notably, mammalian REM2 is expressed in the brain and has been shown to be important for synaptogenesis, as well as activity-dependent dendritic complexity. These findings raise the possibility that RGK proteins may have a role in the synaptic plasticity that underlies memory formation. Drosophila has several genes that encode proteins homologous to the RGK family, including rgk1. Therefore, based on the ample resources available in Drosophila for the investigation of neuronal morphology and functions, Drosophila Rgk proteins will provide a good opportunity to elucidate the function of RGK family proteins (Murakami, 2017).

This study describes the analysis of Drosophila rgk1, which exhibited specific expression in KCs. Rgk1 accumulated at synaptic sites and was required for olfactory aversive memory, making the current study the first to demonstrate the role of an RGK family protein in behavioral plasticity. These data suggest that Rgk1 supports ASM via the suppression of Rac-dependent memory decay, whereas the N-terminal domain has a specific role in ARM formation. Together, these findings indicated that Rgk1 functions as a critical synaptic component that modulates the stability of olfactory memory (Murakami, 2017).

It is proposed that the ITM is genetically divided into three components: the rut-, dnc-, and rgk1-dependent pathways. The rut and dnc pathway act specifically for ASM and ARM, respectively, whereas rgk1 acts for both ASM and ARM, albeit partially. Consistent with this notion, it is noteworthy that the ASM and ARM pathways converge on Rgk1, yet the functional domains may be dissected; the full-length form of Rgk1 is required for ARM, whereas the molecule that lacks the N-terminal domain is capable of generating ASM, which suggests that the protein(s) required for ARM formation may interact with the N-terminal domain of Rgk1 (Murakami, 2017).

The data suggested that Rgk1 acts for both ASM and ARM, whereas the rgk1 deletion mutant, which was shown to be a protein null, exhibited only a partial reduction in ITM; these findings imply that Rgk1 regulates an aspect of each memory component. This idea may be explained by the expression pattern of Rgk1. Rgk1 exhibited exclusive expression and cell-type specificity in the KCs, whereas the memory components have been shown to be regulated by the neuronal network spread outside of the MBs and are encoded by multiple neuronal populations. For example, two parallel pathways exist for ARM and ASM is modulated, not only by MB-extrinsic neurons, but also by the ellipsoid body that localizes outside of the MBs. dnc-dependent ARM requires antennal lobe local neurons and octopamine-dependent ARM requires α'/β' KCs, in neither of which was Rgk1 detected. Therefore, Rgk1 may support a specific part of memory components that exists in a subset of KCs (Murakami, 2017).

The specific expression of Rgk1 in KCs suggests its dedicated role in MB function. Rgk1 exhibited cell-type specificity in KCs from anatomical and functional points of view. Rgk1 is strongly expressed in α/β and γ KCs and weakly expressed in α'/β' KCs and the expression of the rgk1-sh transgene in α/β and γ KCs was sufficient to disrupt memory. Several genes required for memory formation have been shown to be expressed preferentially in the KCs and the notable genes include dunce, rutabaga, and DC0. Although a recent study in KC dendrites showed that the modulation of neurotransmission into the KCs affects memory strength, KC synapses are thought to be the site in which memory is formed and stored. The current analyses with immunostaining and GFP fusion transgenes indicated that Rgk1 is localized to synaptic sites of the KC axons, which raises the possibility that Rgk1 may regulate the synaptic plasticity that underlies olfactory memory. Among the RGK family proteins, Rem2 is highly expressed in the CNS and regulates synapse development through interactions with 14-3-3 proteins, which have been shown to be localized to synapses and are required for hippocampal long-term potentiation and associative learning and memory. In Drosophila, 14-3-3Ζ is enriched in the MBs and is required for olfactory memory. In addition, the C-terminal region of Drosophila Rgk1 contains serine and threonine residues that exhibit homology to binding sites for 14-3-3 proteins in mammalian RGK proteins. Therefore, Rgk1 and 14-3-3Ζ may act together in the synaptic plasticity that underlies olfactory memory (Murakami, 2017).

The roles of RGK family proteins in neuronal functions have been investigated extensively. The current data, when combined with the accumulated data on the function of RGK family proteins, provide novel insights into the mechanism that governs two distinct intermediate-term memories, ASM and ARM. Regarding the regulation of ASM, the data showed that Rgk1 suppressed the forgetting that was facilitated by Rac. Rac is a major regulator of cytoskeletal remodeling. Similarly, mammalian RGK proteins participate in the regulation of cell shape through the regulation of actin and microtubule remodeling. Rgk1 may affect Rac activity indirectly by sharing an event in which Rac also participates because there have been no reports showing that RGK proteins regulate Rac activity directly; further, it was determined that rgk1 transgene expression did not affect the projection defect of KC axons caused by RacV12 induction during development. Therefore, it is suggested that Rgk1 signaling and Rac signaling may merge at the level of downstream effectors in the regulation of forgetting. A member of the mammalian RGK1 proteins, Gem, has been shown to regulate Rho GTPase signaling through interactions with Ezrin, Gimp, and Rho kinase. Rho kinase is a central effector for Rho GTPases and has been shown to phosphorylate LIM-kinase. In Drosophila, the Rho-kinase ortholog DRok has been shown to interact with LIM-kinase. Furthermore, Rac regulates actin reorganization through LIM kinase and cofilin and the PAK/LIM-kinase/cofilin pathway has been postulated to be critical in the regulation of memory decay by Rac. It was shown recently that Scribble scaffolds a signalosome consisting of Rac, Pak3, and Cofilin, which also regulates memory decay. Therefore, Rgk1 may counteract the consequence of Rac activity (i.e., memory decay) through the suppression of the Rho-kinase/LIM-kinase pathway. DRok is a potential candidate for further investigation of the molecular mechanism in which Rgk1 acts to regulate memory decay (Murakami, 2017).

The data indicated that Rgk1 is required for ARM in addition to ASM. It has been shown that Synapsin and Brp specifically regulate ASM and ARM, respectively. The functions of Synapsin and Brp may be differentiated in a synapse by regulating distinct modes of neurotransmission. The exact mechanism has not been identified for this hypothesis; however, the regulation of voltage-gated calcium channels may be one of the key factors that modulate the neurotransmission. Voltage-gated calcium channels are activated by membrane depolarization and the subsequent Ca2+ increase triggers synaptic vesicle release. The regulation of voltage-gated calcium channels has been shown to be important in memory; a β-subunit of voltage-dependent Ca2+ channels, Cavβ3, negatively regulates memory in rodents. Importantly, Brp regulates the clustering of Ca2+ channels at the active zone. Moreover, it has been demonstrated extensively that mammalian RGK family proteins regulate voltage-gated calcium channels. Kir/Gem and Rem2 interact with the Ca2+ channel β-subunit and regulate Ca2+ channel activity. In addition, the ability to regulate Ca2+ channels has been shown to be conserved in Drosophila Rgk1. Therefore, both Brp and Rgk1 may regulate ARM through the regulation of calcium channels, the former through the regulation of their assembly and the latter through the direct regulation of their activity. The finding that Rgk1 localized to the synaptic site and colocalized with Brp lends plausibility to the scenario that Rgk1 regulates voltage-gated calcium channels at the active zone (Murakami, 2017).

Several memory genes identified in Drosophila, including rutabaga, PKA-R, and CREB, have homologous genes that have been shown to regulate behavioral plasticity in other species. The identification of Drosophila rgk1 as a novel memory gene raises the possibility for another conserved mechanism that governs memory. There is limited research regarding the role of RGK proteins at the behavioral level in other species; however, the extensively documented functions of RGK proteins with respect to the regulation of neuronal functions, combined with the data presented in this study regarding Drosophila Rgk1, raise the possibility of an evolutionally conserved function for RGK family proteins in memory (Murakami, 2017).

Memory consolidation is a crucial step for long-term memory (LTM) storage. However, a clear picture of how memory consolidation is regulated at the neuronal circuit level is still lacking. This study took advantage of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). An essential role has been revealed of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. This study used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors (the type 1 DAMB and the type 2 dD2R) in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. It is proposed that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons (Pavlowsky, 2018).

Formation of a memory engram is a multi-step process, from encoding the relevant information to the final storage of memory traces. Describing the neuronal architecture and functions that underlie each step of this process is crucial to understanding memory ability. In Drosophila, a very fine knowledge is available of the anatomy of the mushroom body (MB), the major olfactory integrative brain center, as well as its input and output neurons. The mapping to these circuits of various functional modalities occurring at the different stages of memory encoding, storage, and recall is also quite advanced (Pavlowsky, 2018).

Drosophila MBs are paired structures including ~2,000 intrinsic neurons per brain hemisphere. These neurons receive dendritic input from the antennal lobes through projection neurons in the calyx area on the posterior part of the brain. Their axons form a fascicle, called a peduncle, that traverses the brain to the anterior part, where axons branch to form horizontal and vertical lobes according to three major branching patterns (α/β, α'/β' and γ). MB lobes are tiled by spatially segregated presynaptic projections from dopamine neurons (DANs), on the one hand, and dendrites of MB output neurons (MBONs), on the other hand. DANs and MBONs are matched to form defined anatomical compartments that are increasingly considered as independent functional units. On several of these compartments, it was shown that DAN activity can induce heterosynaptic plasticity at the MB/MBON synapse, which could be a cellular substrate of memory encoding (Pavlowsky, 2018).

In addition to this canonical anatomical motif of the DAN/MB intrinsic neurons/MBON triad, electron microscopy connectome reconstruction in the larval brain has evidenced recently that DANs have direct synaptic connections to their matched MBONs. In the adult, direct DAN-to-MBON synapses have also been observed in several compartments of the MB vertical lobes (Pavlowsky, 2018).

MB activity is regulated by a broad spectrum of neuromodulatory input, among which tonic dopamine signaling plays an important role in the regulation of memory persistence or expression. In particular, it has been shown that sustained rhythmic activity of the MP1 DAN, also named PPL1-γ1pedc and which innervates the γ1 module and the α/β peduncle, is crucial after conditioning to enable the consolidation of both aversive and appetitive long-term memory (LTM), the most stable memory forms that rely on de novo protein synthesis. The MP1 neuron is anatomically matched with the MVP2 neuron, a GABA-ergic MBON that shows a complex arborization. The MVP2 neuron, also named MBON-γ1pedc > α/β, possesses two dendritic domains on the γ1 and peduncle compartments. On the ipsilateral side, MVP2 has presynaptic projections on MB vertical and medial lobes and also targets brain areas outside MB where other MBONs project. In particular, MVP2 neurons mediates a feedforward inhibition of specific MBONs involved in aversive and appetitive memory retrieval. Interestingly, MVP2 neurons also send a presynaptic projection onto the contralateral peduncle, a place of MP1 presynaptic coverage. Hence, the anatomy of the MP1-MVP2 neurons is compatible with the existence of feedback circuitry. This study tested experimentally the existence of such a functional feedback in the context of appetitive LTM formation (Pavlowsky, 2018).

Appetitive memory results from the paired delivery of an odorant and a sugar to starved flies. Only one pairing is sufficient to form both short-term memory (STM) and LTM, but it was shown that these two memory phases stem from distinct properties of the reinforcing sugar: although the sweetness of the sugar is sufficient so that flies form appetitive STM, the formation of LTM requires that the conditioning is made with a caloric sugar. The nutritional value of the reinforcing sugar translates in the fly brain as a post-ingestive sustained rhythmic signaling from MP1 neurons that is necessary to consolidate LTM. At the cellular level, STM and LTM stem from parallel and independent memory traces located in distinct subsets of MB neurons; respectively, γ neurons and α/β neurons. Several MB output circuits have been involved in the retrieval of appetitive STM (MBON-γ2α'1), LTM (MBON-α3, MBON-α1), or both (M4/M6, also named MBON-γ5β'2a/MBON-β'2mp), providing as many candidate synaptic sites of memory encoding (Pavlowsky, 2018).

This work confirmed that post-training MP1 activity is required for LTM formation, but it was shown in addition that this activity must be temporally restricted. MP1 activity is self-regulated through an inhibitory feedback by MVP2 neurons. Immediately after conditioning, the oscillatory activity of MP1 is enhanced and MVP2 is inhibited. After about 30 min, MVP2 is activated, terminating the period of MP1 increased signaling, which, this study shows is a requirement for proper LTM formation. It is proposed that the bidirectional action of this feedback loop is based at the molecular level on the sequential involvement of two antagonist dopamine receptors, the type 1 DAMB and the type 2 dD2R on one side and the metabotropic GABAB-R1 receptor on the other side (Pavlowsky, 2018).

This work describes a functional inhibitory feedback from an MBON, the GABA-ergic MVP2 neuron, to the dopaminergic neuron of the same MB module, the MP1 neuron. Anatomical data from synaptic staining and electron microscopy, as well as the requirement of a specific GABA receptor in MP1 neurons for appetitive LTM, lead to the hypothesis of a direct connection between MVP2 and MP1 neurons, although alternative scenarios featuring plurisynaptic circuits involving additional GABAergic neurons cannot be ruled out at this stage. Using time-resolved manipulation of neuronal activity, it was shown that this feedback circuit is involved in the first hour after appetitive conditioning for LTM formation. It was already known, and confirmed in this study, that the activity of MP1 neurons, in the form of regular calcium oscillations, is necessary in the first 30-45 min after conditioning to build LTM. Strikingly, in the present work, it was shown that, after this initial time period, the activity of MP1 neurons is not merely dispensable but rather deleterious for LTM formation, since activating MP1 neurons from 0.5 hr to 1 hr after conditioning caused an LTM defect (Pavlowsky, 2018).

Conversely, it was found that, in that time interval where MP1 neuron activity is deleterious, MVP2 neurons need to be active for normal LTM performance. Imaging experiments showed that blocking MVP2 neurons increased the persistence of MP1 neuron oscillations, up to more than 1 hr post-conditioning. The same effect was observed when the GABAB-R1 receptor was knocked down in MP1 neurons. Interestingly, blocking MVP2 neurons or GABA-ergic signaling in MP1 neurons mostly affected the frequency and the regularity of MP1 calcium signals, without markedly increasing their amplitude. Hence MVP2 neurons seem to be involved in terminating the period of sustained oscillatory signaling from MP1 neurons rather than merely decreasing MP1 activity. However, in the first 0.5 hr after conditioning, MVP2 neuron activity is not simply dispensable but also deleterious for LTM. Since MVP2 neurons have an inhibitory effect on MP1 activity, it is likely that MVP2 neurons have to be inhibited to let MP1 oscillations occur. Strikingly, this study established that MVP2 neurons are modulated by dopamine signaling through two receptors: DAMB, a type 1 activating receptor; and dD2R, a type 2 inhibitory receptor. Although these two receptors have opposite downstream effects, both are required in MVP2 for normal LTM performance. Overall, the results evidence that the MP1-MVP2 feedback circuit is functionally designed to allow the onset of LTM-gating oscillations only on a precise time windows of about 0.5 hr after conditioning (Pavlowsky, 2018).

It is proposed that MP1 activity is self-regulated through a dual receptor mechanism that controls MVP2 feedback. Initially, the ongoing activity of MP1 neurons inhibits MVP2 neurons through the dD2 receptor, which allows for sustained MP1 activity. In a second step, DAMB is activated in MVP2 neurons to enable the inhibitory feedback that shuts off MP1 oscillations. This model unifies molecular data and the results obtained from time-resolved thermogenetic manipulation of neuronal activity; unfortunately, such temporality of receptor involvement cannot be tested with RNAi-based knockdown (Pavlowsky, 2018).

DAMB and dD2R are two G-protein-coupled dopamine receptors. Although dD2R is a clear homolog of mammalian D2 receptor, and is negatively coupled to cAMP synthesis, the molecular mechanisms downstream of DAMB appear to be more diverse. It was shown that DAMB activation can stimulate cAMP synthesis, similarly to the function of a type 1 receptor, likely through Gβγ-coupled signaling. Surprisingly, it was recently shown that DAMB-mediated dopamine signaling could transiently inhibit the spiking of sleep-promoting neurons through the same G-protein pathway. Additionally, it was shown that DAMB can also activate downstream calcium signaling from intracellular calcium stores. In the current model, MVP2 neurons need, at one point, to be activated to dampen MP1 oscillations, so activating functions of DAMB seem to be more relevant in the present environment. Interestingly, physiological measurements in a heterologous system showed that cAMP activation occurs within tens of minutes, while calcium activation occurs on much shorter timescales. The delayed requirement of MVP2 activity (starting ~30 min after conditioning) seems to be more consistent with an activation of the cAMP pathway. It would be helpful in the future to decipher the molecular mechanism downstream of DAMB involved in this feedback loop. The sequential activation of two distinct dopamine receptors could be due to different affinities for dopamine. Indeed, pharmacological studies show that D2R-like receptors have a higher affinity toward dopamine compared to the D1-like receptors in mammals. However, in the specific case of Drosophila D2R and DAMB, similar dopamine affinities for both receptors were reported (0.5 μM for D2R [52 and 0.1-1 μM for DAMB, although these are all obtained from in vitro preparations of cultured cells. There could be also be subtler differences of activation kinetics based both on the quantity and on the mode of dopamine release by MP1 neurons (Pavlowsky, 2018).

MP1 neurons and MVP2 neurons have been shown to play crucial roles in both aversive and appetitive memories. During aversive conditioning, MP1 neurons mediate the unconditioned stimulus, which is thought to involve dDA1 activation in MB neurons. In a recent report, it was shown that suppressing the activity of MVP2 neurons during an odor presentation leads to the formation of an aversive memory toward this odor. In light of this result, these authors proposed that the role of steady-state MVP2 activity is to prevent the formation of irrelevant memory from insignificant stimuli. Given the role of MP1 in the signaling of negative stimuli during aversive learning, this finding and its interpretation are fully consistent with the existence of an inhibitory feedback from MVP2 neurons to MP1 neurons, as reported in the present work. MP1 neurons are also central in the formation of LTM after conditioning. Tonic signaling through slow oscillations of MP1 neurons gates the formation of aversive LTM after spaced training. The same kind of sustained post-training signaling builds LTM after appetitive conditioning. Both in aversive and appetitive paradigms, this LTM-gating function involves DAMB signaling in MB neurons. After aversive spaced training, it was shown that DAMB activation triggers an upregulation of MB energy metabolism, which starts the consolidation of LTM. Finally, MP1 neurons also regulate the retrieval of appetitive STM. MP1 inhibition in starved flies, through suppressive dNPF signaling, allows integration of the appetitive motivational state with the expression of MB-encoded memory trace during retrieval to allow for the expression of appetitive STM. This involves enhanced feedforward inhibition from MVP2 neurons to the M4/M6 MBONs that mediate appetitive memory retrieval. The fact that MP1 inhibition goes along with enhanced MVP2 activity is consistent with the fact that baseline MP1 activity can drive an inhibition of MVP2 through dD2R, as is reported in this study. This may explain why a knockdown of dD2R in MVP2 neurons, by indiscriminately disturbing this MP1-MVP2 inhibitory link, would impair the odor-specific message carried by M4/M6 neurons for memory retrieval and cause an STM defect. All these findings illustrate how the sophistication of MP1 neuron involvement in memory is tightly linked to the diversity of receptors and neuronal targets that it can activate. A finer understanding of these processes calls for higher resolution physiological measurements to understand how the various dopamine receptors are sensitive to different modalities or kinetics of dopamine release (Pavlowsky, 2018).

Recently, it was shown that acquisition and consolidation of appetitive LTM also rely on a positive-feedback circuit involving the α1 MB compartment, dopaminergic PAM-α1, and glutamatergic MBON-α1 neurons (Ichinose, 2015). Thus, consolidation of appetitive memory involves two different recurrent circuits that share common features, such as the MBON's dual functions in consolidation and retrieval of memory. MP1 neurons are activated after a conditioning with a nutritious sugar, which is necessary for LTM formation. PAM-α1 neurons are activated during conditioning and probably mediate the coincidence detection between sugar intake and odor perception within MB neurons. The recurrent activity of the α1 compartment loop is also necessary for proper LTM formation, presumably to stabilize a nascent memory trace. Interestingly, the electron microscopy reconstruction of the adult MB vertical lobes recently showed that MVP2 neurons form direct synapses with MBONs in the α2 and α3 modules and, probably, in the α1 compartment as well. Therefore, the two feedback circuits may not be independent, and MVP2 neurons may also mediate a feedforward input from the MP1/MVP2 loop to the PAM-α1/MBON-α1 loop. The dD2R-mediated inhibition of MVP2 neurons by MP1 activity immediately after conditioning could, therefore, help in maintaining the recurrent activity in the α1 compartment (Pavlowsky, 2018).

In conclusion, this study shown here that a negative-feedback loop functions to control appetitive LTM formation, likely involving two antagonist dopaminergic receptors. This negative-feedback loop is strikingly similar to one recently described in the mammalian mesolimbic system in which feedback from inhibitory neurons prevents the over-activation of dopaminergic neurons. These two circuits have at least three common features: they rely on the metabotropic receptors DA1 and GABABR1; they comprise dopaminergic and inhibitory neurons, which are monosynaptically connected in mammals, and possibly also in Drosophila; and they are involved in the memory acquisition of motivationally relevant stimuli. These shared properties of negative-feedback loops highlight how similar strategies exist at both the network and molecular levels to regulate certain related behaviors across species (Pavlowsky, 2018).

Amyloid precursor protein (APP), the precursor of amyloid beta peptide, plays a central role in Alzheimer's disease (AD), a pathology characterized by memory decline and synaptic loss upon aging. Understanding the physiological role of APP is fundamental in deciphering the progression of AD, and several studies suggest a synaptic function via protein-protein interactions. Nevertheless, it remains unclear whether and how these interactions contribute to memory. In Drosophila, previous work has shown that APP-like (APPL), the fly APP homolog, is required for aversive associative memory in the olfactory memory center, the mushroom body (MB). The present study shows that APPL is required for appetitive long-term memory (LTM), another form of associative memory, in a specific neuronal subpopulation of the MB, the alpha'/beta' Kenyon cells. Using a biochemical approach, this study identified the synaptic MAGUK (membrane-associated guanylate kinase) proteins X11, CASK, Dlgh2 and Dlgh4 as interactants of the APP intracellular domain (AICD). Next, this study shows that the Drosophila homologs CASK and Dlg are also required for appetitive LTM in the alpha'/beta' neurons. Finally, using a double RNAi approach, it was demonstrated that genetic interactions between APPL and CASK, as well as between APPL and Dlg, are critical for appetitive LTM. In summary, these results suggest that APPL contributes to associative long-term memory through its interactions with the main synaptic scaffolding proteins CASK and Dlg. This function should be conserved across species (Silva, 2020).

AD is the principal neurodegenerative disorder affecting the elderly, and it is characterized by amyloid β (Aβ) deposition derived from proteolytic processing of amyloid precursor protein (APP). A pathological hallmark of AD is a progressive memory decline that correlates intimately with synaptic loss. One of the main hypotheses for the cognitive deficits observed in AD is thus a dysfunction of synapses leading ultimately to synaptic loss and alteration of neural network activity. Therefore, it is essential to understand the physiological role of APP at the synapse. APP is a transmembrane protein expressed on both sides of the synapse (Wang, 2009). The APP extracellular domain can mediate dimerization across the synapse or interact with extracellular matrix components, growth factors and receptor-like proteins. These interactions are involved in synapse stabilization during development and also in regulating synapse plasticity in mature neuronal networks (Montagna, 2017). APP can undergo two types of proteolytic processing, including the non-amyloidogenic pathway, which is initiated by α-secretase and produces a secreted form of APP (sAPPα), and the amyloidogenic pathway, which successively involves β- and then γ-secretase to release Aβ peptide and an APP intracellular C-terminal domain (AICD). Although the manner in which proteolytic processing of APP and its derivatives interferes with neuronal physiology has been extensively studied, little is known about the function of APP intracellular domain at the synapse or its synaptic partners (Silva, 2020).

Studies in mammals suggest that APP can interact via its intracellular domain with synaptic MAGUK proteins such as X11, CASK, or PSD-95. MAGUK proteins are involved in the assembly, maintenance and remodeling of the scaffolding in synaptic compartments mainly via regulation of the targeting of receptors and ion channels to the synapse. Therefore, understanding the interactions between APP and MAGUKs should help decipher the synaptic function of APP (Silva, 2020).

The three mammalian orthologs APP, APLP1 and APLP2 are partially functionally redundant, whereas Drosophila expresses a single APP homolog named APP-like (APPL) that has been implicated in olfactory memory (Bourdet, 2015; Goguel, 2011) and visual memory (Rieche, 2018). APPL is strongly expressed in the adult mushroom body (MB), the main olfactory memory center in insects. Previous work has investigated the function of APPL in Drosophila aversive olfactory memory (Bourdet, 2015; Goguel, 2011; Preat, 2016). However, whether the APP synaptic partners and their interactions might contribute to memory is still unexplored. Several MAGUK homologs in Drosophila have been identified such as dX11, CASK/Caki, and Discs-large (Dlg). Similar to its mammalian counterpart, dX11 binds APPL, and both are necessary for synaptic remodeling at the Drosophila neuromuscular junction (Ashley, 2005). Drosophila CASK regulates CaMKII activity, interacts with dX11. Mammalian Dlg1/SAP97, Dlg2/PSD-93, Dlg3/SAP102 and Dlg4/PSD-95 share similarities with the fly Dlg proteins DlgA and DlgS97), which are encoded by a single dlg gene. Both CASK and Dlg play key roles in neurotransmission, synaptogenesis and plasticity (Silva, 2020).

This study has aimed to decipher the role of APPL and its synaptic partners in appetitive olfactory memory. Additionally, to investigate the AICD interactome, this study used a proteo-liposome recruitment method and found that AICD interacts with the MAGUK synaptic proteins X11, CASK and Dlg. It was then found in flies that APPL, CASK and Dlg are required specifically in the same neuronal subpopulation (the α'/β' KCs) for appetitive LTM. Finally, a double RNAi strategy was used to demonstrate that genetic interaction between APPL and MAGUKs is critical for appetitive LTM. To determine whether this memory deficit could be due to a major disorganization of the synaptic structure, both the pre-synaptic and the post-synaptic sites of MB α'/β' neurons were investigated using confocal immuno-labeling of synaptic proteins (Silva, 2020).

Previous work has showed that APPL is required in the α/β and γ KCs for aversive LTM. Additional work demonstrated that APPL is required for aversive LTM and MTM in the DPM neurons (Turrel, 2017), a pair of serotonergic and GABAergic neurons that project to each of the MB lobes, where they connect both pre- and post-synaptically to the KCs. This study has found that APPL is also required in another form of long-lasting protein synthesis-dependent memory, appetitive LTM, albeit surprisingly in a different subpopulation of KCs, the α'/β' neurons. The requirement of APPL in α'/β' but not in the other KCs for appetitive LTM suggests that it has a specific role in appetitive LTM consolidation, as consolidation of appetitive LTM requires synaptic neurotransmission from α'/β'. Interestingly, recurrent activity of the α'/β'-DPM loop has been described as necessary to consolidate appetitive memories, with LTM eventually being stored in the α/β neurons. An involvement of APPL in memory consolidation may rely on transsynaptic APPL interactions and may also contribute to the molecular support of the α'/β'-DPM loop. Eventually, such a role of APPL in memory consolidation through transsynaptic interaction would be consistent with published research in mammals. Indeed, at the cellular level APP is expressed in pre- and postsynaptic compartments and can form trans-dimers that have been suggested as necessary for synaptic function (Wang, 2009). In addition, perturbation of APP function by intraventricular infusion of an antibody against APP induced memory impairments only when it was performed during the memory consolidation phase of a passive avoidance task. As shown for other synaptic cell adhesion molecules, the regulation of APP expression at the neuronal membrane is critical for hippocampal-dependent memory consolidation in the dentate gyrus, suggesting a potential involvement of APP in synaptic remodeling. Altogether, the present findings combined with research on mammalian models suggest that APP might have a conserved function across species in memory consolidation processes via transsynaptic interactions (Silva, 2020).

This study has shown that in addition to the previously known X11 proteins, the intracellular domain of APP interacts with other scaffolding proteins (CASK, Dlg2/PSD-93 and Dlg4/PSD-95). However, it is not clear whether these proteins interact directly with APP or if their interactions are mediated by the X11 adaptor proteins, as it has been described for CASK. This study demonstrates that the Drosophila homologs of these proteins, i.e. CASK and Dlg, are required in the same neuronal subpopulation as APPL for proper long-term memory. Several arguments in favor of an APPL/X11/CASK/Dlg macromolecular complex can be found in previous studies on mammals or Drosophila. In the Drosophila visual system, the MAGUK complex Lin-7/Dlg/CASK is involved in synaptic stabilization and the interaction between CASK and Dlg proteins has been described as direct (Soukup, 2013). The role of CASK in the recruitment of Dlg1 to the membrane in various cell types has been confirmed in a recent study showing that the N-terminal domain of Dlg1 is critical for its interaction with CASK (Porter, 2019). In mammals, the existence of the APP/X11/CASK ternary complex has also been documented, and the regulation of neuronal excitability through potassium Kir2 channels involves a macromolecular complex consisting of Lin-7/SAP97/CASK/X11. Altogether these studies demonstrate that the interaction between the Dlg, CASK and X11 proteins is both possible and functionally relevant for neuronal physiology. Finally, CASK binds to the Dlg protein SAP97 in mammalian hippocampal neurons to regulate its conformation state and thus its role in glutamate receptor trafficking and insertion at the synapse. Thus, the existence of the APPL/X11/CASK/Dlg macromolecular complex is consistent with previously published reports, and even if this study only demonstrate the genetic interaction between APPL, CASK and Dlg for appetitive LTM, it is likely that such a complex exists in Drosophila α'/β' MB neurons. Complementary studies using genetic tools to impair the interactions between these proteins such as overexpression of an interfering peptide corresponding to the N-terminus of Dlg as in Porter (2019) could bring the confirmation that protein-protein interactions between APPL/X11/CASK/Dlg are required for appetitive LTM via synaptic stabilization of the α'/β' neurons. The present study looked at synaptic organization using confocal microscopy and immuno-labeling of either pre-synaptic proteins of the active zone or post-synaptic scaffold proteins. The levels of the analyzed pre- or post-synaptic proteins were not affected by the concomitant knockdown of APPL and MAGUKs in the adult α'/β' neurons, indicating no obvious alterations in MB synaptic structure. It is noted that as expected this study observed a significant decrease in Dlg levels in the MB calyx upon knockdown of APPL;Dlg in α'/β' neurons, demonstrating the efficacy of the knockdown. To determine the requirement of these proteins for pre- or post-synaptic subtle organization, higher-resolution imaging studies would be required. However, such modification might be difficult to observe if, as suggested by the role of CASK and Dlg proteins, the APPL/X11/CASK/Dlg macromolecular complex is involved in synaptic stabilization specifically when synapses are modified during a plastic event, and not as a basal mechanism for synaptic organization and formation as described for APPL and FasII interactions at the NJM (Silva, 2020).

Interestingly, the Dlg protein SAP97 is involved in trafficking the α-secretase ADAM10 to the synapse through direct interaction, consequently regulating APP processing and the production of the neurotrophic and neuroprotective secreted-APPα fragment. Previous work has shown in Drosophila that a secreted fragment of APPL is involved in aversive memory, as well as KUZBANIAN, the Drosophila homolog of ADAM10 (Bourdet, 2015). Therefore, an APPL/X11/CASK/Dlg supramolecular complex could also be involved in recruiting α-secretase at the synaptic site as well as generating sAPPα (Silva, 2020).

The subcellular localization of APP and MAGUK interactions in the KCs is still an open question. In Drosophila, Dlg and CASK are known to be present at both the pre- and postsynaptic compartments. However, APPL has been described mainly in the neuropil of KCs. Furthermore, the APPL binding protein X11 targets APPL to the axonal compartments and excludes it from MB dendrites via endocytosis. These data suggest that the APPL-MAGUKs complexes would be localized in the α'/β' KC axonal compartment (i.e. the α'/β' lobes), which is importantly also the site of the DPM/KC dialog for appetitive LTM consolidation (Silva, 2020).

In conclusion, this work highlights a novel role of APPL and its synaptic partners in appetitive long-term memory in Drosophila. Genetic interactions between APPL and the MAGUKs are critical for appetitive LTM in the α'/β' KCs, a neuronal sub-population known to be involved in the consolidation of appetitive LTM. A model is proposed in which the role of the interactions between APPL, CASK and Dlg might be the synaptic stabilization of the α'/β'-DPM loop through transsynaptic interactions (Silva, 2020).

Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. This study provides a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. Afferent sensory pathways and a large population of neurons were discovered that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). This was combined with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. It was found that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. This study provides the most detailed view to date of biological circuit motifs that support associative learning (Eschbach, 2020).

To behave adaptively in an ever-changing environment, animals must be able to learn new associations between sensory cues (conditioned stimuli, CS) and rewards or punishments (aversive and appetitive unconditioned stimuli, US), and continuously update previous memories, depending on their relevance and reliability (Eschbach, 2020).

Modulatory neurons (for example, DANs) convey information about rewards and punishments, and provide the teaching signals for updating the valence associated with CS in learning circuits across the animal kingdom (for example, the vertebrate basal ganglia or the insect mushroom body, MB). The co-occurrence of CS and modulatory neuron activity tuned only to the received US can support simple associative memory formation. To account for more complex behavioral phenomena, theories have been developed in which learning can be regulated by previously formed associations. According to reinforcement learning theories, learning is driven by errors between predicted and actual US (prediction errors) which are thought to be represented by the activity of DANs. Indeed, in many model organisms, the responses of modulatory neurons have been shown to be adaptive, including monkeys, rodents and insects. Despite recent progress the basic principles by which DAN activity is adaptively regulated and teaching signals are computed are not well understood (Eschbach, 2020).

A prerequisite for the adaptive regulation of modulatory neuron activity is convergence of afferent pathways that convey information about received US with feedback pathways that convey information about previous experiences. A comprehensive synaptic-resolution connectivity map of the feedback circuits that regulate modulatory neurons would provide a basis for understanding how learning is adaptively regulated by prior learning, but it has previously been out of reach (Eschbach, 2020).

Insects, especially in their larval stages, have small and compact brains that have recently become amenable to large-scale electron microscopy (EM) circuit mapping. Both adult and larval insect stages possess a brain center that is essential for associative learning, the MB. The MB contains neurons called Kenyon cells (KCs) that sparsely encode CS, MB modulatory neurons (collectively called MB input neurons, MBINs) that provide the teaching signals and MB output neurons (MBONs) whose activity represents learnt valences of stimuli. In the Drosophila larva, most modulatory neurons are DANs, some are octopaminergic neurons (OANs) and some have unidentified neurotransmitters (simply called MBINs). Modulatory neurons and MBONs project axon terminals and dendrites, respectively, onto the KC axons in a tiled manner, defining MB compartments, in both adult and larval Drosophila. In adult Drosophila, it has been shown that coactivation of KCs and DANs reduces the strength of the KC-MBON synapse in that compartment. Different compartments have been implicated in the formation of distinct types of memories, for example, aversive and appetitive, or short and long term. However, despite a good understanding of the structure and function of the core components of the MB in both adult and larval Drosophila, the circuits presynaptic to modulatory neurons that regulate their activity have remained relatively uncharacterized (Eschbach, 2020).

This study therefore reconstructed all neurons presynaptic to all modulatory neurons in an EM volume that spans the entire nervous system of a first instar Drosophila larva, in which all the core components of the MB were previously reconstructed. This study also determined which individual modulatory neurons are activated by punishments and reconstructed their afferent US pathways from nociceptive and mechanosensory neurons. The neurotransmitter profiles of some of the neurons in the network were characterized and some of the identified structural connections were functionally confirmed. Finally, a model was developed of the circuit constrained by the connectome, the neurotransmitter data and the functional data, and it was used to explore the computational advantages offered by the recently discovered architectural motifs for performing distinct learning tasks (Eschbach, 2020).

Modulatory neurons (for example, DANs) are key components of higher-order circuits for adaptive behavioral control, and they provide teaching signals that drive memory formation and updating. This study provides a synaptic-resolution connectivity map of a recurrent neural network that regulates the activity of modulatory neurons in a higher-order learning center, the Drosophila larval MB. Some of the recently identified structural pathways were functionally tested, and a model of the circuit was developed to explore the roles of these motifs in different learning tasks (Eschbach, 2020).

A large population was discovered of 61 feedback neuron pairs that provide one- and/or two-step feedback from the MBONs to modulatory neurons. Strikingly, it was found that many modulatory neurons receive more than 50% of their total dendritic input from feedback pathways. These results suggest that prior memories as represented by the pattern of MBON activity can strongly influence modulatory neuron activity (Eschbach, 2020).

Learning and memory systems in vertebrates and insects are often organized into distinct compartments implicated in forming distinct types of memories (for example, aversive and appetitive or short and long term). Interestingly, it was found that the majority of the discovered feedback pathways link distinct memory systems, suggesting that the MB functions as an interconnected ensemble during learning. Thus, prior memories formed about an odor in one compartment can influence the formation and updating of distinct types of memories about that odor in other compartments (Eschbach, 2020).

In adult Drosophila, functional connections between some MBONs and DANs have been reported, and some have been shown to play a role in short-term memory formation, long-term memory consolidation, extinction and reconsolidation, or in synchronizing DAN ensemble activity in a context-dependent manner. In some cases, direct MBON-to-DAN connections have been demonstrated. Although direct connections from several MBONs onto DANs exist in the larva, this study found that indirect connections via the feedback neurons account for a much larger fraction of a modulatory neuron's dendritic input than direct MBON synapses. This suggests that adaptive DAN responses may be largely driven by such indirect feedback (Eschbach, 2020).

Some of the one-step within-compartment feedback motifs that were found are analogous to the feedback motifs so far described for the DANs in the vertebrate midbrain. Although the diversity and the inputs of striatal feedback neurons have not yet been fully explored, in the future it will be interesting to determine whether many of the striatal feedback neurons also link distinct memory systems (Eschbach, 2020).

The use of internal predictions can dramatically increase the flexibility of a learning system. This study reveals candidate circuit motifs that could compute integrated predicted value signals across appetitive and aversive memory systems. A prominent motif that was identified is convergence of excitatory and inhibitory connections from MBONs from compartments of opposite valence onto DANs. In naive animals, odor-evoked MBON excitation in all compartments is thought to be similar. However, associative learning selectively depresses conditioned odor drive to MBONs in compartments where modulatory neuron activation has been paired with the odor. It is proposed that by comparing the conditioned odor-evoked MBON excitation in compartments of opposite valence via cross-compartment feedback connections, modulatory neurons compute an integrated predicted value signal across appetitive and aversive domains (Eschbach, 2020).

Convergence of feedback and US pathways could allow the computation of prediction errors An important aspect of reinforcement learning theories is the idea that modulatory neurons compare predicted and actual US (to compute so-called prediction errors) and drive memory formation or extinction depending on the sign of the prediction error. Although Drosophila modulatory neurons have not yet been directly shown to represent prediction errors, adult and larval Drosophila are capable of extinction, and the current study reveals candidate motifs that could support the comparison of expected and actual US. Modulatory neurons were found to receive convergent input from feedback pathways from MBONs and from US pathways. Modulatory neurons could therefore potentially compute prediction errors by comparing inhibitory drive from the feedback pathways with the excitatory drive from the US pathways, or vice versa. Consistent with this idea, some DANs were observed in this model that are inhibited by US alone and activated by CS+ alone, or vice versa (Eschbach, 2020).

This study also reveals that US pathways and feedback pathways converge at two levels: not only at the modulatory neurons themselves, but also at the FB2Ns. Actual and expected outcomes could therefore also be compared by FB2Ns. A recent study in the mouse ventral tegmental area has found that some pre-DAN neurons encoded only actual or only expected reward, whereas others encoded both variables. Thus, both in vertebrates and in insects, comparing predicted and actual outcomes may be a complex computation involving multiple levels of integration that eventually converge onto an ensemble of modulatory neurons (Eschbach, 2020).

An assumption in many reinforcement learning models is that all modulatory neurons receive a global scalar reward prediction error signal. The current study was able to analyze the comprehensive set of inputs of every individual uniquely identifiable modulatory neuron in a learning center. This revealed that each modulatory neuron receives a unique set of feedback inputs that could enable each neuron to compute a unique set of features. Consistent with this, a diversity of adaptive response types in the modulatory neurons was observed in this model This suggests that instead of computing a single global reward prediction error that is distributed to all modulatory neurons, the network uses a range of distinct compartmentalized and distributed teaching signals (Eschbach, 2020).

The connectivity and modeling studies revealed two architectural features of the circuit that provide input to the modulatory neurons that increase its performance and flexibility on learning tasks. The first is the multilevel feedback architecture that includes not only the previously known direct MBON feedback, but also multiple levels of indirect feedback. The second is the extensive set of cross-compartment connecti,.ons. Modeling suggests that these motifs support improved performance on complex tasks that require the computation of variables such as predictions, prediction errors and context (Eschbach, 2020).

In summary, this study presents a complete circuit diagram of a recurrent network that computes teaching signals in a biological system, providing insights into the architectural motifs that increase its computational power and flexibility. The connectome-constrained model provides numerous predictions that can be tested in the future in a tractable model organism, for which genetic tools can be generated to monitor and manipulate individual neurons. The connectome, together with the functional and modeling studies, therefore provides exciting opportunities for elucidating the biological implementation of reinforcement learning algorithms (Eschbach, 2020).

In Drosophila, the mushroom bodies (MB) constitute the central brain structure for olfactory associative memory. As in mammals, the cAMP/PKA pathway plays a key role in memory formation. In the MB, Rutabaga adenylate cyclase acts as a coincidence detector during associative conditioning to integrate calcium influx resulting from acetylcholine stimulation and G protein activation resulting from dopaminergic stimulation. Amnesiac encodes a secreted neuropeptide required in the MB for two phases of aversive olfactory memory. Previous sequence analysis has revealed strong homology with the mammalian pituitary adenylate cyclase-activating peptide (PACAP). This study examined whether amnesiac is involved in cAMP/PKA dynamics in response to dopamine and acetylcholine co-stimulation in living flies. Experiments were carried out with both sexes, or with either sex. The data show that amnesiac is necessary for the PKA activation process that results from coincidence detection in the MB. Since PACAP peptide is cleaved by the human membrane neprilysin hNEP, an interaction was sought between Amnesiac and Neprilysin 1 (Nep1), a fly neprilysin involved in memory. When Nep1 expression is acutely knocked down in adult MB, memory deficits displayed by amn hypomorphic mutants are rescued. Consistently, Nep1 inhibition also restores normal PKA activation in amn mutant flies. Taken together the results suggest that Nep1 targets Amnesiac degradation in order to terminate its signaling function. This work thus highlights a key role for Amnesiac in establishing within the MB the PKA dynamics that sustain middle-term memory formation, a function modulated by Nep1 (Turrel, 2020).

Associative learning, which temporally pairs a conditioned stimulus (CS) to an unconditioned stimulus (US), is a powerful way of acquiring adaptive behavior. At the molecular and cellular levels, the association between CS and US is mediated by coincidence detection mechanisms that reflect the superadditive activation of a molecular pathway in the presence of both stimuli. One of the major coincidence detectors is the cAMP/PKA pathway, which depends on Type-I adenylate cyclases stimulated by both calcium/calmodulin, via acetylcholine signaling representing the CS, and G-protein coupled to dopamine metabotropic receptors activated by dopaminergic neurons encoding the US (Turrel, 2020).

In Drosophila, the mushroom bodies (MB) constitute the central integrative brain structure for olfactory memory. The MB are composed of 4000 intrinsic neurons called Kenyon cells (KC), and classed into three subtypes whose axons form two vertical (a and a9) and three medial (b, b9, and g) lobes. Using a classical conditioning paradigm in which an odorant (CS) was paired to electric shocks (US), Bouzaiane (2015) revealed that flies are capable of forming six discrete memory phases reflected at the neural network level. Among these phases are middle-term memory (MTM) and long-term memory (LTM), which are both encoded in a/b KC. As in mammals, the fly cAMP/PKA pathway plays a key role in associative memory, wherein the adenylate cyclase Rutabaga (Rut) acts as a coincidence detector in the MB to associate the CS and US pathways (Turrel, 2020).

The amnesiac Drosophila mutant (amn) was isolated in a memory defect behavioral screen. As with other components of the cAMP/PKA pathway involved in Drosophila memory, amn is expressed in the MB. It was recently showen that amn expression in the MB is specifically required for MTM and LTM (Turrel, 2018). amn encodes a neuropeptide precursor with a signal sequence. Sequence analyses suggest the existence of three peptides, with one of them homologous to mammalian pituitary adenylate cyclase-activating peptide (PACAP). PACAP is widely expressed throughout the brain, acting as a neuromodulator or neurotrophic factor through activation of G-protein-linked receptors to regulate a variety of physiological processes through stimulation of cAMP production. Furthermore, PACAP may exert a role in learning and memory (Turrel, 2020).

After its release, a neurotransmitter's action is terminated either by diffusion, re-uptake by the presynaptic neuron, or enzymatic degradation. In contrast, neuropeptide signaling is exclusively terminated by enzymatic degradation. Possible enzyme candidates include neprilysins, type 1 metalloproteinases whose main function is the degradation of signaling peptides at the cell surface (Turner, 2001). Indeed, the human neprilysin hNEP is capable of cleaving a PACAP neuropeptide. Drosophila express four neprilysins that are all required for MTM and LTM, establishing that neuropeptide degradation is a central process for memory formation. Among the four neprilysins, Neprilysin 1 (Nep1) is the only one whose expression is required for MTM in the MBx (Turrel, 2020).

This study aimed to confirm whether AMN intervenes in memory by modulating cAMP concentration, as suggested by its sequence homology. For this, PKA dynamics were analyzed in the MB vertical lobes. The results show that amn mutant brains fail to display PKA activation in the a lobe in response to co-application of dopamine and acetylcholine. Whether Nep is involved in terminating AMN action was examined. Using RNAi, it was shown that Nep1 knock-down restores normal MTM and normal PKA dynamics in amn mutants, establishing a functional interaction between Nep1 and AMN in the MB (Turrel, 2020).

Previous work has shown that AMN expression is required in the MB for Drosophila memory. This study established that AMN expression in the MB is necessary for the synergistic activation of PKA observed on co-stimulation by dopamine and acetylcholine in the a lobe, a process that is thought to mimic the coincidence detection event underlying memory formation. Furthermore, the data demonstrate a functional interaction between AMN and Nep1, suggesting that Nep1 targets AMN degradation, thereby terminating AMN signaling (Turrel, 2020).

Six different aversive memory phases that are spatially segregated have been described in Drosophila (Bouzaiane, 2015). Their formation relies on distinct neuronal circuits, as well as distinct molecular and cellular mechanisms. rut mutants are impaired in specific memory phases, including short-term memory (STM), encoded in g KC, and MTM encoded in a/b KC. It was previously shown that Rut expression restricted to g KC is sufficient to restore STM, but not MTM, in rut mutant flies. It is thus likely that distinct Rut-mediated coincidence detection events occur in parallel in g and a/b KC, resulting in STM and MTM formation, respectively. Interestingly, mutants expressing a reduced amn level display normal STM. Thus, AMN is most likely not required for the coincidence detection process that leads to STM, a process that remains to be identified. Using in vivo imaging, previous work showed that coapplication of dopamine and acetylcholine induces a strong synergistic PKA response, which is Rut dependent and occurs specifically in MB vertical lobes. This study shows that this coincident PKA activation in the a lobe is abolished in amn mutants, while neither calcium signaling nor cAMP signaling following dopaminergic stimulation alone are altered. It is proposed that PKA activation mimics the coincidence detection event that occurs in a/b KC during MTM formation, and that AMN intervenes in this process by enabling a sustained Rut-mediated PKA activation in the MB a lobe (Turrel, 2020).

AMN might thus act at a step that ranges from the initial coincidence detection event that provokes Rut activation, to the final level of PKA activation. This is consistent with previous reports that AMN and DC0, the fly PKA catalytic subunit, act in a common pathway, and that AMN function is upstream of DC0 function. If AMN plays a role posterior to the coincidence detection event, it could be involved in an increase in cAMP concentration through the inhibition of phosphodiesterases that degrade cAMP. Indeed, dopamine receptors positively coupled to adenylate cyclases are equally distributed in all MB lobes as are DC0 and Rut, whereas 100 mM dopamine only induces a PKA response in the a lobe. This spatial control is achieved by the cAMP-specific phosphodiesterase Dunce (Dnc) which preferentially degrades cAMP in the b and g lobes, thus restricting high dopamine-induced PKA activation to the a lobe. AMN could thus be involved in the specific inhibition of Dnc in the a lobe (Turrel, 2020).

One attractive alternative hypothesis is that AMN action could take place at the level of Rut activation itself. Indeed, the fact that one of the AMN peptides is homologous to PACAP suggests that AMN might play a role in activating the adenylate cyclase Rut through G-protein-coupled receptors. This hypothesis fits with sequence prediction , and is supported by studies showing that AMN is functionally related to human PACAP. It was initially reported that Rut is activated by the application of human PACAP-38 (Zhong, 1995), and later shown that bath application of PACAP-38 rescues L-type current deficiency in amnX8 larval muscle fibers. Such rescue is abolished by application of an antagonist to Type-I PACAP-receptor as well as by application of an inhibitor of AC (Turrel, 2020).

Although STM and MTM both rely on the cAMP/PKA pathway, not only these processes occur in separate KC, but while STM is instantaneously acquired, MTM is acquired in a dynamic fashion following a two-step mechanism. It is proposed that AMN function is specifically required in the incremental build-up of MTM by boosting Rut activation following the initial event of coincidence detection, namely the first CS/US association of the training protocol. In this model, this first association results in an initial moderate level of Rut activation, followed by a moderate level of PKA activation (Turrel, 2020).

This moderate level of PKA activation does not mediate MTM formation and is below detection threshold. It is hypothesized that this initial increase in PKA activity, directly or indirectly, triggers the second step of the process, namely AMN secretion, and thus generate an activation loop whereby AMN activates Rut, hence creating a much higher level of Rut activation and subsequent high levels of PKA activation that is observable with the AKAR2 probe. MTM formation would rely on an AMN-dependent PKA-activation loop terminated on AMN degradation by Nep1 (Turrel, 2020).

One previous study has indicated that human PACAP is a substrate for hNEP (Gourlet, 1997), and this present work in Drosophila describes a functional interaction between AMN and Nep1. Importantly, whereas Nep1 knock-down rescues the amn mutant memory phenotype in a genetic context where the AMN level is reduced to ~50% versus wild-type flies (heterozygous for the amn null allele), it fails to do so in a genetic context where AMN is absent (i.e., in flies hemizygous for the amn null allele). Namely, the memory rescue observed on Nep1 inhibition is dependent on the presence of AMN, suggesting that this latter is targeted by Nep1. While a biochemical confirmation of this hypothesis would be welcome, it is technically difficult to achieve. Specifically, not only are AMN antibodies not available, but amn mRNA is expressed at very low levels, indicating that AMN peptide may be very scarce (Turrel, 2020).

The observation that the AMN peptide may be targeted by Nep1 is in agreement with a neuromodulatory function. Once released, a signaling molecule must be removed from its site of action to prevent continued stimulation, and to allow new signals to propagate. If neurotransmitter's action is terminated either by diffusion, re-uptake by the presynaptic neuron, or enzymatic degradation, signaling neuropeptides are specifically removed by degradation. The intensity and duration of neuropeptide-mediated signals are thus controlled via the cleavage of these neuropeptides by peptidases like neprilysins. Despite a few exceptions, neprilysins occur as integral membrane endopeptidases whose catalytic site faces the extracellular compartment. It is hypothesized that on conditioning, AMN is secreted by the KC to participate in Rut activation via G-protein-coupled receptors, and is ultimately removed from the extracellular compartment by Nep1 anchored at the KC membrane. Importantly, AMN expression in the MB restores normal PKA dynamics in amn null mutant flies, suggesting that the AMN peptide secreted by the MB on conditioning should act in an autocrine-like way to sustain Rut activity in the a/b neurons. Interestingly, the effects of neuropeptide transmitters are very diverse and often long-lived, which fits well with the specific involvement of AMN peptide in non-immediate memory phases via sustained PKA activation (Turrel, 2020).

Up to date, fly neprilysins have been involved in several behaviors: in the control of circadian rhythms, via hydrolysis of the pigment dispersing factor neurotransmitter, and in the control of food intake via cleavage of insulin-like regulatory peptides. In the latter study, it was shown that both Neprilysin 4 knock-down and overexpression in the larval CNS cause reduced food intake (Hallier, 2016). In a similar way, this study shows that both Nep1 knock-down and overexpression in a/b KC impairs MTM, consistent with the need for a proper control of AMN levels. It is suggested that Nep1 overexpression results in amn loss of function, whereas Nep1 knock-down causes the prolongation of AMN action, thus generating a prolonged activation of the cAMP/PKA pathway, a process deleterious for memory. This is in agreement with a previous study demonstrating that overexpressing DC0 in the MB impairs MTM (Turrel, 2020).

In conclusion, this study reports an acute role for AMN in memory formation via the PKA pathway in the a/b MB neurons, a function modulated by Nep1. These results thus support a role for AMN as an activating adenylate cyclase peptide, much like the role of PACAP, bringing clarity to the role PACAP may play in memory consolidation in mammals (Turrel, 2020).

Formation of short term memory is energetically costly and by the reason of restricted availability of food or fluctuations in energy expanses, efficient metabolic homeostasis modulating different needs like survival, growth, reproduction, or investment in longer lasting memories is crucial. Whilst equipped with cellular and molecular pre-requisites for formation of a protein synthesis dependent long-term memory (LTM), its existence in the larval stage of Drosophila remains elusive. Considering it from the viewpoint that larval brain structures are completely rebuilt during metamorphosis, and that this process depends completely on accumulated energy stores formed during the larval stage, investing in LTM represents an unnecessary expenditure. However, as an alternative, Drosophila larvae are equipped with the capacity to form a protein synthesis independent so-called larval anaesthesia resistant memory (lARM), which is consolidated in terms of being insensitive to cold-shock treatments. Motivated by the fact that LTM formation causes an increase in energy uptake in Drosophila adults, this study tested the question of whether an energy surplus can induce the formation of LTM in the larval stage. Surprisingly, increasing the metabolic state by feeding Drosophila larvae the disaccharide sucrose directly before aversive olfactory conditioning led to the formation of a protein synthesis dependent longer lasting memory. Moreover, formation of this memory component is accompanied by the suppression of lARM. It was ascertained that insulin receptors (InRs) expressed in the mushroom body Kenyon cells suppresses the formation of lARM and induces the formation of a protein synthesis dependent longer lasting memory in Drosophila larvae. Given the numerical simplicity of the larval nervous system this work offers a unique prospect to study the impact of insulin signaling on the formation of protein synthesis dependent memories on a molecular level (Eschment, 2020).

Active forgetting is an essential component of the memory management system of the brain. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. This study identified a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval (Sadandal, 2021).

Memory formation, consolidation and retrieval are well-known functions that support memory expression; however, the processes that limit these functions -- including forgetting -- are less understood. Forgetting has been characterized as either passive or active, and is crucial for memory removal, flexibility and updating. Memory may be removed completely, resulting in permanent forgetting; or temporarily irretrievable, resulting in transient forgetting (Sadandal, 2021).

One form of active forgetting-known as intrinsic forgetting-involves one dopamine neuron (DAN) that innervates the γ2α'1 compartment of the axons of mushroom body neurons (MBNs) and the dendrites of the downstream, compartment-specific mushroom-body output neurons (MBONs). This DAN resides in a cluster of 12 DANs in each brain hemisphere that is known as the protocerebral posterior lateral 1 (PPL1) cluster. Current evidence indicates that the ongoing activity of these DANs after aversive olfactory conditioning slowly and chronically erodes labile and nonconsolidated behavioural memory, as well as a corresponding cellular memory trace that forms in the MBONs. This intrinsic forgetting mechanism is shaped by external sensory stimulation and sleep or rest, and is mediated by a signalling cascade in the MBNs that is initiated by the activation of the dopamine receptor DAMB, which leads to the downstream activation of the actin-binding protein Cofilin and the postulated reorganization of the synaptic cytoskeleton (Sadandal, 2021).

By contrast, there is little understanding of the mechanisms that arbitrate transient forgetting. Neuropsychological studies of failures or delays in retrieval in humans have primarily focused on lexical access. Phonological blockers or interfering stimuli produce a tip-of-the-tongue state-the failure to recall the appropriate word or phrase. Tip-of-the-tongue states are resolved when the distracting signals dissipate. Several brain regions have been implicated in tip-of-the-tongue states from functional magnetic resonance imaging studies, but the neurobiological mechanisms that produce a temporary state of impaired retrieval are unknown. This study offers an entry point into this area of brain function (Sadandal, 2021).

Memory retrieval has been proposed to consist of an interplay between internal or external cues and memory engrams, with cue-induced reactivation of engrams across multiple regions of the brain facilitating memory expression. But a central question about this process is how interfering stimuli temporarily block memory retrieval, resulting in transient forgetting. This study offers insights into one such mechanism. Behavioural and functional imaging data reveal that PPL1-α2α'2, working through the DAMB receptor expressed in the α2α'2 MBN axonal compartment, mediates the transient forgetting of PSD-LTM. This effect occurs without altering a cellular memory trace in the postsynaptic MBON-α2sc. This process can be triggered by distracting stimuli, illustrating a neural-genetic-environmental interplay that modifies memory expression (Sadandal, 2021).

This study considered why the cellular memory trace remains unaffected by DAN stimulation despite the occurrence of behavioural forgetting. Because blocking synaptic output from MBON-α2sc reduces PSD-LTM expression, the simplest hypothesis posits that cellular memory traces form with conditioning in the MBON in addition to the cytoplasmic Ca2+-based memory trace that was detected in this study. This is expected: neurons undergo broad changes in physiology as they adopt new states, so it is plausible that such plastic mechanisms-especially ones that gate synaptic release-are inactivated by DAN activity while leaving the Ca2+-based memory trace intact (Sadandal, 2021).

The discovery that loss of function of DAMB leads to enhanced PSD-LTM was surprising, because of a previous study reporting that this insult attenuates PSD-LTM. The experiments argue strongly that DAMB functions normally to suppress expression of PSD-LTM. However, this leads to the question of why a receptor involved in transient forgetting would lead to enhanced PSD-LTM when inactivated. Previous experiments have shown that PPL1-α2α'2-similar to PPL1-γ2α'1-exhibits ongoing activity, leading to a slow release of dopamine onto MBNs. This activity should slowly degrade or suppress existing memory so that when the receptor is inactivated memory expression is enhanced (Sadandal, 2021).

PPL1-α2α'2 has no important role in the forgetting of labile nonconsolidated memory. Instead, previous studies have identified a different DAN (PPL1-γ2α'1) as having a role in this process and the apparent erasure of the downstream cellular memory trace-perhaps an indication of 'permanent forgetting'. This process is modulated by internal and external factors, and is mediated by key molecules expressed in the MBN that receive PPL1-γ2α'1 input. No robust decrement was found in expression of PSD-LTM after PPL1-γ2α'1 stimulation, which points to the existence of two separate dopamine-based circuits for permanent and transient forgetting. This functional separation may indicate a fundamental principle in the organization of circuits that mediate several forms of forgetting (Sadandal, 2021).

However, the DAMB receptor is used for both permanent and transient forgetting. DAMB is widely expressed across the MBN axons but alters synaptic plasticity differently across MBN compartments. It is possible that DAMB signalling may be distinct for the two forms of forgetting. DAMB preferentially couples with Gq, the knockdown of which inhibits the potent erasure of memory, but its potential role in transient forgetting is unknown. The scaffolding protein Scribble orchestrates the activities of Rac, Pak and Cofilin, all of which are important for the permanent forgetting pathway. However, Scribble knockdown or inhibition of Rac1 does not enhance the PSD-LTM as is the case in DAMB-knockdown flies, which suggests that this scaffolding signalosome does not have a large role in transient forgetting. In summary, the two distinct forms of forgetting-transient and permanent-share a dopaminergic mechanism and a common dopamine receptor, but differ in upstream and downstream neural circuits and in downstream signalling pathways within MBNs (Sadandal, 2021).

Forgetting is an essential component of the brain's memory management system, providing a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood. This study shows that the memory suppressor gene, sickie, functions in a single dopamine neuron (DAn) by supporting the process of active forgetting in Drosophila. RNAi knockdown (KD) of sickie impairs forgetting by reducing the Ca(2+) influx and DA release from the DAn that promotes forgetting. Coimmunoprecipitation/mass spectrometry analyses identified cytoskeletal and presynaptic active zone (AZ) proteins as candidates that physically interact with Sickie, and a focused RNAi screen of the candidates showed that Bruchpilot (Brp)-a presynaptic AZ protein that regulates calcium channel clustering and neurotransmitter release-impairs active forgetting like sickie KD. In addition, overexpression of brp rescued the impaired forgetting of sickie KD, providing evidence that they function in the same process. Moreover, this study showed that sickie KD in the DAn reduces the abundance and size of AZ markers but increases their number, suggesting that Sickie controls DAn activity for forgetting by modulating the presynaptic AZ structure. These results identify a molecular and circuit mechanism for normal levels of active forgetting and reveal a surprising role of Sickie in maintaining presynaptic AZ structure for neurotransmitter release (Zhang, 2022).

Forgetting, the flip side of memory acquisition and consolidation, is an essential component of the brain's memory management system that provides a balance to memory formation processes by removing unused or unwanted memories, or by suppressing their expression. However, the molecular, cellular, and circuit mechanisms underlying forgetting are poorly understood (Zhang, 2022).

Previous studies showed that dopamine (DA) and its downstream signaling molecules in postsynaptic neurons are essential for active forgetting and transient forgetting in Drosophila. Small subsets of DA neurons (DAn) within the PPL1 cluster of 12 DAn that innervate the Drosophila mushroom body neurons (MBn) mediate forgetting. Blocking the synaptic output from these DAn after learning inhibits forgetting, whereas stimulating the DAn increases forgetting. Moreover, external factors and internal states, such as locomotor activity, stress, and arousal increase the ongoing activity of these DAn and accelerate forgetting. Conversely, sleep or rest after learning, which decreases the ongoing activity of these DAn, inhibits forgetting (Berry, 2015). This DA-based forgetting is mediated by a DA receptor, DAMB, expressed on the postsynaptic MBn, and requires a downstream signaling pathway involving Scribble, Rac1, and Cofilin for actin remolding (Zhang, 2022 and references therein).

A large RNA interference (RNAi) screen of ∼3,500 genes identified sickie as a memory suppressor genes in Drosophila. It was classed as such because knockdown (KD) of sickie led to increased memory expression. Sickie was initially found to be required for the nuclear translocation of Relish for normal innate immune responses using cultured Drosophila S2 cells. Its homologs, NAV2 in humans and Unc53 in Caenorhabditis elegans, were reported to control neurite outgrowth and the anteroposterior directional guidance of some migratory cells. Other studies also suggested that NAV2 is an oncogene whose expression level is closely related to several human tumors. A recent study found that Sickie regulates F-actin-mediated axon growth of Drosophila MBn (Abe, 2014). However, sickie's role in learning and memory was not explored, and its mechanism for memory suppression was unknown (Zhang, 2022).

This study shows that sickie is required in a single DAn for active forgetting, but not for memory acquisition or consolidation. Sickie KD impairs forgetting by reducing the ongoing activity of DAn. Coimmunoprecipitation and mass spectrometry (co-IP/MS) experiments identify presynaptic active zone (AZ) proteins as the top candidates that interact with Sickie. An RNAi screen of the top candidates along with additional experiments reveal that Sickie interacts physically and genetically with Bruchpilot (Brp) to mediate forgetting through the DAn. Moreover, sickie KD was shown to alter the structure of the presynaptic AZ. Taken together, these results suggest a model whereby Sickie maintains the normal structure and function of presynaptic AZ of a single DAn for DA-based forgetting, through its interaction with presynaptic AZ protein Brp and regulating neurotransmitter release (Zhang, 2022).

This study presents data showing that sickie function is required in a single DAn for the active forgetting of olfactory memory. It does this by regulating the ongoing release of DA, by interacting with and altering the function of the important presynaptic protein, Brp, in the maturation or stability of T-bars at presynaptic AZ. It is most interesting that of the dozen or more DAn that innervate the axons of the MBn in defined physical segments, it is the MP1 DAn and its associated target-the heel of the MB neuropil-that has the most pronounced role in the process of active forgetting. This reinforces the conclusion that the 12 DAn in the PPL1 cluster have distinct and specialized functions. Supporting this conclusion, a prior study has shown that TrpA1 activation of MP1 DAn, but not other DAn in the PPL1 cluster, after training is sufficient to induce forgetting. Thus, the specific role for sickie in MP1 DAn for forgetting results from the intersection of sickie's role in regulating ongoing DAn activity and the unique requirement of MP1 DAn for active forgetting. Most importantly, the results identify a player in the process of active forgetting and in the AZ protein machinery that regulates neurotransmitter release (Zhang, 2022).

The results also open the question about the developmental and physiological roles that have been described for sickie. sickie was previously reported to interact genetically with rac1, slingshot, and cofilin to regulate F-actin-mediated axon growth of Drosophila MBn. However, behavioral data after temporal KD and structural data argue against a similar developmental role for sickie in DAn, and point to an additional, physiological role in adult DAn after axon extension. Nevertheless, it is intriguing that sickie genetically interacts with rac1 and cofilin for developmental processes. These two genes are also involved in the MBn for active forgetting. Thus, the genes and their protein products can exhibit functional interactions that depend on cell type and developmental state. Neither Rac1, Slingshot, or Cofilin were observed among the candidates from co-IP/MS data, indicating that either Sickie has no direct physical interaction with these proteins in the adult head, or the interaction is too weak, sparse, or transient to be captured through antibody pulldowns (Zhang, 2022).

However, it is possible that Sickie may mediate more subtle morphological changes in synapse structure. Indeed, putative physical interactors and protein clusters were discovered that point to possible roles in regulating fine synapse morphology. For example, the actin cross-linking protein Actn is among the top 10% of our MS candidates along with other cytoskeletal proteins like α- and &beta-Spec. This observation suggests a role for Sickie in interacting with cytoskeletal proteins to control synapse structure. RNAi KD experiments of these cytoskeletal protein-encoding genes did not uncover a behavioral phenotype, but this could be due to lack of potency of the RNAi used for this focused screen (Zhang, 2022).

Although it cannot be ruled out that sickie KD causes mild morphological changes of the DAn presynaptic terminals by interacting with cytoskeletal proteins to decrease the ongoing activity of the neuron, the results point to altered DA release through Sickie's interaction with Brp. Brp was a top candidate from co-IP/MS experiments; its KD in MP1 DAn produced the same elevated memory phenotype as sickie KD. Brp abundance was reduced in sickie KD flies, and its overexpression rescued the forgetting phenotype of sickie KD. Brp is a component of the T-bar in the presynaptic AZ and is required for the proper clustering of Ca2+ channels at the AZ. It could be that the lack of proper clustering of the Ca2+ channels leads to the decrease in ongoing Ca2+ influx detected in MP1 DAn terminals. Complete loss-of-function of brp dramatically decreases the evoked release at low stimulation frequency, but does not abolish the release, indicating that the protein participates in release but is not an absolute requirement. In a parallel way, sickie KD impairs ongoing DAn release but not electric shock-induced activity. These observations align with one another, leading to the model that sickie KD reduces Brp abundance in MP1 DAn, reducing Ca2+ influx from ongoing activity, reducing DA release during ongoing activity, and impairing forgetting (Zhang, 2022).

In addition to its role in Ca2+ channel clustering, Brp is required for the tethering of synaptic vesicles in the AZ cytomatrix via its C-terminal sequence at the neuromuscular junction of Drosophila larvae, potentially through its genetic interaction with the SNARE regulator, Complexin. This protein network and gene ontology analysis also uncovered a protein cluster for synaptic transmission involving Brp, Syt1, Syn, Dlg1, and RhoGAP100F. Syt1 is a synaptic vesicle protein that is essential for Ca2+-dependent release, whereas Syn functions for synaptic vesicle clustering and synaptic transmission. Thus, these studies may also suggest a role for Sickie in synaptic vesicle trafficking and tethering for neurotransmitter release, either by interacting with Brp or other proteins. Because of the highly conserved structure of the presynaptic AZ and neurotransmitter release machinery across species, the findings suggest a possible but unexplored role for sickie's homologs in neurotransmitter release and forgetting in other species (Zhang, 2022).

Memory formation and forgetting unnecessary memory must be balanced for adaptive animal behavior. While cyclic AMP (cAMP) signaling via dopamine neurons induces memory formation, this study reports that cyclic guanine monophosphate (cGMP) signaling via dopamine neurons launches forgetting of unconsolidated memory in Drosophila. Genetic screening and proteomic analyses showed that neural activation induces the complex formation of a histone H3K9 demethylase, Kdm4B, and a GMP synthetase, Bur, which is necessary and sufficient for forgetting unconsolidated memory. Kdm4B/Bur is activated by phosphorylation through NO-dependent cGMP signaling via dopamine neurons, inducing gene expression, including kek2 encoding a presynaptic protein. Accordingly, Kdm4B/Bur activation induced presynaptic changes. These data demonstrate a link between cGMP signaling and synapses via gene expression in forgetting, suggesting that the opposing functions of memory are orchestrated by distinct signaling via dopamine neurons, which affects synaptic integrity and thus balances animal behavior (Takakura, 2023)

Animal behaviors are shaped by past experiences, which are stored as memories in the brain. Since the surrounding environment changes occasionally, animals should be equipped with neuronal and molecular mechanisms that actively decay memories for the subsequent behavioral adaptation, while retaining robust and reliable memories by a process known as consolidation via gene expression. The active process of memory decay, defined as "forgetting," is well documented at the molecular level in Drosophila. In the olfactory aversive training paradigm, where an odor is associated with electric shocks, associative memory is formed in the olfactory memory center, mushroom body (MB) neurons, mediated by a coincident activation of dopamine neurons. The olfactory aversive memory rapidly undergoes forgetting within 3 h, which is mediated by a member of the Rho GTPase family of small G proteins, Rac1, and an upstream kinase of mitogen-activated protein kinase, Raf. Although inhibition of the Rac1-forgetting pathway prolongs memory, the memory eventually decays, raising the question of whether memory passively decays over time or whether an additional active process of memory decay exists. Elucidating the neuronal and molecular mechanisms underlying the conflicting functions of memory, consolidation, and decay will help to understand the dynamic and adaptive nature of animal behavior (Takakura, 2023).

The mechanisms of memory formation must converge with those of memory decay. Dopamine and its G-protein-coupled receptor activate adenylate cyclase (AC), leading to an increase in cAMP, which is one of the general rules in learning and memory. At the synaptic level in Drosophila, synapses between MB and the postsynaptic neurons, MB output neurons, undergo synaptic depression. How synaptic plasticity in MBs is linked to the memory decay mechanisms: particularly how synaptic depression could be recovered by forgetting mechanisms or other mechanisms of memory decay, if any, remains unclear (Takakura, 2023).

While gene expression has been primarily associated with memory consolidation, this study demonstrated a causal link between gene expression and synaptic plasticity for memory decay, in which activation is independent of Rac1 or Raf. Since this decay follows the time of Rac1-dependent forgetting and is also mechanistically distinct from it, this memory decay has been termed 'gene expression-based forgetting'. Gene expression-based forgetting is mediated by cGMP (cyclic guanine monophosphate) signaling in MB neurons, which is stimulated by dopamine neurons, resulting in gene expression in MB neurons and presynaptic changes. These results demonstrate another role of gene expression via cGMP signaling from dopamine neurons, apart from cAMP signaling, that could fill in the gap in the understanding between memory formation and decay (Takakura, 2023).

Memory undergoes retention and decay, and their imbalance negatively affects behavioral adaptation. Understanding how intracellular mechanisms orchestrate these seemingly opposite functions of memory and how those mechanisms are linked to synaptic plasticity presents challenges in explaining animal behavior. This study focused on gene expression mediated by an epigenetic regulator, Kdm4B. It was demonstrated that gene expression-based forgetting is controlled by cGMP signaling from dopamine neurons through activation of Kdm4B/Bur. Given that cAMP signaling from dopamine neurons is a major pathway for memory formation, these findings suggest a simple model with two signaling pathways for conflicting memory processes: cAMP signaling for memory formation and cGMP signaling for gene expression-based forgetting, both of which are mediated by dopamine neurons (see Activation of Kdm4B/Bur results in presynaptic changes.). Furthermore, the presynaptic changes through activation of Kdm4B/Bur illustrated the pathway from gene expression to synapses (Takakura, 2023).

Memory formation is mediated by the specific sets of MB synapses which are activated by the odor presentation, when dopamine neurons are simultaneously activated, supporting the synapse specificity. In contrast, gene expression-based forgetting may not be equipped with synaptic specificities. Activation of dopamine neurons without activation of MB neurons was sufficient to induce the interaction of Kdm4B/Bur and kek2 mRNA expression. In addition, the expression of Bur-S139E, which binds to Kdm4B, increased the number and size of presynapses in naive flies. In this case, anterograde memory interference could occur: when two learning events happened closely enough, the latter learning could be impaired by the previous activation of gene expression-based forgetting. However, this may not be the case as the latter learning itself activates gene expression-based forgetting, and preactivation of this pathway may not have any additive effects. In addition, retrograde memory interference may also occur, and preexisting memory may be nonspecifically decayed by new learning. This would not be the case if the preexisting memory is consolidated as consolidated memory via spaced training was resistant to the Bur-S139E expression. Thus, although gene expression-based forgetting may occur synapse-nonspecifically, animals would not have significant unanticipated memory interference (Takakura, 2023).

In Drosophila, rapid decay of memory is an active process mediated by dopamine, Rac1, or Raf, which is defined as memory forgetting. While Rac1 targets ASM within 3 h after learning, Kdm4B/Bur-mediated forgetting effectively targets ASM at a later time point. The Rac1- and Raf-mediated memory forgetting pathways did not affect the Kdm4B/Bur interaction, indicating that activation of Kdm4B/Bur is independent of Rac1. However, memory retention was not synergistically enhanced by the expression of Rac1-DN and the knockdown of Kdm4B or bur. This could be a ceiling effect in memory enhancement. Alternatively, Kdm4B/Bur-mediated forgetting could only target synapses destabilized by Rac1. Rac1 activates cofilin and changes the dynamics of the actin cytoskeleton, which may be required for forgetting via Kdm4B/Bur (Takakura, 2023).

The current study highlighted the differential functions of second messenger signaling pathways, cAMP for memory consolidation and cGMP for gene expression-based forgetting through dopamine neurons. A recent report demonstrated that increased soluble guanylyl cyclase (sGC) expression during aging is related to age-related memory impairment, suggesting that cGMP pathway for gene expression-based forgetting can change according to the physiological state. Although regulation of gene expression by Bur acting as GMP synthetase was unexpected, GMP synthetase has been described in gene regulation and control of stability of nuclear protein. It could be important to investigate the biological meaning of the convergence of GMP synthetase to gene expression-mediated forgetting in the future study (Takakura, 2023).

The mushroom body (MB) of Drosophila melanogaster has multiple functions in controlling memory and behavior. This study systematically probed the behavioral contribution of each type of MB output neuron (MBON) by blocking during acquisition, retention, or retrieval of reward or punishment memories. The contribution was evaluated using two conditioned responses: memory-guided odor choice and odor source attraction. Quantitative analysis revealed that these conditioned odor responses are controlled by different sets of MBONs. The valence of memory, rather than the transition of memory steps, has a larger impact on the patterns of required MBONs. Moreover, it was found that the glutamatergic MBONs forming recurrent circuits commonly contribute to appetitive memory acquisition, suggesting a pivotal role of this circuit motif for reward processing. These results provide principles how the MB output circuit processes associative memories of different valence and controls distinct memory-guided behaviors (Ichinose, 2021).

Distinct MBONs are required for odor choice guided by appetitive and aversive memories Neural structures in the central nervous system can have functional roles across a wide range of cognitive tasks. The mushroom body (MB) in Drosophila melanogaster has served as a unique circuit model to study such pleiotropy. So far, many aspects of behavior and physiology, such as sensory processing, learning, and sleep/arousal, have been shown to be under the control of the MB. Particularly, MBs play important roles in different forms of associative learning: appetitive and aversive learning of odor and color. Transient blockade of the MB intrinsic neurons, Kenyon cells (KCs), revealed that neurotransmission from the MB controls not only retrieval but also acquisition and retention of appetitive and aversive olfactory memories. Investigation of MB output should thus be a key to understand how MBs process diverse memory-related functions (Ichinose, 2021).

The anatomy of the MB output is well characterized. The entire MB lobes can be subdivided into 15 compartments, based on the dendritic arbors of 21 types of MB output neurons (MBONs). These MBONs are post-synaptic to KCs and dopamine neurons and project their axons to defined neuropils of the brain. Selective requirement and plasticity in specific KC-MBON synapses during memory processing further corroborate distinct functions of the MB. However, because these studies have been focused on specific MBON subtypes and based on experiments under different conditions, direct comparisons are difficult. Therefore, this study sought to systematically examine the pattern of the MBON requirements under the same experimental condition (Ichinose, 2021).

To this end, each MBON type was blocked using a set of 13 split-GAL4 driver lines that collectively cover all the identified MBON subtypes. The temperature-sensitive, dominant-negative dynamin Shibire^ts1 (Shi^ts1) was expressed by each split-GAL4 driver. Using different temperature-shifting protocols, the blockade of target MBONs was restricted to each step of memory processing-acquisition, retention, or retrieval-or the blockade (permissive control) was omitted. Performance indices (PIs) were calculated based on the conditioned odor choice at 1 h after appetitive or aversive conditioning using sucrose reward or electric shock punishment. Flies were starved for appetitive conditioning and test (Ichinose, 2021).

To quantitatively compare patterns of MBON requirement among different memory steps, an index called the contribution score was introduced. It was calculated by comparing the PI of an experimental group to that of the control genotype without the driver. The experimental PI was normalized to eliminate potential genetic and temperature effects. The score represents the memory loss caused by the MBON blockade. Zero indicates no memory impairment, and one denotes the complete memory loss. An improved performance causes a negative score (Ichinose, 2021).

The MBON contribution scores in the three steps of reward and punishment memories revealed striking distinctions of the requirement patterns: each memory step is mediated by a unique but partially overlapping combination of MBONs. Therefore the similarity of MBON contribution patterns was quantified among the 6 different memory steps. Interestingly, a hierarchical cluster analysis grouped the three steps in reward and punishment memories together. This suggests that the valence of memory, compared to the transition of memory steps, has more influence on the pattern of MBON contribution. Moreover, this study found that MBON contribution patterns are more similar for retrieval and acquisition than retention both in reward and punishment memories. This symmetric result suggests similar MB output usage during encoding and decoding of memory, possibly reflecting the presence of olfactory stimulation (Ichinose, 2021).

Next the behavioral results of each MBON type was projected on a standard brain by representing the contribution scores of all three steps as its intensity. The MBON types were color coded according to the clusters of dopamine neurons (DANs) innervating to them. DANs in the MB originate from the three distinct cell clusters, PAM, PPL1, and PPL2ab. The PAM and PPL1 cluster DANs generally convey the positive and negative valence, respectively, were found in different forms of associative learning. MBONs under the direct control of PAM cluster DANs exhibit remarkably higher contributions to reward than punishment memory. In contrast, those MBONs corresponding to the terminals of the PPL1 or PPL2ab clusters were selectively required for punishment memory. These results generally fit to the local modulation in the MB by individual DANs. Therefore, the valence representation of DANs is globally maintained in the MB output layer for memory-guided odor choice (Ichinose, 2021).

The MBONs can be categorized into three classes based on the neurotransmitters they release: acetylcholine, γ-aminobutyric acid (GABA), and glutamate. To examine the preferential role of neurotransmitters, MBON contributions were grouped according to the neurotransmitter systems presented in different colors. Reward memory preferentially depends on glutamatergic MBONs, whereas the contribution of GABAergic MBON-γ1pedc is most conspicuous in punishment memory. These results are largely consistent with previous investigations about compartment-specific roles of MBONs and DANs (Ichinose, 2021).

Many MBONs have been shown to send their terminals to the dendrites of DANs. These MBON-DAN connections were classified into the recurrent (i.e., MBON dendrites and DAN terminals share the same MB compartment) and the others and found that the recurrent connections are predominantly glutamatergic. Interestingly, all those glutamatergic MBONs with major contributions to reward memory (MBON-α1, -β1, -γ5β'2a, and -β'2mp) form recurrent circuits. These recurrent connections were verified with multiple approaches. Imaging physically expanded immunolabelled brains using a water-dipping objective with a high numerical aperture resolved that Bruchpilot-marked active zones of MBON boutons abut on DAN dendrites, suggesting direct synapses. Furthermore, an anterograde transsynaptic circuit tracing technique, trans-Tango, corroborated the connectivity. A recent connectome study reached the same conclusion. These classes of glutamatergic MBONs are all required during the acquisition of reward memory, and the corresponding DANs are implicated in mediating reward. It is suggested that the glutamate-dopamine recurrent connection in the MB is a circuit motif that controls reward processing (Ichinose, 2021).

It was hypothesized that the MBONs with the glutamatergic feedback motif plays a role in sustaining intense reward signals, as previously proposed for the feedback from MBON-α1 to PAM-α1 neurons. Therefore, the roles of MBON-α1, -β1, -γ5β'2a, and -β'2mp were tested for appetitive memory acquisition by varying conditioning lengths. Interestingly, this study found that MBON-α1 and -β1 were selectively required for longer conditioning. Therefore, it is proposed that reverberating input from these MBONs could sustain DAN activity for prolonged reward presentations (Ichinose, 2021).

Despite a wide variety of behaviors modulated by learning, much attention has been paid to conditioned odor choice in Drosophila olfactory learning. This study focused on memory-guided positioning bias from the upwind odor source, because positioning to the odor source should be ecologically important for efficient maneuver with potential food and danger. Indeed, wild-type flies showed remarkable changes in the distributions with respect to the paired and unpaired odor sources (CS+ and CS-, respectively): in the test of reward memory, flies approach more toward the CS+ source than CS- and vice versa for punishment memory. By introducing different numbers of flies to the T-maze, it was confirmed that a memory-guided distribution bias is not explained by the fly density (Ichinose, 2021).

To evaluate such conditioned odor source attraction, an attraction index (AI) was devised based on receiver operating characteristic (ROC) curve analysis for relative fly distributions in the two arms of the T-maze. AI becomes positive or negative if flies are relatively attracted to or repelled from the CS+ odor source, respectively. As for PIs, AIs in the tests of appetitive and aversive memories were significantly different from zero in the opposite directions. As well as conditioned odor choice, odor source attraction increased with the training durations. Blockade of all three KC subtypes in the MB010B/UAS-shi^ts1 flies abolished conditioned odor source attraction for both reward and punishment memories. These results together indicate that conditioned odor choice and odor source attraction correlate well with each other and are both dependent on the MB (Ichinose, 2021).

Next it was asked whether MBONs are also required for conditioned odor source attraction. To this end, the AI of the Shi^ts1-expressing flies was calculated. Consistent with the observation of the wild-type flies, most of the genotypes showed bi-directional conditioned odor source attraction at the permissive temperature. The transient blockade of certain MBON types attenuated the distribution bias. Because both conditioned odor choice and odor source attraction require the MB output, it was asked whether these two behaviors are controlled by the common circuits. To this end, the contribution scores of AI were calculated and compared to those of PI. Interestingly, no significant correlation was found between these two behavioral variables. For example, the contribution of MBON-α1 during reward memory acquisition was selective to PI, although MBON-β2β'2a was selectively required for AI. These results indicate that conditioned odor choice and attraction are mediated by, at least in part, independent sets of MBONs (Ichinose, 2021).

To understand how the MB differentiates its output in memory steps for odor source attraction, the patterns of MBON contribution were looked into in each phase of reward and punishment memories. In contrast to the case of PI, memory retrieval required a more distinct set of MBONs than acquisition and retention. Indeed, what stood out in the matrix was that requirement of most MBONs is highest during the retrieval of reward memory. The impaired AI in the reward memory retrieval was further examined by comparing the median density profiles of the experimental and control genotypes. This analysis revealed that the MBON blockade altered both, but often differentially, the affinity to the paired odor and the retreat from the unpaired odor source. Altogether, these results highlight the particular importance of the MB output to localize food-predicting odor source and the distinct neuronal regulation for memory-guided odor choice and source attraction (Ichinose, 2021).

Quantitative analysis of MBON contribution resolved distinct patterns during different memory functions. This approach revealed that valence, conveyed by DANs, was a key driving factor to dissociate the circuit usage, corroborating the widely accepted model of compartment-based functional dissociation in the MB. One should, however, be cautious when interpreting each data point, especially negative data, because of the fluctuating nature of the behavioral assays and relatively 'weak' UAS-shi^ts1 strain used in this study. Comprehensive examination of the MB output circuit under the same experimental condition allowed direct and quantitative comparisons of requirements and successfully uncovered the pattern how the MB multiplexes distinct memory functions (Ichinose, 2021).

Interestingly, MBONs that are most required for reward memory acquisition are all glutamatergic and form recurrent circuits by projecting their terminals to dendrites of the corresponding DANs. These results are consistent with a previous proposal of recurrent dopamine reward signals and further expand it as a common circuit motif, which may help to sustain the reward signals. Simplest interpretation of these feedback loops would be mutual potentiation of MBON and DAN activity. Because glutamate and dopamine can also suppress neuronal activity, mutual inhibition between MBONs and DANs may enhance the DAN activity. The glutamatergic recurrent circuits in the MB thus facilitate appetitive memory formation in response to substantial reward input (Ichinose, 2021).

The MB output biases was found not only for odor choice but also their positions toward upwind odor sources. Comparisons of MBON contributions for choice and attraction revealed that the MBON circuit differentially controls these two memory-guided odor responses. Remarkably, it was found that the vast majority of MBONs were required during retrieval of reward memory. Consistently, activation of PAM cluster neurons paired with an odor presentation was reported to trigger plastic changes throughout the MB lobes. Engagement of nearly the whole MB circuit in appetitive memory retrieval might reflect the intricacy of the task: flies need to integrate reward memory, wind direction, and feeding state to execute approach to the food-related odor source. Indeed, the Drosophila MB was shown to play roles in innate odor attraction, airflow, and foraging. Therefore, the rewarded odor may acquire the access to exploit these functions of the MBON circuit. An open question is how different types of information are processed in the MB network and how it is modulated by the past experiences (Ichinose, 2021).

The capacity to use past experience to guide future action is a fundamental and conserved function of the nervous system. Associative memory formation, initiated by the coincident detection of a conditioned stimulus (CS; e.g., odor) and an unconditioned stimulus (US; e.g., sugar reward), leads to a short-lived memory (STM) trace within distinct circuits. Memories can be consolidated into long-term memories (LTMs) through processes that depend on de novo protein synthesis, require structural modifications within the involved neuronal circuits, and might lead to the recruitment of additional ones. Compared with modulation of existing connections, the reorganization of circuits affords the unique possibility of sampling for potential new partners. Nonetheless, only few examples of rewiring associated with learning have been established thus far (Baltruschat, 2021).

The formation and retrieval of olfactory-associative memories in Drosophila require the mushroom body (MB). Within the main MB input compartment, the calyx (MBC), second-order projection neurons (PNs), delivers olfactory information through cholinergic synapses to the intrinsic MB neurons, the Kenyon cells. In the MBC, large, olfactory PN boutons are enwrapped by the claw-like dendrite termini of ∼11 KCs on average, thereby forming characteristic synaptic complexes, the microglomeruli (MGs), which display functional and structural plasticity in adaptation and upon silencing. To start systematically addressing the mechanisms that support memory consolidation, this study sought to investigate the properties of identifiable synaptic MGs in the MB of the adult brain of Drosophila after the establishment of LTMs (Baltruschat, 2021).

Combining behavioral experiments with high-resolution microscopy and functional imaging, this study demonstrates that the consolidation of appetitive olfactory memories closely correlates with an increase in the number of MGs formed by the PNs that deliver the conditioned stimulus and their postsynaptic KC partners. These structural changes result in additional, functional synaptic connections. Thus, the circuit in the calyx of the fly MB reorganizes accompanying the consolidation of associative memories (Baltruschat, 2021).

This study reports input-specific reorganization of the adult MBC circuit associated with the formation of long-term, appetitive memory. By visualizing presynaptic markers in PNs and the KC postsynaptic densities, this study uncovered an increase in the number of PN boutons and, at the same time, reveal that these boutons are enveloped by KC postsynaptic profiles, suggesting that new MGs are formed during memory consolidation. These findings are particularly remarkable, given the high degree of complexity of the MG microcircuits revealed by EM reconstruction and including the dendrite claws of multiple KCs of distinct subtypes. The cellular mechanisms leading to the increased number of odor-specific complex MGs remain to be clarified, but they will require a tight coordination between pre- and postsynaptic partners. In this context, mutations in synaptic proteins or in proteins mediating cell-cell interactions, which specifically block LTM, will be of great interest (Baltruschat, 2021).

It is suggested that remodeling could be driven by intrinsic reactivation of KCs during the consolidation phase or by modulatory inputs into the calyx. In either case, a complex pattern of activation is expected, that might be difficult to reproduce in artificial settings. Although the present observations are limited for technical reasons to the specific case of cVA, the overall density of PN boutons in the MBC increases after appetitive long-term conditioning in honeybees, as well as in leaf-cutting ants after avoidance learning. Based on that and given that the olfactory pathway of cVA is not distinguishable from that of other odors, it is thus suggested that the findings might be generalizable. In comparison with those systems, however, genetic and functional identification of PN subsets were used to reveal that the structural modifications are specific and limited to the PNs conveying the conditioned odor. Importantly, in vivo functional imaging data support the view that the circuit reorganization leads to additional functional MGs responding to the conditioned odor. In addition, they demonstrate a specific change in functional response in the KC dendrites toward the trained odor because the calcium levels drop faster toward baseline after appetitive associative conditioning. The faster decay kinetics and more skewed response toward the onset of the stimulus could contribute to a more-efficient temporal summation of responses or refine the KC response and might be related to inhibitory modifications. An important open question is the effect of the increased number of responding MGs on the pattern of KC activation. KCs respond sparsely to odor input and require the coincident activation of multiples of their claws to produce an action potential. The data might underlie the addition of connections between the active PNs and a set of already-responding KCs, leading to facilitated response to the conditioned odor without changing the set of responding KCs. A recent publication, however, suggests an exciting alternative view. After aversive LTM establishment, the number of KCs responding to the conditioned odor is increased (Delestro, 2020). If it is hypothesized that appetitive conditioning leads to a similar outcome, the data could provide anatomical and functional support to these findings. The pattern of KC response could, thus, be modulated by experience in adulthood and might represent a rich signifier of sensory stimulus and context. Reconstruction of an MG from EM serial sections derived from FAFB dataset (Baltruschat, 2021).

Memory is initially labile but can be consolidated into stable long-term memory (LTM) that is stored in the brain for extended periods. Despite recent progress, the molecular and cellular mechanisms underlying the intriguing neurobiological processes of LTM remain incompletely understood. Using the Drosophila courtship conditioning assay as a memory paradigm, this study showed that the LIM homeodomain (LIM-HD) transcription factor Apterous (Ap), which is known to regulate various developmental events, is required for both the consolidation and maintenance of LTM. Interestingly, Ap is involved in these 2 memory processes through distinct mechanisms in different neuronal subsets in the adult brain. Ap and its cofactor Chip (Chi) are indispensable for LTM maintenance in the Drosophila memory center, the mushroom bodies (MBs). On the other hand, Ap plays a crucial role in memory consolidation in a Chi-independent manner in pigment dispersing factor (Pdf)-containing large ventral-lateral clock neurons (l-LNvs) that modulate behavioral arousal and sleep. Since disrupted neurotransmission and electrical silencing in clock neurons impair memory consolidation, Ap is suggested to contribute to the stabilization of memory by ensuring the excitability of l-LNvs. Indeed, ex vivo imaging revealed that a reduced function of Ap, but not Chi, results in exaggerated Cl- responses to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in l-LNvs, indicating that wild-type (WT) Ap maintains high l-LNv excitability by suppressing the GABA response. Consistently, enhancing the excitability of l-LNvs by knocking down GABAA receptors compensates for the impaired memory consolidation in ap null mutants. Overall, these results revealed unique dual functions of the developmental regulator Ap for LTM consolidation in clock neurons and LTM maintenance in MBs (Inami, 2022).

Neural circuits use homeostatic compensation to achieve consistent behavior despite variability in underlying intrinsic and network parameters. However, it remains unclear how compensation regulates variability across a population of the same type of neurons within an individual and what computational benefits might result from such compensation. These questions were addressed in the Drosophila mushroom body, the fly's olfactory memory center. In a computational model, this study showed that under sparse coding conditions, memory performance is degraded when the mushroom body's principal neurons, Kenyon cells (KCs), vary realistically in key parameters governing their excitability. However, memory performance is rescued while maintaining realistic variability if parameters compensate for each other to equalize KC average activity. Such compensation can be achieved through both activity-dependent and activity-independent mechanisms. Finally, it was shown that correlations predicted by the model's compensatory mechanisms appear in the Drosophila hemibrain connectome. These findings reveal compensatory variability in the mushroom body and describe its computational benefits for associative memory (Abdelrahman, 2021).

This study examined under what conditions interneuronal variability would improve vs. impair associative memory. Using a computational model of the fly mushroom body, it was shown that under sparse coding conditions, associative memory performance is reduced by experimentally realistic variability among KCs in parameters that control neuronal excitability (spiking threshold and the number/strength of excitatory inputs). These deficits arise from unequal average activity levels among KCs. However, memory performance can be rescued by using variability along one parameter to compensate for variability along other parameters, thereby equalizing average activity among KCs. These compensatory models predicted that certain KC features would be correlated with each other, and these predictions were borne out in the hemibrain connectome. In short, this study showed 1) the computational benefits of compensatory variation, 2) multiple mechanisms by which such compensation can occur, and 3) anatomical evidence that such compensation does, in fact, occur. Note that when 'equalizing KC activity,' is said, it does. not mean that all KCs should respond the same to a given odor. Rather, in each responding uniquely to different odors (due to their unique combinations of inputs from different PNs), they should keep their average activity levels the same. That is, while KCs' odor responses should be heterogeneous, their average activity should be homogeneous (Abdelrahman, 2021).

How robust are the connectome analyses of this study? Correlations were found between anatomical proxies for the physiological properties predicted to be correlated in the models (i.e., KCs receiving excitation from more PNs should have weaker excitatory inputs, while KCs receiving more overall excitation should also receive more inhibition). In particular, the number of synapses per connection was measured as a proxy for the strength of a connection. This proxy seems valid based on matching anatomical and electrophysiological data. However, other factors affecting synaptic strength (receptor expression, posttranslational modification of receptors, presynaptic vesicle release, input resistance, etc.) would not be visible in the connectome. Of course, such factors could further enable compensatory variability. It is also worth noting that the connectome data are from only one individual (Abdelrahman, 2021).

The distance between PN-KC synapses and the peduncle is used as a proxy for the passive decay of synaptic currents as they travel to the spike initiation zone. In the absence of detailed compartmental models of KCs, it is hard to predict exactly how much increased distance would reduce the effective strength of synaptic inputs, but it is plausible to assume that signals decay monotonically with distance. Note that calcium signals are often entirely restricted to one dendritic claw. Another caveat is that the posterior boundary of the peduncle is only an estimate (although a plausible one) of the location of the spike initiation zone. However, inaccurate locations should only produce fictitious correlations if the error is correlated with the number of PN-KC synapses per KC (and only in αβ−c and γ-main KCs, not other KCs), which seems unlikely (Abdelrahman, 2021).

This work is consistent with prior work, both theoretical and experimental, showing that compensatory variability can maintain consistent network behavior. However, this study analyzed the computational benefits of equalizing activity levels across neurons in a population (as opposed to across individual animals or over time). A recent preprint showed that equalizing response probabilities among KCs reduces memory generalization, but the current showed that equalizing average activity outperforms equalizing response probabilities. Another model of the mushroom body used compensatory inhibition, in which the strength of inhibition onto each KC was proportional to its average excitation, similar to the inhibitory plasticity model. However, the previous work did not analyze the specific benefits from the compensatory variation; it also set the inhibition strong enough that average net excitation was zero, whereas this study shows that when inhibition is constrained to be only strong enough to reduce KC activity by approximately half (consistent with experimental data), inhibition alone cannot realistically equalize KC activity. In addition, there is experimental support for the current models' predictions that KCs with more PN inputs would have weaker excitatory inputs; when predicting whether calcium influxes in individual claws would add up to cause a suprathreshold response in the whole KC, the most accurate prediction came from dividing the sum of claw responses by the log of the number of claws. However, the functional benefits of this result only become clear with computational models. Finally, the larval mushroom body shows a similar relationship between number and strength of PN-KC connections; the more PN inputs a KC has, the fewer synapses per PN-KC connection; however, again, the larval work did not analyze the computational benefits of this correlation (Abdelrahman, 2021).

This study modeled two forms of compensation: direct correlations between neuronal parameters and activity-dependent homeostasis. Both forms improve performance and predict observed correlations in the connectome. Certainly, activity-dependent mechanisms are plausible as KCs regulate their own activity homeostatically in response to perturbations in activity. Indeed, different KC subtypes use different combinations of mechanisms for homeostatic plasticity, consistent with the different correlations observed in the connectome for different KC subtypes. The activity-dependent models lend themselves to straightforward biological interpretations. Excitatory or inhibitory synaptic weights could be tuned by activity-dependent regulation of the number of synapses per connection or expression/localization of receptors or other postsynaptic machinery. Spiking thresholds could be tuned by altering voltage-gated ion conductances or moving/resizing the spike initiation zone. Such homeostatic plasticity would be akin to the sensory gain control implemented by feedback inhibition but on a slower timescale (Abdelrahman, 2021).

On the other hand, KCs are not infinitely flexible in homeostatic regulation; for example, complete blockade of inhibition causes the same increase in KC activity regardless of whether the blockade is acute (16 to 24 h) or constitutive (throughout life). This apparent lack of activity-dependent down-regulation of excitation suggests that activity-independent mechanisms might contribute to compensatory variation in KCs, as occurs for ion conductances in lobster stomatogastric ganglion neurons. For example, the inverse correlation of w and N arises from the fact that the number of PN-KC synapses per KC increases only sublinearly with increasing numbers of claws (i.e., PN inputs). Perhaps a metabolic or gene regulatory constraint prevents claws from recruiting postsynaptic machinery in linear proportion to their number. (Interestingly, this suppression is stronger in larvae, where the number of PN-KC synapses per KC is actually constant relative to the number of claws). Meanwhile, the correlation between the number of inhibitory synapses and the number of excitatory synapses might be explained if excitatory and inhibitory synapses share bottleneck synaptogenesis regulators on the postsynaptic side. Although activity-dependent compensation produced superior performance in the current model compared with activity-independent compensation thanks to its more effective equalization of KC average activity (most likely because it takes into account the unequal activity of different PNs), activity-dependent mechanisms suffered when the model network switched to a novel odor environment. Given that it is desirable for even a newly eclosed fly to learn well and for flies to learn to discriminate arbitrary novel odors, activity-independent mechanisms for compensatory variation may be more effective in nature (Abdelrahman, 2021).

Compensatory variability to equalize activity across neurons could also occur in other systems. The vertebrate cerebellum has an analogous architecture to the insect mushroom body; cerebellar granule cells are strikingly similar to KCs in their circuit anatomy, proposed role in 'expansion recoding' for improved memory, and even signaling pathways for synaptic plasticity . Whereas cortical neurons' average spontaneous firing rates vary over several orders of magnitude, granule cells are, like KCs, mostly silent at rest, and it is plausible that their average activity levels might be similar (while maintaining distinct responses to different stimuli). Granule cell input synapses undergo homeostatic plasticity, while compartmental models suggest that differences in granule cells' dendritic morphology would affect their activity levels, an effect attenuated by inhibition, raising the possibility that granule cells may also modulate interneuronal variability through activity-dependent mechanisms. Future experiments may test whether compensatory variability occurs in, and improves the function of, the cerebellum or other brain circuits. Finally, activity-dependent compensation may provide useful techniques for machine learning. For example, this study found that performance of a reservoir computing network could be improved if thresholds of individual neurons are initialized to achieve a particular activity probability given the distribution of input activities (Abdelrahman, 2021).

Memory formation and sleep regulation are critical for brain functions in animals from invertebrates to humans. Neuropeptides play a pivotal role in regulating physiological behaviors, including memory formation and sleep. However, the detailed mechanisms by which neuropeptides regulate these physiological behaviors remains unclear. This study report sthat neuropeptide diuretic hormone 31 (DH31) positively regulates memory formation and sleep in Drosophila melanogaster. The expression of DH31 in the dorsal and ventral fan-shaped body (dFB and vFB) neurons of the central complex and ventral lateral clock neurons (LNvs) in the brain was responsive to sleep regulation. In addition, the expression of membrane-tethered DH31 in dFB neurons rescued sleep defects in Dh31 mutants, suggesting that DH31 secreted from dFB, vFB, and LNvs acts on the DH31 receptor in the dFB to regulate sleep partly in an autoregulatory feedback loop. Moreover, the expression of DH31 in octopaminergic neurons, but not in the dFB neurons, is involved in forming intermediate-term memory. These results suggest that DH31 regulates memory formation and sleep through distinct neural pathways (Lyu, 2023).

The coincidence between conditioned stimulus (CS) and unconditioned stimulus (US) is essential for associative learning; however, the mechanism regulating the duration of this temporal window remains unclear. This study found that serotonin (5-HT) bi-directionally regulates the coincidence time window of olfactory learning in Drosophila and affects synaptic plasticity of Kenyon cells (KCs) in the mushroom body (MB). Utilizing GPCR-activation-based (GRAB) neurotransmitter sensors, this study found that Kenyon cell (KC)-released acetylcholine (ACh) activates a serotonergic dorsal paired medial (DPM) neuron, which in turn provides inhibitory feedback to KCs. Physiological stimuli induce spatially heterogeneous 5-HT signals, which proportionally gate the intrinsic coincidence time windows of different MB compartments. Artificially reducing or increasing the DPM neuron-released 5-HT shortens or prolongs the coincidence window, respectively. In a sequential trace conditioning paradigm, this serotonergic neuromodulation helps to bridge the CS-US temporal gap. Altogether, this study reports a model circuitry for perceiving the temporal coincidence and determining the causal relationship between environmental events (Zeng, 2023).

A century ago, Ivan Pavlov proposed the associative conditioning theory, stating as follows: “A … most essential requisite for … a new conditioned reflex lies in a coincidence in time of … the neutral stimulus with … the unconditioned stimulus."1 However, the molecular and circuitry underpinnings that guarantee the maintenance of the coincidence time window have been unknown since then. This study reports that the coincidence time window of olfactory learning in Drosophila is bi-directionally regulated by the 5-HT signal from the single DPM neuron, which forms a feedback inhibitory circuit with the KCs in the MB (Zeng, 2023).

In a natural environment, flies do not experience the precisely controlled conditioned and unconditioned stimuli that can be delivered in a laboratory setting; as a consequence, their learning must be capable of adapting to changing CS/US regimens. Thus, the modulation due to 5-HT signaling improves their ability to successfully extract meaningful cause and effect. Additionally, studies have shown that the DPM neuron is involved in stress, sociality, and aging. Therefore, it is speculated that flies in different brain states shall accordingly exhibit different coincidence time windows due to the changes of serotonergic tone within the MB (Zeng, 2023).

Previously, the DPM neuron was reported to be required specifically during memory consolidation of 3-h middle-term memory after delay conditioning. This study found that the DPM neuron plays a different role in trace conditioning, regulating the coincidence time window during memory formation. Interestingly, people also found that DA has different functions in delay conditioning and trace conditioning of visual learning via distinct receptors (Zeng, 2023).

Another recent finding suggests that the DPM neuron also functions as a bridge between two groups of KCs—encoding visual and olfactory signals, respectively—to improve cross-modal learning. Besides the DPM neuron, there is a serotonergic projection neuron (SPN) innervating DANs in the peduncle of the MB, which gates the formation of long-term memory. Taken together, the 5-HT signals play versatile functions in different computational processes of olfactory learning (Zeng, 2023).

The adenylyl cyclase, rutabaga, and its product, cAMP, have been widely recognized as the key nodes in KCs for olfactory learning, but the regulation of the cAMP signal has not been fully explored. By directly imaging cAMP dynamics with G-Flamp1, it was found that activating the DPM neuron selectively suppressed the tonic level, while the phasic signal remained unchanged, indicating that the cAMP is tightly controlled by the endogenous 5-HT signal (Zeng, 2023).

It also remains unclear how the cAMP-related signaling cascades affect the neurotransmission of KCs. This study found that artificial activation of the Gαi signaling via hM4Di could eliminate physiological stimuli-evoked ACh release and subsequent 5-HT release from the DPM neuron. By contrast, endogenous activation of the Gαi signaling via 5-HT1A—in response to the DPM neuron-released 5-HT—just turned down the phasic and tonic ACh dynamics. These results emphasize the nuance of upstream regulations and downstream functions of the cAMP signal. These results drove the authors to ask how the 5-HT affects intracellular cAMP signaling and regulates the coincidence time window. From the perspective of KCs' ensemble, a computational model suggests that the difference in cAMP levels between odor-responsive KCs and non-responsive KCs determines learning efficiency (Zeng, 2023).

During odor-shock pairing, 5-HT released from the DPM neuron broadly suppresses cAMP in both odor-responsive and non-responsive KCs; thus, 5-HT indeed increases the signal-to-noise ratio and improves learning efficiency. It is hypothesize that this improvement might become more prominent at relatively long ISIs, and in such a way 5-HT extends the coincidence time window. 5-HT serves as an additional timing-regulating factor in the neo-Hebbian learning rule Apart from Drosophila, 5-HT is involved in learning and memory in a wide range of species, including Aplysia, C. elegans, and mammals (Zeng, 2023).

A growing body of evidence supports the notion that 5-HT affects timing during reinforcement learning. Human studies in a trace conditioning paradigm showed that decreasing 5-HT level by tryptophan deprivation specifically impaired learning with a long ISI. By contrast, studies of the nictitating membrane response in rabbits found that the hallucinogenic lysergic acid diethylamide (LSD, a non-selective 5-HT receptor agonist) facilitates learning with a long ISI. These findings are reminiscent of observations in Drosophila in which 5-HT bi-directionally regulates the coincidence time window. Thus, a similar serotonergic mechanism may be recruited by both vertebrates and invertebrates. The classic model of Hebbian plasticity suggests that co-activation of presynaptic and postsynaptic neurons within a short time window enables changes in synaptic plasticity, a phenomenon known as spike timing-dependent plasticity (STDP). Due to the inability of STDP to adequately explain reinforcement learning with a temporal gap, this theoretical framework was updated in the past decade by introducing a third factor encoded by the phasic activity of neuromodulators, mediating reinforcement, surprise, or novelty (Zeng, 2023).

In this updated three-factor neo-Hebbian learning rule, 'co-activation' plants a flag at the synapse called an eligibility trace, which waits for the third factor to implement the change in synaptic strength and determine the direction of that change (i.e., synaptic depression vs. potentiation). The neo-Hebbian learning rule is also applied in the MB of arthropods, where STDP exits between KCs and MBONs, with the dopaminergic reinforcement corresponding to the third factor. However, a putative fourth factor that specifically regulates the length of the eligibility trace remains unknown. Several theories have been proposed suggesting that 5-HT may serve as a timing regulator in a variety of processes, including reinforcement learning (Zeng, 2023).

Consistent with these predictions, this study experimentally showed that 5-HT signaling from the DPM neuron proportionally gates the coincidence time window, therefore serving as a specific timing-regulating factor that provides the missing piece of the puzzle (Zeng, 2023).

Inaccessibility of stored memory in ensemble cells through the forgetting process causes animals to be unable to respond to natural recalling cues. While accumulating evidence has demonstrated that reactivating memory-stored cells can switch cells from an inaccessible state to an accessible form and lead to recall of previously learned information, the underlying cellular and molecular mechanisms remain elusive. The current study used Drosophila as a model to demonstrate that the memory of one-trial aversive olfactory conditioning, although inaccessible within a few hours after learning, is stored in KCαβ and retrievable after mild retraining. One-trial aversive conditioning triggers protein synthesis to form a long-lasting cellular memory trace, approximately 20 days, via creb in KCαβ, and a transient cellular memory trace, approximately one day, via orb in MBON-α3. PPL1-α3 negatively regulates forgotten one-trial conditioning memory retrieval. The current study demonstrated that KCαβ, PPL1-α3, and MBON-α3 collaboratively regulate the formation of forgotten one-cycle aversive conditioning memory formation and retrieval (Wang, 2023).

So far, the role of CO2 in insect behavior has been mostly limited to naive avoidance and attraction. This study reveals an important role for CO2 as a facilitator of olfactory memory. In natural environments, CO2 is a ubiquitous cue, including within the nest of eusocial insects such as ants, termites, or bees8 that can be potentially significant and attractive. It is an attractive cue for insects at food sources and oviposition sites and also plays a key role in host detection for hematophagous insects such as tsetse flies or mosquitoes. Olfactory learning plays a significant role in host preference and disease transmission in blood-feeding insects. Thus, exploring the impact of CO2 on memory processes in these insects would be interesting to develop and improve control strategies to reduce the risk of disease transmission. These findings suggest that CO2 may have an unsuspected impact on the cognition of a broad spectrum of insect species (Maria, 2021).

Prior experience of a stimulus can inhibit subsequent acquisition or expression of a learned association of that stimulus. However, the neuronal manifestations of this learning effect, named latent inhibition (LI), are poorly understood. This study shows that prior odor exposure can produce context-dependent LI of later appetitive olfactory memory performance in Drosophila. Odor pre-exposure forms a short-lived aversive memory whose lone expression lacks context-dependence. Acquisition of odor pre-exposure memory requires aversively reinforcing dopaminergic neurons that innervate two mushroom body compartments-one group of which exhibits increasing activity with successive odor experience. Odor-specific responses of the corresponding mushroom body output neurons are suppressed, and their output is necessary for expression of both pre-exposure memory and LI of appetitive memory. Therefore, odor pre-exposure attaches negative valence to the odor itself, and LI of appetitive memory results from a temporary and context-dependent retrieval deficit imposed by competition with the parallel short-lived aversive memory (Jacob, 2021).

Keeping track of life experience allows animals to benefit from all of their prior knowledge when learning new information and using their memory to direct behavior. Although the subject of great early debate among learning theorists, it is now accepted that learning occurs even without explicit rewards or punishment. Classic experiments showed that rats given the prior opportunity to roam in an empty maze performed better when they were later trained with rewards presented in specific locations (Jacob, 2021).

Becoming familiar with the maze without explicit reinforcement and an obvious initial change in the animal's behavior was called 'latent learning.' Attempts to replicate a facilitating effect of latent learning using classical conditioning led to an unexpected observation. Pre-exposing animals to a stimulus instead often inhibited the ability of the animal to learn using that stimulus--a phenomenon given the name 'latent inhibition' (LI) (Jacob, 2021).

LI has been heavily studied for the last 50 years, and two alternative theories have been proposed to account for the inhibitory effect of stimulus pre-exposure. In the acquisition (A) model, subsequent learning is considered to be impaired because pre-exposure alters the capacity for the stimulus to enter into new associations. In contrast, in the retrieval (R) model, learning is still believed to occur but memory expression is impaired. A strong argument in favor of the R model is the observation that LI often appears to be limited in time, leading to expression of the subsequent learning undergoing 'spontaneous recovery.' Importantly, both theories of latent inhibition assume that something is learned during pre-exposure such as primitive properties of the stimulus including its specific identity, intensity (e.g., concentration), and salience. In addition, LI is often sensitive to the consistency of the context within which the animal is pre-exposed, taught, and tested for memory expression. This led to the proposal that first learning an association between the stimulus and its context makes it difficult for the animal to subsequently associate the stimulus with reinforcement during training. Studying olfactory learning in the relatively small brain of Drosophila has potential to define how LI can operate and reveal an underlying neuronal circuit mechanism. Several earlier studies in both adult flies and larvae demonstrated that repeated exposure to an odor can alter its apparent valence to the fly, either making the fly avoid it more or become unresponsive to it. Although an A model for LI has been reported with appetitive conditioning in the honeybee, a prior study in adult Drosophila did not observe any effect on aversive conditioning following a single odor pre-exposure (Jacob, 2021).

Associative olfactory learning in Drosophila relies on the neuronal circuitry of the mushroom body (MB). Individual odors are represented as activity in sparse and largely non-overlapping subpopulations of the ∼4,000 intrinsic neurons called Kenyon cells (KCs). Positive or negative valence can be assigned to these odor representations by anatomically discrete dopaminergic neurons (DANs) which, via dopamine receptor-directed cyclic AMP (cAMP)-dependent plasticity, modulate the efficacy of KC output synapses onto different downstream mushroom body output neurons (MBONs), whose dendrites occupy the same MB compartment. Aversive learning depresses KC synapses onto MBONs whose activation favors approach, whereas appetitive learning reduces odor-drive to MBONs favoring avoidance. By establishing a skew in the valence of the odor-driven MBON network, learned information subsequently directs either odor avoidance or attraction behavior (Jacob, 2021).

A number of studies indicate that discrete experience is represented as plasticity of different combinations of KC-MBON connections, directed by the engagement of unique combinations of DANs. For example, different types of DANs have been implicated in coding memories for specific rewards (e.g., water, the sweet taste and nutrient value of sugars, the absence of expected shock, and the delayed recognition of safety) (Jacob, 2021).

In contrast, the same PPL1 DANs appear to be required to code aversive memories for electric shock, bitter taste, and heat, although imaging suggests they are activated by temperature decreases and to noxious heat (Jacob, 2021).

By forming and storing conflicting and complementary memories in different places, the fly can more effectively direct its behavior to reflect a history of experience. This study shows that prior odor exposure can temporarily inhibit memory performance after subsequent appetitive learning in Drosophila. This inhibitory effect is sensitive to a change of context across the pre-exposure, training, and testing periods, consistent with it being a form of LI. Odor pre-exposure forms a short-lived odor-specific aversive memory, whose acquisition requires the γ2α'1 and α3 DANs, the latter of which become sensitized to consecutive odor presentation. As a consequence, aversive memory is apparent as a decrease in the odor-evoked response of the corresponding approach-directing γ2α'1 and α3 MBONs. Blocking the α3 MBONs impairs the expression of the aversive odor pre-exposure memory and abolishes LI of appetitive memory. The short-lived presence of a parallel and differently located odor-specific aversive memory therefore temporarily inhibits the retrieval of a subsequently formed appetitive memory for that same odor. These data provide evidence for a context-dependent R model of latent inhibition in Drosophila (Jacob, 2021).

This study has demonstrated a form of latent inhibition (LI) in Drosophila and has identified an underlying neuronal mechanism. Repeated odor presentation can form a labile self-reinforced aversive memory for that odor, that can temporarily compete with the expression of a newly acquired appetitive memory for that same odor. During memory testing, the conditioned odor should therefore activate both the memory of the pre-exposure (odor-self) and that of the appetitive conditioning (odor-sugar). Importantly, the aversive pre-exposure memory is labile, which means that the LI effect is transient. As a result, the appetitive memory performance exhibits 'spontaneous recovery.' These results demonstrate that an R model underlies this form of LI in the fly. An A model is not supported because flies acquire an associative reward memory for the odor after pre-exposures of that odor. Instead, the expression of the learned approach performance is impeded by the co-expression of a competing aversive pre-exposure memory. In further support of this R model, the same pre-exposure regimen caused facilitation of a subsequently acquired aversive olfactory memory. In this instance, the pre-exposure memory adds to the new aversive associative memory, rather than competes with an appetitive memory. Last, pre-exposure to the to be non-reinforced odor (the CS−) enhanced subsequent appetitive memory performance but inhibited aversive memory performance-a logical expectation of the CS− acquiring negative valence during pre-exposure (Jacob, 2021).

A defining feature of LI is sensitivity to the consistency of the context in which the pre-exposure, learning, and testing are carried out. Changing between the clear and paper-lined tubes did not impair LI, suggesting that the flies likely consider these to be a similar context. However, if odor pre-exposure, learning, and testing were performed in different contexts (i.e., a copper grid-lined versus a paper-lined or clear tube) LI was abolished. Most strikingly, LI could be restored if copper grid tubes were used to provide the same context during pre-exposure and testing. In line with prior theories and studies of LI in other animals, these results suggest that flies learn an association between the odor and the context in which it is experienced during the non-reinforced pre-exposure. As a result, the pre-exposure memory gains context-dependence, and experiments show it is not retrieved and therefore does not interfere with the newer appetitive memory if the context is different when memory is tested. The failure to retrieve the pre-exposure memory in a different context manifests as a loss of LI-the appetitive memory is fully expressed. This study therefore reveals that the context-dependency of LI results from the ability (correct context, LI evident) or inability (wrong context, no LI) to retrieve the pre-exposure memory. In addition, context only plays a role in the expression of the pre-exposure memory when it is in conflict with a subsequently acquired appetitive memory. Further work will be required to define what the flies recognize as a 'change of context.' There are many possibilities including background odors, tube/paper texture, relative luminance, and other flies in the group (Jacob, 2021).

This study found that the odor-driven activity of γ2α'1 and α3 DANs increased with repeated odor pre-exposure and that they were required for the formation of the odor pre-exposure memory. In addition, the odor-specific responses of the corresponding MBONs were depressed following pre-exposure. It was therefore concluded that ramping odor-driven DAN activity assigns negative value to the odor itself by depressing odor-specific KC connections onto the γ2α'1 and α3 MBONs. In support of this model, repeated pre-exposure of flies to a lower and less innately aversive odor concentration did not increase the activity of the α3 DANs or form an aversive pre-exposure memory. Importantly, reduced odor activation of the approach-directing γ2α'1 and α3 MBONs is sufficient to account for the aversive nature of pre-exposure memory. Moreover, both expression of pre-exposure memory and LI are abolished if the α3 MBONs are blocked during testing, confirming the model that LI is produced by the expression of the aversive pre-exposure memory competing with that of the associative reward memory believed to be represented as depression of conditioned odor responses of γ5, β'2, and α1 MBONs. Several prior Drosophila studies have documented changes in odor-driven behavior following different regimens of odor exposure, many of which employed longer durations or more trials than those employed here, and that produced shorter-lived inhibitory effects (Jacob, 2021).

One of these studies described odor-driven activity of the PPL1-α'3 DANs, and subsequent depression of odor-specific responses of the α'3 MBONs to underlie how flies can become familiar with an odor following repeated short exposures. In contrast, this study shows that two longer and spaced odor exposures produce an aversive memory that manifests as plasticity of γ2α'1 and α3 DANs and MBONs. Moreover, whereas this study shows a retrieval defect underlies LI of appetitive memory, a reduced attention/familiarity/habituation to the odor following pre-exposures would be expected to result in a subsequent acquisition defect (and A model), likely of both appetitive and aversive learning. It will nevertheless be important to understand how these different types of olfactory experience, and their supporting plasticity mechanisms, are represented and combined in the brain (Jacob, 2021).

LI has often been compared to memory extinction, and the current work in the fly shows that very similar neuronal mechanisms and learning models account for both of these phenomena. Pre-exposure learning in Drosophila appears to follow similar rules to extinction learning following aversive olfactory conditioning: 2 spaced trials with 15-min ITI are more efficient than massed training with 1-min ITI, and in both cases, a resulting parallel opposing odor-nothing memory inhibits the retrieval/expression of the odor-punishment or odor-reward memory. The obvious difference is that the interfering non-reinforced odor memory is formed before learning for LI and after learning for extinction (Jacob, 2021).

These studies of learning, extinction, and LI suggest that flies acquire and store all of their experience (rewarded and unrewarded, punished and unpunished) as parallel memory traces. As a result, when evoked by an appropriate cue, the relevant experiences are compared/combined at the time of retrieval to determine the most fitting behavioral outcome. Such a model is reminiscent of the comparator hypothesis, devised mostly from experiments in rodents. Because recent studies suggest similar processes underlie extinction of fear in flies, rodents, and humans, it seems likely that the form and mechanism of LI described in this study will also be relevant across phyla (Jacob, 2021).

Memory forms when a previously neutral stimulus (CS+) becomes competent to predict a biologically potent stimulus (US). However, if the CS+ is repeatedly presented without the US after the memory formation, this memory will be suppressed by newly formed extinction memory. The striking feature of extinction learning is that it requires repeated trials to robustly form extinction. Extended repetition only yields memories that remain transient in nature, thus imposing challenges in understanding the underlying mechanisms of extinction learning. This study took advantage of the versatile genetic tools and the well-characterized circadian system of Drosophila to link these unique features to clock neurons. Inhibiting the activity of clock neurons blocks the formation of extinction memory. Further investigation attributes this role to a subset of cryptochrome-positive dorsal neurons 1 (DN1s) and their downstream SIFamide neurons. The requirement of clock neurons from a gating mechanism of extinction for a single extinction learning trial robustly causes typical extinction when coupled with acute activation of DN1s, as marked by the initially enhanced but eventually diminished memory suppression. Accordingly, specific neural responses were detected to extinction training in a few DN1s via calcium imaging fulfilled by the TRIC tool, but not in dorsal neuron 2 or dorsolateral neurons. Based on these findings, it is proposed that in extinction of appetitive long-term memory, multiple trials of extinction learning robustly activate DN1 clock neurons to open the gate of extinction, which may contribute to the transient nature of extinction memory (Zhang, 2021).

The current study focused on the extinction of 24-h appetitive long-term memory (apLTM) because only 24-h apLTM shows spontaneous recovery. Examination of extinction with a transient nature revealed that clock neurons are a key component in the mechanism by which multiple trials of extinction learning are required to robustly form the transient extinction memory. The data presented in this study strongly support the proposed model, as multiple trials of extinction learning are required to robustly activate four pairs of CRY-positive, Spl-DN1-Gal4-labeled DN1s to permit extinction. Therefore, this study shows that multi-trial extinction learning is functionally heterogeneous, with multiple trials needed in the robust activation of a subset of clock neurons and a single trial for acquiring the CS-no US association. The coupling of these two factors results in the extinction of the 24-h apLTM, i.e., clock neuron gate extinction (Zhang, 2021).

Four lines of evidence support these conclusions. First, multiple, but not single, trials of extinction learning or the presentation of CS+ for multiple times robustly activates DN1s, but not DN2s or LNds, as detected by accumulating signals for TRIC (a tool for imaging intracellular calcium), an intracellular Ca2+ indicator. Second, suppression of the activity of only CRY-positive, but not CRY-negative, clock neurons, particularly four pairs of CRY-positive, Spl-DN1-Gal4-labeled DN1s, blocked extinction. Third, the artificial activation of Spl-DN1-Gal4-labeled DN1s coupled with a single extinction trial potentiated the transient extinction of 24-h apLTM. Fourth, suppression of the neuronal activity of downstream target SIFa neurons also blocked natural extinction (Zhang, 2021).

The results suggest the role of clock neurons in memory regulation by extinction. In previous studies, the circadian system has been reported to regulate multiple physiological processes. In general, these regulatory effects have been attributed to oscillation of the circadian system. However, this study showed that extinction learning itself can evoke neural responses in DN1s to open the gate for extinction memory, instead of its passive modulation driven by circadian rhythm. Thus, it would be of considerable interest to determine whether such active regulation of clock neurons also occurs in other physiological processes (Zhang, 2021).

Recent studies on the fly's extinction have identified the PPL-1 dopaminergic neurons and γ mushroom body neurons as the key components in mediating the extinction process. In addition, the downstream SIFa neurons are known to send neurites throughout the adult brain. It would be of interest to determine how DN1 neurons, as well as their downstream targets, are related to functions of previously identified neural networks mediating extinction (Zhang, 2021).

A newly formed memory is initially unstable. However, if it is consolidated into the brain, the consolidated memory is stored as stable long-term memory (LTM). Despite the recent progress, the molecular and cellular mechanisms of LTM have not yet been fully elucidated. The fruitfly Drosophila melanogaster, for which various genetic tools are available, has been used to clarify the molecular mechanisms of LTM. Using the Drosophila courtship-conditioning assay as a memory paradigm, previous work identified that the circadian clock gene period (per) plays a vital role in consolidating LTM, suggesting that per-expressing clock neurons are critically involved in LTM. However, it is still incompletely understood which clock neurons are essential for LTM. This study shows that dorsal-lateral clock neurons (LNds) play a crucial role in LTM. Using an LNd-specific split-GAL4 line, this study confirmed that disruption of synaptic transmission in LNds impaired LTM maintenance. On the other hand, induction of per RNAi or the dominant-negative transgene of Per in LNds impaired LTM consolidation. These results reveal that transmitter release and Per function in LNds are involved in courtship memory processing (Suzuki, 2022).

Animals retain some but not all experiences in long-term memory (LTM). Sleep supports LTM retention across animal species. It is well established that learning experiences enhance post-learning sleep. However, the underlying mechanisms of how learning mediates sleep for memory retention are not clear. Drosophila males display increased amounts of sleep after courtship learning. Courtship learning depends on Mushroom Body (MB) neurons, and post-learning sleep is mediated by the sleep-promoting ventral Fan-Shaped Body neurons (vFBs). This study shows that post-learning sleep is regulated by two opposing output neurons (MBONs) from the MB, which encode a measure of learning. Excitatory MBONs-γ2α'1 becomes increasingly active upon increasing time of learning, whereas inhibitory MBONs-β'2mp is activated only by a short learning experience. These MB outputs are integrated by SFS neurons, which excite vFBs to promote sleep after prolonged but not short training. This circuit may ensure that only longer or more intense learning experiences induce sleep and are thereby consolidated into LTM (Lei, 2022).

This study has identified a neural circuit that regulates learning-induced sleep for LTM consolidation. This circuit links neurons essential for learning and memory in Drosophila, the MB neurons, with those critical for post-learning sleep, the vFBs8. It is proposed that only a longer learning experience is sufficient to induce sleep, and thereby be consolidated into LTM. Given that the increasing duration of a learning experience correlates with the total amount of time males spend on futile courtship towards mated females during training, selective activation of vFBs likely depends on the amount or intensity of a learning experience, rather than just its duration. Post-learning sleep induction requires integration of two MB outputs, previously implicated in courtship memory in SFSs. Post-learning activity of the excitatory MBONs-γ2α'1 increases linearly with the duration of the prolonged learning experience. In contrast, activity of the inhibitory MBONs-β'2mp peaks after a short experience sufficient to induce STM. As a result, only when the males court mated females sufficiently long or intensely, the activity of MBONs-γ2α'1 reaches the threshold required to activate SFSs. This in turn leads to activation of vFBs to promote post-learning sleep and the reactivation of those dopaminergic neurons (DANs) that were involved in memory encoding. Consequently, biochemical processes essential for LTM consolidation become engaged (Lei, 2022).

How might MBONs-γ2α'1 and MBONs-β'2mp measure the learning experience to control post-learning sleep? In homeostatic sleep regulation, the potentiation of R2 neurons reflects a measure of sleep loss that is sensed by dFBs, likely in response to the accumulation of byproducts of oxidative stress during sleep loss. In the case of learning-induced sleep, it is envisioned that learning results in lasting changes in the molecular pathways essential for memory formation in the MB. For example, the cAMP pathway along with the dopamine receptor are activated during sleep in a discrete 3-h time window after learning in rodents and Drosophila males lacking a dopamine receptor, and hence unable to learn, do not display increased post-learning sleep. Thus, the accumulation of changes in the cAMP signaling pathway upon increasing learning experience with mated females might lead to the increasing potentiation of MBONs-γ2α'1 and MBONs-β'2mp after learning. Interestingly, MBONs-γ2α'1 and MBONs-β'2mp display distinct temporal activity patterns upon learning which likely reflects their distinct neuronal properties (Lei, 2022).

This study reveals a circuit mechanism that ensures that only persistent, and thus likely significant, learning experiences generate post-learning sleep to consolidate LTM. Recent findings suggest that dFBs, involved in sleep homeostasis, might mediate a paradoxical type of sleep, in humans also called Rapid Eye Movement (REM) sleep. This in conjunction with the current data, provide an opportunity to investigate whether the post-learning sleep, mediated by vFBs, might represent another type of sleep implicated in mammals in memory consolidation (Lei, 2022).

Epitranscriptomic modifications can impact behavior. This study used Drosophila melanogaster to study N(6)-methyladenosine (m⁶A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m(6)A binders. Assays of short term memory in m⁶A mutants reveal neural-autonomous requirements of m⁶A writers working via Ythdf, but not Ythdc1. Furthermore, m⁶A/Ythdf operate specifically via the mushroom body, the center for associative learning. m⁶A from wild-type and Mettl3 mutant heads was mapped, allowing robust discrimination of Mettl3-dependent m⁶A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m⁶A is preferentially deposited on genes with low translational efficiency and that m⁶A does not affect RNA stability. Nevertheless, functional tests indicate a role for m⁶A/Ythdf in translational activation. Altogether, this molecular genetic analyses and tissue-specific m(6)A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway (Kan, 2021).

In an effort to identify factors that regulate memory, the 'epitranscriptome', the multitude of modified bases that exist beyond the standard RNA nucleotides was of primary interest. The most abundant and most well-studied internal modification of mRNA is N6-methyladenosine (m⁶A). While m⁶A has been recognized to exist in mRNA since the 1970s, its functional significance has been elusive until recently. Key advances included (1) techniques to determine individual methylated transcripts, and in particular specific methylated sites, and (2) mechanistic knowledge of factors that install m⁶A ('writers') and mediate their regulatory consequences ('readers'). The core m⁶A methytransferase complex acting on mRNA consists of the Mettl3 catalytic subunit and its heterodimeric partner Mettl14. These associate with other proteins that play broader roles in splicing, mRNA processing and gene regulation, but that are collectively required for normal accumulation of m⁶A (Kan, 2021).

Downstream of the writers, various readers are sensitive to the presence or absence of m⁶A, and thereby mediate differential regulation by this mRNA modification. The most well-characterized readers contain YTH domains, for which atomic insights reveal how a tryptophan-lined pocket selectively binds methylated adenosine and discriminates against unmodified adenosine. In addition, some other proteins were proposed as m⁶A readers, based primarily on preferential in vitro binding to methylated vs. unmethylated RNA probes. In mammals, m⁶A readers confer diverse regulatory fates onto modified transcripts, including splicing and nuclear export via the nuclear reader YTHDC1, and RNA decay via cytoplasmic readers Ythdf1-3. Certain YTHDF and YTHDC2 were also reported to regulate translation via m⁶A under specific contexts (Kan, 2021).

Despite intense efforts into m⁶A mechanisms and genomics using cell systems, genetic analyses of the m⁶A pathway have only begun in earnest in the past few years, mostly in vertebrates. Notably, many studies have revealed sensitivity of the mammalian nervous system to manipulation of m⁶A factors. Mutants in writer (Mettl3 and Mettl14), reader (primarily ythdf1), and eraser (FTO) factors have collectively been shown to exhibit aberrant neurogenesis and/or differentiation. Moreover, these mutants impact neural function and behavior, including during learning and memory paradigms. Overall, these observations may reflect some heightened requirements for m⁶A in neurons, perhaps owing to their unique architectures and/or regulatory needs (Kan, 2021).

Among invertebrates, Caenorhabditis elegans lacks the core m⁶A machinery, but the presence of a Drosophila ortholog of Mettl3 (originally referred to as IME4) opened this model system. While mammals contain multiple members of both nuclear and cytoplasmic YTH domain families, the fly system is simplified in containing only one of each, referred to as Ythdc1 (YT-521B or CG12076) and Ythdf (CG6422), respectively. Recently, several labs established biochemical, genetic, and genomic foundations for studying the m⁶A pathway in Drosophila. Surprisingly, these studies jointly reported that knockout of all core m⁶A writer factors in Drosophila is compatible with viability and largely normal exterior patterning. Nevertheless, mutants of Mettl3, Mettl14, and Ythdc1 exhibit a common suite of molecular and phenotypic defects. These include several behavioral abnormalities as well as aberrant splicing of the master female sex determination factor Sex lethal (Sxl). The suite of locomotor and postural defects in Drosophila m⁶A mutants was again consistent with the notion that the nervous system might be especially sensitive (Kan, 2021).

However, a major open question from these studies concerns the regulatory and biological roles of the sole Drosophila cytoplasmic YTH factor, Ythdf. In contrast to other core m⁶A factors, overt defects were not previously observe in the Ythdf mutants, nor did it seem to exhibit robust m⁶A-specific binding activity. This study used proteomic analyses to reveal Ythdc1 and Ythdf as the major m⁶A-specific binders in Drosophila, and focused biochemical tests show that Ythdf prefers a distinct sequence context than tested previously. Hypothesizing that the nervous system might exhibit particular needs for the m⁶A pathway, a paradigm of aversive olfactory conditioning was used to reveal an m⁶A/Ythdf pathway that is important for STM in older animals. These phenotypic data were complemented with high-stringency maps of methylated transcript sites from fly heads, and it was shown that m⁶A does not impact transcript levels but is preferentially deposited on genes with lower translational efficiency. Nevertheless, functional tests reveal that Mettl3/Ythdf can enhance protein output. Finally, this study showed that physiological Mettl3/Ythdf function is explicitly required within mushroom body neurons to mediate normal conditioned odor memory during aging. Overall, this study provides insights into the in vivo function of this mRNA modification pathway for normal behavior (Kan, 2021).

Despite tremendous interests in the regulatory utilities and biological impacts of mRNA methylation, there has been relatively little study from invertebrate models. Given that the m⁶A pathway seems to have been lost from C. elegans, Drosophila is an ideal choice for this. Since the initial report that Mettl3 mutants affect germline development, it has been shown that Drosophila harbors an m⁶A pathway similar to that of mammals, but simplified in that it has a single nuclear and cytoplasmic YTH reader. Nevertheless, Drosophila has proven to be a useful system to discover and characterize novel m⁶A factors. Expanding the breadth of model systems can increase appreciation for the utilization and impact of this regulatory modification (Kan, 2021).

It is widely presumed, based on mammalian profiling, that metazoan m⁶A is enriched at stop codons and 3' UTRs. However, high-resolution maps indicate that 5' UTRs are by far the dominant location of methylation in mature Drosophila mRNAs. Although further study is required, many of these m⁶A 5' UTR regions coincide with previous embryo miCLIP data, while other miCLIP CIMs calls located in other transcript regions proved usually not to be Mettl3-dependent. Thus, the current data indicate a fundamentally different distribution of m⁶A in Drosophila mRNAs compared to mammals (Kan, 2021).

While mammalian m⁶A clearly elicits a diversity of regulatory consequences, depending on genic and cellular context and other factors, a dominant role is to induce target decay through one or more cytoplasmic YTH readers. This harkens back to classic observations that m⁶A is correlated with preferential transcript decay, and more recent data that loss of m⁶A writers or cytoplasmic YTH readers results in directional upregulation of m⁶A targets. However, several lines of study did not yield convincing evidence for a broad role for the Drosophila m⁶A pathway in target decay. Instead, the dominant localization of m⁶A in fly 5' UTRs is suggestive of a possible impact in translational regulation. Genomic and genetic evidence support the notion that m⁶A is preferentially deposited in transcripts with overall lower translational efficiency, but that m⁶A/Ythdf may potentiate translation. However, it is possible to rationalize a regulatory basis for these apparently opposite trends, if the greater modulatory window of poorly translated loci is utilized for preferred targeting by m⁶A/Ythdf (Kan, 2021).

As is generally the case for mammalian m⁶A, the choice of how appropriate targets are selected for modification, and which gene regions are preferentially methylated, remains to be understood. The minimal context for m⁶A is insufficient to explain targeting, and as mentioned also seems to be different between Drosophila and vertebrates. A further challenge for the future will be to elucidate a mechanism for m⁶A/Ythdf-mediated translational regulation. This will reveal possible similarities or distinctions with the multiple strategies proposed for translational regulation by mammalian m⁶A, which include both cap-independent translation via 5' UTRs during the heat-shock response via eIF383 or YTHDF236; cap-dependent mRNA circularization via Mettl3-eIF3H84; and activity-dependent translational activation in neurons (Kan, 2021).

Recent studies have highlighted neuronal functions of mammalian m⁶A pathway factors. There is a growing appreciation that mouse mutants of multiple components in the m⁶A RNA-modification machinery affect learning and memory. This study provides substantial evidence that, in Drosophila, neural m⁶A is critical for STM. This study specifically focused on STM as this paradigm has been extensively characterized in Drosophila. Mouse studies have almost exclusively examined effects on LTM, and these two memory phases are mechanistically distinct. One main distinction is that LTM requires protein synthesis after training, while STM does not. So, while direct comparisons between the two systems are not possible, it is nevertheless instructive to consider the parallels and distinctions of how m⁶A facilitates normal memory function in these species. This is especially relevant given that both mouse and fly central nervous systems require a cytoplasmic YTH factor for memory (Kan, 2021).

In mice, the m⁶A writer Mettl3 enhance long-term memory consolidation, potentially by promoting the expression of genes such as Arc, c-Fos and others. Another study found that Mettl14 is required for LTM formation and neuronal excitability. Conversely, knockdown of the m⁶A demethylase FTO in the mouse prefrontal cortex resulted in enhanced memory consolidation. Amongst mammalian YTH m⁶A readers, YTHDF1 was shown to induce the translation of m⁶A-marked mRNA specifically in stimulated neurons. In cultured hippocampal neurons, levels of YTHDF1 in the PSD fraction were found to increase by ~30% following KCl treatment. This suggests that YTHDF1 concentration at the synapse could be critical for regulating the expression levels of proteins (such as CaMK2a) involved in synaptic plasticity. Taken together, these studies suggest the m⁶A pathway is a crucial mechanism of LTM consolidation in mammals that optimizes animal behavioral responses (Kan, 2021).

Of note, the genetics and sample sizes possible in Drosophila permit comprehensive, stringent, and anatomically resolved analyses. Thus, this study systematically analyzed all writer and reader factors, and revealed a notable functional segregation, suggesting that the cytoplasmic reader Ythdf is a major effector of Mettl3/Mettl14 m⁶A in memory. Given that Ythdf mutants otherwise exhibit few overt developmental or behavioral defects in normal or sensitized backgrounds (while Ythdc1 mutants generally phenocopy Mettl3/Mettl14 mutants) its role in STM is a surprising insight into the contribution of Ythdf to a critical adaptive function. Moreover, the spatial requirements of m⁶A for STM can be pinpointed by showing that (1) neuronal-specific and MB-specific depletion of Mettl3/Ythdf can induce defective STM, and (2) neuronal and MB-specific restoration of Mettl3 or Ythdf to their respective whole-animal knockouts restores normal STM. Moreover, the fact that Ythdf gain-of-function in the MB can also disrupt STM, but does not generally alter other aspects of development or behavior, points to a homeostatic role of m⁶A regulation in Drosophila learning and memory (Kan, 2021).

It was observed that STM defects in fly m⁶A mutants are age-dependent, which has not been reported in mammals. Although many physiological capacities decline with life history, the observed STM defects seem to be decoupled from other age-related phenotypes, since mutation of Ythdf or neural overexpression of Ythdf can interfere with STM but does not substantially impact lifespan or locomotion. In this regard, Mettl3 and Ythdf are different from classical memory genes such as rutabaga because STM impairment in m⁶A mutants was absent in young flies and only became apparent with progressing age (Kan, 2021).

One interpretation is that there is a cumulative effect of deregulated m⁶A networks that has a progressive impact specific to mushroom-body neurons. To gain further mechanistic insights, future studies will need to examine age-related changes in gene expression and/or translation, in a cell-specific manner. It remains to be seen whether specific deregulated targets downstream of Ythdf have large individual effects, or whether the STM deficits arise from myriad small effects on translation. Ythdf-CLIP and ribosome profiling from the CNS may prove useful to decipher this. Assuming that loss of translational enhancement of m⁶A/Ythdf targets mediates STM defects, one possibility, to be explored in future studies, is that some targets may already be known from prior genetic studies of memory (Kan, 2021).

The second messenger cyclic AMP (cAMP) plays an important role in synaptic plasticity. Although there is evidence for local control of synaptic transmission and plasticity, it is less clear whether a similar spatial confinement of cAMP signaling exists. This study suggests a possible biophysical basis for the site-specific regulation of synaptic plasticity by cAMP, a highly diffusible small molecule that transforms the physiology of synapses in a local and specific manner. By exploiting the octopaminergic system of Drosophila, which mediates structural synaptic plasticity via a cAMP-dependent pathway, this study demonstrates the existence of local cAMP signaling compartments of micrometer dimensions within single motor neurons. In addition, evidence is provided that heterogeneous octopamine receptor localization, coupled with local differences in phosphodiesterase activity, underlies the observed differences in cAMP signaling in the axon, cell body, and boutons (Maiellaro, 2016).

The cyclic AMP (cAMP) pathway plays fundamental roles in the nervous system, where it is prominently involved in synaptic plasticity and memory formation. Previous studies in vertebrate and invertebrate models have shown that cAMP can propagate from dendrites to the cell body of neurons, in line with the properties of a small diffusible molecule. However, a local mode of action for cAMP has also been proposed, whereby cAMP signals are localized to the periphery of neurons, namely, dendrites, creating a cAMP microdomain. While the existence of cAMP microdomains in neuronal dendrites is disputed based on the experimental and theoretical data, very little is known about possible cAMP compartmentation in axons and how this may exert local effects at the presynaptic site. In particular, it is unclear how biochemical signals may spread from presynaptic boutons through the axon (Maiellaro, 2016).

To investigate this question in vivo, the neuromuscular junction (NMJ) of Drosophila melanogaster was used, that displays different forms of synaptic plasticity, many of which are dependent on cAMP signaling. Both structural and functional properties of larval neuromuscular synapses are heterogeneous, varying between boutons belonging to the same motor neuron. How such site-specific synaptic differentiation may be achieved at high spatial resolution is currently unknown, though it is tempting to speculate that local cAMP signals play a role. Thus, the developing Drosophila NMJ is a powerful model to investigate the role of cAMP in synaptic plasticity under physiological conditions (Maiellaro, 2016).

This study focused on glutamatergic type Ib motor neurons, which are structurally regulated via G-protein-coupled receptors (GPCRs) for octopamine. Stimulation of these receptors has been shown to induce synaptic bouton outgrowth via a cAMP- and CREB-dependent pathway. Therefore, this study set out to measure spatiotemporal patterns of octopamine-induced cAMP signals in these neurons. To this end, a FRET (Forster resonance energy transfer)-based sensor for cAMP (Epac1-camps) was expressed that has previously been used to image cAMP levels in central Drosophila neurons. The results reveal that cAMP signals are confined to their initiation site, the individual synaptic bouton, and suggest a highly efficient local mechanism for controlling site-specific synaptic plasticity (Maiellaro, 2016).

This study genetically expressed the cAMP sensor, Epac1-camps, at the Drosophila NMJ to monitor and quantify spatiotemporal cAMP dynamics induced by octopamine stimulation. The results reveal an unexpectedly high degree of cAMP compartmentalization in motor neurons, which may serve as a basis for local synaptic plasticity, with cAMP FRET signals being ultimately limited to single synaptic boutons. Three cAMP signaling compartments were identified within the motor neuron: boutons, axon, and cell body. For each cellular compartment, the particular mechanism responsible for the segregation of cAMP increases is described. Specifically, this study found that boutons constitute the most reactive compartment of the motor neuron in terms of cAMP accumulation. The results demonstrate that the production of cAMP is heterogeneous among boutons, with stronger responses to octopamine in large boutons rather than in smaller ones, possibly related to the increased synaptic strength measured at large boutons. Moreover, the activity and the specific localization of PDEs within the synaptic bouton prevent the propagation of cAMP to the cell body and its diffusion from one bouton to the next (Maiellaro, 2016).

In contrast to the boutons, octopamine receptors seem to be absent or inaccessible in the axon, and PDEs were not detected. Accordingly, cAMP FRET signals recorded in the axon differ from those in boutons in terms of amplitude and kinetics. Hence, the axon emerges as the second independent, but not isolated, cAMP signaling compartment. Interestingly, and in contrast to dendrites, cAMP did not propagate along the axon. It remains to be clarified whether this may relate to functional differences between axons and dendrites, between species, or between specific neuron types. Finally, the cell body was determined as the third cAMP signaling compartment within the motor neuron. The cell body has a very low sensitivity toward octopamine, and it was demonstrated that cAMP FRET signals generated in the cell body are not affected by the cell body's physical isolation from the axon. High PDE activity in the cell body contributes to this local suppression of cAMP signaling and may prevent spillover activation of cAMP effectors in the cell body and in the nucleus (Maiellaro, 2016).

Evidence of cAMP microdomains restricted to single boutons provides a biophysical basis for the local control of synaptic plasticity. Spatially constrained cAMP changes help to establish differences in morphology and synaptic content of boutons, suggesting that local cAMP, bouton structure, and synapse formation are intimately linked. The observed confinement of cAMP supports the notion that individual synaptic boutons may represent largely autonomous signaling units, which can receive and integrate signals independently of the other (Maiellaro, 2016).

The concept that cAMP could act as a local messenger was postulated almost 40 years ago. The existence of cAMP microdomains has been demonstrated in other cell types (e.g., cardiomyocytes). However, in neurons, there are contradicting experimental lines of evidence and simulations concerning the existence of cAMP microdomains. The current data clearly show that cAMP can act as a local messenger upon physiological stimulation of a neuron. The detailed spatiotemporal analysis of the dynamics of cAMP reveals that this messenger can be restricted at the micrometer level to induce highly localized physiological responses (Maiellaro, 2016).

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Zygotically transcribed genes

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