|
Gene name - dunce Synonyms - Cytological map position - 3C11D4 Function - degrades c-AMP Keywords - c-AMP pathway - learning pathway. calcium dependent enzymes |
Symbol - dnc FlyBase ID:FBgn0000479 Genetic map position - 1-3.9 Classification - c-AMP phosphodiesterase Cellular location - cytoplasmic |
| Recent literature | Xiao, C. and Robertson, R.M. (2017). White-cGMP interaction promotes fast locomotor recovery from anoxia in adult
Drosophila. PLoS One 12: e0168361. PubMed ID: 28060942 Summary: Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. It has been previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. This study shows that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery is light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This is further supported by the observations that w-RNAi-induced delay of locomotor recovery is completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promotes fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia. |
Baggett, V., Mishra, A., Kehrer, A. L., Robinson, A. O., Shaw, P. and Zars, T. (2018). Place learning overrides innate behaviors in Drosophila. Learn Mem 25(3): 122-128. PubMed ID: 29449456
Summary: Animals in a natural environment confront many sensory cues. Some of these cues bias behavioral decisions independent of experience, and action selection can reveal a stimulus-response (S-R) connection. How animals use learning to modify S-R relationships is a largely open question. Three sensory stimuli, air, light, and gravity sources were presented to individual Drosophila melanogaster in both naive and place conditioning situations. Flies were tested for a potential modification of the S-R relationships of anemotaxis, phototaxis, and negative gravitaxis by a contingency that associated place with high temperature. With two stimuli, significant S-R relationships were abandoned when the cue was in conflict with the place learning contingency. The role of the dunce (dnc) cAMP-phosphodiesterase and the rutabaga (rut) adenylyl cyclase were examined in all conditions. Both dnc1 and rut2080 mutant flies failed to display significant S-R relationships with two attractive cues, and have characteristically lower conditioning scores under most conditions. Thus, learning can have profound effects on separate native S-R relationships in multiple contexts, and mutation of the dnc and rut genes reveal complex effects on behavior. |
Wiggin, T. D., Hsiao, Y., Liu, J. B., Huber, R. and Griffith, L. C. (2021). Rest Is Required to Learn an Appetitively-Reinforced Operant Task in Drosophila. Front Behav Neurosci 15: 681593. PubMed ID: 34220464
Summary: 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. |
Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. This study asked how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. This study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes were reviewed. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. Factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue are discussed (Ueda, 2012).
It has been demonstrated that cAMP levels are decreased in rut. The results clearly contrast the differential effects of disruptions in synthesis and degradation of cAMP on synaptic function and nerve terminal morphology. Mutations in dnc, including dnc1, dncM11, and dncM14, can lead to severe defects in nerve terminal branching and bouton morphology. Aside from this study, previous reports have documented in identified larval muscles that total bouton numbers and motor terminal branching pattern are severely affected by dnc, but these defects were not detected in rut. A similar situation has been reported in the adult CNS: axon terminal growth in the mushroom body is enhanced in dnc but is not affected in rut. In contrast, rut and dnc mutations both have clear effects on synaptic transmission but in distinct manners. Increased cAMP levels in dnc could enhance transmitter release (as indicated by increased ejp sizes with a minimal disturbance in the temporal precision of the release process. In comparison, rut mutations more severely disrupt temporal control of release, regardless of the rearing temperature. In addition, the rearing temperature affects the amplitude of synaptic transmission in rut, with strongly depressed transmission at high temperature. This likely reflects a decrease in vesicle release because the miniature ejp size was unaltered at different temperatures (data not shown) (Ueda, 2012).
A number of mutant alleles of the rut gene have been described in the literature of developmental studies, but the alleles frequently used in neurogenetic experiments are limited to rut1, rut2, rut3, rut1084, and rut2080. Furthermore, only three mutant alleles have been biochemically characterized in Drosophila: rut1, rut2, and rut3. It should be mentioned that these rut mutations can cause significant decrease in total cAMP synthesis despite the fact that there are at least four adenylyl cyclase (AC) homologous genes that have been identified molecularly and biochemically in Drosophila. This raises the possibility that rut may represent a major AC gene but all AC genes may play differential roles in regulating cAMP levels, depending on their subcellular localization and conditions to activate their actions. As demonstrated in this study as well as in earlier reports, a general pattern of relative severity among several rut mutant alleles is observed across different phenotypes, as represented in the following sequence: rut1 = rut1084 = rut2 = rut3 = WT (Ueda, 2012).
Compared to the AC genes, there appears to be fewer PDE homologous genes and only two genes are known for their cAMP degradation action besides dnc. However, dnc gene products are represented by more than 10 splicing variants as opposed to 2 rut splicing variants. There are a large number of dnc mutant alleles reported in literature but only a small number of them are frequently used in neurogenetic studies, i.e., dnc1, dnc2, dncM11, and dncM14. Interestingly, a consistent pattern of phenotypic severity can be observed across different phenotypes among these four alleles: dncM11 = dncM14 = dnc1 = dnc2 (Ueda, 2012).
A comparison of their effects on a variety of phenotypes includes PDE enzyme activity disruption, defective growth cone motility of cultured neurons, enhanced growth of larval NMJ, enhanced K + and Ca2 + currents in larval muscles, decrease in the larval motor neuron firing frequency upon depolarization, increase in whole-cell ejps or ejcs, and decrease in activity-dependent facilitation of synaptic transmission at larval NMJ, decrease in the habituation rate of olfactory jump response and odor-electric shock association in adult flies, and female sterility. In a different approach, overexpression of a UAS-dnc + transgene in motor neurons results in reduced NMJ growth and decreased ejp size even in larvae reared at room temperature. These phenotypes demonstrated the effects of increased cAMP degradation in contrast to those caused by dnc mutations (Ueda, 2012).
When considering their mechanisms of action, several reported phenotypic effects of dnc alleles may be complicated by the implications of contributions from the genetic background. Notably, the dncM11 mutant line has been reported to affect protein kinase C (PKC) activity in addition to PDE. In addition, the severity of dnc1 may in fact be more extreme than reported, since dnc1 has been shown to be female sterile once a second-site mutation near the dnc locus is removed from the original fertile line. It is possible that many dnc1 lines used in neurogenetic investigations contain this mutation in the background (Ueda, 2012).
A number of experimental paradigms have been used to characterize behavioral and physiological phenotypes of dnc and rut mutants with defined quantitative parameters. For a majority of phenotypes examined, dnc and rut mutations do not lead to opposite effects on these quantitative indices, even though they alter the cAMP levels in opposite directions. Only for certain phenotypes, the dnc and rut mutations affect the parameters in opposite directions. For example, in larval neuromuscular synaptic boutons, mobilization of synaptic vesicles from the reserve pool to exo/endo cycling pool is suppressed in rut and enhanced in dnc. Similarly, the number of docked vesicles at synapses is decreased in rut and increased in dnc. Ca2 + current measured in larval muscles is decreased in rut and increased in dnc. Hyperexcitability-induced overgrowth of larval NMJ can be suppressed by rut but enhanced by dnc. Similarly, dnc and rut mutations exert opposite effects on Kenyon cell axon counts in the mushroom body of developing adult flies. Finally, habituation rate of the giant fiber escape circuit is decreased by rut and increased by dnc (Ueda, 2012).
In contrast, for some other phenotypes, rut alleles have no apparent effects while dnc mutants display clear alterations. For instance, the larval NMJ terminal projection pattern and adult mushroom body axonal terminal growth were altered in dnc but not in rut. Moreover, identified K + currents in larval muscles are increased in dnc but unaltered in rut. In these cases, increased cAMP levels can produce abnormalities but underlying mechanisms may be tolerant to depleted cAMP levels (Ueda, 2012).
For another group of phenotypes, dnc and rut mutations can affect separate parameters and sometimes produce superficially similar effects by altering a parameter in the same direction. Such cases include decreased growth cone motility, irregular action potential firing pattern, and modified intracellular Ca2 + dynamics in cultured neurons. In larval neuromuscular junctions, both dnc and rut decrease synchronicity of synaptic transmitter release and presynaptic facilitation of neuromuscular transmission. During post-eclosion development of adult flies, both dnc and rut mutations enhance the axon terminal growth of mechanosensory cells and decrease the structural and functional adaptation of the olfactory system to odor exposure. Neither dnc nor rut mutants respond to environmental or social deprivation in modifying Kenyon cell axon counts of young adults. Mutations of either dnc or rut decreases habituation rate of the proboscis extension reflex and olfactory avoidance and jump response, and the performance indices of both classical and operant conditioning. Studies on alcohol response have demonstrated increased sensitivity in rut alleles but no apparent change in dnc alleles. Although it is reassuring to observe opposite effects of dnc and rut mutations on some of the quantitative parameters, it should be noted that it is not straightforward in associating most of the indicators with the defective mechanisms directly regulated by cAMP signaling. Dysfunction in AC and PDE may exert opposite effects on some cell biological mechanisms or neural circuit components but can still lead to apparently similar deficiencies of a cellular function or behavioral task (Ueda, 2012).
Some insights may be gained through examining the genetic interactions between dnc and rut in double mutants about how rut AC and dnc PDE are involved in particular aspects of physiological or behavioral plasticity. At the present time, only a limited number of reports document the resultant phenotypes in dnc rut double mutants. Significantly, the majority of the single-mutant phenotypes of dnc or rut mutations do not become less severe in dnc rut double mutants, even though the overall cAMP levels are largely restored. The phenotypes that are not rescued in double mutants include increased bouton size in dnc and impaired synchronicity of transmitter release in larval NMJs, irregular firing of cultured neurons, and habituation and olfactory associative learning of adult flies (Ueda, 2012).
However, a few cases of successful rescue in double mutants have been described. Decreased growth cone motility in dnc and rut neurons in culture can be restored by combining two mutations and the overgrowth and altered projection patterns of dnc larval motor terminals is suppressed in dnc rut. Interestingly, none of the above cases of successful rescue involve opposite effects of dnc and rut single-mutant phenotypes. Notably, both cases of restoration involve a particular allele, rut1. The allele rut1 is different from other alleles with characterized AC enzyme activity (rut2 and rut3) in that the Ca2 + /CaM-dependent activation of AC is eliminated in rut1 flies, but retained in rut2 and rut3. Unlike rut1, the allele rut2 is not able to rescue the dnc mutational effects of enhanced larval NMJ growth and irregular firing in cultured neurons. In the present study of NMJ focal recording, it was clears that rut2 did not affect the precision in release timing (ejc peak time) and ejc amplitudes, although rut1 decreased the temporal precision of release (increased variability in ejc peak time) and the ejc amplitude significantly. It will be helpful if further experiments are performed on additional allele combinations of dnc and rut to delineate the role of Ca2 + -dependent regulation of AC in specific phenotypes of interest (Ueda, 2012).
In addition to peculiarities of enzymatic properties in mutant alleles, e.g., rut1 AC devoid of Ca2 + /calmodulin (CaM) sensitivity, other factors influencing interactions between dnc and rut must be considered. As summarized above, counterbalancing rescue of dnc and rut phenotypes in double mutants is likely to be exceptions rather than a general rule. Therefore, it would be desirable to identify the conditions and factors that could facilitate their counterbalancing interactions, which may provide insights into the orchestration of dnc PDE and rut AC underlying the phenotype of interest (Ueda, 2012).
First, it is important to consider the temporal and spatial characteristics of expression and operation of these enzymes. In the temporal domain, their effects on a variety of phenotypes are mediated through integration among different biochemical pathways and cellular processes, some of which may function with rapid kinetics, whereas others may represents slow accumulation of products through a number of steps. Some of the resultant phenotypes may require continuous adjustment in response to internal or environmental conditions while others may appear relatively permanent and irreversible, possibly associated with developmental events (Ueda, 2012).
The spatial factors to be considered include the cellular expression and subcellular localization of the enzymes. To the best of our knowledge, there is little information about whether dnc PDE and rut AC are colocalized in molecular assemblies or aggregates within certain functional domains in specific neuronal cell types. Close proximity of AC and PDE localization facilitates local regulation of cAMP levels within a short time. Certain cellular processes with slower kinetic steps also facilitate integration of dnc and rut interactions, extending their balancing acts to a broader spatial range (Ueda, 2012).
For the few examples of successful counterbalancing rescue, growth cone motility seems to be a continuous adjustment by cAMP on a time scale of tens of seconds to minutes. This relatively slow kinetics makes it possible to readily manipulate the cAMP signaling pathway, e.g., bath application of db-cAMP increases rut growth cones motility, mimicking dnc counter balancing effects in double-mutant growth cones. Some developmental or maintenance processes, such as axonal path finding, branch formation, target interaction, and synaptogenesis, are also slow adjustment processes (in the order of hours to days). In these cases, restoration of cAMP levels through long-range interactions of AC and PDE may be sufficient to rescue the single-mutant phenotype. For example, dnc defects in larval motor terminal growth are suppressed by rut1 (Ueda, 2012).
In contrast, defects in some physiological properties (K + currents, neuronal firing, and transmitter release timing) and behavioral conditioning (habituation and classical conditioning) cannot be rescued by combining dnc and rut, which sometimes leads to even more extreme deficiencies, e.g., the extremely rapid habituation in dnc rut. One possibility is the requirement of dynamic cAMP regulation within a short time period (millisecond to second range) during which a counterbalancing act is difficult to achieve. Another possible explanation is the requirement of unimpaired cellular machinery laid down during development (e.g., proper channel and receptor localization) and functional connectivity among synaptic partners (inhibitory and excitatory elements in the circuit) underlying behavioral phenotypes under consideration. Deviation from coordinated actions of such subcellular machinery or circuit components will make it difficult to obtain compensatory rescue (Ueda, 2012).
It should be noted that well-defined abnormalities in central fiber projection have been reported in dnc and rut single mutants that reflect the alterations in peripheral motor terminals in larval NMJs. Furthermore, dnc PDE and rut AC are preferentially expressed in mushroom bodies, which are important in odor-associated learning. Therefore, it is reasonable to speculate that defects in higher functions, including classical associative learning and habituation, may involve anatomical defects in the CNS, such as altered dendritic arbors and synaptic connections detectable in certain defined circuits, in addition to potential changes in synaptic physiology (Ueda, 2012).
Cell-specific expression and subcellular localization of AC and PDE isoforms may affect dnc and rut single-mutant, as well as double-mutant phenotypes. These include splicing variants of the dnc and rut gene as well as the products of their homologous genes. Such complexity needs to be considered in the interpretation of dnc and rut interactions in order to appreciate contributions of individual splicing variants and to delineate influence from their homologous genes (Ueda, 2012).
Finally, cross-talk between the cAMP and other signaling pathways can also modify dnc and rut phenotypes. For example, variety of signalling pathways are known to converge onto the CREB transcription factor. It is also established that not only the cAMP cascade but also other signaling pathways, including PKG and CaMKII, can modify larval NMJ physiology and morphology as well as adult habituation, courtship conditioning and classical conditioning. It will be of particular interest to establish the consequences of such genetic interactions across signaling pathways. Double mutant analysis in conjunction with transgenic and genomic approaches remains a powerful and profitable direction for revealing the genetic network underlying neural and behavioral plasticity (Ueda, 2012).
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, previous 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).
The genetic dissection of learning and memory in Drosophila is two decades old. Recently, a great deal of progress has been made towards isolating new mutants as well as a better understanding of those originally isolated. Nighorn's paper reviews the recent developments in the understanding of the structure and function of the gene identified by the first and best-characterized of these mutants, the Drosophila dunce mutant (Nighorn, 1994). R. L. Davis (1996) provides an even more recent review.
Learning in flies is studied using an operant conditioning paradigm involving electric shock and olfactory cues. First an odor (the conditioned stimulus) is paired with electric shock (the unconditioned stimulus). The aversive effects of the shock teach the flies to avoid the odor, or as researchers tend to express it, avoidance of the conditioned stimuli is enhanced. Behavior attributable to learning is measured by testing with a second and different odor. Flies that have learned continue to avoid the conditioned stimulus but not the control odor, both in the absence of the unconditioned stimulus. Proper control includes a second experimental phase: employing the second odor as the conditioned stimulus. When paired with the shock, flies should then avoid the second (control) odor as they did the original conditioned stimulus (Quinn, 1972).
Cyclic AMP has been implicated as the mediator of learning in classical experiments using the marine snail Aplasia (Kandel, 1992). In this system, conditioned stimuli increased Ca++ concentration within neurons. The Ca++ binds to Calmodulin, which in turn binds to adenylate cylase, enhancing the ability of adenylate cyclase to synthesize c-AMP. The Drosophila adenyl cyclase homolog is rutabaga. Mutations in rut result in defective learning. A second gene, dunce, encodes the cAMP phosphodiesterase, the enzyme that degrades cAMP. Mutations in dunce also result in a learning deficit but when combined with the rut mutations in the same fly, the learning defect is reversed. At least in this case it appears that two wrongs make a right. It has been suggested that lower cAMP levels still result in learning despite the double mutation because the cAMP is degraded more slowly.
Protein kinase A, the target of cAMP signaling is a third protein involved in learning . PKA, activated by cAMP, initiates a phosphorylation cascade that in turn activates genes involved in the creation of long term memory. dCREB2, the cyclic AMP response element binding protein is one such gene, a transcription factor associated with long term memory. The Dfos and DJUN genes in Drosophila both have binding sites for CREB, while in mammals, only the fos promoter binds CREB (Davis, 1995, Fagnon, 1995 and Nighorn, 1994). For a discussion of the cellular basis of learning see the rutabaga site.
A novel bioassay system is described that uses Xenopus embryonic myocytes (myoballs) to detect the release of acetylcholine from Drosophila CNS neurons. When a voltage-clamped Xenopus myoball is manipulated into contact with cultured Drosophila 'giant' neurons, spontaneous synaptic current-like events are registered. These events are observed within seconds after contact and are blocked by curare and alpha-bungarotoxin, but not by TTX and Cd2+, suggesting that they are caused by the spontaneous quantal release of acetylcholine (ACh). The secretion occurs not only at the growth cone, but also along the neurite and at the soma, with significantly different release parameters among various regions. The amplitude of these currents displays a skewed distribution. These features are distinct from synaptic transmission at more mature synapses or autapses (a synapse between an axon collateral of a neuron and one of the same neuron's dendrites) formed in this culture system and are reminiscent of the transmitter release process during early development in other preparations. The usefulness of this coculture system in studying presynaptic secretion mechanisms is illustrated by a series of studies on the cAMP pathway mutations, dunce (dnc) and PKA-RI that disrupt a cAMP-specific phosphodiesterase and the regulatory subunit of cAMP-dependent protein kinase A, respectively. These mutations affect the ACh current kinetics, but not the quantal ACh packet, and the release frequency is greatly enhanced by repetitive neuronal activity in dnc, but not wild-type, growth cones. These results suggest that the cAMP pathway plays an important role in the activity-dependent regulation of transmitter release not only in mature synapses as previously shown, but also in developing nerve terminals before synaptogenesis (Yao, 2000).
It is now well established that Drosophila neurons share many molecular components of the transmitter release machinery with vertebrate neurons. The release of neurotransmitter is a multistep process that involves actions of proteins associated with the synaptic vesicle and the plasma membrane, as well as cytoplasmic proteins. Some of these proteins, e.g., synapsin alphaSNAP, and Ca2+ channels are known to be the downstream targets of PKA. Phosphorylation of these proteins may be important for the regulation of vesicle mobilization, docking, and fusion (Yao, 2000).
In Drosophila dnc mutants, increased cAMP levels caused by the disruption of a phosphodiesterase lead to abnormalities in channel function and nerve excitability, synaptic transmission and plasticity, growth cone motility, and nerve arborization. Using the present heterologous detection system, the altered transmitter release process could be examined in developing growth cones of dnc central neurons in isolation from the influence of postsynaptic targets. Examination of PKA-RI neurons suggests that the dnc defects in ACh secretion might be mediated by PKA. These results establish a role for the cAMP cascade in the regulation of the secretion process in developing neurons before synaptogenesis. In light of the profound alterations in synaptic efficacy and activity-dependent modulation observed in mature synapses of dnc mutants, the cAMP pathway may be involved throughout the maturation process of the synapse (Yao, 2000).
The effects of decreased cAMP levels on synaptic transmission have also been extensively studied in Drosophila. Intracellular recordings at the peripheral larval neuromuscular junction have revealed that chronically lowering cAMP causes reduced neurotransmitter release, likely because of reduction of innervation rather than impairment of transmitter release. These results do not contradict results obtained from developing central neurons. It will be important to determine how reduction in cAMP concentration affects neurotransmitter releases in the Drosophila central neurons in future studies (Yao, 2000).
The prolonged ACh currents of dnc and PKA-RI neurons may be attributable to increased ACh diffusion distance and altered presynaptic release mechanisms, as discussed above. A reduced efficiency in the formation of the exocytotic fusion pore and/or a disrupted fusion machinery may account for the prolonged release events for synaptic vesicles containing similar amounts of ACh. Exocytotic efficiency may be regulated by PKA-dependent phosphorylation of vesicular, cytoplasmic, and plasma membrane proteins involved in exocytosis. Additional mutational analysis will be required to identify the specific proteins that are targeted by PKA in this process (Yao, 2000).
Although the spontaneous release in neurons of all genotypes examined
does not require Ca2+ influx, the
activity-dependent increase in release frequency in dnc
neurons after repetitive nerve stimulation appears to depend on the
external Ca2+. It has been proposed that
nerve activity regulates cAMP levels, possibly mediated by
intracellular accumulation of Ca2+ through
repetitive nerve spikes, which can trigger the Ca2+/CaM activation of adenylyl cyclase. The activity-dependent modification of
transmission at mature synapses is altered in
dnc mutants. These
results suggest that the cAMP pathway may mediate such
activity-dependent regulation in developing neurons before synaptogenesis as well, lending support to the notion that the cAMP
pathway is important in a wide variety of neuronal processes throughout development (Yao, 2000).
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 complexity of the dunce locus and its transcription products points to the biological importance of Dunce. The dunce gene encodes multiple RNAs ranging in size from 4.2 to 9.6 kb (1 kb = 10(3) bases). Six different classes have been identified. Exons are distributed over more than 148 kb of genomic DNA, with some exons being used alternatively among the RNAs. The RNAs are transcribed from at least three initiation sites. Some of the heterogeneity is generated by using varying lengths of a 3'-untranslated trailer sequence. The protein variation potentially includes N-terminal differences coded for by transcript-specific 5' exons, internal differences arising from the optional inclusion of a 39 base-pair exon, and the alternative use of two 3' splice sites separated by six base-pairs. An unusual feature of the dunce gene is the presence of at least 7 other genes nested between the dunce exons. The significance of this phenomenon is unknown (Qui, 1991 and Nighorn, 1994).
The deduced amino acid sequence is strikingly homologous to the amino acid sequence of a Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase isolated from bovine brain and more weakly related to the predicted amino acid sequence of a yeast cAMP phosphodiesterase. These homologies, together with prior genetic and biochemical studies, provide unambiguous evidence that dunce+ codes for a phosphodiesterase. In addition, the dunce+ gene product shares a seven-amino acid sequence with a regulatory subunit of cAMP-dependent protein kinase that is predicted to be part of the cAMP binding site. There is also a weak homology between a region of the dunce+ gene product and the egg-laying hormone precursor of Aplysia californica (Chen, 1989).
date revised: 25 September 2021
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.