BIOLOGICAL OVERVIEW

Memory involves synthesis of new proteins. In organisms as diverse as Aplasia, Drosophila and humans, the enzyme cyclic-AMP response element binding protein (CREB) is directly involved in the establishment of the capacity for long term memory (Bailey, 1994). CREB is so named because of the part it plays in binding the cyclic-AMP response element in the promoter of the mammalian proto-oncogene Jun. CREB was originally described as a factor stimulating the transcription of genes in response to growth factors and phorbol esters.

Suppose a Superfly were to exist, one that could learn from a single experience without need for repetition. In fact, such an animal has been created. Precocious long term memory has been observed after a single cycle of training when a transgene for a PKA-responsive activator form of CREB is expressed before training takes place (Yin, 1995b). This activator is one isoform found in multiple splicing of CREB.

It seems that flies need to rest during training periods in order to establish long term memory. Mass training, that is, repeated experience without rest, fails to produce long term memory. Spaced training, training with rest periods between repeated tests, produces two functionally independent forms of consolidated memory: long term memory (LTM) and anesthesia resistant memory (ARM). LTM is disrupted by cyclohexamide, a drug that blocks protein synthesis, in contrast to ARM, which remains unaffected. LTM, and not ARM is disrupted by induced expression of a dominant negative transgene of CREB (Yin, 1995b).

Understanding of gene activation in the memory pathway comes from an analysis of transcriptional regulation of the Jun gene. A classic model for the formation of heterodimers is the Jun/Fos heterodimer, the predominant form of the gene regulator AP-1 (activator protein-1). Analysis of the Drosophila Jun promoter reveals binding sites for AP-1, CREB and DTF1, a protein that acts as an enhancer binding factor of the D2 promoter of Antennapedia (Perkins, 1988a and b). Thus Jun is regulated by JUN itself combined with FOS and by two other proteins: DTF1 and CREB. Three of these proteins (FOS, JUN and CREB) are responsive to the phosphorylation cascade that results from cAMP signaling. Additional information on the role of cyclic-AMP in fly memory (Davis, R. L., 1996) will be found in the dunce and rutabaga sites.

The role of dCREB2 (CrebB-17A) in circadian rhythms has been examined. dCREB2 activity cycles with a 24 hr rhythm in flies, both in a light:dark cycle and in constant darkness. A mutation in dCREB2 shortens circadian locomotor rhythm in flies and dampens the oscillation of period, a known clock gene. Cycling dCREB2 activity is abolished in a period mutant, indicating that dCREB2 and Period affect each other and suggesting that the two genes participate in the same regulatory feedback loop. It is proposed that dCREB2 supports cycling of the Period/Timeless oscillator. These findings support CREB's role in mediating adaptive behavioral responses to a variey of environmental stimuli (stress, growth factors, drug addiction, circadian rhythms, and memory formation) in mammals and long-term memory formation and circadian rhythms in Drosophila (Belvin, 1999).

To measure dCREB2 activity in vivo, transgenic Drosophila lines were constructed carrying the luciferase reporter gene driven by an enhancer element comprised of consensus CREB binding sites. Three cAMP response elements (CREs), 5'-TGACGTCA-3', were placed upstream of the TATA box region of the hsp70 gene promoter, followed by the luciferase reporter gene. This sequence was flanked by the scs and scs' insulator elements to reduce potential positional effects caused by the random insertion site of the transgene. The transfected lines are referred to as CRE-luc lines. A mutant CRE-luc reporter construct (mCRE-luc) was also generated in which the consensus CRE sites were mutated to TGAAATCA. dCREB2 protein binds this mutant CRE site with at least 20-fold lower affinity in gel shift experiments. This construct is otherwise identical to wild-type CRE-luc (Belvin, 1999).

The expression of luciferase in the wild-type CRE-luc flies oscillates in a 24 hr rhythm, both in a light:dark cycle and in constant darkness. The main peak of activity occurs just after lights out, with the nadir just before the main peak. Since this rhythmic transcription pattern is sustained in constant darkness, it is regulated by the circadian system, rather than simply being a response to light. In light:dark conditions, a second peak is observed in the middle of the day; however, these two peaks gradually blend together under conditions of constant darkness. This pattern is very similar to that seen for per activity. The per-luc reporter also exhibits a similar secondary peak under light:dark conditions, even though per RNA peaks only once per cycle. It is likely that the secondary peaks of both reporters, which occur during the day, are due to a light response of luciferase rather than a circadian response. The expression level of the mCRE-luc reporter is drastically reduced relative to the wild-type reporter, indicating that the CRE sites mediate the high-level expression of the wild-type reporter (Belvin, 1999).

These experiments show that dCREB2 activity is under circadian control, but they do not show whether dCREB2 plays a role in maintaining the rhythms or is just responsive to them. The A dCREB2 mutation, S162, one that allows for survival of a few flies to adulthood, was used to address this question. To test for behavioral effects of the mutation, S162 escaper males were assayed for circadian locomotor activity. The flies were tested for 10 days in constant darkness to determine whether they display normal circadian fluctuations in activity. Of the 34 S162 mutants tested, 13 (38%) were arrhythmic while the 21 that were rhythmic had a short period averaging 22.8 hr. None of these flies had a wild-type 24 hr rhythm. The high percentage of arrhythmicity is typical of mutations that affect period length. To verify that this behavioral phenotype is specific for the S162 mutation, the phenotype was rescued by induction of the hs-dCREB2-10 transgene. To induce the transgene, larvae and pupae were subjected to a daily 60 min heat pulse of 37°C during development. All of the rescued flies (12/12) were rhythmic, and they all displayed normal circadian locomotor rhythms of 23.5-24 hr. This demonstrates that the short period phenotype is caused by the S162 mutation rather than a second site mutation elsewhere on the chromosome. It also shows the involvement of dCREB2 in the timing of the clock (Belvin, 1999).

If S162 is indeed acting in the clock, then it should affect the per clock gene. The effects of S162 on two different per-dependent reporters were examined. The first is a transcriptional fusion with a 4.2 kb fragment of the per promoter upstream of the luciferase reporter gene, referred to as per-luc. The second is a translational fusion containing the same promoter fragment, plus the 5' untranslated region and the first 2.4 kb of the per coding region fused in frame to the luciferase gene, referred to as BG-luc. When the expression of these reporters was compared, it was found that the BG-luc reporter cycles much more robustly than the per-luc reporter, consistent with the interpretation that there are at least two mechanisms contributing to the cycling of Per: one mediated by the promoter, and the other(s) mediated by sequences in either the per transcript or Per protein itself. S162 affects the two reporters differently. The S162 mutation reduces both the expression level and cycling pattern of the per-luc reporter. However, its effect on the BG-luc reporter is weaker. In S162 flies, the BG-luc reporter maintains a robust cycling pattern, although its expression level and amplitude are reduced. The peak in the mutant background also occurs in advance of the peak in wild-type flies, consistent with the short period phenotype of these flies (Belvin, 1999).

In order to demonstrate a direct effect of the S162 mutation on the clock, its effects on the Per protein itself were examined. Wild-type and S162 flies were entrained on a 12 hr light:12 hr dark cycle and aliquots were frozen every 2 hr throughout the cycle. Head extracts were prepared and analyzed by Western blot using an antibody directed against Per. Per is present at very low levels at ZT 6 and ZT 8 (Zeitgeber time; 6 and 8 hours after lights on), increasing through the lights of period to peak levels before lights on, which occurs at ZT 0. A corresponding change in phosphorylation, and protein mobility, accompanies the change in absolute levels, with Per becoming more highly phosphorylated as it accumulates. This temporal pattern of Per is altered in the S162 mutant background, where Per is present at more equal levels throughout the circadian cycle. At the peak time, ZT 20, the amount of Per is at least comparable to that in wild-type flies; however, it decreases less at ZT 6 and ZT 8, when Per is virtually absent in wild-type flies. At these trough periods of Per expression, a discrete doublet protein band persists in S162, perhaps representing preservation of certain phosphorylated forms. There also seems to be a general increase in the amount of Per protein throughout the cycle in the mutant flies. The change in both per-luc expression and Per protein levels in the S162 mutant background demonstrates that Per activity is under the influence of the dCREB2 gene. The effects of S162 on Tim protein were assayed in the same experiment. The effect of S162 on Tim is much more subtle than the effect on Per. The Tim protein appears to accumulate slightly sooner in the mutant than in the wild type (ZT 12 versus ZT 14); however, its overall oscillation remains fairly normal (Belvin, 1999).

What is the biological significance of dCREB2's participation in the clock? Since a mutation in the dCREB2 gene affects the clock, the possibility exists that stimuli that activate CREB could affect circadian rhythmicity. This is clearly true for light pulses that reset the clock when delivered at the appropriate times during the nighttime period and have been shown to induce CREB phosphorylation in mammals. What about 'noncircadian' stimuli? One example of this phenomenon is work showing that rats can learn an association between air puffs delivered to the eye (conditioned stimulus) and light (unconditioned stimulus). After acquisition, the conditioned stimulus alone, when delivered during the nighttime period, phase shifts the clock. This suggests that there may be much more overlap in the conditioning circuitry and the circadian circuitry than is generally appreciated. Since the molecules within all of these neurons are similar, the molecular pathways may also overlap. CREB-mediated transcription occurs in response to stimuli that induce stress, long-term memory formation, and growth factor responses. What is the significance of the fact that this transcription factor also responds to circadian signals, which happen regularly over a 24 hr period? One speculation is that the cyclical pattern in CREB activity means that there are optimal periods during the 24 hr cycle for CREB-responsive physiological processes. One physiological process for which there is evidence that the nighttime period is important is the consolidation of long-term memory formation. Over the years, a large number of experiments, primarily on rodents and humans, suggest a possible involvement of some aspect of nighttime sleep in the consolidation of memory. A unifying interpretation of the physiological and behavioral data is that the brain utilizes the sleep period to 'replay' plasticity-related events, thereby ensuring their total consolidation and maintenance. This replay occurs during sleep, when there is minimal external input into the brain. It is speculated that the circadian system controls some of the neuronal activity that occurs during the sleep period. This activity in turn leads to activation of CREB-responsive transcription, which may be important in consolidating and maintaining preexisting circuits and memories. Experiments to test these types of ideas are underway (Belvin, 1999 and references).

dCREB2-mediated enhancement of memory formation

CREB-responsive transcription has an important role in adaptive responses in all cells and tissue. In the nervous system, it has an essential and well established role in long-term memory formation throughout a diverse set of organisms. Activation of this transcription factor correlates with long-term memory formation and disruption of its activity interferes with this process. Most convincingly, augmenting CREB activity in a number of different systems enhances memory formation. In Drosophila, a sequence rearrangement in the original transgene used to enhance memory formation has been a source of confusion. This rearrangement prematurely terminates translation of the full-length protein, leaving the identity of the 'enhancing molecule' unclear. This report shows that a naturally occurring, downstream, in-frame initiation codon is used to make a dCREB2 protein off of both transgenic and chromosomal substrates. This protein is a transcriptional activator and is responsible for memory enhancement. A number of parameters can affect enhancement, including the short-lived activity of the activator protein, and the time-of-day when induction and behavioral training occur. The results reaffirm that overexpression of a dCREB2 activator can enhance memory formation and illustrate the complexity of this behavioral enhancement (Tubon, 2013).

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).

The 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).

Nuclear gating of a Drosophila dCREB2 activator is involved in memory formation

The transcription factor CREB is an important regulator of many adaptive processes in neurons, including sleep, cellular homeostasis, and memory formation. The Drosophila dCREB2 family includes multiple protein isoforms generated from a single gene. Overexpression of an activator or blocker isoform has been shown to enhance or block memory formation, but the molecular mechanisms underlying these phenomena remain unclear. In this study isoform-specific antibodies and new transgenic flies were generated to track and manipulate the activity of different dCREB2 isoforms during memory formation. It was found that nuclear accumulation of a dCREB2 activator-related species, p35+, is dynamically regulated during memory formation. Furthermore, various dCREB2 genetic manipulations that enhance or block memory formation correspondingly increase or decrease p35+ levels in the nucleus. Finally, it was shown that overexpression of S6K can enhance memory formation and increase p35+ nuclear abundance. Taken together, these results suggest that regulation of dCREB2 localization may be a key molecular convergence point in the coordinated host of events that lead to memory formation (Fropf, 2013).

Surprisingly, the antibodies showed that the majority of dCREB2 protein, regardless of structure and apparent mobility on protein gels, is localized in the cytoplasmic compartment. This finding is true for both the blocker and activator isoforms. The blocker primarily consists of the 'backbone' structure (exons 1, 3, 5 and 7), which migrates as a doublet species around 38-40 kD. When the alternative exons (2, 4 and/or 6) are spliced into this backbone, the apparent mobility of the resulting proteins does not change dramatically. The activator-related molecular species (p24, p28, p30, p35+, p60; Tubon, 2013) also are enriched in the cytoplasm, and this is most clearly demonstrated when nuclear and cytoplasmic samples are loaded on a per head-equivalent basis. All of the dCREB2 protein isoforms contain the amino acid residues RRKKK in their basic regions, and this sequence has been shown to be necessary and sufficient to localize mammalian CREB proteins into the nucleus. Since the mammalian CREB protein is mostly found in the nucleus, the cytoplasmic enrichment for dCREB2 protein is unexpected. This localization is maintained even when transgenes are induced and different dCREB2 protein isoforms are overexpressed. These data suggest that a significant fraction of dCREB2-encoded proteins are tethered in the cytoplasmic compartment through an unknown mechanism (Fropf, 2013).

Since dCREB2 is a transcription factor, its primary function occurs in the nuclear compartment. Previous studies have demonstrated that the majority of dCREB2 protein exists with serine 231 (the fly equivalent to the mammalian serine 133 residue) phosphorylated in the basal state. This contrasts sharply with the mammalian CREB protein, which exists in the nucleus in a largely unphosphorylated state, awaiting activity-dependent signaling mechanisms that activate it. Taken together, the cytoplasmic localization of dCREB2 protein and the phosphorylation of serine 231 in the basal state suggest strongly that the rate-limiting step in activation of dCREB2 probably involves increasing the nuclear abundance of the activator species. This could at least partially occur through an increase in nuclear entry (Fropf, 2013).

A number of results support the view that p35+ is an endogenous, dCREB2-specific protein product. Different dCREB2-specific antibodies recognize it on western blots. p35+ is decreased or missing when head extracts are made from the S162 mutant fly. When blocker-encoding transgenes are induced, they affect the nuclear levels of p35+. Finally, when the 807 (activator-encoding) transgene is induced, p35+ increases. Although the 807-encoded protein is transgenic and not endogenous, the primary product has an apparent mobility of 24 kD, but when expressed in flies increases the levels of both p28 and p35+ bands (Fropf, 2013).

Three independent lines of evidence support the interpretation that p35+ represents an activator-related species. First, there is a correlation between memory formation and nuclear abundance of p35+. Forward, but not backward, paired training produces long-term memory and results in a greater than 3-fold increase in nuclear p35+. Weaker training (5xS instead of 10xS), which produces less robust memory, produces a smaller, non-significant trend towards an increase. Second, there is a strong correlation between memory formation and a qualitative increase in p35+ in the nucleus. Genetic manipulations of dCREB2 that enhance memory formation (807 induction) increase nuclear abundance of p35+, ones that interfere with long-term memory formation (induction of 17-2 and 936) prevent an increase, while a control manipulation (induction of 581) does not interfere with either memory formation or affect p35+ levels. Third, manipulating a totally different protein (S6K) results in a parallel set of correlations. Increasing the amount of the S6K protein enhances memory formation and increases p35+ nuclear abundance (Fropf, 2013).

What is the nature of the p35+ isoform? Mammalian CREB has been shown to be SUMOylated and o-glycosylated, and these mod ifications decrease the apparent mobility of the resulting proteins on gels. This study has evidence that dCREB2 protein can be o-glycosylated, and one of those species migrates around 35 kD. Future experiments will be needed to determine if is p35+ o-glycosylated, and how this would affect protein interactions, subcellular localization, or transcriptional activity (Fropf, 2013).

Since a nuclear increase in p35+ correlates so well with memory formation, it is believed that p35+ (or its predecessor species p24, p28 and/or p30) enter the nucleus from the cytoplasm, where it is tethered in a heterodimer with other dCREB2 proteins. Transgenic experiments support this view. The 807 transgene makes p24/p28, overexpression of which increases nuclear abundance of p35+ and can enhance memory formation. The 936 transgene makes a protein that differs in two of the five residues that constitute the nuclear localization signal, which renders the induced protein exclusively cytoplasmic. Induction of 936 decreases nuclear p35+ levels, and blocks memory formation. Taken together, these data are consistent with the view that 936 blocks memory formation through a cytoplasmic tethering mechanism, where the overexpressed protein binds to, and prevents the nuclear entry of, the endogenous activator species. The induced 17-2 protein is able to form heterodimers with the activator, but is less efficient at staying in the cytoplasm. However, it is still able to interfere with memory formation, since a heterodimeric species (activator:17-2) is less transcriptionally potent. The 17-2 and 581 transgenes differ in two leucine residues in the leucine zipper. These amino acid changes are known to disrupt the ability of the mutant (581) protein to form dimers. Therefore, the overexpressed 581 protein cannot form a dimer with the activator, does not prevent a nuclear increase in p35+ after training, and does not affect memory formation. Finally, the overexpressed 568 protein is mostly in the nucleus, and it is speculated that it can outcompete activator-containing dimers for binding to CRE sites. Therefore, it blocks memory formation but does not decrease nuclear p35+ abundance. This mechanism of action is identical to the actions of the ICER and S-CREM blockers (Fropf, 2013).

Although the data from the transgenic experiments supports the hypothesis that nuclear entry of the activator is rate-limiting for transcription and memory formation, other possibilities. For example, it is possible that there exists a constant rate of activator import and export from the nucleus, and that memory formation transiently decreases export, thus increasing nuclear abundance. Another possibility is that behavioral training could transiently increase the nuclear stability of the activator protein. More detailed experimentation would be needed to determine the relative contribution of each of these possible mechanisms. However, transgenic experiments argue that regulating nuclear import of the activator is at least partially involved in controlling the strength and duration of dCREB2-responsive transcription (Fropf, 2013).

This study has shown that inducing S6K stimulates nuclear abundance of p35+ and enhances memory formation. These data are the first demonstration that S6K overexpression can enhance long-term memory. Although the correlation between memory formation and an increase in the nuclear abundance of p35+ is clear, the underlying molecular mechanisms are not known. Behavioral training could stimulate translation of the activator protein or decrease its turnover. In addition, training might stimulate nuclear entry and/or decrease nuclear export of the activator protein. Similarly, it is not clear if induction of S6K stimulates translation (as this pathway is known to do), affects nuclear entry/export, or modulates protein stability. The kinetics of the increase in dCREB2 protein in both the nuclear and cytoplasmic compartments cannot distinguish among these possibilities. However, taken together with transgenic experiments, the simplest interpretation is that behavioral training and S6K at least affect nuclear entry of an activator isoform (Fropf, 2013).

One question that arises from the data is why the different antibodies do not recognize a more common pattern of bands. There are nine different dCREB2 protein isoforms reported in the literature. Six out of the nine isoforms contain the bZIP region, while the remaining three use alternative splice sites that result in termination of translation prior to the bZIP region (Tubon, 2013). In addition, there is strong evidence that internal translation initiation can occur at one or more of the three late, in-frame ATG codons just upstream of the bZIP (near the C-terminus of the protein). Therefore, the current estimate is that there are at least six to nine distinct coding isoforms made off of this gene that would contain the bZIP region (Fropf, 2013).

There are seven known, identified phosphorylation sites that are conserved between the mammalian CREB and dCREB2 proteins. In addition, mammalian CREB has been shown to be acetylated, SUMOylated, o-glycosylated, and subject to oxidation-reduction responsive modifications. Almost all of these sites that have been identified on the mammalian protein are conserved on the fly molecules. The possible combinations between protein coding capacity and modification state is very large. For reasons that are not clear, each of seven antibodies that were made, and one mammalian antibody that that this study has used extensively, recognizes some common bands, and some unique ones. It is believe that the combinatorial, [coding + post-translational state] of the protein affects antibody affinity, but the bases for these differences are unclear. However, over the years, the general impression is that certain antibodies recognize certain subsets of bands, and that comparisons need to be made across all of the antibodies to validate specific bands as belonging to dCREB2. Therefore, it is likely that the total number of bands that can be resolved on protein gels is extremely large, and somewhat limited by the availability and relative affinity of each individual antibody (Fropf, 2013).

This report has demonstrated that dCREB2 protein species are enriched in the cytoplasmic compartment. However, at least one species, p35+, increases in nuclear abundance in response to behavioral signals and S6K induction. Moreover, genetic manipulations that block nuclear entry of p35+ also block memory formation. The correlation between behavioral training that produces long-term memory and an increase in p35+ nuclear abundance is consistent with the possibility that nuclear entry is the rate-limiting step for dCREB2-responsive transcription (Fropf, 2013).

A kinase-dependent feedforward loop affects CREBB stability and long term memory formation

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).

Long-term memory engram cells are established by c-Fos/CREB transcriptional cycling

Training-dependent increases in c-fos have been used to identify engram cells encoding long-term memories (LTMs). However, the interaction between transcription factors required for LTM, including CREB and c-Fos, and activating kinases such as phosphorylated ERK (pERK) in the establishment of memory engrams has been unclear. Formation of LTM of an aversive olfactory association in flies requires repeated training trials with rest intervals between trainings. This study finds that prolonged rest interval-dependent increases in pERK induce transcriptional cycling between c-Fos and CREB in a subset of KCs in the mushroom bodies, where olfactory associations are made and stored. Preexisting CREB is required for initial c-fos induction, while c-Fos is required later to increase CREB expression. Blocking or activating c-fos-positive engram neurons inhibits memory recall or induces memory-associated behaviors. These results suggest that c-Fos/CREB cycling defines LTM engram cells required for LTM (Miyashita, 2018).

This study has found that activation of CREB is only part of a c-Fos/CREB cycling program that occurs in specific cells to generate memory engrams. Previous studies have shown that LTM is encoded in a subset of neurons that are coincidently activated during training. The data suggest that these coincidently activated neurons differ from other neurons because they activate c-Fos/CREB cycling, which then likely induces expression of downstream factors required for memory maintenance. Thus, memory engram cells can be identified by the colocalization of c-Fos, CREB, and pERK activities. Inhibiting synaptic outputs from these neurons suppresses memory-associated behaviors, while artificial activation of these neurons induces memory-based behaviors in the absence of the conditioned stimulus (Miyashita, 2018).

The importance of rest intervals during training for formation of LTM is well known. 10x spaced training produces LTM in flies, while 48x massed trainings, which replace rest intervals with further training, does not. It has been shown that pERK is induced in brief waves after each spaced training trial, and it has been proposed that the number of waves of pERK activity gates LTM formation. While the current results are generally consistent with previous studies, this study found that LTM is formed after 48x massed training in CaNB2/+ and PP1/+ flies, which show sustained pERK activity instead of wave-like activity. Thus, it is suggest that either sustained pERK activity or several bursts of pERK activity are required, first to activate endogenous CREB, then to activate induced c-Fos, and later to activate induced CREB (Miyashita, 2018).

In this study, 10x massed training of CaNB2/+ flies produces an intermediate form of protein synthesis-dependent LTM that declines to baseline within 7 days. This result is consistent with results from a previous study, which identified two components of LTM: an early form that decays within 7 days and a late form that lasts more than 7 days. 10x massed training takes the same amount of time as 3x spaced training, which is insufficient to produce 7-day LTM and instead produces only the early form of LTM from preexisting dCREB2. It is proposeed that long-lasting LTM requires increased dCREB2 expression generated from c-Fos/CREB cycling. This increased dCREB2 expression allows engram cells to sustain expression of LTM genes for more than 7 days (Miyashita, 2018).

Although it is proposed that c-Fos/CREB cycling forms a positive feedback loop, this cycling does not result in uncontrolled increases in c-Fos and dCREB2. Instead, spaced training induces an early dCREB2-dependent increase in c-fos and other LTM-related genes, and subsequent c-Fos/CREB cycling maintains this increase and sustains LTM. It is believed that c-Fos/CREB cycling does not cause uncontrolled activation, because dCREB2 activity depends on an increase in the ratio of activator to repressor isoforms. The data indicate that splicing to dCREB2 repressor isoforms is delayed relative to expression of activator isoforms, leading to a transient increase in the activator-to-repressor ratio during the latter half of spaced training. However, the ratio returns to basal by the 10th training cycle, suggesting that the splicing machinery catches up to the increase in transcription. The transience of this increase prevents uncontrolled activation during c-Fos/CREB cycling and may explain the ceiling effect observed in which training in excess of 10 trials does not further increase LTM scores or duration (Miyashita, 2018).

Why does ERK activity increase during rest intervals, but not during training? ERK is phosphorylated by MEK, which is activated by Raf. Amino acid homology with mammalian B-Raf suggests that Drosophila Raf (DRaf) is activated by cAMP-dependent protein kinase (PKA) and deactivated by CaN. The current results indicate that ERK activation requires D1-type dopamine receptors and rut-AC, while a previous study demonstrates that ERK activation also requires Ca2+ influx through glutamate NMDA receptors. Thus, training-dependent increases in glutamate and dopamine signaling may activate rut-AC, which produces cAMP and activates PKA. PKA activates the MAPK pathway, resulting in ERK phosphorylation. At the same time, training-dependent increases in Ca2+/CaM activate CaN and PP1 to deactivate MEK signating in increased ERK activation during the rest interval after training (Miyashita, 2018).

This study examined the role of ERK phosphorylation and activation in LTM and did not observe significant effects of ERK inhibition in short forms of memory. However, a previous study reported that ERK suppresses forgetting of 1-hr memory, suggesting that ERK may have separate functions in regulating STM and LTM. c-Fos/CREB cycling distinguishes engram cells from non-engram cells, and it is suggested that this cycling functions to establish and maintain engrams. However, studies in mammals indicate that transcription and translation after fear conditioning is required for establishing effective memory retrieval pathways instead of memory storage. Thus, c-Fos/CREB cycling may be required for establishment and maintenance of engrams or for retrieval of information from engrams (Miyashita, 2018).

The engram cells identified in this study consist of α/β KCs, a result consistent with previous studies demonstrating the importance of these cells in LTM. Although some α/β neurons are seen expressing high amounts of dCREB2 in naive and massed trained animals (6.5% ± 0.5% of pERK-positive cells in massed trained animals), few c-fos-positive cells are seen and no overlap between c-fos expression and dCREB2 in these animals. After spaced training, the percentage of cells that express both c-fos and dCREB2 jumps to 18.9% ± 1.2%, and these cells fulfill the criteria for engram cells, because they are reactivated upon recall and influence memory-associated behaviors. The phosphatase pathway may predominate during training, inhibiting ERK phosphorylation. However, phosphatase activity may deactivate faster at the end of training compared to the Rut/PKA activity, While some mammalian studies suggest that neurons that express high amounts of CREB are preferentially recruited to memory engrams, this study found that the percentage of neurons that express high dCREB2 and low c-fos remains relatively unchanged between massed trained and spaced trained flies. Furthermore, this study finds that the increase in neurons expressing high amounts of dCREB2 after spaced training corresponds to the increase in c-Fos/CREB cycling engram cells. Thus, in flies, LTM-encoding engram cells might not be recruited from cells that previously expressed high amounts of dCREB2 but instead may correspond to cells in which c-Fos/CREB cycling is activated by coincident odor and shock sensory inputs (Miyashita, 2018).

Mushroom body subsets encode CREB2-dependent water-reward long-term memory in Drosophila

Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. This study shows that acute inhibition of protein synthesis in the mushroom body (MB) αβ or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αβ or γ neurons also disrupts wLTM. The MB αβ and γ neurons can be further classified into five different neuronal subsets including αβ core, αβ surface, αβ posterior, γ main, and γ dorsal. This stuyd observed that the neurotransmission from αβ surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αβ core, αβ posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αβ surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αβ surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. These results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila (Lee, 2020).

CREB-dependent gene transcription is critical for memory formation, especially LTM, in both vertebrates and invertebrates. Several previous studies in Drosophila suggest that LTM formation requires CREB-dependent gene transcription followed by de novo protein synthesis. Moreover, it has been reported that CREB2 activity in both αβ and α'β' neurons is critical for appetitive LTM produced by sugar-reward conditioning. This study observed that wLTM formation requires de novo protein synthesis in the αβ and γ neurons. Moreover, adult stage-specific expression of the CREB2 repressor (dCREB2b) in αβ or γ neurons disrupts wLTM, whereas CREB2 activity in α'β' neurons is dispensable. Food or water deprivation is necessary to induce the motivational drive in flies to form sugar- or water-reward LTM since different motivational drives are critical for distinct memories. It has been shown that individual internal motivational inputs for sugar and water are delivered via distinct MB input neurons, which finally induce sugar- or water-reward LTM in different MB neuron subsets. Previous studies together with the current findings suggest that CREB2 activity is required for both sugar- and water-reward LTMs, however, these LTMs are processed in different MB circuits (Lee, 2020).

Neurotransmission from αβ neurons is required for both shock-punitive and sugar-reward LTMs retrieval, whereas neurotransmission from γ neurons is dispensable for retrieval of both shock-punitive and sugar-reward LTMs. However, a previous study showed that the neurotransmission from αβ and γ neurons is required for wLTM retrieval suggesting that wLTM is different from the other types of olfactory associative LTMs at MB circuit levels. The αβ and γ neurons are classified into αβ core, αβ surface, αβ posterior, γ main, and γ dorsal neuron subsets according to the morphology of their axons (Aso, 2014). It has been shown that neurotransmission from the combination of MB αβ surface and αβ posterior subsets is necessary for the retrieval of both shock-punitive and sugar-rewarded LTMs. Another study suggests that the neurotransmission from αβ surface and αβ core subsets is required for the retrieval of sugar-reward LTM. These results imply that several different subsets of αβ neurons participate in the retrieval of Drosophila sugar-reward LTM. This study showed that neurotransmission only from αβ surface is necessary for wLTM retrieval, whereas the αβ core and αβ posterior subdivisions are dispensable. Contrary to the sugar reward conditioning in which γ neurons are dispensable for the retrieval of sugar-reward LTM, wLTM retrieval requires neurotransmission from γ dorsal but not from γ main neuron subset. Taken together, these results imply that γ dorsal neuronal activity is specifically required for wLTM retrieval but not for sugar-reward LTM. Adult stage-specific expression of dCREB2b or blocking de novo protein synthesis in αβ surface and γ dorsal neurons disrupts wLTM, further suggesting the crucial role of αβ surface and γ dorsal neurons in Drosophila wLTM process (Lee, 2020).

A previous study suggests that PAM-β'1 neurons convey the water-rewarding event as the US signal to the MB β' lobes, and the neurotransmission in α'β' neurons is required for wLTM consolidation (Wu, 2017). How the wLTM is transferred from α'β' neurons and finally stored in αβ surface and γ dorsal neurons through system consolidation is still unclear. In both shock-punitive and sugar-reward LTMs, the neurotransmission in α'β', γ, and, αβ neurons is required for at least 3 hours after conditioning, but the expression of 24-hour shock-punitive or sugar-rewarded memories only requires neurotransmission in αβ neurons. In addition, the expression of DopR1, a D1-like dopamine receptor, in the γ neurons is sufficient to fully support the shock-punitive STM and LTM in DopR1 mutant background (dumb²),hβ' neurons. Another possibility is that the activity from α'β' neurons is transmitted to αβ surface and γ dorsal neurons via the relevant α'β' MBONs and their downstream neurons. Therefore, it is noteworthy to test the physiological roles of DPM neurons and α'β' MBONs during consolidation phase of wLTM (Lee, 2020).

A significant increase in cellular calcium response to training odor in the αβ surface neurons, but not in other αβ neuron subsets, was observed 24-hour after water-reward conditioning. These results are consistent with the behavioral study showing that neurotransmission from αβ surface neurons is required for wLTM retrieval, whereas the αβ core and αβ posterior neuron subsets are dispensable. This training-induced increased calcium response was abolished in water-sated or dCREB2b expressing flies. A previous study showed that the fly forms α-lobe branch-specific aversive LTM trace 24-hour after odor/shock conditioning. In a recent study, the α-lobe branch-specific aversive anesthesia-resistant memory (ARM) trace 3-hour after odor/shock conditioning was also observed. Intriguingly, it was found that both α- and β- lobes of the surface neurons show increased calcium response to training odor 24-hour after odor/water conditioning, suggesting that the wLTM trace is not specific to the α-lobe branch (Lee, 2020).

An increased GCaMP response to training odor in MB γ neurons is observed at 24-hour after ten sessions of spaced odor/shock training, and this increased calcium response is abolished by expressing dCREB2b in γ neurons throughout the fly development. This study found that γ main neuron subset shows an evoked calcium response to both odors, but no further increased calcium response to the training odor at 24-hour after water-reward conditioning was observed. A previous study suggests that γ dorsal neurons respond to visual stimuli, which is required for visual, but not for aversive olfactory memory in Drosophila. However, this study observed a decreased calcium response to odor stimuli in the γ dorsal lobe, which is consistent with the electrophysiological study showing slow inhibitory responses to odor stimuli in the γ dorsal neurons. Interestingly, a further decrease was observed in calcium responses to the training odor in the γ dorsal neurons 24-hour after water-reward conditioning. This training-induced additional decrease in the calcium response is abolished in the water-sated or acutely dCREB2b expressing flies, suggesting a type of wLTM trace in the γ dorsal neurons different from the αβ surface neurons. Since blocking neurotransmission from the γ dorsal neuron subset during memory retrieval disrupts wLTM, why the γ dorsal neurons show additionally suppressed calcium response to training odor, needs to be answered. One possible explanation is that odor/water association alters the olfactory response of MB neurons to the training odor, and this change can be represented by an increased or decreased calcium response as compared to the response of the non-training odor (memory traces). The training-induced differences in odor responsive levels in the MB allow the flies to distinguish two odors by increasing the contrast and perform appropriate behavioral output during testing. However, shits abolishes the increased or decreased training-odor responses thereby eliminating the contrast between odors, and consequently, the flies could not distinguish two odors and make appropriate behavioral output during testing (Lee, 2020).

In conclusion, this study has shown that αβ surface and γ dorsal neuron subsets regulate Drosophila wLTM. Blocking neurotransmission from αβ surface or γ dorsal neurons only abolishes wLTM retrieval but does not affect the olfactory acuity or water preference in thirsty flies. Further, adult stage-specific expression of dCREB2b or blocking de novo protein synthesis in αβ surface and γ dorsal neurons disrupts wLTM. Different dynamics of cellular wLTM traces are formed in the αβ surface and γ dorsal neurons, which are blocked by dCREB2b expression. Taken together, these results reveal a small population of MB neurons that encode wLTM in the brain and provide a broader view of the olfactory memory process in fruit flies (Lee, 2020).

CREBA and CREBB in two identified neurons gate long-term memory formation in Drosophila

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).

CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila

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).

GENE STRUCTURE

PROTEIN STRUCTURE

Structural Domains

CrebB-17A consists of seven isoforms (alternatively spliced forms). The isoforms fall into two categories. In one category, four of the seven isoforms have the basic leucine zipper domain of exon 7. In the second category the b zip domain is absent. The basic zip domain consists of a basic activation domain followed by a leucine zipper domain. A short amino acid motif is a target for phosphorylation by kinases (P-box). The P-box is located carboxyl terminal to a glutamate-rich region (Yin 1994).

date revised: 29 August 2023

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