Synapsin is a phosphoprotein reversibly associated with synaptic vesicles. Synapsin function in mediating synaptic activity was investigated during intense stimulation at Drosophila motor boutons. Electron microscopy analysis of synapsin⁻ boutons demonstrated that synapsin maintains vesicle clustering over the periphery of the bouton. Cyclosporin A pretreatment disrupted peripheral vesicle clustering, presumably due to increasing synapsin phosphorylated state. Labeling recycling vesicles with a fluorescent dye FM1-43 followed by photoconversion of the dye into electron dense product demonstrated that synapsin deficiency does not affect mixing of the reserve and recycling vesicle pools but selectively reduces the size of the reserve pool. Intense stimulation produced a significant increase in vesicle abundance and vesicle redistribution toward the central core of synapsin ⁺ boutons, while in synapsin⁻ boutons the area occupied by vesicles did not change and the increase in vesicle numbers was not as prominent. However, intense stimulation produced an increase in basal release at synapsin⁻ but not in synapsin⁺ boutons, suggesting that synapsin may direct vesicles to the reserve pool. Finally, synapsin deficiency inhibited an increase in quantal size and formation of endosome-like cisternae, which was activated either by intense electrical stimulation or by high K⁺ application. Taken together, these results elucidate a novel synapsin function, specifically, promoting vesicle reuptake and reserve pool formation upon intense stimulation (Akbergenova, 2010).

The question of how neuronal terminals maintain synaptic transmission during intense neuronal stimulation remains fascinating and controversial. It is now understood that synaptic vesicles undergo a series of preparatory steps to be properly activated for neurotransmitter release, and, according to their functional state, vesicles can be subdivided into several functional pools (Schneggenburger, 2002 for review). The recycling pool maintains exo/endocytosis at moderate stimulation paradigms, and it is refilled by newly recycled vesicles, while the reserve pool is a depot of synaptic vesicles which contribute to release only during intense stimulation (Rizzoli, 2005 for review). Multiple pathways have been proposed for recycling of synaptic vesicles upon exocytosis, but the relationship between these pathways and functional vesicle pools is not fully understood (Akbergenova, 2010).

At the majority of synapses, the recycling and reserve pools are spatially intermixed (Rizzoli, 2005 for review). However, at Drosophila Ib type motor boutons, optical studies relying on vesicle staining with FM1-43 dye lead to a view that the recycling pool occupies the periphery of the bouton and the reserve pool is spread towards its central core (Kidokoro, 2004; Kuromi, 2005; Verstreken, 2005). This view has been recently revised by two ultrastructural studies (Akbergenova, 2009; Denker, 2009) demonstrating that at this synapse vesicles generally cluster over the periphery of the bouton, but the reserve and recycling pools are spatially intermixed. Furthermore, it was demonstrated that intense stimulation produces an increase in vesicle abundance, as well as vesicle redistribution towards the central core of the bouton (Akbergenova, 2009a). This form of plasticity was associated with an increased synaptic enhancement during a subsequent stimulation (Akbergenova, 2010).

Vesicle recycling may follow different routes or pathways, including active zone recycling, clathrin-mediated endocytosis, endosomal intermediates, or bulk membrane uptake. An intense synaptic activity is often associated with the recycling pathway that involves a formation of endosome-like structures or cisternae. Recently, it was recognized that endosome-like structures may be capable of exocytosis, and that this process may produce enlarged neurosecretory quanta. Thus, formation of endosome-like cisternae and enlarged vesicles is likely to represent a pathway that enhances synaptic efficacy (Akbergenova, 2010).

In summary, a neuronal terminal may sustain an intense stimulation by mobilizing reserve vesicles into the recycling pathway, by formation of extra vesicles, or by formation of enlarged vesicles and quanta. The goal of this study was to understand how these mechanisms are regulated by a synaptic vesicle protein synapsin (Akbergenova, 2010).

Synapsins, abundant and highly conserved family of phoshoproteins reversibly associated with vesicles, have been implicated in maintaining the reserve pool (Bloom, 2003; Akbergenova, 2007; Gitler, 2008) and regulating mobilization of vesicles into the recycling pool (Chi, 2003; Cousin, 2003; Menegon, 2006; Baldelli, 2007). In their dephosphorylated form, synapsins attach to synaptic vesicles and trigger actin polymerization (Bloom, 2003), while synapsin phosphorylation causes its dissociation from vesicles (Hosaka, 1999). Synapsin-dependent regulation of the reserve and recycling vesicle pools was found to be critical during sustained stimulation (Humeau, 2001). Consistently, synapsin deficiency produces enhanced synaptic depression (Rosahl, 1995; Gitler, 2004; Samigullin, 2004; Akbergenova, 2010 and references therein).

To investigate the role of synapsin in activity dependent plasticity at the Drosophila motor boutons, advantage was taken of the synapsin knockout [synapsin⁻] fly (Godenschwege, 2004) that was shown to have defects in complex behavior, learning and memory (Michels, 2005). This study has investigated how synaptic ultrastructure and activity is regulated upon intense stimulation in synapsin⁻ boutons (Akbergenova, 2010).

Synapsin functions in enabling a nerve terminal to maintain and enhance its activity during sustained stimulation. This synapsin function is likely to be mediated by several mechanisms. First, synapsin maintains vesicle organization that leaves void and, possibly, reserve spaces; these reserve spaces may be filled with extra vesicles upon intense stimulation. Second, synapsin is likely to participate in the process of vesicle recycling more directly, by directing the extra vesicles formed upon intense stimulation into the reserve pool. Finally, synapsin is required for an activation of the recycling pathway that produces enlarged vesicles/quanta upon maintained depolarization (Akbergenova, 2010).

Synapsin functioning is responsible for peripheral vesicle organization in Drosophila Ib motor boutons. Normally, vesicles in these boutons cluster over the periphery of the bouton leaving void spaces. This study has demonstrated that this vesicle organization is disrupted in synapsin null mutants. Furthermore, synapsin, similar to vesicles, has a peripheral distribution. This result suggests that synapsin may perform its function by binding to vesicles and tethering them at a peripheral cluster. In support of this idea, it was also demonstrated that a treatment by a calcineurin inhibitor CycA disrupts both peripheral vesicle clustering and the peripheral distributiuon of synapsin (Akbergenova, 2010).

The latter findings compel revisiting an earlier hypothesis that CycA treatment mobilizes reserve vesicles into the recycling pathway. The above interpretation was largely based upon the observation that FM1-43 fluorescence spread towards the center of the bouton when a mild stimulation paradigm was employed in the presence of CycA. The data suggests a different interpretation for this CycA action. It is known that CycA application completely blocks synapsin dephosphorylation at sites 4,5,6 (Jovanovic, 2001), which are controlled by MAP kinase/calcineurin activity. Interestingly, one of these sites (S62) is conserved between mammals and Drosophila (Genbank, COBALT alignment). Thus, it is likely that one of the consequences of CycA treatment is an increase in synapsin phosphorylated forms, and, consequentially, synapsin dispersion from vesicles followed by a dispersion of vesicles over the entire bouton (Akbergenova, 2010).

The synapsin function in maintaining vesicle clustering appears to be rather general among different types of organisms and synapses. Indeed, the studies performed at the giant lamprey synapse showed that synapsin neutralization disrupts vesicle clustering (Bloom, 2003; Hilfiker, 1999). Vesicle clustering is widely observed at mammalian CNS synapses even though regularities in vesicle distribution are not immediately evident. In synapsin null mutants vesicles are generally depleted (Rosahl, 1995; Li, 1995; Gitler, 2004; this study), and the degree of depletion may depend on the terminal region in relation to the plasma membrane. A tomography study (Siksou, 2007) demonstrated that vesicles are linked into clusters by a filamentous matrix, and that synapsin belongs to these clusters. Thus, the general synapsin function in vesicle clustering is evident. This study suggests an important physiological function of this clustering. Specifically, it suggests that synapsin may enable nerve terminals to maintain reserve spaces that can be filled by newly formed vesicles upon intense stimulation (Akbergenova, 2010).

This study has demonstrated that synapsin is a critical player in the process of structural potentiation, a form of plasticity which produces formation of extra vesicles upon intense stimulation (Akbergenova, 2009). It is thought that the major underlying mechanism of this structural potentiation is excess endocytosis (Engisch, 1998) that may be accelerated by intense synaptic activity (Wu, 2005). The possibility that a part of the newly formed vesicle pool is produced by anterograde axonal vesicle trafficking cannot be completely excluded (Akbergenova, 2010).

Hypothetically, actin/synapsin interactions could contribute to the latter mechanism by reducing vesicle mobility and promoting vesicle capturing in the terminals, thus enhancing vesicle supply via axonal transport. However, recent studies indicate that this possibility is unlikely. There is, in contrast emerging evidence that synapsin may contribute to endocytosis upon intense stimulation. In particular, nerve stimulation has been shown to produce synapsin immunoreactivity in the region of endocytic zones (Bloom, 2003). Furthermore, sustained stimulation of synapsin null terminals produced enhanced synaptic depression, as was observed in several preparations (Rosahl, 1995; Gitler, 2004; Samigullin, 2004), including Drosophila NMJ (this study). It is therefore suggested that a synapsin-dependent mechanism may enhance endocytosis upon sustained intense activity and direct the newly formed vesicles into the reserve pool (Akbergenova, 2010).

This study clearly demonstrates a critical role of synapsin in the latter mechanism, namely directing newly formed vesicles into the reserve pool, as well as maintaining the reserve pool. First, FM1-43 photoconversion experiments demonstrated that the reserve pool is diminished in synapsin⁻ terminals, while the recycling pool is unaffected. Second, recordings of synaptic activity from potentiated boutons demonstrated that the vesicles newly formed upon potentiation are directed into the reserve pool in WT but not in synapsin⁻ terminals. These results directly demonstrate synapsin functioning in forming and maintaining the reserve pool and suggest that this function is carried out via mediating compensatory endocytosis during sustained activity (Akbergenova, 2010).

Synapses may respond to maintained depolarization by activation of a recycling pathway that produces numerous endosome-like cisternae. This endosome-like structures are likely to represent a mixture of intermediates from three pathways, including bulk membrane uptake. Some of the endosome-like structures may fuse with the presynaptic membrane (Akbergenova, 2010).

Recent rigorous study at the calyx synapse demonstrated that intense activity initiates compound vesicle fusion, which is followed by formation of enlarged vesicles, subsequent release of enlarged neurosecretory quanta, and a compensatory bulk membrane reuptake. This study at the Drosophila NMJ produced similar conclusions, showing no FM1-43 staining at some of the enlarged vesicles (presumably formed by compound vesicle fusion), a strong and uniform FM1-43 uptake at other endosome-like structures (presumably produced by bulk endocytosis), and a non-uniform staining pattern (presumably, a fusion of stained and non-stained vesicles). The formation of enlarged vesicles produces an increase in quantal size that is likely to represent a mechanism for enhancing synaptic activity in response to a sustained stimulation. Indeed, enlarged vesicles/quanta appear as a result of enhanced motor activity in vivo, implying a physiological significance of this mechanism (Akbergenova, 2010).

This study has demonstrated that both formation of enlarged vesicles/cisternae and release of enlarged quanta critically depend on the presence of synapsin. Synapsin⁻ terminals lack the ability to increase the quantal size in response to sustained depolarization, since the formation of enlarged vesicles/cisternae is inhibited in synapsin⁻ terminals. It is important to note that synapsin deficiency inhibits activity-dependent formation of both endosome-like structures and vesicles. This finding, rules out the possibility that extra vesicles produced upon intense stimulation could be formed from endosome-like cisternae (Akbergenova, 2010).

Thus this study reveals a novel synapsin function, specifically, enhancing synaptic transmission via an increase in the size of a neurosecretory quantum. Together with earlier studies demonstrating that the formation of enlarged vesicles/cisternae depends on actin polymerization, these results may elucidate a specific molecular pathway of the compound vesicle fusion, suggesting that some of the observed endosome-like structures may be formed via an actin/synapsin-dependent association of synaptic vesicles (Akbergenova, 2010).

In summary, this study has demonstrated that the ability of a synapse to respond to a sustained stimulation is compromised in synapsin⁻ terminals in several ways. First, synapsin⁻ terminals have a diminished reserve pool and a limited ability to sustain synaptic activity in response to continuous stimulation. Second, synapsin⁻ terminals have a reduced ability to increase vesicle abundance in response to intense stimulation. Third, the vesicles newly formed in response to an intense stimulation are not directed to the reserve pool in the absence of synapsin. Finally, synapsin is required for an activation of a recycling pathway that produces enlarged vesicles/quanta in response to a maintained depolarization (Akbergenova, 2010).

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. This assumption was tested by an approach termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake it was found that only approximately 1%-5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ~5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. It is concluded that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in an accompanying paper (Denker, 2011a).

It is hypothesized that a physical barrier may inhibit the release of the majority of the vesicles, perhaps by lowering their mobility. Synapsin has been proposed to bind to synaptic vesicles and/or to the cytoskeleton, thus acting as a 'glue' keeping vesicles in a clustered (possibly inactive) state (see Cesca, 2011). To test whether vesicle clustering may affect their release in vivo, synapsin-null Drosophila larvae (Godenschwege, 2004) were studied. Indeed, fluorescence recovery after photobleaching (FRAP) experiments revealed that the vesicles were significantly more mobile in synapsin-null animals. The vesicle labeling was also much stronger in vivo, with approximately 30% of the vesicles labeled at 2-4 h after injection, allowing the conclusion that synapsin is one potential marker for the inactive vesicles (Denker, 2011a).

Because synapsin is not the only molecule cross-linking vesicles, it is not unexpected that a substantial nonreleasing pool of vesicles persists in the synapsin-null animals. Importantly, synapsin is a soluble molecule, rather than an integral component of the vesicles, and therefore it is not likely to constitute a permanent vesicle tag. Thus, an intermixing of the active and inactive vesicles should take place over time, although it seems to be on a very slow scale, of a few hours (Denker, 2011a)

It is suggested that most vesicles do not participate directly in neurotransmitter release and vesicle recycling. The number of vesicles functioning in vitro has been much discussed in the last decades, with many conflicting studies. Generally, NMJ works conclude that many or most vesicles can be used under stimulation, although some vesicle pools are used preferentially, whereas the remaining vesicles constitute a reserve pool. One group of studies has concluded that a substantial proportion of the vesicles in hippocampal neurons remain unused even during strong nonphysiological stimulation, albeit other investigations suggested that all vesicles may be releasable (Denker, 2011a and references therein)

The classic reserve pool of the literature typically comprises 50%-80% of all vesicles. This concept can hardly apply to an in vivo situation in which this study found more than 90% to be 'in reserve' or else to serve another purpose. It is difficult to imagine physiological situations that would require synapses to release substantially more vesicles at any one time. For example, the ventral muscles of the Drosophila larva would certainly be used more frequently if the larva attempted to avoid a predator, but with Drosophila movement being inherently slow, no great rates of activity could ever be attained. Similarly, stress increases the use of vesicles in locusts, but even in this life-or-death situation the jumping behavior is infrequent. Importantly, because predation eventually results in the death of all locusts analyzed, no higher stimulation could take place in vivo -- no life situation could have induced a stronger need for movement (and hence vesicle recycling). Finally, the rates of activity cannot increase endlessly/continually, at least for the NMJs, because the muscles would stop responding (Denker, 2011a).

The reserve vesicles seem to be clustered and are therefore rather immobile. They would simply stay linked to other vesicles and/or to the cytoskeleton (perhaps via synapsin), at tens or hundreds of nanometers from the active zones. This hypothesis is in agreement with two recent findings on vesicle mobility and recycling: first, only a small pool of vesicles is mobile in cultured hippocampal synapses, with all other vesicles immobile ('fixed'). The fixed vesicles did not become more mobile upon physiological stimulation, suggesting that they may not be involved in neurotransmitter release and recycling under normal circumstances. Second, in the same preparation only few active (mobile) vesicles have their membranes optimally sorted via endosomes. All other vesicles were found to be reluctant to release and did not seem to use endosomes when forced to release via prolonged stimulation in vitro. It is tempting to hypothesize that the reason why most synaptic vesicles do not sort their membranes optimally (via endosomes) is that these vesicles are not normally intended for recycling (in vivo) (Denker, 2011a).

The current data do not exclude the existence of synapses that use most of their vesicles. They also clearly indicate that the pool tags are not permanent, with the active and inactive vesicles intermixing slowly over time, so that in a strict sense all vesicles may be eventually used in neurotransmitter release. Additionally, the intermixing might be faster at higher temperatures (at least for invertebrates, which were maintained at 21°C in these experiments). However, at any point in time only a small proportion of the vesicles recycle. It is therefore unlikely that physiological activity would deplete all vesicles at one time, which is the only event that would justify the need for a large reserve pool of vesicles. In a related study (Denker, 2011b), the hypothesis is presented that these vesicles support release indirectly, functioning primarily as a molecular buffer for proteins involved in vesicle recycling. Thus, all vesicles seem to have some role in synaptic physiology, although only a minority is actually involved in neurotransmitter release at any one time (Denker, 2011a).

Synapsin is an evolutionarily conserved, presynaptic vesicular phosphoprotein. This study asked where and how synapsin functions in associative behavioral plasticity. Upon loss or reduction of synapsin in a deletion mutant or via RNAi, respectively, Drosophila larvae are impaired in odor-sugar associative learning. Acute global expression of synapsin and local expression in only the mushroom body, a third-order 'cortical' brain region, fully restores associative ability in the mutant. No rescue is found by synapsin expression in mushroom body input neurons or by expression excluding the mushroom bodies. On the molecular level, it was found that a transgenically expressed synapsin with dysfunctional PKA-consensus sites cannot rescue the defect of the mutant in associative function, thus assigning synapsin as a behaviorally relevant effector of the AC-cAMP-PKA cascade. It is therefore suggested that synapsin acts in associative memory trace formation in the mushroom bodies, as a downstream element of AC-cAMP-PKA signaling. These analyses provide a comprehensive chain of explanation from the molecular level to an associative behavioral change (Michels, 2011).

The associative defect in the syn⁹⁷-mutant (Michels, 2005) can be phenocopied by an RNAi-mediated knock-down of synapsin, and can be rescued by acutely restoring synapsin. In terms of site of action, locally restoring synapsin in the mushroom bodies fully restores associative ability, whereas restoring synapsin in the projection neurons does not. If synapsin is restored in wide areas of the brain excluding the mushroom bodies, learning ability is not restored either. Therefore, it was concluded that a synapsin-dependent memory trace is located in the mushroom bodies, and suggest that this likely is the only site where such a trace is established regarding odor-sugar short-term memory in larval Drosophila. In terms of mode of action, it was found that a synapsin protein that carries dysfunctional PKA sites cannot rescue the syn⁹⁷-mutant learning defect. It is therefore suggested that synapsin functions as a downstream element of AC-cAMP-PKA signaling in associative function (Michels, 2011).

Arguably, the Rutabaga type I adenylyl cyclase acts as a detector of the coincidence between an aminergic reinforcement signal (appetitive learning: octopamine; aversive learning: dopamine) and the odor-specific activation of the mushroom body neurons. Initially, this notion had been based on mutant and biochemical analyses in Drosophila and physiology in Aplysia. Indeed, activation of mushroom body neurons in temporal coincidence with dopamine application increases cAMP levels in wild-type, but not AC-deficient flies (rut²⁰⁸⁰), and a corresponding AC-dependence of PKA activation has been shown by mushroom body costimulation with octopamine. However, the downstream effects of the AC-cAMP-PKA cascade has remained clouded. This study suggests that, similar to the situation in snails, one of these PKA effectors is synapsin, such that synapsin phosphorylation allows a transient recruitment of synaptic vesicles from the reserve pool to the readily releasable pool. A subsequent presentation of the learned odor could then draw upon these newly recruited vesicles. This scenario also captures the lack of additivity of the syn⁹⁷ and rut²⁰⁸⁰ mutations in adult odor-shock associative function, and the selective defect of the syn⁹⁷-mutation in short- rather than longer-term memory (Michels, 2011).

Given that the memory trace established in the current paradigm likely is localized to few cells relative to the brain as a whole, given that these are transient, short-term memory traces, and given the possibility of dephosphorylation, it is not unexpected that Nuwal (2011) did not uncovered either predicted PKA site of synapsin as being phosphorylated in a biochemical approach, using whole-brain homogenates from untrained adult Drosophila (for similar results in Drosophila embryos see Zhai, 2008). Given the likely spatial and temporal restriction of these events in vivo, immunohistological approaches are warranted to see whether, where, and under which experimental conditions synapsin phosphorylated at either of its PKA sites indeed can be detected (Michels, 2011).

Interestingly, the evolutionarily conserved N-terminal PKA-1 site undergoes ADAR-dependent mRNA editing, which despite the genomically coded RRFS motif yields a protein carrying RGFS. This editing event, as judged from whole-brain homogenates, occurs for most but not all synapsin and, as suggested by in vitro assays of an undecapeptide with bovine PKA, may reduce phosphorylation rates by PKA. Given that the successfully rescuing UAS-syn construct codes the edited RGFS sequence, it should be interesting to see whether this rescue is conferred by residual phosphorylation at PKA-1, and/or by phosphorylation of the evolutionarily nonconserved PKA-2 site. Last, one may ask whether an otherwise wild-type synapsin protein featuring a nonedited RRFS motif is also rescuing associative function (Michels, 2011).

In any event, the finding that the PKA consensus sites of synapsin are required to restore learning in the syn⁹⁷-mutant is the first functional argument to date, in any experimental system, to suggest synapsin as an effector of the AC-cAMP-PKA cascade in associative function (Michels, 2011).

In Drosophila, aversive associative memory of an odor consists of heterogeneous components with different stabilities. This study reports that Bruchpilot (Brp), a ubiquitous presynaptic active zone protein, is required for olfactory memory. Brp was shown before to facilitate efficient vesicle release, particularly at low stimulation frequencies. Transgenic knockdown in the Kenyon cells of the mushroom body, the second-order olfactory interneurons, revealed that Brp is required for olfactory memory. Brp in the Kenyon cells preferentially functions for anesthesia-resistant memory. Another presynaptic protein, Synapsin, has been shown previously to be required selectively for the labile anesthesia-sensitive memory, which is less affected in brp knockdown. Thus, consolidated and labile components of aversive olfactory memory can be dissociated by the function of different presynaptic proteins (Knapek, 2011).

In flies, middle-term olfactory memory after a single training cycle comprises functionally dissociable forms of memory: the labile ASM and the stable ARM. In contrast to ASM, the molecular basis of ARM formation is poorly understood. Only a few molecules have been shown to be important for ARM so far. This study demonstrated that Brp in the Kenyon cells of the mushroom body is preferentially required for ARM. Although there is no apparent developmental defect in the downregulation of Brp (Wagh, 2006), that study does not specify the requirement for ARM in the adult (Knapek, 2011).

The Brp protein is specifically localized to the active zone at the presynaptic terminals, in which it forms electron dense projections. Interestingly, the Radish protein that is also required for ARM is highly enriched in the lobes of the mushroom body. Thus, Brp and Radish might interact at the active zones to regulate neurotransmission underlying ARM (Knapek, 2011).

Several memory mutants have been shown to have a selective phenotype in ASM. Consistent with the parallel memory formation of ASM and ARM, the brp knockdown and a rutabaga mutation caused an additive memory deficit. Interestingly, Synapsin is required for ASM preferentially, and the null mutation caused no augmentation of the memory phenotype in the rutabaga single mutant (Knapek, 2010). Thus, the complementary forms of memory might recruit differential signaling mechanisms that rely on distinct presynaptic machineries (Knapek, 2011).

In a current model of memory dynamics, ARM gradually develops after training, whereas ASM occupies the largest part of early memory and decays more quickly. Although Radish and Brp are selectively required for ARM measured at 3 h after training, flies lacking either of the proteins are impaired also in immediate memory. By applying cold anesthesia for STM, this study found that STM does contain a significant ARM component. The consistent requirement of Brp for short-term and 3 h ARM may contribute to a synaptic mechanism of memory that is stable against amnesic treatment (Knapek, 2011).

If ARM and ASM were formed at the same synapses of Kenyon cells, how could the two synaptic proteins Brp and Synapsin dissociate these different forms of memory? Notably, Brp and Synapsin are meant to be required for distinct components of action potential-evoked vesicle release. In vertebrates, Synapsin has been shown to be particularly important for recruitment of synaptic vesicles from reserve pools at high stimulation frequencies (Pieribone, 1995; Rosahl, 1995; Gitler, 2004; Sun, 2006). Consistently, synapsin mutants show normal quantal content (number of synaptic vesicles released per action potential) at moderate action potential frequencies. Similarly, Drosophila Synapsin maintains the reserve pool of vesicles and mediates mobilization of the reserve pool during intense stimulation (Akbergenova, 2007). The brp null mutant in contrast shows decreased quantal contents particularly in response to the first arrival of an action potential. Vesicle release after subsequently following high-frequency spikes however is less affected, suggesting the importance in vesicle release at low-frequency stimulation. The two different modes of neurotransmission (e.g., different release probabilities during high- vs low-frequency stimulation) could therefore differentiate ASM and ARM, even if the traces of these different forms of memory resided in the same synapses of the Kenyon cells (Knapek, 2011).

Alternatively, the memory traces of ASM and ARM could be spatially separated within the same neurons, i.e., localized at different synapse populations. In the lobes, Kenyon cell axons have multiple compartments that are intersected by transverse extrinsic neurons. This study found that brp knockdown in the α/β neurons affected ARM. This is consistent with a previous report, in which inhibition of the output of the α/β neurons impaired ARM. Interestingly, inhibition of a specific type of dopaminergic neurons that synapse onto another restricted compartment of the β lobe selectively affected ASM (Aso, 2010). Thus, associative plasticity underlying ASM and ARM could be formed by stimulating different synapses of the same neurons. This hypothesis may be tested in future by the identification of extrinsic neurons that are specifically required for ARM and corresponding functional imaging of memory traces (Knapek, 2011).

Living organisms need to search for and ingest nutritional chemicals, and gustation plays a major role in detecting and discriminating between chemicals present in the environment. Using Drosophila as a model organism, it was asked whether animals have the ability to evaluate the nutritional value of sugars. In flies, chemosensilla on the tarsi and labellum are the gustatory organs used to discriminate between edible and nonedible compounds. It was noticed that Drosophila do not assign nutritional values to all sweet chemicals. D-arabinose is sweet to flies, but it provides them with no nutrition. By contrast, the sugar alcohol D-sorbitol is not sensed as sweet, but flies can live on it. Behavioral and electrophysiological measurements were performed to confirm these gustatory and feeding responses. It was found that Drosophila can learn the nutritional value of nonsweet D-sorbitol when it is associated with an odor cue. The learning process involved the synapsin molecule, suggesting that a neuronal mechanism is involved. It is proposed that Drosophila uses neural machinery to detect, evaluate, and learn the nutritional value of foods after ingestion (Fujita, 2011).

In an insect body, D-sorbitol is synthesized from D-glucose. It is then stored in the body during the diapause period and used for energy conservation and cold stability. Because D-sorbitol provides energy and supports other functions in flies, it is clear that flies need to recognize and ingest D-sorbitol. The feeding behavior of Drosophila is induced by sequential stimulation of taste cells in the tarsus on the legs, labellum on the proboscis, and interpseudotracheal papilla located inside the labellum. D-sorbitol was found to invoked feeding without stimulating external taste cells (Fujita, 2011).

Tests were performed to see whether a neural system was involved in learning. To this end, the syn⁹⁷ mutant, which is a null mutant of the synapsin gene that encodes a protein necessary for synaptic function, was used. The gene is associated with reduced learning ability in adult flies and larvae. syn⁹⁷ mutant flies showed significantly reduced learning ability, demonstrating that a neural system is involved in nutritional value learning (Fujita, 2011).

How do flies detect D-sorbitol and start feeding behavior? Is it possible that pharyngeal taste cells are involved? If these taste cells play a major role in regulating ingestion and in sensing D-sorbitol, flies would consume a large amount of D-sorbitol once feeding starts. But no evidence for this was found in the feeding assay. As food deprivation increases, spontaneous extension of the proboscis may initiate feeding on D-sorbitol-containing agar medium, and after ingestion the internal sensor might send a positive signal to initiate feeding behavior, despite providing no initial external gustatory input. Evidence is available to support the hypothesis that flies control feeding behavior by a putative internal sensor. poxn flies with no external taste cells also survive on standard medium. These flies may recognize the nutritional value of substances or other factors in sugar without sensing sugar sweetness. In fact, Gr64 null mutant flies have no glucose receptors but can survive on glucose-containing medium. It has been postulated that an internal sensor must exist to monitor the hemolymph sugar level, and a putative receptor molecule has been identified. Such a nutrient body sensor mechanism might inhibit feeding behavior if the ingested sugar provides no nutrition. A similar phenomenon was reported in mutant mice with deficient taste reception that regulate feeding behavior by providing feedback on the nutritional state in the body after ingestion. It has been proposed that a dopaminergic neural reward system is involved (Fujita, 2011).

This study reveals that flies learn the nutritional value of sugar, but the process by which they learn this is still unclear. Gustatory receptor neurons send axons to the subesophageal ganglion. It remains to be elucidated how information from the subesophageal ganglion is transferred to the central brain. A detailed functional anatomy of the subesophageal ganglion may help to elucidate the neural network of taste learning. Future studies could elucidate how the nutritional state is internally sensed and how that the information is processed in the fly brain (Fujita, 2011).

Synapse remodeling is a widespread and fundamental process that underlies the formation of neuronal circuitry during development and in adaptation to physiological and/or environmental changes. However, the mechanisms of synapse remodeling are poorly understood. Synapses at the neuromuscular junction (NMJ) in Drosophila larvae undergo dramatic and extensive remodeling during metamorphosis to generate adult-specific synapses. To explore the molecular and cellular processes of synapse elimination, confocal microscopy, live imaging, and electron microscopy (EM) of NMJ synapses were performed during the early stages of metamorphosis in Drosophila in which the expressions of selected genes were genetically altered. It is reported that the localization of the postsynaptic scaffold protein Disc large (Dlg) becomes diffuse first and then undetectable, as larval muscles undergo histolysis, whereas presynaptic vesicles aggregate and are retrogradely transported along axons in synchrony with the formation of filopodia-like structures along NMJ elaborations and retraction of the presynaptic plasma membrane. EM revealed that the postsynaptic subsynaptic reticulum vacuolizes in the early stages of synapse dismantling concomitant with diffuse localization of Dlg. Ecdysone is the major hormone that drives metamorphosis. Blockade of the ecdysone signaling specifically in presynaptic neurons by expression of a dominant-negative form of ecdysone receptors delayed presynaptic but not postsynaptic dismantling. However, inhibition of ecdysone signaling, as well as ubiquitination pathway or apoptosis specifically in postsynaptic muscles, arrested both presynaptic and postsynaptic dismantling. These results demonstrate that presynaptic and postsynaptic dismantling takes place through different mechanisms and that the postsynaptic side plays an instructive role in synapse dismantling (Liu, 2010).

The Drosophila NMJ is an attractive model system for studying synaptogenesis but has only rarely been exploited to study synapse elimination. The present study used the Drosophila NMJ to study synapse elimination in the early stages of metamorphosis during which extensive synapse elimination occurs. This study unveiled distinct presynaptic and postsynaptic dismantling processes. Presynaptic elimination is characterized by the formation of prominent filopodial structures. The presynaptic membrane then retracts toward the nerve-muscle contact site with decreased bouton number and enlarged bouton size, accompanied by SV aggregation and retrograde axonal transport of SVs. It is worth pointing out that the precise timing of synapse dismantling revealed by immunostaining and live imaging is different, and this is probably attributable to the fact that the samples were analyzed under different conditions. For example, animals were kept at 25°C for immunostaining but were maintained at 20°C during live imaging. It is well known that filopodia are present in growth cones and play an important role in neurite outgrowth. The filopodia-like structures observed during synapse elimination presumably sense and explore the environment. The data also demonstrate that retrograde axonal transport plays an important role in presynaptic elimination. It is expected that the retrogradely transported synaptic constituents are reused to form adult-specific synaptic connections, although the final fate of the motor neuron MN4a innervating muscle 4 has not been determined. During the metamorphic period from 4 to 11 h APF we examined for the complete synapse elimination, neither disrupted synaptic microtubules were observed as reported for the local synapse disassembly in Drosophila larvae nor was an axonal 'retraction bulb' as seen during mammalian NMJ synapse elimination; a retraction bulb appears when a presynaptic terminal is detached mostly or completely from the postsynaptic specialization (Liu, 2010).

The first signs of postsynaptic elimination were the blurred and diffuse localization of postsynaptic markers Dlg and CD8-GFP-Shaker at 4 h APF, followed by a more expanded distribution of GluRs and vacuolization of SSR in 6 h APF. The postsynaptic components of Dlg and CD8-GFP-Shaker were almost completely eliminated at 9 h APF. Completion of the synapse dismantling process starting from the diffusion of postsynaptic Dlg at 4 h APF takes ~7 h. It is remarkable to note that the patterns of elimination of postsynaptic Dlg and GluRs are different; Dlg is eliminated by a diffusion-degradation process, whereas no diffusion of GluRs was observed before degradation, indicating that they are eliminated by different mechanisms. The differential elimination of Dlg and GluRs is consistent with the previous finding that the synaptic localization of GluR IIA is independent of Dlg. Interestingly, as for Drosophila GluRs, mammalian NMJ postsynaptic acetylcholine receptors are eliminated without the intermediate process of diffusion (Liu, 2010).

Synapse elimination is distinct from axonal and dendritic pruning. In Drosophila, the pruning of axonal and dendritic processes during metamorphosis closely resembles the pathological process of Wallerian degeneration, a process in which part of the axon separated from the nucleus of the neuron degenerates. In both axonal pruning of central mushroom body neurons and dendritic elimination of peripheral sensory neurons, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration. It is conceivable that synapse elimination and pruning of neuronal processes are closely interconnected, but the time course of the two discrete processes has yet to be determined. It has been shown that specific E2/E3 ubiquinating enzymes and caspases (i.e., UbcD1-Diap1-Dronc) are involved in dendritic pruning. But it is unknown whether those molecules also participate in NMJ synapse elimination. However, this study has demonstrated that disruption of ubiquitination and apoptosis pathways on the postsynaptic side arrests synaptic elimination. It will be of great interest to identify the specific ubiquinating enzymes and caspases that participate in synapse dismantling during metamorphosis. It is worth noting that glial cells play an important role in axonal pruning of mushroom body neurons and olfactory receptor neurons. This study found no evidence to suggest that glial cells play a role in NMJ synapse elimination (Liu, 2010).

Synapse disassembly or instability in Drosophila NMJ terminals have been described. However, this process is fundamentally different from that reported in this paper in several aspects. First, local synapse disassembly, the disassembly of distal synaptic boutons or a branch of the whole synaptic terminals of a motor neuron, a process that occurs during synapse growth in larval development was examined in previous papers, whereas this paper studied the elimination of complete NMJ 4 synapses in synchrony with muscle histolysis during metamorphosis. Also, the synapse elimination reported in this study is different from that of mammalian NMJ synapses; the former involves muscle destruction, whereas the latter does not. Second, the processes of local versus general synapse disassembly are different. In local synapse disassembly, presynaptic dismantling precedes postsynaptic dismantling: the presynaptic microtubule cytoskeleton retracts first, followed by the elimination of synaptic release machinery (i.e., the vesicle-associated protein synapsin) and ultimately the disassembly of the postsynaptic apparatus including the postsynaptic GluRs and the scaffold Dlg. However, in the elimination of complete synaptic terminals during metamorphosis, postsynaptic dismantling starts first, followed by presynaptic dismantling. Third, disrupting the dynactin complex destabilizes local synapses, leading to more synaptic 'footprints' that are defined as the withdrawal of presynaptic components from clearly defined postsynaptic specialization containing Dlg was studied previously. It was argued that the dynactin complex functions locally within presynaptic terminals to maintain synapse stability. Although the current study not examined synaptic footprints during metamorphosis, this study reports that disrupting the dynactin complex in presynaptic neurons delays presynaptic dismantling specifically, whereas the postsynaptic components disassemble normally, indicating that dynactin-mediated retrograde axonal transport is required for presynaptic elimination. These results demonstrate that the dynactin complex functions differently in distinct cellular contexts (Liu, 2010).

Two independent lines of evidence indicate that the postsynaptic rather than presynaptic side plays an instructive role in synapse elimination during metamorphosis. First, immunostaining showed that postsynaptic dismantling precedes presynaptic elimination by ~1 h. Second, blockade of retrograde axonal transport and ecdysone signaling specifically in presynaptic neurons delayed presynaptic dismantling only but postsynaptic dismantling proceeded normally. However, inactivation of ecdysone signaling, ubiquitination, or apoptosis pathways in postsynaptic muscles arrested both presynaptic and postsynaptic dismantling. It is noted that synapse elimination is closely correlated with muscle destruction. Indeed, muscle histolysis might be the primary cause of NMJ synapse elimination during metamorphosis. These results together indicate that postsynaptic elimination is independent of presynaptic elimination, but presynaptic elimination depends on postsynaptic elimination; in other words, postsynaptic elimination triggers presynaptic elimination. This hypothesis is supported by a previous report that local ecdysone treatment of the hawkmoth, Manduca sexta, to induce local muscle degeneration results in loss of synaptic contacts in the treated region, whereas neighboring NMJ synapses remain intact. The current data are also consistent with mounting evidence from mammalian studies supporting a major role for the postsynaptic side in synapse elimination. However, the downstream targets of the ubiquitination, apoptosis, or ecdysone pathways in the postsynaptic muscles that are crucial for initiating presynaptic dismantling are currently unknown. It will be of great interest to identify these targets (Liu, 2010).

Odor-shock memory in Drosophila consists of heterogeneous components each with different dynamics. A null mutant for the evolutionarily conserved synaptic protein Synapsin entails a memory deficit selectively in early memory, leaving later memory as well as sensory motor function unaffected. Notably, a consolidated memory component remaining after cold-anesthesia is not impaired, suggesting that only anesthesia-sensitive memory (ASM) depends on Synapsin. The lack of Synapsin does not further impair the memory deficit of mutants for the rutabaga gene encoding the type I adenylyl cyclase. This suggests that cAMP signaling, through a Synapsin-dependent mechanism, may underlie the formation of a labile memory component (Knapek, 2010).

The inclusion of the memory phenotype of the synapsin mutant in rutabaga may suggest Synapsin as a part of the cAMP signaling cascade and, if they would function in the same cells, as a potential downstream target of PKA. This is consistent with physiological studies demonstrating that cAMP signaling is selectively required for the recruitment of the reserve pool vesicles (Kuromi, 2000; Kuromi, 2002), as well as with the selective requirement of rutabaga for ASM, but not for ARM (Isabel, 2004). Both results fit very well with the physiology and memory of the synapsin mutant (Akbergenova, 2007). These lines of argument suggest that cAMP/PKA signaling might regulate the reserve-pool vesicle recruitment through Synapsin and that this process might underlie ASM (Knapek, 2010).

Optical detection of the lipophylic dye FM1-43 and focal recordings of quantal release were employed to investigate how synapsin affects vesicle cycling at the neuromuscular junction of synapsin knockout (Syn KO) Drosophila. Loading the dye employing high K+ stimulation, which presumably involves the recycling pool of vesicles in exo/endocytosis, stained the periphery of wild type (WT) boutons, while in Syn KO the dye was redistributed towards the center of the bouton. When endocytosis was promoted by cyclosporin A pretreatment, the dye uptake was significantly enhanced in WT boutons, and entire boutons were stained, suggesting staining of the reserve vesicle pool. In Syn KO boutons, the same loading paradigm produced fainter staining and significantly faster destaining. When the axon was stimulated electrically, a distinct difference in dye loading patterns was observed in WT boutons at different stimulation frequencies: a low stimulation frequency (3 Hz) produced a ring-shaped staining pattern, while at a higher frequency (10 Hz) the dye was redistributed towards the center of the bouton and the fluorescence intensity was significantly increased. This difference in staining patterns was essentially disrupted in Syn KO boutons, although synapsin did not affect the rate of quantal release. Stimulation of the nerve in the presence of bafilomycin, the blocker of the transmitter uptake, produced significantly stronger depression in Syn KO boutons. These results suggest that synapsin maintains the reserve pool of vesicles and segregation between the recycling and reserve pools, and that it mediates mobilization of the reserve pool during intense stimulation (Akbergenova, 2007).