Adenylyl cyclase encoded by AC78C participates in sugar perception in Drosophila melanogaster.

In gustatory receptor neurons (GRNs) in Drosophila melanogaster, Gr5a and one of the Gr64s encode sugar receptors with seven transmembrane domains. Previously, we have shown that the responses to various sugars are depressed in DGsalpha mutant flies (Ueno et al., 2006). Because DGsalpha is a homolog of Gs, we hypothesized that the sugar receptors are coupled to adenylyl cyclase (AC) in Drosophila. The aim of this study was to identify the AC that participates in sugar perception. Here, we found that an AC inhibitor, MDL-12330A, depressed the response in GRNs to trehalose as well as sucrose; that an AC gene, AC78C, was expressed in the sugar-sensitive GRNs; that RNAi against AC78C depressed the electrical response in GRNs to sucrose; and that the sugar response in GRNs, as well as sugar intake in a behavioral assay in an AC78C mutant, was depressed at low sugar concentrations. We conclude that AC78C, via cAMP, participates in the sugar-taste signaling pathway at the low concentration range.

Delayed synaptic transmission in Drosophila cacophonynull embryos.

Ca(2+) influx through the Drosophila N-type Ca(2+) channel, encoded by cacophony (cac), triggers fast synaptic transmission. We now ask whether the cac Ca(2+) channel is the Ca(2+) channel solely dedicated for fast synaptic transmission. Because the cac(null) mutation is lethal, we used cac(null) embryos to address this question. At the neuromuscular junction in HL3 solution, no fast synchronous synaptic transmission was detected on nerve stimulation. When the wild-type cac gene was introduced in the cac(null) background, fast synaptic transmission recovered. However, even in cac(null) embryos, nerve stimulation infrequently induced delayed synaptic events in the minority of cells in 1.5 mM [Ca(2+)](e) and in the majority of cells in 5 mM [Ca(2+)](e). The number of delayed quantal events per stimulus was greater in 5 mM [Ca(2+)](e) than in 1.5 mM. Thus the delayed release is [Ca(2+)](e) dependent. Plectreurys toxin II (PLTXII) (10 nM; a spider toxin analog) depressed the frequency of delayed events, suggesting that voltage-gated Ca(2+) channels, other than cac Ca(2+) channels, are contributing to them. However, delayed events were not affected by 50 microM La(3+). The frequency of miniature synaptic currents in cac(null) embryos was approximately 1/2 of control, whereas in high K(+) solutions, it was approximately 1/135. The hypertonicity response was approximately 1/10 of control. These findings indicate that the number of release-ready vesicles is smaller in cac(null) embryos. Taken together, the cac Ca(2+) channel is indispensable for fast synaptic transmission in normal conditions, and another type of Ca(2+) channel, the non-cac, PLTXII-sensitive Ca(2+) channel, is contributing to delayed release in cac(null) embryos.

Nerve-evoked synchronous release and high K+ -induced quantal events are regulated separately by synaptotagmin I at Drosophila neuromuscular junctions.

The distal Ca(2+)-binding domain of synaptotagmin I (Syt I), C2B, has two Ca(2+)-binding sites. To study their function in Drosophila, pairs of aspartates were mutated to asparagines and the mutated syt I was expressed in the syt I-null background (P[syt I(B-D1,2N)] and P[syt I(B-D3,4N)]). We examined the effects of these mutations on nerve-evoked synchronous synaptic transmission and high K(+)-induced quantal events at embryonic neuromuscular junctions. The P[syt I(B-D1,2N)] mutation virtually abolished synaptic transmission, whereas the P[syt I(B-D3,4N)] mutation strongly reduced but did not abolish it. The quantal content in P[syt I(B-D3,4N)] increased with the external Ca(2+) concentration, [Ca(2+)](e), with a slope of 1.86 in double-logarithmic plot, whereas that of control was 2.88. In high K(+) solutions the quantal event frequency in P[syt I(B-D3,4N)] increased progressively with [Ca(2+)](e) between 0 and 0.15 mM as in control. In contrast, in P[syt I(B-D1,2N)] the event frequency did not increase progressively between 0 and 0.15 mM and was significantly lower at 0.15 than at 0.05 mM [Ca(2+)](e). The P[syt I(B-D1,2N)] mutation inhibits high K(+)-induced quantal release in a narrow range of [Ca(2+)](e) (negative regulatory function). When Sr(2+) substituted for Ca(2+), nerve-evoked synchronous synaptic transmission was severely depressed and delayed asynchronous release was appreciably increased in control embryos. In high K(+) solutions with Sr(2+), the quantal event frequency was higher than that in Ca(2+) and increased progressively with [Sr(2+)](e) in control and in both mutants. Sr(2+) partially substitutes for Ca(2+) in synchronous release but does not support the negative regulatory function of Syt I.

Gsalpha is involved in sugar perception in Drosophila melanogaster.

In Drosophila melanogaster, gustatory receptor genes (Grs) encode G-protein-coupled receptors (GPCRs) in gustatory receptor neurons (GRNs) and some olfactory receptor neurons. One of the Gr genes, Gr5a, encodes a sugar receptor that is expressed in a subset of GRNs and has been most extensively studied both molecularly and physiologically, but the G-protein alpha subunit (Galpha) that is coupled to this sugar receptor remains unknown. Here, we propose that Gs is the Galpha that is responsible for Gr5a-mediated sugar-taste transduction, based on the following findings: First, immunoreactivities against Gs were detected in a subset of GRNs including all Gr5a-expressing neurons. Second, trehalose-intake is reduced in flies heterozygous for null mutations in DGsalpha, a homolog of mammalian Gs, and trehalose-induced electrical activities in sugar-sensitive GRNs were depressed in those flies. Furthermore, expression of wild-type DGsalpha in sugar-sensitive GRNs in heterozygotic DGsalpha mutant flies rescued those impairments. Third, expression of double-stranded RNA for DGsalpha in sugar-sensitive GRNs depressed both behavioral and electrophysiological responses to trehalose. Together, these findings indicate that DGsalpha is involved in trehalose perception. We suggest that sugar-taste signals are processed through the Gsalpha-mediating signal transduction pathway in sugar-sensitive GRNs in Drosophila.

Experience-dependent formation and recruitment of large vesicles from reserve pool.

The sizes and contents of transmitter-filled vesicles have been shown to vary depending on experimental manipulations resulting in altered quantal sizes. However, whether such a presynaptic regulation of quantal size can be induced under physiological conditions as a potential alternative mechanism to alter the strength of synaptic transmission is unknown. Here we show that presynaptic vesicles of glutamatergic synapses of Drosophila neuromuscular junctions increase in size as a result of high natural crawling activities of larvae, leading to larger quantal sizes and enhanced evoked synaptic transmission. We further show that these larger vesicles are formed during a period of enhanced replenishment of the reserve pool of vesicles, from which they are recruited via a PKA- and actin-dependent mechanism. Our results demonstrate that natural behavior can induce the formation, recruitment, and release of larger vesicles in an experience-dependent manner and hence provide evidence for an additional mechanism of synaptic potentiation.

External Ca2+ dependency of synaptic transmission in drosophila synaptotagmin I mutants.

To resolve some of differences in reports on the function of Synaptotagmin I (Syt I), we re-examined synaptic transmission at the neuromuscular junction of Drosophila embryos that have mutations in the Syt I gene (syt I). Two major questions addressed were which Ca2+ binding domain, C2A or C2B, sense Ca2+ and is Syt I a negative regulator of spontaneous vesicle fusion. Synaptic currents were induced by nerve stimulation or by high K+ treatment in external solutions containing various Ca2+ concentrations. In a null allele, syt I(AD4), synchronous synaptic currents were rarely observed but not abolished. The quantal content was about 1/60 of control but increased linearly with [Ca2+](e) with a slope of 0.95 (N) in the double logarithmic plot, in contrast to 3.01 in control. The slope of 1.06 in an allele, syt I(AD1), which lacks the second Ca2+ binding domain, C2B, was not different from in syt I(AD4). In another allele, syt I(AD3), in which one amino acid in C2B is mutated, synchronous synaptic transmission was also impaired and N was 1.54, which is significantly smaller than in control. In high K+ saline, the [Ca2+](e) dependency of vesicle release in syt I(AD4) was lower than in controls, whereas that in syt I(AD3) was even lower than in syt I(AD4), suggesting that syt I(AD3) is inhibiting vesicle fusion. These findings led us to conclude that C2B, not C2A, senses Ca2+, and Syt I is a negative regulator of vesicle fusion.

Exocytosis and endocytosis of synaptic vesicles and functional roles of vesicle pools: lessons from the Drosophila neuromuscular junction.

To maintain synaptic transmission during intense neuronal activities, the synaptic vesicle (SV) pool at release sites is effectively replenished by recruitment of SVs from the reserve pool and/or by endocytosis. The authors have studied dynamics of SVs using a fluorescence dye, FM1-43, which is incorporated into SVs during endocytosis and released by exocytosis. Drosophila is one of the most suitable preparations for genetic and pharmacological analyses, and this provides a useful model system. The authors found at the neuromuscular junctions of Drosophila that exocytosis and endocytosis of SVs are triggered by Ca(2+) influx through distinct routes and that selective inhibition of exocytosis or endocytosis resulted in depression of synaptic transmission with a distinct time course. They identified two SV pools in a single presynaptic bouton. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low-frequency stimulation and locates close to release sites in the periphery of boutons, whereas the reserve pool (RP) is loaded and unloaded only during high-frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the quantal content of evoked release, suggesting that SVs in the ECP are primarily involved in synaptic transmission. SVs in the RP are recruited to synaptic transmission by a process involving the cAMP/PKA cascade during high-frequency stimulation. Cytochalasin D blocked this recruitment process, suggesting involvement of filamentous actin. Endocytosed SVs replenish the ECP during stimulation and the RP after tetanic stimulation. Replenishment of the ECP depends on Ca(2+) influx from external solutions, and that of the RP is initiated by Ca(2+) release from internal stores. Thus, SV dynamics is closely involved in modulation of synaptic efficacy and influences synaptic plasticity.

Functional role of a novel ternary complex comprising SRF and CREB in expression of Krox-20 in early embryos of Xenopus laevis.

Krox-20, originally identified as a member of "immediate-early" genes, plays a crucial role in the formation of two specific segments in the hindbrain during early development of the vertebrate nervous system. Here we cloned a genomic sequence of Xenopus Krox-20 (XKrox-20) and studied functions of a promoter element in the flanking sequence and associated transcription factors, which function in early Xenopus embryos. Using the luciferase reporter assay system, we showed that the 5' flanking sequence was sufficient to induce luciferase activities when the reporter construct was injected into embryos at the eight-cell stage. Deletion and mutagenesis analyses of the 5' flanking sequence revealed a minimal promoter element that included two known subelements, a CArG-box and cAMP response element (CRE) within a stretch of 22 bp nucleotide sequence (-72 to -51 from the transcription initiation site), both of which were essential for the promoter activity. The gel mobility shift assay indicated that these two subelements bound to some components in whole cell extracts prepared from stage 20 Xenopus embryos. Antibody supershift and competition experiments revealed that these components in cell extracts were serum response factor (SRF) and a member of CRE binding protein (CREB) family proteins that bound the CArG-box and CRE, respectively. They appeared to assemble on the minimal promoter element to produce a novel ternary complex. When we injected mRNA of a dominant-negative version of Xenopus SRF (XSRFDeltaC) into animal pole blastomeres at the eight-cell stage, expression of XKrox-20 in the nervous system as well as the minimal promoter activity was strongly suppressed. Suppression by XSRFDeltaC was counteracted by coexpressed wild-type XSRF. These results indicate that XSRF functions as an endogenous activator of XKrox-20 by forming a ternary complex with CREB on the minimal promoter element.

Synaptic vesicle pools and plasticity of synaptic transmission at the Drosophila synapse.

Our knowledge on the Drosophila neuromuscular synapse is rapidly expanding. Thus, this synapse offers an excellent model for studies of the molecular mechanism of synaptic transmission and synaptic plasticity. Two synaptic vesicle (SV) pools have been identified and characterized using a fluorescent styryl dye, FM1-43, to stain SVs. They are termed the exo/endo cycling pool (ECP), which corresponds to the readily releasable pool (RRP) defined electrophysiologically, and the reserve pool (RP). These two pools were identified first in a temperature-sensitive paralytic mutant, shibire, and subsequently confirmed in wild-type larvae. The ECP participates in synaptic transmission during low frequency firing of presynaptic nerves and locates in the periphery of presynaptic boutons in the vicinity of release sites, while SVs in the RP spread toward the center of boutons and are recruited only during tetanic stimulation. These two pools are separately replenished by endocytosis. Cyclic AMP facilitates recruitment of SVs from the RP to the ECP. Activation of presynaptic metabotropic glutamate receptors recruits SVs from the RP and enhances SV release by elevation of the cAMP level. Memory mutants that have defects in the cAMP/PKA cascade, dunce and rutabaga, exhibit reduced levels of recruitment of synaptic SVs from the RP to the ECP and have limited short-term synaptic plasticity. SV mobilization between the two pools could be a key step for changes in synaptic efficacy. Since a variety of mutants that have distinct defects in synaptic transmission are available for detailed studies of synaptic function, this direction of approach in Drosophila seems promising.

A clock gene, period, plays a key role in long-term memory formation in Drosophila.

The cAMP-responsive transcription factor, CREB, is required for formation of long-term memory (LTM) in Drosophila melanogaster and regulates transcription of a circadian clock gene, period (per). Involvement of CREB both in LTM and circadian rhythm raises the possibility that per also plays a role in LTM. Assaying the experience-dependent courtship inhibition in male flies as a measure for LTM, we show here that per mutants are defective in LTM formation. This defect was rescued by induction of a wild-type per transgene in a per-null mutant, and overexpression of per enhanced LTM formation in the wild-type background. Furthermore, we found that synaptic transmission through per-expressing cells is most likely to be required during retrieval of LTM. In contrast, mutations in other clock genes (timeless, dClock, and cycle) did not affect LTM formation. Thus, independent of the core oscillator of circadian clock, per plays a key role in LTM formation.

Repetitive exposures to nicotine induce a hyper-responsiveness via the cAMP/PKA/CREB signal pathway in Drosophila.

Nicotine, in addition to acute effects, has long-lasting effects on mammalian behaviors, such as those leading to addiction. Here we present genetic and pharmacological evidence in Drosophila suggesting that repetitive exposures to nicotine induce a hyper-responsiveness through synthesis of new protein(s) via CREB-mediated gene transcription. Single exposure to volatilized nicotine dose-dependently inhibited the startle-induced climbing response. Compared with this effect of nicotine in wild-type flies, it was stronger in dunce, which has defective phosphodiesterase, and in wild-type flies treated with a phosphodiesterase inhibitor, whereas it was weaker in DC0, which has defective protein kinase A (PKA), and in wild-type flies treated with a PKA blocker. Thus, the effect of nicotine is enhanced by a mechanism involving the cAMP/PKA cascade. However, in wild-type flies, an increase in head cAMP was not detected within 2 min after single exposure to nicotine, during which the nicotine effect on the behavior was maximal. In wild-type flies, after repetitive exposures to nicotine, the nicotine effect was significantly enhanced and the head cAMP was elevated. The responsiveness to nicotine at second exposure increased with a 4 h interval but not with a 2 h interval, suggesting that the observed hyper-responsiveness was not due to accumulation of residual nicotine. Both enhancement of the nicotine effect and elevation of cAMP during repetitive exposures to nicotine were blocked by a protein synthesis inhibitor. Induction of a dominant negative CREB transgene also blocked the enhancement, suggesting that CREB-mediated gene transcription is required for the hyper-responsiveness.

Signaling through Gs alpha is required for the growth and function of neuromuscular synapses in Drosophila.

Although synapses are assembled in a highly regulated fashion, synapses once formed are not static structures but continue to expand and retract throughout the life of an organism. One second messenger that has been demonstrated to play a critical role in synaptic growth and function is cAMP. Here, we have tested the idea that signaling through the heterotrimeric G protein, Gs, plays a coincident role with increases in intracellular Ca(+2) in the regulation of adenylyl cyclases (ACs) during synaptic growth and in the function of synapses. In larvae containing a hypomorphic mutation in the dgs gene encoding the Drosophila Gs alpha protein, there is a significant decrease in the number of synaptic boutons and extent of synaptic arborization, as well as defects in the facilitation of synaptic transmission. Microscopic analysis confirmed that Gs alpha is localized at synapses both pre- and postsynaptically. Restricted expression of wild-type Gs alpha either pre- or postsynaptically rescued the mutational defects in bouton formation and defects in the facilitation of synaptic transmission, indicating that pathways activated by Gs alpha are likely to be involved in the reciprocal interactions between pre- and postsynaptic cells required for the development of mature synapses. In addition, this Gs alpha mutation interacted with fasII, dnc, and hyperexcitability mutants in a manner that revealed a coincident role for Gs alpha in the regulation of cAMP and FASII levels required during growth of these synapses. Our results demonstrate that Gs alpha-dependent signaling plays a role in the dynamic cellular reorganization that underlies synaptic growth.

Ca2+ influx through distinct routes controls exocytosis and endocytosis at drosophila presynaptic terminals.

Endocytosis of synaptic vesicles follows exocytosis, and both processes require external Ca(2+). However, it is not known whether Ca(2+) influx through one route initiates both processes. At larval Drosophila neuromuscular junctions, we separately measured exocytosis and endocytosis using FM1-43. In a temperature-sensitive Ca(2+) channel mutant, cacophony(TS2), exocytosis induced by high K(+) decreased at nonpermissive temperatures, while endocytosis remained unchanged. In wild-type larvae, a spider toxin, PLTXII, preferentially inhibited exocytosis, whereas the Ca(2+) channel blockers flunarizine and La(3+) selectively depressed endocytosis. None of these blockers affected exocytosis or endocytosis induced by a Ca(2+) ionophore. Evoked synaptic potentials were depressed regardless of stimulus frequency in cacophony(TS2) at nonpermissive temperatures and in wild-type by PLTXII, whereas flunarizine or La(3+) gradually depressed synaptic potentials only during high-frequency stimulation, suggesting depletion of synaptic vesicles due to blockade of endocytosis. In shibire(ts1), a dynamin mutant, flunarizine or La(3+) inhibited assembly of clathrin at the plasma membrane during stimulation without affecting dynamin function.

Presynaptic impairment of synaptic transmission in Drosophila embryos lacking Gs(alpha).

Gs(alpha) is a subunit of the heterotrimeric G-protein complex, expressed ubiquitously in all types of cells, including neurons. Drosophila larvae, which have mutations in the Gs(alpha) gene, are lethargic, suggesting an impairment of neuronal functions. In this study, we examined synaptic transmission at the neuromuscular synapse in Gs(alpha)-null (dgsR60) embryos shortly before they hatched. At low-frequency nerve stimulation, synaptic transmission in mutant embryos was not very different from that in controls. In contrast, facilitation during tetanic stimulation was minimal in dgsR60, and no post-tetanic potentiation was observed. Miniature synaptic currents (mSCs) were slightly smaller in amplitude and less frequent in dgsR60 embryos in normal-K+ saline. In high-K+ saline, mSCs with distinctly large amplitude occurred frequently in controls at late embryonic stages, whereas those mSCs were rarely observed in dgsR60 embryos, suggesting a developmental defect in the mutant. Using the Gal4-UAS expression system, we found that these phenotypes in dgsR60 were caused predominantly by lack of Gs(alpha) in presynaptic neurons and not in postsynaptic muscles. To test whether Gs(alpha) couples presynaptic modulator receptors to adenylyl cyclase (AC), we examined the responses of two known G-protein-coupled receptors in dgsR60 embryos. Both metabotropic glutamate and octopamine receptor responses were indistinguishable from those of controls, indicating that these receptors are not linked to AC by Gs(alpha). We therefore suggest that synaptic transmission is compromised in dgsR60 embryos because of presynaptic defects in two distinct processes; one is uncoupling between the yet-to-be-known modulator receptor and AC activation, and the other is a defect in synapse formation.

The role of integrins in the modulation of neurotransmitter release from motor nerve terminals by stretch and hypertonicity.

Integrins are found at most or all synapses and play a variety of roles. At frog neuromuscular junctions, mechanical tension on integrins due to muscle stretch or hypertonicity causes a powerful modulation of release efficacy. Understanding the mechanism(s) of integrin-mediated modulation will likely further our understanding of mechanisms of neurotransmitter release. The modulation of evoked release with stretch occurs with no detectable delay, does not adapt, and bypasses the Ca(2+) triggering step in vesicle fusion. It depends primarily on integrin bonds to native ligands and requires that one or more proteins in the link between integrins and vesicle fusion be dephosphorylated. Hypertonicity, studied in both frog and Drosophila terminals, causes a larger but slower phasic-tonic change in spontaneous release, which is also Ca(2+)-independent and mostly dependent on integrins, but not dependent on the phosphorylation state of molecules in its pathway of action. In Drosophila, the integrin-dependent component involves the cAMP/PKA pathway, and is absent in mutants lacking PKA. Both stretch and hypertonicity responses in frog terminals are enhanced by agents that elevate PKA levels, suggesting that, in frogs, the cAMP/PKA cascade primarily determines the size of the pool of vesicles available for release by the integrin-mediated mechanism and is not a direct intermediary in the modulation. Evoked release is affected little or even inhibited by hypertonicity. In Drosophila, the inhibition can be explained by a decrease in Ca(2+) influx. The effect of hypertonicity on evoked release in frogs may similarly be a balance between mechanisms that enhance spontaneous release and those that suppress I (Ca).

Overexpression of a CREB repressor isoform enhances the female sexual receptivity in Drosophila.

The cAMP responsive transcriptional factor, CREB, is highly conserved among animal species, and its activity affects their behavior. In Drosophila melanogaster, one of alternatively spliced products of the CREB gene, dCREB2-a, is a transcriptional activator, while another isoform, dCREB2-b, is a repressor of dCREB2-a. Here, we demonstrate that overexpression of dCREB2-b in virgin females enhances their sexual receptivity. We studied the role of dCREB2 in female mating behavior using two transgenic lines, hs-dCREB2-a and hs-dCREB2-b, which overexpress respective products with heat-shock treatment. Wild-type males started their courtship behavior and mated more quickly with heat-shocked hs-dCREB2-b females than with non-heat-shocked hs-dCREB2-b females. Overexpression of dCREB2-a in females affected neither their courtship behavior nor mating frequency. The effects of overexpressed dCREB2-b were not due to elevated locomotor activities of heat-shocked females nor due to more vigorous courtship behavior of paired wild-type males. CREB might be involved in female sexual behavior of animals.

Biphasic modulation of synaptic transmission by hypertonicity at the embryonic Drosophila neuromuscular junction.

Puff-application of hypertonic saline (sucrose added to external saline) causes a transient increase in the frequency of spontaneous miniature synaptic currents (mSCs) at the neuromuscular junctions of Drosophila embryos. The frequency gradually returns to pre-application levels. External Ca(2+) is not needed for this response, but it may modify it. At 50 mM added sucrose, for example, enhanced spontaneous release was observed only in the presence of external Ca(2+), suggesting that Ca(2+) augments the response. In a high-K(+) solution, in which the basal mSC frequency was elevated, higher sucrose concentrations produced an increase in mSC frequency that was followed (during and after the hypertonic exposure) by depression, with the magnitude of both effects increasing with hypertonicity between 100 and 500 mM. Evoked release by nerve stimulation showed only depression in response to hypertonicity. We do not believe that the depression of spontaneous or evoked release can be explained by the depletion of releasable quanta, however, since the frequency of quantal release did not reach levels compatible with this explanation and the enhancement and depression could be obtained independent of one another. In a mutant lacking neuronal synaptobrevin, only the depression of mSC frequency was induced by hypertonicity. Conversely, only the enhancing effect was observed in wild-type embryos when the mSC frequency was elevated with forskolin in Ca(2+)-free saline. In cultured embryonic Drosophila neurons, Ca(2+) signals that were induced by high K(+) and detected by Fura-2, were reduced by hypertonicity, suggesting that the depressing response is due to a direct effect of hypertonicity on Ca(2+) influx.

Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse.

After synaptic vesicles (SVs) undergo exocytosis, SV pools are replenished by recycling SVs at nerve terminals. At Drosophila neuromuscular synapses, there are two distinct SV pools (i.e., the exo/endo cycling pool (ECP), which primarily maintains synaptic transmission, and the reserve pool (RP), which participates in synaptic transmission only during tetanic stimulation). Labeling endocytosed vesicular structures with a fluorescent styryl dye, FM1-43, and measuring intracellular Ca2+ concentrations with a Ca2+ indicator, rhod-2, we show here that the ECP is replenished by SVs endocytosed during stimulation, and this process depends on external Ca2+. In contrast, the RP is refilled after cessation of tetanus by a process mediated by Ca2+ released from internal stores.

Hypertonicity-induced transmitter release at Drosophila neuromuscular junctions is partly mediated by integrins and cAMP/protein kinase A.

The frequency of quantal transmitter release increases upon application of hypertonic solutions. This effect bypasses the Ca(2+) triggering step, but requires the presence of key molecules involved in vesicle fusion, and hence could be a useful tool for dissecting the molecular process of vesicle fusion. We have examined the hypertonicity response at neuromuscular junctions of Drosophila embryos in Ca(2+)-free saline. Relative to wild-type, the response induced by puff application of hypertonic solution was enhanced in a mutant, dunce, in which the cAMP level is elevated, or in wild-type embryos treated with forskolin, an activator of adenylyl cyclase, while protein kinase A (PKA) inhibitors decreased it. The response was also smaller in a mutant, DC0, which lacks the major subunit of PKA. Thus the cAMP/PKA cascade is involved in the hypertonicity response. Peptides containing the sequence Arg-Gly-Asp (RGD), which inhibit binding of integrins to natural ligands, reduced the response, whereas a peptide containing the non-binding sequence Arg-Gly-Glu (RGE) did not. A reduced response persisted in a mutant, myospheroid, which expresses no integrins, and the response in DC0 was unaffected by RGD peptides. These data indicate that there are at lease two components in the hypertonicity response: one that is integrin mediated and involves the cAMP/PKA cascade, and another that is not integrin mediated and does not involve the cAMP/PKA cascade.