Drosophila Neuromuscular Junction Research Articles

Actions of MDMA at glutamatergic neuromuscular junctions.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.

Developmental stage-dependent modulation of synapses by postsynaptic expression of activated calcium/calmodulin-dependent protein kinase II

In Drosophila neuromuscular junctions, there is a unique system which consists of two neighboring muscles (M6 and M7) innervated by the same neurons and a gene of interest can be expressed in only M6 or in both muscles by GAL4-upstream activating sequence expression system. By using this system, we previously demonstrated that expression of activated calcium/calmodulin-dependent protein kinase II (CaMKII) in the muscle cell promotes coordinated maturation of pre- and postsynaptic sites of larvae just after hatching (JAH larvae) in a synapse-specific manner. Here we show that the promotive effects are no longer seen in the older larvae, 8–10 h after hatching (8 h AH larvae). Morphological studies indicate that CaMKII activation in fact reduces postsynaptic sites at 8 h AH. This is opposite to the effect observed in JAH larvae. These results suggest that the mode of CaMKII function switches during development, and that regulation of postsynaptic CaMKII activity is necessary for normal synaptic development. Finally, we report that in 8 h AH but not in JAH larvae, synapses on M7, in which CaMKII activity is not manipulated, are affected by the expression of activated CaMKII in M6. This suggests the interesting possibility that at certain developmental stages only, modification of synapses on one target cell can influence the synapses on another target cell innervated by the same neurons.

Morphology and molecular organization of the adult neuromuscular junction of Drosophila.

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5-1.5 microm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8-5.5 microm) to an elongate terminal (80-100 microm long) with few branches and contiguous swellings (3-15 microm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II-immunoreactivity is not detectable at type I terminals, while DLG-immunoreactivity is observed at the synapse; 3) a Shaker-GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle-specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs.

Presynaptic N-type Calcium Channels Regulate Synaptic Growth

Voltage-gated calcium channels couple changes in membrane potential to neuronal functions regulated by calcium, including neurotransmitter release. Here we report that presynaptic N-type calcium channels not only control neurotransmitter release but also regulate synaptic growth at Drosophila neuromuscular junctions. In a screen for behavioral mutants that disrupt synaptic transmission, an allele of the N-type calcium channel locus (Dmca1A) was identified that caused synaptic undergrowth. The underlying molecular defect was identified as a neutralization of a charged residue in the third S4 voltage sensor. RNA interference reduction of N-type calcium channel expression also reduced synaptic growth. Hypomorphic mutations in syntaxin-1A or n-synaptobrevin, which also disrupt neurotransmitter release, did not affect synapse proliferation at the neuromuscular junction, suggesting calcium entry through presynaptic N-type calcium channels, not neurotransmitter release per se, is important for synaptic growth. The reduced synapse proliferation in Dmca1A mutants is not due to increased synapse retraction but instead reflects a role for calcium influx in synaptic growth mechanisms. These results suggest N-type channels participate in synaptic growth through signaling pathways that are distinct from those that mediate neurotransmitter release. Linking presynaptic voltage-gated calcium entry to downstream calcium-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.

Retrograde Gbb signaling through the Bmp type 2 receptor wishful thinking regulates systemic FMRFa expression in Drosophila.

Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). We demonstrate here that mutations in wishful thinking (wit) a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. We show that Gbb, a Bmp ligand expressed in the neurohemal organ provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Finally, we show that supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. We propose that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity.

Retrograde Control of Synaptic Transmission by Postsynaptic CaMKII at the Drosophila Neuromuscular Junction

Retrograde signaling plays an important role in synaptic homeostasis, growth, and plasticity. A retrograde signal at the neuromuscular junction (NMJ) of Drosophila controls the homeostasis of neurotransmitter release. Here, we show that this retrograde signal is regulated by the postsynaptic activity of Ca 2+/calmodulin-dependent protein kinase II (CaMKII). Reducing CaMKII activity in muscles enhances the signal and increases neurotransmitter release, while constitutive activation of CaMKII in muscles inhibits the signal and decreases neurotransmitter release. Postsynaptic inhibition of CaMKII increases the number of presynaptic, vesicle-associated T bars at the active zones. Consistently, we show that glutamate receptor mutants also have a higher number of T bars; this increase is suppressed by postsynaptic activation of CaMKII. Furthermore, we demonstrate that presynaptic BMP receptor wishful thinking is required for the retrograde signal to function. Our results indicate that CaMKII plays a key role in the retrograde control of homeostasis of synaptic transmission at the NMJ of Drosophila.

The BMP Homolog Gbb Provides a Retrograde Signal that Regulates Synaptic Growth at the Drosophila Neuromuscular Junction

We show that the BMP ortholog Gbb can signal by a retrograde mechanism to regulate synapse growth of the Drosophila neuromuscular junction (NMJ). gbb mutants have a reduced NMJ synapse size, decreased neurotransmitter release, and aberrant presynaptic ultrastructure. These defects are similar to those we observe in mutants of BMP receptors and Smad transcription factors. However, whereas these BMP receptors and signaling components are required in the presynaptic motoneuron, Gbb expression is required in large part in postsynaptic muscles; gbb expression in muscle rescues key aspects of the gbb mutant phenotype. Consistent with this notion, we find that blocking retrograde axonal transport by overexpression of dominant-negative p150/Glued in neurons inhibits BMP signaling in motoneurons. These experiments reveal that a muscle-derived BMP retrograde signal participates in coordinating neuromuscular synapse development and growth.

Two synaptic vesicle pools, vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction.

Drosophila neuromuscular junctions ( D NMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K(+). The ECP locates close to release sites in the periphery of presynaptic boutons. 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 efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca(2+). Mobilization of internal Ca(2+) stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and D NMJs are suitable for further studies.

The relationship of neuromuscular synapse elimination to synaptic degeneration and pathology: Insights from WldSand other mutant mice

Neuromuscular synapse elimination, Wallerian degeneration and peripheral neuropathies are not normally considered as related phenomena. However, recent studies of mutant and transgenic mice, particularly the Wld(S) mutant-in which orthograde degeneration is delayed following axotomy-suggest that re-evaluation of possible links between natural, traumatic and pathogenic regression of synapses may be warranted. During developmental synapse elimination from polyneuronally innervated junctions, some motor nerve terminals progressively and asynchronously vacate motor endplates. A form of asynchronous synapse withdrawal, strongly resembling synapse elimination, also occurs from mononeuronally-innervated motor endplates following axotomy in young adult Wld(S) mutant mice. A similar pattern is observed in skeletal muscles of several neuropathic mutants, including mouse models of dying-back neuropathies, motor neuron disease and-remarkably-models of neurodegenerative diseases such as Huntington's and Alzheimer's diseases. Taken together with recent analysis of synaptic remodelling at neuromuscular junctions in Drosophila, a strong candidate for a common regulatory mechanism in these diverse conditions is one based on protein ubiquitination/deubiquitination. Axotomised neuromuscular junctions in Wld(S) mutant mice offer favourable experimental opportunities for examining developmental mechanisms of synaptic regression, that may also benefit our understanding of how degeneration in the synaptic compartment of a neuron is initiated, and its role in progressive, whole-cell neuronal degeneration.

Postsynaptic activation of calcium/calmodulin-dependent protein kinase ii promotes coordinated pre- and postsynaptic maturation of drosophila neuromuscular junctions

The interaction between a neuron and its target cell(s) is essential for the development of synapses. To elucidate the role of target cells in synaptogenesis, the activity of postsynaptic calcium/calmodulin-dependent protein kinase II (CaMKII) was manipulated in a mosaic manner and its specific effect was examined at the developing Drosophila neuromuscular junction. We found that postsynaptic expression of constitutively active CaMKII augmented the amplitude of excitatory synaptic currents (ESCs) and the frequency of miniature ESCs. It also promoted morphological maturation of presynaptic as well as postsynaptic specializations, which presumably underlie the enhancement of synaptic activities. Expression of an inhibitory peptide of CaMKII in the postsynaptic cell partially affected the synaptic maturation. These results suggest two significant functions of postsynaptic CaMKII in synaptogenesis–retrograde modulation of presynaptic properties and coordinated regulation of pre- and postsynaptic maturation.

Effects of m-CPP in altering neuronal function: blocking depolarization in invertebrate motor and sensory neurons but exciting rat dorsal horn neurons

The compound m-chlorophenylpiperazine (m-CPP) is used clinically to manipulate serotonergic function, though its precise mechanisms of actions are not well understood. m-CPP alters synaptic transmission and neuronal function in vertebrates by non-selective agonistic actions on 5-HT 1 and 5-HT 2 receptors. In this study, we demonstrated that m-CPP did not appear to act through a 5-HT receptor in depressing neuronal function in the invertebrates (crayfish and Drosophila). Instead, m-CPP likely decreased sodium influx through voltage-gated sodium channels present in motor and primary sensory neurons. Intracellular axonal recordings showed that m-CPP reduced the amplitude of the action potentials in crayfish motor neurons. Quantal analysis of excitatory postsynaptic currents, recorded at neuromuscular junctions (NMJ) of crayfish and Drosophila, indicated a reduction in the number of presynaptic vesicular events, which produced a decrease in mean quantal content. m-CPP also decreased activity in primary sensory neurons in the crayfish. In contrast, serotonin produces an increase in synaptic strength at the crayfish NMJ and an increase in activity of sensory neurons; it produces no effect at the Drosophila NMJ. In the rat spinal cord, m-CPP enhances the occurrence of spontaneous excitatory postsynaptic potentials with no alteration in evoked currents.

Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity

BackgroundThe transcription factor AP1 mediates long-term plasticity in vertebrate and invertebrate central nervous systems. Recent studies of activity-induced synaptic change indicate that AP1 can function upstream of CREB to regulate both CREB-dependent enhancement of synaptic strength as well as CREB-independent increase in bouton number at the Drosophila neuromuscular junction (NMJ). However, it is not clear from this study if AP1 functions autonomously in motor neurons to directly modulate plasticity.ResultsHere, we show that Fos and Jun, the two components of AP1, are abundantly expressed in motor neurons. We further combine immunohistochemical and electrophysiological analyses with use of a collection of enhancers that tightly restrict AP1 transgene expression within the nervous system to show that AP1 induction or inhibition in, but not outside of, motor neurons is necessary and sufficient for its modulation of NMJ size and strength.ConclusionBy arguing against the possibility that AP1 effects at the NMJ occur via a polysynaptic mechanism, these observations support a model in which AP1 directly modulates NMJ plasticity processes through a cell autonomous pathway in the motor neuron. The approach described here may serve as a useful experimental paradigm for analyzing cell autonomy of genes found to influence structure and function of Drosophila motor neurons.

Long-term in vitro maintenance of neuromuscular junction activity of Drosophila larvae

The larval Drosophila neuromuscular junction (NMJ) has proven to be an excellent system to test fundamental aspects of synaptic transmission, such as relationships among ion channel function, subtypes of glutamate receptors, and the functions of synaptic proteins in the presynaptic compartment. Recent advances in understanding bi-directional communication between nerves and muscles of Drosophila are helping uncover developmental as well as maintenance cues that could be applicable to all chemical synapses. The development of HL3 medium makes it possible to record synaptic responses at NMJs for prolonged periods of time. We demonstrate that media commonly used to culture CNS neurons and imaginal disks of Drosophila such as Schneider's and M3 completely block glutamatergic synaptic transmission at the NMJ. The depressed postsynaptic excitatory junction potentials (EJPs) partially recover from exposure to such media shortly after switching to the HL3 medium. Preliminary results from NMJs of filleted 3rd instar larvae for 4 days in vitro bathed in a modified HL3 medium show great promise. The resting membrane potential and the EJP amplitudes after 4 days in vitro are normal. These results demonstrate the possibility for chronic studies of developmental regulation in culture, which in some cases are impractical in the whole animal.

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.

Development of Drosophila larval neuromuscular junctions: maintaining synaptic strength

In spite of the available information about the development of Drosophila neuromuscular junctions, the correlation between nerve terminal morphology and maintenance of synaptic strength has still not been systematically addressed throughout larval development. We characterized the growth of the abdominal longitudinal muscle 6 (m6) and the motor terminals Ib and Is that innervate it within segment 4. In addition, we measured the evoked excitatory junction potential (EJP) amplitudes while the Ib and Is axons were selectively recruited. Regression analysis with natural log transformation of response variables indicated that the developmental curves for m6 and the motor axons Ib and Is were best fitted as second order polynomial regressions during larval development. Initially Is terminals are longer and possess more synaptic varicosities at the first instar stage. The Is terminals also grow faster in subsequent developmental stages. The growth of nerve terminals and their target m6 are not proportional although tightly correlated. This results in a larger average muscle area innervated by a single varicosity as the animal develops. The amplitudes of the EJPs of Ib and Is neurons show no developmental difference in their amplitudes from the first to the late third larval instar. The Is axon consistently produced larger EJPs than the Ib axon at each developmental stage. The time constants for both rising and decay phases of EJPs increase exponentially throughout larval development. The results presented not only help in quantifying the normal development of Drosophila neuromuscular junctions, but also provide a framework for future investigations to properly interpret developmental abnormalities that may occur in various mutants.

Regulation of synaptic connectivity: levels of Fasciclin II influence synaptic growth in the Drosophila CNS.

Much of our understanding of synaptogenesis comes from studies that deal with the development of the neuromuscular junction (NMJ). Although well studied, it is not clear how far the NMJ represents an adequate model for the formation of synapses within the CNS. Here we investigate the role of Fasciclin II (Fas II) in the development of synapses between identified motor neurons and cholinergic interneurons in the CNS of Drosophila. Fas II is a neural cell adhesion molecule homolog that is involved in both target selection and synaptic plasticity at the NMJ in Drosophila. In this study, we show that levels of Fas II are critical determinants of synapse formation and growth in the CNS. The initial establishment of synaptic contacts between these identified neurons is seemingly independent of Fas II. The subsequent proliferation of these synaptic connections that occurs postembryonically is, in contrast, significantly retarded by the absence of Fas II. Although the initial formation of synaptic connectivity between these neurons is seemingly independent of Fas II, we show that their formation is, nevertheless, significantly affected by manipulations that alter the relative balance of Fas II in the presynaptic and postsynaptic neurons. Increasing expression of Fas II in either the presynaptic or postsynaptic neurons, during embryogenesis, is sufficient to disrupt the normal level of synaptic connectivity that occurs between these neurons. This effect of Fas II is isoform specific and, moreover, phenocopies the disruption to synaptic connectivity observed previously after tetanus toxin light chain-dependent blockade of evoked synaptic vesicle release in these neurons.

Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development.

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including betaPS and its alpha partners (alphaPS1, alphaPS2, alphaPS3/alphaVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. betaPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory betaPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in betaPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that betaPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.

Dynactin Is Necessary for Synapse Stabilization

We present evidence that synapse retraction occurs during normal synaptic growth at the Drosophila neuromuscular junction (NMJ). An RNAi-based screen to identify the molecular mechanisms that regulate synapse retraction identified Arp-1/centractin, a subunit of the dynactin complex. Arp-1 dsRNA enhances synapse retraction, and this effect is phenocopied by a mutation in P150/Glued, also a dynactin component. The Glued protein is enriched within the presynaptic nerve terminal, and presynaptic expression of a dominant-negative Glued transgene enhances retraction. Retraction is associated with a local disruption of the synaptic microtubule cytoskeleton. Electrophysiological, ultrastructural, and immunohistochemical data support a model in which presynaptic retraction precedes disassembly of the postsynaptic apparatus. Our data suggests that dynactin functions locally within the presynaptic arbor to promote synapse stability.

Linking to Liprin

Synaptic development requires coordinated process in both pre- and post-synaptic regions. However, much less is understood about pre-synaptic structure and function. Kaufmann et al. show that an intracellular protein called liprin interacts with the cytoplasmic domain of the receptor-type protein tyrosine phosphatase LAR in the pre-synaptic active zone of the Drosophila neuromuscular junction (NMJ). liprin was detected on both sides of the synaptic cleft but LAR was only detected in the pre-synaptic NMJ. Genetic analysis revealed that both proteins are required for normal NMJ morphogenesis, as absence of either caused a decrease in synapse size and bouton number. Absence of liprin prevented an increase in NMJ bouton number observed upon LAR overexpression, indicating that liprin acts downstream of LAR. Synaptic transmission and pre-synaptic vesicle release was also impaired in liprin mutants. Wyszinsku et al. also report that in rat brain, liprin associated in a multi-complex with the adaptor protein GRIP and LAR. GRIP was also associated with AMPA receptors, indicting a role for liprin post-synaptic function. Disruption of the GRIP-liprin interaction disrupted AMPA receptor clustering. Hence, liprin appears fundamental to organizing structure and function of developing synapses. N. Kaufmann, J. DeProto, R. Ranjan, H. Wan, D. Van Vactor, Drosophila liprin-α and the receptor phosphatase Dlar control synapse morphogenesis. Neuron 34: 27-38 (2002).[Online Journal]M. Wyszynski, E. Kim, A.W. Dunah, M. Passafaro, J.G. Valtschanoff, C. Serr-Pages, M. Streuhli, R.J. Weinberg, M. Sheng, Interaction between GRIP and liprin-α/SYD2 is required for AMPA receptor targeting. Neuron 34: 39-54 (2002).[Online Journal]

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction.

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.