Neurotransmitter Release Mechanisms Research Articles

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Synapsins I and II Are Not Required for β-Cell Insulin Secretion: Granules Must Pool Their Own Weight

Neurotransmitter release mechanisms studied in Caenorhabditis elegans

The process of regulated exocytosis has received considerable interest as a key component of synaptic transmission. Fusion of presynaptic vesicles and the subsequent release of their neurotransmitter contents is driven by a series of interactions between evolutionarily conserved proteins. Key insights into the molecular mechanisms of vesicle fusion have come from research using genetic model systems such as the nematode worm Caenorhabditis elegans. We review here the current knowledge regarding regulated exocytosis at the C. elegans synapse and future research directions involving this model organism.

Impact of Synaptic Neurotransmitter Concentration Time Course on the Kinetics and Pharmacological Modulation of Inhibitory Synaptic Currents

The time course of synaptic currents is a crucial determinant of rapid signaling between neurons. Traditionally, the mechanisms underlying the shape of synaptic signals are classified as pre- and post-synaptic. Over the last two decades, an extensive body of evidence indicated that synaptic signals are critically shaped by the neurotransmitter time course which encompasses several phenomena including pre- and post-synaptic ones. The agonist transient depends on neurotransmitter release mechanisms, diffusion within the synaptic cleft, spill-over to the extra-synaptic space, uptake, and binding to post-synaptic receptors. Most estimates indicate that the neurotransmitter transient is very brief, lasting between one hundred up to several hundreds of microseconds, implying that post-synaptic activation is characterized by a high degree of non-equilibrium. Moreover, pharmacological studies provide evidence that the kinetics of agonist transient plays a crucial role in setting the susceptibility of synaptic currents to modulation by a variety of compounds of physiological or clinical relevance. More recently, the role of the neurotransmitter time course has been emphasized by studies carried out on brain slice models that revealed a striking, cell-dependent variability of synaptic agonist waveforms ranging from rapid pulses to slow volume transmission. In the present paper we review the advances on studies addressing the impact of synaptic neurotransmitter transient on kinetics and pharmacological modulation of synaptic currents at inhibitory synapses.

Porosome: the universal secretory portal in cells

In the past 50 years it was believed that during cell secretion, membrane-bound secretory vesicles completely merge at the cell plasma membrane resulting in the diffusion of intra-vesicular contents to the cell exterior and the compensatory retrieval of the excess membrane by endocytosis. This explanation made no sense or logic, since following cell secretion partially empty vesicles accumulate as demonstrated in electron micrographs. Furthermore, with the 'all or none' mechanism of cell secretion by complete merger of secretory vesicle membrane at the cell plasma membrane, the cell is left with little regulation and control of the amount of content release. Moreover, it makes no sense for mammalian cells to possess such 'all or none' mechanism of cell secretion, when even single-cell organisms have developed specialized and sophisticated secretory machinery, such as the secretion apparatus of Toxoplasma gondii, the contractile vacuoles in paramecium, or the various types of secretory structures in bacteria. Therefore, in the 1960's, experimental data concerning neurotransmitter release mechanisms by B. Katz and B. Folkow brilliantly hypothesized that limitation of the quantal packet may be set by the nerve membrane, in which case the size of the packet may actually correspond to just a fraction of the vesicle content. This conundrum in the molecular mechanism of cell secretion was finally resolved in 1997 following discovery of the porosome, the universal secretory machinery in the cell. Porosomes are supramolecular lipoprotein structures at the cell plasma membrane, where membrane-bound secretory vesicles transiently dock and fuse to release inravesicular contents to the outside during cell secretion. In the past decade, the composition of the porosome, its structure and dynamics at nanometer (nm) resolution and in real time, and its functional reconstitution into artificial lipid membrane, have all been elucidated. Since porosomes in exocrine and neuroendocrine cells measure 100-180 nm, and only 20-45% increase in porosome diameter is demonstrated following the docking and fusion of secretory vesicles (0.2-1.2 μm in diameter), it is concluded that secretory vesicles "transiently" dock and fuse, rather than completely merge at the base of the porosome complex to release their contents to the outside. In agreement, it has been demonstrated that "secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells"; that "single synaptic vesicles fuse transiently and successively without loss of identity"; and that "zymogen granule (the secretory vesicle in exocrine pancreas) exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity". In this review, the discovery of the porosome, resulting in a paradigm shift in our understanding of cell secretion, is briefly presented. Biomedical Reviews 2010; 21: 1-15.

Ectopic vesicular neurotransmitter release along sensory axons mediates neurovascular coupling via glial calcium signaling

Neurotransmitter release generally is considered to occur at active zones of synapses, and ectopic release of neurotransmitters has been demonstrated in a few instances. However, the mechanism of ectopic neurotransmitter release is poorly understood. We took advantage of the intimate morphological and functional proximity of olfactory receptor axons and specialized glial cells, olfactory ensheathing cells (OECs), to study ectopic neurotransmitter release. Axonal stimulation evoked purinergic and glutamatergic Ca(2+) responses in OECs, indicating ATP and glutamate release. In axons expressing synapto-pHluorin, stimulation evoked an increase in synapto-pHluorin fluorescence, indicative of vesicle fusion. Transmitter release was dependent on Ca(2+) and could be inhibited by bafilomycin A1 and botulinum toxin A. Ca(2+) transients in OECs evoked by ATP, axonal stimulation, and laser photolysis of NP-EGTA resulted in constriction of adjacent blood vessels. Our results indicate that ATP and glutamate are released ectopically by vesicles along axons and mediate neurovascular coupling via glial Ca(2+) signaling.

Release of neurotransmitters from glia

There is no question about the fact that astrocytes and other glial cells release neurotransmitters that activate receptors on neurons, glia and vascular cells, and that calcium is an important second messenger regulating the release. This occurs in cell culture, tissue slice and in vivo. Negative results from informative experiments designed to test the mechanism of calcium-dependent neurotransmitter release from astrocytes and the ensuing effects on synaptic transmission, have been cited as evidence calling into question whether astrocytes release neurotransmitters under normal circumstances with effects on synaptic transmission. The special feature section in this issue of Neuron Glia Biology addresses these issues and other aspects of neurotransmitter release from astrocytes in communicating with neurons and glial cells. Together these studies suggest that application of vocabulary and concepts developed for synaptic communication between neurons can lead to confusion and apparent paradoxes with respect to communication by extracellular signaling molecules released from glia in response to functional activity.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Glia as transmitter sources and sensors in health and disease

Glial cells express a bewildering array of neurotransmitter receptors. To illustrate the complexity of expression, we have assayed non-glutamatergic neurotransmitter receptor mRNA in isolated rat optic nerve, a preparation devoid of neurons and neuronal synapses and from which relatively pure “glial” RNA can be isolated. Of the 44 receptor subunits examined which span the GABA-A, nicotinic, adreno- and glycine receptor families, over three quarters were robustly expressed in this mixed population of white matter glial cells, with several expressed at higher levels than found in control whole brain RNA. In addition to the complexity of glial receptor expression, numerous neurotransmitter release mechanisms have been identified. We have focused on glutamate release from astrocytes, which can occur via at least seven distinct pathways and which is implicated in excitotoxic injury and are neurons and glia. Recent findings suggest that non-glutamatergic receptors can also mediate acute glial injury are also discussed.

Pathophysiological Aspects of Temporal Lobe Epilepsy and the Role of P2X Receptors

In the central nervous system (CNS), ATP is released from vesicles at nerve terminals in a frequency- dependent manner and can activate P2 receptors widely expressed and distributed in the CNS. In addition to interacting with P2 receptors, ATP can be rapidly hydrolyzed to adenosine to activate P1 receptors modulating neuronal transmission. Thus, complex synaptic interactions in the CNS are modulated by P2 and P1 receptors. This review focuses on the role of P2X receptors in temporal lobe epilepsy. P2X receptors are cationic-selective channels gated by extracellular ATP. Seven subunits (P2X1-7) are expressed throughout the central nervous system and are involved with modulatory mechanisms of neurotransmitter release, hyperexcitability, intracellular calcium influx, cell-cell communication, neuroprotection, and cell death. This review discusses the current data regarding the involvement of P2 receptors in the pathophysiology of tempo- ral lobe epilepsy (TLE).

Remote Homology between Munc13 MUN Domain and Vesicle Tethering Complexes

Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca2+-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.

Tonic Activation of GABAB Receptors Reduces Release Probability at Inhibitory Connections in the Cerebellar Glomerulus

In the cerebellum, granule cells are inhibited by Golgi cells through GABAergic synapses generating complex responses involving both phasic neurotransmitter release and the establishment of ambient gamma-aminobutyric acid (GABA) levels. Although at this synapse the mechanisms of postsynaptic integration have been clarified to a considerable extent, the mechanisms of neurotransmitter release remained largely unknown. Here we have investigated the quantal properties of release during repetitive neurotransmission, revealing that tonic GABA(B) receptor activation by ambient GABA regulates release probability. Blocking GABA(B) receptors with CGP55845 enhanced the first inhibitory postsynaptic current (IPSC) and short-term depression in a train while reducing trial-to-trial variability and failures. The changes caused by CGP55845 were similar to those caused by increasing extracellular Ca(2+) concentration, in agreement with a presynaptic GABA(B) receptor modulation of release probability. However, the slow tail following IPSC peak demonstrated a remarkable temporal summation and was not modified by CGP55845 or extracellular Ca(2+) increase. This result shows that tonic activation of presynaptic GABA(B) receptors by ambient GABA selectively regulates the onset of inhibition bearing potential consequences for the dynamic regulation of signal transmission through the mossy fiber-granule cell pathway of the cerebellum.

Effects of zonisamide on neurotransmitter release associated with inositol triphosphate receptors

To clarify the antiepileptic mechanisms of zonisamide (ZNS), we determined the interaction between ZNS and inositol-1,4,5-triphosphate receptor (IP3R) on exocytosis of GABA and glutamate in rat frontal cortex using microdialysis. ZNS increased basal GABA release, but not glutamate, concentration-dependently, and reduced concentration-dependently K +-evoked GABA and glutamate releases. Inhibition and activation of IP3R reduced and enhanced basal and K +-evoked GABA releases, respectively. The K +-evoked glutamate release was reduced and enhanced by IP3R antagonist and agonist, respectively, whereas basal glutamate release was increased by IP3R agonist but not affected by IP3R antagonist. Under extracellular Ca 2+ depletion, IP3R agonist increased basal GABA and glutamate releases. The latter effects of IP3R agonist were weakly enhanced by ZNS, but such stimulatory action of ZNS was abolished by extracellular Ca 2+ depletion. In contrast, ZNS inhibited the stimulatory effect of IP3R agonist on K +-evoked release. The stimulatory effect of IP3R agonist on basal release was regulated by N-type voltage-sensitive Ca 2+ channel (VSCC) rather than P- and L-type VSCCs, whereas the stimulatory effect of IP3R agonist on K +-evoked release was regulated by P- and L-type VSCCs rather than N-type VSCC. These results suggest that ZNS-activated N-type VSCC enhances IP3R-associated neurotransmitter release during resting stage, whereas ZNS-induced suppression of P- and L-type VSCCs possibly attenuates IP3R-associated neurotransmitter release during neuronal hyperexcitability. Therefore, the combination of both of these two actions of ZNS on IP3R-associated neurotransmitter release mechanism seems to be involved, at least in part, in the mechanisms of antiepileptic and neuroprotective actions of ZNS.

Influence of Integrin-blocking Peptide on Gadolinium- and Hypertonic Shrinking-induced Neurotransmitter Release in Rat Brain Synaptosomes

Polyvalent cations and hypertonic shrinking of presynaptic endings lead to calcium-independent exocytosis in various synapses. In the present study we have investigated the contribution of integrins to this phenomenon. It was found that hypertonic shrinking, polyvalent cations ruthenium red and gadolinium results in dose-dependent calcium-independent neurotransmitter release in rat brain synaptosomes. The exocytotic mechanism of neurotransmitter release induced by 300 microM gadolinium was additionally verified by the fluorescent dye FM2-10. We found that 200 microM of RGDS peptide, an inhibitor of integrins, decreased polyvalent gadolinium-induced [3H]D: -aspartate release by 26%. This compound had no effect upon hypertonicity-induced release. The peptide RGES, a negative control for RGDS; genistein, an inhibitor of tyrosine kinases; and citrate, an inhibitor of lanthanides-induced aggregation were ineffective in both cases. Therefore, we have shown that integrins did not influence hypertonicity-evoked [3H]D: -aspartate release, but partially mediated that evoked by gadolinium ions.

Carbamazepine prevents breakdown of neurotransmitter release induced by hyperactivation of ryanodine receptor

To clarify the mechanisms of the pharmacological action of carbamazepine (CBZ), we determined the effect of CBZ on GABA and glutamate release associated with the ryanodine receptor (Ryr)-sensitive Ca 2+-induced Ca 2+-releasing system (CICR) in the rat hippocampus using microdialysis. The therapeutically relevant concentration of CBZ increased basal GABA release without affecting basal glutamate release; however, K +-evoked releases were concentration-dependently reduced by CBZ. Lower-concentration ryanodine increased basal and K +-evoked releases of GABA and glutamate in a concentration dependent manner, whereas higher-concentration ryanodine reduced them. These inflection points in the concentration-response curves of ryanodine for neurotransmitter release (critical concentrations) were shifted to the left by K +-evoked stimulation. The critical concentration of ryanodine in GABA release was lower than that in glutamate release. During the resting stage, the critical concentrations of ryanodine were unaffected by inhibition of L-type, N-type and P-type voltage-sensitive Ca 2+ channels (VSCCs) but were prevented by CBZ; however, during the neuronal hyperexcitable stage, the critical concentration was increased by CBZ, L-type and P-type VSCC inhibitors but not the N-type VSCC inhibitor. Therefore, a therapeutically relevant concentration of CBZ protects against the breakdown of the neurotransmitter release mechanism induced by hyperactivation of Ryr via inhibition of L-type and P-type VSCCs as well as inhibition of Ryr-sensitive CICR. These actions of CBZ appear to be involved, at least partially, in its anti-seizure mechanisms.

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology

Bernard Katz, quantal transmitter release and the foundations of presynaptic physiology

Hyposmolarity evokes norepinephrine efflux from synaptosomes by a depolarization‐ and Ca2+‐dependent exocytotic mechanism

Osmolarity reduction (20%) elicited 3H-norepinephrine (NE) efflux from rat cortical synaptosomes. The hyposmotic NE release resulted from the following events: (i) a Na+-dependent and La3+-, Gd3+- and ruthenium red-sensitive depolarization; (ii) a cytosolic Ca2+ ([Ca2+]i) rise with contributions from external Ca2+ influx and internal Ca2+ release, probably through the mitochondrial Na+-Ca2+ exchanger; and (iii) activation of a [Ca2+]i-evoked, tetanus toxin (TeTX)-sensitive, PKC-modulated NE efflux mechanism. This sequence was established from results showing a drop in the hyposmotic [Ca2+]i rise by preventing depolarization with La3+, and by the inhibitory effects of Ca2+-free medium (EGTA; 50%), CGP37157 (the mitochondrial Na+-Ca2+ exchanger blocker; 48%), EGTA + CGP37157 or by EGTA-AM (> 95% in both cases). In close correspondence with these effects, NE efflux was 92% decreased by Na+ omission, 75% by La3+, 47% by EGTA, 50% by CGP37157, 90% by EGTA + CGP37157 and 88% by EGTA-AM. PKC influenced the intracellular Ca2+ release and, mainly through this action, modulated NE efflux. TeTX suppressed NE efflux. The K+-stimulated NE release, studied in parallel, was unaffected by Na+ omission, or by La3+, Gd3+ or ruthenium red. It was fully dependent on external Ca2+, insensitive to CGP37157 and abolished by TeTX. These results suggest that the hyposmotic events, although different from the K+-evoked depolarization and [Ca2+]i rise mechanisms, are able to trigger a depolarization-dependent, Ca2+-dependent and TeTX-sensitive mechanism for neurotransmitter release.

Young Brains on Lead: Adult Neurological Consequences?

Gilbert and colleagues examine the effect of chronic lowlevel lead exposure, occurring during development, on hippocampal neurogenesis and spatial learning in adulthood. The effects of lead exposure on cognitive and behavioral deficits in children leading to learning and memory impairments is well established, whereas there is little information on the long-term (adult) consequences of gestationally initiated lead exposure. One important issue that Gilbert and colleagues assess is the long-term consequences of chronic developmental low-level lead exposure in adult offspring. Secondly, Gilbert and colleagues are the first to report the effect of lead exposure on hippocampal neurogenesis. The work by Gilbert and colleagues is intriguing, in view of the fact that this paper attempts to integrate the consequences of developmental exposure to lead, known for its ability to impair cognitive functions, such as learning and memory, with a dysfunction in adult hippocampal neurogenesis. Chronic lead exposure initiated during development and continuing through adulthood (E16–PND75) resulted in a reduced survival of newly generated neurons in the dentate gyrus, but no effects on spatial learning and memory as assessed by the Morris water maze (MWM). As in other studies, discontinuation of lead at weaning did not result in observable differences from control animals in the chosen endpoint. Generally, the effects of developmental lead exposure on neurobehavioral deficits may or may not persist, and may be subtly manifested, but this paper emphasizes that lead exposure does have neurological consequences in the long term. Up until the early 1900s, lead poisoning was viewed largely as a disease of adults due to occupational exposure. However, the particular sensitivity of children to lead exposure quickly became recognized. In combination with higher rates of gastrointestinal tract absorption, the nervous systems of children are more vulnerable to the toxic effects of lead, making childhood exposure a great concern. Thus, much effort has been put into public awareness campaigns and government interventions to reduce the level of this environmental toxicant. Although mean national blood lead levels have decreased dramatically over the past 30 years in the United States, elevated levels are still found in specific areas, affecting mainly low-income, urban children and those living in older housing (Meyer et al., 2005). The most significant sources of lead continue to be old paint in homes built before 1978, lead pipes placed before the 1930s, and soil along highways and heavily traveled roads. A bimodal distribution of elevated blood lead levels in humans shows a peak for children (1–5 years of age) and a second one for adults >50 years of age (Pirkle et al., 1998). Therefore, despite intensive efforts to reduce lead use, exposure continues to be a major public health problem. Due to lead’s effects on children, the major focus of the literature has been on the acute effects of lead exposure in developing animals. Although there is considerable consensus about the toxic effects of high levels of lead, the effects of lowlevel lead exposure are less clear. Human epidemiological studies have consistently found a strong association between lead and IQ. However, there is still considerable debate on these manifestations, due to highly confounding variables such as socioeconomic status and quality of parenting. Animal studies have confirmed a significant interaction between developmental lead exposure and the nature and persistence of neurotoxic effects manifested in cognitive defects such as learning and memory, although these effects are highly ageand dose-dependent (Finkelstein et al., 1998). Numerous investigations focusing on the mechanism of lead neurotoxicity have found a wide array of effects, including apoptosis, excitotoxicity, interference with neurotransmitter storage and release mechanisms, alterations in second messengers, and damage to mitochondria (Lidsky and Schneider, 2003). Although there is no unifying mechanism, lead’s ability to substitute for calcium, and possibly zinc, is a factor common to many of its toxic actions. The disruption of dopaminergic functioning, which is involved in motor control and attention, as well as memory and executive functioning, can produce a host of behavioral problems, including attention deficit hyperactivity disorder and alterations in cognition (Brown et al., 1997). Also, lead has effects on glutamatergic transmission, which is a major player in both development and 1 To whom correspondence should be addressed. E-mail: bolivar@ wadsworth.org.

Three Toxins Are Better Than One

Understanding molecular mechanisms of neurotransmitter release and short-term synaptic plasticity is one of the central questions in neuroscience. Sakaba et al. studied the roles of SNARE proteins in neurotransmitter release using clostridial neurotoxins. A detailed kinetic analysis of the action of several toxins revealed that the kinetics of transmitter release differs, depending on which SNARE proteins were cleaved. Toxins cleaving synaptobrevin and syntaxin reduced the number of fusion-competent vesicles without changing the Ca 2+ sensitivity of the release apparatus of remaining vesicles. In contrast, toxins cleaving the C terminal of SNAP-25 reduced intracellular Ca 2+ sensitivity of vesicle fusion, which suggests that the C terminal is important for driving rapid fusion. Furthermore, toxins cleaving synaptobrevin lead to a modification of the coupling between Ca 2+ channels and release-competent vesicles. T. Sakaba, A. Stein, R. Jahn, E. Neher, Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage. Science 309 , 491-494 (2005). [Abstract] [Full Text]

Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3‐kinase

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor-mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1) on neurotransmitter release were studied in rat PC12 cells. Brief treatment with EGF and IGF-1 enhanced Ca2+-dependent dopamine release in a concentration-dependent manner. EGF activated both mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase) pathways, and the EGF-dependent enhancement of DA release was suppressed by a MAPK kinase inhibitor as well as by PI3-kinase inhibitors. In striking contrast, IGF-1 activated the PI3-kinase pathway but not the MAPK pathway, and IGF-1-dependent enhancement was suppressed by a PI3-kinase inhibitor but not by a MAPK kinase inhibitor. The enhanced green fluorescent protein-tagged pleckstrin homology (PH) domain of protein kinase B, which selectively binds to phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, was translocated to the plasma membrane after treatment with either EGF or NGF. By contrast, no significant redistribution was induced by IGF-1. These results indicate that PI3-kinase participates in the enhancement of neurotransmitter release by two distinct mechanisms: EGF and NGF activate PI3-kinase in the plasma membrane, whereas IGF-1 activates PI3-kinase possibly in the intracellular membrane, leading to enhancement of neurotransmitter release in a MAPK-dependent and -independent manner respectively.

Properties of Short-Term Synaptic Depression at Larval Neuromuscular Synapses in Wild-Type and Temperature-Sensitive Paralytic Mutants ofDrosophila

The larval neuromuscular synapse of Drosophila serves as an important model for genetic and molecular analysis of synaptic development and function. Further functional characterization of this synapse, as well as adult neuromuscular synapses, will greatly enhance the impact of this model system on our understanding of synaptic transmission. Here we describe a form of short-term synaptic depression observed at larval, but not adult, neuromuscular synapses and explore the underlying mechanisms. Larval neuromuscular synapses exhibited a form of short-term depression that was strongly dependent on stimulation frequency over a narrow range of low frequencies (0.1-1 Hz). This form of synaptic depression, referred to here as low-frequency short-term depression (LF-STD), results from an activity-dependent reduction in neurotransmitter release. However, in contrast to the predictions of depletion models, the degree of depression was independent of the initial level of neurotransmitter release over a range of extracellular calcium concentrations. This conclusion was confirmed in two temperature-sensitive (TS) paralytic mutants, cacophony and shibire, which exhibit reduced neurotransmitter release resulting from conditional disruption of presynaptic calcium channels and dynamin, respectively. Higher stimulation frequencies (40 or 60 Hz) produced two components of depression that appeared to include LF-STD as well as a more conventional component of short-term depression. These findings reveal novel properties of short-term synaptic depression and suggest that complementary genetic analysis of larval and adult neuromuscular synapses will further define the in vivo mechanisms of neurotransmitter release and short-term synaptic plasticity.