Quantal Size Research Articles

Cholesterol Stabilizes the Fusion Pore of Rat Chromaffin Granules before Its Rapid Dilation

Changes in cellular cholesterol level affect transmitter release but the role of cholesterol in the fusion machinery is not well understood. Using carbon fiber amperometry, we examined whether changes in cellular cholesterol level has any direct effect on the release of catecholamines from individual chromaffin granules. To avoid any possible effect of cholesterol perturbation on ion channels, exocytosis was stimulated directly via whole-cell dialysis of a Ca2+-buffered solution. Cellular cholesterol level was either reduced by ∼30% (via a cholesterol synthesis inhibitor and extracellular application of a cholesterol extractor) or increased by ∼3 fold (via loading of cholesterol). Changes in cellular cholesterol level did not affect the rate of exocytosis, the quantal size or the kinetic parameters of the main amperometric spikes (which reflect the rapid release during and after the rapid dilation of the fusion pore). In contrast, cholesterol perturbation affected the amperometric foot signals (which reflect the catecholamine leakage via a semi-stable fusion pore). Reduction of cellular cholesterol destabilized the fusion pore while it was flickering (resulting in a decrease in the proportion of “stand-alone foot” signal) and before the onset of rapid dilation (resulting in a shortening of the foot signal). Elevation in cellular cholesterol level had opposite effects, suggesting that cholesterol elevation increased the stability of the semi-stable fusion pores. Acute extraction of cholesterol from the cytosolic side of the plasma membrane (via whole-cell dialysis of a cholesterol extractor) also shortened the foot signal and reduced the proportion of “stand-alone-foot” signals. However, acute extracellular application of cholesterol or its extractor did not affect the amperometric signals. We suggest that cholesterol on the cytosolic leaflet of the vesicular membranes constrained the fusion pores of chromaffin granules before the onset of rapid dilation.

Plasticity between Neuronal Pairs in Layer 4 of Visual Cortex Varies with Synapse State

In neocortex, the induction and expression of long-term potentiation (LTP) and long-term depression (LTD) vary depending on cortical area and laminae of presynaptic and postsynaptic neurons. Layer 4 (L4) is the initial site of sensory afference in barrel cortex and primary visual cortex (V1) in which excitatory inputs from thalamus, L6, and neighboring L4 cells are integrated. However, little is known about plasticity within L4. We studied plasticity at excitatory synaptic connections between pairs and triplets of interconnected L4 neurons in guinea pig V1 using a fixed delay pairing protocol. Plasticity outcomes were heterogeneous, with some connections undergoing LTP (n = 7 of 42), some LTD (n = 19 of 42), and some not changing (n = 16 of 42). Although quantal analysis revealed both presynaptic and postsynaptic plasticity expression components, reduction in quantal size (a postsynaptic property) contributing to LTD was ubiquitous, whereas in some cell pairs, this change was overridden by an increase in the probability of neurotransmitter release (a presynaptic property) resulting in LTP. These changes depended on the initial reliability of the connections: highly reliable connections depressed with contributions from presynaptic and postsynaptic effects, and unreliable connections potentiated as a result of the predominance of presynaptic enhancement. Interestingly, very strong, reliable pairs of connected cells showed little plasticity. Pairs of connected cells with a common presynaptic or postsynaptic L4 cell behaved independently, undergoing plasticity of different or opposite signs. Release probability of a connection with initial 100% failure rate was enhanced after pairing, potentially avoiding silencing of the presynaptic terminal and maintaining L4-L4 synapses in a broader dynamic range.

PACAP/PAC 1R signaling modulates acetylcholine release at neuronal nicotinic synapses

Neuropeptides collaborate with conventional neurotransmitters to regulate synaptic output. Pituitary adenylate cyclase-activating polypeptide (PACAP) co-localizes with acetylcholine in presynaptic nerve terminals, is released by stimulation, and enhances nicotinic acetylcholine receptor- (nAChR-) mediated responses. Such findings implicate PACAP in modulating nicotinic neurotransmission, but relevant synaptic mechanisms have not been explored. We show here that PACAP acts via selective high-affinity G-protein coupled receptors (PAC 1Rs) to enhance transmission at nicotinic synapses on parasympathetic ciliary ganglion (CG) neurons by rapidly and persistently increasing the frequency and amplitude of spontaneous, impulse-dependent nicotinic excitatory postsynaptic currents (sEPSCs). Of the canonical adenylate cyclase (AC) and phospholipase-C (PLC) transduction cascades stimulated by PACAP/PAC 1R signaling, only AC-generated signals are critical for synaptic modulation since the increases in sEPSC frequency and amplitude were mimicked by 8-Bromo-cAMP, blocked by inhibiting AC or cAMP-dependent protein kinase (PKA), and unaffected by inhibiting PLC. Despite its ability to increase agonist-induced nAChR currents, PACAP failed to influence nAChR-mediated impulse-independent miniature EPSC amplitudes (quantal size). Instead, evoked transmission assays reveal that PACAP/PAC 1R signaling increased quantal content, indicating that it modulates synaptic function by increasing vesicular ACh release from presynaptic terminals. Lastly, signals generated by the retrograde messenger, nitric oxide- (NO-) are critical for the synaptic modulation since the PACAP-induced increases in spontaneous EPSC frequency, amplitude and quantal content were mimicked by NO donor and absent after inhibiting NO synthase (NOS). These results indicate that PACAP/PAC 1R activation recruits AC-dependent signaling that stimulates NOS to increase NO production and control presynaptic transmitter output at neuronal nicotinic synapses.

Rab3 GTPase Lands Bruchpilot

Active zones are the sites of neurotransmitter release, but their assembly mechanisms are poorly understood. In this issue of Neuron, Graf et al. perform a genetic screen in Drosophila and uncover a novel role for the Rab3 GTPase in organizing the active zone at the neuromuscular junction.

Endocannabinoids contribute to metabotropic glutamate receptor-mediated inhibition of GABA release onto hippocampal CA3 pyramidal neurons in an isolated neuron/bouton preparation

Retrograde synaptic signaling by endogenous cannabinoids (endocannabinoids) is a recently discovered form of neuromodulation in various brain regions. In hippocampus, it is well known that endocannabinoids suppress presynaptic inhibitory neurotransmitter release in CA1 region. However, endocannabinoid signaling in CA3 region remains to be examined. Here we investigated whether presynaptic inhibition can be caused by activation of postsynaptic group I metabotropic glutamate receptors (mGluRs) and following presynaptic cannabinoid receptor type 1 (CB1 receptor) using mechanically dissociated rat hippocampal CA3 pyramidal neurons with adherent functional synaptic boutons. Application of group I mGluR agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) reversibly suppressed spontaneous inhibitory postsynaptic currents (IPSCs). In the presence of tetrodotoxin (TTX), frequency of miniature IPSCs was significantly reduced by DHPG, while there were no significant changes in minimum quantal size and sensitivity of postsynaptic GABAA receptors to the GABAA receptor agonist muscimol, indicating that this suppression was caused by a decrease in GABA release from presynaptic nerve terminals. Application of CB1 synthetic agonist WIN55212-2 (mesylate(R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholino)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthyl)methanone) or endocannabinoid 2-arachidonoylglycerol also suppressed the spontaneous IPSC. The inhibitory effect of DHPG on spontaneous IPSCs was abolished by SR-141716 (5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), a CB1 receptor antagonist. Furthermore, postsynaptic application of GDP-βS blocked the DHPG-induced inhibition of spontaneous IPSCs, indicating the involvement of endcannabinoid-mediated retrograde synaptic signaling. These results provide solid evidence for retrograde signaling from postsynaptic group I mGluRs to presynaptic CB1 receptors, which induces presynaptic inhibition of GABA release in rat hippocampal CA3 region.

Reliability and Precision of the Mouse Calyx of Held Synapse

Traditionally, the calyx of Held synapse is viewed as a highly reliable relay in the sound localization circuit of the auditory brainstem, with every presynaptic action potential triggering a postsynaptic action potential in vivo. However, this view is at odds with slice recordings that report large short-term depression (STD). To investigate the reliability and precision of this synapse, we compared slice and in vivo recordings from medial nucleus of the trapezoid body neurons of young adult mice. We show that the extracellularly recorded complex waveform can be used to estimate both presynaptic release and postsynaptic excitability. Whereas under standard slice conditions the synapse underwent large STD, both extracellular and whole-cell recordings indicated that in vivo the size of the EPSPs was independent of recent history. The estimated quantal content was typically <20 in vivo, much lower than in the resting synapse under standard slice conditions. However, due to the large quantal size and summation of EPSPs, the safety factor of this synapse was generally still sufficiently large and postsynaptic failures were observed only infrequently in vivo. When present, failures were typically due to stochastic fluctuations in EPSP size or postsynaptic spike depression. In vivo, the calyx of Held synapse thus functions as a tonic synapse. The price it pays for its low release probability is an increase in jitter and synaptic latency and occasional postsynaptic failures.

Development of multisensory convergence in the Xenopus optic tectum.

The adult Xenopus optic tectum receives and integrates visual and nonvisual sensory information. Nonvisual inputs include mechanosensory inputs from the lateral line, auditory, somatosensory, and vestibular systems. While much is known about the development of visual inputs in this species, almost nothing is known about the development of mechanosensory inputs to the tectum. In this study, we investigated mechanosensory inputs to the tectum during critical developmental stages (stages 42-49) in which the retinotectal map is being established. Tract-tracing studies using lipophilic dyes revealed a large projection between the hindbrain and the tectum as early as stage 42; this projection carries information from the Vth, VIIth, and VIIIth nerves. By directly stimulating hindbrain and visual inputs using an isolated whole-brain preparation, we found that all tectal cells studied received both visual and hindbrain input during these early developmental stages. Pharmacological data indicated that the hindbrain-tectal projection is glutamatergic and that there are no direct inhibitory hindbrain-tectal ascending projections. We found that unlike visual inputs, hindbrain inputs do not show a decrease in paired-pulse facilitation over this developmental period. Interestingly, over this developmental period, hindbrain inputs show a transient increase followed by a significant decrease in the alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)/N-methyl-D-aspartate (NMDA) ratio and show no change in quantal size, both in contrast to visual inputs. Our data support a model by which fibers are added to the hindbrain-tectal projection across development. Nascent fibers form new synapses with tectal neurons and primarily activate NMDA receptors. At a time when retinal ganglion cells and their tectal synapses mature, hindbrain-tectal synapses are still undergoing a period of rapid synaptogenesis. This study supports the idea that immature tectal cells receive converging visual and mechanosensory information and indicates that the Xenopus tectum might be an ideal preparation to study the early development of potential multisensory interactions at the cellular level.

Roles of Cholesterol in Vesicle Fusion and Motion

Although it is well established that exocytosis of neurotransmitters and hormones is highly regulated by numerous secretory proteins, such as SNARE proteins, there is an increasing appreciation of the importance of the chemophysical properties and organization of membrane lipids to various aspects of the exocytotic program. Based on amperometric recordings by carbon fiber microelectrodes, we show that deprivation of membrane cholesterol by methyl- β-cyclodextrin not only inhibited the extent of membrane depolarization-induced exocytosis, it also adversely affected the kinetics and quantal size of vesicle fusion in neuroendocrine PC12 cells. In addition, total internal fluorescence microscopy studies revealed that cholesterol depletion impaired vesicle docking and trafficking, which are believed to correlate with the dynamics of exocytosis. Furthermore, we found that free cholesterol is able to directly trigger vesicle fusion, albeit with less potency and slower kinetics as compared to membrane depolarization stimulation. These results underscore the versatile roles of cholesterol in facilitating exocytosis.

The Pre/Post LTP Debate

The pre/post debate involves the question of whether long-term potentiation (LTP) is mediated by enhancement of release, enhancement of postsynaptic receptors, or both. Recent papers have presented evidence for purely postsynaptic or purely presynaptic changes, and a paper by Ahmed and Siegelbam (in this issue of Neuron) suggests a mechanism by which release is enhanced. This debate is increasingly constrained by technical advances that allow central synapses to be studied with increasing precision. A possible of way of reconciling conflicting evidence is suggested.

Hippocampal distribution of vesicular glutamate transporter 1 in patients with temporal lobe epilepsy

Vesicular glutamate transporters (VGLUTs) are responsible for loading synaptic vesicles with glutamate, determining the phenotype of glutamatergic neurons, and have been implicated in the regulation of quantal size and presynaptic plasticity. We analyzed VGLUT subtype expression in normal human hippocampus and tested the hypothesis that alterations in VGLUT expression may contribute to long-term changes in glutamatergic transmission reported in patients with temporal lobe epilepsy (TLE). VGLUT immunohistochemistry, immunofluorescence, in situ hybridization, Western blotting, and quantitative polymerase chain reaction (qPCR) were performed on biopsies from TLE patients without (non-HS) and with hippocampal sclerosis (HS) and compared to autopsy controls and rat hippocampus. VGLUT1 expression was compared with synaptophysin, neuropeptide Y (NPY), and Timm's staining. VGLUT1 was the predominant VGLUT in human hippocampus and appeared to be localized to presynaptic glutamatergic terminals. In non-HS hippocampi, VGLUT1 protein levels were increased compared to control and HS hippocampi in all subfields. In HS hippocampi VGLUT1 expression was decreased in subfields with severe neuronal loss, but strongly up-regulated in the dentate gyrus, characterized by mossy fiber sprouting. VGLUT1 is used as marker for glutamatergic synapses in the human hippocampus. In HS hippocampi VGLUT1 up-regulation in the dentate gyrus probably marks new glutamatergic synapses formed by mossy fiber sprouting. Our data indicate that non-HS patients have an increased capacity to store glutamate in vesicles, most likely due to an increase in translational processes or upregulation of VGLUT1 in synapses from afferent neurons outside the hippocampus. This up-regulation may increase glutamatergic transmission, and thus contribute to increased extracellular glutamate levels and hyperexcitability.

Impaired dopamine release and synaptic plasticity in the striatum of Parkin−/− mice

Parkin is the most common causative gene of juvenile and early-onset familial Parkinson's diseases and is thought to function as an E3 ubiquitin ligase in the ubiquitin-proteasome system. However, it remains unclear how loss of Parkin protein causes dopaminergic dysfunction and nigral neurodegeneration. To investigate the pathogenic mechanism underlying these mutations, we used parkin-/- mice to study its physiological function in the nigrostriatal circuit. Amperometric recordings showed decreases in evoked dopamine release in acute striatal slices of parkin-/- mice and reductions in the total catecholamine release and quantal size in dissociated chromaffin cells derived from parkin-/- mice. Intracellular recordings of striatal medium spiny neurons revealed impairments of long-term depression and long-term potentiation in parkin-/- mice, whereas long-term potentiation was normal in the Schaeffer collateral pathway of the hippocampus. Levels of dopamine receptors and dopamine transporters were normal in the parkin-/- striatum. These results indicate that Parkin is involved in the regulation of evoked dopamine release and striatal synaptic plasticity in the nigrostriatal pathway, and suggest that impairment in evoked dopamine release may represent a common pathophysiological change in recessive parkinsonism.

Dysfunction of the Dentate Basket Cell Circuit in a Rat Model of Temporal Lobe Epilepsy

Temporal lobe epilepsy is common and difficult to treat. Reduced inhibition of dentate granule cells may contribute. Basket cells are important inhibitors of granule cells. Excitatory synaptic input to basket cells and unitary IPSCs (uIPSCs) from basket cells to granule cells were evaluated in hippocampal slices from a rat model of temporal lobe epilepsy. Basket cells were identified by electrophysiological and morphological criteria. Excitatory synaptic drive to basket cells, measured by mean charge transfer and frequency of miniature EPSCs, was significantly reduced after pilocarpine-induced status epilepticus and remained low in epileptic rats, despite mossy fiber sprouting. Paired recordings revealed higher failure rates and a trend toward lower amplitude uIPSCs at basket cell-to-granule cell synapses in epileptic rats. Higher failure rates were not attributable to excessive presynaptic inhibition of GABA release by activation of muscarinic acetylcholine or GABA(B) receptors. High-frequency trains of action potentials in basket cells generated uIPSCs in granule cells to evaluate readily releasable pool (RRP) size and resupply rate of recycling vesicles. Recycling rate was similar in control and epileptic rats. However, quantal size at basket cell-to-granule cell synapses was larger and RRP size smaller in epileptic rats. Therefore, in epileptic animals, basket cells receive less excitatory synaptic drive, their pools of readily releasable vesicles are smaller, and transmission failure at basket cell-to-granule cell synapses is increased. These findings suggest dysfunction of the dentate basket cell circuit could contribute to hyperexcitability and seizures.

The Unitary Event Underlying Multiquantal EPSCs at a Hair Cell's Ribbon Synapse

EPSCs at the synapses of sensory receptors and of some CNS neurons include large events thought to represent the synchronous release of the neurotransmitter contained in several synaptic vesicles by a process known as multiquantal release. However, determination of the unitary, quantal size underlying such putatively multiquantal events has proven difficult at hair cell synapses, hindering confirmation that large EPSCs are in fact multiquantal. Here, we address this issue by performing presynaptic membrane capacitance measurements together with paired recordings at the ribbon synapses of adult hair cells. These simultaneous presynaptic and postsynaptic assays of exocytosis, together with electron microscopic estimates of single vesicle capacitance, allow us to estimate a single vesicle EPSC charge of approximately -45 fC, a value in close agreement with the mean postsynaptic charge transfer of uniformly small EPSCs recorded during periods of presynaptic hyperpolarization. By thus establishing the magnitude of the fundamental quantal event at this peripheral sensory synapse, we provide evidence that the majority of spontaneous and evoked EPSCs are multiquantal. Furthermore, we show that the prevalence of uniquantal versus multiquantal events is Ca2+ dependent. Paired recordings also reveal a tight correlation between membrane capacitance increase and evoked EPSC charge, indicating that glutamate release during prolonged hair cell depolarization does not significantly saturate or desensitize postsynaptic AMPA receptors. We propose that the large EPSCs reflect the highly synchronized release of multiple vesicles at single presynaptic ribbon-type active zones through a compound or coordinated vesicle fusion mechanism.

Differences in the quantal release of catecholamines in chromaffin cells of rat embryos and their mothers

Studies on the bulk catecholamine release from fetal and neonatal rat adrenals, adrenal slices, or isolated chromaffin cells stimulated with high K(+), hypoxia, hypercapnia, or acidosis are available. However, a study analyzing the kinetics of quantal secretion is lacking. We report here such a study in which we compare the quantal release of catecholamines from immature rat embryo chromaffin cells (ECCs) and their mothers' (MCCs). Cell challenging with a strong depolarizing stimulus (75 mM K(+)) caused spike bursts having the following characteristics. ECCs released more multispike events and wave envelopes than MCCs. This, together with narrower single-spike events, a faster decay, and a threefold smaller quantal size suggest a faster secretory machinery in ECCs. Furthermore, with a milder stimulus (25 mM K(+)) enhanced Ca(2+) entry by L-type Ca(2+) channel activator BAY K 8644 did not change the kinetic parameters of single spikes in ECCs; in contrast, augmentation of Ca(2+) entry increased spike amplitude and width, quantal size, and decay time in MCCs. This suggests that in mature MCCs, the last exocytotic steps are more tightly regulated than in immature ECCs. Finally, we found that quantal secretion was fully controlled by L-type voltage-dependent Ca(2+) channels (VDCCs) in ECCs, whereas both L- and non-L VDCCs (N and PQ) contributed equally to secretion control in MCCs. Our results have the following physiological, pharmacological, and clinical relevance: 1) they may help to better understand the regulation of adrenal catecholamine release in response to stress during fetal life and delivery; 2) if clinically used, L-type Ca(2+) channel blockers may augment the incidence of sudden infant death syndrome (SIDS); and 3) so-called Ca(2+) promotors or activators of Ca(2+) entry through L-type VDCCs may be useful to secure a healthy catecholamine surge upon violent stress during fetal life, at birth, or to prevent the SIDS in neonates at risk.

Compound vesicle fusion increases quantal size and potentiates synaptic transmission

Exocytosis at synapses generally refers to fusion between vesicles and the plasma membrane1. Although compound fusion between vesicles2,3 was proposed at ribbon-type synapses4,5, whether it exists, how it is mediated, and what role it plays at conventional synapses remain unclear. Here we addressed this issue at a nerve terminal containing conventional active zones. High potassium application and high frequency firing induced giant capacitance up-steps reflecting exocytosis of vesicles larger than regular ones, followed by giant down-steps reflecting bulk endocytosis. They also induced giant vesicle-like structures, as observed with electron microscopy, and giant miniature EPSCs (mEPSCs) reflecting more transmitter release. Calcium and its sensor for vesicle fusion, synaptotagmin, were required for these giant events. After high frequency firing, calcium/synaptotagmin-dependent mEPSC size increase was paralleled by calcium/synaptotagmin-dependent post-tetanic potentiation (PTP). These results suggest that calcium/synaptotagmin mediates compound fusion between vesicles, that exocytosis of compound vesicles increases quantal size which enhances synaptic strength and thus contributes to the generation of PTP, and that exocytosed compound vesicles may be retrieved via bulk endocytosis. We suggest to include a new vesicle cycling route, compound exocytosis followed by bulk endocytosis, into models of synapses, where currently only vesicle fusion with the plasma membrane is considered (Fig. S1)1.

SNAT2 Amino Acid Transporter Is Regulated by Amino Acids of the SLC6 γ-Aminobutyric Acid Transporter Subfamily in Neocortical Neurons and May Play No Role in Delivering Glutamine for Glutamatergic Transmission

System A transporters SNAT1 and SNAT2 mediate uptake of neutral alpha-amino acids (e.g. glutamine, alanine, and proline) and are expressed in central neurons. We tested the hypothesis that SNAT2 is required to support neurotransmitter glutamate synthesis by examining spontaneous excitatory activity after inducing or repressing SNAT2 expression for prolonged periods. We stimulated de novo synthesis of SNAT2 mRNA and increased SNAT2 mRNA stability and total SNAT2 protein and functional activity, whereas SNAT1 expression was unaffected. Increased endogenous SNAT2 expression did not affect spontaneous excitatory action-potential frequency over control. Long term glutamine exposure strongly repressed SNAT2 expression but increased excitatory action-potential frequency. Quantal size was not altered following SNAT2 induction or repression. These results suggest that spontaneous glutamatergic transmission in pyramidal neurons does not rely on SNAT2. To our surprise, repression of SNAT2 activity was not limited to System A substrates. Taurine, gamma-aminobutyric acid, and beta-alanine (substrates of the SLC6 gamma-aminobutyric acid transporter family) repressed SNAT2 expression more potently (10x) than did System A substrates; however, the responses to System A substrates were more rapid. Since ATF4 (activating transcription factor 4) and CCAAT/enhancer-binding protein are known to bind to an amino acid response element within the SNAT2 promoter and mediate induction of SNAT2 in peripheral cell lines, we tested whether either factor was similarly induced by amino acid deprivation in neurons. We found that glutamine and taurine repressed the induction of both transcription factors. Our data revealed that SNAT2 expression is constitutively low in neurons under physiological conditions but potently induced, together with the taurine transporter TauT, in response to depletion of neutral amino acids.

Evoking and Resolving Quantal Neurotransmitter Release on a Microchip

A microchip that facilitates in-vitro electrical and electrochemical measurements of individual cells and cell clusters was fabricated using surface micromachining and thick film technologies. In the present study, the device was applied towards the detection of exocytotic events from electrically stimulated rat pheochromocytoma (PC12) cells. Using device microfluidics, cells were positioned in a recording chamber over a 5 μm × 10 μm gold working electrode (WE). Channel dimensions (10 μm deep × 10 μm wide) ensured a tight fit for the ∼12 μm diameter PC12 cells in the chamber resulting in direct contact of the cells with the WE. This proximity allowed for quantal resolution of catecholamine release events from the cells and corresponding analysis of release kinetics and quantal size. Cells were stimulated through the application of sinusoidal voltage waveforms across axially-positioned, extracellular electrodes. In this manner, patterned extracellular gradients were generated across the cell thereby resulting in membrane depolarization. To facilitate interpretation of the stimulating electric field in relation to the cell and subsequent dopamine release, quasi-static electromagnetic FEM models were generated using COMSOL Multiphysics software. Upon depolarization, simultaneous chronoamperometric recordings at the WE confirmed stimulus-triggered dopamine release from the cells with a small subset of cells exhibiting release that modulated with the depolarizing cycle of the sinusoidal stimulus. It is anticipated that such a chip could provide a semi-automated alternative to the conventional, labor-intensive carbon fiber electrode (CFE) approach to neurotransmitter measurement.This work was supported in part by the National Institutes of Health, NIDCD R01 DC04928 and by National Science Foundation, IGERT NSF DGE-9987616.

Synchronous Versus Asynchronous Contributions to Frequency-induced Synaptic Depletion in Zebrafish

Paired spinal motorneuron and target muscle recordings were used to examine transmitter depletion and subsequent recovery during high frequency stimulation. The skeletal muscle is sufficiently compact that the unitary synaptic events (∼400pA average) were fully resolved through whole cell voltage clamp. Also, the evoked endplate current reflected the sum of 20Hz led to drops in quantal content and eventual failure, with no associated change in unitary quantal size. The time required to deplete 80% of the transmitter corresponded to 25 sec at 20Hz, 10 sec at 50Hz and 5 sec at 100 Hz. Recovery occurred abruptly after a 40 sec rest and the rate of recovery was calcium-dependent. Analysis of the depletion profile during 100 Hz stimulation revealed two partially overlapping processes. Initially, all release was synchronous upon repolarization of the motorneuron action potential. With continued stimulation the release was delayed and asynchronous with respect to the action potential. The quantal size underlying both synchronous and asynchronous modes was unchanged during the depletion and following recovery. At 100 Hz the synchronous endplate current converted to asynchronous unitary openings in a manner that was qualitatively reciprocal. Moreover, traces composed principally of synchronous events had few asynchronous events and conversely traces with the largest numbers of asynchronous events lacked synchronous release. This reciprocity suggested that the two modes share common release sites. However, measurements of total synchronous and asynchronous events during the time of maximal overlap revealed a period of facilitation, suggesting that the synchronous and asynchronous modes were capable of additivity and might represent different release sites. Experiments are ongoing to distinguish between these alternative possibilities.

Exit chloride, enter glutamate

The in vitro reconstitution of vesicular glutamate transport reveals that the transporter itself antiports chloride ions and suggests that extracellular chloride concentrations may determine neurotransmitter refilling and quantal size.

Calcium/synaptotagmin-mediated Compound Fusion Increases Quantal Size And Causes Post-tetanic Potentiation At Synapses

Exocytosis at synapses generally refers to fusion between vesicles and the plasma membrane. Although fusion between vesicles, known as compound fusion, occurs in non-neuronal secretory cells and has recently been proposed at ribbon-type synapses, it remains unclear whether it exists, how it is mediated, and what role it plays at the vast majority of synapses, where release occurs at conventional active zones. Here we addressed this issue in rats and mice at a large nerve terminal containing conventional active zones. High potassium application induced giant capacitance up-steps at the release face of nerve terminals, which were larger than the membrane capacitance of regular vesicles. These giant up-steps were not comprised of several smaller steps, nor were they bulk endocytic vesicles that had re-fused. High potassium application also induced giant vesicle-like structures in nerve terminals and giant miniature EPSCs (mEPSCs) that reflected release of a large amount of transmitter. The giant up-steps, giant vesicle-like structures, and giant mEPSCs were abolished by removing the extracellular calcium or by knocking out synaptotagmin II, the calcium sensor mediating fusion at calyces. These results suggest that calcium binding with synaptotagmin II mediates compound fusion and increases quantal size. Compound fusion significantly contributed to the generation of a widely observed synaptic plasticity, post-tetanic potentiation (PTP) of the EPSC, because 1) action potential trains that generated PTP also evoked giant up-steps and increased the mEPSC amplitude, 2) the time course and the degree of the mEPSC amplitude increase paralleled those of PTP, and 3) both the mEPSC amplitude increase and PTP were abolished by the calcium buffer EGTA or synaptotagmin II knockout. Our finding may be of wide application because intense nerve activity, PTP, and giant miniature currents occur in physiological conditions at many synapses.