Synapsin-Dependent Vesicle Recruitment Modulated by Forskolin, Phorbol Ester and Ca2+ in Mouse Excitatory Hippocampal Synapses

Repeated release of transmitter from presynaptic elements depends on stimulus-induced Ca2+ influx together with recruitment and priming of synaptic vesicles from different vesicle pools. We have compared three different manipulations of synaptic strength, all of which are known to increase short-term synaptic efficacy through presynaptic mechanisms, in the glutamatergic CA3-to-CA1 stratum radiatum synapse in the mouse hippocampal slice preparation. Synaptic responses elicited from the readily releasable vesicle pool during low-frequency synaptic activation (0.1 Hz) were significantly enhanced by both the adenylate cyclase activator forskolin, the priming activator β-phorbol-12,13-dibutyrate (PDBu) and 4 mM [Ca2+]o′ whereas during 20 Hz stimulation, the same manipulations reduced the time needed to reach the peak and increased the magnitude of the resulting frequency facilitation. In contrast, paired-pulse facilitations were unchanged in the presence of forskolin, decreased by 4 mM [Ca2+]o and essentially abolished by PDBu. The subsequent delayed response enhancement (DRE) responses, elicited during continuous 20 Hz stimulations and mediated by recruited vesicles, were enhanced by forskolin, essentially unchanged by PDBu and slightly decreased by 4 mM [Ca2+]o· Similar experiments done on slices devoid of the vesicle-associated synapsin I and II proteins indicated that synapsin I/II-induced enhancements of vesicle recruitment were restricted to Ca2+-induced frequency facilitations and forskolin-induced enhancements of the early DRE phase, whereas the proteins had minor effects during PDBu-treatment and represented constraints on late Ca2+-induced responses. The data indicate that in these glutamatergic synapses, the comparable enhancements of single synaptic responses induced by these biochemical mechanisms can be transformed during prolonged synaptic stimulation into highly distinct short-term plasticity patterns, which are partly dependent on synapsins I/II

Calmodulin mediates rapid recruitment of fast-releasing synaptic vesicles at a calyx-type synapse.

In many synapses, depletion and recruitment of releasable synaptic vesicles contribute to use-dependent synaptic depression and recovery. Recently it has been shown that high- frequency presynaptic stimulation enhances recovery from depression, which may be mediated by Ca2+. We addressed this issue by measuring quantal release rates at the calyx of Held synapse and found that transmission is mediated by a heterogeneous population of vesicles, with one subset releasing rapidly and recovering slowly and another one releasing reluctantly and recovering rapidly. Ca2+ promotes refilling of the rapidly releasing synaptic vesicle pool and calmodulin inhibitors block this effect. We propose that calmodulin- dependent refilling supports recovery from synaptic depression during high-frequency trains in concert with rapid recovery of the slowly releasing vesicles

Multiple roles of calcium ions in the regulation of neurotransmitter release.

The intracellular calcium concentration ([Ca2+]) has important roles in the triggering of neurotransmitter release and the regulation of short-term plasticity (STP). Transmitter release is initiated by quite high concentrations within microdomains, while short-term facilitation is strongly influenced by the global buildup of “residual calcium.” A global rise in [Ca2+] also accelerates the recruitment of release-ready vesicles, thereby controlling the degree of short-term depression (STD) during sustained activity, as well as the recovery of the vesicle pool in periods of rest. We survey data that lead us to propose two distinct roles of [Ca2+] in vesicle recruitment: one accelerating “molecular priming” (vesicle docking and the buildup of a release machinery), the other promoting the tight coupling between releasable vesicles and Ca2+ channels. Such coupling is essential for rendering vesicles sensitive to short [Ca2+] transients, generated during action potentials

MCTP is an ER-resident calcium sensor that stabilizes synaptic transmission and homeostatic plasticity.

Presynaptic homeostatic plasticity (PHP) controls synaptic transmission in organisms from Drosophila to human and is hypothesized to be relevant to the cause of human disease. However, the underlying molecular mechanisms of PHP are just emerging and direct disease associations remain obscure. In a forward genetic screen for mutations that block PHP we identified mctp (Multiple C2 Domain Proteins with Two Transmembrane Regions). Here we show that MCTP localizes to the membranes of the endoplasmic reticulum (ER) that elaborate throughout the soma, dendrites, axon and presynaptic terminal. Then, we demonstrate that MCTP functions downstream of presynaptic calcium influx with separable activities to stabilize baseline transmission, short-term release dynamics and PHP. Notably, PHP specifically requires the calcium coordinating residues in each of the three C2 domains of MCTP. Thus, we propose MCTP as a novel, ER-localized calcium sensor and a source of calcium-dependent feedback for the homeostatic stabilization of neurotransmission

PHYSIOLOGICAL AND ANATOMICAL ASSESSMENT OF SYNAPSES AT THE CRAYFISH NEUROMUSCULAR JUNCTION

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of neuromuscular junctions since the preparation is identifiable from animal to animal and the nerve terminals are visible using a vital dye. This allows ease in finding a suitable site to record from in each preparation and offers the ability to relocate it anatomically. Marking a recorded site and rebuilding it through electron microscopy gives good detail of synaptic struture for assesment.In the first of these studies, low output sites known as stems (which lie between varicosities) were used to reduce n (number of release sites) in order to minimize synaptic complexity so individual quantal events could be analyzed by their unique parameters (area, peak, tau, rise time and latency). This was in attempt to uncover specific quantal signatures that could be traced back to the structure of the area recorded. It was found that even at stem regions synaptic structure is still complex having multiple synapses each of which could harbor a number of AZs. This gives insight as to how quantal analysis should be treated. Even low output synapses n must be treated at the AZ level.Synaptic depression was studied at the crayfish extensor muscle. By depressing the phasic neuron and recording from the muscle it appears thatdepression is a presynaptic phenomenon. The use of 5-HT gave insight to vesicular dynamics within the nerve terminal, by delaying depression and increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no change in numbers of dock or RRP vesicles. Short term facilitation and vesicular dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which blocks the glutamate transporter. In this study I showed differential mechanisms that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is depleted as shown by reduced EPSPs, but recovered with 5-HT application. The understanding of vesicle dynamics in any system has relevance for all chemical synapses

THE REGULATION AND PACKAGING OF SYNAPTIC VESICLES RELATED TO RECRUITMENT WITHIN CRAYFISH AND FRUIT FLY NEUROMUSCULAR JUNCTIONS: VARIATIONS IN LOW- AND HIGH-OUTPUT TERMINALS

Glutamate is the main excitatory neurotransmitter in the CNS and at the neuromuscular junctions (NMJs) of invertebrate. The characteristic similarities to CNS glutamatergic synapses in vertebrate and the anatomical simplicity of invertebrate NMJs favor the investigation of glutamatergic synaptic functions in this system. This dissertation mainly aimed to physiologically separate two functional vesicle groups, the reserve pool (RP) and readily releasable pool (RRP) within presynaptic nerve terminals of Procambarus Clarkii and Drosophila melanogaster. This was addressed in part by blocking the vesicular glutamate transporter (VGlut) with bafilomycin A1. Various frequencies of motor nerve stimulation, exposure time, and concentration of bafilomycin A1 were examined. The low-output tonic opener NMJs in crayfish exposed to 4μM bafilomycin A1 and 20Hz continuous stimulation decreased the EPSP amplitude to 50% in ∼30min with controls lasting 3h. After activity and bafilomycin A1-induced synaptic depression, the EPSPs were rapidly revitalized by serotonin (5-HT, 1μM) in the crayfish preparations. The 5-HT action can be blocked almost completely with a PLC inhibitor, but partially with a cAMP activator. The higher output synapses of the larval Drosophila NMJ when stimulated at 1Hz or 5Hz and exposed to 4μM of bafilomycin A1 showed a depression rate of 50% within ∼10min with controls lasting ∼40min. After low frequency depression and/or exposure to bafilomycin A1 a burst of higher frequency (10Hz) can recruit vesicles from the RP to the RRP. Physiological differences in low- (tonic like) and high-output (phasic like) synapses match many of the expected anatomical features of these terminals, part of this dissertation highlights physiological differences and differential modulation and/or extent of the vesicles in a RP for maintaining synaptic output during evoked depression of the RRP in crayfish abdomen extensor preparation. With the use of bafilomycin A1, the tonic terminal is fatigue resistant due to a large RRP, whereas the phasic depresses rapidly upon continuous stimulation. Upon depression of the tonic terminal, 5-HT has a large RP to act on to recruit vesicles to the RRP; whereas, the phasic terminal, 5-HT can recruit RP vesicles to the RRP prior to synaptic depression but not after depression

Transmitter release from cochlear hair cells is phase locked to cyclic stimuli of different intensities and frequencies

The auditory system processes time and intensity through separate brainstem pathways to derive spatial location as well as other salient features of sound. The independent coding of time and intensity begins in the cochlea, where afferent neurons can fire action potentials at constant phase throughout a wide range of stimulus intensities. We have investigated time and intensity coding by simultaneous presynaptic and postsynaptic recording at the hair cell-afferent synapse from rats. Trains of depolarizing steps to the hair cell were used to elicit postsynaptic currents that occurred at constant phase for a range of membrane potentials over which release probability varied significantly. To probe the underlying mechanisms, release was examined using single steps to various command voltages. As expected for vesicular release, first synaptic events occurred earlier as presynaptic calcium influx grew larger. However, synaptic depression produced smaller responses with longer first latencies. Thus, during repetitive hair cell stimulation, as the hair cell is more strongly depolarized, increased calcium channel gating hurries transmitter release, but the resulting vesicular depletion produces a compensatory slowing. Quantitative simulation of ribbon function shows that these two factors varied reciprocally with hair cell depolarization (stimulus intensity) to produce constant synaptic phase. Finally, we propose that the observed rapid vesicle replenishment would help maintain the vesicle pool, which in turn would equilibrate with the stimulus intensity (and therefore the number of open Ca 2+ channels), so that for trains of different levels the average phase will be conserved.Fil: Goutman, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

The coupling between synaptic vesicles and Ca²⁺ channels determines fast neurotransmitter release

SummaryIn order to release neurotransmitter synchronously in response to a presynaptic action potential, synaptic vesicles must be both release competent and located close to presynaptic Ca2+ channels. It has not been shown, however, which of the two is the more decisive factor. We tested this issue at the calyx of Held synapse by combining Ca2+ uncaging and electrophysiological measurements of postsynaptic responses. After depletion of the synaptic vesicles that are responsible for synchronous release during action potentials, uniform elevation of intracellular Ca2+ by Ca2+ uncaging could still elicit rapid release. The Ca2+ sensitivity of remaining vesicles was reduced no more than 2-fold, which is insufficient to explain the slow-down of the kinetics of release (10-fold) observed during a depolarizing pulse. We conclude that recruitment of synaptic vesicles to sites where Ca2+ channels cluster, rather than fusion competence, is a limiting step for rapid neurotransmitter release in response to presynaptic action potentials

Astrocyte-mediated short-term synaptic depression in the rat hippocampal CA1 area: two modes of decreasing release probability

<p>Abstract</p> <p>Background</p> <p>Synaptic burst activation feeds back as a short-term depression of release probability at hippocampal CA3-CA1 synapses. This short-term synaptic plasticity requires functional astrocytes and it affects both the recently active (< 1 s) synapses (post-burst depression) as well as inactive neighboring synapses (transient heterosynaptic depression). The aim of this study was to investigate and compare the components contributing to the depression of release probability in these two different scenarios.</p> <p>Results</p> <p>When tested using paired-pulses, following a period of inactivity, the transient heterosynaptic depression was expressed as a reduction in the response to only the first pulse, whereas the response to the second pulse was unaffected. This selective depression of only the first response in a high-frequency burst was shared by the homosynaptic post-burst depression, but it was partially counteracted by augmentation at these recently active synapses. In addition, the expression of the homosynaptic post-burst depression included an astrocyte-mediated reduction of the pool of release-ready primed vesicles.</p> <p>Conclusions</p> <p>Our results suggest that activated astrocytes depress the release probability via two different mechanisms; by depression of vesicular release probability only at inactive synapses and by imposing a delay in the recovery of the primed pool of vesicles following depletion. These mechanisms restrict the expression of the astrocyte-mediated depression to temporal windows that are typical for synaptic burst activity.</p