BDNF increases release probability and the size of a rapidly recycling vesicle pool within rat hippocampal excitatory synapses

Exerting its actions pre-, post- and peri-synaptically, brain-derived neurotrophic factor (BDNF) is one of the most potent modulators of hippocampal synaptic function. Here, we examined the effects of BDNF on a rapidly recycling pool (RRP) of vesicles within excitatory synapses. First, we estimated vesicular release in hippocampal cultures by performing FM4-64 imaging in terminals impinging on enhanced green fluorescent protein (eGFP)-labelled dendritic spines – a hallmark of excitatory synapses. Consistent with a modulation of the RRP, BDNF increased the evoked destaining rate of FM4-64 only during the initial phase of field stimulation. Multiphoton microscopy in acute hippocampal slices confirmed these observations by selectively imaging the RRP, which was loaded with FM1-43 by hyperosmotic shock. Slices exposed to BDNF showed an increase in the evoked and spontaneous rates of FM1-43 destaining from terminals in CA1 stratum radiatum, mostly representing excitatory terminals of Schaffer collaterals. Variance-mean analysis of evoked EPSCs in CA1 pyramidal neurons further confirmed that release probability is increased in BDNF-treated slices, without changes in the number of independent release sites or average postsynaptic quantal amplitude. Because BDNF was absent during dye loading, imaging, destaining and whole-cell recordings, these results demonstrate that BDNF induces a long-lasting enhancement in the probability of transmitter release at hippocampal excitatory synapses by modulating the RRP. Since the endogenous BDNF scavenger TrkB-IgG prevented the enhancement of FM1-43 destaining rate caused by induction of long-term potentiation in acute hippocampal slices, the modulation of a rapidly recycling vesicle pool may underlie the role of BDNF in hippocampal long-term synaptic plasticity.     

Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation

In addition to maintaining the extracellular glutamate concentration at low ambient levels, high-affinity glutamate transporters play a direct role in synaptic transmission by speeding the clearance of glutamate from the synaptic cleft and limiting the extent to which transmitter spills over between synapses. Transporters are expressed in both neurons and glia, but glial transporters are likely to play the major role in removing synaptically released glutamate from the extracellular space. The role of transporters in synaptic transmission has been studied directly by measuring synaptically activated, transporter-mediated currents (STCs) in neurons and astrocytes. Here we record from astrocytes in the CA1 region of hippocampal slices and elicit STCs with high-frequency (100 Hz) stimulus trains of varying length to determine whether transporters are overwhelmed by stimuli that induce long-term potentiation. We show that, at near-physiological temperatures (34 degrees C), high-frequency stimulation (HFS) does not affect the rate at which transporters clear glutamate from the extrasynaptic space. Thus, although spillover between synapses during "normal" stimulation may compromise the absolute synapse specificity of fast excitatory synaptic transmission, spillover is not exacerbated during HFS. Transporter capacity is diminished somewhat at room temperature (24 degrees C), although transmitter released during brief, "theta burst" stimulation is still cleared as quickly as following a single stimulus, even when transport capacity is partially diminished by pharmacological means.     

Discharge of the readily releasable pool with action potentials at hippocampal synapses

A readily releasable pool (RRP) of synaptic vesicles has been identified at hippocampal synapses with application of hypertonic solution. RRP size correlates with important properties of synaptic function such as release probability. However, a discrepancy in RRP size has been reported depending on the method used to evoke synaptic release. This study was undertaken to determine quantitative relationships between the RRP defined with hypertonic solution and that released with trains of action potentials. We find that asynchronous release at cell culture synapses contributes significantly to the discharge of the RRP with trains of action potentials and that RRP size is the same when elicited by either nerve stimuli or hypertonic challenge.     

Potentiation phase of spike timing-dependent neuromodulation by a serotonergic interneuron involves an increase in the fraction of transmitter release

In the mollusk, Tritonia diomedea, the serotonergic dorsal swim interneuron (DSI) produces spike timing-dependent neuromodulation (STDN) of the synaptic output of ventral swim interneuron B (VSI) resulting in a biphasic, bidirectional change of synaptic strength characterized by a rapid heterosynaptic potentiation followed by a more prolonged heterosynaptic depression. This study examined the mechanism underlying the potentiation phase of STDN. In the presence of 4-aminopyridine, which blocks the depression phase and enhances transmitter release from VSI, rapidly stimulating VSI led to a steady-state level of transmitter depletion during which potentiation by DSI or serotonin (5-HT) was eliminated. Cumulative plots of excitatory postsynaptic currents were used to estimate changes in the size and replenishment rate of the readily releasable pool (RRP) and the fraction of release. 5-HT application increased transmitter release without altering replenishment rate. The magnitude of 5-HT-evoked potentiation correlated with the increase in the fraction of release. A phenomenological model of the synapse further supported the hypothesis that 5-HT-induced potentiation was caused by an increase in the fraction of release and correctly predicted no change in frequency facilitation. A dynamic version of the model correctly predicted the effect of DSI stimulation under a variety of conditions. Finally, depletion of internal Ca(2+) stores with cyclopiazonic acid showed that Ca(2+) from internal stores is necessary for the 5-HT-induced potentiation. The data indicate that 5-HT released from DSI increases the fraction of the RRP discharged during VSI action potentials using a mechanism that involves Ca(2+) extrusion from internal stores, resulting in time- and state-dependent neuromodulation.     

Kinetic isolation of a slowly recovering component of short-term depression during exhaustive use at excitatory hippocampal synapses

This study examines the kinetics of the longest lasting form of short-term depression at excitatory hippocampal synapses. After initial depletion of the readily releasable pool (RRP), continued 20-Hz stimulation was found to be fast enough to maximally drive presynaptic neurotransmitter exocytosis; maximal is defined here as the rate needed to maintain the RRP in a nearly empty steady state. Induction of depression proceeded in two distinct phases. The first was caused by RRP depletion, whereas the second is shown to reflect the progressive reduction of the overall rate at which new vesicles are supplied to the RRP and is termed "supply-rate depression." Supply-rate depression is identified further with the emergence, during heavy use, of a rate-limiting vesicle trafficking step that slows the timing of RRP replenishment by switching from a fast (tau congruent with 7 s) to a slow (tau congruent with 1 min) vesicle supply mechanism. Both mechanisms apparently follow first-order kinetics. After the induction of the maximum amount of depression, individual synapses were able to output only <1 quantum of neurotransmitter per synapse per second, matching previous predictions based on cell biological measurements of synaptic vesicle cycling. Surprisingly, the onset of supply-rate depression occurred with a marked delay, not having a detectable impact on synaptic function until after several seconds of continuous use. The delayed onset is not consistent with traditional vesicle trafficking models, but may be important for limiting the impact of supply-rate depression to pathological episodes and might function as a native antiepilepsy device.     

Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system

Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 μM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 μM) by high K⁺ or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2–30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 μM) as well as nominally zero external Ca²⁺ (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca²⁺]_i increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be ∼27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.     

Augmentation controls the fast rebound from depression at excitatory hippocampal synapses

Short-term plasticity occurs at most central chemical synapses and includes both positive and negative components, but the principles governing interaction between components are largely unknown. The residual Ca²⁺ that persists in presynaptic terminals for several seconds after repetitive use is known to enhance neurotransmitter release under artificial, low probability of release conditions where depression is absent; this is termed augmentation. However, the full impact of augmentation under standard conditions at synapses where depression dominates is not known because of possibly complicated convolution with a variety of potential depression mechanisms. This report shows that residual Ca²⁺ continues to have a large enhancing impact on release at excitatory hippocampal synapses recovering from depression, including when only recently recruited vesicles are available for release. No evidence was found for gradual vesicle priming or for fast refilling of a highly releasable subdivision of the readily releasable pool (RRP). And decay of enhancement matched the clearance of residual Ca²⁺, thus matching the behavior of augmentation when studied in isolation. Because of incomplete RRP replenishment, synaptic strength was not typically increased above baseline when residual Ca²⁺ levels were highest. Instead residual Ca²⁺ caused single pulse release probability to rebound quickly from depression and then depress quickly during subsequent bursts of activity. Together, these observations can help resolve discrepancies in recent timing estimates of recovery from depression. Additionally, in contrast to results obtained under reduced release conditions, augmentation could be driven to a maximal level, occluding paired-pulse facilitation and other mechanisms that increase release efficiency.     

Changes in the readily releasable pool of transmitter and in efficacy of release induced by high-frequency firing at Aplysia sensorimotor synapses in culture

Synaptic transmission at the sensory neuron-motor neuron synapses of Aplysia, like transmission at many synapses of both vertebrates and invertebrates, is increased after a short burst of high-frequency stimulation (HFS), a phenomenon known as posttetanic potentiation (PTP). PTP is generally attributable to an increase in transmitter release from presynaptic neurons. We investigated whether changes in the readily releasable pool of transmitter (RRP) contribute to the potentiation that follows HFS. We compared the changes in excitatory postsynaptic potentials (EPSPs) evoked with action potentials to changes in the RRP as estimated from the asynchronous transmitter release elicited by a hypertonic solution. The changes in the EPSP were correlated with changes in the RRP, but the changes matched quantitatively only at connections whose initial synaptic strength was greater than the median for all experiments. At weaker connections, the increase in the RRP was insufficient to account for PTP. Weaker connections initially released a smaller fraction of the RRP with each EPSP than stronger ones, and this fraction increased at weaker connections after HFS. Moreover, the initial transmitter release in response to the hypertonic solution was accelerated after HFS, indicating that the increase in the efficacy of release was not restricted to excitation-secretion coupling. Modulation of the RRP and of the efficacy of release thus both contribute to the enhancement of transmitter release by HFS.     

Progressive potentiation of peptide release during a neuronal discharge

1. In response to electrical stimulation, the bag cell neurons of Aplysia generate an afterdischarge that lasts 20-40 min. During this afterdischarge several neuroactive peptides are released. We have now studied the time course of release of two of these peptides, egg-laying hormone (ELH) and acidic peptide (AP). For the collection of released peptides, the artery to the bag cell clusters was perfused. The medium surrounding the clusters (artificial seawater, ASW) was completely exchanged at 5-min intervals before, during, and after stimulation of an afterdischarge. Peptides released into the external medium were analyzed with the use of high-pressure liquid chromatography. 2. Before stimulation, no detectable ELH and AP were found in the external medium. After the onset of an afterdischarge, the amount of ELH and AP released increased progressively until 15-20 min of firing. Toward the conclusion of an afterdischarge, the release of ELH and AP returned to control levels. 3. In contrast to the pattern of release of the peptides, the firing rate of the bag cell neurons is maximal within the first minute of afterdischarge and thereafter declines. 4. Release of the peptides from axonal varicosities occurs within the vascularized connective-tissue sheath that covers the clusters of bag cell neurons. Experiments were therefore carried out to establish whether the observed time course of release is affected by diffusion of the peptides through the vasculature into the external medium and, in particular, to determine whether the maximal rate of release at 15-20 min into the afterdischarge could be accounted for by a delay in transport of peptides from the neurites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic release probability is increased in hippocampal neurons from ASIC1 knockout mice

Acid-sensing ion channels (ASICs) are H(+)-gated channels that produce transient cation currents in response to extracellular acid. ASICs are expressed in neurons throughout the brain, and ASIC1 knockout mice show behavioral impairments in learning and memory. The role of ASICs in synaptic transmission, however, is not thoroughly understood. We analyzed the involvement of ASICs in synaptic transmission using microisland cultures of hippocampal neurons from wild-type and ASIC knockout mice. There was no significant difference in single action potential (AP)-evoked excitatory postsynaptic currents (EPSCs) between wild-type and ASIC knockout neurons. However, paired-pulse ratios (PPRs) were reduced and spontaneous miniature EPSCs (mEPSCs) occurred at a higher frequency in ASIC1 knockout neurons compared with wild-type neurons. The progressive block of NMDA receptors by an open channel blocker, MK-801, was also faster in ASIC1 knockout neurons. The amplitude and decay time constant of mEPSCs, as well as the size and refilling of the readily releasable pool, were similar in ASIC1 knockout and wild-type neurons. Finally, the release probability, which was estimated directly as the ratio of AP-evoked to hypertonic sucrose-induced charge transfer, was increased in ASIC1 knockout neurons. Transfection of ASIC1a into ASIC1 knockout neurons increased the PPRs, suggesting that alterations in release probability were not the result of developmental compensation within the ASIC1 knockout mice. Together, these findings demonstrate that neurons from ASIC1 knockout mice have an increased probability of neurotransmitter release and indicate that ASIC1a can affect presynaptic mechanisms of synaptic transmission.     

GABA(B)-receptor modulation of short-term synaptic depression at an excitatory input to murine hippocampal CA3 pyramidal neurons

GABA(B) agonists inhibit excitatory transmission to hippocampal CA3 neurons during low frequency stimulation. We examined whether GABA(B) receptor activation can also enhance synaptic efficacy, when investigated at an input with high initial release probability. Short-term depression of field excitatory postsynaptic potential (EPSP) amplitude was observed during trains of stimuli applied to associational/commissural inputs (10-50 Hz; 22 degrees C). Baclofen (10 microM) reduced the amplitude of initial EPSPs in a train, and also reduced the degree of short-term depression. EPSPs recorded late in a train were significantly larger in baclofen than those recorded in control solution. These dual effects were mimicked by another selective GABA(B) agonist (SKF 97541, 10 microM), and abolished by a GABA(B)-selective antagonist (SCH 50911, 20 microM). The effects of baclofen were similar at a higher recording temperature (32 degrees C), where short-term depression was observed at higher stimulation frequencies. These results are consistent with the idea that a reduction of transmitter release probability could increase the fidelity of high-frequency transmission at this input, an effect that could help account for excitatory effects of GABA(B) agonists in some seizure models.     

A delayed response enhancement during hippocampal presynaptic plasticity in mice

High frequency afferent stimulation of chemical synapses often induces short-term increases in synaptic efficacy, due to increased release probability and/or increased supply of readily releasable synaptic vesicles. This may be followed by synaptic depression, often caused by vesicle depletion. We here describe an additional, novel type of delayed and transient response enhancement phase which occurred during prolonged stimulation at 5–20 Hz frequency of excitatory glutamatergic synapses in slices from the adult mouse CA1 hippocampal region. This second enhancement phase, which was most clearly defined at physiological temperatures and essentially absent at 24°C, was dependent on the presence of F-actin filaments and synapsins I and/or II, and could not be ascribed to changes in presynaptic action potentials, inhibitory neurotransmission or glutamate receptor desensitization. Time course studies showed that the delayed response phase interrupted the synaptic decay 3–4 s after stimulus train initiation and continued, when examined at 5–10 Hz frequencies, for approximately 75 stimuli before decay. The novel response enhancement, probably deriving from a restricted pool of synaptic vesicles, may allow maintenance of synaptic efficacy during prolonged periods of excitatory synaptic activity.     

Inhibition of presynaptic Na(+)/K(+)-ATPase reduces readily releasable pool size at the avian end-bulb of Held synapse

A glutamatergic end-bulb synapse in the avian nucleus magnocellularis relays temporal sound information from the auditory nerve. Here, we show that presynaptic Na⁺/K⁺-ATPase (NKA) activity at this synapse contributes to the maintenance of the readily releasable pool (RRP) of vesicles, thereby preserving synaptic strength. Whole-cell voltage clamp recordings were made from chick brainstem slices to examine the effects of NKA blocker dihydroouabain (DHO) on synaptic transmission. DHO suppressed the amplitude of EPSCs in a dose-dependent manner. This suppression was caused by a decrease in the number of neurotransmitter quanta released because DHO increased the coefficient of variation of EPSC amplitude and reduced the frequency but not the amplitude of miniature EPSCs. Cumulative plots of EPSC amplitude during a stimulus train revealed that DHO reduced the RRP size without affecting vesicular release probability. DHO did not affect [Ca²⁺]_i-dependent processes, such as the paired-pulse ratio or recovery time course from the paired-pulse depression, suggesting a minimal effect on Ca²⁺ concentration in the presynaptic terminal. Using mathematical models of synaptic depression, we further demonstrated the contribution of RRP size to the synaptic strength during a high-frequency stimulus train to highlight the importance of presynaptic NKA in the auditory synapse.     

Dynamics of the readily releasable pool during post-tetanic potentiation in the rat calyx of Held synapse

The size of the readily releasable pool (RRP) of vesicles was measured in control conditions and during post-tetanic potentiation (PTP) in a large glutamatergic terminal called the calyx of Held. We measured excitatory postsynaptic currents evoked by a high frequency train of action potentials in slices of 4–11-day-old rats. After a tetanus the cumulative release during such a train was enlarged by approximately 50%, indicating that the size of the RRP was increased. The amount of enhancement depended on the duration and frequency of the tetanus and on the age of the rat. After the tetanus, the size of the RRP decayed more slowly ( t _1/2= 10 versus 3 min) back to control values than the release probability. This difference was mainly due to a very fast initial decay of the release probability, which had a time constant compatible with an augmentation phase (τ≈ 30 s). The overall decay of PTP at physiological temperature was not different from room temperature, but the increase in release probability ( P _r) was restricted to the first minute after the tetanus. Thereafter PTP was dominated by an increase in the size of the RRP. We conclude that due to the short lifetime of the increase in release probability, the contribution of the increase in RRP size during post-tetanic potentiation is more significant at physiological temperature.     

A simple depletion model of the readily releasable pool of synaptic vesicles cannot account for paired-pulse depression

Paired-pulse depression (PPD) is a form of short-term plasticity that plays a central role in processing of synaptic activity and is manifest as a decrease in the size of the response to the second of two closely timed stimuli. Despite mounting evidence to the contrary, PPD is still commonly thought to reflect depletion of the pool of synaptic vesicles available for release in response to the second stimulus. Here it is shown that PPD cannot be accounted for by depletion at excitatory synapses made by hippocampal neurons because PPD is unaffected by changes in the fraction of the readily releasable pool (RRP) released by the first of a pair of pulses.     

Rapid translocation of Zn(2+) from presynaptic terminals into postsynaptic hippocampal neurons after physiological stimulation.

Zn(2+) is found in glutamatergic nerve terminals throughout the mammalian forebrain and has diverse extracellular and intracellular actions. The anatomical location and possible synaptic signaling role for this cation have led to the hypothesis that Zn(2+) is released from presynaptic boutons, traverses the synaptic cleft, and enters postsynaptic neurons. However, these events have not been directly observed or characterized. Here we show, using microfluorescence imaging in rat hippocampal slices, that brief trains of electrical stimulation of mossy fibers caused immediate release of Zn(2+) from synaptic terminals into the extracellular microenvironment. Release was induced across a broad range of stimulus intensities and frequencies, including those likely to induce long-term potentiation. The amount of Zn(2+) release was dependent on stimulation frequency (1-200 Hz) and intensity. Release of Zn(2+) required sodium-dependent action potentials and was dependent on extracellular Ca(2+). Once released, Zn(2+) crosses the synaptic cleft and enters postsynaptic neurons, producing increases in intracellular Zn(2+) concentration. These results indicate that, like a neurotransmitter, Zn(2+) is stored in synaptic vesicles and is released into the synaptic cleft. However, unlike conventional transmitters, it also enters postsynaptic neurons, where it may have manifold physiological functions as an intracellular second messenger.     

Long-term adaptation in lobster motor neurons and compensation of transmitter release by synergistic inputs

Earlier studies with crayfish have shown that chronic increases in neural activity, by electrical stimulation, cause a long-lasting reduction in the amount of transmitter released at low stimulus frequencies or at the beginning of a stimulus train. When such chronic stimulation is applied to phasic extensor motor neurons of the lobster abdomen, a similar change in transmitter release is apparent, as indicated by a decrease in excitatory postsynaptic potential (EPSP) size at 0.1 Hz. However, the EPSPs from unstimulated axons which innervate the same target muscle from a different nerve increase in size. Thus, activity-dependent reduction in transmitter release at one set of synapses appears to be compensated for by increased synaptic efficacy from less active synergistic inputs. The mechanism of such compensation is not known.     

Activity-dependent release of adenosine inhibits the glutamatergic synaptic transmission and plasticity in the hypothalamic hypocretin/orexin neurons

The hypocretin (orexin) neurons in the lateral hypothalamus play a crucial role in the promotion of arousal. Adenosine, an endogenous sleep-promoting factor, modulates both neuronal excitatory and synaptic transmission in the CNS. In this study, the involvement of endogenous adenosine in the regulation of excitatory glutamatergic synaptic transmission to hypocretin neurons was investigated in the hypothalamic slices from transgenic mice by using different frequencies of stimulation. A train of low-frequency stimulation (0.033, 1 Hz) had no effect on the amplitude of evoked excitatory postsynaptic currents (evEPSCs) in hypocretin neurons. Blockade of adenosine A1 receptors with selective A1 receptor antagonist 8-cyclopentyltheophylline (CPT), the amplitude of evEPSCs did not change during 0.033 and 1 Hz stimuli. When the frequency of stimulation was increased upto 2 Hz, a time-dependent depression of amplitude was recorded in hypocretin neurons. Administration of CPT caused no significant change in depressed synaptic response induced by 2 Hz stimulus. While depression induced by 10 and 100 Hz stimuli was partially inhibited by the CPT but not by the selective A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine. Further findings have demonstrated that high-frequency stimulation could induce long-term potentiation (LTP) of glutamatergic synaptic transmission to hypocretin neurons in acute hypothalamic slices. The experiments with CPT suggested that A1 receptor antagonist could facilitate the induction of LTP, indicating that endogenous adenosine, acting through A1 receptors, may suppress the induction of LTP of excitatory synaptic transmission to hypocretin neurons. These results suggest that in the hypothalamus, endogenous adenosine will be released into extracellular space in an activity-dependent manner inhibiting both basal excitatory synaptic transmission and LTP in hypocretin neurons via A1 receptors. Our data provide further support for the notion that hypocretin neurons in the lateral hypothalamus may be another important target involved in the endogenous adenosine modulating the sleep and wakefulness cycle in the mammalian CNS.     

Synaptic transmission and plasticity are modulated by nonmuscle myosin II at the neuromuscular junction of Drosophila

The synaptic vesicle population in a nerve terminal is traditionally divided into subpopulations according to physiological criteria; the readily releasable pool (RRP), the recycling pool, and the reserve pool. It is recognized that the RRP subserves synaptic transmission evoked by low-frequency neural activity and that the recycling and reserve populations are called on to supply vesicles as neural activity increases. Here we investigated the contribution of nonmuscle myosin II (NMMII) to synaptic transmission with emphasis on the role a motor protein could play in the supply of vesicles. We used Drosophila genetics to manipulate NMMII and assessed synaptic transmission at the larval neuromuscular junction. We observed a positive correlation between synaptic strength at low-frequency stimulation and NMMII expression: reducing NMMII reduced the evoked response, while increasing NMMII increased the evoked response. Further, we found that NMMII contributed to the spontaneous release of vesicles differentially from evoked release, suggesting differential contribution to these two release mechanisms. By measuring synaptic responses under conditions of differing external calcium concentration in saline, we found that NMMII is important for normal synaptic transmission under high-frequency stimulation. This research identifies diverse functions for NMMII in synaptic transmission and suggests that this motor protein is an active contributor to the physiology of synaptic vesicle recruitment.     

Release kinetics, quantal parameters and their modulation during short-term depression at a developing synapse in the rat CNS

We have characterized developmental changes in the kinetics and quantal parameters of action potential (AP)-evoked neurotransmitter release during maturation of the calyx of Held synapse. Quantal size ( q ) and peak amplitudes of evoked EPSCs increased moderately, whereas the fraction of vesicles released by single APs decreased. During synaptic depression induced in postnatal day (P) 5–7 synapses by 10–100 Hz stimulation, q declined rapidly to 40–12% of its initial value. The decrease in q was generally smaller in more mature synapses (P12–14), but quite severe for frequencies ≥ 300 Hz. The stronger decline of q in immature synapses resulted from a slower recovery from desensitization, presumably due to delayed glutamate clearance. Recovery from this desensitization followed an exponential time course with a time constant of ∼480 ms in P5–7 synapses, and sped up > 20-fold during maturation. Deconvolution analysis of EPSCs revealed a significant acceleration of the release time course during development, which was accompanied by a 2-fold increase of the peak release rate. During long 100 Hz trains, more mature synapses were able to sustain average rates of 8–10 quanta s⁻¹ per active zone for phasic release. The rates of asynchronous vesicle release increased transiently > 35-fold immediately after such stimuli and decayed rapidly with an exponential time constant of ∼50 ms to low resting levels of spontaneous release. However, even following extended periods of 100 Hz stimulation, the amount of asynchronous release was relatively minor with peak rates of less than 5% of the average rate of synchronous release measured at steady state during the tetani. Therefore, a multitude of mechanisms seems to converge on the generation of fast, temporally precise and reliable high-frequency transmission at the mature calyx of Held synapse.