Relative roles of different mechanisms of depression at the mouse endbulb of Held

Several mechanisms can underlie short-term synaptic depression, including vesicle depletion, receptor desensitization, and changes in presynaptic release probability. To determine which mechanisms affect depression under physiological conditions, we studied the synapse formed by auditory nerve fibers onto bushy cells in the anteroventral cochlear nucleus (the "endbulb of Held") using voltage-clamp recordings of brain slices from P15-P21 mice near physiological temperatures. Depression of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) excitatory postsynaptic currents (EPSCs) showed two phases of recovery. The fast component of depression for the AMPA EPSC was eliminated by cyclothiazide and aniracetam, suggesting it results from desensitization. The fast component of depression for the NMDA EPSC was reduced by the low-affinity antagonist l-AP5, suggesting it results from saturation. The remaining depression in AMPA and NMDA components is identical and therefore presynaptic in origin. It is likely to result from presynaptic vesicle depletion. Recovery from depression after trains of activity was slowed by the application of EGTA-AM, suggesting that the endbulb has a residual-calcium-dependent form of recovery. We developed a model that incorporates depletion, desensitization, and calcium-dependent recovery. This model replicated experimental findings over a range of experimental conditions. The model further indicated that desensitization plays only a minor role during prolonged activity, in large part because presynaptic release is so depleted. Thus depletion appears to be the dominant mechanism of depression at the endbulb during normal activity. Furthermore, calcium-dependent recovery at the endbulb is critical to prevent complete rundown during high activity and to preserve the reliability of information transmission.     

Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission

Short-term synaptic depression during repetitive activity is a common property of most synapses. Multiple mechanisms contribute to this rapid depression in neurotransmission including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca²⁺ channels or use-dependent inhibition of release machinery by presynaptic receptors. In addition, synaptic depression can arise from a rapid reduction in the number of vesicles available for release. This reduction can be countered by two sources. One source is replenishment from a set of reserve vesicles. The second source is the reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough then it can replenish vesicles during a brief burst of action potentials and play a substantial role in regulating the rate of synaptic depression. In the last 5 years, we have examined the impact of synaptic vesicle reuse on neurotransmission using fluorescence imaging of synaptic vesicle trafficking in combination with electrophysiological detection of short-term synaptic plasticity. These studies have revealed that synaptic vesicle reuse shapes the kinetics of short-term synaptic depression in a frequency-dependent manner. In addition, synaptic vesicle recycling helps maintain the level of neurotransmission at steady state. Moreover, our studies showed that synaptic vesicle reuse is a highly plastic process as it varies widely among synapses and can adapt to changes in chronic activity levels.     

Short-term synaptic depression and recovery at the mature mammalian endbulb of Held synapse in mice

The endbulb of Held synapses between the auditory nerve fibers (ANF) and cochlear nucleus bushy neurons convey fine temporal information embedded in the incoming acoustic signal. The dynamics of synaptic depression and recovery is a key in regulating synaptic transmission at the endbulb synapse. We studied short-term synaptic depression and recovery in mature (P22-38) CBA mice with stimulation rates that were comparable to sound-driven activities recorded in vivo. Synaptic depression in mature mice is less severe ( approximately 40% at 100 Hz) than reported for immature animals and the depression is predominately due to depletion of releasable vesicles. Recovery from depression depends on the rate of activity and accumulation of intracellular Ca(2+) at the presynaptic terminal. With a regular stimulus train at 100 Hz in 2 mM external [Ca(2+)], the recovery from depletion was slow (tau(slow), approximately 2 s). In contrast, a fast (tau(fast), approximately 25 ms), Ca(2+)-dependent recovery followed by a slower recovery (tau(slow), approximately 2 s) was seen when stimulus rates or external [Ca(2+)] increased. In normal [Ca(2+)], recovery from a 100-Hz Poisson-like train is rapid, suggesting that Poisson-like trains produce a higher internal [Ca(2+)] than regular trains. Moreover, the fast recovery was slowed by approximately twofold in the presence of calmidazolium, a Ca(2+)/calmodulin inhibitor. Our results suggest that endbulb synapses from high spontaneous firing rate auditory nerve fibers normally operate in a depressed state. The accelerated synaptic recovery during high rates of activity is likely to ensure that reliable synaptic transmission can be achieved at the endbulb synapse.     

Kinetics and activation of postsynaptic kainate receptors at thalamocortical synapses: role of glutamate clearance

Kainate (KA) receptor-mediated excitatory postsynaptic currents (EPSCs) exhibit slow kinetics at the great majority of synapses. However, native or heterologously expressed KA receptors exhibit rapid kinetics in response to agonist application. One possibility to explain this discrepancy is that KA receptors are extrasynaptic and sense glutamate diffusing from the synaptic cleft. We investigated this by studying the effect of three manipulations that change glutamate clearance on evoked KA EPSCs at thalamocortical synapses. First, we used high-frequency stimulation to increase extrasynaptic glutamate levels. This caused an apparent increase in the relative contribution of the KA EPSC to transmission and slowed the decay kinetics. However, scaling and summing the EPSC evoked at low frequency reproduced this, demonstrating that the effect was due to postsynaptic summation of KA EPSCs. Second, we applied inhibitors of high-affinity glutamate transport. This caused a depression in both AMPA and KA EPSC amplitude due to the activation of a presynaptic glutamatergic autoreceptor. However, transport inhibitors had no selective effect on the amplitude or kinetics of the KA EPSC. Third, to increase glutamate clearance, we raised temperature during recordings. This shortened the decay of both the AMPA and KA components and increased their amplitudes, but this effect was the same for both. Therefore these data provide evidence against glutamate diffusion out of the synaptic cleft as the mechanism for the slow kinetics of KA EPSCs. Other possibilities such as interactions of KA receptors with other proteins or novel properties of native synaptic heteromeric receptors are required to explain the slow kinetics.     

Synaptic physiology in the cochlear nucleus angularis of the chick

Nucleus angularis (NA), one of the two cochlear nuclei in birds, is important for processing sound intensity for localization and most likely has role in sound recognition and other auditory tasks. Because the synaptic properties of auditory nerve inputs to the cochlear nuclei are fundamental to the transformation of auditory information, we studied the properties of these synapses onto NA neurons using whole cell patch-clamp recordings from auditory brain stem slices from embryonic chickens (E16-E20). We measured spontaneous excitatory postsynaptic currents (EPSCs), and evoked EPSCs and excitatory postsynaptic potentials (EPSPs) by using extracellular stimulation of the auditory nerve. These excitatory EPSCs were mediated by AMPA and N-methyl-D-aspartate (NMDA) receptors. The spontaneous EPSCs mediated by AMPA receptors had submillisecond decay kinetics (556 micros at E19), comparable with those of other auditory brain stem areas. The spontaneous EPSCs increased in amplitude and became faster with developmental age. Evoked EPSC and EPSP amplitudes were graded with stimulus intensity. The average amplitude of the EPSC evoked by minimal stimulation was twice as large as the average spontaneous EPSC amplitude (approximately 110 vs. approximately 55 pA), suggesting that single fibers make multiple contacts onto each postsynaptic NA neuron. Because of their small size, minimal EPSPs were subthreshold, and we estimate at least three to five inputs were required to reach threshold. In contrast to the fast EPSCs, EPSPs in NA had a decay time constant of approximately 12.5 ms, which was heavily influenced by the membrane time constant. Thus NA neurons spatially and temporally integrate auditory information arriving from multiple auditory nerve afferents.     

Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor-1

Short-term synaptic plasticity is a defining feature of neuronal activity, but the underlying molecular mechanisms are poorly understood. Depression of synaptic activity might be due to limited vesicle availability, whereas facilitation is thought to result from elevated calcium levels. However, it is unclear whether the strength and direction (facilitation versus depression) of plasticity at a given synapse result from preexisting synaptic strength or whether they are regulated by separate mechanisms. Here we show, in rat hippocampal cell cultures, that increases in the calcium binding protein neuronal calcium sensor-1 (NCS-1) can switch paired-pulse depression to facilitation without altering basal synaptic transmission or initial neurotransmitter release probability. Facilitation persisted during high-frequency trains of stimulation, indicating that NCS-1 can recruit 'dormant' vesicles. Our results suggest that NCS-1 acts as a calcium sensor for short-term plasticity by facilitating neurotransmitter output independent of initial release. We conclude that separate mechanisms are responsible for determining basal synaptic strength and short-term plasticity.     

Frequency-dependent shift from paired-pulse facilitation to paired-pulse depression at unitary CA3-CA3 synapses in the rat hippocampus

Paired recordings between CA3 interconnected pyramidal neurons were used to study the properties of short-term depression occurring in these synapses under different frequencies of presynaptic firing (   n = 22  ). In stationary conditions (0.05-0.067 Hz) pairs of presynaptic action potentials (50 ms apart) evoked EPSCs whose amplitude fluctuated from trial to trial with occasional response failures. In 15/20 cells, paired-pulse ratio (PPR) was characterized by facilitation (PPF) while in the remaining five by depression (PPD). Increasing stimulation frequency from 0.05-0.067 Hz to 0.1-1 Hz induced low frequency depression (LFD) of EPSC amplitude with a gradual increase in the failure rate. Overall, 9/12 cells at 1 Hz became almost 'silent'. In six cells in which the firing rate was sequentially shifted from 0.05 to 0.1 and 1 Hz, changes in synaptic efficacy were so strong that PPR shifted from PPF to PPD. The time course of depression of EPSC1 could be fitted with single exponentials with time constants of 98 and 36 s at 0.1 and 1 Hz, respectively. In line with the inversion of PPR at 1 Hz, the time course of depression of EPSC2 was faster than EPSC1 (7 s). Recovery from depression could be obtained by lowering the frequency of stimulation to 0.025 Hz. These results could be explained by a model that takes into account two distinct release processes, one dependent on the residual calcium and the other on the size of the readily releasable pool of vesicles.     

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.     

Dopamine modulates synaptic transmission in the nucleus of the solitary tract

10.1152/jn.00224.2002. Dopamine (DA) modulates the cardiorespiratory reflex by peripheral and central mechanisms. The aim of this study was to examine the role of DA in synaptic transmission of the nucleus tractus solitarius (NTS), the major integration site for cardiopulmonary reflexes. To examine DA's role, we used whole cell, voltage-clamp recordings in a rat horizontal brain stem slice. Solitary tract stimulation evoked excitatory postsynaptic currents (EPSCs) that were reduced to 70 +/- 5% of control by DA (100 microM). The reduction in EPSCs by DA was accompanied by a decrease in the paired pulse depression ratio with little or no change in input resistance or EPSC decay, suggesting a presynaptic mechanism. The D1-like agonist SKF 38393 Br (30 microM) did not alter EPSC amplitude, whereas the D2-like agonist, quinpirole HCl (30 microM), depressed EPSCs to 73 +/- 4% of control. The D2-like receptor antagonist, sulpiride (20 microM), abolished DA modulation of EPSCs. Most importantly, sulpiride alone increased EPSCs to 131 +/- 10% of control, suggesting a tonic D2-like modulation of synaptic transmission in the NTS. Examination of spontaneous EPSCs revealed DA reversibly decreased the frequency of events from 9.4 +/- 2.2 to 6.2 +/- 1.4 Hz. Sulpiride, however, did not alter spontaneous events. Immunohistochemistry of NTS slices demonstrated that D2 receptors colocalized with synaptophysin and substance P, confirming a presynaptic distribution. D2 receptors also localized to cultured petrosal neurons, the soma of presynaptic afferent fibers. In the petrosal neurons, D2 was found in cells that were TH-immunopositive, suggesting they were chemoreceptor afferent fibers. These results demonstrate that DA tonically modulates synaptic activity between afferent sensory fibers and secondary relay neurons in the NTS via a presynaptic D2-like mechanism.     

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.     

A role for short-term synaptic facilitation and depression in the processing of intensity information in the auditory brain stem

The nature of the synaptic connection from the auditory nerve onto the cochlear nucleus neurons has a profound impact on how sound information is transmitted. Short-term synaptic plasticity, by dynamically modulating synaptic strength, filters information contained in the firing patterns. In the sound-localization circuits of the brain stem, the synapses of the timing pathway are characterized by strong short-term depression. We investigated the short-term synaptic plasticity of the inputs to the bird's cochlear nucleus angularis (NA), which encodes intensity information, by using chick embryonic brain slices and trains of electrical stimulation. These excitatory inputs expressed a mixture of short-term facilitation and depression, unlike those in the timing nuclei that only depressed. Facilitation and depression at NA synapses were balanced such that postsynaptic response amplitude was often maintained throughout the train at high firing rates (>100 Hz). The steady-state input rate relationship of the balanced synapses linearly conveyed rate information and therefore transmits intensity information encoded as a rate code in the nerve. A quantitative model of synaptic transmission could account for the plasticity by including facilitation of release (with a time constant of approximately 40 ms), and a two-step recovery from depression (with one slow time constant of approximately 8 s, and one fast time constant of approximately 20 ms). A simulation using the model fit to NA synapses and auditory nerve spike trains from recordings in vivo confirmed that these synapses can convey intensity information contained in natural train inputs.     

Contribution of presynaptic Na(+) channel inactivation to paired-pulse synaptic depression in cultured hippocampal neurons

Paired-pulse depression (PPD) of synaptic transmission is important for neuronal information processing. Historically, depletion of the readily releasable pool of synaptic vesicles has been proposed as the major component of PPD. Recent results suggest, however, that other mechanisms may be involved in PPD, including inactivation of presynaptic voltage-dependent sodium channels (NaChs), which may influence coupling of action potentials to transmitter release. In hippocampal cultures, we have examined the potential role and relative contribution of presynaptic NaCh inactivation in excitatory postsynaptic current (EPSC) PPD. Based on current- and voltage-clamp recordings from somas, our data suggest that NaCh inactivation could potentially participate in PPD. Paired stimulation of somatic action potentials (20- to 100-ms interval) results in subtle changes in action potential shape that are mimicked by low concentrations of tetrodotoxin (TTX) and that appear to be generated by a combination of fast and slow recovery from NaCh inactivation. Dilute concentrations of TTX dramatically depress glutamate release. However, we find evidence for only minimal contribution of NaCh inactivation to EPSC PPD under basal conditions. Hyperpolarization of presynaptic elements to speed recovery from inactivation or increasing the driving force on Na(+) ions through active NaChs had minimal effects on PPD while more robustly reversing the effects of pharmacological NaCh blockade. On the other hand, slight depolarization of the presynaptic membrane potential, by elevating extracellular [K(+)](o), significantly increased PPD and frequency-dependent depression of EPSCs during short trains of action potentials. The results suggest that NaCh inactivation is poised to modulate EPSC amplitude with small tonic depolarizations that likely occur with physiological or pathophysiological activity.     

Analysis of 5-HT-induced short-term facilitation at Aplysia sensorimotor synapse during bursts: increased synaptic gain that does not require ERK activation

The 5-HT-induced synaptic plasticity of Aplysia sensorimotor synapses has typically been probed by firing a single presynaptic spike. In this study, 5-HT-induced synaptic plasticity was probed with brief bursts of spikes (10 Hz, 1 s), which are more behaviorally relevant stimuli. Because such bursts provide a greater challenge to the release machinery than single spikes, their use may reveal additional aspects of synaptic modulation, and, in particular, the role of extracellular signal-regulated protein kinase (ERK), which has recently been implicated in several examples of short- and long-term synaptic plasticity. Excitatory postsynaptic currents (EPSCs) were characterized by their amplitudes. In addition, two kinetic measurements, time to peak and decay time constant, were determined for the initial and last EPSCs of each burst. Application of 5-HT produced a uniform increase in gain by facilitating each EPSC elicited during a burst of spikes without affecting the kinetics of the initial or last EPSC. These data suggest that short-term facilitation during a burst is mediated largely by processes such as those that affect the size of the releasable pool or rate of vesicle mobilization rather than by an increase in the duration of the presynaptic action potential. An ERK cascade inhibitor (U0126) had no effect on the 5-HT-mediated facilitation of either the initial EPSC or EPSCs elicited late in the burst.     

Residual bound Ca2+ can account for the effects of Ca2+ buffers on synaptic facilitation

Facilitation is a transient stimulation-induced increase in synaptic response, a ubiquitous form of short-term synaptic plasticity that can regulate synaptic transmission on fast time scales. In their pioneering work, Katz and Miledi and Rahamimoff demonstrated the dependence of facilitation on presynaptic Ca(2+) influx and proposed that facilitation results from the accumulation of residual Ca(2+) bound to vesicle release triggers. However, this bound Ca(2+) hypothesis appears to contradict the evidence that facilitation is reduced by exogenous Ca(2+) buffers. This conclusion led to a widely held view that facilitation must depend solely on the accumulation of Ca(2+) in free form. Here we consider a more realistic implementation of the bound Ca(2+) mechanism, taking into account spatial diffusion of Ca(2+), and show that a model with slow Ca(2+) unbinding steps can retain sensitivity to free residual Ca(2+). We demonstrate that this model agrees with the facilitation accumulation time course and its biphasic decay exhibited by the crayfish inhibitor neuromuscular junction (NMJ) and relies on fewer assumptions than the most recent variants of the free residual Ca(2+) hypothesis. Further, we show that the bound Ca(2+) accumulation is consistent with Kamiya and Zucker's experimental results, which revealed that photolytic liberation of a fast Ca(2+) buffer decreases the synaptic response within milliseconds. We conclude that Ca(2+) binding processes with slow unbinding times (tens to hundreds of milliseconds) constitute a viable mechanism of synaptic facilitation at some synapses and discuss the experimental evidence for such a mechanism.     

Voltage-clamp analysis of posttetanic potentiation of the mossy fiber to CA3 synapse in hippocampus

1. Short-term changes in synaptic efficacy were studied at the mossy fiber (MF) to CA3 (MF-CA3) synapse in the in vitro hippocampus. Monosynaptic excitatory postsynaptic currents (EPSCs) were recorded before and during posttetanic potentiation (PTP) with the use of intracellular recording and single-electrode voltage-clamp (SEVC) techniques. 2. Repetitive stimulation (100 Hz for 1 s) of the MF synaptic inputs to CA3 pyramidal cells resulted in PTP averaging 170 +/- 19% (SE, n = 42) over control and decaying with a time constant (tau p) of 59.7 +/- 5 s(n = 23). Reproducible episodes of PTP could be recorded if low stimulus intensities were used. Also, after MF tetanization, a faster component, termed augmentation, preceded PTP but could not be accurately resolved within the experimental protocol; only estimates of this component are included. 3. Biophysical parameters of the EPSC that were monitored before and during PTP included synaptic conductance (G), synaptic reversal potential (Erev), decay time constant (tau EPSC), and input resistance of the postsynaptic cell. During PTP the EPSC synaptic conductance increased from 9.8 to 32.7 nS (P less than 0.02, n = 6), whereas there was no statistical change in Erev (-6.0 compared with -6.7 mV, n = 6), tau EPSC (4.3 compared with 4.5 ms, n = 9), or postsynaptic input resistance (59 compared with 63 M omega, n = 12). 4. A presynaptic contribution to PTP was studied directly by observing changes in transmitter release during PTP. Presynaptic mechanisms were assessed by determining the ratio of evoked synaptic excitatory postsynaptic potentials (EPSPs) over the total number of stimuli (EPSP-to-stimuli ratio). The ratio of EPSP to stimuli changed from 0.64 to 0.90 (P less than 0.01, n = 7) during PTP. A reduction in the number of synaptic failures can only be explained by a presynaptic mechanism. No assumptions concerning the statistical distribution of transmitter release were necessary because no statistical parameters were determined. 5. Changes in postsynaptic cell properties do not appear to contribute to PTP studied under the present experimental conditions. Direct stimulation of the postsynaptic neuron via the intracellular recording electrode (20-100 Hz/1 s) failed to produce potentiation of the EPSC; in fact, a slight depression was observed at 50 and 100 Hz direct stimulation. Likewise, the postsynaptic input resistance and synaptic Erev did not change during PTP. 6. The specific N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (APV, 20 microM) had no effect on either the magnitude or duration of PTP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synapsin regulates Basal synaptic strength, synaptic depression, and serotonin-induced facilitation of sensorimotor synapses in Aplysia

Synapsin is a synaptic vesicle-associated protein implicated in the regulation of vesicle trafficking and transmitter release, but its role in heterosynaptic plasticity remains elusive. Moreover, contradictory results have obscured the contribution of synapsin to homosynaptic plasticity. We previously reported that the neuromodulator serotonin (5-HT) led to the phosphorylation and redistribution of Aplysia synapsin, suggesting that synapsin may be a good candidate for the regulation of vesicle mobilization underlying the short-term synaptic plasticity induced by 5-HT. This study examined the role of synapsin in homosynaptic and heterosynaptic plasticity. Overexpression of synapsin reduced basal transmission and enhanced homosynaptic depression. Although synapsin did not affect spontaneous recovery from depression, it potentiated 5-HT-induced dedepression. Computational analysis showed that the effects of synapsin on plasticity could be adequately simulated by altering the rate of Ca(2+)-dependent vesicle mobilization, supporting the involvement of synapsin not only in homosynaptic but also in heterosynaptic forms of plasticity by regulating vesicle mobilization.     

Properties of short-term synaptic depression at larval neuromuscular synapses in wild-type and temperature-sensitive paralytic mutants of Drosophila

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.     

LTP regulates burst initiation and frequency at mossy fiber-granule cell synapses of rat cerebellum: experimental observations and theoretical predictions

Long-term potentiation (LTP) is a synaptic change supposed to provide the cellular basis for learning and memory in brain neuronal circuits. Although specific LTP expression mechanisms could be critical to determine the dynamics of repetitive neurotransmission, this important issue remained largely unexplored. In this paper, we have performed whole cell patch-clamp recordings of mossy fiber-granule cell LTP in acute rat cerebellar slices and studied its computational implications with a mathematical model. During LTP, stimulation with short impulse trains at 100 Hz revealed earlier initiation of granule cell spike bursts and a smaller nonsignificant spike frequency increase. In voltage-clamp recordings, short AMPA excitatory postsynaptic current (EPSC) trains showed short-term facilitation and depression and a sustained component probably generated by spillover. During LTP, facilitation disappeared, depression accelerated, and the sustained current increased. The N-methyl-d-aspartate (NMDA) current also increased. In agreement with a presynaptic expression caused by increased release probability, similar changes were observed by raising extracellular [Ca(2+)]. A mathematical model of mossy fiber-granule cell neurotransmission showed that increasing release probability efficiently modulated the first-spike delay. Glutamate spillover, by causing tonic NMDA and AMPA receptor activation, accelerated excitatory postsynaptic potential (EPSP) temporal summation and maintained a sustained spike discharge. The effect of increasing neurotransmitter release could not be replicated by increasing receptor conductance, which, like postsynaptic manipulations enhancing intrinsic excitability, proved very effective in raising granule cell output frequency. Independent regulation of spike burst initiation and frequency during LTP may provide mechanisms for temporal recoding and gain control of afferent signals at the input stage of cerebellar cortex.     

The pool of fast releasing vesicles is augmented by myosin light chain kinase inhibition at the calyx of Held synapse

Synaptic strength is determined by release probability and the size of the readily releasable pool of docked vesicles. Here we describe the effects of blocking myosin light chain kinase (MLCK), a cytoskeletal regulatory protein thought to be involved in myosin-mediated vesicle transport, on synaptic transmission at the mouse calyx of Held synapse. Application of three different MLCK inhibitors increased the amplitude of the early excitatory postsynaptic currents (EPSCs) in a stimulus train, without affecting the late steady-state EPSCs. A presynaptic locus of action for MLCK inhibitors was confirmed by an increase in the frequency of miniature EPSCs that left their average amplitude unchanged. MLCK inhibition did not affect presynaptic Ca²⁺ currents or action potential waveform. Moreover, Ca²⁺ imaging experiments showed that [Ca²⁺]_i transients elicited by 100-Hz stimulus trains were not altered by MLCK inhibition. Studies using high-frequency stimulus trains indicated that MLCK inhibitors increase vesicle pool size, but do not significantly alter release probability. Accordingly, when AMPA-receptor desensitization was minimized, EPSC paired-pulse ratios were unaltered by MLCK inhibition, suggesting that release probability remains unaltered. MLCK inhibition potentiated EPSCs even when presynaptic Ca²⁺ buffering was greatly enhanced by treating slices with EGTA-AM. In addition, MLCK inhibition did not affect the rate of recovery from short-term depression. Finally, developmental studies revealed that EPSC potentiation by MLCK inhibition starts at postnatal day 5 (P5) and remains strong during synaptic maturation up to P18. Overall, our data suggest that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release at a CNS synapse.     

Short-term synaptic plasticity, simulation of nerve terminal dynamics, and the effects of protein kinase C activation in rat hippocampus

Phorbol esters are hypothesised to produce a protein kinase C (PKC)-dependent increase in the probability of transmitter release via two mechanisms: facilitation of vesicle fusion or increases in synaptic vesicle number and replenishment. We used a combination of electrophysiology and computer simulation to distinguish these possibilities. We constructed a stochastic model of the presynaptic contacts between a pair of hippocampal pyramidal cells that used biologically realistic processes and was constrained by electrophysiological data. The model reproduced faithfully several forms of short-term synaptic plasticity, including short-term synaptic depression (STD), and allowed us to manipulate several experimentally inaccessible processes. Simulation of an increase in the size of the readily releasable vesicle pool and the time of vesicle replenishment decreased STD, whereas simulation of a facilitation of vesicle fusion downstream of Ca²⁺ influx enhanced STD. Because activation of protein kinase C with phorbol ester enhanced STD of EPSCs in rat hippocampal slice cultures, we conclude that an increase in the sensitivity of the release process for Ca²⁺ underlies the potentiation of neurotransmitter release by PKC.