Fast vesicle reloading and a large pool sustain high bandwidth transmission at a central synapse.

What limits the rate at which sensory information can be transmitted across synaptic connections in the brain? High-frequency signalling is restricted to brief bursts at many central excitatory synapses, whereas graded ribbon-type synapses can sustain release and transmit information at high rates. Here we investigate transmission at the cerebellar mossy fibre terminal, which can fire at over 200 Hz for sustained periods in vivo, yet makes few synaptic contacts onto individual granule cells. We show that connections between mossy fibres and granule cells can sustain high-frequency signalling at physiological temperature. We use fluctuation analysis and pharmacological block of desensitization to identify the quantal determinants of short-term plasticity and combine these with a short-term plasticity model and cumulative excitatory postsynaptic current analysis to quantify the determinants of sustained high-frequency transmission. We show that release is maintained at each release site by rapid reloading of release-ready vesicles from an unusually large releasable pool of vesicles (approximately 300 per site). Our results establish that sustained vesicular release at high rates is not restricted to graded ribbon-type synapses and that mossy fibres are well suited for transmitting broad-bandwidth rate-coded information to the input layer of the cerebellar cortex.

Estimation of quantal parameters with multiple-probability fluctuation analysis.

The functional properties of central synapses are difficult to study because they can be modulated either presynaptically or postsynaptically, each connection has multiple contacts and release at each contact is stochastic. Moreover, studying central synapses with electrophysiology is complicated by the fact that synapses are often remote from the recording site and signals are often difficult to resolve above the noise. This together with the fact that central synapses often have few release sites and have nonuniform quantal parameters makes classical quantal analysis methods difficult to apply. Here, we discuss an alternative approach, multiple-probability fluctuation analysis (MPFA), which can be used to estimate nonuniform quantal parameters from fits of the relationship between the variance and mean amplitude of postsynaptic responses, recorded at different release probabilities. We illustrate the experimental protocols and the analysis procedure that should be followed to perform MPFA and interpret the estimated parameters.

Rapid vesicular release, quantal variability, and spillover contribute to the precision and reliability of transmission at a glomerular synapse.

The amplitude and shape of EPSC waveforms are thought to be important determinants of information processing and storage in the brain, yet relatively little is known about the origins of EPSC variability or how it affects synaptic signaling. We investigated the stochastic determinants of AMPA receptor-mediated EPSC variability at cerebellar mossy fiber-granule cell (MF-GC) connections by combining multiple-probability fluctuation analysis (MPFA) and deconvolution methods. The properties of MF connections with a single release site and the effects of the rapidly equilibrating competitive antagonist kynurenic acid on EPSCs suggest that receptors are not saturated by glutamate during a quantal event and that quanta sum linearly over a wide range of release probabilities. MPFA revealed an average of five vesicular release sites per MF-GC connection. Our results show that the time course of vesicular release is rapid (decay, tau = 75 micros) and independent of release probability, introducing little jitter in the shape or timing of the quantal component of the EPSC at physiological temperature. Moreover, the peak vesicular release rate per release site after an action potential (AP) (approximately 3 ms(-1)) is substantially higher than previously reported for central synapses. Interaction of amplitude fluctuations arising from quantal release and quantal size with the slower, low variability spillover-mediated current produce substantial variability in EPSC shape. Our simulations of MF-GC transmission suggest that quantal variability and transmitter spillover extend the voltage from which AP threshold can be crossed, improving reliability, and that fast vesicular release allows precise signaling across MF connections with heterogeneous weights.

Errors in the estimation of the variance: implications for multiple-probability fluctuation analysis.

Synapses play a crucial role in information processing in the brain. Amplitude fluctuations of synaptic responses can be used to extract information about the mechanisms underlying synaptic transmission and its modulation. In particular, multiple-probability fluctuation analysis can be used to estimate the number of functional release sites, the mean probability of release and the amplitude of the mean quantal response from fits of the relationship between the variance and mean amplitude of postsynaptic responses, recorded at different probabilities. To determine these quantal parameters, calculate their uncertainties and the goodness-of-fit of the model, it is important to weight the contribution of each data point in the fitting procedure. We therefore investigated the errors associated with measuring the variance by determining the best estimators of the variance of the variance and have used simulations of synaptic transmission to test their accuracy and reliability under different experimental conditions. For central synapses, which generally have a low number of release sites, the amplitude distribution of synaptic responses is not normal, thus the use of a theoretical variance of the variance based on the normal assumption is not a good approximation. However, appropriate estimators can be derived for the population and for limited sample sizes using a more general expression that involves higher moments and introducing unbiased estimators based on the h-statistics. Our results are likely to be relevant for various applications of fluctuation analysis when few channels or release sites are present.

An ID-like current that is downregulated by Ca2+ modulates information coding at CA3-CA3 synapses in the rat hippocampus.

Voltage-gated K+ channels localised on presynaptic nerve terminals control information coding by modulating presynaptic firing and synaptic efficacy in target neurones. We found that at CA3-CA3 connections in hippocampal slice cultures, a fast-activating, slowly inactivating K+ conductance similar to the so-called delay current (ID) is responsible for the delayed appearance of the first spike upon membrane depolarisation, for action potential repolarisation and for modulation of transmitter release. The ID-like current was downregulated by intracellular Ca2+, as indicated by the increased delay in the appearance of the first action potential following either the block of Ca2+ flux through voltage-dependent Ca2+ channels with Cd2+ or replacement of the bathing solution with one devoid of Ca2+. In both cases, this effect was reversed by blocking this conductance with a low concentration of 4-aminopyridine (4-AP, 10-50 muM). Application of 4-AP shortened the delay to the first spike generation, prevented the effect of Cd2+ and increased the spike duration. The earlier appearance of the first action potential was also observed in the presence of dendrotoxin-1 (100 nM). In voltage-clamp experiments larger currents were recorded in the absence of extracellular Ca2+, thus confirming the downregulation of the ID-like current by Ca2+ due to the positive shift of its inactivation. Spike broadening was associated with an enhancement of synaptic efficacy in target neurones, as assessed by the increase in EPSC amplitude and in the percentage of successes. Moreover, in the presence of 4-AP, EPSCs appeared with a longer latency and were more scattered. This conductance is therefore crucial for setting the timing and strength of synaptic transmission at CA3-CA3 connections. It is conceivable that switching off ID by increasing intracellular Ca2+ following activity-dependent processes may facilitate network synchronisation and crosstalk between CA3 pyramidal cells, leading to seizure activity.

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.

BK potassium channels control transmitter release at CA3-CA3 synapses in the rat hippocampus.

Large conductance calcium- and voltage-activated potassium channels (BK channels) activate in response to calcium influx during action potentials and contribute to the spike repolarization and fast afterhyperpolarization. BK channels targeted to active zones in presynaptic nerve terminals have been shown to limit calcium entry and transmitter release by reducing the duration of the presynaptic spike at neurosecretory nerve terminals and at the frog neuromuscular junction. However, their functional role in central synapses is still uncertain. In the hippocampus, BK channels have been proposed to act as an 'emergency brake' that would control transmitter release only under conditions of excessive depolarization and accumulation of intracellular calcium. Here we demonstrate that in the CA3 region of hippocampal slice cultures, under basal experimental conditions, the selective BK channel blockers paxilline (10 microM) and iberiotoxin (100 nM) increase the frequency, but not the amplitude, of spontaneously occurring action potential-dependent EPSCs. These drugs did not affect miniature currents recorded in the presence of tetrodotoxin, suggesting that their action was dependent on action potential firing. Moreover, in double patch-clamp recordings from monosynaptically interconnected CA3 pyramidal neurones, blockade of BK channels enhanced the probability of transmitter release, as revealed by the increase in success rate, EPSC amplitude and the concomitant decrease in paired-pulse ratio in response to pairs of presynaptic action potentials delivered at a frequency of 0.05 Hz. BK channel blockers also enhanced the appearance of delayed responses, particularly following the second action potential in the paired-pulse protocol. These results are consistent with the hypothesis that BK channels are powerful modulators of transmitter release and synaptic efficacy in central neurones.