Counting Vesicular Release Events Reveals Binomial Release Statistics at Single Glutamatergic Synapses

Abstract

Many central glutamatergic synapses contain a single presynaptic active zone and a single postsynaptic density. However, the basic functional properties of such "simple synapses" remain unclear. One important step toward understanding simple synapse function is to analyze the number of synaptic vesicles released in such structures per action potential, but this goal has remained elusive until now. Here, we describe procedures that allow reliable vesicular release counting at simple synapses between parallel fibers and molecular layer interneurons of rat cerebellar slices. Our analysis involves local extracellular stimulation of single parallel fibers and deconvolution of resulting EPSCs using quantal signals as template. We observed a reduction of quantal amplitudes (amplitude occlusion) in pairs of consecutive EPSCs due to receptor saturation. This effect is larger (62%) than previously reported and primarily reflects receptor activation rather than desensitization. In addition to activation-driven amplitude occlusion, each EPSC reduces amplitudes of subsequent events by an estimated 3% due to cumulative desensitization. Vesicular release counts at simple synapses follow binomial statistics with a maximum that varies from 2 to 10 among experiments. This maximum presumably reflects the number of docking sites at a given synapse. These results show striking similarities, as well as significant quantitative differences, with respect to previous results at simple GABAergic synapses.

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…1A) (35). As in previous studies (9,29), we obtained uniform event amplitudes in response to train stimulation, indicating that a single synaptic connection was stimulated. Using a mean quantal excitatory postsynaptic current (EPSC) trace obtained from delayed asynchronous responses as a template, we deconvolved multiquantal EPSCs to estimate the number of SVs released by each stimulus ( Fig.…”

“…1 B and C). We fixed the extracellular Ca 2+ concentration to 3 mM to optimize SV counting (9). Plots of mean released SV number as a function of stimulus number revealed a facilitation-depression sequence at both 2 wk and 4 wk (Fig.…”

“…Despite these advances, electrophysiological methods remain the most rapid and sensitive method to assess SV exocytosis at central synapses such as the calyx of Held (7) or cerebellar mossy fiber terminals (8). In recent years, electrophysiological recordings performed at special synapses containing a single AZ and a single postsynaptic density (PSD), called "simple synapses," revealed a fixed maximum in the number of SVs that can be released per AZ by a short calcium stimulus (9)(10)(11), providing a direct evaluation of the number of docking sites per AZ. This number ranges between 1 and 6 at synapses between molecular layer interneurons (MLIs) of the cerebellum and between 3 and 6 at synapses between parallel fibers (PFs) and MLIs (9)(10)(11).…”

“…For this purpose, we compare simple PF-MLI synapses at postnatal day 13 (P13)-P16 and P26-P29 (respectively called "2-wk data" and "4-wk data" hereafter). At 4 wk, however, the growing density of PF-MLI innervation jeopardized the method of stimulation of individual PFs used previously to obtain simple synapse recordings (9). Therefore, instead of using extracellular stimulation in the molecular layer, we used in the present work either local extracellular stimulation in the granule cell (GC) layer or paired GC-MLI recordings (Fig.…”

“…S1) (9), and we derived from these fits functional estimates of the number of docking sites, N D . To improve the accuracy of N D estimates, N D was determined for the stimulus number corresponding to maximal release probability at the peak of synaptic facilitation (9). Plotting N D as a function of age revealed a significant decrease (Fig.…”