Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such "simple synapses" indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Ca v 2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Ca v 2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1-10) and 2.03 at 4 wk (range: 1-4), whereas the mean numbers of Ca v 2.1 clusters were 2.84 at 2 wk (range: 1-8) and 2.37 at 4 wk (range: 1-5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm 2 to 0.0234 μm 2 ), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels.neurotransmitter release | active zone | calcium channel | release site | parallel fiber I n presynaptic terminals, the active zone (AZ) represents a highly specialized structure allowing the binding and Ca 2+ -dependent release of synaptic vesicles (SVs) (1). Fluctuation analysis of synaptic signals suggests the presence of one or several functional units per AZ (2, 3). These units are either called "release sites" or "docking sites," and their number per AZ represents the maximum SV output that this structure can provide per action potential (AP). In recent years, optical methods have been developed to record single-SV release (4). Notably, studies using superresolution and total internal reflection fluorescence imaging in functioning central synapses have provided information on the kinetics (5) and subsynaptic localization (6) of single-SV docking and release. 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 (9-11), providing a direct evaluation of the number of docking sites per AZ. This number ran...

Glutamate-induced excitotoxicity, the most common pathological mechanism leading to neuronal death, may occur even with normal levels of glutamate if it coincides with a persistent enhancement of neuronal excitability. Neurons expressing nitric oxide (NO) synthase (NOS-I), which is upregulated in many human chronic neurodegenerative diseases, are highly susceptible to neurodegeneration. We hypothesized that chronic production of NO in damaged neurons may increase their intrinsic excitability via modulation of resting or "leak" K ϩ currents. Peripheral XIIth nerve injury in adult rats induced de novo NOS-I expression and an increased incidence of lowthreshold motor units, the latter being prevented by chronic inhibition of the neuronal NO/cGMP pathway. Accordingly, sustained synthesis of NO maintained an enhanced basal activity in injured motoneurons that was slowly reverted (over the course of 2-3 h) by NOS-I inhibitors. In slice preparations, persistent, but not acute, activation of the NO/cGMP pathway evoked a robust augment in motoneuron excitability independent of synaptic activity. Furthermore, chronic activation of the NO/cGMP pathway fully suppressed TWIK-related acid-sensitive K ϩ (TASK) currents through a protein kinase G (PKG)-dependent mechanism. Finally, we found evidence for the involvement of this long-term mechanism in regulating membrane excitability of motoneurons, because their pH-sensitive currents were drastically reduced by nerve injury. This NO/cGMP/PKG-mediated modulation of TASK conductances might represent a new pathological mechanism that leads to hyperexcitability and sensitizes neurons to excitotoxic damage. It could explain why de novo expression of NOS-I and/or its overexpression makes them susceptible to neurodegeneration under pathological conditions.

Central mammalian synapses release synaptic vesicles in dedicated structures called docking/release sites. It has been assumed that when voltage-dependent calcium entry is sufficiently large, synaptic output attains a maximum value of one synaptic vesicle per action potential and per site. Here we use deconvolution to count synaptic vesicle output at single sites (mean site number per synapse: 3.6). When increasing calcium entry with tetraethylammonium in 1.5 mM external calcium concentration, we find that synaptic output saturates at 0.22 vesicle per site, not at 1 vesicle per site. Fitting the results with current models of calcium-dependent exocytosis indicates that the 0.22 vesicle limit reflects the probability of docking sites to be occupied by synaptic vesicles at rest, as only docked vesicles can be released. With 3 mM external calcium, the maximum output per site increases to 0.47, indicating an increase in docking site occupancy as a function of external calcium concentration.