Influence of spatially segregated IP3-producing pathways on spike generation and transmitter release in Purkinje cell axons.

It has been known for a long time that inositol-trisphosphate (IP3) receptors are present in the axon of certain types of mammalian neurons, but their functional role has remained unexplored. Here we show that localized photolysis of IP3 induces spatially constrained calcium rises in Purkinje cell axons. Confocal immunohistology reveals that the axon initial segment (AIS), as well as terminals onto deep cerebellar cells, express specific subtypes of Gα/q and phospholipase C (PLC) molecules, together with the upstream purinergic receptor P2Y1. By contrast, intermediate parts of the axon express another set of Gα/q and PLC molecules, indicating two spatially segregated signaling cascades linked to IP3 generation. This prompted a search for distinct actions of IP3 in different parts of Purkinje cell axons. In the AIS, we found that local applications of the specific P2Y1R agonist MRS2365 led to calcium elevation, and that IP3 photolysis led to inhibition of action potential firing. In synaptic terminals on deep cerebellar nuclei neurons, we found that photolysis of both IP3 and ATP led to GABA release. We propose that axonal IP3 receptors can inhibit action potential firing and increase neurotransmitter release, and that these effects are likely controlled by purinergic receptors. Altogether our results suggest a rich and diverse functional role of IP3 receptors in axons of mammalian neurons.

Physiological involvement of presynaptic L-type voltage-dependent calcium channels in GABA release of cerebellar molecular layer interneurons.

While high threshold voltage-dependent Ca2+ channels (VDCCs) of the N and P/Q families are crucial for evoked neurotransmitter release in the mammalian CNS, it remains unclear to what extent L-type Ca2+ channels (LTCCs), which have been mainly considered as acting at postsynaptic sites, participate in the control of transmitter release. Here, we investigate the possible role of LTCCs in regulating GABA release by cerebellar molecular layer interneurons (MLIs) from rats. We found that BayK8644 (BayK) markedly increases mIPSC frequency in MLIs and Purkinje cells (PCs), suggesting that LTCCs are expressed presynaptically. Furthermore, we observed (1) a potentiation of evoked IPSCs in the presence of BayK, (2) an inhibition of evoked IPSCs in the presence of the LTCC-specific inhibitor Compound 8 (Cp8), and (3) a strong reduction of mIPSC frequency by Cp8. BayK effects are reduced by dantrolene, suggesting that ryanodine receptors act in synergy with LTCCs. Finally, BayK enhances presynaptic AP-evoked Ca2+ transients and increases the frequency of spontaneous axonal Ca2+ transients observed in TTX. Taken together, our data demonstrate that LTCCs are of primary importance in regulating GABA release by MLIs.

Concerted Interneuron Activity in the Cerebellar Molecular Layer During Rhythmic Oromotor Behaviors.

Molecular layer interneurons (MLIs, stellate and basket cells) of the cerebellar cortex are linked together by chemical and electrical synapses and exert a potent feedforward inhibition on Purkinje cells. The functional role of MLIs during specific motor tasks is uncertain. Here, we used two-photon imaging to study the patterns of activity of neighboring individual MLIs in the Crus II region of awake female mice during two types of oromotor activity, licking and bruxing, using specific expression of the genetically encoded calcium indicator protein GCaMP6s. We found that both stellate and basket cells engaged in synchronized waves of calcium activity during licking and bruxing, with high degrees of correlation among the signals collected in individual MLIs. In contrast, no calcium activity was observed during whisking. MLI activity tended to lag behind the onset of sustained licking episodes, indicating a regulatory action of MLIs during licking. Furthermore, during licking, stellate cell activity was anisotropic: the coordination was constant along the direction of parallel fibers (PFs), but fell off with distance along the orthogonal direction. These results suggest a PF drive for Ca2+ signals during licking. In contrast, during bruxing, MLI activity was neither clearly organized spatially nor temporally correlated with oromotor activity. In conclusion, MLI activity exhibits a high degree of correlation both in licking and in bruxing, but spatiotemporal patterns of activity display significant differences for the two types of behavior.SIGNIFICANCE STATEMENT It is known that, during movement, the activity of molecular layer interneurons (MLIs) of the cerebellar cortex is enhanced. However, MLI-MLI interactions are complex because they depend both from excitatory electrical synapses and from potentially inhibitory chemical synapses. Accordingly, the pattern of MLI activity during movement has been unclear. Here, during two oromotor tasks, licking and bruxism, individual neighboring MLIs displayed highly coordinated activity, showing that the positive influences binding MLIs together are predominant. We further find that spatiotemporal patterns differ between licking and bruxing, suggesting that the precise pattern of MLI activity depends on the nature of the motor task.

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

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. SIGNIFICANCE STATEMENT: It is generally accepted that the output signal of individual central synapses saturates at high release probability, but it remains unclear whether the source of saturation is presynaptic, postsynaptic, or both presynaptic and postsynaptic. To clarify this and other issues concerning the function of synapses, we have developed new recording and analysis methods at single central glutamatergic synapses. We find that individual release events engage a high proportion of postsynaptic receptors (62%), revealing a larger component of postsynaptic saturation than anticipated. Conversely, we also find that the number of released synaptic vesicles is limited at each active zone. Altogether, our results argue for both presynaptic and postsynaptic contributions to signal saturation at single glutamatergic synapses.

Measuring the firing rate of high-resistance neurons with cell-attached recording.

Cell-attached recording is extensively used to study the firing rate of mammalian neurons, but potential limitations of the method have not been investigated in detail. Here we perform cell-attached recording of molecular layer interneurons in cerebellar slices from rats and mice, and we study how experimental conditions influence the measured firing rate. We find that this rate depends on time in cell-attached mode, on pipette potential, and on pipette ionic composition. In the first minute after sealing, action currents are variable in shape and size, presumably reflecting membrane instability. The firing rate remains approximately constant during the first 4 min after sealing and gradually increases afterward. Making the pipette potential more positive leads to an increase in the firing rate, with a steeper dependence on voltage if the pipette solution contains K(+) as the main cation than if it contains Na(+). Ca(2+) imaging experiments show that establishing a cell-attached recording can result in an increased somatic Ca(2+) concentration, reflecting an increased firing rate linked to an increase in the pipette-cell conductance. Pipette effects on cell firing are traced to a combination of passive electrical coupling, opening of voltage- and Ca(2+)-sensitive K(+) channels (BK channels) after action potentials, and random activation of voltage-insensitive, presumably mechanosensitive, cationic channels. We conclude that, unless experimental conditions are optimized, cell-attached recordings in small neurons may report erroneous firing rates.

KCa1.1 channels contribute to optogenetically driven post-stimulation silencing in cerebellar molecular layer interneurons.

Using cell-attached recordings from molecular layer interneurons (MLI) of the cerebellar cortex of adult mice expressing channel rhodopsin 2, we show that wide-field optical activation induces an increase in firing rate during illumination and a firing pause when the illumination ends (post-stimulation silencing; PSS). Significant spike rate changes with respect to basal firing rate were observed for optical activations lasting 200 ms and 1 s as well as for 1 s long trains of 10 ms pulses at 50 Hz. For all conditions, the net effect of optical activation on the integrated spike rate is significantly reduced because of PSS. Three lines of evidence indicate that this PSS is due to intrinsic factors. Firstly, PSS is induced when the optical stimulation is restricted to a single MLI using a 405-nm laser delivering a diffraction-limited spot at the focal plane. Secondly, PSS is not affected by block of GABA-A or GABA-B receptors, ruling out synaptic interactions amongst MLIs. Thirdly, PSS is mimicked in whole-cell recording experiments by step depolarizations under current clamp. Activation of Ca-dependent K channels during the spike trains appears as a likely candidate to underlie PSS. Using immunocytochemistry, we find that one such channel type, KCa1.1, is present in the somato-dendritic and axonal compartments of MLIs. In cell-attached recordings, charybdotoxin and iberiotoxin significantly reduce the optically induced PSS, while TRAM-34 does not affect it, suggesting that KCa1.1 channels, but not KCa3.1 channels, contribute to PSS.

Axonal speeding: shaping synaptic potentials in small neurons by the axonal membrane compartment.

The role of the axonal membrane compartment in synaptic integration is usually neglected. We show here that in interneurons of the cerebellar molecular layer, where dendrites are so short that the somatodendritic domain can be considered isopotential, the axonal membrane contributes a significant part of the cell input capacitance. We examine the impact of axonal membrane on synaptic integration by cutting the axon with two-photon illumination. We find that the axonal compartment acts as a sink for signals generated at fast conductance synapses, thus increasing the initial decay rate of corresponding synaptic potentials over the value predicted from the resistance-capacitance (RC) product of the cell membrane; signals generated at slower synapses are much less affected. This mechanism sharpens the spike firing precision of fast glutamatergic inputs without resorting to multisynaptic pathways.

Somatic calcium level reports integrated spiking activity of cerebellar interneurons in vitro and in vivo.

We examined the relationship between somatic Ca²âº signals and spiking activity of cerebellar molecular layer interneurons (MLIs) in adult mice. Using two-photon microscopy in conjunction with cell-attached recordings in slices, we show that in tonically firing MLIs loaded with high-affinity Ca²âº probes, Ca²âº-dependent fluorescence transients are absent. Spike-triggered averages of fluorescence traces for MLIs spiking at low rates revealed that the fluorescence change associated with an action potential is small (1% of the basal fluorescence). To uncover the relationship between intracellular Ca²âº concentration ([Ca²âº](i)) and firing rates, spikes were transiently silenced with puffs of the GABA(A) receptor agonist muscimol. [Ca²âº](i) relaxed toward basal levels following a single exponential whose amplitude correlated to the preceding spike frequency. The relaxation time constant was slow (2.5 s) and independent of the probe concentration. Data from parvalbumin (PV)-/- animals indicate that PV controls the amplitude and decay time of spike-triggered averages as well as the time course of [Ca²âº](i) relaxations following spike silencing. The [Ca²âº](i) signals were sensitive to the L-type Ca²âº channel blocker nimodipine and insensitive to ryanodine. In anesthetized mice, as in slices, fluorescence traces from most MLIs did not show spontaneous transients. They nonetheless responded to muscimol iontophoresis with relaxations similar to those obtained in vitro, suggesting a state of tonic firing with estimated spiking rates ranging from 2 to 30 Hz. Altogether, the [Ca²âº](i) signal appears to reflect the integral of the spiking activity in MLIs. We propose that the muscimol silencing strategy can be extended to other tonically spiking neurons with similar [Ca²âº](i) homeostasis.

Reiterative infusions of MSCs improve pediatric osteogenesis imperfecta eliciting a pro-osteogenic paracrine response: TERCELOI clinical trial.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of cases are due to mutations in the COL1A1 or COL1A2 genes, which encode type I collagen. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. METHODS: To overcome the short-term effect of MSCs therapy, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs, administered in a 2.5-year period, in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in nonimmunosuppressed OI patients. The host response to MSCs was studied by analyzing the sera from OI patients, collected before, during, and after the cell therapy. RESULTS: We first demonstrated that the sequential administration of MSCs was safe and improved the bone parameters and quality of life of OI patients along the cell treatment plus 2-year follow-up period. Moreover, the study of the mechanism of action indicated that MSCs therapy elicited a pro-osteogenic paracrine response in patients, especially noticeable in the patient affected by severe OI. CONCLUSIONS: Our results demonstrate the feasibility and potential of reiterative MSCs infusion for two pediatric OI and highlight the paracrine response shown by patients as a consequence of MSCs treatment.

Activation of metabotropic glutamate receptors induces periodic burst firing and concomitant cytosolic Ca2+ oscillations in cerebellar interneurons.

Little is known about the generation of slow rhythms in brain neuronal circuits. Nevertheless, a few studies, both from reconstituted systems and from hippocampal slices, indicate that activation of metabotropic glutamate receptors (mGluRs) could generate such rhythms. Here we show in rat cerebellar slices that after either release of glutamate by repetitive stimulation, or direct stimulation of type 1 mGluRs, molecular layer interneurons exhibit repetitive slow Ca(2+) transients. By combining cell-attached patch-clamp recording with Ca(2+) imaging, we show that the regular Ca(2+) transients (mean frequency, 35 mHz induced by 2 microm quisqualate in the presence of ionotropic glutamate receptor blockers) are locked with bursts of action potentials. Nevertheless, the Ca(2+) transients are not blocked by tetrodotoxin, indicating that firing is not necessary to entrain oscillations. The first Ca(2+) transient within a train is different in several ways from subsequent transients. It is broader than the subsequent transients, displays a different phase relationship to associated spike bursts, and exhibits a distinct sensitivity to ionic and pharmacological manipulations. Whereas the first transient appears to involve entry of Ca(2+) ions through transient receptor potential channel-like channels and secondarily activated L-type Ca(2+) channels, subsequent transients rely mostly on an exchange of Ca(2+) ions between the cytosol and D-myo-inositol-1,4,5-triphosphate-sensitive intracellular Ca(2+) stores. The slow, highly regular oscillations observed in the present work are likely to drive pauses in postsynaptic Purkinje cells, and could play a role in coordinating slow oscillations involving the cerebello-olivar circuit loop.

Differentially poised vesicles underlie fast and slow components of release at single synapses.

In several types of central mammalian synapses, sustained presynaptic stimulation leads to a sequence of two components of synaptic vesicle release, reflecting the consecutive contributions of a fast-releasing pool (FRP) and of a slow-releasing pool (SRP). Previous work has shown that following common depletion by a strong stimulation, FRP and SRP recover with different kinetics. However, it has remained unclear whether any manipulation could lead to a selective enhancement of either FRP or SRP. To address this question, we have performed local presynaptic calcium uncaging in single presynaptic varicosities of cerebellar interneurons. These varicosities typically form "simple synapses" onto postsynaptic interneurons, involving several (one to six) docking/release sites within a single active zone. We find that strong uncaging laser pulses elicit two phases of release with time constants of ∼1 ms (FRP release) and ∼20 ms (SRP release). When uncaging was preceded by action potential-evoked vesicular release, the extent of SRP release was specifically enhanced. We interpret this effect as reflecting an increased likelihood of two-step release (docking then release) following the elimination of docked synaptic vesicles by action potential-evoked release. In contrast, a subthreshold laser-evoked calcium elevation in the presynaptic varicosity resulted in an enhancement of the FRP release. We interpret this latter effect as reflecting an increased probability of occupancy of docking sites following subthreshold calcium increase. In conclusion, both fast and slow components of release can be specifically enhanced by certain presynaptic manipulations. Our results have implications for the mechanism of docking site replenishment and the regulation of synaptic responses, in particular following activation of ionotropic presynaptic receptors.

Molecular layer interneurons in the cerebellum encode for valence in associative learning.

The cerebellum plays a crucial role in sensorimotor and associative learning. However, the contribution of molecular layer interneurons (MLIs) to these processes is not well understood. We used two-photon microscopy to study the role of ensembles of cerebellar MLIs in a go-no go task where mice obtain a sugar water reward if they lick a spout in the presence of the rewarded odorant and avoid a timeout when they refrain from licking for the unrewarded odorant. In naive animals the MLI responses did not differ between the odorants. With learning, the rewarded odorant elicited a large increase in MLI calcium responses, and the identity of the odorant could be decoded from the differential response. Importantly, MLIs switched odorant responses when the valence of the stimuli was reversed. Finally, mice took a longer time to refrain from licking in the presence of the unrewarded odorant and had difficulty becoming proficient when MLIs were inhibited by chemogenetic intervention. Our findings support a role for MLIs in learning valence in the cerebellum.

Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo.

Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions.

Targeted Next-Generation Sequencing in Patients with Suggestive X-Linked Intellectual Disability.

X-linked intellectual disability (XLID) is known to contribute up to 10% of intellectual disability (ID) in males and could explain the increased ratio of affected males observed in patients with ID. Over the past decade, next-generation sequencing has clearly stimulated the gene discovery process and has become part of the diagnostic procedure. We have performed targeted next-generation sequencing of 82 XLID genes on 61 non-related male patients with suggestive non-syndromic XLID. These patients were initially referred to the molecular genetics laboratory to exclude Fragile X Syndrome. The cohort includes 47 male patients with suggestive X-linked family history of ID meaning that they had half-brothers or maternal cousins or uncles affected; and 14 male patients with ID and affected brothers whose mothers show skewed X-inactivation. Sequencing data analysis identified 17 candidate variants in 16 patients. Seven families could be re-contacted and variant segregation analysis of the respective eight candidate variants was performed: HUWE1, IQSEC2, MAOA, MED12, PHF8, SLC6A8, SLC9A6, and SYN1. Our results show the utility of targeted next-generation sequencing in unravelling the genetic origin of XLID, especially in retrospective cases. Variant segregation and additional studies like RNA sequencing and biochemical assays also helped in re-evaluating and further classifying the genetic variants found.

Targeted Next-Generation Sequencing in a Large Cohort of Genetically Undiagnosed Patients with Neuromuscular Disorders in Spain.

The term neuromuscular disorder (NMD) includes many genetic and acquired diseases and differential diagnosis can be challenging. Next-generation sequencing (NGS) is especially useful in this setting given the large number of possible candidate genes, the clinical, pathological, and genetic heterogeneity, the absence of an established genotype-phenotype correlation, and the exceptionally large size of some causative genes such as TTN, NEB and RYR1. We evaluated the diagnostic value of a custom targeted next-generation sequencing gene panel to study the mutational spectrum of a subset of NMD patients in Spain. In an NMD cohort of 207 patients with congenital myopathies, distal myopathies, congenital and adult-onset muscular dystrophies, and congenital myasthenic syndromes, we detected causative mutations in 102 patients (49.3%), involving 42 NMD-related genes. The most common causative genes, TTN and RYR1, accounted for almost 30% of cases. Thirty-two of the 207 patients (15.4%) carried variants of uncertain significance or had an unidentified second mutation to explain the genetic cause of the disease. In the remaining 73 patients (35.3%), no candidate variant was identified. In combination with patients' clinical and myopathological data, the custom gene panel designed in our lab proved to be a powerful tool to diagnose patients with myopathies, muscular dystrophies and congenital myasthenic syndromes. Targeted NGS approaches enable a rapid and cost-effective analysis of NMD- related genes, offering reliable results in a short time and relegating invasive techniques to a second tier.

Osmotic tension as a possible link between GABA(A) receptor activation and intracellular calcium elevation.

Intracellular calcium concentration rises have been reported following activation of GABA(A) receptors in neonatal preparations and attributed to activation of voltage-dependent Ca(2+) channels. However, we show that, in cerebellar interneurons, GABA(A) agonists induce a somatodendritic Ca(2+) rise that persists at least until postnatal day 20 and is not mediated by depolarization-induced Ca(2+) entry. A local Ca(2+) elevation can likewise be elicited by repetitive stimulation of presynaptic GABAergic afferent fibers. We find that, following GABA(A) receptor activation, bicarbonate-induced Cl(-) entry leads to cell depolarization, Cl(-) accumulation, and osmotic tension. We propose that this tension induces the intracellular Ca(2+) rise as part of a regulatory volume decrease reaction. This mechanism introduces an unexpected link between activation of GABA(A) receptors and intracellular Ca(2+) elevation, which could contribute to activity-driven synaptic plasticity.

Excitatory effects of GABA in established brain networks.

Although GABA remains the predominant inhibitory neurotransmitter of the brain, there are numerous recent examples of excitatory actions of GABA. These actions can be classified in two broad categories: phasic excitatory effects, as follow single activation of GABAergic afferents, and sustained excitatory effects, as follow prolonged activation of GABA(A) receptors. Evidence reviewed here indicates that, contrary to common belief, these effects are not restricted to embryonic or neonatal preparations.

Presynaptic calcium stores and synaptic transmission.

Following the gradual recognition of the importance of intracellular calcium stores for somatodendritic signaling in the mammalian brain, recent reports have also indicated a significant role of presynaptic calcium stores. Ryanodine-sensitive stores generate local, random calcium signals that shape spontaneous transmitter release. They amplify spike-driven calcium signals in presynaptic terminals, and consequently enhance the efficacy of transmitter release. They appear to be recruited by an association with certain types of calcium-permeant ion channels, and they induce specific forms of synaptic plasticity. Recent research also indicates a role of inositoltrisphosphate-sensitive presynaptic calcium stores in synaptic plasticity.

Developmental changes in parvalbumin regulate presynaptic Ca2+ signaling.

Certain interneurons contain large concentrations of specific Ca2+-binding proteins (CBPs), but consequences on presynaptic Ca2+ signaling are poorly understood. Here we show that expression of the slow CBP parvalbumin (PV) in cerebellar interneurons is cell specific and developmentally regulated, leading to characteristic changes in presynaptic Ca2+ dynamics (Ca(i)). Using whole-cell recording and fluorescence imaging, we studied action potential-evoked Ca(i) transients in axons of GABA-releasing interneurons from mouse cerebellum. At early developmental stages [postnatal days 10-12 (P10-P12)], decay kinetics were significantly faster for basket cells than for stellate cells, whereas at P19-P21 both interneurons displayed fast decay kinetics. Biochemical and immunocytochemical analysis showed parallel changes in the expression levels and cellular distribution of PV. By comparing wild-type and PV(-/-) mice, PV was shown to accelerate the initial decay of action potential-evoked Ca(i) signals in single varicosities and to introduce an additional slow phase that summates during bursts of action potentials. The fast initial Ca(i) decay accounts for a previous report that PV elimination favors synaptic facilitation. The slow decay component is responsible for a pronounced, PV-dependent, delayed transmitter release that we describe here at interneuron-interneuron synapses after presynaptic bursts of action potentials. Numerical simulations account for the effect of PV on Ca(i) kinetics, allow estimates for the axonal PV concentration (approximately 150 microm), and predict the time course of volume-averaged Ca(i) in the absence of exogenous buffer. Overall, PV arises as a major contributor to presynaptic Ca(i) signals and synaptic integration in the cerebellar cortex.

Actin- and Myosin-Dependent Vesicle Loading of Presynaptic Docking Sites Prior to Exocytosis.

Variance analysis of postsynaptic current amplitudes suggests the presence of distinct docking sites (also called release sites) where vesicles pause before exocytosis. Docked vesicles participate in the readily releasable pool (RRP), but the relation between docking site number and RRP size remains unclear. It is also unclear whether all vesicles of the RRP are equally release competent, and what cellular mechanisms underlie RRP renewal. We address here these questions at single glutamatergic synapses, counting released vesicles using deconvolution. We find a remarkably low variance of cumulative vesicle counts during action potential trains. This, combined with Monte Carlo simulations, indicates that vesicles transit through two successive states before exocytosis, so that the RRP is up to 2-fold higher than the docking site number. The transition to the second state has a very rapid rate constant, and is specifically inhibited by latrunculin B and blebbistatin, suggesting the involvement of actin and myosin.