The coupling distance between presynaptic Ca(2+) influx and the sensor for vesicular transmitter release determines speed and reliability of synaptic transmission. Nanodomain coupling (<100 nm) favors fidelity and is employed by synapses specialized for escape reflexes and by inhibitory synapses involved in synchronizing fast network oscillations. Cortical glutamatergic synapses seem to forgo the benefits of tight coupling, yet quantitative detail is lacking. The reduced transmission fidelity of loose coupling, however, raises the question whether it is indeed a general characteristic of cortical synapses. Here we analyzed excitatory parallel fiber to Purkinje cell synapses, major processing sites for sensory information and well suited for analysis because they typically harbor only a single active zone. We quantified the coupling distance by combining multiprobability fluctuation analyses, presynaptic Ca(2+) imaging, and reaction-diffusion simulations in wild-type and calretinin-deficient mice. We found a coupling distance of <30 nm at these synapses, much shorter than at any other glutamatergic cortical synapse investigated to date. Our results suggest that nanodomain coupling is a general characteristic of conventional cortical synapses involved in high-frequency transmission, allowing for dense gray matter packing and cost-effective neurotransmission.

Key points• Endogenous Ca 2+ binding proteins such as calbinding-D28k (CB) and parvalbumin (PV) are considered important regulators of short-term synaptic plasticity.• Cerebellar Purkinje neurons express large amounts of CB and PV and are laterally connected by inhibitory synapses that show paired-pulse facilitation (PPF) during high-frequency activation.• We report quantal synaptic release parameters of these synapses in wild-type and in CB and PV knock-out mice; evidence is provided that these synapses operate at nanodomain influx-release coupling.• We find that PPF is independent of CB and PV, using a combination of paired electrophysiological recordings, synaptic Ca 2+ imaging and numerical computer simulations.• Our results suggest that PPF during high-frequency activation results from slow Ca 2+ unbinding from the sensor for transmitter release, which is reminiscent of the 'active Ca 2+ ' mechanism of PPF suggested by Katz and Miledi in 1968.Abstract Paired-pulse facilitation (PPF) is a dynamic enhancement of transmitter release considered crucial in CNS information processing. The mechanisms of PPF remain controversial and may differ between synapses. Endogenous Ca 2+ buffers such as parvalbumin (PV) and calbindin-D28k (CB) are regarded as important modulators of PPF, with PV acting as an anti-facilitating buffer while saturation of CB can promote PPF. We analysed transmitter release and PPF at intracortical, recurrent Purkinje neuron (PN) to PN synapses, which show PPF during high-frequency activation (200 Hz) and strongly express both PV and CB. We quantified presynaptic Ca 2+ dynamics and quantal release parameters in wild-type (WT), and CB and PV deficient mice. Lack of CB resulted in increased volume averaged presynaptic Ca 2+ amplitudes and in increased release probability, while loss of PV had no significant effect on these parameters. Unexpectedly, none of the buffers significantly influenced PPF, indicating that neither CB saturation nor residual free Ca 2+ ([Ca 2+ ] res ) was the main determinant of PPF. Experimentally constrained, numerical simulations of Ca 2+ -dependent release were used to estimate the contributions of [Ca 2+ ] res , CB, PV, calmodulin (CaM), immobile buffer fractions and Ca 2+ remaining bound to the release sensor after the first of two action potentials ('active

Endogenous Ca2+-binding proteins affect synaptic transmitter release and short-term plasticity (STP) by buffering presynaptic Ca2+ signals. At parallel-fiber (PF)-to-Purkinje neuron (PN) synapses in the cerebellar cortex loss of calretinin (CR), the major buffer at PF terminals, results in increased presynaptic Ca2+ transients and an almost doubling of the initial vesicular releases probability (pr). Surprisingly, however, it has been reported that loss of CR from PF synapses does not alter paired-pulse facilitation (PPF), while it affects presynaptic Ca2+ signals as well as pr. Here, we addressed this puzzling observation by analyzing the frequency- and Ca2+-dependence of PPF at unitary PF-to-PN synapses of wild-type (WT) and CR-deficient (CR−/−) mice using paired recordings and computer simulations. Our analysis revealed that PPF in CR−/− is indeed smaller than in the WT, to a degree, however, that indicates that rapid vesicle replenishment and recruitment of additional release sites dominate the synaptic efficacy of the second response. These Ca2+-driven processes operate more effectively in the absence of CR, thereby, explaining the preservation of robust PPF in the mutants.

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The efficacy of neocortical synapses to transmit during bursts of action potentials (APs) increases during development but the underlying mechanisms are largely unclear. We investigated synaptic efficacy at synapses between layer 5 pyramidal neurons (L5PNs) during development, using paired recordings, presynaptic two-photon Ca 2+ imaging, and numerical simulations. Our data confirm a developmental increase in paired-pulse ratios (PPRs). Independent of age, Ca 2+ imaging revealed no AP invasion failures and linear summation of presynaptic Ca 2+ transients, making differences in Ca 2+ signaling an unlikely reason for developmental changes in PPR. Cumulative excitatory postsynaptic current (EPSC) amplitudes indicate that neither the size of the readily-releasable pool (RRP) nor replenishment rates were different between age groups, while the time-courses of depression differed significantly. At young synapses, EPSCs depressed rapidly to near steady-state during the first four APs, and synaptic failures (F syn) increased from 0 to 30%. At mature synapses this drop was significantly slower and strongly biphasic, such that near steady-state depression was reached not before 18 APs with F syn remaining between 0 and 5%. While young synapses reliably transmitted during pairs of APs, albeit with strong depression, mature synapses maintained near 100% transfer efficacy with significantly less depression during high-frequency bursts of APs. Our analysis indicates that at mature synapses a replenishment pool (RepP) is responsible for their high efficacy during bursting activity, while this RepP is functionally immature at young synapses. Hence, our data provide evidence that the functional maturation of a RepP underlies increasing synaptic efficacy during the development of an excitatory cortical synapse.

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)‐to‐Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning‐relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF‐to‐PC synapses. Microglia enrich in the PC layer at pruning‐relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX3CR1) signaling during postnatal development using genetic ablation of the CX3CR1 receptor and an in‐depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX3CR1 signaling is not mandatory for correct cerebellar circuit formation. Main Points Ablation of CX3CR1 results in a transient migration defect in cerebellar microglia. CX3CR1 is not required for functional pruning of cerebellar climbing fibers. Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX3CR1‐deficient mice.

The composition of voltage-gated Ca2+channel (Cav) subtypes that gate action potential (AP)-evoked release changes during development of mammalian CNS synapses. Cav2.2 and Cav2.3 lose their function in gating evoked release during postnatal synapse maturation. In mature boutons, Cav2.1 currents provide the almost exclusive trigger for evoked release and Cav2.3 currents are required for the induction of presynaptic long term potentiation. However, the functional significance of Cav2.2 remained elusive in mature boutons, although they remain present at active zones and continue contributing significantly to presynaptic Ca2+influx. Here, we addressed the functional significance of Cav2.2 and Cav2.3 at mature parallel-fiber (PF) to Purkinje-neuron synapses of mice of either sex. These synapses are known to exhibit the corresponding developmental Cavsubtype changes in gating release. We addressed two hypotheses, namely that Cav2.2 and Cav2.3 are involved in triggering spontaneous glutamate release and that they are engaged in vesicle recruitment during repetitive evoked release. We found that spontaneous miniature release is Ca2+-dependent. However, experiments with Cavsubtype-specific blockers excluded spontaneous opening of Cavs as Ca2+source for spontaneous glutamate release. Thus, neither Cav2.2 nor Cav2.3 control spontaneous release from PF boutons. Furthermore, vesicle recruitment during brief bursts of APs was also independent of Ca2+influx through Cav2.2 and Cav2.3. However, Cav2.2 but not Cav2.3 currents significantly boosted vesicle recruitment during sustained high-frequency synaptic transmission. Thus, in mature PF boutons Cav2.2 channels are specifically required to sustain synaptic transmission during prolonged neuronal activity.Significance statement:At young CNS synapses action potential evoked release is gated via three subtypes of voltage gated Ca2+channels, Cav2.1, Cav2.2 and Cav2.3. During postnatal maturation, Cav2.2 and Cav2.3 lose their function in gating evoked release, such that at mature synapses Cav2.1 provide the almost exclusive source for triggering evoked release. Cav2.3 currents are required for the induction of presynaptic long term potentiation. However, the function of the still abundant Cav2.2 in mature boutons remained largely elusive. Here, we studied mature cerebellar parallel-fiber synapses and found that Cav2.2 do not control spontaneous release. However, Ca2+influx through Cav2.2 significantly boosted vesicle recruitment during trains of action potentials. Thus, Cav2.2 in mature parallel-fiber boutons participate in sustaining synaptic transmission during prolonged activity.

The postnatal development of cerebellar climbing fiber (CF) to Purkinje neuron (PN) synapses is characterized by a substantial pruning during the first 3 weeks after birth, switching from multiple- to single-CF innervation. Previous studies suggested that CF maturation is governed by bidirectional changes of synaptic plasticity. The strengthening of surviving “winner” CFs, which translocate from the PN soma to the dendrite, is thought to be guided by long-term potentiation (LTP), while weakening of to-be-eliminated “loser” CFs, which remain on the soma, was proposed to be due to long-term depression (LTD). However, there are conflicting results from previous studies, whether or not strengthening of winner and weakening of loser CFs during postnatal development is accompanied by changes in short-term plasticity and, thus, whether pre- or postsynaptic forms of LTD and LTP are operational. We, therefore, analyzed the developmental profile of paired-pulse depression (PPD) in “weak” and “strong” CFs in 3–21-day old Igsf9 -eGFP mice, which allow visual identification of GFP-labeled CFs. We found that in 3–8-day old mice strong CFs are marked by a stronger PPD compared to weak CFs. Surprisingly, PPD of strong CFs eases during maturation, while PPD in weak CFs remains unchanged. This easing of PPD is neither due to changes in presynaptic influx-release coupling nor to an increased saturation of postsynaptic receptors. Thus, our results imply that synaptic contacts of CFs show distinct features of PPD depending on their affiliation to winner or loser CFs and depending on their somatic or dendritic location.