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

Tight coupling between Ca 2ϩ channels and the sensor for vesicular transmitter release at the presynaptic active zone (AZ) is crucial for high-fidelity synaptic transmission. It has been hypothesized that a switch from a loosely coupled to a tightly coupled transmission mode is a common step in the maturation of CNS synapses. However, this hypothesis has never been tested at cortical synapses. We addressed this hypothesis at a representative small cortical synapse: the synapse connecting mouse cerebellar cortical parallel fibers to Purkinje neurons. We found that the slow Ca 2ϩ chelator EGTA affected release significantly stronger at immature than at mature synapses, while the fast chelator BAPTA was similarly effective in both groups. Analysis of paired-pulse ratios and quantification of release probability (p r ) with multiple-probability fluctuation analysis revealed increased facilitation at immature synapses accompanied by reduced p r . Ca v 2.1 Ca 2ϩ channel immunoreactivity, assessed by quantitative high-resolution immuno-electron microscopy, was scattered over immature boutons but confined to putative AZs at mature boutons. Presynaptic Ca 2ϩ signals were quantified with two-photon microscopy and found to be similar between maturation stages. Models adjusted to fit EGTA dose-response curves as well as differential effects of the Ca 2ϩ channel blocker Cd 2ϩ indicate looser and less homogenous coupling at immature terminals compared with mature ones. These results demonstrate functionally relevant developmental tightening of influx-release coupling at a single AZ cortical synapse and corroborate developmental tightening of coupling as a prevalent phenomenon in the mammalian brain.

Highlights d Influx-release coupling tightens during development of L5 pyramidal neuron synapses d The vesicular release probability remains high during development d Ca 2+ influx dynamics do not change during development d Ca v 2.1, Ca v 2.2 microdomains develop toward Ca v 2.1

Spatial relationships between Ca channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Ca2.1 and Ca2.2 channels, alters the localization of Ca2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca-channel pharmacology in electrophysiological and two-photon Ca imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca influx through Ca2.1 and Ca2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Ca2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Ca2.1 and Ca2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains.