Developmental Tightening of Cerebellar Cortical Synaptic Influx-Release Coupling

Tight coupling between Ca²⁺ 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 Ca²⁺ 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_v2.1 Ca²⁺ channel immunoreactivity, assessed by quantitative high-resolution immuno-electron microscopy, was scattered over immature boutons but confined to putative AZs at mature boutons. Presynaptic Ca²⁺ 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 Ca²⁺ channel blocker Cd²⁺ 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.