Developmental Tightening of Cerebellar Cortical Synaptic Influx-Release Coupling

Abstract

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.

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…This tighter VGCC arrangement presumably allows for more efficient coupling between Ca 2+ entry and SV release at 4 wk compared with 2 wk and is consistent with the finding that the blocking efficiency of EGTA on transmitter release decreases with age at the calyx of Held (34, 52, 53; review: ref. 16), at PF-PC synapses (32), and at amacrine cell synapses (54). Likewise, recent morphological data show an age-dependent tightening of the VGCC-SV arrangement in AZs of the fly neuromuscular junction (23).…”

Developmental synaptic changes at the transient olivocochlear-inner hair cell synapse

“…The fact that, as of P4 -P7, Ca 2ϩ influx through L-type VGGCs, apart from activating BK channels, can also reach the release sites, whereas at P9 -P11, it cannot strongly suggests that at the early stages there is less compartmentalization of the presynaptic proteins that compose the action potential evoked-release machinery. Even though other experimental approaches should be used to give further support to this hypothesis at the MOC-IHC synapse, spatial tightening during development between the proteins involved in the release process has been described both at the calyx of Held synapse (Fedchyshyn and Wang, 2005;Leão and von Gersdorff, 2009) and at cerebellar cortical synapses (Baur et al, 2015).…”

Distinct nanoscale calcium channel and synaptic vesicle topographies contribute to the diversity of synaptic function

“…In cortical glutamatergic synapses, the amplitude of presynaptic Ca 2+ transients is positively correlated with release probability (Holderith et al, 2012; Koester and Johnston, 2005). We therefore measured the amplitude of single AP-evoked Ca 2+ transients (sAPCaTs) in boutons of weak GC to Purkinje cell (PC) synapses (P v ∼ 0.2; Baur et al, 2015; Valera et al, 2012) and in boutons of strong SC-SC synapses (P v 0.3-0.8; Arai and Jonas, 2014; Pulido et al, 2015). GC and SC were whole-cell patch-clamped with a pipette containing Alexa-594 (10 μM) and the Ca 2+ indicator Fluo-5F (100 μM) in the internal solution (Figure 1A, C).…”

“…3). Grey zone in F indicates mean±SEM of published P v (Baur et al, 2015). Light blue boxes indicate simulated coupling distances compatible with experiments.…”

“…To reduce computational time simulations were performed using the 2ms window which is sufficient to account for 80% of total release events for the slower EZM (50 nm), tested against a 4 ms simulation (Figure S5). Deconvolution of GC-SC EPSCs with quantal responses (Abrahamsson et al, 2012) revealed that the peak of evoked EPSC, which was used to estimate release probability (Baur et al, 2015) at GC synapses, occurs at a similar percentage (82%) of the release integral. The simulator was run on the TARS cluster of Institut Pasteur, Paris, France.…”