Significance: The inferior olive, one of the major source of inputs to the cerebellum, sends climbing fibers to Purkinje cells, the key processing units of cerebellar-dependent motor control. Using an optogenetic strategy, we demonstrate that Purkinje cells disinhibit their climbing-fiber afferents via a poly-synaptic circuit. These findings identify a functional closed-loop organization in the olivo-cerebellar circuits that is potentially important for cerebellar motor learning.

Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structure-function analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.

Neurotransmitter release is a highly efficient secretory process exhibiting resistance to fatigue and plasticity attributable to the existence of distinct pools of synaptic vesicles (SVs), namely a readily releasable pool and a reserve pool from which vesicles can be recruited after activity. Synaptic vesicles in the reserve pool are thought to be reversibly tethered to the actin-based cytoskeleton by the synapsins, a family of synaptic vesicle-associated phosphoproteins that have been shown to play a role in the formation, maintenance, and regulation of the reserve pool of synaptic vesicles and to operate during the post-docking step of the release process. In this paper, we have investigated the physiological effects of manipulating synapsin levels in identified cholinergic synapses ofAplysia californica. When endogenous synapsin was neutralized by the injection of specific anti-synapsin antibodies, the amount of neurotransmitter released per impulse was unaffected, but marked changes in the secretory response to high-frequency stimulation were observed, including the disappearance of post-tetanic potentiation (PTP) that was substituted by post-tetanic depression (PTD), and increased rate and extent of synaptic depression. Opposite changes on post-tetanic potentiation were observed when synapsin levels were increased by injecting exogenous synapsin I. Our data demonstrate that the presence of synapsin-dependent reserve vesicles allows the nerve terminal to release neurotransmitter at rates exceeding the synaptic vesicle recycling capacity and to dynamically change the efficiency of release in response to conditioning stimuli (e.g., post-tetanic potentiation). Moreover, synapsin-dependent regulation of the fusion competence of synaptic vesicles appears to be crucial for sustaining neurotransmitter release during short periods at rates faster than the replenishment kinetics and maintaining synchronization of quanta in evoked release.

The mossy fiber (MF)–granule cell (GC) pathway conveys multiple modalities of information to the cerebellar cortex, converging on Purkinje cells (PC), the sole output of the cerebellar cortex. Recentin vivoexperiments have shown that activity in GCs varies from tonic firing at a few hertz to phasic bursts >500 Hz. However, the responses of parallel fiber (PF)–PC synapses to this wide range of input frequencies are unknown, and there is controversy regarding several frequency-related parameters of transmission at this synapse. We performed recordings of unitary synapses and combined variance–mean analysis with a carefully adapted extracellular stimulation method in young and adult rats. We show that, although the probability of release at individual sites is low at physiological calcium concentration, PF–PC synapses release one or more vesicles with a probability of 0.44 at 1.5 mm[Ca₂₊]_e. Paired-pulse facilitation was observed over a wide range of frequencies; it renders burst inputs particularly effective and reproducible. These properties are primarily independent of synaptic weight and age. Furthermore, we show that the PF–PC synapse is able to sustain transmission at very high frequencies for tens of stimuli, as a result of accelerated vesicle replenishment and an apparent recruitment of release site vesicles, which appears to be a central mechanism of paired-pulse facilitation at this synapse. These properties ensure that PF–PC synapses possess a dynamic range enabling the temporal code of MF inputs to be transmitted reliably to the PC.