RIM-BP2 regulates Ca2+ channel abundance and neurotransmitter release at hippocampal mossy fiber terminals.

Synaptic vesicles dock and fuse at the presynaptic active zone (AZ), the specialized site for transmitter release. AZ proteins play multiple roles such as recruitment of Ca2+ channels as well as synaptic vesicle docking, priming, and fusion. However, the precise role of each AZ protein type remains unknown. In order to dissect the role of RIM-BP2 at mammalian cortical synapses having low release probability, we applied direct electrophysiological recording and super-resolution imaging to hippocampal mossy fiber terminals of RIM-BP2 knockout (KO) mice. By using direct presynaptic recording, we found the reduced Ca2+ currents. The measurements of excitatory postsynaptic currents (EPSCs) and presynaptic capacitance suggested that the initial release probability was lowered because of the reduced Ca2+ influx and impaired fusion competence in RIM-BP2 KO. Nevertheless, larger Ca2+ influx restored release partially. Consistent with presynaptic recording, STED microscopy suggested less abundance of P/Q-type Ca2+ channels at AZs deficient in RIM-BP2. Our results suggest that the RIM-BP2 regulates both Ca2+ channel abundance and transmitter release at mossy fiber synapses.

Mechanistic insights into cAMP-mediated presynaptic potentiation at hippocampal mossy fiber synapses.

Presynaptic plasticity is an activity-dependent change in the neurotransmitter release and plays a key role in dynamic modulation of synaptic strength. Particularly, presynaptic potentiation mediated by cyclic adenosine monophosphate (cAMP) is widely seen across the animals and thought to contribute to learning and memory. Hippocampal mossy fiber-CA3 pyramidal cell synapses have been used as a model because of robust presynaptic potentiation in short- and long-term forms. Moreover, direct presynaptic recordings from large mossy fiber terminals allow one to dissect the potentiation mechanisms. Recently, super-resolution microscopy and flash-and-freeze electron microscopy have revealed the localizations of release site molecules and synaptic vesicles during the potentiation at a nanoscale, identifying the molecular mechanisms of the potentiation. Incorporating these growing knowledges, we try to present plausible mechanisms underlying the cAMP-mediated presynaptic potentiation.

Ca-dependence of synaptic vesicle exocytosis and endocytosis at the hippocampal mossy fibre terminal.

KEY POINTS: We used presynaptic capacitance measurements at the hippocampal mossy fibre terminal at room temperature to measure Ca-dependence of exo- and endocytotic kinetics. The readily releasable pool (RRP) of synaptic vesicles was released with a time constant of 30-40 ms and was sensitive to Ca buffers, BAPTA and EGTA. Our data suggest that recruitment of the vesicles to the RRP was Ca-insensitive and had a time constant of 1 s. In addition to the RRP, the reserve pool of vesicles, which had a similar size to RRP, was depleted during repetitive stimulation. Our data suggest that synaptic vesicle endocytosis was also Ca-insensitive. ABSTRACT: Hippocampal mossy fibre terminals comprise one of the cortical terminals, which are sufficiently large to be accessible by patch clamp recordings. To measure Ca-dependence of exo- and endocytotic kinetics quantitatively, we applied presynaptic capacitance measurements to the mossy fibre terminal at room temperature. The time course of synaptic vesicle fusion was slow, with a time constant of tens of milliseconds, and was sensitive to Ca buffers EGTA and BAPTA, suggesting a loose coupling between Ca channels and synaptic vesicles. The size of the readily-releasable pool (RRP) of synaptic vesicles was relatively insensitive to Ca buffers. Once the RRP was depleted, it was recovered by a single exponential with a time constant of ∼1 s independent of the presence of Ca buffers, suggesting Ca independent vesicle replenishment. In addition to the RRP, the reserve pool of vesicles was released slowly during repetitive stimulation. Endocytosis was also insensitive to Ca buffers and had a slow time course, excluding the involvement of rapid vesicle cycling in vesicle replenishment. Although mossy fibre terminals are known to have various forms of Ca-dependent plasticity, some features of vesicle dynamics are robust and Ca-insensitive.

Hapln4/Bral2 is a selective regulator for formation and transmission of GABAergic synapses between Purkinje and deep cerebellar nuclei neurons.

Purkinje cells (PCs) convey the sole output of the cerebellar cortex to the deep cerebellar nuclei (DCN). DCN neurons are enwrapped in densely organized extracellular matrix structures, known as perineuronal nets (PNNs). PNNs are typically found around fast-spiking GABAergic interneurons expressing parvalbumin but interestingly also exist surrounding other neurons, such as the neurons in the DCN and medial nucleus of the trapezoid body, which are the post-synaptic neurons of large axo-somatic synapses adapted for fast signaling. This characteristic localization prompted the hypothesis that PNNs might play a role in the maintenance and formation of large fast-signaling synapses. To elucidate the role of the PNN at these synapses, we investigated the electrophysiological and morphological properties of DCN synapses in hyaluronan and proteoglycan binding link protein 4 (Hapln4/Bral2) knockout (KO) mice around postnatal day (P)14. Hapln4/Bral2 is important for PNN structure, as it stabilizes the interaction between hyaluronan and proteoglycan. Here, using immunohistochemistry we show that Hapln4/Bral2 localized closely with GABAergic terminals. In DCN neurons of Hapln4/Bral2 KO mice, inhibitory synaptic strengths were reduced as compared to those in wild-type mice, whereas the properties of excitatory synapses were unaffected. The reduced IPSC amplitudes were mainly because of reduced numbers of releasable vesicles. Moreover, Hapln4/Bral2 deficiency reduced the number of PC GABAergic terminals in the DCN. These results demonstrate that Hapln4/Bral2 is a PNN component that selectively contributes to formation and transmission of PC-DCN synapses in the cerebellum. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.