RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation-inhibition (E-I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation-inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 ß-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation.

pOpsicle: An all-optical reporter system for synaptic vesicle recycling combining pH-sensitive fluorescent proteins with optogenetic manipulation of neuronal activity.

pH-sensitive fluorescent proteins are widely used to study synaptic vesicle (SV) fusion and recycling. When targeted to the lumen of SVs, fluorescence of these proteins is quenched by the acidic pH. Following SV fusion, they are exposed to extracellular neutral pH, resulting in a fluorescence increase. SV fusion, recycling and acidification can thus be tracked by tagging integral SV proteins with pH-sensitive proteins. Neurotransmission is generally activated by electrical stimulation, which is not feasible in small, intact animals. Previous in vivo approaches depended on distinct (sensory) stimuli, thus limiting the addressable neuron types. To overcome these limitations, we established an all-optical approach to stimulate and visualize SV fusion and recycling. We combined distinct pH-sensitive fluorescent proteins (inserted into the SV protein synaptogyrin) and light-gated channelrhodopsins (ChRs) for optical stimulation, overcoming optical crosstalk and thus enabling an all-optical approach. We generated two different variants of the pH-sensitive optogenetic reporter of vesicle recycling (pOpsicle) and tested them in cholinergic neurons of intact Caenorhabditis elegans nematodes. First, we combined the red fluorescent protein pHuji with the blue-light gated ChR2(H134R), and second, the green fluorescent pHluorin combined with the novel red-shifted ChR ChrimsonSA. In both cases, fluorescence increases were observed after optical stimulation. Increase and subsequent decline of fluorescence was affected by mutations of proteins involved in SV fusion and endocytosis. These results establish pOpsicle as a non-invasive, all-optical approach to investigate different steps of the SV cycle.

Endophilin A and B Join Forces With Clathrin to Mediate Synaptic Vesicle Recycling in Caenorhabditis elegans.

Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles-LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.