Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.
Aim A key feature of the mammalian retina is the segregation of visual information in parallel pathways, starting at the photoreceptor terminals. Cone photoreceptors establish synaptic contacts with On bipolar and horizontal cells at invaginating, ribbon‐containing synaptic sites, whereas Off bipolar cells form flat, non‐ribbon‐containing contacts. The cytomatrix protein Bassoon anchors ribbons at the active zone, and its absence induces detachment of ribbons from the active zone. In this study we investigate the impact of a missing Bassoon on synaptic transmission at the first synapse of the visual system. Methods Release properties of cone photoreceptors were studied in wild‐type and mutant mouse retinae with a genetic disruption of the presynaptic cytomatrix protein Bassoon using whole‐cell voltage‐clamp recordings. Light and electron microscopy revealed the distribution of Ca2+ channels and synaptic vesicles, respectively, in both mouse lines. Results Whole‐cell recordings from postsynaptic horizontal cells of the two mouse lines showed that the presence of Bassoon (and a ribbon) enhanced the rate of exocytosis during tonic and evoked release by increasing synaptic vesicle pool size and replenishment rate, while at the same time slowing synaptic vesicle release. Furthermore, the number of Cav1.4 channels and synaptic vesicles was significantly higher at wild‐type than at Bassoon mutant synaptic sites. Conclusion The results of our study demonstrate that glutamate release from cone photoreceptor terminals can occur independent of a synaptic ribbon, but seems restricted to active zones, and they show the importance of a the synaptic ribbon in sustained and spatially and temporally synchronized neurotransmitter release.
RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1β are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM-Munc13-RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis. RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.
Purpose Cone photoreceptors of the retina use a sophisticated ribbon-containing synapse to convert light-dependent changes in membrane potential into release of synaptic vesicles (SVs). We aimed to study the functional and structural maturation of mouse cone photoreceptor ribbon synapses during postnatal development and to investigate the role of the synaptic ribbon in SV release. Methods We performed patch-clamp recordings from cone photoreceptors and their postsynaptic partners, the horizontal cells during postnatal retinal development to reveal the functional parameters of the synapses. To investigate the occurring structural changes, we applied immunocytochemistry and electron microscopy. Results We found that immature cone photoreceptor terminals were smaller, they had fewer active zones (AZs) and AZ-anchored synaptic ribbons, and they produced a smaller Ca 2+ current than mature photoreceptors. The number of postsynaptic horizontal cell contacts to synaptic terminals increased with age. However, tonic and spontaneous SV release at synaptic terminals stayed similar during postnatal development. Multiquantal SV release was present in all age groups, but mature synapses produced larger multiquantal events than immature ones. Remarkably, at single AZs, tonic SV release was attenuated during maturation and showed an inverse relationship with the appearance of anchored synaptic ribbons. Conclusions Our developmental study suggests that the presence of synaptic ribbons at the AZs attenuates tonic SV release and amplifies multiquantal SV release. However, spontaneous SV release may not depend on the presence of synaptic ribbons or voltage-sensitive Ca 2+ channels at the AZs.
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