Robust syntaxin-4 immunoreactivity in mammalian horizontal cell processes

Abstract

Horizontal cells mediate inhibitory feed-forward and feedback communication in the outer retina; however, mechanisms that underlie transmitter release from mammalian horizontal cells are poorly understood. Toward determining whether the molecular machinery for exocytosis is present in horizontal cells, we investigated the localization of syntaxin-4, a SNARE protein involved in targeting vesicles to the plasma membrane, in mouse, rat, and rabbit retinae using immunocytochemistry. We report robust expression of syntaxin-4 in the outer plexiform layer of all three species. Syntaxin-4 occurred in processes and tips of horizontal cells, with regularly spaced, thicker sandwich-like structures along the processes. Double labeling with syntaxin-4 and calbindin antibodies, a horizontal cell marker, demonstrated syntaxin-4 localization to horizontal cell processes; whereas, double labeling with PKC antibodies, a rod bipolar cell~RBC! marker, showed a lack of co-localization, with syntaxin-4 immunolabeling occurring just distal to RBC dendritic tips. Syntaxin-4 immunolabeling occurred within VGLUT-1-immunoreactive photoreceptor terminals and underneath synaptic ribbons, labeled by CtBP20RIBEYE antibodies, consistent with localization in invaginating horizontal cell tips at photoreceptor triad synapses. Vertical sections of retina immunostained for syntaxin-4 and peanut agglutinin~PNA! established that the prominent patches of syntaxin-4 immunoreactivity were adjacent to the base of cone pedicles. Horizontal sections through the OPL indicate a one-to-one co-localization of syntaxin-4 densities at likely all cone pedicles, with syntaxin-4 immunoreactivity interdigitating with PNA labeling. Pre-embedding immuno-electron microscopy confirmed the subcellular localization of syntaxin-4 labeling to lateral elements at both rod and cone triad synapses. Finally, co-localization with SNAP-25, a possible binding partner of syntaxin-4, indicated co-expression of these SNARE proteins in the same subcellular compartment of the horizontal cell. Taken together, the strong expression of these two SNARE proteins in the processes and endings of horizontal cells at rod and cone terminals suggests that horizontal cell axons and dendrites are likely sites of exocytotic activity.

Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments

“…1B,C), as previously reported (Hirano et al, 2007;Morgans et al, 1996). Syntaxin 3 was also detected in the calycal processes (CPs) (Fig.…”

Functional and structural impact of the loss of the leucine-rich repeat protein LRIT1 in the mouse retina.

“…These data suggest LRIT1 is likely not expressed in cone OFF bipolar cells. Previous work has shown that the horizontal cell markers calbindin, syntaxin-4, and GluA2 all localize distal to PNA, which is similar to our data for LRIT1 and PNA (133,136), suggesting LRIT1 may be localized on the horizontal cell invaginating contacts at cone terminals. Our observed LRIT1 staining pattern with mGluR6 and GPR179 is consistent …”

“…Prior work has shown that the ON bipolar cell markers LRIT3 (78), NYX (63,73), mGluR6 (130), and TRPM1 (131) show a similar localization. Previous studies also showed that the horizontal cell markers calbindin and syntaxin-4 localize adjacent to piccolino or RIBEYE crescents, respectively (132,133). Piccolino is another synaptic ribbon marker shown to colocalize with RIBEYE (134,135).…”

Specialized synaptic pathway for chromatic signals beneath S-cone photoreceptors is common to human, Old and New World primates

“…The question addressed was if primate horizontal cells may employ a feed-forward pathway from horizontal cells directly to cone bipolar cells. Syntaxin-4 has been shown to be expressed by horizontal cells in different mammals and it was used as an indicator for vesicular GABA release in horizontal cells [3–5]. In macaque monkeys, syntaxin-4 was found in HI and HII horizontal cells postsynaptic to all photoreceptor types, but highly enriched beneath short (S-) wavelength sensitive cones, co-localized with dendritic processes of HII horizontal cells.…”