Cellular distribution and subcellular localization of molecular components of vesicular transmitter release in horizontal cells of rabbit retina

Abstract

The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of gamma-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca(2+)-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells.

Fig. 1

Fluorescence micrographs of labeling produced by antibodies to (A) complexin I/II, (B) syntaxin-1, and (C) synapsin I (P610) in vertical sections of rabbit retina. Note the prominent immunoreactivity in the plexiform layers for all three synaptic proteins. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar = 20 μm in A–C.

Fig. 2

Localization of CPX I/II to horizontal cells and amacrine cells. A–C: Co-localization of (A) CPX I/II with (B) calbindin, a horizontal cell marker, showed clear double labeling (arrows) of somata and processes in the OPL (a stack of optical slices projected onto a single plane in order to visualize fully the thin horizontal cell process in the OPL; C). Pre-embedding immunoelectron microscopic labeling for CPX I/II shows dark granular DAB reaction product in horizontal cell processes at (D) cone and (E,F) rod photoreceptor synapses. Arrowheads point to the synaptic ribbons in the photoreceptor terminals. G–I: Double-label experiments with (G) CPX I/II and (H) GABA antibodies demonstrated the localization of CPX I/II to some GABAergic amacrine cell bodies (I, arrows). J–L: Double-label experiments with (J) CPX I/II and (K) glycine antibodies showed the localization of CPX I/II to some glycinergic amacrine cell bodies (L, arrows). Note that there are numerous amacrine cell bodies that do not express CPX I/II. OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer; Hc, horizontal cell. Scale bar = 20 μm in C (applies to A–C); 20 μm in I (applies to G–I); 20 μm in L (applies to J–L); 1 μm in D; 0.3 μm in E,F.

Fig. 3

Cellular and subcellular localization of syntaxin-1 to horizontal cells and their processes and endings in photoreceptor synapses. (A) Syntaxin-1 (red) co-localized with (B) calbindin (green) in horizontal cell bodies and processes in the OPL, as well as some amacrine cell bodies (arrows) in the INL. In addition to horizontal cells, calbindin immunoreactivity was present in a subtype of bipolar cell and amacrine cells in the rabbit retina. Pre-embedding immunoelectron microscopy with the HPC-1 monoclonal antibody produced dark, granular DAB immunolabeling for syntaxin-1 in horizontal cell endings at (D,E) cone and (F–H) rod photoreceptor synapses. OPL, outer plexiform layer; IPL, inner plexiform layer; Hc, horizontal cell; Bc, bipolar cell. Arrowheads point to the synaptic ribbons in the photoreceptor terminals. Scale bar = 20 μm in C (applies to A–C; 1 μm in E (applies to D,E); 0.3 μm in G (applies to F,G); 0.3 μm in H.

Fig. 4

CPX I/II and syntaxin-1 are expressed in the same horizontal cells. A: Vertical section of rabbit retina stained for CPX I/II (red) and syntaxin-1 (green). Note the co-localization in horizontal and amacrine cell bodies. B–D: Higher magnification view of horizontal cells shows the co-localization of CPX I/II (B) and syntaxin-1 (C) in processes and endings (arrows) of horizontal cells (D). Note the membrane localization of syntaxin-1 to the plasma membrane of horizontal cells. OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; Hc, horizontal cell. Scale bar = 20 μm in A; 10 μm in D (applies to B–D).

Fig. 5

Synapsin I and syntaxin-1 co-localize in horizontal cells. A–C: Vertical section of rabbit retina stained for synapsin I (red) and syntaxin-1 (green). A: Synapsin I immunoreactivity is present in horizontal and bipolar cells. B: Syntaxin-1 co-localizes with synapsin I in horizontal cells (C). Note that the co-localization appears to extend into the protrusions coming off of horizontal cell processes (arrows). D,E: Electron micrographs of synapsin I immunoreactivity show localization to horizontal cell endings within rod photoreceptor terminals. OPL, outer plexiform layer; Hc, horizontal cell; Bc, bipolar cell. Scale bar = 10 μm in C (applies to A–C); 0.4 μm in D,E.