Abstract
Purpose: :
Horizontal cells (HCs) mediate inhibitory feedforward and feedback in the outer retina at bipolar cell dendrites and photoreceptor terminals; however, the mechanisms that underlie transmitter release from mammalian HCs are poorly understood. The localization of a vesicular GABA transporter (VGAT) to HC processes in primate and rodent retinae suggested that mammalian HCs release transmitter in a vesicular manner. To determine whether components of the molecular machinery for vesicular transmitter release are present in HCs, we studied the localization of syntaxin–4 (syx–4), a SNARE protein, in mouse, rat and rabbit retinae.
Methods: :
We determined the immunolabeling pattern of syx–4 (Chemicon) by indirect immunofluorescence immunohistochemistry on vertical and horizontal sections of adult retina with antibodies (Ab) to cell type–specific markers: mouse monoclonal Ab to calbindin (Sigma), PKC (Biodesign), SNAP–25 (Synaptic Systems), and a guinea pig Ab to VGLUT1 (Chemicon). FITC–conjugated peanut agglutinin (PNA, Vector Labs) was used to identify cone pedicles. The sections were examined by confocal microscopy.
Results: :
We report robust expression of syx–4 in the outer plexiform layer (OPL) of all three species. Syx–4 occurred in the processes and tips of HCs, with thicker sandwich–like structures along the processes. Double labeling with Abs to syx–4 and calbindin, a HC marker, demonstrated syx–4 localization to HC processes. Double labeling with Abs to syx–4 and PKC, a rod bipolar cell (RBC) marker, showed a lack of co–localization, with syx–4 immunolabeling occurring just distal to RBC dendritic tips. To evaluate syx–4 localization within photoreceptor terminals, double labeling with Abs to syx–4 and VGLUT1, present in both rod and cone photoreceptor terminals, was carried out. Syx–4 immunolabeling occurred within VGLUT1–immunoreactive terminals, consistent with invaginating HC tips. Vertical sections of retina immunostained for syx–4 and PNA established that the prominent patches of syx–4 immunoreactivity are adjacent to cone pedicle bottoms, with HC tips inserting into the terminals. Horizontal sections through the OPL confirmed the one–to–one co–localization of syx–4 densities at cone pedicles. Finally, co–localization studies with an Ab to SNAP–25, a binding partner of syx–4, indicated co–expression of these SNARE proteins in HC processes and tips.
Conclusions: :
These studies indicate that syx–4 occurs in horizontal cell processes and tips in mammalian outer retina. The co–localization of syx–4 with SNAP–25 provides further support for the vesicular release of transmitter.
Keywords: horizontal cells • microscopy: light/fluorescence/immunohistochemistry • synapse