| Horizontal cells play a critical role in visual processing by modifying signals sent from photoreceptors to bipolar cells at the synaptic triad, the first site of visual information transfer in the retina. It is at this synapse that horizontal cells contribute to light adaptation, luminance contrast, and color opponency. They are thought to release the inhibitory neurotransmitter gamma-amino-butryic acid (GABA) to shape the receptive field properties of photoreceptors and bipolar cells. However, the underlying mechanisms governing GABA release are poorly understood.;This study tested the hypothesis that mammalian horizontal cells release GABA via regulated vesicular exocytosis. Previous studies have established the presence of spherical, clear-core vesicles in mammalian horizontal cells processes, as well GABA and its synthetic enzyme glutamate decarboxylase isoform 65 (GAD65) in mammalin horizontal cells. In this study we used the guinea pig retina, an emerging model of retinal studies, to identify key vesicular and synaptic proteins involved in regulated exocytosis and to demononstrate recycling vesicles in mammalian horizontal cells. Immunocytochemistry of vertical sections and acutely dissociated horizontal cells revealed that guinea pig horizontal cells possess the molecular machinery to support synaptic transmission. These include the vesicular GABA transporter (VGAT) located in horizontal cell tips, the vesicular proteins synapsin I, synaptic vesicle protein 2A (SV2A) and synaptotagmin-2, as well as the SNARE proteins SNAP-25, syntaxins-1a and -4, the vesicle-associated membrane protein 1 (VAMP-1), and the SNARE-associated protein complexin I/II. Moreover, VAMP-1 expression, which has not previously been identified in the horizontal cells of any mammalian species, was confirmed by single cell RT-PCR from laser captured horizontal cells. Finally, the presence of recycling GABAergic vesicles was demonstrated in retinal slices with VGAT antibodies that, with a depolarizing stimulus, bound to the lumenal portion of the vesicle. Furthermore, vesicle recycling was shown to be a calcium-dependent process.;Taken together, this study supports the hypothesis that mammalian horizontal cells release the transmitter GABA through a vesicular release mechanism. These findings in the guinea pig suggest that a vesicular mechanism of release in mammalian horizontal cells may be a highly conserved process present in many different mammalian species. |
