Abstract
Feedback inhibition by horizontal cells regulates rod and cone photoreceptor calcium channels that control their release of the neurotransmitter glutamate. This inhibition contributes to synaptic gain control and the formation of the center-surround antagonistic receptive fields passed on to all downstream neurons, which is important for contrast sensitivity and color opponency in vision. In contrast to the plasmalemmal GABA transporter found in non-mammalian horizontal cells, there is evidence that the mechanism by which mammalian horizontal cells inhibit photoreceptors involves the vesicular release of the inhibitory neurotransmitter GABA. Historically, inconsistent findings of GABA and its biosynthetic enzyme, L-glutamate decarboxylase (GAD) in horizontal cells, and the apparent lack of surround response block by GABAergic agents diminished support for GABA's role in feedback inhibition. However, the immunolocalization of the vesicular GABA transporter (VGAT) in the dendritic and axonal endings of horizontal cells that innervate photoreceptor terminals suggested GABA was released via vesicular exocytosis. To test the idea that GABA is released from vesicles, we localized GABA and GAD, multiple SNARE complex proteins, synaptic vesicle proteins, and Cav channels that mediate exocytosis to horizontal cell dendritic tips and axonal terminals. To address the perceived relative paucity of synaptic vesicles in horizontal cell endings, we used conical electron tomography on mouse and guinea pig retinas that revealed small, clear-core vesicles, along with a few clathrin-coated vesicles and endosomes in horizontal cell processes within photoreceptor terminals. Some small-diameter vesicles were adjacent to the plasma membrane and plasma membrane specializations. To assess vesicular release, a functional assay involving incubation of retinal slices in luminal VGAT-C antibodies demonstrated vesicles fused with the membrane in a depolarization- and calcium-dependent manner, and these labeled vesicles can fuse multiple times. Finally, targeted elimination of VGAT in horizontal cells resulted in a loss of tonic, autaptic GABA currents, and of inhibitory feedback modulation of the cone photoreceptor Cai, consistent with the elimination of GABA release from horizontal cell endings. These results in mammalian retina identify the central role of vesicular release of GABA from horizontal cells in the feedback inhibition of photoreceptors.
Highlights
Horizontal cells receive synaptic input from thousands of photoreceptors and feedback this broad spatial information back to photoreceptors as well as feeding it forward to bipolar cells to generate receptive field surrounds (Thoreson and Mangel, 2012)
Whereas mammalian horizontal cells lack plasmalemmal GABA Transporter (GAT) (Johnson et al, 1996; Guo et al, 2009), our laboratory and others showed the presence of the vesicular inhibitory amino acid/GABA transporter (VIAAT/VGAT) in mammalian horizontal cells in mouse (Figure 4A), rat, rabbit and primate retina, where VGAT immunostaining is concentrated in the endings that insert into the rod and cone photoreceptor terminals (Figure 4A, arrows, Haverkamp et al, 2000; Cueva et al, 2002; Jellali et al, 2002; Johnson et al, 2003; Hirano et al, 2005, 2007, 2011; Guo et al, 2010; Lee and Brecha, 2010)
The evidence for GABA as the horizontal cell neurotransmitter is the presence of GABA immunoreactivity and of GABA synthesizing enzymes (GAD65 and/or GAD67), and postsynaptic targets bearing GABA receptors as well as autoreceptors on horizontal cells
Summary
Horizontal cells receive synaptic input from thousands of photoreceptors and feedback this broad spatial information back to photoreceptors as well as feeding it forward to bipolar cells to generate receptive field surrounds (Thoreson and Mangel, 2012).
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