Targeted Deletion of Vesicular GABA Transporter from Retinal Horizontal Cells Eliminates Feedback Modulation of Photoreceptor Calcium Channels

Abstract

The cellular mechanisms underlying feedback signaling from horizontal cells to photoreceptors, which are important for the formation of receptive field surrounds of early visual neurons, remain unsettled. Mammalian horizontal cells express a complement of synaptic proteins that are necessary and sufficient for calcium-dependent exocytosis of inhibitory neurotransmitters at their contacts with photoreceptor terminals, suggesting that they are capable of releasing GABA via vesicular release. To test whether horizontal cell vesicular release is involved in feedback signaling, we perturbed inhibitory neurotransmission in these cells by targeted deletion of the vesicular GABA transporter (VGAT), the protein responsible for the uptake of inhibitory transmitter by synaptic vesicles. To manipulate horizontal cells selectively, an iCre mouse line with Cre recombinase expression controlled by connexin57 (Cx57) regulatory elements was generated. In Cx57-iCre mouse retina, only horizontal cells expressed Cre protein, and its expression occurred in all retinal regions. After crossing with a VGAT flox/flox mouse line, VGAT was selectively eliminated from horizontal cells, which was confirmed immunohistochemically. Voltage-gated ion channel currents in horizontal cells of Cx57-VGAT Ϫ/Ϫ mice were the same as Cx57-VGAT ϩ/ϩ controls, as were the cell responses to the ionotropic glutamate receptor agonist kainate, but the response to the GABA A receptor agonist muscimol in Cx57-VGAT Ϫ/Ϫ mice was larger. In contrast, the feedback inhibition of photoreceptor calcium channels, which in control animals is induced by horizontal cell depolarization, was completely absent in Cx57-VGAT Ϫ/Ϫ mice. The results suggest that vesicular release of GABA from horizontal cells is required for feedback inhibition of photoreceptors. Key words: Ca channels; Cx57-iCre; GABA receptors; inhibitory feedback; retinal horizontal cells; synaptic vesicles Significance StatementFeedback inhibition by horizontal cells regulates the photoreceptor calcium channels responsible for the release of the neurotransmitter glutamate. This feedback inhibition contributes to the formation of the receptive field surrounds of early visual neurons, which is important for contrast sensitivity and color opponency in vision. The cellular mechanisms underlying feedback inhibition are not fully understood. One disputed mechanism for mammalian horizontal cell inhibitory output involves the vesicular release of the inhibitory neurotransmitter GABA. By selectively deleting the transporter that normally loads GABA into vesicles in horizontal cells, we show that horizontal cells lose their ability to modulate photoreceptor calcium signals. These findings implicate a role for the vesicular release of GABA in mediating inhibitory feedback.

Invaginating Presynaptic Terminals in Neuromuscular Junctions, Photoreceptor Terminals, and Other Synapses of Animals

“…For example, Kramer and Davenport (2015) favor the proton/pH mechanism, at least for mammals, perhaps in combination with the ephaptic mechanism; in contrast, Gardner et al (2015) favor the ephaptic mechanism, perhaps in combination with the GABA mechanism. And as noted, Hirano et al (2016) show evidence supporting a GABA mechanism. However, this latter mechanism seems to be indirect, with GABA released from horizontal cells and activating autoreceptors on the horizontal cells, possibly driving a pH-based feedback mechanism (Liu et al 2013; Hirano et al 2016); a GABA autoreceptor-based mechanism also might be present in lower vertebrates (Klooster et al 2004; Endeman et al 2012).…”

“…And as noted, Hirano et al (2016) show evidence supporting a GABA mechanism. However, this latter mechanism seems to be indirect, with GABA released from horizontal cells and activating autoreceptors on the horizontal cells, possibly driving a pH-based feedback mechanism (Liu et al 2013; Hirano et al 2016); a GABA autoreceptor-based mechanism also might be present in lower vertebrates (Klooster et al 2004; Endeman et al 2012). …”

“…f Diagram illustrating three theories for the feedback mechanism of horizontal cell processes on the retinal terminal synapse: GABA, protons (H + is pH) and ephaptic transmission (ΔV; an electrical field effect at close range; Gardner et al 2015; Kramer and Davenport 2015). The involvement of GABA is supported by recent studies (Hirano et al 2016; also Liu et al 2013), but they suggest that GABA from horizontal cell processes activates autoreceptors directly on the horizontal cell processes, probably indirectly mediating the pH-based feedback mechanism (Color figure online)…”

“…7). Hirano et al (2016) show that vesicular release of GABA from mouse horizontal cells appears to be necessary for the modulation of calcium channels of the photoreceptor terminals (see also Hirano et al 2005). But we also have noted that most of the invaginating horizontal processes in vertebrates do not seem to contain many vesicles, and several studies suggest that GABA is released from these invaginated horizontal cell processes via non-vesicular GABA transport (Haverkamp et al 2000; Gardner et al 2015; Kramer and Davenport 2015).…”

Horizontal cells expressing melanopsin x are novel photoreceptors in the avian inner retina

“…Several mechanisms for HC feedback to photoreceptors have been proposed. HCs contain GAD, the synthesizing GABA enzyme (44,45), and exhibit a direct GABAergic synaptic connection to ON and OFF bipolar cells (46) and to visual photoreceptors (47), showing that vesicular GABA release from HCs is required for feedback inhibition of photoreceptors.…”