The physiological actions of biogenic amine and amino-acid neurotransmitters are terminated by their removal from the synaptic cleft by specific high-affinity transport proteins. The members of the Na+- and Cl−-dependent neurotransmitter transporter family expressed in bovine retina and responsible for the uptake of biogenic amine and amino-acid neurotransmitters were identified using a reverse transcriptase-polymerase chain reaction-based approach. cDNA clones encoding bovine homologues of glycine (GLYT-1), γ-aminobutyric acid (GAT-1) creatine (CreaT), and orphan (NTT4) transporters were identified using this strategy. The expression pattern of mRNAs encoding these proteins in the retina was determined by in situ hybridization histochemistry GLYT-1 CreaT NTT4 and GAT-1 mRNAs were expressed in the retina by cells in the inner nuclear inner plex, iform and ganglion cell layers They were not expressed at detectable levels in the photoreceptor cells whose cell bodies are in the outer nuclear layer and are the most abundant cell type in the retina GLYT-1 mRNA was present exclusively in the proximal inner nuclear layer GAT-1 mRNA was localized to both the inner nuclear and ganglion cell layers CreaT mRNA was expressed in all cell types in the retina except photoreceptors and NTT4 mRNA was expressed by a sub subpoulation of cells in the ganglion cell laver. Elucidation of the expression pattern of these neurotransmitter transporter mRNAs in the retina provides a basis for studies of the role of glycine γ-aminobutyric acid and creatine transporters in retinal function.

GABA-mediated interactions between horizontal cells (HCs) and bipolar cells (BCs) transform signals within the image-processing circuitry of distal retina. To further understand this process, we have studied the GABA-driven membrane responses from isolated retinal neurons. Papain-dissociated retinal cells from adult zebrafish were exposed to GABAergic ligands while transmembrane potentials were monitored with a fluorescent voltage-sensitive dye (oxonol, DiBaC4(5)). In HCs hyperpolarizing, ionotropic GABA responses were almost never seen, nor were responses to baclofen or glycine. A GABA-induced depolarization followed by after hyperpolarization (dep/AHP) occurred in 38% of HCs. The median fluorescence increase (dep component) was 0.17 log units, about 22 mV. HC dep/AHP was not blocked by bicuculline or picrotoxin. Muscimol rarely evoked dep/AHP responses. In BCs picrotoxin sensitive, hyperpolarizing, ionotropic GABA and muscimol responses occurred in most cells. A picrotoxin insensitive dep/AHP response was seen in about 5% of BCs. The median fluorescence increase (dep component) was 0.18 log units, about 23 mV. Some BCs expressed both muscimol-induced hyperpolarizations and GABA-induced dep/AHP responses. For all cells, the pooled Hill fit to median dep amplitudes, in response to treatments with a GABA concentration series, gave an apparent k of 0.61 μM and an n of 1.1. The dep/AHP responses of all cells required both extracellular Na+ and Cl−, as dep/AHP was blocked reversibly by Li+ substituted for Na+ and irreversibly by isethionate substituted for Cl−. All cells with dep/AHP responses in zebrafish have the membrane physiology of neurons expressing GABA transporters. These cells likely accumulate GABA, a characteristic of GABAergic neurons. We suggest Na+ drives GABA into these cells, depolarizing the plasma membrane and triggering Na+, K+-dependent ATPase. The ATPase activity generates AHP. In addition to a GABA clearance function, these large-amplitude transporter responses may provide an outer plexiform layer GABA sensor mechanism.

As a part of ongoing efforts to understand the cholinergic circuitry in the mammalian retina, we studied the coexpression of nicotinic acetylcholine receptors (nAChRs) and gamma-aminobutyric acid (GABA), the GABA transporter 1 (GAT-1), or choline acetyltransferase (ChAT) immunoreactivity in the rabbit retina. Double-label experiments with monoclonal antibody 210 (mAb 210) against nAChRs and antibodies against GABA revealed that several populations of GABA-containing amacrine, displaced amacrine, and ganglion cells displayed nAChR immunoreactivity. Some of them also exhibited ChAT immunoreactivity and were identified as the cholinoceptive starburst cells. Other GABAergic amacrine cells positive for mAb 210 were not cholinergic. Simultaneous visualization of mAb 210 and GAT-1 immunoreactivity revealed that 10% of GAT-1 immunoreactive amacrine cells contained nAChRs. Ninety-nine percent of the GAT-1 labeled cells demonstrated GABA immunoreactivity, but only 75% of the GABAergic cells were outlined by GAT-1 staining. Neither population of starburst cells exhibited GAT-1 immunoreactivity. Thus, mAb 210 expressing, GAT-1 positive cells in the rabbit retina constitute a noncholinergic subset of GABAergic amacrine cells. Taken together, our results suggest that some GABAergic amacrine cells are cholinoceptive, raising the possibility that ACh, acting through nAChRs, can modulate the release of GABA in the rabbit retina.

The physiological function of GABA transporters and GABA receptors in retinal horizontal cells (HCs) under dark- and light-adapted conditions were studied by whole-cell voltage clamp and intracellular recording techniques in retinal slices and whole-mounted isolated retinas of the larval tiger salamander. Puff application of GABA in picrotoxin elicited a NO-711 (a potent GABA transporter blocker)-sensitive inward current that did not exhibit a reversal potential in the physiological range, consistent with the idea that these HCs contain electrogenic GABA transporters. Application of GABA in NO-711 elicited a chloride current in HCs; about half of the current was suppressed by bicuculline or I4AA (a GABAC receptor antagonist), and the remaining half was suppressed by bicuculline + I4AA or picrotoxin. In whole-mount retinas, NO-711, bicuculline, I4AA, or picrotoxin hyperpolarized the HCs and enhanced the light responses under dark-adapted conditions, and blocked the time-dependent recovery of HC membrane potential and light responses during background illumination. Based on the parallel conductance model, GABA released in darkness mediates a chloride conductance about three times greater than the leak conductance or the glutamate-gated cation conductance. About half of this chloride conductance is mediated by GABAA receptors, and the other half is mediated by GABAC receptors. These results suggest that GABA released from HCs through the NO-711-sensitive GABA transporters activates GABAA and GABAC receptors, resulting in chloride conductance increase which leads to a HC depolarization and reduction of the light response. Additionally, GABA transporters also mediate GABA release in background light that is responsible for the recovery of HC membrane potential and light responses.

We studied the GABA sensitivity of horizontal cells in the isolated goldfish retina. After the glutamatergic input to the horizontal cells was blocked with DNQX, GABA depolarized the monophasic and biphasic horizontal cells. The pharmacology of these GABA-induced depolarizations was tested with the GABA receptor antagonists bicuculline-methiodide and picrotoxin, the GABA transporter agonist nipecotic acid, and the GABA transporter antagonist SKF 89976-A. The GABA-induced responses of monophasic horizontal cells consisted of two components; one with characteristics of GABA-gated chloride channels, and one with characteristics of GABA transporters. In biphasic horizontal cells, we only found evidence for GABA-gated chloride channels. The results show that monophasic horizontal cells in goldfish contain the two components of a positive feedback loop (GABA transporters and GABA-gated chloride channels), as described in salamander. Furthermore, our results indicate that the monophasic horizontal cells may project directly to the biphasic horizontal cells, via an excitatory GABAergic pathway. We propose that the function of these GABAergic systems in horizontal cells is to abolish cone dominance in bipolar cells surround in the dark-adapted retina.