Abstract
Purpose: :
To identify GABA signaling pathways for horizontal and bipolar cells in the outer plexiform layer of the zebrafish retina.
Methods: :
Retinas removed from adult zebrafish eyes were papain–dissociated and plated on poly–D–lysine–coated plastic culture dishes. Dissociated horizontal cells (HCs) and bipolar cells (BCs) were identified morphologically (Connaughton & Dowling, 1998). Changes in membrane potential induced by GABA, selective GABAergic ligands, or treatments affecting GABA responses were obtained by photometry using the fluorescent voltage–sensitive dye oxonol (DiBaC4(5), 80 nM; Nelson et al, 2003). Only the dendro–somatic GABA responses of axotomized BCs were considered. HC recordings were mainly of the HB type (Connaughton et al, 2004).
Results: :
Most BCs (194/289, 67%) hyperpolarized in response to GABA (1–20 µM, but HCs almost never did (2/73, 3%). A subpopulation of HCs (27/73, 37%) either depolarized and/or responded with after hyperpolarization (dep/AHP) in response to GABA, as did a minor group of BCs (13/289; 4%). In GABA depolarized BCs or HCs the mean fluorescence increase was .24 log units (N=27), about 30 mV (Nelson et al, 1999). Muscimol (10 µM), an iontropic GABA agonist, readily hyperpolarized BCs (56/91, 62%), but was ineffective at evoking dep/AHP in either HCs (3/44, 7%) or BCs (2/91, 2%). GABA–induced hyperpolarizations in BCs were blocked or partially blocked by picrotoxin (24/24, 100%); however, the dep/AHP GABA response, wherever seen in dissociated retinal neurons, was not (0/16, 0%). Dep/AHP responses required both extracellular Na+ and Cl–. They were reversibly blocked by Li+ substitution for Na+ (15/16; 94%). In some cases (8/16, 50%) a hyperpolarizing GABA response was unmasked by this substitution. Na isethionate substituted for NaCl resulted in irreversible loss of GABA dep/AHP (13/14, 93%). Among 14 dep/AHP responses studied with GABA concentration series, the median concentration for half saturation of response was 0.7 µM.
Conclusions: :
Ionotropic GABA receptors are expressed on most zebrafish BCs, but not HCs. Many HCs and rarely, BCs, express an Na+ –dependent, Cl– –dependent, picrotoxin–insensitive, and muscimol–resistant membrane transporter for GABA. A depolarized Na+ gradient drives GABA into these cells, depolarizing the plasma membrane and triggering Na+, K+–dependent ATPase generating AHP. In effect many HCs and a few BCs in zebrafish exhibit a GABA–selective, Na+–dependent excitation within the outer plexiform layer.
Keywords: horizontal cells • neurotransmitters/neurotransmitter systems • bipolar cells