GABA-activated whole-cell currents in isolated retinal ganglion cells.
1. We have begun to analyze neurotransmitter-activated conductances in retinal ganglion cells by measuring the response of single voltage-clamped adult goldfish ganglion cells to gamma-aminobutyric acid (GABA). Here we describe 1) our method of identifying ganglion cells in vitro after their dissociation from papain-treated retinas, and 2) the response of these cells to GABA in the tight-seal whole cell configuration of the patch-clamp method (cf. 41) after 1-4 days of primary cell culture. 2. Ganglion cell somata were backfilled in situ by injections of horseradish peroxidase (HRP) into the optic nerve. After dissociation of the retinas containing these cells, HRP reaction product was localized to cells that retained the size, shape, and an intracellular organelle characteristic of ganglion cells in situ. These features enabled us thereafter to identify ganglion cells in vitro without retrograde marker transport. 3. GABA (3-10 microM) elicited inward currents and substantial noise increases in almost all ganglion cells at negative holding potentials. Reversal potential measurements in salines containing different chloride concentrations indicated that GABA produces a chloride-selective conductance increase in ganglion cells. Bicuculline (10 microM) reversibly inhibited ganglion cell GABA responses. Baclofen (10 microM) alone elicited no responses in ganglion cells. 4. Noise analysis of GABA-activated whole cell currents yielded elementary conductance estimates of 16 pS, with a slow time constant of 30 ms plus a faster component of 1-2 ms. No significant voltage dependence of these values was observed between -20 and -80 mV. 5. We have thus devised a means of identifying ganglion cells dissociated from adult retinas, identified GABAA receptors (cf. 16) on these cells, and found that the responses mediated by these receptors resemble those found in other regions of central nervous system (CNS). These results are consistent with the notion that GABA may function as an inhibitory transmitter at synapses on ganglion cells.
Ishida
A T
AT
Jules Stein Eye Institute, University of California, Los Angeles 90024.
Cohen
B N
BN
D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002452:Cell Count; D002469:Cell Separation; D006054:Goldfish; D006735:Horseradish Peroxidase; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects; D012160:Retina / Q000502:physiology*; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D005680:gamma-Aminobutyric Acid; D006735:Horseradish Peroxidase; D001640:Bicuculline