GABA Agonist Linked to a Quantum Dot Activates GABA–A and GABA–C Receptor Currents in Intact Retinal Slice

Abstract

Abstract: : Purpose: Light evokes strong inhibition of ganglion cells through GABAergic chloride currents, but the location of the receptors can not be identified in living tissue. We have linked a fluorescent quantum dot (QD) to the GABA receptor agonist TACA to evoke GABAergic currents and to visually determine the location of these receptors. Prior to use as a optical probe in living tissue, the efficacy of the ligand–QD must be determined. The activation of receptor ion currents by biotinylated–TACA and TACA–QD was recorded and compared to free TACA, a potent GABA–A and GABA–C receptor agonist, to determine whether the added steric bulk of biotin and the QD diminished bioactivity of the ligand. Methods: Whole–cell patch clamp recordings were obtained from salamander retinal ganglion cells in slice. 20 mM Mg⁺⁺ Ringer’s was locally perfused to block release of neurotransmitter from presynaptic terminals. TACA, biotinylated–TACA and TACA–QD were focally applied in the IPL adjacent to the recorded cell through a small pressure ejection (puff) pipette to evoke receptor ion currents at different holding potentials which were then blocked with 100 µM bicuculline and 150 µM picrotoxin. Results: Whole cell currents evoked by the focal application of TACA, biotinylated–TACA and TACA–QD were similar in amplitude and time course. Current–voltage relations of agonist–evoked currents showed reversal potentials near the calculated reversal potential for chloride. Whole cell currents in response to each agonist also showed similar sensitivity to pharmacological block by 100 µM bicuculline and 100 µM bicuculline with 150 µM picrotoxin. Conclusions: Biotinylated–TACA and TACA–QD activate GABA–A and GABA–C currents in retinal slice with bioactivity comparable to that of free TACA. Neurotransmitter coupled–QDs hold good potential as combined optical and physiological probes for investigating synaptic activity in intact neural tissue.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • ganglion cells • inhibitory receptors