Synaptic Interactions Between Starburst Cells Under Dual Patch-Clamp Recording in Newborn Rabbit Retinal Wholemounts

Abstract

Abstract: : Purpose: Cholinergic (starburst) amacrine cells play an important role in the formation of spontaneous retinal waves during development. This study was designed to understand the mechanism of synaptic interaction between starburst cells during the early stage of synaptogenesis in the mammalian retina. Methods: Dual patch-clamp recordings were performed on pairs of displaced starburst amacrine cells in a wholemount preparation of perinatal rabbit retina. Intracellular free Ca²⁺ concentration in individual staburst cells was imaged with Ca²⁺ indicator dyes loaded into the cells by a gene gun. Results: When neighboring starburst cells were voltage-clamped in pairs, depolarizing steps given to one starburst cell evoked synaptic current responses in the other starburst cell. The responses ranged in amplitude from 20 to 400 pA, and their properties changed with age. Between the ages of E29 and E30, the synaptic responses were mainly cholinergic in nature, because they were inwardly rectifying and sensitive to nicotinic antagonists. Immediately after birth (E31), however, two synaptic current components became detectable, with physiological and pharmacological properties consistent with cholinergic and GABAergic currents, respectively. The cholinergic component was inward at –70 mV (the equilibrium potential for Cl^-) and persisted in the presence of picrotoxin. The GABA component was outward at 0 mV and +45 mV, resistant to nicotinic antagonists, and blocked by picrotoxin. Both cholinergic and GABAergic currents were blocked by CdCl₂, suggesting a Ca²⁺-dependent release mechanism for both transmitters. The waveform of GABA currents at +45 mV also displayed characteristics consistent with quantal release. In addition to well-correlated pre- and post-synaptic currents, asynchronous release of neurotransmitter, lasting many seconds after the termination of presynaptic depolarization, was also seen in some starburst cells. Conclusions: The results demonstrate at the single cell level that starburst cells in the postnatal rabbit retina are capable of releasing both ACh and GABA, and that both transmitters are released by a vesicular mechanism. Starburst cells use both ACh an GABA to communicate among themselves in the postnatal retina. The onset of GABAergic synaptic communication between starburst cells correlates the beginning of GABAergic inhibition of retinal waves in rabbit.

Keywords: amacrine cells • acetylcholine • synapse