Spontaneous release of acetylcholine affects the physiological nicotinic responses of rat retinal ganglion cells in culture

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Abstract
Nictonic cholinergic responses have been previously documented in mammalian retinal ganglion cells. In the present study, dissociated retinal cells were densely plated and grown in a specific batch of rat serum. When held at negative potentials during whole-cell recording with a patch electrode, the retinal ganglion cells located close to presumptive cholinergic amacrine cells in these cultures were found to respond to acetylcholine (ACh; 20–200 microM) with an apparent outward current in the presence of physiological salines on both sides of the membrane. Other nicotinic agonists (nicotine, carbachol) produced the same effect. Conductance measurements revealed that this apparent outward current was actually a decrease in a tonic inward current. Nicotinic antagonists such as d-turbocurarine (10 microM) and dihydro- beta-erythroidine (20 microM), when applied in dishes that had never been exposed to exogenous ACh, produced a similar decrease in a tonic inward current. The reversal potential of the tonic current suppressed by ACh was similar to the nicotinic current previously studied in these central neurons. Furthermore, purified acetylcholinesterase was capable of modulating the tonic inward current of the retinal ganglion cells. Lowering an excised patch of muscle into dense areas of the retinal cultures activated nicotinic channels in the muscle membrane, indicating the presence of endogenous ACh in the culture fluid. Divalent cations such as Co2+ blocked the tonic inward current of retinal ganglion cells in these cultures. Finally, direct biochemical measurements indicated that low levels of endogenous ACh (on the order of 0.5 microM near putative cholinergic amacrine cells) were present in the retinal cultures. Taken together, these results show that ACh was being spontaneously released into these cultures, resembling at least to some degree the tonic leakage of ACh found in the intact retina. This concentration of ACh was capable of tonically depolarizing the membrane potential of retinal ganglion cells exposed to this dosage. This culture system allows the study of trophic effects of ACh on a central mammalian neuron in a precisely controlled extracellular environment.