Electrophysiological characterization of a nicotinic acetylcholine receptor on leech neuropile glial cells

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Abstract
Ion-selective double-barrelled microelcetrodes were used to measure the activities of intracellular K+, Na+, Cl−, and H+ (a, a, a, pHi) and membrane potential (Em) in neuropile glial cells as well as extracellular K+ activity (a) in the neuropile of the leech, Hirudo medicinalis, during bath application of carbachol. As measured with conventional single-barrelled microelectrodes, acetylcholine (ACh), nicotine, carbachol, tetramethylammonium (TMA), and choline elicited concentration-dependent (10−6–5 × 10−3 M) transient membrane depolarizations of up to 60 mV amplitude whereas muscarine (10−6 –10−3 M) did not affect Em. α-Bungarotoxin (10−7 M), decamethonium (10−5 M), d-tubocurarine (5 × 10−5 M), and strychnine (5 × 10−5 M) blocked the carbachol depolarization by about 90%. Atropine (5 × 10−5 M) blocked the response by about 75%, whereas hexaethonium was only effective at millimolar concentrations. Average baseline levels of a in the neuropile and of a, a, and a in the neuropile glial cells were about 3, 70, 10, and 7 mM, respectively. During the carbachol depolarization a and a transiently increased, whereas a decreased. In contrast, a rise of a and a fall of a were observed during glial depolarizations in solutions with elevated K+ concentration. A increased during both the carbachol- and the K+ -induced depolarization. During carbachol, pHi transiently fell by about 0.2 units from its average baseline level of 6.9, whereas an alkalinization of small amplitude was observed in high-K+ solutions. Bath-applied choline, TMA, and decamethonium rapidly accumulated in the neuropile glial cells as intracellulary monitored with double-barrelled microelectrodes filled with Corning K+ exchanger resin, which is highly selective for these agents. The results suggest that leech neuropile glial cells have a nicotinic ACh receptor coupled to a cation channel. It is hypothesized that this channel might also be permeable to choline, TMA, and decamethonium.