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

Abstract

Ion-selective double-barrelled microelcetrodes were used to measure the activities of intracellular K⁺, Na⁺, Cl⁻, and H⁺ (a, a, a, pH_i) and membrane potential (E_m) 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 E_m. 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, pH_i 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.