Dose-dependence of the excitatory effects of acetylcholine on common snail neurons after orthodromic tetanization.

Abstract

Studies of the cellular mechanisms of learning and memory often use neuronal analogs of these processes. Post-tetanic changes in the reactivity of nerve cells provide one such model. Orthodromic tetanic stimulation of nerves evokes increases in the cholinosensitivity of the bodies of neurons LPa3 and RPa3 in the common snail [3]. The likely mechanism for post-tetanic increases in neuron cholinosensitivity may consist of changes in the number of binding centers for the mediator in the cholinoreceptor molecule, which is characterized by the Hill coefficient [4]. The aim of the present work was to compare the Hill coefficient for somatic extrasynaptic cholinoreceptors on neurons LPa3 and RPa3 in the common snail before and during post-tetanic increases in the cholinosensitivity of these cells. The Hill coefficient was calculated by constructing dose-response curves for the effects of acetylcholine (ACh) on neurons. Experiments were conducted using semi-intact “CNS-visceral sac” preparations from the common snail Helix lucorum. Methods for obtaining these preparations, applying acetylcholine, and recording transmembrane currents have been described in detail elsewhere [3]. Intracellular electrodes were made from Pyrex glass and filled with 2 M KCl or 2 M potassium acetate (electrode resistance was 20.61 ± 0.04 MΩ). The mean amplitude of the excitatory postsynaptic current was 18.0 ± 3.0 nA. Local iontophoretic application of ACh was to the dorsal surface of the cells from glass micropipettes filled with 1 M ACh chloride (Sigma, USA) and having a resistance of 37.29 ± 0.04 MΩ. Cationic phoretic currents were 0.1‐1.5 μA with injections lasting 0.2‐2 sec. The mean amplitude of the ACh response was 14.9 ± 1.6 nA, with a latency of 1.5 ± 0.2 sec. Electrical tetanic stimulation of the intestinal nerve was applied via Nichrome electrodes (resistance 200 MΩ) using square-wave current impulses (10.5 mA, 100 msec); tetanization lasted 1‐2 min (1‐2 per sec). The Hill coefficient was determined as the tangent of the angle of the slope on plots of the relationship between the magnitude of the response and concentration of agonist in double logarithmic coordinates at the lowest concentrations [4]. The measure of the quantity of ACh applied to membranes used in the present work was the strength of the phoretic current through the ACh-containing injection pipette. The size of the phoretic current was directly proportional to the quantity of acetylcholine released in strictly uniform conditions (phoresis time, micropipette resistance) in each individual experiment. Results were obtained from 37 neurons (17 LPa3 and 20 RPa3) in 37 preparations. Cell membrane potentials was ‐50.31 ± 0.05 mV. Recording and measurement of the amplitudes of ACh-evoked currents (ACh currents) were performed using a IBM-486 PC running the program CONAN 3.0. Results were analyzed statistically used in Excel, Stadia, and Statistica 4.3. The significance of effects was assessed using Student’s and Wilcoxon’s tests. The strength of the iontophoretic current was selected at the beginning of the experiment such that the amplitude of the ACh response was maximal. The interval between mediator injections was 10 min. The mean of the three maximal ACh responses was taken as 100%. The dose-response relationship was then determined for the effects of ACh: ACh was injected by phoretic currents of increasing magnitude (0.1‐1.5 μA). After control applications of ACh, experimental series were performed in which tetanic stimulation was applied to the intestinal nerve (1‐2 per sec, 1‐2 min).