364

Brain Research. 141 (1978) 364-370 t~ EIsevierrNorth-Holland Biomedical Press

Muscimol, GABA and picrotoxin: effects on membrane conductance of a crustacean neuron

N. HORI*, K. I K E D A * and E. ROBERTS

Division of Neurosciences, City o! Hope National Medical Center. 1500 Last Duarte Road. Duarte. Calif. 91010 ,'U.S.,4.)

(Accepted September 7th, 1977}

Inhibitory synapses utilizing ~-aminobutyric acid (GABA) as transmitter play important roles in the nervous function of various invertebrate and vertebrate organisms. In order to develop a specific pharmacology of the GABA system, it is necessary to have a quantitatively responding test system in which effects of G A B A can be monitored. The crayfish stretch receptor provides a conveniently isolated. single-neuron preparation with inhibitory innervation suitable for quantitative intracellular study on postsynaptic membrane conductance in response to applied substances. We have previously investigated several chemicals which interact with the receptor site at this synapse and report herein a study of the comparative actions of muscimol and G A B A and of the effects of picrotoxin on these actions. Muscimol is an lsoxazole compound 4, structurally similar to GABA, which was originally isolated from the mushroom A m a n i t a muscarla, and which has an hallucinogenic action in man 25. The slowly adapting stretch receptor neuron with innervating nerves and attached receptor muscle was dissected from the second abdominal tergum o f Proc a m b a r u s clarkii and mounted in a chamber. The slowly adapting receptor muscle was clamped at both ends in order to maintain constant tension throughout the experiment. The tension was kept at the level where no depolarization of receptor neuron membrane was observed in van Harreveld's solution 22. The preparation was perfused with the solution with and without varying concentrations of test substances_ The pH of all solutions was kept between 7.4 and 7.6 with sodium bicarbonate or Tris. maleate buffers, and the temperature of the chamber and solutions employed was maintained between 19 and 21 °C. The neuron was impaled with two glass pipette microelectrodes filled either with 0.6 M K~SO4 or 1.5 M K-citrate. Constant hyperpolarizing current pulses of 500 msec duration were applied through one electrode every 2 sec. The applied current was displayed on an oscilloscope through an operational amplifier of the

* Present address: Department of Pharmacology, Dental School, Kyushu University, Fukuoka, Japan. ** To whom correspondence and reprint requests should be addressed.

365 current-voltage conversion type. The resulting potential across the membrane was detected by the other electrode and simultaneously displayed on the oscilloscope. The current-voltage relationship (i-v curve) was obtained by applying the current pulse of various magnitudes and recording the resulting voltage change. The input conductance was measured as the slope of the i-v curve, which was confirmed to be linear at the levels between the resting membrane potential and the hyperpolarized level by the applied current. In each instance, conductance measurements were made before and after application of the test solution, and the results were averaged. The latter values were subtracted from the conductances observed during the application of the test solution to obtain the value for conductance change produced by the solution. Typical results from a single experiment with muscimol, homomuscimol and GABA are shown in Fig. 1. By assuming the law of mass action behavior 3, a modification 14 of the Langmuir equation was employed : l Ag

I Ka Agmax. (1 i ~ )

where Ag max. is the maximal conductance change produced by the test substance: Ag, the conductance change at concentration C; Ka, the dissociation constant; and n, the number of molecules of substance interacting with each receptor unit. The results for GABA (similar results were obtained in 15 experiments) and for muscimol were

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Fig. 1. Dose-conductance change relationship in the postsynapic membrane of crayfish slowly adapting stretch receptor neuron produced by GABA (O), muscimol (×), and homomuscimol ( ) . The curves for muscimol and homomuscimol were theoretical, plotted on the assumption that one molecule of these substances was bound to one receptor complex, while that for GABA assumed two molecules per receptor site.

366 best approximated by theoretical curves, assuming n == 2 and n -- I. respectively. The part o f the curve available for h o m o m u s c i m o l also was best approximated by a theoretical curve in which n was assumed to be 1. Muscimol began to exert its effects on conductance at concentrations approximately one-tenth of those required for G A B A , the maximal conductance changes produced by both substances being the same. An examination o f the above equation shows that. if n is chosen properly, the plot of l / A g vs. 1 rCn should gwe a straight line. Such plots o f data from several experiments other than the one shown in Fig. l gave straight lines when n was assumed to be l for muscimol and 2 for G A B A . The latter results are consistent with the interpretation that two molecules o f G A B A and one o f muscimol are required to react with each receptor unit involved in the conductance increase and that both substances are interacting with the same unit. In further support o f this supposition, it was shown in a number o f experiments that the maximal conductance change produced by either G A B A or muscimol in a particular preparation was not exceeded when the other substance was added in varying concentrations. The conductance changes induced by the application o f either G A B A or muscimol were suppressed by picrotoxin. Fig. 2 shows the effect o f picrotoxin on the conductance change caused by G A B A . assuming n =-- 2 for G A B A . The conductance changes caused by various concentrations of G A B A in the presence of 4 l0 -6 M picrotoxin fall on a straight line. as do those without picrotoxin. Further. the lines intersect the ordinate at the same point ( I / A g max.), suggesting that picrotoxin is competitive with G A B A at the receptor site. The affinity is decreased in the presence o f picrotoxin, as shown by a decreased intercept ( l/Ka) on the abscissa. The conductance change caused by muscimol was similarly suppressed by picrotoxin. As shown in Fig. 3, the conductance changes caused by various concentrations of muscitool alone or in the presence o f 2 ~ 10 -6 M picrotoxin fall on straight lines when n is

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Muscimol, GABA and picrotoxin: effects on membrane conductance of a crustacean neuron.

364 Brain Research. 141 (1978) 364-370 t~ EIsevierrNorth-Holland Biomedical Press Muscimol, GABA and picrotoxin: effects on membrane conductance of...
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