Internal perfusion studies demonstrating GABA-induced chloride responses in frog primary afferent neurons

Abstract

Cl- current in gamma-aminobutyric acid (GABA)-sensitive neurons of the frog dorsal root ganglia was separated from other ionic components (i.e., Na+, Ca2+, and K+ currents) using a suction pipette technique, which allowed internal perfusion and current clamp. The GABA-induced depolarization response increased slightly on substituting Na+ by tris(hydroxymethyl)amino-methane+ or Ca2+ by Mg2+ in the external solution. Additional replacement of external and internal K+ with Cs+ further enhanced the GABA response. The GABA response was virtually unaffected when the internal perfusate contained, in addition to 60 mM Cl-, large organic anions such as isethionate-, aspartate-, and citrate- (each 70 mM) as well as aspartate- and GABA (each 35 mM). The reversal potential of GABA-induced Cl- response (EGABA) was equal to Cl- equilibrium potential (ECl) and behaved as a simple Cl- electrode following changes of external and internal Cl- concentrations. These observations indicate the adequacy of internal perfusion. The GABA-induced Cl- response increased in a sigmoidal dose-dependent manner, in which the threshold GABA concentration was around 10(-7) M and the GABA response reached ECl at 10(-4) M. When GABA concentration was higher than 6 X 10(-6) M, the responses were always accompanied by a rapid desensitization.