INITIALLY EXPRESSED EARLY RAT EMBRYONIC GABA(A) RECEPTOR CL- ION CHANNELS EXHIBIT HETEROGENEOUS CHANNEL PROPERTIES

We have studied the earliest expression of GABA-induced Cl- channels i n the rat embryonic dorsal spinal cord (DSC) using in situ hybridizati on, immunocytochemistry, flow cytometry and electrophysiology. At embr yonic day 13 (E13) cells in the dorsal region are still proliferating. In situ hybridization consistently showed transcripts encoding only t hree GABA(A) receptor subunits (alpha(4), beta(1) and gamma(1)); immun ocytochemistry both in tissue sections and in acutely isolated cells i n suspension demonstrated the expression of the corresponding proteins and also revealed staining for other subunits (alpha(2), alpha(3), be ta(3), gamma(2)) In patch-recordings performed in cells acutely isolat ed from the dorsal cord, responses to GABA were detected in 356 out of 889 cells. GABA-evoked responses, which often displayed the opening o f a few channels, were mediated by Cl- ions, were inhibited by bicucul line and picrotoxin, and potentiated by benzodiazepines. Taken togethe r, these observations indicate that Cl- channels likely involve GABAA type receptors. Fluctuation analysis revealed channel kinetics consist ing of three exponential components (tau s: approximate to 1, 9 and 90 ms) and a wide variety of inferred unitary conductance values, rangin g between 4 and 40 pS. A comparison of these results with observations in other, later embryonic cell types and recombinant receptors sugges ts that most of the earliest E13 DSC GABAA receptors may include alpha (3) subunit. These GABAA receptor Cl- channels may be activated physio logically as both GABA synthesizing enzymes and GABA are present in th e E13 dorsal cord.