Initially expressed early rat embryonic GABA(A) receptor Cl- ion channels exhibit heterogeneous channel properties

We have studied the earliest expression of GABA-induced CI- channels in the rat embryonic dorsal spinal cord (DSC) using in situ hybridization, immunocytochemistry, flow cytometry and electrophysiology. At embryonic day 13 (E13) cells in the dorsal region are still proliferating. In situ hybridization consistently showed transcripts encoding only three GABAA receptor subunits (alpha4, beta1 and gammal); immunocytochemistry both in tissue sections and in acutely isolated cells in suspension demonstrated the expression of the corresponding proteins and also revealed staining for other subunits (alpha2, alpha3, beta3, gamma2). In patch-recordings performed in cells acutely isolated from the dorsal cord, responses to GABA were detected in 356 out of 889 cells. GABA-evoked responses, which often displayed the opening of a few channels, were mediated by CI- ions, were inhibited by bicuculline and picrotoxin, and potentiated by benzodiazepines. Taken together, these observations indicate that CI- channels likely involve GABAA type receptors. Fluctuation analysis revealed channel kinetics consisting of three exponential components (Ts: approximately 1,9 and 90 ms) and a wide variety of inferred unitary conductance values, ranging between 4 and 40 pS. A comparison of these results with observations in other, later embryonic cell types and recombinant receptors suggests that most of the earliest E13 DSC GABAA receptors may include alpha3 subunit. These GABAA receptor Cl- channels may be activated physiologically as both GABA synthesizing enzymes and GABA are present in the E13 dorsal cord.