Expression of heteromeric glycine receptor-channels in rat spinal cultures and inhibition by neuroactive steroids

Ionotropic glycine receptors were studied in cultured spinal cord neurons prepared from 17-day-old rat embryos, using whole-cell patch clamp electrophysiology. Glycine receptors of 3–17 days in vitro were characterized via subtype-specific channel blockade by micromolar picrotoxin and cyanotriphenylborate, as well as nanomolar strychnine. Potentiation by nanomolar tropisetron indicated coexpression of β with α subunits. The neuroactive steroids pregnenolone sulfate and dehydroepiandrosterone sulfate, as well as alphaxalone and its 3β epimer betaxalone inhibited the chloride current with IC50 values of 19, 46, 16 and 208 μM, respectively, with no potentiation. Reverse transcription polymerase chain reaction and immunocytochemistry demonstrated mRNAs and proteins of α1, α2, α3 and β subunits in rat spinal cord cultures. In conclusion, neuroactive steroids, both positive and negative modulators of γ-aminobutyric-acidA receptors, inhibited heteromeric glycine receptors at micromolar concentrations.