Differential effects of two major neurosteroids on cerebellar and cortical GABA(A) receptor binding and function

Abstract

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABA(A)) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [(3)H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [(3)H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC(50)=150nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [(3)H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABA(A) receptors containing α6 and δ subunits. In contrast, [(3)H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC(50) ~80nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [(3)H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [(3)H]EBOB binding with EC(50) ~0.5μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1μM GABA in cerebellar granule cells, for 5α-THDOC with EC(50,1) ~16nM and EC(50,2) ~1.3μM. Differences in peak current enhancements in the absence minus presence of 0.1mM furosemide corresponding to α6ßδ GABA(A) receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABA(A) receptor populations.