Figure 2.

GABA_A receptor α5 subunit expression and α5 receptor-mediated tonic currents are upregulated in cortical pyramidal neurons from HCN1 knock-out mice. A, By qRT-PCR of cortical RNA, we found selective upregulation of GABA_A α5 subunit transcripts in HCN1 knock-out (KO) mice (*p < 0.05 vs WT); expression of α1–α4, α6, β1–β3, γ1–γ3, and δ subunits was unaltered. B, Coronal forebrain sections from wild-type (WT) and HCN1 knock-out mice were hybridized with ³³P-labeled cRNA probes for the GABA_A α5 subunit; expression was higher in cortex of HCN1 knock-out mice (arrow). Note that hippocampal expression of α5 subunit mRNA is comparatively high and not obviously altered in HCN1 knock-out mice. C, Coronal brain sections of cortex from wild-type and HCN1 knock-out mice were immunostained with antibodies to GABA_A α5 subunit (red; left) and NeuN (green; middle) and images were overlaid (right); note that α5 subunit immunoreactivity is higher in cortical layer 5 of the HCN1 knock-out than in that region of wild-type mice (arrows). D, High-power confocal images of cortical layer 5 (from C) illustrating the enhanced GABA_A α5 subunit immunofluorescence; note that immunoreactivity is not typically associated with cell somata but is present mostly in the neuropil. E, Voltage-clamp recordings of tonic GABAergic currents under control conditions and with 20 μm L-655,708 and 30 μm bicuculline in cortical pyramidal neurons from wild-type and HCN1 knock-out mice. F, Averaged L-655,708-inhibited currents and fractional contribution of α5 subunit currents to total (i.e., bicuculline-inhibited) tonic GABA_A current in control and HCN1 knock-out cells (*p < 0.05 vs WT).