Homeostatic regulation of synaptic excitability: tonic GABA(A) receptor currents replace I(h) in cortical pyramidal neurons of HCN1 knock-out mice

Abstract

Homeostatic control of synaptic efficacy is often mediated by dynamic regulation of excitatory synaptic receptors. Here, we report a novel form of homeostatic synaptic plasticity based on regulation of shunt currents that control dendritosomatic information transfer. In cortical pyramidal neurons from wild-type mice, HCN1 channels underlie a dendritic hyperpolarization-activated cationic current (I(h)) that serves to limit temporal summation of synaptic inputs. In HCN1 knock-out mice, as expected, I(h) is reduced in pyramidal neurons and its effects on synaptic summation are strongly diminished. Unexpectedly, we found a markedly enhanced bicuculline- and L-655,708-sensitive background GABA(A) current in these cells that could be attributed to selective upregulation of GABA(A) alpha5 subunit expression in the cortex of HCN1 knock-out mice. Strikingly, despite diminished I(h), baseline sublinear summation of evoked EPSPs was unchanged in pyramidal neurons from HCN1 knock-out mice; however, blocking tonic GABA(A) currents with bicuculline enhanced synaptic summation more strongly in pyramidal cells from HCN1 knock-out mice than in those cells from wild-type mice. Increasing tonic GABA(A) receptor conductance in the context of reduced I(h), using computational or pharmacological approaches, restored normal baseline synaptic summation, as observed in neurons from HCN1 knock-out mice. These data indicate that upregulation of alpha5 subunit-mediated GABA(A) receptor tonic current compensates quantitatively for loss of dendritic I(h) in cortical pyramidal neurons from HCN1 knock-out mice to maintain normal synaptic summation; they further imply that dendritosomatic synaptic efficacy is a controlled variable for homeostatic regulation of cortical neuron excitability in vivo.

Figure 1.

Tonic GABA_A receptor currents are increased in cortical pyramidal neurons from HCN1 knock-out mice. A, Tonic and synaptic GABA_A receptor currents in cortical pyramidal neurons from wild-type and HCN1 knock-out (KO) mice were recorded at a holding potential of −70 mV during application of isoflurane (0.6 mm) and bicuculline (30 μm). All-points histograms, shown at the left of each current trace, were obtained for 30–60 s epochs and fitted with Gaussian functions to obtain tonic current levels under different conditions. In this and other experiments, recordings of inward GABA_A currents were obtained in the presence of glutamate receptor blockers (CNQX, 10 μm; APV 50 μm) and using pipettes with elevated intracellular Cl. B, Total tonic GABA_A current was determined as the bicuculline-inhibited current, and quantified from Gaussian fits to all-points histograms. Averaged values of isoflurane-activated and bicuculline-inhibited currents were ∼2-fold larger in cells from HCN1 knock-out mice (see text).

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).

Figure 3.

Tonic GABA_A currents mediated by α1 and δ subunits are not increased in cortical pyramidal cells from HCN1 knock-out (KO) mice. A, B, Effects of zolpidem (A, 100 nm) and 5-THDOC (B, 100 nm) on tonic currents in cortical pyramidal neurons from wild-type and HCN1 knock-out mice. The drug-activated currents were quantified from Gaussian fits to all-points histograms, and averaged values were not different between genotypes (see text).

Figure 4.

GABA_A α5 subunit-mediated tonic currents are enhanced in cortical pyramidal cells from HCN1 knock-out mice under conditions of reduced GABA transporter activity with saturating GABA concentrations. A, Effect of the GABA transport inhibitor NO-711 (5 μm) on GABA currents in cortical neurons from wild-type (WT) and HCN1 knock-out (KO) mice. B, Summary data illustrating that averaged NO-711-activated currents (left) and the total bicuculline-inhibited currents during NO-711 application (right) are enhanced in cortical pyramidal neurons from HCN1 knock-out mice. (p < 0.05, n = 7). C, Effect of a fixed concentration of GABA (5 μm) in the presence of the GABA transport inhibitor NO-711 (5 μm) on tonic GABA currents in cortical neurons from wild-type and HCN1 knock-out mice. D, Averaged data illustrating that both the α5 subunit-mediated component of maximal tonic GABA_A current (left) and the total tonic GABA_A current (right) were larger in cortical pyramidal neurons from HCN1 knock-out mice. (p < 0.05, n = 9 and 6).

Figure 5.

Increased tonic GABA_A currents maintain normal EPSP summation properties in cortical pyramidal neurons from HCN1 knock-out mice. A, Reconstruction of a biocytin-filled cortical pyramidal neuron illustrating schematically the stimulating and recording configuration. B, Sample voltage traces of EPSPs obtained in cortical pyramidal neurons from wild-type (top) and HCN1 knock-out (bottom) mice in response to 40 Hz, 7 V stimulation under control conditions, after incubation in bicuculline (30 μm) or ZD-7288 (50 μm). EPSPs are aligned to initial membrane potential to highlight enhanced temporal summation of EPSPs associated with bicuculline or ZD-7288. C, Summation properties of evoked EPSPs (EPSP5/EPSP1 ratio) under control conditions and in the presence of 30 μm bicuculline or 50 μm ZD-7288 in wild-type (WT) or HCN1 knock-out (KO) mice. D, Enhancement of EPSP summation by bicuculline and ZD-7288 (percentage of control) in cells from wild-type and HCN1 knock-out mice. Note that whereas blocking HCN channels with ZD-7288 had a prominent effect on synaptic summation in cortical neurons from WT mice, EPSP summation was most strongly enhanced by blocking GABA_A currents in cells from HCN1 knock-out mice. (*p < 0.05 vs control).

Figure 6.

Effects of upregulated GABA_A currents on EPSP summation are observed in cortical pyramidal neurons from HCN1 knock-out mice with unperturbed intracellular Cl⁻. A, Cortical pyramidal neurons from wild-type (top) and HCN1 knock-out (KO) mice (bottom) were recorded using gramicidin perforated patch to preserve [Cl⁻]i; aligned voltage traces of evoked EPSPs obtained in response to 40 Hz, 7 V stimulation under control conditions, after incubation in bicuculline (30 μm) or ZD-7288 (50 μm). B, Averaged EPSP5/EPSP1 summation ratio (top) and percentage enhancement (bottom) in cells from wild-type (WT) and HCN1 knock-out mice under control conditions and in the presence of 30 μm bicuculline or 50 μm ZD-7288. As with whole-cell recordings, blocking HCN channels with ZD-7288 had a prominent effect on synaptic summation in cortical neurons from WT mice, whereas EPSP summation was most strongly enhanced by blocking GABA_A currents in cells from HCN1 knock-out mice. (*p < 0.05 vs control).

Figure 7.

Computer simulation and pharmacological manipulation recapitulates effects of increased tonic GABA_A currents on EPSP summation properties in cortical pyramidal neuron model. A, B, Simulation of EPSP summation properties (40 Hz) was obtained using an existing NEURON model of a cortical pyramidal cell (from Day et al., 2005), with Kir conductance set to zero and the membrane potential adjusted to −70 mV in all conditions using simulated DC. The existing model for the wild-type (WT) pyramidal neuron (A) was modified to include a small tonic GABA_A-like conductance (1/10 the size of HCN conductance); for the HCN1 knock-out (KO) cell simulation (B), the HCN conductance was decreased by 75% with a −10 mV shift in V _1/2 and the tonic GABA_A conductance was increased by a factor of 2.5. The HCN and GABA_A conductances were eliminated to mimic effects of ZD-7288 and bicuculline. C, Values obtained for EPSP5:EPSP1 (top) and percentage change in EPSP summation ratio (bottom). Note that effects of eliminating I _h and tonic GABA_A currents on EPSP summation were reversed in the simulations of wild-type and HCN1 cortical pyramidal neurons, reproducing results obtained experimentally. D, In pyramidal cells from wild-type mice, diazepam (1 μm) approximately doubled the tonic GABA_A current. E, EPSPs were evoked in wild-type cortical pyramidal neurons under control conditions (C), after blocking I _h with ZD-7288 (Z, 50 μm) and after activating tonic GABA_A current with diazepam (D, 1 μm) in the continued presence of ZD-7288. Top, Aligned voltage traces of evoked EPSPs under the indicated conditions. Bottom, Values obtained for EPSP5:EPSP1 (left) and percentage change in EPSP summation ratio (right). Note that the enhanced EPSP summation associated with block of I _h in wild-type neurons was reversed by pharmacologically activating tonic GABA_A currents.