Protein kinase Cdelta regulates ethanol intoxication and enhancement of GABA-stimulated tonic current

Abstract

Ethanol alters the distribution and abundance of PKCdelta in neural cell lines. Here we investigated whether PKCdelta also regulates behavioral responses to ethanol. PKCdelta(-/-) mice showed reduced intoxication when administered ethanol and reduced ataxia when administered the nonselective GABA(A) receptor agonists pentobarbital and pregnanolone. However, their response to flunitrazepam was not altered, suggesting that PKCdelta regulates benzodiazepine-insensitive GABA(A) receptors, most of which contain delta subunits and mediate tonic inhibitory currents in neurons. Indeed, the distribution of PKCdelta overlapped with GABA(A) delta subunits in thalamus and hippocampus, and ethanol failed to enhance tonic GABA currents in PKCdelta(-/-) thalamic and hippocampal neurons. Moreover, using an ATP analog-sensitive PKCdelta mutant in mouse L(tk(-)) fibroblasts that express alpha4beta3delta GABA(A) receptors, we found that ethanol enhancement of GABA currents was PKCdelta-dependent. Thus, PKCdelta enhances ethanol intoxication partly through regulation of GABA(A) receptors that contain delta subunits and mediate tonic inhibitory currents. These findings indicate that PKCdelta contributes to a high level of behavioral response to ethanol, which is negatively associated with risk of developing an alcohol use disorder in humans.

Figure 1.

Acute responses to ethanol in PKCδ^−/− mice. A, Ethanol induced much more ataxia in PKCδ^+/+ mice compared with PKCδ^−/− mice (n = 8 for each genotype; *p < 0.05 compared with PKCδ^+/+ mice at the same time by Tukey's tests). B, Ethanol-induced hypothermia was greater in PKCδ^+/+ mice compared with PKCδ^−/− mice (n = 8 for each genotype; *p < 0.05 compared with PKCδ^+/+ mice at the same time by Tukey's tests). C, Blood ethanol clearance after administration of 4.0 g/kg ethanol was similar in both genotypes (n = 12 for each genotype).

Figure 2.

Expression of PKCδ in mouse brain. A, B, Immunoperoxidase staining for PKCδ immunoreactivity in sagittal brain sections from PKCδ^+/+ (A) and PKCδ^−/− (B) mice. C–E, High-power micrographs of (C) thalamus, (D) thalamocortical fibers in layer IV of cerebral cortex, and (E) cerebellar cortex from a PKCδ^+/+ mouse. ML, Molecular layer; GCL, granule cell layer. F, G, PKCδ immunoreactivity was moderate in cell bodies of pyramidal neurons, dentate gyrus granule cells and molecular layer interneurons of the hippocampus (F), and in the central amygdala (CeA) (G). H, I, Scattered PKCδ immunoreactivity was observed in the caudate–putamen (CPu), but none was observed in the nucleus accumbens (NAc) or the ventral tegmental area (VTA). Scale bars: (in B) A, B, 1 mm; (in E) C–E, 50 μm; (in I) F–I, 250 μm.

Figure 3.

A–E, Ataxia induced by drugs that act at GABA_A and NMDA receptors. Mice were tested for their ability to remain for 3 min on a rotarod treadmill rotating at a constant velocity of 20 rpm before and after intraperitoneal injection of pentobarbital (A), pregnanolone (B), flunitrazepam (C), ketamine (D), or MK-801 (E). n = 8 (A, B, D) and n = 16 (C) for each genotype. In E, n = 7 for PKCδ^+/+ and n = 8 for PKCδ^−/− mice.

Figure 4.

Colocalization of GABA_A α4 and δ subunit immunoreactivity with PKCδ in mouse brain. A, Coronal sections through the hippocampus and thalamus showing immunoreactivity for PKCδ (red), α4 subunits (green), and their colocalization (yellow) in hippocampus and thalamus. B, Similar sections showing immunoreactivity for PKCδ (red), δ subunits (green), and their colocalization (yellow) in hippocampus and thalamus. C, Sections of cerebellar cortex showing PKCδ immunoreactivity in the Purkinje cell and molecular layers, which did not colocalize with δ subunit immunoreactivity present in the granule cell layer. Scale bars: A, B, 500 μm for low-power images, 50 μm for high-power images; C, 50 μm.

Figure 5.

Ethanol enhances tonic inhibitory GABA currents in neurons from PKCδ^+/+ but not PKCδ^−/− mice. A, Sample tonic current traces recorded in a thalamic relay neuron from a PKCδ^+/+ (left) and a PKCδ^−/− (right) mouse. These currents were completely blocked by the nonselective GABA_A receptor antagonist SR95531 (SR). B, Paired plots of tonic current amplitudes before and after the addition of 30 mm ethanol to the perfusate of thalamic neurons from PKCδ^+/+ (left) and PKCδ^−/− (right) mice. Filled circles (left) and squares (right) with error bars indicate mean ± SEM current values. C, Mean ± SEM values for tonic current amplitudes in PKCδ^+/+ (n = 6) and PKCδ^−/− (n = 12) thalamic relay (TR) neurons before (control) and after addition of 30 mm ethanol. D, Tonic current in PKCδ^+/+ (n = 9) and PKCδ^−/− (n = 7) dentate gyrus granule cell (DGGC) neurons and E, in PKCδ^+/+ (n = 5) and PKCδ^−/− (n = 9) ML interneurons (ML) before and after 30 mm ethanol. C–E, *p < 0.05 compared with the control condition in PKCδ^+/+ neurons by Bonferroni tests.

Figure 6.

PKCδ regulates ethanol sensitivity of α4β3δ GABA_A receptors expressed in L(tk⁻) cells. A, Western blot analysis of PKCδ and actin immunoreactivity in HEK293, Neuro2A, L(tk⁻), and CHO cells showing less PKCδ abundance in L(tk⁻) cells relative to actin when compared with CHO and HEK293 cells. B, Western blot showing overexpression of PKCδ in L(tk⁻) cells after transfection with as-PKCδ. C, D, Ethanol enhancement of EC₂₀ GABA-stimulated currents in L(tk⁻) cells that express α4β3δ receptors. *p < 0.05 compared with cells not transfected with as-PKCδ at the same concentration of ethanol by Bonferroni tests. E, Inhibition of as-PKCδ, a commercial mixture of PKC isozymes (PKC) (Calbiochem) and wild-type PKCδ by the PP1 analog 1NaPP1. Nonlinear regression analysis showed significant differences (< 0.0001) between log EC₅₀ values (M) for 1NaPP1 inhibition of as-PKCδ (−6.81 ± 0.02) versus wild type PKCδ (−4.06 ± 0.04) or the mixture of PKC isozymes (−4.59 ± 0.04). F, L(tk⁻) cells that express α4β3δ receptors and as-PKCδ were treated with 0.1 μm GABA and 30 mm ethanol. Ethanol reversibly enhanced the GABA-stimulated current and addition of 10 μm 1NaPP1 reduced this effect of ethanol