Ischemic insult to cerebellar Purkinje cells causes diminished GABAA receptor function and allopregnanolone neuroprotection is associated with GABAA receptor stabilization

Abstract

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down-regulation of GABA(A) receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen-glucose deprivation (OGD) caused a decline in functional GABA(A) receptors, within the first hour of re-oxygenation. Decreased amplitude of miniature inhibitory post-synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABA(A) receptors and quantitative Western blot analysis demonstrated the loss of GABA(A) receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABA(A) receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABA(A) receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia-induced decline in GABA(A) receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABA(A) receptors in PC, whereas ALLO prevents the decline in GABA(A) receptors and protects against ischemia-induced damage. Thus, interventions which prevent ischemia-induced decline in GABA(A) receptors may represent a novel neuroprotective strategy.

Fig.1. Oxygen-glucose deprivation causes a significant reduction in synaptic and total GABA_A receptor current

A.) Representative trace depicting response of a single cell to 1s applications of increasing concentrations of GABA, indicated by solid bar. B.) Concentration-response relationships for control and OGD cells. Each concentration-response relation was normalized to individual cell's maximal current response, data presented are the average of 5–6 cells. C.) Representative responses to 1s application of 1mM GABA from control (left) and OGD (right). D.) Quantification of GABA_A receptor current density. E.) Representative traces of mIPSCs, recorded in the presence of CNQX and TTX under control (top) and OGD (bottom) conditions. F.) Quantification of mIPSC amplitude. OGD treated cells exposed to 2hr OGD and data collected within initial 1hr of re-oxygenation. The number of experiments is indicated in each bar. Data are mean ± SEM. * P < 0.05 vs. control cells.

Fig.2. Oxygen-glucose deprivation causes a rapid and sustained loss of GABA_A receptor protein that can be prevented by ALLO

A.) Representative Western blot for GABA_A receptor α₁ subunit protein from a control cell and OGD cells after 0, 30, 60, or 180 minutes re-oxygenation, indicated above each lane. B.) Representative Western blot for GABA_A receptor α₁ subunit protein from control, OGD and OGD+ALLO treated samples following 1hr re-oxygenation. ALLO cells received 15min pre-treatment with 10nM or 1µM ALLO, and ALLO remained present during OGD and re-oxygenation. β-actin loading control shown in lower panel C.) Quantification of normalized GABA_A receptor α₁ subunit protein, normalized to β-actin loading control and expressed as percent of average control level. The number of experiments is indicated in each bar. Data are mean ± SEM. * P < 0.05 vs. control cells.

Fig.3. ALLO prevents the oxygen-glucose deprivation induced loss of GABA_AR current but diazepam does not

OGD treated cells exposed to 2 hr OGD and data collected within initial 1 hr of re-oxygenation. A.) Representative responses to 1s application of 1mM GABA from a control, OGD and OGD+ALLO cell. ALLO cells received 15 min pre-treatment with 10 nM ALLO, and ALLO remained present during OGD and removed prior to recording. B.) Quantification of measured current density for conditions in A. C.) Representative current responses to 1s application of 1mM GABA from a control, OGD, and OGD+DZP cell. DZP cells received 15min pre-treatment with 2µM DZP, and DZP remained present during OGD and removed prior to recording. B.) Quantification of measured current density for conditions in C. The number of experiments is indicated in each bar. Data are mean ± SEM. * P < 0.05 vs. control cells.

Fig.4. ALLO protects cerebellar PC against global cerebral ischemia in vivo

A.) Representative FluoroJade B staining of cerebellar section from a vehicle treated mouse 48hr after CA/CPR. Injured PC stain bright green and red box indicates region expanded to right. GC= granule cell layer, PC= Purkinje cell layer, ML= molecular layer. B.) Representative FluoroJade B staining of cerebellar section from an ALLO treated mouse 48hr after CA/CPR. Injured PC stain bright green and red box indicates region expanded to right. C.) Quantification of ALLO protection of cerebellar PC. Percentage of FluoroJade positive PC determined by dividing the number of bright green PC by total number of PC in the section. The number of experiments is indicated in each bar. Data are mean ± SEM. * P < 0.05 vs. control cells.

Fig.5. ALLO prevents ischemia-induced decline in cerebellar GABA_A receptor α₁ protein

A.) Representative Western blot of cerebellar GABA_A receptor α₁ subunit protein from control (sham), vehicle and ALLO treated mice 48hr after CA/CPR. β-actin loading control shown in lower panel B.) Quantification of effect of ALLO on ischemia-induced decline in GABA_A receptor α₁ subunit protein, normalized to β-actin loading control and expressed as percent of control level. The number of experiments is indicated in each bar. Data are mean ± SEM. * P < 0.05 vs. control cells.

Fig.6. ALLO prevents ischemia-induced decline in PC GABA_A receptor α₁ protein

A.) Representative staining with GABA_A receptor α₁ subunit antibody of cerebellar section from sham operated mouse. B,C.) Representative staining with GABA_A receptor α₁ subunit antibody of cerebellar sections from vehicle (B) or ALLO (C) treated mice 48hr after CA/CPR. GC= granule cell layer, PC= Purkinje cell layer, ML= molecular layer.