Administration of acute and chronic EtOH induces an increase in GABA_AR α2 and γ1 subunit surface levels. (A) Representative Western blots for α2, γ1, and β-actin after cell-surface cross-linking (left) and quantification (right) 24 hours after a single intoxicating dose of EtOH in the DG. MANOVA, Wilks’ λ = 0.226, P = 0.0055, n = 5. tot, amount of total protein; int, amount of internal protein. (B) Quantification of the total amount of α2 and γ1 protein measured in DG 24 hours after EtOH intoxication. Wilks’ λ = 0.554, P = 0.126, n = 5. ctrl, control. (C) Representative Western blots for α2, γ1, and β-actin after cell-surface cross-linking (left) and quantification (right) 24 hours after a single intoxicating dose of EtOH in the CA1. Wilks’ λ = 0.443, P = 0.0255, n = 6. (D) Quantification of the total amount of α2 and γ1 protein measured in CA1 24 hours after EtOH intoxication. Wilks’ λ = 0.478, P = 0.0754, n = 5. (E) Representative Western blots for α2, γ1, and β-actin after cell-surface cross-linking (left) and quantification (right) after CIE in the DG + CA1. Wilks’ λ = 0.246, P = 0.0073, n = 5. (F) Quantification of the total amount of α2 and γ1 protein measured after CIE in DG + CA; tot, total amount of protein; int, intracellular protein content. Wilks’ λ = 0.457, P = 0.141, n = 4. Controls are set as 1.0%. Data are mean ± S.E.M. *Significant difference (P < 0.05, calculated within the MANOVA model) between the treatment versus the control.

γ1-Receptors contain α2 and β1 subunits and bind to gephyrin. Solubilized membrane proteins from DG+CA1 slices after CIE were co-immunoprecipitated with antibodies against A. γ1, γ2, or IgG and immunoblotted with antibodies against α1, α2, α4, α5, γ1, and γ2 or B. β1, β2, β3, or IgG and immunoblotted with antibodies against γ1, γ2, β1, β2 and β3 or C. γ1, γ2, and IgG and immunoblotted with antibodies against gephyrin, γ1, and γ2 on the same blot. For C, co-IP experiments were performed after membrane proteins were reversibly cross-linked in acute slices (see Material and Methods). Note, that the β1 antibody gives a signal with β2 and β3; and β3 with β1.

A second pulse of EtOH reveals further changes in GABA_AR physiology, pharmacology, and plasticity, correlated with anxiety-like behavior. (A) “Two-pulse” experimental protocol. Treatment regimen is indicated by black arrows. 1E: First EtOH oral gavage (5 g/kg); 2E: second EtOH gavage. Gray arrows show time points when patch-clamp recordings from DGCs in slices were performed. 1E1h: 1 hour after first EtOH gavage; 1E48h: 48 hours after first EtOH; 2E1h: 1 hour after second EtOH gavage; 2E48h: 48 hours after second EtOH. (B) Effects of acute EtOH (50 mM) applied in the recording chamber on I_tonic and mIPSCs. High chloride (135 mM CsCl, refer to Materials and Methods section) patch solution was used. The cells were whole-cell voltage-clamped at −70 mV. Gray dashed line is the basal I_tonic level after picrotoxin (PTX, 100 μM) application. Representative recordings are shown (left) with the averaged mIPSCs (right) before (black trace) and during (gray trace) EtOH perfusion. ctrl, control (drinking water). (C) Summary of acute EtOH-induced changes in I_tonic and mIPSCs. Gray dashed line represents E0 (set as 100%). *P < 0.05, significant difference between EtOH treatment versus control in I_tonic; #, P < 0.05, significant difference between EtOH treatment versus control in mIPSC area (n = 5 or 6/group), one-way ANOVA followed by Dunnett’s multiple comparison method comparing three treatment groups with one control group. (D) Representative Western blots for GABA_AR α4 and γ1 subunits with their β-actin signals (all from the same blot) after cell-surface cross-linking from control (ctrl), 1E48h, 2E1h, and 2E48h. tot, amount of total protein; int, amount of internal protein. (E) Quantification of surface levels measured by cross-linking experiments. Data are mean ± S.E.M., n = 4–10 rats. *P < 0.05 significant decrease versus 1E48h and 2E48h, one-way ANOVA, followed by Holm-Sidak’s (pairwise) multiple comparisons test. (F) Anxiety assayed by elevated plus maze (EPM, n = 6/group). Open bars are the time rats spent in open arms (% of total 5-minute monitor time); solid bars are the time spent in closed arms in respective treatment groups. *P < 0.05; time spent in open arm of EtOH treatment groups versus control group (drinking water), one-way ANOVA followed by Dunnett’s multiple comparison method comparing the four treatment groups with a single control. Note that % time spent in the closed arms is complementary to that in the open arms. Significant % time increase in open arms is equivalent to significant % time decrease in closed arms. Therefore, we omitted statistical tests on time spent in closed arms.

Pattern analysis reveals different kinetic patterns of mIPSCs after CIE versus CIV and α4KO versus WT. (A) Sample recordings of mIPSCs from DGCs. (B) Classifications of patterns of mIPSCs. a, b, c, and d: Averaged mIPSC patterns during recordings before (solid line) and after (dotted line) EtOH application on the hippocampal slice. All averaged mIPSC event patterns were detected by templates from recordings from CIV and CIE rats or α4KO and WT mice using the optimally scaled template algorithm in software DataView (V9.3). The event no. ratios (abundance) of classified mIPSC patterns are shown in Table 1. The scale is for all panels. (C) Responses of mIPSCs charge transfer (area) to acute EtOH application in DGCs. Averaged mIPSC area (mean ± S.E.M.) of different patterns before (empty columns) and after (solid columns) EtOH application. CIV (control); α4KO: α4 subunit knockout mice. E0: without EtOH; E50, in the presence of 50 mM EtOH. *P < 0.05, the area of a pattern in the presence of 50 mM EtOH is statistically different from that of 0 EtOH (two-way repeated measures ANOVA followed by Sidak’s method for pairwise multiple comparisons). n = 5 neurons from three rats/group. n = 4 neurons from two mice/group.