Figure 5: Fmr1 KO prevents ethanol-induced altered GABABR expression. | Nature Communications

Figure 5: Fmr1 KO prevents ethanol-induced altered GABABR expression.

From: FMRP regulates an ethanol-dependent shift in GABABR function and expression with rapid antidepressant properties

Figure 5

(ac) Western blot analysis of GABABR1 and GABABR2 in wild type (WT) and Fmr1 KO C57BL/6 hippocampal synaptoneurosomes after vehicle (Veh; saline i.p., 30 min) or ethanol (ETOH; 2.5 g kg−1 i.p., 30 min) treatment. (a) Pseudocoloured representative western blots showing band intensity, and normalized optical densities to WT–vehicle are reported below each image (lookup table, below western blot). Western blots were normalized to the loading control, α-Tubulin. No change was found in b GABABR1 after ethanol treatment in either genotype as shown by ethanol/vehicle comparison. A significant increase in c GABABR2 expression was observed in WT mice after ethanol, but no change was observed in Fmr1 KO mice (shown as ethanol/vehicle). WT ETOH/Veh: GABABR1=1.15±0.21; GABABR2=1.22±0.01. Fmr1 KO ETOH/Veh: GABABR1=0.94±0.04; GABABR2=0.95±0.03. Experiment was repeated three times. Significance determined by Student’s t-test. Values represent mean±s.e.m. Representative immunofluorescent images (dg) and quantification summaries (h,i) of dendritic expression of GABABR1 and GABABR2 from WT and Fmr1 KO primary mouse hippocampal cultures normalized to MAP2. (h) GABABR1 expression was not changed in either genotype after 2-h treatment with vehicle (Veh; H2O), ethanol (ETOH; 30 mM), or ethanol and cycloheximide (30 mM ETOH+50 μM CHX). WT GABABR1: Veh=1.00±0.04, n=44 dendrites; ETOH=1.07±0.04, n=29 dendrites; ETOH+CHX=1.22±0.05, n=34 dendrites. Fmr1 KO GABABR1: Veh=1.09±0.03, n=72 dendrites; ETOH=1.12±0.03, n=41 dendrites; ETOH+CHX=1.42±0.07, n=43 dendrites. (i) GABABR2 expression in WT neurons increased after ethanol (ETOH; 30 mM, 2 h) compared with vehicle (Veh; H2O, 2 h) treatment, and was rescued with co-treatment of cycloheximide (CHX; 50 μM, 2 h). GABABR2 expression in Fmr1 KO dendrites was not significantly altered between neurons treated with Veh, ETOH, or ETOH+CHX. WT GABABR2: Veh=1.00±0.03, n=41 dendrites; ETOH=1.28±0.06, n=40 dendrites; ETOH+CHX=0.99±0.05, n=33 dendrites. Fmr1 KO GABABR2: Veh=1.46±0.05, n=73 dendrites; ETOH=1.56±0.04, n=45 dendrites; ETOH+CHX=1.63±0.07, n=36 dendrites. Significance determined by two-way analysis of variance with Tukey’s post test. Value represent mean±s.e.m. Scale bars, 5 μm. Uncropped version of western blots, with size markers are available in Supplementary Fig. 7c.

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