Fig 2: l-Lactate reverses DAB-induced reduction in cocaine craving and plasticity-related proteins after prolonged withdrawal.(A) Experimental timeline. (B) The number of responses during drug seeking tests in rats injected with DAB (300 pmol per side) and l-lactate (100 nmol per side) into the CeA on WD30 (n = 8 per group). Two-way RM ANOVA (F2,21 = 9.885, P < 0.001), post hoc test, P < 0.001. (C) Western blot analysis of p-ERK1/2, t-ERK1/2, GluA1, GluA2, and GluA3 in the CeA of rats injected with vehicle + vehicle, DAB + vehicle, or DAB + l-lactate (n = 6 per group). p-ERK1/2, one-way ANOVA (F2,15 = 38.534, P < 0.001), post hoc test (vehicle + vehicle WD30 versus DAB + vehicle WD30, P < 0.001; DAB + vehicle WD30 versus DAB + l-lactate WD30, P < 0.001). t-ERK1/2, one-way ANOVA (F2,15 = 0.616, P = 0.553). GluA1, one-way ANOVA (F2,15 = 32.150, P < 0.001), post hoc test (vehicle + vehicle WD30 versus DAB + vehicle WD30, P < 0.001; DAB + vehicle WD30 versus DAB + l-lactate WD30, P < 0.001). GluA2, one-way ANOVA (F2,15 = 37.722, P < 0.001), post hoc test (vehicle + vehicle WD30 versus DAB + vehicle WD30, P < 0.001; DAB + vehicle WD30 versus DAB + l-lactate WD30, P < 0.001). GluA3, one-way ANOVA (F2,15 = 0.905, P = 0.426). Data are presented as mean ± SEM. ***P < 0.001. V + V, vehicle + vehicle group; D + V, DAB + vehicle group; D + L, DAB + l-lactate group.

Fig 3: OGD/R decreases GRIA1 and GRIA2 expression following OGD/R in male hippocampal slices, but OGD/R does not significantly affect female GRIA1 and GRIA2 expression in hippocampal slices. Utilizing rt-qPCR, we examined the expression of GRIA1 (GluA1), GRIA2 (GluA2), and GRIA3 (GluA3) with OGD/R treatment. (A) Expression of GRIA1 in male and female hippocampal slices with OGD/R treatment, normalized to normoxic control group and ActB (β-Actin) (N = 3). (B) Expression of GRIA2 in male and female hippocampal slices with OGD/R treatment, normalized to normoxic control group and ActB (β-Actin) (N = 3). (C) Expression of GRIA3 in male and female hippocampal slices with OGD/R treatment, normalized to normoxic control group and ActB (β-Actin) (N = 3). * p < 0.05; ** p < 0.01; ns denotes no significance. ANOVA with Sidak post hoc test comparing OGD/R values (2−ΔΔCt ) corresponding to normoxic control. Data are expressed as mean ± SEM.

Fig 4: Effect of general anesthesia on membrane expression levels of AMPA receptors in the cerebellum in aged App-KI mice. Mean (+ SEM) protein expression ratio (normalized with the HSP60 expression levels) of GluA1 (A), GluA2 (B), GluA3 (C) and GluA4 (D) subunits in the crude membrane fractions in each group, WT-Air (n = 3), WT-Des (n = 3), WT-Sev (n = 3), App-Air (n = 5), App-Des (n = 5) and App-Sev (n = 5) group, are shown. Individual values were normalized with the mean values of WT-Air group as 1. *p < 0.050; †p < 0.100

Fig 5: Disruption of astrocytic MCT4 in the CeA impairs incubation of cocaine craving.(A) Experimental timelines. (B) Western blot analysis of MCT2 and MCT4 in the CeA from rats injected with ODNs (n = 5 per group). MCT2, unpaired t test (t8 = 0.267, P = 0.796). MCT4, Mann-Whitney test (U = 0, P = 0.008). (C) The number of responses during drug seeking tests in rats with intra-CeA injections of ODNs on WD30 (n = 6 per group). Two-way RM ANOVA (F1,10 = 17.620, P = 0.002), post hoc test, P < 0.001. (D) Representative images of immunofluorescent staining of rAAV-GFaABC1D-shMCT4RNA-mCherry in the CeA. (Top) AAV, red; GFAP, green; DAPI, blue. (Bottom) AAV, red; NeuN, green; DAPI, blue. Scale bars, 500 μm (left) and 50 μm (right). (E) Representative traces of sEPSCs in CeA neurons from rats injected with rAAV-GFaABC1D-shMCT4RNA-mCherry or control virus. Scale bars, 200 ms and 20 pA. (F and G) sEPSC amplitude and frequency (n = 25 neurons for GFaABC1D-SCR group and n = 27 neurons for GFaABC1D-shMCT4RNA group). Amplitude, unpaired t test (t50 = 0.316, P = 0.753). Frequency, Mann-Whitney test (U = 187, P = 0.005). (H) Self-administration training. (n = 10 per group). (I) The number of responses during drug seeking tests in rats with intra-CeA injections of rAAV-GFaABC1D-shMCT4RNA-mCherry or control virus (n = 10 per group). Two-way RM ANOVA (F1,18 = 4.506, P = 0.048), post hoc test, P = 0.016. (J) Western blot analysis of MCT4, GluA1, GluA2, and GluA3 in the CeA from rats injected with rAAV-GFaABC1D-shMCT4RNA-mCherry or control virus (n = 6 per group). MCT4, unpaired t test (t10 = 3.362, P = 0.007). GluA1, Mann-Whitney test (U = 5, P = 0.041). GluA2, unpaired t test (t10 = 3.279, P = 0.008). GluA3, unpaired t test (t10 = 3.975, P = 0.003). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.