Figure 1
Chemogenetic inhibition of LHb neurons alleviates hyperalgesia of rats withdrawn from chronic repeated voluntary ethanol drinking. (A) Mean paw withdrawal latency is significantly shorter in rats at 24 h withdrawal from voluntary drinking ethanol in the intermittent two-bottle choice paradigm (EtOH-WD) in comparison to ethanol-naïve control rats (CTRL). Unpaired t-test, t = 3.9, ***p < 0.001. Nrat = 24/group. (B) The schematic of location of recorded neurons. A camera captured image of the coronal section containing the LHb (box) and CCD camera captured IR image of the LHb for lose cell-attached patch-clamping recording (right corner). (C,D) Representative traces (C) and summary data (D) show increased spontaneous firing of LHb neurons in ethanol-withdrawn rats. Unpaired t-test, t = 3.789, ***p < 0.001. Numbers of neurons are indicated. (E) hm4Di-mCherry expression in LHb neurons after viral vector injection and immunofluorescence of the neuronal marker NeuN. A strong hM4Di-mCherry expression overlaps with NeuN. (F) Bath-applied CNO (10 μM) sharply inhibited spontaneous firings of LHb neurons infected with AAV-CaMKIIa-hM4Di-mCherry viruses. (G) After systemic CNO injection (1 mg/kg, i.p.), EtOH-WD rats infected with hm4Di showed an increased paw withdrawal latency than those infected with eGFP. Two-way ANOVA, for group F2,29 = 4.021, P = 0.0288, post-hoc: *p < 0.05, **p < 0.01 vs GFP + CNO; #p < 0.05, ##p < 0.01 vs. hm4Di + Veh; Nrat = 8–12/group. (H,I) Pretreating hM4Di-rats with systemic injection of CNO (1 mg/kg, i.p.) reduced ethanol intake relative to saline injection, whereas eGFP hM3Dq mice were unaffected. For 2 h, Unpaired t-test, t = 2.796, *p < 0.05, Fig. 2D, Nrat = 8/group; For 24 h, Unpaired t-test, t = 2.587, *p < 0.05, Nrat = 8/group.
