Downregulation of M-channels in lateral habenula mediates hyperalgesia during alcohol withdrawal in rats

Abstract

Hyperalgesia often occurs in alcoholics, especially during abstinence, yet the underlying mechanisms remain elusive. The lateral habenula (LHb) has been implicated in the pathophysiology of pain and alcohol use disorders. Suppression of m-type potassium channels (M-channels) has been found to contribute to the hyperactivity of LHb neurons of rats withdrawn from chronic alcohol administration. Here, we provided evidence that LHb M-channels may contribute to hyperalgesia. Compared to alcohol naïve counterparts, in male Long-Evans rats at 24-hours withdrawal from alcohol administration under the intermittent access paradigm for eight weeks, hyperalgesia was evident (as measured by paw withdrawal latencies in the Hargreaves Test), which was accompanied with higher basal activities of LHb neurons in brain slices, and lower M-channel protein expression. Inhibition of LHb neurons by chemogenetics, or pharmacological activation of M-channels, as well as overexpression of M-channels' subunit KCNQ3, relieved hyperalgesia and decreased relapse-like alcohol consumption. In contrast, chemogenetic activation of LHb neurons induced hyperalgesia in alcohol-naive rats. These data reveal a central role for the LHb in hyperalgesia during alcohol withdrawal, which may be due in part to the suppression of M-channels and, thus, highlights M-channels in the LHb as a potential therapeutic target for hyperalgesia in alcoholics.

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Chemogenetic inhibition of LHb neurons alleviates hyperalgesia of rats withdrawn from chronic repeated voluntary ethanol drinking.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. N_rat = 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 (C,D) Representative traces (C) and summary data (D) show increased spontaneous firing of LHb neurons in ethanol-withdrawn rats.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 F_2,29 = 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; N_rat = 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, N_rat = 8/group; For 24 h, Unpaired t-test, t = 2.587, *p < 0.05, N_rat = 8/group.

Figure 2

Negative correlation between Paw withdrawal latency (PWL) and Ethanol intake. The changes in PWL induced by LHb manipulation correlated significantly with changes in ethanol intake. Data are presented from rats in GFP/hm4Di expressed group (A,B) and YFP/KCNQ3 expressed group (C) in the LHb. R² and p values are indicated in the plot. (A,B) N_rat = 8/group, (C) N_rat = 8–9/group.

Figure 3

Chemogenetic activation of the LHb induces thermal hyperalgesia in Naïve rats. (A,B) Representative figures (A) and pooled data (B) showing that c-fos expression in the LHb is increased after CNO systemic application in hM3Dq overexpressed rats. Unpaired t-test, t = 4.891, **p < 0.01. N_rat = 4–5/group. (C) Bath-applied CNO (10 μM) sharply increased the firing rate of LHb neurons infected with AAV-CaMKIIa-hM3Dq-mCherry viruses. (D) After systemic CNO injection (1 mg/kg, i.p.), rats infected with hM3Dq showed a decreased paw withdrawal latency than those infected with eGFP. Two-way ANOVA, for group F_2,30 = 3.583, P = 0.0402, for Time F_4,120 = 4.534, P = 0.0019, post-hoc: *p < 0.05, **p < 0.01 vs GFP + CNO; ^#p < 0.05, ^##p < 0.01 vs. hm3Dq+Veh; N_rat = 9–12/group.

Figure 4

Intra-LHb M-channel activators reduces thermal hyperalgesia in EtOH-WD rats. (A–C) Representative expression of  KCNQ2 (A), KCNQ3 (B) and pooled results (C) of Western blots show the reduced KCNQ2 and KCNQ3 expression in the LHb from ethanol-withdrawn rats compared to CTRL rats. Note an even greater loss of KCNQ3 expression in the EtOH-WD rats. Unpaired t-test, p < 0.05, ***p < 0.001. N_rat = 6/group. Full-length blots are presented in Supplementary Fig. 1. (D) Cresyl violet-stained brain sections show the accurate guide cannula placements above the LHb. Scale bar = 1.0 mm. (E, F) Intra-LHb retigabine and ICA-27243 increased the paw withdrawal latency (PWL) in EtOH-WD rats (Two-way ANOVA, for Drug F_2,35 = 0.0017, P = 0.0017, for Time F_4,140 = 0.6.256, P = 0.0001, post-hoc: **p < 0.01 vs. Retigabine, ^#p < 0.05, ^###p < 0.001 vs. ICA-27243, N_rat = 19(aCSF), 12(Retigabine), 7(ICA-27243).

Figure 5

Overexpression of KCNQ3 in the LHb of EtOH-WD rats attenuated thermal hyperalgesia and ethanol consumption. (A) KCNQ3-YFP expression after viral vector injection and immunofluorescence of the neuronal marker, NeuN. A Strong signal of KCNQ3-YFP overlaps with NeuN. Scale bar: 10 μm. (B) Example and pooled results of Western blots show the increased KCNQ3 expression in the LHb from EtOH-WD rats injected with KCNQ3-eYFP viruses compared to that injected with eYFP control viruses. Unpaired t-test, t = 3.18, *p < 0.05, N_rat = 4/group. Full-length blots are presented in Supplementary Fig. 1. (C) CCD camera captured IR (left) and ET-DSRed filtered fluorescence (right) image of the LHb neuron after viral injection for patch-clamp recording. (D,E) Sample traces (D) and pooled data (E) showing increased inward current relaxations in neurons of EtOH-WD rats after KCNQ3-eYFP virus injection. Two-way ANOVA, for YFP/KCNQ F_1,17 = 5.485, P = 0.0316, post-hoc: **p < 0.01. N_cell = 9–10/group. Scale bar: 200 pA and 200 ms. (F) Paw withdrawal latency was significantly increased after KCNQ3 overexpression in the LHb. Two-way ANOVA, for Before/After F_1,16 = 9.211, P = 0.0079, for Interaction F_1,16 = 16.06, P = 0.0010, post-hoc: ***p < 0.001 vs. Before, ^#p < 0.05 vs. YFP, N_rat = 8–10/group. (G) KCNQ3-eYFP-overexpressing rats showed reduced ethanol intake relative to eYFP-overexpressing rats. Unpaired t-test, t = 2.342, *p < 0.05. N_rat = 8–9/group.