Endocannabinoid signaling in hypothalamic circuits regulates arousal from general anesthesia in mice
Haixing Zhong, Li Tong, Ning Gu, Fang Gao, Yacheng Lu, Rou-gang Xie, Jingjing Liu, Xin Li, Richard Bergeron, Lisa E. Pomeranz, Ken Mackie, Feng Wang, Chun-Xia Luo, Yan Ren, Sheng-Xi Wu, Zhongcong Xie, Lin Xu, Jinlian Li, Hailong Dong, Lize Xiong, Xia Zhang
Haixing Zhong, Li Tong, Ning Gu, Fang Gao, Yacheng Lu, Rou-gang Xie, Jingjing Liu, Xin Li, Richard Bergeron, Lisa E. Pomeranz, Ken Mackie, Feng Wang, Chun-Xia Luo, Yan Ren, Sheng-Xi Wu, Zhongcong Xie, Lin Xu, Jinlian Li, Hailong Dong, Lize Xiong, Xia Zhang
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Research Article Neuroscience

Endocannabinoid signaling in hypothalamic circuits regulates arousal from general anesthesia in mice

Abstract

Consciousness can be defined by two major attributes: awareness of environment and self, and arousal, which reflects the level of awareness. The return of arousal after general anesthesia presents an experimental tool for probing the neural mechanisms that control consciousness. Here we have identified that systemic or intracerebral injection of the cannabinoid CB1 receptor (CB1R) antagonist AM281 into the dorsomedial nucleus of the hypothalamus (DMH) — but not the adjacent perifornical area (Pef) or the ventrolateral preoptic nucleus of the hypothalamus (VLPO) — accelerates arousal in mice recovering from general anesthesia. Anesthetics selectively activated endocannabinoid (eCB) signaling at DMH glutamatergic but not GABAergic synapses, leading to suppression of both glutamatergic DMH-Pef and GABAergic DMH-VLPO projections. Deletion of CB1R from widespread cerebral cortical or prefrontal cortical (PFC) glutamatergic neurons, including those innervating the DMH, mimicked the arousal-accelerating effects of AM281. In contrast, CB1R deletion from brain GABAergic neurons or hypothalamic glutamatergic neurons did not affect recovery time from anesthesia. Inactivation of PFC-DMH, DMH-VLPO, or DMH-Pef projections blocked AM281-accelerated arousal, whereas activation of these projections mimicked the effects of AM281. We propose that decreased eCB signaling at glutamatergic terminals of the PFC-DMH projection accelerates arousal from general anesthesia through enhancement of the excitatory DMH-Pef projection, the inhibitory DMH-VLPO projection, or both.

Authors

Haixing Zhong, Li Tong, Ning Gu, Fang Gao, Yacheng Lu, Rou-gang Xie, Jingjing Liu, Xin Li, Richard Bergeron, Lisa E. Pomeranz, Ken Mackie, Feng Wang, Chun-Xia Luo, Yan Ren, Sheng-Xi Wu, Zhongcong Xie, Lin Xu, Jinlian Li, Hailong Dong, Lize Xiong, Xia Zhang

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Figure 7

PFC-DMH-Pef/VLPO activation enhances arousal.

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PFC-DMH-Pef/VLPO activation enhances arousal. (A) A sample trace and sum...
(A) A sample trace and summary histogram show that CNO induces rapid depolarization of membrane potential and increases firing rate of DMH neurons with red mCherry fluorescence after intra-VLPO and intra-DMH injection of PRV-Cre and AAV-hM3Dq-mCherry (hM3Dq), respectively. (B–E, I, and J) Rats (B–E), vGLUT2-iCreERT2 mice (I), or vGLUT1-iCreERT2 mice (J) received an i.p. (B–E), intra-Pef (I), or intra-DMH injection (J) of vehicle or CNO 1–2 weeks after receiving intra-DMH (B and C) or intra-PFC (D, E, I, and J) injection of AAV-mCherry (Control) or AAV-hM3Dq-mCherry (hM3Dq) with or without intra-VLPO (B), intra-Pef (C), or intra-DMH (D and E) injection of PRV-Cre. The hM3Dq animals receiving CNO show a significant decrease in recovery time (P < 0.05 or P < 0.01) relative to each of the other 3 groups (B–D, I, and J), but the hM3Dq animals injected with AM281 (AM; 3 mg/kg, i.p.) or its vehicle do not show a significant difference (P = 0.3601) in recovery time (E). (F–H) After intra-DMH injection of hM3Dq into vGLUT2-iCreERT2 mice, the DMH neurons containing both vGLUT2-driven iCre and mCherry are visible as fluorescent-labeling cells. (F: scale bar: 100 μm). Sample traces (G) and summary histograms (H) show that with patch clamp recording of Pef neurons and stimulation of DMH input axons, isoflurane reduces and increases the first EPSC amplitude and PPR, respectively, which are reversed by CNO. All summary graphs show mean ± SEM; n, number of animals in each group. *P < 0.05 and **P < 0.01 vs. baseline or Veh, Student’s t test (A and E) or Tukey’s post-hoc test after 1-way ANOVA (B: F3,20 = 5.8893, P < 0.01; C: F3,16 = 6.328, P < 0.01; D: F3,22 = 12.65, P < 0.01; H: left, F2,15 = 9.045, P < 0.01; right, F2,15 = 7.492, P < 0.01; I: F3,16 = 8.044, P < 0.01; J: F3,32 = 11.77, P < 0.01).