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 6

PFC-DMH-Pef/VLPO inactivation counteracts AM281’s effects on arousal.

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PFC-DMH-Pef/VLPO inactivation counteracts AM281’s effects on arousal. (A...
(A) The AAV-DREADD-mCherry employs the FLEX Switch strategy utilizing 2 pairs of heterotypic, antiparallel loxP-type recombination sites to achieve Cre-mediated transgene inversion and expression. ITR, inverted terminal repeat; phSyn1, promoter of human synapsin I; WPRE, woodchuck hepatitis posttranscriptional regulatory element; Poly (A), polyadenylic acid tail. (B) Illustration of intra-VLPO and intra-DMH injection of PRV-Cre and DREADD, respectively, and CNO activation of DREADD. (C) Coronal brain section shows red mCherry fluorescence in bilateral DMH after intra-DMH and intra-VLPO injection of DREADD and PRV-Cre, respectively. Scale bar: 500 μm. (D) Representative trace (left) and summary histogram (right) show that bath application of CNO induces rapid hyperpolarization of the membrane potential and decreases the firing rate in red fluorescence–labeled DMH neurons after intra-VLPO and intra-DMH injection of PRV-Cre and AAV-hM4Di-mCherry, respectively. (E–G) Rats received an i.p. injection of vehicle (Veh) or CNO with or without AM281 injection (3 mg/kg, i.p.) at 1–2 weeks after receiving both intra-DMH (E and F) or intra-PFC (G) injection of AAV-mCherry (Control) or AAV-hM4Di-mCherry (hM4Di) and intra-VLPO (E), intra-Pef (F), or intra-DMH (G) injection of PRV-Cre. The control rats receiving both CNO and AM281 show a significant decrease in recovery time (P < 0.05) relative to each of other 5 groups (E–G). All summary graphs show mean ± SEM; n, number of rats in each group. *P < 0.05 vs. baseline (Base) or Veh group, Student’s t test (D) or Tukey’s post-hoc test after 1-way ANOVA (E: F5,32 = 4.929, P < 0.01; F: F5,28 = 5.767, P < 0.01; G: F5,25 = 7.485, P < 0.01).