Zhang-Shu B.S. Med Li

Background: Glutamatergic neurons in the supramammillary nucleus (SuM) have been recently identified as a key node in arousal system, yet their role in regulating general anesthesia remains unclear. The aim of the current study is to examine the role of the glutamatergic supramammillary neurons and their projections to the medial septum in mediating the effects of isoflurane anesthesia. Methods: Fiber photometry recording was used to determine the changes in calcium signals of glutamatergic neurons in the SuM during isoflurane anesthesia. Optogenetic and chemogenetic approaches were employed to manipulate SuM glutamatergic neuron activity, and the effects on cortical activity, behavioral responses, and physiologic parameters—including pupil diameter, respiratory rate, and blood pressure—were examined in anesthetized mice. Both male and female mice were used in this study. Results: The activities of SuM glutamatergic neurons decreased during isoflurane anesthesia and recovered after the emergence. Optogenetic activation of these neurons enhanced cortical activity, decreasing electroencephalogram delta power (mean ± SD, prestimulation vs. stimulation: 51.35 ± 7.26% vs. 32.08 ± 10.48%, n = 8, P = 0.002) and burst suppression ratio (81.82 ± 7.83% vs. 44.53 ± 28.62%, n = 8, P = 0.002). Furthermore, optogenetic activation altered physiologic parameters including enlarged pupil diameter (prestimulation vs. stimulation: 1.05 ± 0.08% vs. 1.95 ± 0.46%, n = 8, P < 0.001), increased respiratory rate (0.98 ± 0.08% vs. 1.57 ± 0.39%, n = 10, P < 0.001) and elevated blood pressure and induced behavioral responses including increased arousal scores and accelerated emergence (light off vs. light on, 171.40 ± 56.39 s to 59.88 ± 27.18 s, n = 8, P = 0.007). Moreover, chemogenetic activation produced similar effects, whereas inhibition led to opposite effects. Finally, optogenetically activating SuM glutamatergic terminals projecting to the medial septum mimicked the effects of activating SuM glutamatergic soma and increased the activity of medial septum glutamatergic neurons. Conclusions: This study identifies glutamatergic neurons of the SuM as key neural substrates regulating isoflurane anesthesia and facilitating emergence through their projections to the medial septum.

Background: Recent evidence indicates that general anesthesia and sleep–wake behavior share some overlapping neural substrates. γ-Aminobutyric acid–mediated (GABAergic) neurons in the central amygdala have a high firing rate during wakefulness and play a role in regulating arousal-related behaviors. The objective of this study was to investigate whether central amygdala GABAergic neurons participate in the regulation of isoflurane general anesthesia and uncover the underlying neural circuitry. Methods: Fiber photometry recording was used to determine the changes in calcium signals of central amygdala GABAergic neurons during isoflurane anesthesia in Vgat-Cre mice. Chemogenetic and optogenetic approaches were used to manipulate the activity of central amygdala GABAergic neurons, and a righting reflex test was used to determine the induction and emergence from isoflurane anesthesia. Cortical electroencephalogram (EEG) recording was used to assess the changes in EEG spectral power and burst–suppression ratio during 0.8% and 1.4% isoflurane anesthesia, respectively. Both male and female mice were used in this study. Results: The calcium signals of central amygdala GABAergic neurons decreased during the induction of isoflurane anesthesia and were restored during the emergence. Chemogenetic activation of central amygdala GABAergic neurons delayed induction time (mean ± SD, vehicle vs. clozapine-N-oxide: 58.75 ± 5.42 s vs. 67.63 ± 5.01 s; n = 8; P = 0.0017) and shortened emergence time (385.50 ± 66.26 s vs. 214.60 ± 40.21 s; n = 8; P = 0.0017) from isoflurane anesthesia. Optogenetic activation of central amygdala GABAergic neurons produced a similar effect. Furthermore, optogenetic activation decreased EEG delta power (prestimulation vs. stimulation: 46.63 ± 4.40% vs. 34.16 ± 6.47%; n = 8; P = 0.0195) and burst–suppression ratio (83.39 ± 5.15% vs. 52.60 ± 12.98%; n = 8; P = 0.0003). Moreover, optogenetic stimulation of terminals of central amygdala GABAergic neurons in the basal forebrain also promoted cortical activation and accelerated behavioral emergence from isoflurane anesthesia. Conclusions: The results suggest that central amygdala GABAergic neurons play a role in general anesthesia regulation, which facilitates behavioral and cortical emergence from isoflurane anesthesia through the GABAergic central amygdala–basal forebrain pathway.