GABAergic Neurons in the Central Amygdala Promote Emergence from Isoflurane Anesthesia in Mice

Abstract

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.

Fig. 1.

The calcium signals of central amygdala (CeA) γ-aminobutyric acid–mediated (GABAergic) neurons change during isoflurane anesthesia. (A) Schematic diagram of the experimental setup for fiber photometry recording. (B) Schematic diagram of virus injection and optical fiber implantation. AAV-hSyn-DIO-GCaMP6m was injected into the CeA of Vgat-Cre mice, and optical fibers were implanted above the CeA. (C) Representative image showing the GCaMP6m fluorescence in the CeA of Vgat-Cre mice (scale bar, 250 μm). (D through F) The change in calcium signals of CeA GABAergic neurons during 1.4% isoflurane (Iso)-induced anesthesia. (D) Heatmap illustration of calcium signals aligned to the initiation and termination of isoflurane exposure. Each row plots one trial, and a total of eight trials are illustrated. Color scale at the right indicates ∆ F/F peri-event plot of the average calcium signals transients. (E) Time courses of calcium signals before, during, and after 1.4% isoflurane anesthesia (n = 8). Thick line indicates the mean, and area of shadow indicates SD. (F) Statistical chart of changes in calcium signal before, during, and after 1.4% isoflurane exposure (n = 8). (G) Heatmaps for the calcium signals of CeA GABAergic neurons during 1.4% isoflurane induced loss of righting reflex (LORR) (n = 6). (H) Quantification of calcium signal changes during the transition of LORR (n = 6). (I) Statistical results showing changes of calcium signals before and after LORR (n = 6). (J) Heatmaps for the calcium signals of CeA GABAergic neurons during recovery of righting (RORR) (n = 6). (K) Quantification of calcium signal changes during the transition of RORR (n = 6). (L) Statistical results showing changes of calcium signals before and after RORR (n = 6). Statistical comparisons were conducted using one-way repeated-measures ANOVA in F followed by the Bonferroni post hoc test. H and I were conducted using paired t tests. Data are presented as mean ± SD. *P < 0.05; **P < 0.01. BLA, basolateral amygdalar complex; LH, lateral hypothalamus; MGP, medial globus pallidus.

Fig. 2.

Chemogenetic activation of central amygdala (CeA) γ-aminobutyric acid–mediated (GABAergic) neurons delays the induction and accelerates emergence from isoflurane general anesthesia. (A) Experimental strategy for chemogenetic activation of CeA GABAergic neurons. AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry was injected into the CeA of Vgat-Cre mice. (B) Representative image showing the expression of AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry in the CeA of Vgat-Cre mice. (C) Representative images of mCherry/c-Fos immunofluorescence in the CeA after vehicle or clozapine-N-oxide (CNO) treatment. (D) Statistical results for the effects of activating CeA GABAergic neurons on induction time of 1.4% isoflurane (n = 8 for mCherry and hM3Dgroup). (E) Statistical results for the effects of activating CeA GABAergic neurons on the isoflurane concentrations at which hM3D mice loss of righting reflex (LORR) occurred (n = 11). (F) Dose–response curves showing the percentages of hM3D mice showing LORR with CNO or vehicle injection when isoflurane concentration was gradually increased (n = 11). (G) Statistical results for the effects of activating CeA GABAergic neurons on emergence time after isoflurane anesthesia (n = 8 for mCherry and hM3Dgroup). (H) Statistical results for the effects of activating CeA GABAergic neurons on the isoflurane concentrations at which hM3D mice occurred recovery of righting reflex (RORR; n = 11). (I) Dose–response curves showing the percentages of hM3D mice showing RORR with CNO or vehicle injection when isoflurane concentration was gradually decreased (n = 11). Statistical comparisons were conducted using two-way repeated-measures ANOVA followed by the Bonferroni post hoc test (D and E) or Wilcoxon rank-sum test (F and H). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Optogenetic activation of central amygdala (CeA) γ-aminobutyric acid–mediated (GABAergic) neurons delays the induction and accelerates the emergence from isoflurane general anesthesia. (A) Experimental strategy for optogenetic activation of CeA GABAergic neurons. AAV2/9-hEF1a-DIO-ChR2-mCherry was injected into the CeA of Vgat-Cre mice, and optical fibers were planted above the CeA. (B) Representative image showing the expression of AAV2/9-hEF1a-DIO-ChR2-mCherry in the CeA of Vgat-Cre mice. (C) Representative image of mCherry/c-Fos immunofluorescence in the CeA with or without blue light stimulation. (D) The effect of activating central amygdala GABAergic neurons on arousal score induced by optostimulation in mice under isoflurane anesthesia (n = 8 for ChR2 and mCherry group). (E) The effect of optogenetic activation of CeA GABAergic neurons on induction time of 1.4% isoflurane (n = 8 for ChR2 and mCherry group). (F) The effect of optogenetic activation of CeA GABAergic neurons on emergence time after isoflurane anesthesia (n = 8 for ChR2 and mCherry group). (G) Statistical results for the isoflurane concentrations at which mice occurred loss of righting reflex (LORR) with or without blue light stimulation of the CeA in ChR2 group (n = 8). (H) Dose–response curve shows the percentages of mice showing LORR when isoflurane concentration was gradually increased in ChR2 group (n = 8). (I) Statistical results for the isoflurane concentrations at which mice occurred recovery of righting reflex (RORR) with or without blue light stimulation of the CeA in ChR2 group (n = 8). (J) Dose–response curve shows the percentages of mice showing RORR when isoflurane concentration was gradually decreased in ChR2 group (n = 8). Statistical comparisons were conducted using two-way repeated-measures ANOVA followed by the Bonferroni post hoc test (E and F) or Wilcoxon rank-sum test (D, G, and I). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 4.

Optogenetic stimulation of central amygdala (CeA) γ-aminobutyric acid–mediated (GABAergic) neurons induces cortical activation during isoflurane anesthesia. (A) Representative EEG (electroencephalogram)/EMG (electromyogram) traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of the central amygdala in a ChR2 mouse under 0.8% isoflurane anesthesia. (B) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of the CeA in an mCherry mouse under 0.8% isoflurane anesthesia. (C) Relative EEG power of before (gray), during (blue), and after (green) the optostimulation period in ChR2 mice under 0.8% isoflurane anesthesia (n = 8). (D) Relative EEG power of before (gray), during (blue), and after (green) the optostimulation period in mCherry mice under 0.8% isoflurane anesthesia (n = 8). (E) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) in ChR2 mice under 1.4% isoflurane anesthesia. (F) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) in mCherry mice under 1.4% isoflurane anesthesia. (G) Statistical results of burst–suppression ratio changes before (gray), during (blue), and after (green) optostimulation under 1.4% isoflurane anesthesia in ChR2 mice (n = 8). (H) Statistical results of burst–suppression ratio changes before (gray), during (blue), and after (green) optostimulation under 1.4% isoflurane anesthesia in mCherry mice (n = 8). Two-way repeated-measures ANOVA (C and D) or one-way repeated-measures ANOVA (G and H) followed by the Bonferroni post hoc test was used for statistical analysis. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 5.

Optogenetic activation of γ-aminobutyric acid–mediated (GABAergic) central amygdala (CeA)–basal forebrain (BF) pathway delays the induction and accelerates emergence from isoflurane general anesthesia. (A) Diagrams showing the experimental strategy for optogenetic activation of the GABAergic CeA-BF pathway. AAV2/9-hEF1a-DIO-ChR2-mCherry was injected into the CeA of Vgat-Cre mice, and optical fibers were planted above the BF. (B) Schematic of coronal section illustrating the implantation of optical fibers in the BF after the microinjection of AAV into the CeA. (C) Fluorescence of AAV2/9-hEF1a-DIO-ChR2-mCherry in the BF and traces of fiber implantation. (D) The effect of optostimulating CeA-BF pathway on arousal score of mice under isoflurane anesthesia (n = 8 for mCherry and ChR2 group). (E) The effect of optostimulating CeA-BF pathway on induction time of 1.4% isoflurane (n = 8 for mCherry and ChR2 group). (F) The effect of optostimulating CeA-BF pathway on emergence time after isoflurane anesthesia (n = 8 for mCherry and ChR2 group). (G) Statistical results for the isoflurane concentrations at which mice loss of righting reflex (LORR) occurred with or without blue light stimulation of CeA-BF pathway in ChR2 group (n = 8). (H) Dose–response curve shows the percentages of mice showing LORR when isoflurane concentration was gradually increased in ChR2 group (n = 8). (I) Statistical results for the isoflurane concentrations at which mice recovery of righting reflex (RORR) occurred with or without blue light stimulation of CeA-BF pathway in ChR2 group (n = 8). (J) Dose–response curve shows the percentages of mice showing RORR when isoflurane concentration was gradually decreased in ChR2 group (n = 8). Statistical comparisons were conducted using Wilcoxon rank-sum test (D, G, and I), or two-way repeated-measures ANOVA was used for statistical analysis, followed by Bonferroni post hoc test (E and F). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 6.

Optogenetic stimulation of γ-aminobutyric acid–mediated (GABAergic) central amygdala (CeA)–basal forebrain (BF) pathway induces cortical activation during isoflurane anesthesia. (A) Representative EEG (electroencephalogram)/EMG (electromyogram) traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of CeA-BF pathway in a BF-ChR2 mouse under 0.8% isoflurane anesthesia. (B) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of CeA-BF pathway in a BF-mCherry mouse under 0.8% isoflurane anesthesia. (C) Relative EEG power of before (gray), during (blue), and after (green) optostimulation (30 Hz, 10 ms, 120 s) period in BF-ChR2 mice under 0.8% isoflurane anesthesia (n = 8). (D) Relative EEG power of before (gray), during (blue), and after (green) optostimulation (30 Hz, 10 ms, 120 s) period in BF-mCherry mice under 0.8% isoflurane anesthesia (n = 8). (E) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of CeA-BF pathway in a BF-ChR2 mouse under 1.4% isoflurane anesthesia. (F) Representative EEG/EMG traces (top) and EEG spectrograms power (bottom) before, during, and after optostimulation (30 Hz, 10 ms, 120 s) of CeA-BF pathway in a BF-mCherry mouse under 1.4% isoflurane anesthesia. (G) Statistical results showing burst–suppression ratio of before (gray), during (blue), and after (green) optostimulation period in basal forebrain–ChR2 mice under 1.4% isoflurane anesthesia (n = 8). (H) Statistical results showing burst–suppression ratio of before (gray), during (blue), and after (green) optostimulation period in basal forebrain–mCherry mice under 1.4% isoflurane anesthesia (n = 8). Data are presented as mean ± SD. Asterisks in C, D, G, and H indicate significant differences (*P < 0.05; **P < 0.01; ***P < 0.001). Two-way repeated-measures ANOVA (C and D) was used for statistical analysis, or one-way repeated-measures ANOVA (G and H) followed by the Bonferroni post hoc test.