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

The central nucleus of the amygdala has extensive connections with other brain structures mediating arousal and plays an important role in arousal-related behaviors Several lines of evidence indicate a role for the central nucleus of the amygdala in the regulation of general anesthesia, but the underlying mechanisms remain incompletely understood

In mice, activity of γ-aminobutyric acid–mediated (GABAergic) neurons in the central nucleus of the amygdala decreased during the induction of isoflurane anesthesia and was restored during emergence Both chemogenetic and optogenetic activation of GABAergic neurons in the central nucleus of the amygdala delayed induction time and shortened recovery from isoflurane anesthesia Optogenetic stimulation of GABAergic terminal projections from the central nucleus of the amygdala to the basal forebrain also promotes recovery, suggesting that this pathway plays a role in mediating the effects of isoflurane anesthesia

In all experiments, male and female Vgat-IRES-Cre mice weighing 22 to 28 g (8 to 12 weeks old) were used in this study. The Vgat-IRES-Cre mice used in this study were from The Jackson Laboratory (USA) and housed in standard laboratory conditions (temperature, 25° ± 0.5°C; humidity, 55 ± 5%). The lighting environment was maintained on a 12-h light/dark cycle (lights on at 7:00 am). The animals had free access to water and food. Behavioral experiments were conducted during the daytime, and the mice were randomly assigned to different groups using a random number table to minimize the effect of systematic factors on experimental results. The experimenters who conducted the behavioral tests were blinded to the group allocation. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fujian Medical University (FJMU IACUC 2023-0318).

number table to minimize the effect of systematic factors on experimental results. The experimenters who conducted the behavioral tests were blinded to the group allocation. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Fujian Medical University (FJMU IACUC 2023-0318). The experimental mice were anesthetized with 1.0 to 2.0% isoflurane during surgery. They were placed on a stereotaxic apparatus (RWD Life Science, China) for skull exploration and horizontal adjustment. After that, using a skull drill, a cranial window was opened directly above the central amygdala for virus injection and optic fiber implantation. Four virus types, namely AAV-hSyn-DIO-GCaMP6m for fiber photometry (Taitool, China), AAV2/9-hEF1a-DIO-ChR2-mCherry for optogenetics (Taitool), AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry for chemogenetics (Taitool), and AAV2/9-hSyn-DIO-mCherry for control (Taitool), were bilaterally injected into the central amygdala (anteroposterior = –1.20 mm; mediolateral = ±2.75 mm; dorsoventral = –4.85 mm) of the Vgat-Cre mice. After the injection of AAV (100 to 150 nl for each site), the glass pipette was held stationarily for 10 min and then withdrawn slowly. After virus injection, optical fibers for optogenetic stimulation or fiber photometry recordings were placed above the central amygdala or basal forebrain (anteroposterior = 0.00 mm; mediolateral = ±1.40 mm; dorsoventral = –5.50 mm) region. Electroencephalogram (EEG)/electromyogram (EMG) recording electrodes were implanted into the skull and trapezius muscle and secured with dental cement. Throughout and after the surgery, the mice were kept at 37°C until they fully recovered. Carprofen (4 mg/kg) was used for postoperative pain, which was dissolved in saline and injected subcutaneously every 12 h for 2 days after surgery.

were implanted into the skull and trapezius muscle and secured with dental cement. Throughout and after the surgery, the mice were kept at 37°C until they fully recovered. Carprofen (4 mg/kg) was used for postoperative pain, which was dissolved in saline and injected subcutaneously every 12 h for 2 days after surgery. A two-color fiber recording system (Inper Vista, Inper, China) was used to record the population calcium signals of central amygdala GABAergic neurons in the mice. The apparatus emitted two lights, that is, excitation light (470-nm blue light) and control light (410-nm violet light). Before the recording of fluorescence signal, laser light was used to irradiate the optical fiber for 1.5 to 2.0 h to reduce the self-fluorescence of optical fiber. We conducted fiber photometry recording experiment in an anesthesia-induction chamber (length × width × height = 23 × 10 × 17 cm), which was connected to an anesthesia vaporizer (R580S; RWD Life Science, China) and anesthesia monitor (BeneView T5; Mindray, China). The mice were connected to the fiber photometry system and EEG/EMG recording system, placed in the induction chamber, and allowed to acclimate to it. The calcium and EEG/EMG signals were recorded in real time. The experiments were conducted for a continuous 90 min (30 min before the start of isoflurane delivery, 30 min of isoflurane delivery, and 30 min after the cessation of isoflurane delivery). At the 30th minutes, 1.4% isoflurane was delivered with pure oxygen. At the 60th minute, the delivery of isoflurane was stopped. The pure oxygen was delivered at 1.5 l/min throughout the experiment, and the concentration of isoflurane was monitored with an anesthesia monitor with an accuracy of 0.1%. The average ΔF/F values of the mice were calculated for the three periods, namely before anesthesia, during anesthesia, and after anesthesia.

pped. The pure oxygen was delivered at 1.5 l/min throughout the experiment, and the concentration of isoflurane was monitored with an anesthesia monitor with an accuracy of 0.1%. The average ΔF/F values of the mice were calculated for the three periods, namely before anesthesia, during anesthesia, and after anesthesia. For chemogenetic experiments, the mice were intraperitoneally injected with 1.0 mg/kg clozapine-N-oxide (C4759; LKT, USA) or an equivalent volume of saline solution 1 h before isoflurane induction. The mice were gently placed in an anesthesia induction chamber filled with 1.4% isoflurane. The chamber was gently rotated 90° with a 10-s interval, and the time from exposure to 1.4% isoflurane to the occurrence of loss of righting reflex was recorded and defined as the induction time. After 30 min of exposure to isoflurane, the mice were gently taken out from the induction chamber and placed in a supine position in room air, and the time from exposure to room air to the occurrence of recovery of righting reflex was recorded and defined as the emergence time.1,2 In the experiments of the loss of righting reflex dose–response curve, the isoflurane concentration started from 0.5% and was increased by 0.1% every 15 min until loss of righting reflex occurred. In the experiments of the recovery of righting reflex dose–response curve, the isoflurane concentration was kept at 1.4% at the beginning and was decreased by 0.1% every 15 min until recovery of righting reflex occurred, and the concentration at that time was recorded.

ery 15 min until loss of righting reflex occurred. In the experiments of the recovery of righting reflex dose–response curve, the isoflurane concentration was kept at 1.4% at the beginning and was decreased by 0.1% every 15 min until recovery of righting reflex occurred, and the concentration at that time was recorded. For optogenetic experiments, a laser generator (Inper Venus, Inper) was used to apply blue light (3 to 5 mW, 30 Hz, 10 ms) until loss of righting reflex or recovery of righting reflex occurred, as previously described.1 In the experiments of the loss of righting reflex dose–response curve, the mice were initially exposed to 0.5% isoflurane for 15 min, followed by optostimulation for 60 s. During the optostimulation, the chamber was rotated 90°, and the mice were observed for the occurrence of loss of righting reflex. During induction, the isoflurane concentration was increased by 0.1% every 15 min until the mice showed loss of righting reflex, and the concentration was recorded. In the experiments of the recovery of righting reflex dose–response curve, the mice were first placed in an anesthesia-induction chamber filled with 1.4% isoflurane for 15 min, followed by optostimulation for 60 s. During the optostimulation, the chamber was rotated 90°, and the mice were observed for the occurrence of recovery of righting reflex. If recovery of righting reflex did not occur, the concentration was decreased by 0.1%, and the process was repeated until recovery of righting reflex occurred, and the concentration was recorded.

optostimulation, the chamber was rotated 90°, and the mice were observed for the occurrence of recovery of righting reflex. If recovery of righting reflex did not occur, the concentration was decreased by 0.1%, and the process was repeated until recovery of righting reflex occurred, and the concentration was recorded. We evaluated the effect of optostimulation (30 Hz, 10 ms, 60 s) on behavioral responses in mice under lighter isoflurane anesthesia. Briefly, the mice were placed in the anesthesia-induction chamber and exposed to 1.4% isoflurane for 10 min. After 1.4% isoflurane anesthesia induction, the concentration of isoflurane in the chamber was adjusted to 0.6%. If the mice exhibited any signs of recovery of righting reflex, the isoflurane concentration was increased by 0.1% until the concentration was sufficient to maintain the mice showing loss of righting reflex. The mice were kept at a certain isoflurane concentration for 30 min, and then 60-s optostimulation was provided to the mice. The behavioral response of the mice was observed during the optostimulation period. Based on the observed behavior, the arousal level of the mice was scored in line with previously established criteria.1,12

re kept at a certain isoflurane concentration for 30 min, and then 60-s optostimulation was provided to the mice. The behavioral response of the mice was observed during the optostimulation period. Based on the observed behavior, the arousal level of the mice was scored in line with previously established criteria.1,12 For EEG/EMG recording with optogenetic manipulations, after connecting to the EEG amplifier and laser stimulation device, the mice were placed in the anesthesia-induction chamber for a 5-min adaptation period. Subsequently, a continuous flow of 0.8% or 1.4% isoflurane was administered with oxygen into the chamber for spectrum and burst–suppression ratio experiments, respectively. After maintaining the isoflurane concentration for 30 min, a 120-s optostimulation (3 to 5 mW, 30 Hz, 10 ms) was given. After the optostimulation, EEG recording continued for an extended 10 min. Throughout the experiment, EEG/EMG signals were recorded, and a heating pad was used to maintain the core body temperature of the animals.

ning the isoflurane concentration for 30 min, a 120-s optostimulation (3 to 5 mW, 30 Hz, 10 ms) was given. After the optostimulation, EEG recording continued for an extended 10 min. Throughout the experiment, EEG/EMG signals were recorded, and a heating pad was used to maintain the core body temperature of the animals. For spectrum and burst–suppression ratio analysis, the recording and analysis of EEG/EMG signals in all optogenetic experiments were conducted using SleepSign software (Kissei Comtec, Japan). The EEG/EMG signals were amplified and sampled at a rate of 128 Hz. For 0.8% isoflurane anesthesia, the total power changes were segmentally calculated (120 s before, during, and after the optostimulation) using the fast Fourier transform. The EEG frequency bands were defined as follows: delta, 0.5 to 4.0 Hz; theta, 4.0 to 7.0 Hz; alpha, 8.0 to 15.0 Hz; and beta, 16.0 to 30.0 Hz.1,13 In the burst–suppression ratio experiment, the isoflurane concentration in the induction chamber was 1.4%. All of the obtained EEG signal data were converted to text format, and we calculated the relative changes of burst–suppression ratio 120 s before, during, and after the optostimulation using Matlab R2019b software (MathWorks, USA).1,13

on ratio experiment, the isoflurane concentration in the induction chamber was 1.4%. All of the obtained EEG signal data were converted to text format, and we calculated the relative changes of burst–suppression ratio 120 s before, during, and after the optostimulation using Matlab R2019b software (MathWorks, USA).1,13 For c-Fos staining, the mice were administered 1 mg/kg clozapine-N-oxide or subjected to blue optostimulation for 1 h (3 to 5 mW, 10 Hz, 10 ms, 30 s off and 30 s on) and were then deeply anesthetized with sodium pentobarbital. Once the mice reached a deep anesthesia state, they underwent cardiac perfusion with phosphate-buffered saline and 4% paraformaldehyde. After removing the mouse brains, they were fixed in 4% paraformaldehyde for 12 h. Subsequently, the brains were sequentially stored in 20% and 30% sucrose solutions in phosphate buffer until they sank to the bottom of the solution. The brains were embedded in optimal cutting temperature compound (Sakura, Japan) and frozen, and 30-μm-thick coronal sections were obtained using a cryostat (Leica CM1950, Germany). The sections were washed three times with 0.01 M phosphate-buffered saline to remove the optimal cutting temperature compound. The slices were then incubated in 0.7% Triton X-100 at room temperature for 1 h to permeabilize the cell membranes. Afterward, the sections were incubated with primary antibodies in phosphate-buffered saline with Tween (Leagene, China) overnight. After three washes with phosphate-buffered saline, the sections were incubated with a 488-nm–labeled secondary antibody (1:1,000, 111-545-003, The Jackson Laboratory) for 2 h. Finally, the images were captured using a fluorescence microscope (DMi8, Leica).

hosphate-buffered saline with Tween (Leagene, China) overnight. After three washes with phosphate-buffered saline, the sections were incubated with a 488-nm–labeled secondary antibody (1:1,000, 111-545-003, The Jackson Laboratory) for 2 h. Finally, the images were captured using a fluorescence microscope (DMi8, Leica). In this study, we used 58 male mice and 37 female mice for the central amygdala soma experiments. Histologic analysis was performed at the end of all experiments to check the location of the virus expression. Thirty-three mice with inaccurate virus injection sites, inaccurate placement of optic fibers, poor recovery, or death after virus injection were excluded, and the remaining 62 mice were included in the data analysis. For central amygdala–basal forebrain experiments, we used 17 male mice and 18 female mice. Ten mice with inaccurate injection sites or poor recovery were excluded, and 25 mice were used for data analysis. In this study, no a priori statistical power calculation was performed, and sample sizes were determined based on our previous experience with assay variance and experimental feasibility. The experimenters conducting all behavioral tests were blinded to group allocation. All presented data in this study are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, USA). The normal distribution of data was tested using the Shapiro–Wilk test. Statistical significance was evaluated using a two-tailed unpaired t test, paired t test, two-tailed Wilcoxon rank-sum test, and one-way or two-way repeated-measures ANOVA followed by Bonferroni multiple-comparison tests. Data were considered statistically significant when P was lower than 0.05. Significance annotations were as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. All graphical layouts were created using Adobe Illustrator for typesetting and design purposes.

The experimental mice were anesthetized with 1.0 to 2.0% isoflurane during surgery. They were placed on a stereotaxic apparatus (RWD Life Science, China) for skull exploration and horizontal adjustment. After that, using a skull drill, a cranial window was opened directly above the central amygdala for virus injection and optic fiber implantation. Four virus types, namely AAV-hSyn-DIO-GCaMP6m for fiber photometry (Taitool, China), AAV2/9-hEF1a-DIO-ChR2-mCherry for optogenetics (Taitool), AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry for chemogenetics (Taitool), and AAV2/9-hSyn-DIO-mCherry for control (Taitool), were bilaterally injected into the central amygdala (anteroposterior = –1.20 mm; mediolateral = ±2.75 mm; dorsoventral = –4.85 mm) of the Vgat-Cre mice. After the injection of AAV (100 to 150 nl for each site), the glass pipette was held stationarily for 10 min and then withdrawn slowly. After virus injection, optical fibers for optogenetic stimulation or fiber photometry recordings were placed above the central amygdala or basal forebrain (anteroposterior = 0.00 mm; mediolateral = ±1.40 mm; dorsoventral = –5.50 mm) region. Electroencephalogram (EEG)/electromyogram (EMG) recording electrodes were implanted into the skull and trapezius muscle and secured with dental cement. Throughout and after the surgery, the mice were kept at 37°C until they fully recovered. Carprofen (4 mg/kg) was used for postoperative pain, which was dissolved in saline and injected subcutaneously every 12 h for 2 days after surgery.

A two-color fiber recording system (Inper Vista, Inper, China) was used to record the population calcium signals of central amygdala GABAergic neurons in the mice. The apparatus emitted two lights, that is, excitation light (470-nm blue light) and control light (410-nm violet light). Before the recording of fluorescence signal, laser light was used to irradiate the optical fiber for 1.5 to 2.0 h to reduce the self-fluorescence of optical fiber. We conducted fiber photometry recording experiment in an anesthesia-induction chamber (length × width × height = 23 × 10 × 17 cm), which was connected to an anesthesia vaporizer (R580S; RWD Life Science, China) and anesthesia monitor (BeneView T5; Mindray, China). The mice were connected to the fiber photometry system and EEG/EMG recording system, placed in the induction chamber, and allowed to acclimate to it. The calcium and EEG/EMG signals were recorded in real time. The experiments were conducted for a continuous 90 min (30 min before the start of isoflurane delivery, 30 min of isoflurane delivery, and 30 min after the cessation of isoflurane delivery). At the 30th minutes, 1.4% isoflurane was delivered with pure oxygen. At the 60th minute, the delivery of isoflurane was stopped. The pure oxygen was delivered at 1.5 l/min throughout the experiment, and the concentration of isoflurane was monitored with an anesthesia monitor with an accuracy of 0.1%. The average ΔF/F values of the mice were calculated for the three periods, namely before anesthesia, during anesthesia, and after anesthesia.

For chemogenetic experiments, the mice were intraperitoneally injected with 1.0 mg/kg clozapine-N-oxide (C4759; LKT, USA) or an equivalent volume of saline solution 1 h before isoflurane induction. The mice were gently placed in an anesthesia induction chamber filled with 1.4% isoflurane. The chamber was gently rotated 90° with a 10-s interval, and the time from exposure to 1.4% isoflurane to the occurrence of loss of righting reflex was recorded and defined as the induction time. After 30 min of exposure to isoflurane, the mice were gently taken out from the induction chamber and placed in a supine position in room air, and the time from exposure to room air to the occurrence of recovery of righting reflex was recorded and defined as the emergence time.1,2 In the experiments of the loss of righting reflex dose–response curve, the isoflurane concentration started from 0.5% and was increased by 0.1% every 15 min until loss of righting reflex occurred. In the experiments of the recovery of righting reflex dose–response curve, the isoflurane concentration was kept at 1.4% at the beginning and was decreased by 0.1% every 15 min until recovery of righting reflex occurred, and the concentration at that time was recorded.

We evaluated the effect of optostimulation (30 Hz, 10 ms, 60 s) on behavioral responses in mice under lighter isoflurane anesthesia. Briefly, the mice were placed in the anesthesia-induction chamber and exposed to 1.4% isoflurane for 10 min. After 1.4% isoflurane anesthesia induction, the concentration of isoflurane in the chamber was adjusted to 0.6%. If the mice exhibited any signs of recovery of righting reflex, the isoflurane concentration was increased by 0.1% until the concentration was sufficient to maintain the mice showing loss of righting reflex. The mice were kept at a certain isoflurane concentration for 30 min, and then 60-s optostimulation was provided to the mice. The behavioral response of the mice was observed during the optostimulation period. Based on the observed behavior, the arousal level of the mice was scored in line with previously established criteria.1,12

For EEG/EMG recording with optogenetic manipulations, after connecting to the EEG amplifier and laser stimulation device, the mice were placed in the anesthesia-induction chamber for a 5-min adaptation period. Subsequently, a continuous flow of 0.8% or 1.4% isoflurane was administered with oxygen into the chamber for spectrum and burst–suppression ratio experiments, respectively. After maintaining the isoflurane concentration for 30 min, a 120-s optostimulation (3 to 5 mW, 30 Hz, 10 ms) was given. After the optostimulation, EEG recording continued for an extended 10 min. Throughout the experiment, EEG/EMG signals were recorded, and a heating pad was used to maintain the core body temperature of the animals.

For c-Fos staining, the mice were administered 1 mg/kg clozapine-N-oxide or subjected to blue optostimulation for 1 h (3 to 5 mW, 10 Hz, 10 ms, 30 s off and 30 s on) and were then deeply anesthetized with sodium pentobarbital. Once the mice reached a deep anesthesia state, they underwent cardiac perfusion with phosphate-buffered saline and 4% paraformaldehyde. After removing the mouse brains, they were fixed in 4% paraformaldehyde for 12 h. Subsequently, the brains were sequentially stored in 20% and 30% sucrose solutions in phosphate buffer until they sank to the bottom of the solution. The brains were embedded in optimal cutting temperature compound (Sakura, Japan) and frozen, and 30-μm-thick coronal sections were obtained using a cryostat (Leica CM1950, Germany). The sections were washed three times with 0.01 M phosphate-buffered saline to remove the optimal cutting temperature compound. The slices were then incubated in 0.7% Triton X-100 at room temperature for 1 h to permeabilize the cell membranes. Afterward, the sections were incubated with primary antibodies in phosphate-buffered saline with Tween (Leagene, China) overnight. After three washes with phosphate-buffered saline, the sections were incubated with a 488-nm–labeled secondary antibody (1:1,000, 111-545-003, The Jackson Laboratory) for 2 h. Finally, the images were captured using a fluorescence microscope (DMi8, Leica).

In this study, we used 58 male mice and 37 female mice for the central amygdala soma experiments. Histologic analysis was performed at the end of all experiments to check the location of the virus expression. Thirty-three mice with inaccurate virus injection sites, inaccurate placement of optic fibers, poor recovery, or death after virus injection were excluded, and the remaining 62 mice were included in the data analysis. For central amygdala–basal forebrain experiments, we used 17 male mice and 18 female mice. Ten mice with inaccurate injection sites or poor recovery were excluded, and 25 mice were used for data analysis. In this study, no a priori statistical power calculation was performed, and sample sizes were determined based on our previous experience with assay variance and experimental feasibility. The experimenters conducting all behavioral tests were blinded to group allocation. All presented data in this study are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, USA). The normal distribution of data was tested using the Shapiro–Wilk test. Statistical significance was evaluated using a two-tailed unpaired t test, paired t test, two-tailed Wilcoxon rank-sum test, and one-way or two-way repeated-measures ANOVA followed by Bonferroni multiple-comparison tests. Data were considered statistically significant when P was lower than 0.05. Significance annotations were as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. All graphical layouts were created using Adobe Illustrator for typesetting and design purposes.

To investigate whether the activity of central amygdala GABAergic neurons correlates with isoflurane anesthesia, fiber photometry was used to detect the calcium signals of these neurons in freely behaving mice (fig. 1A). AAV-hSyn-DIO-GCaMP6m was injected into the central amygdala of the Vgat-Cre mice, and optical fibers were implanted above the central amygdala (fig. 1B). The expression of GCaMP6m was observed in the central amygdala at 4 weeks after virus transfection (fig. 1C). The heatmap and peri-event plot showed that the calcium signals of central amygdala GABAergic neurons decreased rapidly at the beginning, followed by a slow decline after exposure to 1.4% isoflurane (fig. 1, D and E). The calcium signals were 20% lower than the baseline after 30 min of isoflurane exposure, and were gradually restored after the cessation of isoflurane exposure (fig. 1, D and E). One-way repeated-measures ANOVA revealed that the calcium signals during the 30-min isoflurane exposure were significantly decreased compared with the signals before isoflurane exposure (before vs. during: from 1.03 ± 1.48% to –12.09 ± 4.80%; P = 0.0009; fig. 1F). After the cessation of isoflurane, the calcium signals were significantly increased compared with those during isoflurane exposure (during vs. after: from –12.09 ± 4.80% to –4.18 ± 3.00%; P = 0.0028; fig. 1F).

nals before isoflurane exposure (before vs. during: from 1.03 ± 1.48% to –12.09 ± 4.80%; P = 0.0009; fig. 1F). After the cessation of isoflurane, the calcium signals were significantly increased compared with those during isoflurane exposure (during vs. after: from –12.09 ± 4.80% to –4.18 ± 3.00%; P = 0.0028; fig. 1F). 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.

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. Next, we further analyzed the calcium signals of central amygdala GABAergic neurons during the induction and emergence from isoflurane anesthesia, according to a previous study.14 For the induction, we analyzed two consecutive periods, namely the pre–loss of righting reflex period (–30 to 0 s, where 0 s represents the time of occurrence of loss of righting reflex) and the post–loss of righting reflex period (from 0 s to 30 s). The heatmap and peri-event plot showed that the calcium signals began to decline before the occurrence of loss of righting reflex (fig. 1, G and H). The paired t test showed that the calcium signals of central amygdala GABAergic neurons significantly decreased after loss of righting reflex (before loss of righting reflex vs. after loss of righting reflex: from 1.17 ± 4.96% to –4.81 ± 4.51%; n = 6; P = 0.0005; fig. 1I). For the emergence, we analyzed the calcium signals of the pre–recovery of righting reflex period (–30 to 0 s, where 0 s represents the time of occurrence of recovery of righting reflex) and post–recovery of righting reflex period (from 0 s to 30 s). The heatmap and peri-event plot showed that the calcium signals began to rise before the occurrence of recovery of righting reflex and peaked near the occurrence of recovery of righting reflex (fig. 1, J and K). The paired t test showed that the calcium signals significantly increased after recovery of righting reflex (pre–recovery of righting reflex vs. post–recovery of righting reflex: from –7.51 ± 4.99% to –1.02 ± 2.29%; n = 6; P = 0.01; fig. 1L). Taken together, these findings demonstrate a close relationship between the activity of central amygdala GABAergic neurons and isoflurane anesthesia, implying that central amygdala GABAergic neurons may take part in the regulation of isoflurane anesthesia.

from –7.51 ± 4.99% to –1.02 ± 2.29%; n = 6; P = 0.01; fig. 1L). Taken together, these findings demonstrate a close relationship between the activity of central amygdala GABAergic neurons and isoflurane anesthesia, implying that central amygdala GABAergic neurons may take part in the regulation of isoflurane anesthesia. To determine whether central amygdala GABAergic neurons participate in the regulation of isoflurane anesthesia, we first examined the effect of chemogenetic activation of central amygdala GABAergic neurons on isoflurane anesthesia. AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry was injected into the bilateral central amygdala of mice (fig. 2A), and a strong expression of hM3D(Gq)-mCherry was observed in the central amygdala approximately 4 weeks later (fig. 2B; Supplemental Digital Content 1, fig. S1, https://links.lww.com/ALN/D732). Immunohistochemical experiments showed that c-Fos protein was intensively expressed in hM3D(Gq)-mCherry–positive neurons of the central amygdala after 1.0 mg/kg clozapine-N-oxide injection, whereas little c-Fos protein was expressed after vehicle injection (fig. 2C), indicating the activation of central amygdala GABAergic neurons by chemogenetics. To investigate the effects of chemogenetic activation of central amygdala GABAergic neurons on the induction of isoflurane anesthesia, we conducted the righting reflex test in mice, considering that the loss of righting reflex in rodents is regarded as a surrogate for loss of consciousness in humans.15,16 Two-way ANOVA showed that 1.0 mg/kg clozapine-N-oxide injection significantly delayed the induction time of 1.4% isoflurane anesthesia in the hM3D group (vehicle vs. clozapine-N-oxide: from 58.75 ± 5.42 s to 67.63 ± 5.01 s; n = 8; P = 0.0017; fig. 2D). In the mCherry group, clozapine-N-oxide injection did not alter the induction time of isoflurane anesthesia. The change in isoflurane sensitivity was detected by gradually changing isoflurane concentration. Our analysis showed that the intraperitoneal injection of 1.0 mg/kg clozapine-N-oxide induced a higher concentration of isoflurane to induce anesthesia in the hM3D group (vehicle vs. clozapine-N-oxide: from 0.69 ± 0.07% to 0.80 ± 0.09%; n = 11; P = 0.0039; fig. 2E). Additionally, the loss of righting reflex dose–response curve after clozapine-N-oxide injection shifted to the right, with an increase in the EC50 for loss of righting reflex from 0.64% (95% CI, 0.636 to 0.643%) to 0.75% (95% CI, 0.740 to 0.758%; vehicle vs. clozapine-N-oxide; fig. 2F).

%; n = 11; P = 0.0039; fig. 2E). Additionally, the loss of righting reflex dose–response curve after clozapine-N-oxide injection shifted to the right, with an increase in the EC50 for loss of righting reflex from 0.64% (95% CI, 0.636 to 0.643%) to 0.75% (95% CI, 0.740 to 0.758%; vehicle vs. clozapine-N-oxide; fig. 2F). 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.

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. Next, we investigated the effects of chemogenetic activation of central amygdala GABAergic neurons on the emergence from isoflurane anesthesia, considering that the recovery of righting reflex in rodents is regarded as a surrogate for recovery of consciousness in humans.15,16 Two-way ANOVA showed that the hM3D group mice showed a significant decrease in the time required for emergence from 1.4% isoflurane anesthesia after receiving a 1.0 mg/kg clozapine-N-oxide injection (vehicle vs. clozapine-N-oxide: from 385.50 ± 66.26 s to 214.60 ± 40.21 s; n = 8; P = 0.0017; fig. 2G). The mCherry group mice did not show significant changes in the emergence time after clozapine-N-oxide injection. When the isoflurane concentration was gradually decreased, hM3D group mice recovered from anesthesia at a higher isoflurane concentration after clozapine-N-oxide injection (vehicle vs. clozapine-N-oxide: from 0.47 ± 0.09% to 0.62 ± 0.09%; n = 11; P = 0.0039; fig. 2H). The recovery of righting reflex dose–response curve after clozapine-N-oxide injection shifted to the right, with an increase in the EC50 for recovery of righting reflex from 0.51% (95% CI, 0.488 to 0.539%) to 0.66% (95% CI, 0.655 to 0.669%; vehicle vs. clozapine-N-oxide; fig. 2I). These results suggest that chemogenetic activation of central amygdala GABAergic neurons contributes to impeding induction and facilitating emergence from isoflurane anesthesia.

r recovery of righting reflex from 0.51% (95% CI, 0.488 to 0.539%) to 0.66% (95% CI, 0.655 to 0.669%; vehicle vs. clozapine-N-oxide; fig. 2I). These results suggest that chemogenetic activation of central amygdala GABAergic neurons contributes to impeding induction and facilitating emergence from isoflurane anesthesia. Compared with chemogenetics, optogenetics allows for precise and real-time modulation of neuronal activity. Thus, the optogenetic approach was chosen to activate central amygdala GABAergic neurons to further validate their effects on isoflurane anesthesia. AAV2/9-hEF1a-DIO-ChR2-mCherry was bilaterally injected into the central amygdala of mice (fig. 3A), and the expression of ChR2-mCherry was observed in the central amygdala approximately 4 weeks later (fig. 3B; Supplemental Digital Content 2, fig. S2, https://links.lww.com/ALN/D733). Immunohistochemistry results showed strong expression of c-Fos protein in ChR2-positive neurons after optostimulation (fig. 3C), indicating effective activation of central amygdala GABAergic neurons by optogenetics. To test the effect of activating central amygdala GABAergic neurons on the initiation of anesthesia emergence, we recorded the behavioral responses of the mice and determined the arousal score, which is widely used to estimate the arousal level of mice.1,12,13 Our results showed that optogenetic activation of central amygdala GABAergic neurons remarkably induced behavioral changes and significantly improved arousal scores in the ChR2 group (Supplemental Video File 1, https://links.lww.com/ALN/D738). Specifically, we observed body (including limbs, heads, and tails) movements (eight of eight), recovery of righting reflex in the majority of mice (seven of eight), and crawling in some mice (four of eight) (Supplemental Digital Content 3, table 1, https://links.lww.com/ALN/D734). In control mCherry mice, optostimulation of the central amygdala did not induce behavioral changes (Supplemental Video File 2, https://links.lww.com/ALN/D739). Two-way ANOVA revealed that the ChR2 group showed an increase in arousal scores (mCherry vs. ChR2: from 0.50 ± 0.53 to 8.38 ± 1.19; n = 8; P = 0.0002; fig. 3D). Next, we tested the effect of optogenetic activation of central amygdala GABAergic neurons on the induction and emergence times from isoflurane anesthesia. Data analysis indicated that optostimulation significantly prolonged the induction time in the ChR2 group (no light vs.

to 8.38 ± 1.19; n = 8; P = 0.0002; fig. 3D). Next, we tested the effect of optogenetic activation of central amygdala GABAergic neurons on the induction and emergence times from isoflurane anesthesia. Data analysis indicated that optostimulation significantly prolonged the induction time in the ChR2 group (no light vs. blue light: from 46.88 ± 5.44 s to 57.25 ± 5.73 s; n = 8; P = 0.0102; fig. 3E) and shortened the emergence time (no light vs. blue light: from 449.30 ± 129.20 s to 255.80 ± 120.50 s; n = 8; P = 0.0034; fig. 3F). Optogenetic stimulation did not significantly alter the induction and emergence times in the mCherry group (fig. 3, E and F).

46.88 ± 5.44 s to 57.25 ± 5.73 s; n = 8; P = 0.0102; fig. 3E) and shortened the emergence time (no light vs. blue light: from 449.30 ± 129.20 s to 255.80 ± 120.50 s; n = 8; P = 0.0034; fig. 3F). Optogenetic stimulation did not significantly alter the induction and emergence times in the mCherry group (fig. 3, E and F). 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.

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. Furthermore, we tested the effects of optogenetic activation of central amygdala GABAergic neurons on the isoflurane sensitivity. Wilcoxon rank-sum test revealed that blue light stimulation of central amygdala GABAergic neurons required a higher isoflurane concentration to induce anesthesia in the ChR2 group (no light vs. blue light: from 0.71 ± 0.06% to 0.84 ± 0.09%; n = 8; P = 0.0078; fig. 3G). Additionally, light stimulation of central amygdala GABAergic neurons resulted in the right shift of the loss of righting reflex dose–response curve, with the EC50 increasing from 0.66% (95% CI, 0.662 to 0.666%) to 0.78% (95% CI, 0.773 to 0.789%; no light vs. blue light; fig. 3H). Furthermore, light stimulation of central amygdala GABAergic neurons made ChR2 group mice recovery of righting reflex occur at a higher isoflurane concentration (no light vs. blue light: 0.50 ± 0.05% to 0.69 ± 0.08%; n = 8; P = 0.0078; fig. 3I). The recovery of righting reflex dose–response curve shifted to the right, and the EC50 increased from 0.55% (95% CI, 0.550 to 0.551%) to 0.75% (95% CI, 0.745 to 0.755%; no light vs. blue light; fig. 3J). These results, in conjunction with the chemogenetic results, support the hypothesis that central amygdala GABAergic neurons promote behavioral emergence from isoflurane anesthesia.

he right, and the EC50 increased from 0.55% (95% CI, 0.550 to 0.551%) to 0.75% (95% CI, 0.745 to 0.755%; no light vs. blue light; fig. 3J). These results, in conjunction with the chemogenetic results, support the hypothesis that central amygdala GABAergic neurons promote behavioral emergence from isoflurane anesthesia. Cortical EEG can effectively reflect the state of cortical activity and provide information about consciousness.17 To investigate the effects of activating central amygdala GABAergic neurons on cortical activity, we analyzed the EEG changes after optogenetic activation (30 Hz, 10 ms, 120 s) of these neurons under 0.8% and 1.4% isoflurane anesthesia. The experimental results showed that the ChR2 group exhibited a rapid transformation of cortical EEG from a high-amplitude and low-frequency pattern to a low-amplitude and high-frequency pattern after the optostimulation was given during 0.8% isoflurane anesthesia (fig. 4A; Supplemental Video File 3, https://links.lww.com/ALN/D740), and EEG gradually returned to the prestimulation state after the termination of optostimulation. In the mCherry group, EEG signals did not show obvious change after optostimulation (fig. 4B; Supplemental Video File 4, https://links.lww.com/ALN/D741). The analysis of EEG spectral power revealed that optostimulation potently changed EEG power in the delta, alpha, and beta bands in the ChR2 group. Specifically, delta power was significantly decreased (prestimulation vs. stimulation: from 46.63 ± 4.40% to 34.16 ± 6.47%; n = 8; P = 0.0195), while alpha power was significantly increased (prestimulation vs. stimulation: from 16.33 ± 3.74% to 21.20 ± 2.81%; n = 8; P = 0.0363) and beta power was significantly increased (prestimulation vs. stimulation: from 8.278 ± 1.92% to 15.92 ± 4.26%; n = 8; P = 0.0130; fig. 4C). In the mCherry group, optostimulation did not produce a significant change in EEG delta, alpha, or beta power (fig. 4D). To further illustrate the power changes in different frequency bands, we conducted a detailed analysis of the normalized power density of the EEG signals within the 0- to 30-Hz range (Supplemental Digital Content 4, fig. S3, https://links.lww.com/ALN/D735).

gnificant change in EEG delta, alpha, or beta power (fig. 4D). To further illustrate the power changes in different frequency bands, we conducted a detailed analysis of the normalized power density of the EEG signals within the 0- to 30-Hz range (Supplemental Digital Content 4, fig. S3, https://links.lww.com/ALN/D735). 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.

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. Burst–suppression oscillation is defined as an EEG pattern that includes alternating periods of high-amplitude slow waves (bursts) and flat EEG (suppression).18 It has been reported that high doses of anesthetic drugs induce EEG burst–suppression patterns in rodents and humans,19 and the burst–suppression ratio is an important indicator reflecting the depth of isoflurane anesthesia. We explored the effects of optogenetic activation of central amygdala GABAergic neurons on cortical burst–suppression ratio in mice under deep anesthesia with 1.4% isoflurane. Our results showed that the cortical EEG transited from burst–suppression oscillation mode to a low-amplitude, high-frequency state after the optostimulation was given in the ChR2 group (fig. 4E; Supplemental Video File 5, https://links.lww.com/ALN/D742). Cortical EEG gradually returned to burst–suppression oscillation mode similar to prestimulation after the termination of optostimulation. In the mCherry group, optostimulation did not obviously change EEG signals (fig. 4F; Supplemental Video File 6, https://links.lww.com/ALN/D743). Statistical analysis revealed that optostimulation significantly decreased the burst–suppression ratio in the ChR2 group (prestimulation vs. stimulation: from 83.39 ± 5.15% to 52.60 ± 12.98%; n = 8; P = 0.0003; fig. 4G). After the termination of optostimulation, the burst–suppression ratio was significantly restored (stimulation vs. poststimulation: from 52.60 ± 12.98% to 79.52 ± 5.56%; n = 8; P = 0.0007; fig. 4G). In the mCherry group, the burst–suppression ratio did not significantly change after optostimulation (fig. 4H). These findings demonstrate that optogenetic activation of central amygdala GABAergic neurons significantly facilitates cortical activity in mice, with significant alteration in the EEG spectrum and reduction in the burst–suppression ratio.

–suppression ratio did not significantly change after optostimulation (fig. 4H). These findings demonstrate that optogenetic activation of central amygdala GABAergic neurons significantly facilitates cortical activity in mice, with significant alteration in the EEG spectrum and reduction in the burst–suppression ratio. The results of neuroanatomical studies show that the GABAergic central amygdala projects to many anesthesia-related structures, such as the basal forebrain, lateral hypothalamus, and lateral habenula.6 Among these structures, the basal forebrain is a key component of the ascending arousal system and has been implicated in the regulation of general anesthesia.20 Thus, we selected the basal forebrain as the downstream target of the GABAergic central amygdala and investigated the effect of manipulating the GABAergic central amygdala–basal forebrain circuit on isoflurane anesthesia. To achieve this, AAV2/9-hEF1a-DIO-ChR2-mCherry was bilaterally injected into the central amygdala, and optical fibers were implanted above the basal forebrain (fig. 5, A to C). Similar to the experimental methods described earlier (refer to the result section of “Optogenetic Activation of Central Amygdala GABAergic Neurons Delays the Induction and Accelerates Emergence from Isoflurane Anesthesia”), we tested the effect of optogenetic activation of the GABAergic central amygdala–basal forebrain circuit on arousal scores, loss of righting reflex, and recovery of righting reflex. Our research results showed that activation of the GABAergic central amygdala–basal forebrain circuit remarkably induced behavioral changes in the ChR2 mice and improved their arousal scores. Specifically, we observed body (including limbs, heads, and tails) movements in all mice (eight of eight), recovery of righting reflex in some mice (two of eight), and crawling in some mice (two of eight; Supplemental Digital Content 5, table 2, https://links.lww.com/ALN/D736). Statistical analysis showed the arousal scores significantly increased (mCherry vs. ChR2: from 0.38 ± 0.52 to 6.13 ± 1.96; n = 8; P < 0.0001; fig. 5D).

overy of righting reflex in some mice (two of eight), and crawling in some mice (two of eight; Supplemental Digital Content 5, table 2, https://links.lww.com/ALN/D736). Statistical analysis showed the arousal scores significantly increased (mCherry vs. ChR2: from 0.38 ± 0.52 to 6.13 ± 1.96; n = 8; P < 0.0001; fig. 5D). 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.

on 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. Furthermore, optogenetic activation of the GABAergic central amygdala–basal forebrain circuit effectively affected the isoflurane anesthesia induction time and emergence time in mice. We showed that light stimulation significantly prolonged the induction time in the ChR2 group (no light vs. blue light: from 53.50 ± 5.16 s to 66.75 ± 8.05 s; n = 8; P = 0.0049; fig. 5E) and shortened the emergence time (no light vs. blue light: from 369.88 ± 108.30 s to 189.75 ± 71.19 s; n = 8; P = 0.0004; fig. 5F). The mCherry group mice did not show significant changes in the induction and emergence time after light stimulation (fig. 5, E and F). Finally, we tested the effect of optogenetic activation of the GABAergic central amygdala–basal forebrain circuit on isoflurane sensitivity. Statistical analysis showed that light stimulation of the GABAergic central amygdala–basal forebrain circuit required a higher isoflurane concentration to induce anesthesia in the ChR2 group (no light vs. blue light: from 0.69 ± 0.06% to 0.81 ± 0.06%; n = 8; P = 0.0156; fig. 5G). Light stimulation of the GABAergic central amygdala–basal forebrain circuit resulted in the right shift of the loss of righting reflex dose–response curve, with the EC50 increasing from 0.64% (95% CI, 0.635 to 0.639%) to 0.76% (95% CI, 0.762 to 0.766%; no light vs. blue light; fig. 5H). In addition, light stimulation of the GABAergic central amygdala–basal forebrain circuit made ChR2 group mice recovery of righting reflex occur at a higher isoflurane concentration (no light vs. blue light: from 0.49 ± 0.08% to 0.60 ± 0.08%; n = 8; P = 0.0016; fig. 5I). The recovery of righting reflex dose–response curve shifted to the right, and the EC50 increased from 0.53% (95% CI, 0.523 to 0.546%) to 0.65% (95% CI, 0.645 to 0.655%; no light vs. blue light; fig. 5J). These findings indicate that activation of the GABAergic central amygdala–basal forebrain circuit prolongs the induction time, shortens the recovery time, and reduces the sensitivity of isoflurane anesthesia.

ased from 0.53% (95% CI, 0.523 to 0.546%) to 0.65% (95% CI, 0.645 to 0.655%; no light vs. blue light; fig. 5J). These findings indicate that activation of the GABAergic central amygdala–basal forebrain circuit prolongs the induction time, shortens the recovery time, and reduces the sensitivity of isoflurane anesthesia. To assess the effect of manipulating the GABAergic central amygdala–basal forebrain circuit on cortical activity, we optogenetically activated this circuit under 0.8% and 1.4% isoflurane anesthesia and respectively analyzed the change in EEG power and burst–suppression ratio using the same experimental paradigm mentioned in the result part of “Optogenetic Activation of Central Amygdala GABAergic Neurons Enhances Cortical Activity during Isoflurane Anesthesia.” Our experimental results showed that, similar to the results of central amygdala activation, optostimulation of this circuit immediately interrupted the high-amplitude and low-frequency EEG pattern and produced a low-amplitude and high-frequency pattern in the ChR2 group during 0.8% isoflurane anesthesia (fig. 6A; Supplemental Video File 7, https://links.lww.com/ALN/D744). In the mCherry group, optostimulation did not obviously change EEG signals (fig. 6B; Supplemental Video File 8, https://links.lww.com/ALN/D745). EEG spectral analysis showed that optostimulation induced a significant decrease in delta power (prestimulation vs. stimulation: from 44.91 ± 6.25% to 35.22 ± 7.45%; P = 0.0207) and a significant increase in alpha power (prestimulation vs. stimulation: from 15.71 ± 3.61% to 19.65 ± 5.91%; P = 0.0329; fig. 6C). In the mCherry group, optostimulation did not produce a significant change in EEG delta, theta, alpha, or beta power (fig. 6D). We also analyzed the normalized power density of the mice EEG signals within 0 to 30 Hz in detail, and demonstrated the power changes in each frequency band during optostimulation (Supplemental Digital Content 6, fig. S4, https://links.lww.com/ALN/D737).

icant change in EEG delta, theta, alpha, or beta power (fig. 6D). We also analyzed the normalized power density of the mice EEG signals within 0 to 30 Hz in detail, and demonstrated the power changes in each frequency band during optostimulation (Supplemental Digital Content 6, fig. S4, https://links.lww.com/ALN/D737). 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.

ane 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. Next, we determined the effect of optogenetic activation of the GABAergic central amygdala–basal forebrain circuit on the burst–suppression ratio under 1.4% isoflurane anesthesia. Our experimental results showed that the administration of optostimulation immediately interrupted the cortical burst–suppression oscillation mode and produced a low-amplitude, high-frequency state in the ChR2 group (fig. 6E; Supplemental Video File 9, https://links.lww.com/ALN/D746), which was similar to the results of the central amygdala activation. In the mCherry group, optostimulation of the basal forebrain did not obviously change EEG signals (fig. 6F; Supplemental Video File 10, https://links.lww.com/ALN/D747). Statistical analysis revealed that the burst–suppression ratio was significantly reduced after the optostimulation in the ChR2 group (prestimulation vs. stimulation: from 82.29 ± 5.10% to 47.92 ± 13.73%; n = 8; P = 0.0012; fig. 6G) and gradually restored to prestimulation level after the termination of optostimulation (stimulation vs. poststimulation: from 47.92 ± 13.73% to 67.71 ± 13.36%; n = 8; P = 0.0660; fig. 6G). In the mCherry group, the burst–suppression ratio did not significantly change after the optostimulation of the basal forebrain (fig. 6H). These results indicate that optogenetic activation of the GABAergic central amygdala–basal forebrain circuit promotes the activity of the cerebral cortex and reduces the depth of isoflurane anesthesia.

To determine whether central amygdala GABAergic neurons participate in the regulation of isoflurane anesthesia, we first examined the effect of chemogenetic activation of central amygdala GABAergic neurons on isoflurane anesthesia. AAV2/9-hSyn-DIO-hM3D(Gq)-mCherry was injected into the bilateral central amygdala of mice (fig. 2A), and a strong expression of hM3D(Gq)-mCherry was observed in the central amygdala approximately 4 weeks later (fig. 2B; Supplemental Digital Content 1, fig. S1, https://links.lww.com/ALN/D732). Immunohistochemical experiments showed that c-Fos protein was intensively expressed in hM3D(Gq)-mCherry–positive neurons of the central amygdala after 1.0 mg/kg clozapine-N-oxide injection, whereas little c-Fos protein was expressed after vehicle injection (fig. 2C), indicating the activation of central amygdala GABAergic neurons by chemogenetics. To investigate the effects of chemogenetic activation of central amygdala GABAergic neurons on the induction of isoflurane anesthesia, we conducted the righting reflex test in mice, considering that the loss of righting reflex in rodents is regarded as a surrogate for loss of consciousness in humans.15,16 Two-way ANOVA showed that 1.0 mg/kg clozapine-N-oxide injection significantly delayed the induction time of 1.4% isoflurane anesthesia in the hM3D group (vehicle vs. clozapine-N-oxide: from 58.75 ± 5.42 s to 67.63 ± 5.01 s; n = 8; P = 0.0017; fig. 2D). In the mCherry group, clozapine-N-oxide injection did not alter the induction time of isoflurane anesthesia. The change in isoflurane sensitivity was detected by gradually changing isoflurane concentration. Our analysis showed that the intraperitoneal injection of 1.0 mg/kg clozapine-N-oxide induced a higher concentration of isoflurane to induce anesthesia in the hM3D group (vehicle vs. clozapine-N-oxide: from 0.69 ± 0.07% to 0.80 ± 0.09%; n = 11; P = 0.0039; fig. 2E). Additionally, the loss of righting reflex dose–response curve after clozapine-N-oxide injection shifted to the right, with an increase in the EC50 for loss of righting reflex from 0.64% (95% CI, 0.636 to 0.643%) to 0.75% (95% CI, 0.740 to 0.758%; vehicle vs. clozapine-N-oxide; fig. 2F).

Compared with chemogenetics, optogenetics allows for precise and real-time modulation of neuronal activity. Thus, the optogenetic approach was chosen to activate central amygdala GABAergic neurons to further validate their effects on isoflurane anesthesia. AAV2/9-hEF1a-DIO-ChR2-mCherry was bilaterally injected into the central amygdala of mice (fig. 3A), and the expression of ChR2-mCherry was observed in the central amygdala approximately 4 weeks later (fig. 3B; Supplemental Digital Content 2, fig. S2, https://links.lww.com/ALN/D733). Immunohistochemistry results showed strong expression of c-Fos protein in ChR2-positive neurons after optostimulation (fig. 3C), indicating effective activation of central amygdala GABAergic neurons by optogenetics. To test the effect of activating central amygdala GABAergic neurons on the initiation of anesthesia emergence, we recorded the behavioral responses of the mice and determined the arousal score, which is widely used to estimate the arousal level of mice.1,12,13 Our results showed that optogenetic activation of central amygdala GABAergic neurons remarkably induced behavioral changes and significantly improved arousal scores in the ChR2 group (Supplemental Video File 1, https://links.lww.com/ALN/D738). Specifically, we observed body (including limbs, heads, and tails) movements (eight of eight), recovery of righting reflex in the majority of mice (seven of eight), and crawling in some mice (four of eight) (Supplemental Digital Content 3, table 1, https://links.lww.com/ALN/D734). In control mCherry mice, optostimulation of the central amygdala did not induce behavioral changes (Supplemental Video File 2, https://links.lww.com/ALN/D739). Two-way ANOVA revealed that the ChR2 group showed an increase in arousal scores (mCherry vs. ChR2: from 0.50 ± 0.53 to 8.38 ± 1.19; n = 8; P = 0.0002; fig. 3D). Next, we tested the effect of optogenetic activation of central amygdala GABAergic neurons on the induction and emergence times from isoflurane anesthesia. Data analysis indicated that optostimulation significantly prolonged the induction time in the ChR2 group (no light vs.

Cortical EEG can effectively reflect the state of cortical activity and provide information about consciousness.17 To investigate the effects of activating central amygdala GABAergic neurons on cortical activity, we analyzed the EEG changes after optogenetic activation (30 Hz, 10 ms, 120 s) of these neurons under 0.8% and 1.4% isoflurane anesthesia. The experimental results showed that the ChR2 group exhibited a rapid transformation of cortical EEG from a high-amplitude and low-frequency pattern to a low-amplitude and high-frequency pattern after the optostimulation was given during 0.8% isoflurane anesthesia (fig. 4A; Supplemental Video File 3, https://links.lww.com/ALN/D740), and EEG gradually returned to the prestimulation state after the termination of optostimulation. In the mCherry group, EEG signals did not show obvious change after optostimulation (fig. 4B; Supplemental Video File 4, https://links.lww.com/ALN/D741). The analysis of EEG spectral power revealed that optostimulation potently changed EEG power in the delta, alpha, and beta bands in the ChR2 group. Specifically, delta power was significantly decreased (prestimulation vs. stimulation: from 46.63 ± 4.40% to 34.16 ± 6.47%; n = 8; P = 0.0195), while alpha power was significantly increased (prestimulation vs. stimulation: from 16.33 ± 3.74% to 21.20 ± 2.81%; n = 8; P = 0.0363) and beta power was significantly increased (prestimulation vs. stimulation: from 8.278 ± 1.92% to 15.92 ± 4.26%; n = 8; P = 0.0130; fig. 4C). In the mCherry group, optostimulation did not produce a significant change in EEG delta, alpha, or beta power (fig. 4D). To further illustrate the power changes in different frequency bands, we conducted a detailed analysis of the normalized power density of the EEG signals within the 0- to 30-Hz range (Supplemental Digital Content 4, fig. S3, https://links.lww.com/ALN/D735).

The results of neuroanatomical studies show that the GABAergic central amygdala projects to many anesthesia-related structures, such as the basal forebrain, lateral hypothalamus, and lateral habenula.6 Among these structures, the basal forebrain is a key component of the ascending arousal system and has been implicated in the regulation of general anesthesia.20 Thus, we selected the basal forebrain as the downstream target of the GABAergic central amygdala and investigated the effect of manipulating the GABAergic central amygdala–basal forebrain circuit on isoflurane anesthesia. To achieve this, AAV2/9-hEF1a-DIO-ChR2-mCherry was bilaterally injected into the central amygdala, and optical fibers were implanted above the basal forebrain (fig. 5, A to C). Similar to the experimental methods described earlier (refer to the result section of “Optogenetic Activation of Central Amygdala GABAergic Neurons Delays the Induction and Accelerates Emergence from Isoflurane Anesthesia”), we tested the effect of optogenetic activation of the GABAergic central amygdala–basal forebrain circuit on arousal scores, loss of righting reflex, and recovery of righting reflex. Our research results showed that activation of the GABAergic central amygdala–basal forebrain circuit remarkably induced behavioral changes in the ChR2 mice and improved their arousal scores. Specifically, we observed body (including limbs, heads, and tails) movements in all mice (eight of eight), recovery of righting reflex in some mice (two of eight), and crawling in some mice (two of eight; Supplemental Digital Content 5, table 2, https://links.lww.com/ALN/D736). Statistical analysis showed the arousal scores significantly increased (mCherry vs. ChR2: from 0.38 ± 0.52 to 6.13 ± 1.96; n = 8; P < 0.0001; fig. 5D).

To assess the effect of manipulating the GABAergic central amygdala–basal forebrain circuit on cortical activity, we optogenetically activated this circuit under 0.8% and 1.4% isoflurane anesthesia and respectively analyzed the change in EEG power and burst–suppression ratio using the same experimental paradigm mentioned in the result part of “Optogenetic Activation of Central Amygdala GABAergic Neurons Enhances Cortical Activity during Isoflurane Anesthesia.” Our experimental results showed that, similar to the results of central amygdala activation, optostimulation of this circuit immediately interrupted the high-amplitude and low-frequency EEG pattern and produced a low-amplitude and high-frequency pattern in the ChR2 group during 0.8% isoflurane anesthesia (fig. 6A; Supplemental Video File 7, https://links.lww.com/ALN/D744). In the mCherry group, optostimulation did not obviously change EEG signals (fig. 6B; Supplemental Video File 8, https://links.lww.com/ALN/D745). EEG spectral analysis showed that optostimulation induced a significant decrease in delta power (prestimulation vs. stimulation: from 44.91 ± 6.25% to 35.22 ± 7.45%; P = 0.0207) and a significant increase in alpha power (prestimulation vs. stimulation: from 15.71 ± 3.61% to 19.65 ± 5.91%; P = 0.0329; fig. 6C). In the mCherry group, optostimulation did not produce a significant change in EEG delta, theta, alpha, or beta power (fig. 6D). We also analyzed the normalized power density of the mice EEG signals within 0 to 30 Hz in detail, and demonstrated the power changes in each frequency band during optostimulation (Supplemental Digital Content 6, fig. S4, https://links.lww.com/ALN/D737).

Recent evidence has shown that neural substrates regulating sleep–wake are often involved in the loss and recovery of consciousness during general anesthesia,21 and many overlapping neural subpopulations regulating sleep–wake and general anesthesia have been reported, such as the GABAergic neurons in the basal forebrain,1 orexinergic neurons in lateral hypothalamic area,22 and glutamatergic neurons in parabrachial nucleus.23 Our previous studies showed that optogenetic activation of the glutamatergic ventral tegmental area–central amygdala pathway potently promotes defense-related wakefulness in mice.7 In addition, Zhao et al. demonstrated that the glutamatergic paraventricular thalamus–central amygdala pathway modulates acute stress-induced heightened wakefulness in mice and that chemogenetic inhibition of this pathway decreases acute stress-induced wakefulness.24 Our current study revealed that central amygdala GABAergic neurons facilitated behavioral and cortical emergence from general anesthesia. It further demonstrated that sleep–wake and general anesthesia regulation might share some common neural substrates, suggesting that studying sleep–wake circuits could help us to understand the underlying mechanisms of general anesthesia.

ic neurons facilitated behavioral and cortical emergence from general anesthesia. It further demonstrated that sleep–wake and general anesthesia regulation might share some common neural substrates, suggesting that studying sleep–wake circuits could help us to understand the underlying mechanisms of general anesthesia. Using in vivo calcium imaging, Hua et al.25 showed that specific subpopulations of central amygdala GABAergic neurons exhibit increased calcium signals during isoflurane induction and maintenance. These results are not in accordance with ours, where the calcium signals of central amygdala GABAergic neurons displayed a potent decrease during isoflurane anesthesia. These contradictory outcomes may be ascribed to the heterogeneity of central amygdala GABAergic neurons.26 Gene expression studies have suggested a wide genetic diversity of central amygdala GABAergic neurons, with different subtypes expressing various molecules, such as calcitonin receptor-like receptor, neurotensin, serotonin receptor 2a, corticotropin-releasing factor, protein kinase C-δ (PKC-δ), and somatostatin.27 It is quite possible that different subtypes of central amygdala neurons may produce different reactions to isoflurane anesthesia. That is, isoflurane may decrease calcium signals in the majority of subtypes of central amygdala GABAergic neurons but increase calcium signals in the minority of subtypes of central amygdala GABAergic neurons. The specific effects of isoflurane on different subtypes of central amygdala neurons need further clarification. In addition, a previous study has shown the phenomenon of mutual inhibition among different subtypes of central amygdala neurons.28 Subtypes of central amygdala neurons regulate specific behaviors through mutual inhibition.29 For example, in a study on conditioned fear, Haubensak et al.30 found that PKC-δ–positive central amygdala neurons inhibited neuronal output in the central medial amygdala and produced mutual inhibition with PKC-δ–negative neurons in the lateral central amygdala. We speculated that isoflurane-inhibited and isoflurane-activated neurons could regulate isoflurane anesthesia through a similar pattern of mutual inhibition: isoflurane-inhibited neurons may promote anesthesia emergence, at least partly, through the inhibition of isoflurane-activated neurons, although the specific modulatory effect of isoflurane-inhibited neurons on isoflurane-activated neurons needs to be further studied.

sia through a similar pattern of mutual inhibition: isoflurane-inhibited neurons may promote anesthesia emergence, at least partly, through the inhibition of isoflurane-activated neurons, although the specific modulatory effect of isoflurane-inhibited neurons on isoflurane-activated neurons needs to be further studied. Neuroanatomical results show that the GABAergic central amygdala abundantly innervates multiple brain areas associated with general anesthesia, including the basal forebrain, lateral hypothalamus, lateral habenula, and bed nucleus of the stria terminalis.11 The basal forebrain is a key structure of the ascending arousal system31 and has been implicated in general anesthesia.20 In a recent study, Cai et al.20 reported that basal forebrain somatostatin-positive neurons promoted propofol and isoflurane anesthesia. Chemogenetic activation of basal forebrain somatostatin-positive neurons not only shortened induction and prolonged maintenance time of anesthesia but also produced a greater suppression of cortical activity during anesthesia. Previous studies have indicated that basal forebrain somatostatin-positive neurons promote sleep and suppress neighboring cholinergic and glutamatergic neurons, both of which promote wakefulness.32 In the current study, we demonstrated that the GABAergic central amygdala exerted its antianesthesia effect through its projection to the basal forebrain. Central amygdala GABAergic neurons may regulate the anesthesia emergence, at least partially, through their inhibition of downstream basal forebrain somatostatin-positive neurons. The specific modulating effects of central amygdala GABAergic neurons on basal forebrain somatostatin-positive neurons and this modulation on anesthesia emergence still need to be confirmed.

e anesthesia emergence, at least partially, through their inhibition of downstream basal forebrain somatostatin-positive neurons. The specific modulating effects of central amygdala GABAergic neurons on basal forebrain somatostatin-positive neurons and this modulation on anesthesia emergence still need to be confirmed. Although our results showed that the basal forebrain is a crucial downstream nucleus for central amygdala GABAergic neurons to facilitate anesthesia emergence, we still found that the antianesthesia effect of central amygdala GABAergic neurons cannot be fully imitated by the GABAergic central amygdala–basal forebrain circuit. The effect of the GABAergic central amygdala–basal forebrain circuit on behavioral and cortical emergence was weaker than that of central amygdala GABAergic soma. According to the results of a neuroanatomical study,11 the GABAergic central amygdala may mediate the antianesthesia effect via multiple downstream targets in addition to the basal forebrain. Other downstream targets, such as the lateral hypothalamus, lateral habenula, and bed nucleus of the stria terminalis, may also participate in the antianesthesia effect of central amygdala GABAergic neurons. This one-to-many arrangement, which allows the GABAergic central amygdala to communicate with multiple downstream targets simultaneously, may partly explain the strong anesthesia emergence–promoting effect of central amygdala GABAergic neurons. The specific roles of other downstream targets of the GABAergic central amygdala in general anesthesia need to be further investigated in future studies.

ommunicate with multiple downstream targets simultaneously, may partly explain the strong anesthesia emergence–promoting effect of central amygdala GABAergic neurons. The specific roles of other downstream targets of the GABAergic central amygdala in general anesthesia need to be further investigated in future studies. The central amygdala receives projections from many arousal-related brain regions,33 such as the ventral tegmental area, paraventricular nucleus of the thalamus,34 and lateral parabrachium.35 Among these brain regions, the ventral tegmental area is particularly noteworthy because the dopaminergic neurons in the ventral tegmental area have been proven to play an important role in the regulation of wakefulness and general anesthesia.12,36,37 In the study by Taylor et al., optogenetic activation of the ventral tegmental area’s dopaminergic neurons was found to strongly promote the emergence from isoflurane anesthesia and produce behavioral activation in mice, including leg, head, and whisker movements, as well as righting and walking.12 Neuroanatomical evidence has revealed that dopaminergic neurons in the ventral tegmental area send abundant projection to the central amygdala.38 Recent findings have further shown that photostimulation of the ventral tegmental area’s dopaminergic terminals in the central amygdala promotes arousal in mice and significantly decreases the latency from non–rapid eye movement sleep to wakefulness.39 Considering the arousal-promoting effects of central amygdala–projecting outputs from the dopaminergic ventral tegmental area, the dopaminergic ventral tegmental area may serve as the upstream target in mediating the anesthetic emergence effect of the central amygdala, but further research is needed to confirm this assumption.

ing the arousal-promoting effects of central amygdala–projecting outputs from the dopaminergic ventral tegmental area, the dopaminergic ventral tegmental area may serve as the upstream target in mediating the anesthetic emergence effect of the central amygdala, but further research is needed to confirm this assumption. Interestingly, previous clinical studies have reported a positive correlation between preoperative anxiety and anesthetic consumption.40 Patients with higher anxiety scores require higher doses of propofol to achieve appropriate sedation and anesthesia, indicating the inherent antianesthesia property of stress and anxiety.41,42 The central amygdala is a key region involved in regulating anxiety and stress-related behaviors.43,44 Neuroimaging studies revealed that patients with anxiety disorders show an exaggerated amygdala response compared with the healthy control group.45 Optogenetic activation of the central amygdala enhances emotional and anxiety-like behaviors in rodents.46 Our current results demonstrated that optogenetic activation of central amygdala GABAergic neurons promotes the emergence from isoflurane general anesthesia. Hence, we speculated that the overexcitation of central amygdala GABAergic neurons may be the underlying mechanism behind why higher dosage of anesthetic drugs is needed in patients with stress and anxiety. Selective inhibition of central amygdala GABAergic neurons may provide a promising avenue for reducing anesthetic dosage and thus improving anesthetic safety in patients with stress and anxiety.

s may be the underlying mechanism behind why higher dosage of anesthetic drugs is needed in patients with stress and anxiety. Selective inhibition of central amygdala GABAergic neurons may provide a promising avenue for reducing anesthetic dosage and thus improving anesthetic safety in patients with stress and anxiety. The current study has some limitations. First, we merely investigated the regulatory effect of central amygdala γ-aminobutyric acid (GABA) neurons on isoflurane anesthesia but did not investigate the effects of these neurons on general anesthesia induced by other general anesthetics. Considering that recent studies have proved one neural substrate can regulate general anesthesia induced by various anesthetic drugs,23,47 we speculate that central amygdala GABA neurons may also regulate general anesthesia induced by other general anesthetics, such as sevoflurane, propofol, and ketamine, although some studies showed that different general anesthetics may act at different molecular targets and neural circuits and have distinct effects on cortical activity.48–50 The specific effects of central amygdala GABA neurons on other general anesthetics require further research. Second, we did not measure the changes in physiologic indicators during induction and emergence from general anesthesia, such as the minute ventilation and cardiac output, which heavily influence the process of anesthesia induction and emergence.51,52 We proposed that the activation of central amygdala GABA neurons may cause changes in the minute ventilation and cardiac output of mice during anesthesia induction and emergence. We are in the process of constructing the methods to detect these physiologic indicators and will try to take the measurements in the future. Third, previous studies have demonstrated that there are differences in the central amygdala between mice and humans of different sexes.53,54 Whether sex differences play a role in the effect of the GABAergic central amygdala or central amygdala–basal forebrain pathway on anesthetic emergence needs to be further explored.

rd, previous studies have demonstrated that there are differences in the central amygdala between mice and humans of different sexes.53,54 Whether sex differences play a role in the effect of the GABAergic central amygdala or central amygdala–basal forebrain pathway on anesthetic emergence needs to be further explored. This study was supported by the following grants: the National Natural Science Foundation of China (Beijing, China; 82471503 to Dr. L. Chen, 82271529 to Dr. Cai); Joint Funds for the Innovation of Science and Technology in Fujian Province, China (Fuzhou, China; 2021Y9005 to Dr. L. Chen); the Natural Science Foundation of Fujian Province, China (Fuzhou, China; 2024J01582 to Dr. Cai); Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research, Fujian Medical University (Fuzhou, China; FKLDSR-202304 to Dr. Cai); and Innovation Training and Entrepreneurship Plan for College Students of Fujian Medical University (202310392018 to Dr. L. Chen). The authors declare no competing interests.

This study was supported by the following grants: the National Natural Science Foundation of China (Beijing, China; 82471503 to Dr. L. Chen, 82271529 to Dr. Cai); Joint Funds for the Innovation of Science and Technology in Fujian Province, China (Fuzhou, China; 2021Y9005 to Dr. L. Chen); the Natural Science Foundation of Fujian Province, China (Fuzhou, China; 2024J01582 to Dr. Cai); Fujian Key Laboratory of Drug Target Discovery and Structural and Functional Research, Fujian Medical University (Fuzhou, China; FKLDSR-202304 to Dr. Cai); and Innovation Training and Entrepreneurship Plan for College Students of Fujian Medical University (202310392018 to Dr. L. Chen).

Supplemental Digital Content 1: Figure S1, expression extent of hM3D(Gq)-mCherry, https://links.lww.com/ALN/D732 Supplemental Digital Content 2: Figure S2, expression extent of ChR2-mCherry, https://links.lww.com/ALN/D733 Supplemental Digital Content 3: Table S1, arousal scores of central amygdala optostimulation, https://links.lww.com/ALN/D734 Supplemental Digital Content 4: Figure S3, power density of central amygdala optostimulation, https://links.lww.com/ALN/D735 Supplemental Digital Content 5: Table S2, arousal scores of central amygdala–basal forebrain optostimulation, https://links.lww.com/ALN/D736 Supplemental Digital Content 6: Figure S4, power density of central amygdala–basal forebrain optostimulation, https://links.lww.com/ALN/D737 Supplemental Video File 1: Arousal score of central amygdala ChR2 mouse, https://links.lww.com/ALN/D738 Supplemental Video File 2: Arousal score of central amygdala mCherry mouse, https://links.lww.com/ALN/D739 Supplemental Video File 3: EEG power of central amygdala ChR2 mouse under 0.8% isoflurane, https://links.lww.com/ALN/D740 Supplemental Video File 4: EEG power of central amygdala mCherry mouse under 0.8% isoflurane, https://links.lww.com/ALN/D741 Supplemental Video File 5: Burst–suppression ratio of central amygdala ChR2 mouse under 1.4% isoflurane, https://links.lww.com/ALN/D742 Supplemental Video File 6: Burst–suppression ratio of central amygdala mCherry mouse under 1.4% isoflurane, https://links.lww.com/ALN/D743 Supplemental Video File 7: EEG power of basal forebrain ChR2 mouse under 0.8% isoflurane, https://links.lww.com/ALN/D744

Supplemental Video File 5: Burst–suppression ratio of central amygdala ChR2 mouse under 1.4% isoflurane, https://links.lww.com/ALN/D742 Supplemental Video File 6: Burst–suppression ratio of central amygdala mCherry mouse under 1.4% isoflurane, https://links.lww.com/ALN/D743 Supplemental Video File 7: EEG power of basal forebrain ChR2 mouse under 0.8% isoflurane, https://links.lww.com/ALN/D744 Supplemental Video File 8: EEG power of basal forebrain mCherry mouse under 0.8% isoflurane, https://links.lww.com/ALN/D745 Supplemental Video File 9: Burst–suppression ratio of basal forebrain ChR2 mouse under 1.4% isoflurane, https://links.lww.com/ALN/D746 Supplemental Video File 10: Burst–suppression ratio of basal forebrain mCherry mouse under 1.4% isoflurane, https://links.lww.com/ALN/D747