Neuroscience

Hypothalamic CRH Neurons Modulate Sevoflurane Anesthesia and The Post-anesthesia Stress Responses

Shan Jiang
Lu Chen
Wei-Min Qu
Zhi-Li Huang author has email address
Chang-Rui Chen author has email address

Department of Pharmacology, School of Basic Medical Sciences; State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Institutes of Brain Science, Fudan University, Shanghai, China

https://doi.org/10.7554/eLife.90191.3

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Figures and data

Whole-brain mapping of active neurons during the post-anesthesia period.
A. Protocol for c-fos staining. Animals were exposed to sevoflurane or oxygen for 30 min and were sacrificed 3 h after the treatment. B. Quantification of the number of c-fos+ cells per mm² in different brain regions (n = 4, unpaired two-tailed t-test, *p < 0.05, **p < 0.01). AON, anterior olfactory nucleus; ARC, arcuate hypothalamic nucleus; DRD, dorsal raphe nucleus, dorsal part; IO, inferior olive; LC, locus coeruleus; LDTg, laterodorsal tegmental nucleus; LHb, lateral habenular nucleus; LSD, lateral septal nucleus, dorsal part; LSI, lateral septal nucleus, intermediate part; LSV, lateral septal nucleus, ventral part; MVPO, medioventral periolivary nucleus; PAG, periaqueductal gray; Pir, piriform cortex; Pn, pontine nuclei; PVH, paraventricular nucleus of the hypothalamus; PVT, paraventricular thalamus; RC, raphe cap; RVL, rostroventrolateral reticular nucleus; Sol, nucleus of the solitary tract; Tu, olfactory tubercle; VMPO, ventromedial preoptic nucleus. C. Representative images of c-fos staining in the PVH, LS, LC, and Sol for a control and an experimental animal exposed to sevoflurane GA. Scale bar, 500 µm (left), 250 µm (right, enlarged). D. Representative images showing colocalization of CRH immunoreactivity and mCherry expression (upper panel, scale bar = 100 µm), c-fos and mCherry expression (bottom panel, scale bar = 100 µm) in the PVH of CRH-Cre mice. Arrowheads indicate co-labeled neurons. E. Quantification of the percentage of mCherry+ cells in the c-fos+ population (green) and the percentage of c-fos+ cells in the mCherry+ population (red) in the PVH.

Population activities of PVH^CRH neurons in response to sevoflurane GA.
A. Diagram of the virus injection, EEG/EMG electrode, and optic fiber implantation sites of CRH-Cre mice. B. jGCaMP7b/DAPI immunofluorescence in CRH neurons and track of the optic fiber implanted above the PVH; scale bar, 200 μm. Viral expression of jGCaMP7b and placement of the fiber-optic probe above the PVH. C. Schematic of the recording configuration. D-E. Time courses of Ca²⁺ signals following sevoflurane anesthesia D and quantification of Ca²⁺ signal changes before, during, and after (post 1 and post 2 periods) sevoflurane inhalation (E, n = 4, F (3, 12) = 61.49, p < 0.001, one-way ANOVA with Tukey’s post-hoc test; pre vs. during, pre vs. post 1, during vs. post 2, post 1 vs. post 2, p**< 0.001; pre vs. post 2, p = 0.0894; during vs. post 1, p = 0.2012). Freq, frequency; LORR, loss of right reflexing; RORR, recovery of right reflexing; Sevo, sevoflurane. F. Time percentage of self-grooming before, during, and after (post 1 and post 2 periods) sevoflurane inhalation (n = 4, F (3, 12) = 76.87, p < 0.001, one-way repeated measures ANOVA with Tukey’s post-hoc test; pre vs. during, pre vs. post 1, p = 0.0005; pre vs. post 2 during vs. post 2, post 1 vs. post 2, p** <0.0001; during vs. post 1, p > 0.9999).

Characterizations of sevoflurane GA-induced grooming.
A. Quantification of incorrect transitions with respect to the cephalocaudal sequence of stereotypic grooming patterns in the post-anesthesia period (n = 4, two-way ANOVAs followed by Sidak’s test, F (1, 6) = 8.646, p = 0.0259, 0–50 min: O₂ vs sevo, p = 0.9545, t = 0.2767, df = 12, 95% CI = -12.34 to 15.34; 50–100 min, O₂ vs sevo, p = 0.0055, t = 3.753, df = 12, 95% CI = -34.19 to -6.504). B. Number of interrupted bouts in the post-anesthesia period (n = 4, two-way ANOVAs followed by Sidak’s test, F (1, 6) = 19.14, p = 0.0047, 0–50 min: O₂ vs sevo, p = 0.4770, t = 1.139, df = 12, 95% CI = -19.45 to 7.446; 50–100 min, O₂ vs sevo, **p < 0.01, t = 6.969, df = 12, 95% CI = -50.15 to -23.26). C. Six representative grooming models, including spontaneous (control), swimming, water spray, physical attack, body restraint, and sevoflurane GA-induced grooming. D-F. The mean bout duration (D, F (5, 18) = 34.52, p < 0.0001), transitions per bout (E, F (5, 18) = 30.32, p < 0.0001), and grooming frequency (bouts per min, F, F (5, 18) = 7.935, p =0.0004) varied across the models (n = 4, one-way ANOVA with Tukey’s post-hoc test. *p < 0.05; **p < 0.01). G-H. 3-D plot of bout frequency, bout duration, and transitions per bout (G); the percentage of time spent grooming different body parts (H). The dimension of the symbol along an axis is defined by the SD of the corresponding parameter.

Chemogenetic modulation of PVH^CRH neurons bidirectionally altered induction of and emergence from sevoflurane GA.
A. Schematic of AAV-DIO-hM3Dq-mCherry or AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry injected into the PVH of CRH-Cre mice. B. Left: representative images of mCherry/c-fos immunofluorescence in CRH neurons after vehicle or CNO treatment; scale bars, 200 μm. Right: magnified images are shown; scale bar, 200 μm. Arrowheads indicate co-labeled neurons. C. Timelines of sevoflurane anesthesia-related behavioral tests measuring induction time (LORR) and emergence time (RORR). D-E. Dose-response curves showing the percentages of mice exhibiting LORR in response to incremental sevoflurane concentrations for the vehicle and CNO groups. Inset: the sevoflurane concentrations at which each mouse exhibited LORR are shown (D, hM3Dq group, n = 10, paired t-test, p = 0.0411, t = 4.714, df = 9, 95% CI = 0.05 to 0.16; E, hM4Di group, n = 8, paired t-test, p = 0.0021, t = 9.375, df = 7, 95% CI = -0.266 to -0.1589). F. Induction time with 2% sevoflurane exposure after intraperitoneal injections of vehicle or CNO for 1 h (mCherry group, n = 7, paired t-test, p = 0.847, t = 0.2014, df = 6, 95% CI = -47.77 to 56.35; hM3Dq group, n = 8, paired t-test, p = 0.0498, t = 5.545, df =7, 95% CI = 32.26 to 80.24; hM4Di group, n = 8, paired t-test, p = 0.0060, t = 4.573, df =7, 95% CI = -104.1 to -33.14). G. Emergence time with 2% sevoflurane exposure for 1 h after intraperitoneal injections of vehicle or CNO (mCherry group, n = 7, paired t-test, p = 0.8298, t = 0.9286, df =6, 95% CI = -19.15 to 42.58; hM3Dq group, n = 8, paired t-test, p = 0.0048, t = 4.057, df =7, 95% CI = -43.72 to -11.53; hM4Di group, n = 8, paired t-test, p = 0.0057, t = 3.922, df =7, 95% CI = 24.77 to 99.98).

Optogenetic stimulation of PVH^CRH neurons induced cortical activation and behavioral emergence during continuous steady-state sevoflurane GA.
A-B. Typical examples of EEG, EMG, and EEG power spectral in a mouse injected with AAV-DIO-mCherry(A) or AAV-DIO-ChR2-mCherry (B) following acute photostimulation (30 Hz, 5 ms, 60 s) during continuous steady-state sevoflurane GA. Time 0 indicates the beginning of photostimulation. The blue shadow indicates the 60-s duration of blue light stimulation. C-D. Left: normalized group power spectral densities from PVH^CRH-mCherry (C) or PVH^CRH-ChR2 (D) mice with pre photostimulation (gray) and photostimulation (blue). Dark blue lines in d indicate the power band with significant difference. Right: differences between normalized group power spectral densities from PVH^CRH-mCherry (C) or PVH^CRH-ChR2 (D). E-F. Power percentage changes in cortical EEG before (gray) and during (blue) photostimulation at 30 Hz in PVH^CRH-mCherry (E) or PVH^CRH-ChR2 mice (F) during continuous steady-state sevoflurane GA (n = 6, two-way ANOVAs followed by Sidak’s test, *p < 0.05).

Optogenetic stimulation of PVH^CRH neurons induced cortical activation during burst-suppression oscillations induced by deep sevoflurane GA.
A-B. Typical examples of EEG, EMG, and EEG power spectral in a mouse injected with AAV-DIO-mCherry (A) or AAV-DIO-ChR2-mCherry (B) following acute photostimulation (30 Hz, 5 ms, 60 s) during burst-suppression oscillations. Time 0 indicates the beginning of photostimulation. The blue shadow indicates the 60-s duration of blue light stimulation. C-D. Top left: normalized group power spectral densities from PVH^CRH-mCherry mice (C) and PVH^CRH-ChR2 (D) with pre photostimulation (gray) and photostimulation (blue); Top right: differences between normalized group power spectral densities from PVH^CRH-mCherry mice (C) and PVH^CRH-ChR2 mice (D). Bottom left: normalized group power spectral densities from PVH^CRH-mCherry mice (C) and PVH^CRH-ChR2 mice (D) with pre photostimulation (gray) and post photostimulation (green); bottom right: differences between normalized group power spectral densities from PVH^CRH-mCherry mice (C) and PVH^CRH-ChR2 mice (D). Dark blue lines in panel d indicate the power band with significant difference. E, G. Power percentage changes in cortical EEG before (gray), during (blue), and post (green) photostimulation in PVH^CRH-mCherry (E) or PVH^CRH-ChR2 (G) mice during burst-suppression oscillations. F, H. BSR change before (gray), during (blue), and post (green) photostimulation in PVH^CRH-mCherry (G) or PVH^CRH-ChR2 (H) mice during burst-suppression oscillations (n = 6, two-way ANOVAs followed by Sidak’s test, *p < 0.05, **p < 0.01; Pre-stim vs. Stim, p < 0.01, df = 15, 95% CI = 17.36 to 41.97; Pre-stim vs. Post-stim, p < 0.01, df = 15, 95% CI = 23.53 to 48.14). Stim, stimulation; Freq, frequency.

Chemogenetic inhibition of PVH^CRH neurons alleviated the stress response after sevoflurane GA.
A. Schedule of sacrificing mice for c-fos quantification, collecting blood samples from mice subjected to sevoflurane or pure oxygen for CRH, and CORT measurement and behavior testing (OFT and EPM). B. The schedule of the video recording after inhalation of sevoflurane or pure oxygen for 30 min. C. Quantification of the number of c-fos-positive neurons in the PVH (n = 4, saline: O₂ vs. sevo, p = 0.0017, t = 3.523, df = 12, 95% CI = -164.6 to -9.424; CNO: O₂ vs. sevo, p = 0.7122, t = 0.2632, df = 12, 95% CI = -71.08 to 84.08). D-E. Serum CRH (D, n = 4, saline: O₂ vs. sevo, p = 0.0456, t = 2.604, df = 12, 95% CI = -3.585 to -0.03; CNO: O₂ vs. sevo, p = 0.0077, t = 3.567, df = 12, 95% CI = -4.255 to -0.705) and CORT (E, n = 4, saline: O₂ vs. sevo, p = 0.0032, t = 4.059, df = 12, 95% CI = -44.73 to -10.19; CNO: saline: O₂ vs. sevo, p = 0.9066, t = 0.4024, df = 12, 95% CI = -19.99 to 14.55) levels following the protocol in (a). F-H. Time percentage of self-grooming (F, n = 7, saline: O₂ vs. sevo, p < 0.001, t = 7.23, df = 24, 95% CI = -33.26 to -16.76,; CNO: O₂ vs. sevo, p = 0.9915, t = 0.1171, df = 24, 95% CI = -8.656 to 7.846), moving distances in the central areas of OFT (G, n = 7, saline (: O₂ vs. sevo, p = 0.0014, t = 3.892, df = 24, 95% CI = 5.269 to 21.95; CNO: O₂ vs. sevo, p = 0.7291, t = 0.7183, df = 24, 95% CI = -10.85 to 5.829) and time percentage of staying in the open arms of the EPM (H, n = 7, saline: O₂ vs. sevo, p = 0.0007, t = 4.166, df = 24, 95% CI = 3.649 to 13.42; CNO: O₂ vs. sevo, p = 0.4626, t = 1.137, df = 24, 95% CI = -7.217 to 2.559) after inhalation of sevoflurane or pure oxygen and administration of saline or CNO. Statistical comparisons were conducted using two-way ANOVA followed by Sidak’s tests. *p < 0.05, **p < 0.01, n.s., no significant differences). I. Representative heatmaps of OFT and EPM after inhalation of sevoflurane or pure oxygen and administration of saline or CNO.

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