Sex-specific effects of acute stress on emergence from isoflurane anesthesia via brainstem circuitry in mice

Background: Preoperative stress is a well-documented phenomenon in surgical patients and leads to clinically significant variability in anesthetic requirements. This variability poses important challenges for safe anesthesia practice, as stress may alter the function of neural circuits targeted by general anesthetics. However, the specific mechanisms linking stress to changes in anesthetic efficacy remain unclear. This study aimed to investigate how acute stress affects the efficacy of isoflurane anesthesia and to uncover the associated neural circuitry and receptor mechanisms. Methods: Using 30-min restraint stress in mice, we assessed isoflurane anesthesia through both loss and recovery of righting reflex alongside EEG spectral analysis. Circuit mechanisms were identified through immunofluorescence staining, fiber photometry, and viral-based optogenetics/chemogenetics. Pharmacological and shRNA approaches were carried out to verify receptor involvement in stress-driven modulation of isoflurane anesthesia. Results: Acute stress reduced isoflurane anesthesia depth and shortened emergence time in male mice (15.78 ± 3.17 vs. 6.84 ± 1.77 min; P = 0.0034), whereas these effects were absent in females. c-Fos immunofluorescence staining and fiber photometry revealed heightened activation of locus coeruleus norepinephrine (LC NE ) neurons during anesthesia following restraint stress in males. Pharmacological lesioning or chemogenetic inhibition of LC NE neurons abolished these stress-induced effects (EYFP + Stress vs. hM4Di + Stress: 7.76 ± 1.20 vs. 20.22 ± 5.93 min, P < 0.0001). Downstream tracing showed LC NE neurons projecting to GABAergic neurons in the dorsal raphe nucleus (DRN). Optogenetic or chemogenetic inhibition of the LC NE →DRN GABA circuit, as well as pharmacological or genetic disruption of α1-adrenergic receptors (α1-ARs) in DRN neurons, eliminated stress-induced modulation of anesthesia in males (Scramble + Stress vs. shRNA + Stress: 8.06 ± 1.94 vs. 23.38 ± 4.61 min, P = 0.0002). Conclusions: This study identifies the LC NE →DRN GABA circuit and α1-ARs in DRN GABA neurons as critical mediators in acute stress-induced emergence acceleration from isoflurane anesthesia in male mice.