General anesthesia-activated neurons in the central amygdala mediate antinociception: Distinct roles in acute versus chronic phases of nerve injury

Background: General anesthesia (GA), such as isoflurane, induces analgesia (loss of pain) and loss of consciousness through mechanisms that are not fully understood. A distinct population of GABAergic neurons has been recently identified in the central amygdala (CeA) that can be activated by general anesthesia (CeA GA ) and exert antinociceptive functions. In this study, we aim to explore the underlying cellular mechanisms of CeA GA neurons across different phases of nerve injury-induced nociceptive sensitization in mice. Methods: This study used 107 mice, including 57 males and 50 females. We induced c-Fos activation in their brains using 1.2% isoflurane and validated Fos expression via RNAscope in situ hybridization. Unlike previous studies using the CANE method, we labeled CeA GA neurons (tdTomato + ) with the Fos-TRAP2 method. We then performed ex vivo electrophysiological recordings to assess the properties of both Fos-positive/CeA GA neurons and Fos-negative CeA neurons. Using chemogenetic strategy to selectively activate the CeA GA neurons, we investigated pain-like behaviors and associated comorbidities in mice after spared nerve injury (SNI). Results: Isoflurane induced robust Fos expression in CeA GABAergic neurons. Electrophysiological recordings in brain slices revealed that compared to Fos-negative CeA neurons, CeA GA neurons had higher excitability and exhibited distinct patterns of action potentials. Chemogenetic activation of Fos-TRAPed CeA GA neurons increased nociceptive thresholds in naïve mice and in mice 2 weeks post-SNI, but demonstrated modest antinociception 8 weeks post-SNI. Finally, Fos-negative CeA neurons, but not CeA GA neurons, exhibited increased excitability in the chronic phase of SNI, which was correlated with a downregulation of K + -Cl − cotransporter-2 (KCC2) in the CeA (sham vs. SNI 8 weeks). Conclusions: These results validate the antinociceptive power of CeA GA neurons using a different approach. Additionally, we highlight distinct roles of CeA GA neurons in governing physiological pain, acute pain, and the transition to chronic pain through KCC2 dysregulation.