Astrocytic Connexin43 Channels Are Essential for Breathing Pattern Stabilization in the preBötzinger Complex

ABSTRACT Objective Astrocytes within the preBötzinger complex (preBötC) critically regulate respiratory rhythmogenesis and pattern formation. However, the molecular mechanisms underlying their contributions remain poorly understood. This study aims to investigate whether connexin 43 (Cx43) channels, a prominent subtype of connexin proteins expressed in preBötC astrocytes, are essential for stabilizing breathing patterns. Methods We employed a multidisciplinary approach, integrating whole‐body plethysmography, in vivo fiber photometry, phrenic nerve discharge (PND) recordings, photostimulation, RNAscope fluorescence in situ hybridization, and RNA sequencing to elucidate the functional role of Cx43 channels in respiratory regulation. Results Elevated activation levels of preBötC astrocytes were synchronized with specific respiratory events, including sighs and transiently augmented breathing. RNA‐sequencing analysis demonstrated that Gja1 (encoding Cx43) was identified as the predominant connexin transcript in preBötC astrocytes. Photostimulation of preBötC astrocytes significantly increased PND frequency in anesthetized mice, an effect replicated by pharmacological blockade of Cx43 hemichannels. Conditional knockdown of astrocytic Gja1 in the preBötC considerably increased resting breathing frequency and minute ventilation. Blockade of Cx43 hemichannels enhanced astrocytic activation and induced ATP accumulation around somatostatin‐expressing preBötC neurons (preBötC SST ). Furthermore, Cx43 hemichannel blockade activated preBötC SST neurons, an effect mediated by P2Y1 but not P2X receptors. Conclusion We identify an astrocyte‐to‐neuron signaling cascade involving Cx43 hemichannel‐dependent ATP release, P2Y1 receptor activation on preBötC SST neurons, and subsequent modulation of respiratory motor output. These findings establish Cx43 hemichannels as critical molecular determinants for stabilizing breathing patterns.