Dorsal Raphe Serotonin Neurons Mediate CO2-Induced Arousal from Sleep

Arousal from sleep in response to CO₂ is a critical protective phenomenon. Dysregulation of CO₂-induced arousal contributes to morbidity and mortality from prevalent diseases, such as obstructive sleep apnea and sudden infant death syndrome. Despite the critical nature of this protective reflex, the precise mechanism for CO₂-induced arousal is unknown. Because CO₂ is a major regulator of breathing, prevailing theories suggest that activation of respiratory chemo- and mechano-sensors is required for CO₂-induced arousal. However, populations of neurons that are not involved in the regulation of breathing are also chemosensitive. Among these are serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) that comprise a component of the ascending arousal system. We hypothesized that direct stimulation of these neurons with CO₂ could cause arousal from sleep independently of enhancing breathing. Dialysis of CO₂-rich acidified solution into DRN, but not medullary raphe responsible for modulating breathing, caused arousal from sleep. Arousal was lost in mice with a genetic absence of 5-HT neurons, and with acute pharmacological or optogenetic inactivation of DRN 5-HT neurons. Here we demonstrate that CO₂ can cause arousal from sleep directly, without requiring enhancement of breathing, and that chemosensitive 5-HT neurons in the DRN critically mediate this arousal. Better understanding mechanisms underlying this protective reflex may lead to interventions to reduce disease-associated morbidity and mortality.

SIGNIFICANCE STATEMENT Although CO₂-induced arousal is critical to a number of diseases, the specific mechanism is not well understood. We previously demonstrated that serotonin (5-HT) neurons are important for CO₂-induced arousal, as mice without 5-HT neurons do not arouse to CO₂. Many have interpreted this to mean that medullary 5-HT neurons that regulate breathing are important in this arousal mechanism. Here we found that direct application of CO₂-rich aCSF to the dorsal raphe nucleus, but not the medullary raphe, causes arousal from sleep, and that this arousal was lost with genetic ablation or acute inhibition of 5-HT neurons. We propose that 5-HT neurons in the dorsal raphe nucleus can be activated directly by CO₂ to cause arousal independently of respiratory activation.