Functional modulation of retrotrapezoid neurons drives fentanyl-induced respiratory depression

The primary cause of death from opioid overdose is opioid-induced respiratory depression (OIRD), characterized by severe suppression of respiratory rate, destabilized breathing patterns, hypercapnia, and heightened risk of apnea. The retrotrapezoid nucleus (RTN), a critical chemosensitive brainstem region in the rostral ventrolateral medullary reticular formation, contains Phox2b⁺/neuromedin-B (Nmb) propriobulbar neurons. These neurons, stimulated by CO₂/H⁺, regulate breathing to prevent respiratory acidosis. Since the RTN shows limited expression of opioid receptors, we expected that opioid-induced hypoventilation should activate these neurons to restore ventilation and stabilize arterial blood gases. However, the ability of the RTN to stimulate ventilation during OIRD has never been tested. We used optogenetic and pharmacogenetic approaches, to activate and inhibit RTN Phox2b⁺/Nmb⁺ neurons before and after fentanyl administration. As expected, fentanyl (500 µg/kg ip) suppressed respiratory rate and destabilized breathing. Before fentanyl, optogenetic stimulation of Phox2b⁺/Nmb⁺ or chemogenetic inhibition of Nmb⁺ cells increased and decreased breathing activity, respectively. Surprisingly, optogenetic stimulation after fentanyl administration caused a significantly greater increase in breathing activity compared with prefentanyl levels. In contrast, chemogenetic inhibition of RTN Nmb neurons caused profound hypoventilation and breathing instability after fentanyl. The results suggest that fentanyl does not inhibit the ability of Phox2b⁺/Nmb⁺ cells within the RTN region to stimulate breathing. Thus, this study highlights the potential of stimulating RTN neurons as a possible therapeutic approach to restore respiratory function in cases of opioid-induced respiratory depression (OIRD).NEW & NOTEWORTHY Opioid-induced respiratory depression (OIRD) suppresses breathing and destabilizes ventilation. Using optogenetic and chemogenetic tools, we demonstrated that stimulating retrotrapezoid nucleus (RTN) Phox2b⁺/Nmb⁺ neurons enhances breathing, even after fentanyl administration, whereas their inhibition exacerbates hypoventilation. These findings reveal that RTN neurons retain their ability to drive ventilation during OIRD, highlighting their potential as a therapeutic target to restore respiratory function in opioid overdose cases.