Unraveling the Role of Enkephalin Signaling in Respiratory Control

Opioid-induced respiratory depression (OIRD) is the primary cause of death during opioid overdose characterized by a dramatic decrease in respiratory frequency and regularity. Opioid drugs cause neural depression in the ventral respiratory column (VRC) of the medulla via activation of mu-opioid receptors (MORs) that partially contribute to breathing cessation. Nonetheless, little is known about the role of endogenous opioid signaling in respiratory modulation. This study investigates the role of endogenous MORs ligands, enkephalins encoded by the Penk gene, in breathing control. To test whether enkephalin-expressing neurons of the preBӧtzinger Complex (preBӧtC) — the inspiratory rhythm-generating region of the VRC — are necessary for breathing rhythmogenesis, we permanently silenced the region’s Penk+ population via AAV1 injections encoding tetanus toxin light chain. Using whole-body plethysmography, we recorded the animals' respiratory performance within 14 days post-injection. During the second week of testing, we observed unexpected mortality in some experimental but not control animals; post-hoc analysis of breathing traces revealed the development of severe tachypnea in succumbed mice. These data suggest that Penk+ preBӧtC cells are integral for normal respiration. Still, these observations did not assess the specific contribution of enkephalin, as opposed to other types of signaling, to breathing stability. To directly dissect the effects of enkephalin on respiratory rhythm, we utilized optogenetics and pharmacological approaches in brainstem slice preparation from neonatal mice. Brief optogenetic activation of Penk+ neurons in the preBӧtC evoked population bursts while prolonged stimulation increased the rhythm frequency. However, the application of MOR antagonist Naloxone did not affect the network’s responses to light stimulations indicating the lack of MOR/enkephalin involvement in mediating these excitatory effects. High fidelity and temporal coordination of evoked bursts implicated glutamatergic transmission as the primary mechanism of excitation. Likewise, we were able to optogenetically drive respiratory rhythm in urethane-anesthetized spontaneously breathing adult mice by stimulating the preBӧtC. Further, there was a small but significant difference in breathing responses in the presence of Naloxone compared to control conditions. We hypothesized that longitudinal projections to the VRC that might be disrupted during slice preparation contributed enkephalin to the respiratory circuits in the whole-body preparation. We utilized recombinase-dependent reporter mice crosses and retrograde AAV tracers to identify brain-wide Penk inputs to the preBӧtC. Using conventional and light sheet fluorescence microscopy, we located regions in the pons, midbrain, and forebrain that send direct enkephalinergic projections to the VRC. Functional characterizations of these projections will potentially uncover state-dependent mechanisms of respiratory modulation via endogenous opioids. Untimely, this study will help elucidate the role of enkephalin signaling in breathing control and may establish therapeutic targets for preventing OIRD. R01HL166317, R00HL145004. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.