Enkephalinergic and Tachykinergic Neurons Project to the Neural Pacemaker for Breathing—the preBötzinger Complex

Opioids remain essential for strong pain relief, yet the neural systems that regulate pain are closely tied to those that control breathing. As a result, opioid-induced respiratory depression and apnea are the leading causes of death in overdoses—a crisis intensified by the rise of synthetic opioids, which contributed to roughly 80, 000 deaths in the United States in 2023. To mitigate this life-threatening side effect, we need a deeper understanding of the specific circuits that couple pain and breathing, and how these circuits respond to synthetic opioids such as fentanyl. The preBötzinger Complex (preBötC) is the central brainstem locus that generates the respiratory rhythm. It contains transcriptionally diverse neurons, including populations expressing the μ-opioid receptor (Oprm1) and the tachykinin receptor (Tacr1), both of which influence breathing when manipulated. We hypothesize that brain regions expressing the endogenous opioid ligand enkephalin (Penk) and the endogenous NK1 ligand tachykinin (Tac1) form functional circuits with the preBötC that modulate breathing and/or pain. To map these circuits, we used a dual-recombinase, dual-transgenic approach to achieve both transcript-type and projection specificity. PenkCre/Cre or Tac1Cre/Cre mice were crossed with Rosa26CreONFlpON-tdTomato (Ai65D) mice, which express tdTomato only when both Cre and Flp are present. Offspring received injections of retroAAV-FlpO into the preBötC, restricting tdTomato labeling to neurons that express Penk or Tac1 (via Cre) and project to the injected region (via retrograde FlpO). Using this strategy, we found that the preBötC receives substantial synaptic input from enkephalinergic (Penk) and tachykinergic (Tac1) neurons distributed across the brain, including brainstem nuclei involved in respiration and nociception such as the Kölliker-Fuse nucleus and the nucleus of the solitary tract. Current work focuses on two directions: (1) combining immunostaining with light-sheet imaging to determine how these identified neurons respond to fentanyl, and (2) using optogenetic stimulation and photometry recordings of Penk- or Tac1-expressing neurons in regions that project to the preBötC to test their functional roles in breathing and pain modulation. R01 HL1660317 & R00 HL145004. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.