Effects of xylazine and opioids on prefrontal cortex inhibitory transmission - Xylazine is now highly prevalent in illicit supplies of opioids and poses an emerging threat to public health. While the compound is used in veterinary medicine for its sedative effects mediated by α2 adrenergic receptors, little is known about its acute or persistent effects on brain circuits involved in substance use disorders. This proposal is directly motivated by a recent Notice of Special Interest (NOSI) issued by the National Institute on Drug Abuse, “Xylazine: Understanding its Use and the Consequences” (NOT-DA-24- 012). The studies outlined in this proposal aim to address the xylazine’s cell signaling profiles and interactions with opioids at the circuit level. Human studies and preclinical models indicate the prefrontal cortex (PFC) is a key area involved in affective disturbances, amotivation, and cravings in opioid use disorder (OUD). Opioid drugs primarily act through the µ opioid receptor (MOR), which reduces feedforward GABA transmission within the PFC. We provide preliminary data corroborating these findings with the specific MOR agonist DAMGO. By contrast, the effects of xylazine on PFC function are unknown. We performed analogous electrophysiology experiments using bath application of xylazine and found a pronounced reduction in inhibitory postsynaptic currents. Furthermore, we found that xylazine reduces inhibitory transmission within the PFC through the σ1 receptor, independent from actions at the adrenergic or opioid receptors. In addition, xylazine’s ability to reduce IPSCs was blunted in slices from mice that underwent oxycodone dependence. Together, these preliminary findings support the hypothesis that xylazine and opioids synergistically reduce PFC inhibitory transmission through distinct actions on shared neural circuits. Here, we outline a series of innovative, circuit- specific electrophysiology experiments to test this hypothesis. Aim 1: Test the hypothesis that xylazine and opioids exert synergistic effects on PFC inhibitory transmission through distinct molecular mechanisms. Results from these experiments will determine whether xylazine and opioids exert synergistic effects and will refine our understanding of xylazine’s cell signaling actions under neurotypical conditions and following opioid dependence. Aim 2: Test the hypothesis that xylazine and opioids inhibit overlapping GABAergic circuits. We will leverage the recent development of viral constructs to access and manipulate MOR-expressing cells (i.e., AAV-mMORp-Cre-mCherry). We will next use a combination of intersectional viral tools to isolate transmission from MOR-expressing excitatory cells, MOR-expressing inhibitory cells, and MOR- negative inhibitory cells to define whether xylazine and MOR agonists regulate fast transmission from overlapping cell types in PFC. Impact: Results from these studies have the potential to be tremendously influential in how we conceptualize xylazine-opioid co-use. These results could also increase or enthusiasm for the development of MOR/σ1 bifunctional ligands or σ1 antagonists for the treatment of OUD or pain.
