Program Director/Principal Investigator (Last, First, Middle): Ozra Dehkordi, Ph.D ABSTRACT The most frequent cause of death associated with opioid overdose is respiratory depression. The respiratory depressant effects of opioids are mainly mediated by G-protein-gated inward rectifying potassium channels subunit 2 (GIRK2) expressed by the respiratory rhythm generating neurons of the pre-Bötzinger complex. GIRK2 are also known to be implicated in opioid-induced analgesia. However, biochemical and metabolic changes associated with opioid activation of GIRK2 in the pre-Bötzinger and brainstem pain sensitive sites such as rostral ventral medulla (RVM), is still unknown. In the present study in mice, we hypothesize that 1) the metabolomic changes in the RVM and pre-Bötzinger complex associated with long-term subcutaneous morphine administration, is due to activation of GIRK2 channels expressed by pain sensitive cells of the RVM and neurokinin-1 receptor (NK-1R) expressing rhythm generating cells of the pre-Bötzinger complex; and that 2) this activation leads to an imbalance between excitatory and inhibitory neurotransmitters and their metabolites in the immediate vicinity of pre-Bötzinger complex and RVM. We will test this hypothesis using in vivo proton magnetic resonance spectroscopy (1H MRS) in wild-type and GIRK2 heterozygous (GIRK2+/-) mutant mice. We will analyze changes in the concentration of glutamate, glutamine, GABA, glycine and other metabolites in the pre-Bötzinger complex and RVM before and 6-7 days after subcutaneous implantation of morphine (75 mg pellets) and/ or placebo pellets (control). The results will provide valuable information regarding cellular and biochemical changes associated with morphine activation of GIRK2 at the pre-Bötzinger and RVM. The results also has the potential to enable the study of brain metabolites involved in opioid-induced analgesia at the RVM and opioid-induced respiratory depression at the pre- Bötzinger complex. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page