1H MRS-based metabolite changes at ventral respiratory control centers of the medulla oblongata following administration of morphine in wild-type and GIRK2 mutant mice

Respiratory depression is the leading cause of death in opioid overdose and is closely associated with the development of tolerance following repeated morphine use. However, the neurochemical adaptations in brainstem regions that regulate breathing, particularly under chronic opioid exposure, remain poorly understood. G-protein-gated inwardly rectifying potassium (GIRK) channels, especially the GIRK2 subunit, are expressed in rhythm-generating neurons of the pre-Bötzinger complex and have been implicated in opioid-induced respiratory depression. Nonetheless, their specific role in morphine-induced neurochemical changes is not yet fully defined. In this study, <i>in vivo</i> proton magnetic resonance spectroscopy (¹H MRS) was used in mice to assess morphine-induced metabolite changes in ventral brainstem regions encompassing the pre-Bötzinger complex. Wild-type mice were compared with GIRK2 heterozygous (GIRK2⁺/^-) mutants. Baseline levels of several metabolites including glutamate (Glu), myo-inositol (Ins), N-acetylaspartate plus N-acetylaspartylglutamate (NAA + NAAG), and glutamate plus glutamine (Glu + Gln) differed significantly between GIRK2⁺/^- and wild-type mice. Despite these baseline differences, many of morphine's effects on metabolite levels were similar in the wild-type and GIRK2⁺/^- mice. Morphine increased phosphocreatine (PCr) in both genotypes, while total creatine (Cr + PCr) decreased only in the wild-type mice. Glutamine levels increased significantly in both groups. Notably, NAA decreased in wild-type but increased in GIRK2⁺/^- mice, whereas NAA + NAAG decreased in both. These findings demonstrate that chronic morphine exposure induces substantial neurochemical changes in brainstem respiratory centers. Although the GIRK2⁺/ ^- mutation altered some of the metabolite responses, it does not fully block morphine's effects, highlighting the complexity of opioid-induced adaptations in the respiratory control networks.