Sex differences in neurotransmitter levels in different brain regions after acute and chronic morphine treatment in mice

Background and Purpose Pain management is a major health burden. Pain results from the integration of the nociceptive message and neuronal communication relying on neurotransmitters such as glutamate, γ-aminobutyric acid (GABA), dopamine, noradrenaline and serotonin in brain regions including the periaqueductal gray (PAG), the nucleus accumbens (Nac), the caudate-putamen (Cpu) and the amygdala. Morphine remains the gold standard painkiller for severe pain via the activation of the mu opioid receptors. However, among side effects, morphine chronic treatment lead to antinociceptive tolerance. As antinociceptive tolerance might be linked to neurotransmission dysregulation, we have compared various neurotransmitter concentrations in acute vs chronic morphine conditions in the amygdala, PAG, Cpu, and the Nac of male and female mice. Experimental approach Sex differences in morphine antinociception and tolerance were assessed using the tail-immersion test. The behavioural effects of acute and chronic morphine treatments, as well as sex differences in the levels of dopamine, serotonin, noradrenaline, glutamate and GABA in the amygdala, PAG, Cpu, and the Nac were determined by an absolute quantification LC-MS/MS approach using the isotopic dilution method. Key results This study indicates, as previously reported, that female mice are less sensitive to morphine and develop morphine antinociceptive tolerance earlier than males (ED50 of 5.5±0.24 days vs 1.54±0.11 days, respectively). However, the rate at which the tolerance developed did not differ between both sex. We have found major differences in dopamine, serotonin, noradrenaline, glutamate and GABA levels between female and male mice in the amygdala, PAG, Cpu, and the Nac. Finally, no major effect of anti-nociceptive tolerance induced by chronic morphine was observed compared to acute administration of morphine. Conclusion Neurotransmitter differences are attributable mainly to sex differences in pain-related CNS regions. However, the impacts of morphine anti-nociceptive tolerance on dopamine, serotonin, noradrenaline, glutamate and GABA contents appeared to be limited. ### Competing Interest Statement The authors have declared no competing interest. * 5-HT2C receptor : 5-hydroxy tryptamine receptor2C ACN : acetonitrile CID : collision gas CNS : central nervous system Cpu : caudate-putamen CT : group of mice treated acutely with morphine ED50 : half-maximal effective dose GABA : γ-Aminobutyric acid IS : internal standard LC-MS/MS : liquid chromatography coupled to tandem mass spectrometry LSC : lumbar spinal cord MOR : mu-opioid receptor MPE : maximal possible effect MRM : multiple reaction monitoring mode Nac : nucleus accumbens PAG : periaqueductal gray TIT : tail immersion test TOL : group of mice groups treated chronically with morphine UGT : UDP-glucuronosyl-transferase VTA : ventral tegmental area