Centre for Neuropsychopharmacology, Division of Brain Sciences, Imperial College London, London, UK.
Centre for Neuroinflammation and Neurodegeneration, Division of Brain Sciences, Imperial College London, Hammersmith Hospital, London, UK.
J Cereb Blood Flow Metab. 2014 Oct;34(10):1604-12. doi: 10.1038/jcbfm.2014.117. Epub 2014 Jul 9.
Understanding the cellular processes underpinning the changes in binding observed during positron emission tomography neurotransmitter release studies may aid translation of these methodologies to other neurotransmitter systems. We compared the sensitivities of opioid receptor radioligands, carfentanil, and diprenorphine, to amphetamine-induced endogenous opioid peptide (EOP) release and methadone administration in the rat. We also investigated whether agonist-induced internalization was involved in reductions in observed binding using subcellular fractionation and confocal microscopy. After radioligand administration, significant reductions in [(11)C]carfentanil, but not [(3)H]diprenorphine, uptake were observed after methadone and amphetamine pretreatment. Subcellular fractionation and in vitro radioligand binding studies showed that amphetamine pretreatment only decreased total [(11)C]carfentanil binding. In vitro saturation binding studies conducted in buffers representative of the internalization pathway suggested that μ-receptors are significantly less able to bind the radioligands in endosomal compared with extracellular compartments. Finally, a significant increase in μ-receptor-early endosome co-localization in the hypothalamus was observed after amphetamine and methadone treatment using double-labeling confocal microscopy, with no changes in δ- or κ-receptor co-localization. These data indicate carfentanil may be superior to diprenorphine when imaging EOP release in vivo, and that alterations in the ability to bind internalized receptors may be a predictor of ligand sensitivity to endogenous neurotransmitter release.
了解正电子发射断层扫描神经递质释放研究中观察到的结合变化背后的细胞过程，可能有助于将这些方法转化为其他神经递质系统。我们比较了阿片受体放射性配体卡芬太尼和二苯哌啶与安非他命诱导的内源性阿片肽（EOP）释放和美沙酮给药在大鼠中的敏感性。我们还研究了激动剂诱导的内化是否参与了观察到的结合减少，使用亚细胞分级分离和共焦显微镜。在放射性配体给药后，在美沙酮和安非他命预处理后，观察到 [(11)C]卡芬太尼而不是 [(3)H]二苯哌啶摄取的显著减少。亚细胞分级分离和体外放射性配体结合研究表明，安非他命预处理仅降低了总 [(11)C]卡芬太尼结合。在与内化途径相似的缓冲液中进行的体外饱和结合研究表明，与细胞外隔室相比，μ-受体在内涵体中结合放射性配体的能力显著降低。最后，使用双标记共焦显微镜观察到安非他命和美沙酮处理后下丘脑 μ-受体-早期内涵体的共定位显著增加，而 δ-或 κ-受体的共定位没有变化。这些数据表明，在体内成像 EOP 释放时，卡芬太尼可能优于二苯哌啶，并且内化受体结合能力的改变可能是配体对内源性神经递质释放敏感性的预测指标。
