Online Preservation for In Vivo Microdialysis with MS~3 Quantification Dynamic Monitoring of Endogenous Opioids in the Anterior Cingulate Cortex

摘要

Endogenous opioids and their receptors are important in multiple biological circuits including those associated with pain, reward, addiction, drug abuse, stress, and affective disorders. The dynamic monitoring of endogenous opioid peptides (EOPs) in the mammalian central nervous system has been difficult due to their low concentrations, high susceptibility to degradation, and the significant expense and specialization of available monitoring techniques. To address the role of EOPs in chronic pain, and expand dynamic in vivo measurements of EOPs to cortical regions, we developed on-line preservation microdialysis with nano liquid-chromatography and tandem mass-spectrometry (nano LC-MSn). This approach was employed to prevent peptide degradation during collection from awake and freely moving rats. A commercially available microchip-based electrospray ionization source facilitated ultra-trace measurements of enkephalins (limit of detection 1.5 amol) and overcame difficulties associated with previously employed LC-MS instrumentation. This allowed in vivo measurement of leucine and methionine enkephalin, and endomorphin II in the rat anterior cingulate cortex at estimated concentrations of 46 pM, 160 pM, and 1.4 nM, respectively. Additionally, significant stimulated release occurred for enkephalin (> 150% baseline). This approach can be used to quantitatively monitor release of endogenous opioids in physiological settings for pharmacological and behavioral challenge of EOP dynamics.