We have developed a novel technique to measure basal changes of dopamine and its analytes in response to amphetamine and other pharmacological agents. Activation of protein kinase C-β enhances extracellular dopamine in the presence of amphetamine by enhancing the reverse transport of dopamine and internalizing the D2 autoreceptor. We previously demonstrated that PKCβ inhibitors block amphetamine-stimulated dopamine efflux in rat striatum in vitro.  In this study, we utilized in vivo microdialysis with LCMS for detection in live, behaving rats to assess the effect of the PKCβinhibitors enzastaurin and ruboxistaurin on amphetamine-stimulated increases in monoamines and their metabolites. Our novel technique can measure over 30 neuorchemicals in freely behaving rats using microdialysis with LCMS for detection. Previous techniques can only measure one analyte at a time using HPLC or voltammetry.  A 30 min perfusion of the nucleus accumbens core  with 1 uM enzastaurin or 1 uM ruboxistaurin reduced amphetamine-stimulated efflux of dopamine and its metabolite 3-methoxytyramine by approximately 50%.  The inhibitors also significantly reduced extracellular levels of  norepinephrine and its metabolite normetanephrine after amphetamine. The stimulation of locomotor behavior by amphetamine, measured simultaneously with the analytes, was comparably reduced by the PKCb inhibitors. These inhibitors are specific for dopamine and their metabolites.  Ruboxistaurin also attenuated cocaine stimulated extracellular dopamine, a process that would not be dependent upon DAT reversal. In order to see if this process was D2 autoreceptor mediated, we examined the effect of ruboxistaurin on cocaine activation when D2 receptors were blocked with raclopride.  The inhibitory effect of ruboxistaurin was reduced in the presence of cocaine and raclopride, suggesting that ruboxistaurin action involved D2 autoreceptors.   Using a stable isotope label retrodialysis procedure, we determined that ruboxistaurin had no effect on basal levels of dopamine, norepinephrine, glutamate, or GABA. Our results support the utility of using PKCβ inhibitors to reduce the effects of amphetamine and cocaine.<br/>

LC-MS Method to detect neurotransmitters <i>in vivo</i> during period of drug abuse

254th National Meeting (2017)

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LC-MS Method to detect neurotransmitters in vivo during period of drug abuse