Viral Strategies for Targeting and Manipulating Opioidergic Neuronal Populations for Cell Specific Study and Therapeutic Intervention

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      With concurrent global epidemics of opioid use and chronic pain disorders, there is a critical need to identify, target, observe and manipulate specific cell populations in the peripheral and central nervous systems expressing the mu-opioid receptor (MOR). To gain long-term access to neural cells types and circuits involved in modulating pain, analgesia and addiction, we have developed a catalog of MOR specific promoter constructs (MORsp) for driving transgene expression in MOR+ cells compatible with use in AAV vectors. Working off the mouse Oprm1 promoter, we created a pAAV1-mMORsp-eYFP construct and confirmed its expression in cultured rat cortical neurons. To validate MOR+ cell-type selectivity in vivo, we performed intracranial injections into several structures implicated in pain-processing, opioid analgesia and drug reward in C57BL6/J mice, including: PFC, insula, amygdala, VTA, PAG and spinal cord. Robust expression of the reporter tag was observed in all regions, and in situ hybridization studies demonstrated eYFP transcript signal overlaps heavily with endogenous Oprm1 mRNA expression patterns. Comparable levels of reporter were also observed in the CeA and VTA of injected Sprague-Dawley rats and common tree shrews. Next, MORsp construct variants designed to drive expression of the chemogenetic receptor hM4Di and calcium activity indicator GCaMP6f were injected into mouse spinal cord and amygdala, respectively. Behavioral responses to noxious mechanical and heat stimuli were markedly decreased in hM4Di-expressing animals following DREADD agonist administration, and noxious heat stimuli produced robust GCaMP6f-mediated calcium transients that were morphine-sensitive. In addition to mouse promoter based constructs, we created a human Oprm1 promoter variant and confirmed its selective expression in OPRM1+ neurons in macaque PFC, insula, and amygdala. In total, our new MORsp toolkit provides researchers cell-type specific genetic access to monitor and functionally manipulate opioidergic neural circuits across a range of vertebrate species and translational screening models for pain and addiction. Grant support from Harnessing cortical neuromodulation to disrupt pain perception (1DP2GM140923-01) T32 Translational Addiction Research Fellowship Program (5T32DA028874-11).
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