Fig. 1: Development and validation of murine and human mu opioid receptor promoter (MORp) driven viral constructs.

a DNA sequence for the murine Oprm1 (upper) and human OPRM1 (lower) promoter regions, including the approximate locations of several transcriptional elements such as repressor and activator transcription factors (TFs), transcription start sites and promoter elements, as determined via PROMO and Eukaryotic Promoter Database & UCSC Genome Browser analyses of the murine and human genes (Supplementary Fig. 1). The promoter region encoded by the four murine promoter constructs (mMORp1-4) and single human promoter construct (hMORp) are depicted beneath each sequence map. b mMORp and hMORp construct designs and packaging schema within adeno-associated viral (AAV) vectors of multiple different capsid serotypes. c Transduction efficacy from initial in vivo intracranial injections of the mMORp1-4-eYFP constructs into C57BL/6J mouse medial prefrontal cortex (mPFC), scale bar = 500 μm. d mPFC expression pattern with AAV1-mMORp1-eYFP across mPFC subregions, including the cingulate (Cg1), prelimbic (PL) and infralimbic (IL) cortex, scale bar = 200 μm. e Higher magnification images of the mPFC following transduction with the mMORp1 viral construct. Amplification of the eYFP signal, along with staining for both neuronal (NeuN) and microglial (Iba1) markers demonstrate selective transduction of neurons, with staining for additional glial markers to further verify this shown in Supplementary Fig. 5. Cortical layer division markers (Layers 1–6) highlight viral spread and efficiency, scale bar = 100 μm. f Overlap of mMORp1-eYFP viral expression and endogenous mu opioid receptor (MOR) immunoreactivity within the central amygdala (CeA) (denoted by blue outline), but not surrounding amygdalar subregions of a C57BL/6J mouse, using a knock-out mouse-validated anti-MOR antibody (Supplementary Fig. 3). Basolateral amygdala (BLA), medial anterodorsal amygdala (MeAd), medial anteroventral amygdala (MeAv), basomedial amygdala (BMA), dorsal entopeduncular nucleus (EPd), ventral entopeduncular nucleus (EPv), intercalcated cells (ITCs), globus pallidus externa (GPe), substantia innominota (SI), and lateral hypothalamus (LH); scale bar = 200 µm. g RNAscope FISH in the CeA of a mMORp1-eYFP injected mouse examining co-localized of Oprm1 and eYfp mRNA transcripts. CeA co-localization of AAV5-mMORp1-hM4Di-mCherry and Oprm1^2A-Cre:Sun1-sfGFP reporter nuclei (h) or anti-Cre staining (i). Co-expression of AAV5-mMORp1-hM4Di-mCherry with anti-Cre immunoreactive cells in the CeA of Oprm1^Cre mice (j), and the dorsomedial striatum (DMS) (k), mPFC (l) and ventral tegmental area (VTA, m) of Oprm1^2A-Cre mice. n Averaged number of mMORp1-mCherry+/anti-Cre+ cells compared to mMORp1-mCherry+/anti-Cre− cells quantified from successfully transduced brain regions of interest (from left or right hemispheres, or both) of Oprm1^Cre and Oprm1^2A-Cre:Sun1 mice within the CeA (~90.2%, N = 5, n = 9), DMS (~89.65%, N = 5, n = 7), mPFC (~88.28%, N = 3, n = 4), and VTA (~85.24%, N = 5, n = 8). Detailed information of total counts and quantification for each region within individual mouse lines can be found in Supplementary Fig. 4. Scale bar = 100 μm for g–m. White arrow heads denote cells in which co-labeling for Oprm1 and EYFP transcript (g) or mMORp1-mCherry and anti-Cre signal (h–m) is observed.