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. 2023 Sep 13;14:5632. doi: 10.1038/s41467-023-41407-2

Fig. 5. mMORp driven recombinases and PHP.eB/PHP.S capsid packaging support intersectional viral strategies for gaining genetic access to CNS and PNS opioidergic cells/circuits.

Fig. 5

C57BL/6J mice injected with a 9 µl:1 µl mix of AAV8-mMORp-mCherry-IRES-Cre and a Cre-dependent AAV9-hDlx-FLEx-eGFP with intersectional expression in putative MOR+/GABAergic neurons in mPFC (a) and somatosensory cortex (S1, b). Insets in a and b show higher magnification images of hDlx-FLEx-eGFP cells (green) overlap with mMORp-mCherry-IRES-Cre cells (magenta); scale bars = 200 μm and 100 μm for insets. C57BL/6J mice injected in mPFC (c) or S1 (d) with a 9 µl:1 µl mix of AAV1-mMORp-FlpO and Flp-dependent AAV9-Ef1α-fDIO-mCherry. Inset high magnification images show mCherry+ cells. e CNS expression in representative sections from the spinal cord (coronal) and brain (sagittal along the medial-lateral axis relative to Bregma; coronal sections are shown in Supplementary Fig. 10) of C57BL/6J mice injected retro-orbitally with AAV.PHP.eB-mMORp-eYFP virus; scale bars = 500 μm (spinal cord sections) and 1000 µm (sagittal sections). f PNS dorsal root ganglia (DRG) FISH from C57BL/6J mice with intracerebroventrical injection of either AAV.PHP.S-mMORp-eYFP (upper) or AAV.PHP.S-CAG-tdTomato (lower). Custom cDNA probes targeting Rbfox3 (NeuN), Oprm1, EYFP and tdTomato transcripts; scale bars = 50 μm. g Quantification of total Oprm1+ cells co-labeled for tdTomato in control animals (N = 5 mice, n = 14 ROIs) compared to total Oprm1+ cells co-labeled for EYFP in PHP.S-mMORp-eYFP injected mice (N = 4 mice, n = 11 ROIs) across treatment groups (two tailed unpaired t-test with Welsh’s correction, P = 0.0437). h Summary quantification of the percent total number of cells positive for EYFP transcript (i.e., transduced by the AAV.PHP.s-mMORp-eYFP virus) that were also either positive for Oprm1 transcript (mMORp-eYFP/Oprm1+, 82.5%) or negative for Oprm1 transcript (mMORp-eYFP+ only, 17.5%) to demonstrate AAV.PHP.s-mMORp-eYFP specificity within mouse DRG neurons. Data presented as means ± SEM, *P < 0.05.