In this study, female mice homozygous for the wild-type (AA) or mutant (GG) allele of the Oprm1 A112G SNP (orthologous to the common human variant, A118G) underwent an experimenter-administered morphine dependence protocol (escalating doses of morphine sulfate [20, 40, 60, 80, and 100 mg/kg, i.p., twice daily] or saline control). Twenty-four hours following the final dose of morphine, mice were sacrificed for RNAscope analysis. The goal of the RNAscope was to validate genotype-specific differentially expressed genes previously identified in this model using spatial transcriptomics, as described in the associated manuscript. Midline sagittal sections were stained for Cocaine and amphetamine regulated transcript prepropeptide using RNAscope probes and reagents (ACD Bio). Whole-slide images of one tissue section per mouse are provided.

To model opioid dependence and withdrawal, mice with Oprm1 AA or GG alleles were randomly assigned to chronic morphine (escalating doses of morphine sulfate (20, 40, 60, 80, and 100 mg/kg, i.p., twice daily) This escalating morphine dosing regimen is widely established in rodent models to reliably induce opioid dependence while minimizing acute toxicity, enabling the  study of chronic adaptation mechanisms^46,47.  Control mice received saline (3 mice x 2 genotypes x 2 treatments = 12 total mice).  This sample size aligns with established standards for high-resolution spatial transcriptomics studies, where tissue-wide sampling across hundreds of fields of view per animal provides robust statistical power even with relatively small animal cohorts^48,49.

An additional cohort of adult female mice were exposed to escalating morphine or saline but** mice remained in the home cage for 24 hours following the final morphine or saline injection. (n= 14 AA-SAL, 10 GG-SAL, 16 AA-MOR, 16 GG-MOR). Mice were then placed in a clear plastic arena and observed for 20 minutes for jumping, a well characterized index of spontaneous somatic opioid withdrawal.

RNAscope

Immediately following spontaneous withdrawal scoring, mice were quickly euthanized by live cervical dislocation. Brains were rapidly dissected and flash-frozen in isopentane. For slide preparation, flash-frozen brains were equilibrated to -20 degrees C in Tissue-Tek Optimal Cutting Temperature Compound and sectioned at 20um on a Leica CM3050 cryostat. Midline sagittal section corresponding to lateral coordinates 0.475–0.674 mm (Allen Mouse Brain Common Coordinate Framework CCFv3) were dry-mounted onto SuperFrost Plus microscope slides (n=3/group).

Prepared slides were hybridized with RNAscope Probe Mm-Cartpt (Cat No. 432001) using the RNAscope Multiplex Fluorescent Assay v2 (Cat No. 323100, ACD Bio). Slides were removed from −80 °C and fixed in pre-chilled 10% NBF (HT5011) at 4°C for 1 hour, then rinsed twice with 1× PBS. Sections were then dehydrated by sequential immersion in 50% ethanol (~5 min), 70% ethanol (~5 min), 100% ethanol (~5 min), and 100% ethanol (~5 min) at room temperature (RT).

A hydrophobic barrier was created using an ImmEdge hydrophobic barrier pen and allowed to dry completely. For optimal results, hydrogen peroxide, target retrieval, and protease treatments were skipped. Slides were submerged in 1× RNAscope wash buffer for ~5 min at RT, then inserted into the HybEZ humidity control tray and incubated with each probe mixture for ~2 hours at 40 °C. Before continuing, slides were washed once with 1× wash buffer for 2 min at RT. Tissue sections were then hybridized at 40 °C in AMP 1 (~30 min), AMP 2 (~30 min), and AMP 3 (~15 min), with two washes in 1× RNAscope wash buffer between each incubation.

To develop the HRP-C1 signal for Mm-Cartpt, tissue sections were incubated in RNAscope Multiplex FL v2 HRP-C1 for ~15 min at 40 °C, followed by two washes (~2 min each) in 1× RNAscope wash buffer. Sctions were then completely covered with diluted fluorophore for labeling the C1 probe and incubated for 30 min at 40 °C (Opal 690 dye, FP149700, 1:1500 in TSA buffer). Tissue sections were then incubated in HRP blocker for ~15 min at 40 °C. Finally, tissue sections were washed twice for 2 min in 1× RNAscope wash buffer, counterstained with DAPI reagent for 30 seconds, and mounted using ProLong Gold Antifade Mountant. Slides were allowed to dry for ~8 hours in the dark.

The Leica Stellaris 5 Confocal Microscope was used to evaluate the samples at 20X magnification and collect composite, whole-slide images. Analysis was performed in CellProfiler 4.2.8⁵⁰ (Broad Institute). Images were cropped to the regions of interest (nucleus accumbens, frontal cortex) and separated by channel. Nuclei were segmented by DAPI staining. Cartpt images were converted to binary by automated thresholding. Total area of Cartpt staining and number of Cartpt+ nuclei was computed per image, as well as the mean intensity of Cartpt staining within DAPI nuclei.