2.1 Animals
Adult (4-6 months old) male and female wild-type (WT) C57BL/6J mice (20–45g; The Jackson Laboratory, Bar Harbor, ME) and Oprm1conditional knockout mice (Oprm1 cKO) were housed under optimal laboratory conditions with a 12-hr light/dark cycle and free access to food and water. Mice were ear-marked at the time of weaning (21 days after birth), and tail biopsies were used for PCR genotyping. Mice were group-housed (one to five animals per cage) and maintained in a temperature- and humidity-controlled environment (18-26˚C with 30-70% relative humidity).
The Cre/LoxP recombination system was used to generate Oprm1 cKO mice in which MOPs were deleted specifically from primary sensory neurons. We crossed Oprm1 floxed mice, provided by Pr. Claire Gaveriaux-Ruff (University of Strasbourg, Strasbourg, France), andPirt-Cre mice, provided by Dr. Xinzhong Dong (Johns Hopkins University, Baltimore MD, USA). Pirt is a modulator of several TRP channels and is expressed specifically in all primary sensory neurons in the DRG, but not in any CNS neurons. The Cre recombinase is under control of the Pirt promoter and expressed exclusively in >95% of all DRG neurons (Kim et al., 2008). One WT allele of the Pirt gene must be present in order for the Cre recombinase to use the Pirt promoter (Pirt-Cre+/-). Animals homozygous for the floxed allele (Oprm1fl/fl) and heterozygous for thePirt-Cre (Pirt-Cre+/-) transgene were selected for this study. Control WT animals were homozygous for both Oprm1and Pirt (Pirt+/+ Oprm1+/+).
Behavioral experiments were carried out with an equal number of age- and sex-matched adult WT and Oprm1 cKO mice. Animals were handled and acclimatized to laboratory conditions starting at least 1 week before being used in experimental procedures. All tests were carried out between 9:00 a.m. and 5:00 p.m. The experimental protocols were approved by the Animal Care and Use Committee of Johns Hopkins University and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals to ensure minimal animal use and discomfort. Animals were randomly assigned into treatment groups, and investigators were blinded to treatment assignment and outcome assessment. The number of mice in each group is designated in the figure legends. The sample size in each group was determined based on our previous studies with similar experimental protocols (Tiwari et al., 2018; Tiwari et al., 2020). Animal studies are reported in compliance with the ARRIVE guidelines and with the recommendations made by the British Journal of Pharmacology(Kilkenny, Browne, Cuthill, Emerson & Altman, 2010; McGrath, McLachlan & Zeller, 2015).