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).