2.14.2 Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).
Results 3.1 MOP expression is selectively deleted in DRG neurons and reduced in the spinal cord of Oprm1 cKO miceTo selectively delete MOP expression in primary sensory neurons, we crossed Oprm1 floxed mice and Pirt-Cre mice. LoxP sites were inserted upstream of Oprm1 exon 2 and downstream of exon 3 (Weibel et al., 2013). The Cre-mediated recombination deleted the floxed exons 2 and 3. Genotyping PCR was used to select animals that were heterozygous for the Pirt-Cretransgene (Pirt-Cre+/-) and homozygous for the nullOprm1 allele (Oprm1fl/fl; Figure 1 ). Immunoblotting results indicated that MOP produced two protein bands (approximately 50 and 55 kDa) in all tissues tested from WT mice, potentially due to glycosylation (Huang, Chen & Liu-Chen, 2015). In Oprm1 cKO mice, however, quantification analysis revealed a marked reduction in the level of MOP protein in the DRG (17.5%), relative to that in WT mice. MOP protein expression levels in the spinal cord, small intestine, and periaqueductal gray tissues did not differ significantly between genotypes (Figure 2A, B) . Immunofluorescence imaging of DRG sections showed that MOP antibody stained a significantly smaller subset of neurons in MOP cKO mice (3.75 ± 2.25 cells) relative to that in WT animals (27.00 ± 2.75 cells). Immunofluorescence imaging of spinal cord sections revealed that MOP immunoreactivity was primarily distributed in the superficial laminae of the spinal dorsal horn in WT mice. In Oprm1 cKO mice, MOP immunoreactivity was significantly reduced to 58.91 ± 12.84% in the superficial dorsal horn and 75.88 ± 16.54% in deeper laminar neurons, relative to that in WT animals (Figure 2C-F) . MOP immunoreactivity was higher in deep laminae of cKO spinal cord than in superficial laminae. These data indicate that MOPs are removed exclusively from primary sensory neurons and their central terminals in the spinal cord of Oprm1 cKO mice.3.2 MOP agonists inhibit calcium currents in naïve DRG neuronsPrior evidence suggests that MOP agonism diminishes neuronal excitability via G-protein-dependent inhibition of HVA calcium currents (Dolphin & Scott, 1989; Rusin & Moises, 1995; Tan, Groszer, Tan, Pandya, Liu & Xie, 2003). To elucidate the involvement of MOP activation in DALDA application-dependent pain inhibition, we examined whole-cell patch-clamp electrophysiological recordings of HVA-ICain adult WT mouse DRG neurons (Figure 3 ). Bath-application of 1 µM DALDA (Figure 3A-C, top panels; t(146) = 11.84, unpaired t-test) and 1 µM morphine (Figure 3A-C, lower panels; t(106) = 16.75, unpaired t-test) significantly inhibited HVA-ICa in naïve, small-diameter DRG neurons.3.3 Absence of MOPs in primary sensory neurons does not affect physiological response to noxious stimuli
WT and cKO mice showed no significant difference in behavioral response to acute thermal and mechanical stimuli. PWL to radiant heat was 14.90 ± 1.43 s in WT mice and 14.72 ± 0.78 s in Oprm1 cKO mice (Figure 4A ). In the hot plate test, response latency was 13.70 ± 0.56 s in WT mice and 13.48 ± 0.45 s in the Oprm1 cKO group(Figure 4B) . The von Frey assay for mechanical sensitivity (0.4 g filament) revealed a 35 ± 2.24% PWF in WT mice and 34 ± 2.21% PWF inOprm1 cKO mice (Figure 4C ). No sex differences in behavioral response to nociceptive stimuli were observed in either genotype (Supplemental Figure 1A-C) .
Additional studies confirmed that the absence of MOPs in primary sensory neurons does not affect gross motor function. Thus, the animals’ behavioral response to nociceptive stimuli was not influenced by peripheral MOP-mediated motor deficits. Baseline motor coordination, ataxia, and balance, measured as the latency to fall from an accelerating rotarod, did not significantly differ between Oprm1cKO and WT mice (Figure 4D) . Interestingly, significant differences were observed between groups in the open field test(Figure 4E, F and Supplemental Figure 1E) . WT mice traveled a total distance of 4135 ± 199.40 cm, whereas Oprm1 cKO mice traveled only 2660 ± 175.00 cm. WT mice spent approximately 7.52 ± 1.02% of their time in the center of the open field apparatus, with an average of 67.92 ± 10.79 center zone entries, whereas Oprm1 cKO mice spent only 4.42 ± 0.98% of their time in the center, with an average of 32.13 ± 5.94 entries. No significant differences were observed in the body weight of males or females between genotypes (not shown), and no sex-dependent differences were observed in motor activity(Supplemental Figure 1D-F) .