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) .