Figure Legends
Figure 1. Mouse genotypes. (A) Genetic makeup of mice
used for this study: conditional knockout (cKO) =
Pirt-Cre+/- Oprm1fl/fl and wild-type
(WT) = Pirt-Cre+/+ Oprm1+/+.(B) Representative images of ethidium bromide-stained agarose
gels show PCR genotyping of Pirt-Cre (top) and Oprm1(bottom) from tail snip tissue samples. Higher band in Pirt-Crepanel indicates Pirt-Cre allele, and lower band indicates WT
allele (i.e. Pirt gene without Cre [Pirt-Cre+/-]).
Single band in Oprm1 panel indicates null gene (i.e. floxed
allele [Oprm1fl/fl]).
Figure 2. Oprm1 conditional knockout (cKO) selectively
deletes mu-opioid receptors (MOPs) in dorsal root ganglion (DRG)
neurons. Representative western blot images (A) and
quantification (B) of MOP protein levels in the DRG, spinal
cord (SC), small intestine, and periaqueductal gray (PAG) (N= 4-7 mice
per group). The amount of MOP protein (50 and 55 kDa) was normalized to
that of GAPDH (37 kDa) in the same sample, and the mean MOP level in
wild-type (WT) mice was considered to be 1. Data are shown as means ±
SEM. Protein levels were evaluated with Welch’s t-test.
Immunofluorescence-stained images and quantification of MOP expression
in DRG (C, D) and spinal cord dorsal horn (E, F) in
male Oprm1fl/fl Pirt-Cre+/- and WT
mice (N=2-3 mice per group, 2-3 DRG sections per mouse). Arrow direction
indicates dorsal side. Scale bar for DRG: 20 μm, and scale bar for
spinal cord: 50 μm. *p <0.05.
Figure 3. Bath application of DALDA and morphine reduces the
mean amplitude of HVA-ICa in cultured small-diameter DRG
neurons. (A) Representative traces of HVA-ICain response to bath application of 1 µM DALDA (Upper panel, N= 4; Black
trace, pre-DALDA mean HVA-ICa from 0-60 s; Gray trace,
post-DALDA mean HVA-ICa from 120-145 s) and 1 µM
morphine (Lower panel, N= 3; Black trace, pre-morphine mean
HVA-ICa from 0-60 s; Gray trace, post-morphine mean
HVA-ICa from 120-145 s). Scale bars: 200 pA, 10 ms.(B) Upper panel: A single bath application of 1 µM DALDA
produced a mean reduction in HVA-ICa magnitude. Lower
panel: A single bath application of 1 µM morphine produced a mean
reduction in HVA-ICa magnitude. Horizontal black bars
indicate the duration of drug exposure. (C) Upper panel: Bath
application of 1 µM DALDA significantly reduced HVA-ICa(t(146) = 11.84, unpaired t-test). Lower panel: Bath
application of 1 µM morphine significantly reduced
HVA-ICa (t(106) = 16.75, unpaired
t-test). *p <0.05.
Figure 4. Absence of mu-opioid receptors (MOPs) in primary
sensory neurons does not affect behavioral response to acute pain.(A) Paw withdrawal latency in response to radiant heat
stimulus. (B) Response latency to hot plate. (C) Paw
withdrawal frequency in response to low force (0.07 g) and high force
(0.4 g) von Frey monofilaments. (D) Latency to fall during
rotarod test. (E) Total distance traveled and (F)number of entries into the center of the open field test apparatus byOprm1 cKO and wild-type (WT) mice. (A-D) N = 10; (E, F) N = 16.
Equal numbers of male and female mice were used. Data are shown as means
± SEM and were compared by Welch’s t-test. *p <0.05.
Figure 5. Systemic DALDA and morphine do not inhibit responses
to acute nociceptive stimuli in MOP cKO mice. Wild-type and MOP cKO
(Oprm1fl/fl Pirt-Cre+/-) mice were
injected subcutaneously with 5 mg∙kg-1 DALDA or
morphine. Time course of the effects of DALDA (A) and morphine(B) on the paw withdrawal latency in response to radiant heat
stimuli and the paw withdrawal frequency in response to high force (0.4
g) von Frey filaments. N= 10 with 5 males and 5 females. Data are shown
as means ± SEM. *p <0.05 compared to pre-drug (within
the same genotype); #p <0.05 compared to wild-type
(same time point) by repeated measures two-way ANOVA with Bonferroni’s
post hoc test.
Figure 6. Absence of peripheral MOPs abolishes analgesic effect
of systemic DALDA and attenuates analgesic effect of systemic morphine
under persistent inflammatory pain conditions. (A , B )
Wild-type and MOP cKO (Oprm1fl/flPirt-Cre+/-) mice were injected subcutaneously with 5
mg∙kg-1 DALDA or morphine. Time course of the effects
of DALDA (A) and morphine (B) on paw withdrawal
latency in response to radiant heat stimuli. N = 10, with 5 males and 5
females. (C , D ) Wild-type and MOP cKO mice received a
20-μL subcutaneous injection of 1 mg∙mL-1 CFA in one
hind paw. Time course of the effects on thermal hyperalgesia(C) and paw edema (D) . Data are shown as means ± SEM.
*p <0.05 compared to pre-drug or pre-CFA (within the
same genotype); #p <0.05 compared to wild-type (same
time point) by repeated measures two-way ANOVA with Bonferroni’s post
hoc test.
Figure 7. Nerve injury induces bilateral mechanical allodynia
in the absence of peripheral MOPs. Time course of the effects of
SNIt-induced neuropathic pain on ipsilateral and
contralateral paw withdrawal frequency in response to (A) low
force (0.07 g) and (B) high force (0.4 g) von Frey
monofilaments in Oprm1 cKO and wild-type mice. N = 10 with 5
males and 5 females. Data are shown as means ± SEM.
*p <0.05 (ipsilateral) and† p <0.05 (contralateral) compared to
pre-surgery (within the same genotype);# p <0.05 (ipsilateral) and
‡p <0.05 (contralateral) compared to wild-type (same
time point) by repeated measures two-way ANOVA with Bonferroni’s post
hoc test.
Figure 8. Absence of peripheral MOPs abolishes analgesic effect
produced by systemic DALDA and attenuates analgesic effect produced by
systemic morphine in mice with neuropathic pain. Wild-type and MOP cKO
(Oprm1fl/fl Pirt-Cre+/-) mice were
injected subcutaneously with 5 mg∙kg-1 DALDA or
morphine. Time course of the effects of DALDA (A) and morphine(B) on paw withdrawal frequency in response to low force (0.07
g) and high force (0.4 g) von Frey monofilaments. N = 10, with 5 males
and 5 females. Data are shown as means ± SEM. *p <0.05
(ipsilateral) and † p <0.05 (contralateral)
compared to pre-drug (within the same genotype); #p <0.05 (ipsilateral) and ‡p <0.05
(contralateral) compared to WT (same time point) by repeated measures
two-way ANOVA with Bonferroni’s post hoc test.