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.