Compensatory Activation of Cannabinoid CB2 Receptor Inhibition of GABA Release in the Rostral Ventromedial Medulla in Inflammatory Pain

Abstract

The rostral ventromedial medulla (RVM) is a relay in the descending pain modulatory system and an important site of endocannabinoid modulation of pain. Endocannabinoids inhibit GABA release in the RVM, but it is not known whether this effect persists in chronic pain states. In the present studies, persistent inflammation induced by complete Freund's adjuvant (CFA) increased GABAergic miniature IPSCs (mIPSCs). Endocannabinoid activation of cannabinoid (CB1) receptors known to inhibit presynaptic GABA release was significantly reduced in the RVM of CFA-treated rats compared with naive rats. The reduction in CFA-treated rats correlated with decreased CB1 receptor protein expression and function in the RVM. Paradoxically, the nonselective CB1/CB2 receptor agonist WIN55212 inhibited GABAergic mIPSCs in both naive and CFA-treated rats. However, WIN55212 inhibition was reversed by the CB1 receptor antagonist rimonabant in naive rats but not in CFA-treated rats. WIN55212-mediated inhibition in CFA-treated rats was blocked by the CB2 receptor-selective antagonist SR144528, indicating that CB2 receptor function in the RVM is increased during persistent inflammation. Consistent with these results, CB2 receptor agonists AM1241 and GW405833 inhibited GABAergic mIPSC frequency only in CFA-treated rats, and the inhibition was reversed with SR144528. When administered alone, SR144528 and another CB2 receptor-selective antagonist AM630 increased mIPSC frequency in the RVM of CFA-treated rats, indicating that CB2 receptors are tonically activated by endocannabinoids. Our data provide evidence that CB2 receptor function emerges in the RVM in persistent inflammation and that selective CB2 receptor agonists may be useful for treatment of persistent inflammatory pain.

Significance statement: These studies demonstrate that endocannabinoid signaling to CB1 and CB2 receptors in adult rostral ventromedial medulla is altered in persistent inflammation. The emergence of CB2 receptor function in the rostral ventromedial medulla provides additional rationale for the development of CB2 receptor-selective agonists as useful therapeutics for chronic inflammatory pain.

Keywords: GABAergic transmission; RVM; endocannabinoids; persistent inflammation; whole-cell patch clamp.

Figure 1.

GABA release in the RVM is increased during persistent inflammation. A, Representative traces of mIPSCs in RVM slices of naive rats. B, Representative traces of mIPSCs in RVM slices of CFA-treated rats. C, D, Summary data of mIPSC frequency and amplitude recorded from DERM-A594-labeled neurons showing that mIPSC frequency is increased in CFA-treated compared with naive RVM (Mann–Whitney, U = 8.50) **p = 0.001 without a change in mIPSC amplitude (unpaired t test, t₍₁₉₎ = 0.75), p = 0.46. E, F, Summary data of mIPSC frequency and amplitude recorded from unlabeled RVM neurons showing that mIPSC frequency is increased in CFA-treated rats compared with naive rats (Mann–Whitney U = 8.0), **p = 0.001 without a change in mIPSC amplitude (unpaired t test, t₍₁₇₎ = 1.20). p = 0.25.

Figure 2.

Reduced endocannabinoid inhibition of presynaptic GABA release in persistent inflammation. A, Representative traces showing mIPSC frequency in the absence and presence of the CB1 antagonist rimonabant (RIM) in RVM neurons of naive rats. B, Representative traces showing mIPSC frequency in the absence and presence of the CB1 antagonist RIM in RVM neurons of CFA-treated animals. C, Summary data showing the increase in mIPSC frequency in the presence of rimonabant recorded from naive RVM neurons (two-way ANOVA, interaction, F_(1,17) = 28.66). **p < 0.001. p < 0.01 (Sidak's multiple comparison). There was no significant change in mIPSC frequency in RVM neurons from CFA-treated rats. p > 0.05 (Sidak's multiple comparison). D, Mean amplitudes determined from Gaussian fits to mIPSC amplitude histograms were not different in the presence of rimonabant between naive and CFA-treated RVM neurons (two-way ANOVA, interaction, F_(1,17) = 0.071), p = 0.79.

Figure 3.

Inhibition of FAAH reduces mIPSC frequency in naive but not CFA-treated rats. A, Cumulative frequency plot and summary data showing that the FAAH inhibitor URB597 increases interevent interval (IEI; left) and decreases mIPSC frequency in naive RVM neurons (right; Wilcoxon matched-pairs signed rank test, W = −28). *p = 0.016. n = 8. B, There were no significant differences in mIPSC amplitude (paired t test, t₍₇₎ = 1.05) p = 0.33. C, Cumulative frequency plot and summary data showing that the FAAH inhibitor URB597 has a reduced effect on IEI (left) and mIPSC frequency in RVM neurons from CFA-treated rats (right; Wilcoxon matched-pairs signed rank test, W = −19). p = 0.063, n = 9. D, There were no significant differences in the Gaussian mean of mIPSC amplitude distributions (paired t test, t₍₈₎ = 0.48). p = 0.65. E, Summary bar graph comparing the percentage inhibition by URB597 between RVM neurons from naive and CFA-treated rats (unpaired t test, t₍₁₅₎ = 2.91). *p = 0.011. F, mIPSC amplitude was not different in the presence of URB597 in naive compared with CFA-treated rats (unpaired t test, t₍₁₅₎ = 0.55). p = 0.59.

Figure 4.

Persistent inflammation does not change CB1 and CB2 mRNA levels but decreases CB1 protein levels. A, Relative quantities of mRNA for CB1 receptor expression in the RVM are not changed during persistent inflammation (unpaired t test, t₍₈₎ = 1.31). p = 0.6. Expression levels were compared with mapk6 internal control. B, Representative Western blot image and summary data show that the CB1 protein levels for the band running at 62 kDa are reduced in RVM from CFA compared with naive rats (unpaired t test, t₍₅₎ = 5.67). *p = 0.002. n = 3 or 4 rats/group. Other bands were not significantly changed (p > 0.05). Bands were analyzed relative to β-actin bands and then normalized to naive. C, Relative quantities of mRNA for CB2 receptor expression in the RVM are not changed after 5 d of inflammation (unpaired t test, t₍₈₎ = 0.46). p = 0.66. D, Representative Western blot image and summary data show that the CB2 protein levels for the band running at 40 kDa were the same for RVM tissue from naive and CFA-treated rats (unpaired t test, t₍₄₎ = 2.0). p > 0.05. n = 3 rats/group. Other bands were also not significantly changed (p > 0.05). Bands were analyzed relative to β-actin bands and then normalized to naive.

Figure 5.

CB1 receptor function is reduced in RVM neurons from CFA-treated rats. A, B, Summary data showing the change in mIPSC frequency in the presence of WIN55212 and after addition of the CB1 receptor antagonist rimonabant in RVM neurons from naive and CFA-treated rats (two-way repeated-measures ANOVA, interaction, F_(4,60) = 5.27, p = 0.001; Sidak's multiple comparisons test). **p < 0.01. Rimonabant reversed WIN55212 inhibition of mIPSC frequency in RVM neurons from naive but not in CFA-treated rats. C, The effect of WIN55212 was greater in slices from naive rats (unpaired t test, t₍₃₁₎ = 2.12, normalized to control mIPSC frequencies). *p = 0.04. D, The effect of WIN55212 was also greater in slices from naive rats (unpaired t test, t₍₃₁₎ = 5.15, normalized to mIPSC frequencies in the presence of rimonabant). **p < 0.0001.

Figure 6.

The selective CB2 antagonist SR144528 blocks WIN55212 effects in CFA-treated but not naive rats. A, WIN55212 reduces mIPSC frequency in naive RVM neurons in the presence of the CB2 antagonist SR144528 (3 μm) (Wilcoxon matched-pairs signed rank test, W = −28). *p = 0.02. B, WIN55212 did not inhibit mIPSC frequency in CFA-treated rats in the presence of SR144528 (Wilcoxon matched-pairs signed rank test, W = 12). p = 0.38. C, The effect of WIN55212 in the presence of CB2 antagonist SR144528 was greater in RVM neurons from naive compared with CFA-treated rats (unpaired t test, t₍₁₂₎ = 6.26). **p < 0.0001. Data are normalized to mIPSC frequency in the presence of SR144528.

Figure 7.

The selective CB2 agonists AM1241 and GW405833 inhibit GABA release in RVM neurons from CFA-treated rats. A, The CB2 agonist AM1241 (3 μm) significantly inhibits mIPSC frequency in neurons from CFA-treated but not in naive rats (two-way repeated-measures ANOVA, interaction, F_(2,30) = 9.90, p = 0.0005; Dunnett's multiple comparisons test). *p < 0.05. B, The percentage inhibition of mIPSC frequency by AM1241 was greater in RVM slices from CFA-treated compared with naive rats (unpaired t test, t₍₁₅₎ = 3.99). **p = 0.0012. Data are normalized to control mIPSC frequency before AM1241 superfusion. C, The CB2 agonist GW405833 (1 μm) significantly inhibits mIPSC frequency in neurons from CFA-treated but not in naive rats (two-way repeated-measures ANOVA, interaction, F_(2,22) = 14.23, p = 0.0001; Dunnett's multiple comparisons test). *p < 0.05. D, The percentage inhibition of mIPSC frequency by GW405833 was greater in RVM slices from CFA-treated compared with naive rats (unpaired t test, t₍₁₁₎ = 4.54). **p = 0.001. Data are normalized to control mIPSC frequency before GW405833 superfusion.

Figure 8.

Endocannabinoid tone is present in the RVM of CFA-treated rats. A, The CB2-selective antagonist SR144528 increases mIPSC frequency in CFA-treated rats but not naive rats (two-way repeated measure ANOVA, interaction, F_(1,13) = 5.84, p = 0.03, Sidak's multiple comparison). *p < 0.05. B, The CB2-selective antagonist AM630 also increases mIPSC frequency in CFA-treated rats without an effect in naive rats (two-way repeated measure ANOVA, interaction, F_(1,26) = 4.28, p = 0.049, Sidak's multiple comparison). *p < 0.05.