Monoacylglycerol Lipase Protects the Presynaptic Cannabinoid 1 Receptor from Desensitization by Endocannabinoids after Persistent Inflammation

Abstract

Cannabinoid-targeted pain therapies are increasing with the expansion of cannabis legalization, however, their efficacy may be limited by pain-induced adaptations in the cannabinoid system. Cannabinoid receptor subtype 1 (CB1R) inhibition of spontaneous, GABAergic miniature IPSCs (mIPSCs) and evoked IPSCs (eIPSCs) in the ventrolateral periaqueductal gray (vlPAG) were compared in slices from naive and inflamed male and female Sprague Dawley rats. Complete Freund's Adjuvant (CFA) injections into the hindpaw induced persistent inflammation. In naive rats, exogenous cannabinoid agonists robustly reduce both eIPSCs and mIPSCs. After 5-7 d of inflammation, the effects of exogenous cannabinoids are significantly reduced because of CB1R desensitization via GRK2/3, as function is recovered in the presence of the GRK2/3 inhibitor, Compound 101 (Cmp101). Inhibition of GABA release by presynaptic μ-opioid receptors in the vlPAG does not desensitize with persistent inflammation. Unexpectedly, while CB1R desensitization significantly reduces the inhibition produced by exogenous agonists, depolarization-induced suppression of inhibition protocols that promote 2-arachidonoylglycerol (2-AG) synthesis exhibit prolonged CB1R activation after inflammation. 2-AG tone is detected in slices from CFA-treated rats when GRK2/3 is blocked, suggesting an increase in 2-AG synthesis after persistent inflammation. Inhibiting 2-AG degradation with the monoacylglycerol lipase (MAGL) inhibitor JZL184 during inflammation results in the desensitization of CB1Rs by endocannabinoids that is reversed with Cmp101. Collectively, these data indicate that persistent inflammation primes CB1Rs for desensitization, and MAGL degradation of 2-AG protects CB1Rs from desensitization in inflamed rats. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapeutics targeting MAGL and CB1Rs.SIGNIFICANCE STATEMENT Presynaptic G-protein-coupled receptors are resistant to desensitization. Here we find that persistent inflammation increases endocannabinoid levels, priming presynaptic cannabinoid 1 receptors for desensitization on subsequent addition of exogenous agonists. Despite the reduced efficacy of exogenous agonists, endocannabinoids have prolonged efficacy after persistent inflammation. Endocannabinoids readily induce cannabinoid 1 receptor desensitization if their degradation is blocked, indicating that endocannabinoid concentrations are maintained at subdesensitizing levels and that degradation is critical for maintaining endocannabinoid regulation of presynaptic GABA release in the ventrolateral periaqueductal gray during inflammatory states. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapies.

Keywords: GPCR; desensitization; endocannabinoid; inflammation; periaqueductal gray; presynaptic.

Figure 1.

Persistent inflammation reduces WIN-mediated inhibition of GABA release. A, Representative traces of eIPSCs isolated in NBQX recorded from vlPAG neurons in baseline (5 μm; teal), the cannabinoid receptor agonist WIN (3 μm; black) and the CB1R-selective antagonist RIM (3 μm; blue) from naive (left) and CFA-treated (right) animals. B, Percentage of inhibition of eIPSC amplitude by WIN compared with the average of baseline and RIM recovery in slices from naive and CFA-treated rats (unpaired t test; t₍₁₄₎ = 5.34, p < 0.0001). C, D, Representative traces of mIPSCs recorded from vlPAG neurons in baseline containing TTX (500 nm) and NBQX (5 μm), WIN (3 μm), and RIM (3 μm) from slices of naive (C) or CFA-treated rats (D). E, mIPSC frequency at baseline, WIN, and RIM from slices of naive and CFA-treated rats. (F) WIN percentage inhibition of mIPSC frequency from naive and CFA-treated rats (unpaired t test; t₍₁₀₎ = 4.65, p = 0.0005). Error bars represent the SEM, dots indicate individual recordings, and numbers represent the number of rats represented per bar.

Figure 2.

Persistent inflammation does not affect MOR suppression of GABA release. A, Representative eIPSC traces at baseline: NBQX, 5 μm (teal); DAMGO, 1 μm (black), and naloxone, 1 μm (blue). B, Percentage inhibition of eIPSCs by DAMGO in naive (black bar) and CFA-treated (red bar) conditions (unpaired t test; t₍₁₀₎ = 0.32, p = 0.8). C, Spontaneous mIPSC frequency in slices from naive (black) or CFA-treated (red) animals during baseline, DAMGO administration (1 μm), and naloxone (1 μm). D, DAMGO inhibition of mIPSC frequency inhibition (unpaired t test: t₍₉₎ = 1.11, p = 0.3). Error bars represent the SEM, dots indicate individual recordings, and numbers represent the number of rats represented per bar.

Figure 3.

Cannabinoid receptor binding is unaffected by persistent inflammation. A, Representative traces of eIPSCs recorded from vlPAG neurons in baseline (NBQX 5 μm; teal), cannabinoid agonist CP55,940 (CP55; 3 μm; black), and CB1-selective antagonist AM251 (3 μm; blue) from naive and CFA-treated rats. B, Percentage inhibition of eIPSC amplitude by CP55,940 in vlPAG slices from naive or CFA-treated rats (unpaired t test; t₍₉₎ = 7.8, p < 0.0001). Error bars represent the SEM, dots indicate individual recordings, and numbers represent the number of rats per bar. C, Representative radioligand binding saturation curve with ^[3H]CP55,940 and vlPAG tissue from naive (black) and CFA-treated (red) rats (vlPAG from 8 rats pooled per curve; statistics run on an average of 3 curves). Naive B_max, 708 ± 126 fmol/mg; CFA B_max, 785 ± 61 fmol/mg; unpaired t test: t₍₄₎ = 0.55, p = 0.6. Naive K_d = 1.7 ± 0.5 nmol; CFA K_d = 1.8 ± 0.4 nmol; unpaired t test: t₍₄₎ = 0.27, p = 0.8.

Figure 4.

CB1R function is sustained throughout 5 h WIN-induced activation. A, Inhibition of mIPSC frequency in recordings from vlPAG neurons during 30 min of WIN exposure (3 μm; n = 6 cells from 6 rats). Data are normalized to mIPSC frequency during baseline in TTX (500 nm) and NBQX (5 μm). WIN (3 μm) reduced mIPSC frequency over the first 10 min of drug application. Frequency remained reduced for the entirety of the 30 min drug application and was reversed by RIM (3 μm). B, eIPSC amplitude with bath application of CB1R-selective antagonist RIM (3 μm) after 15 min in WIN (3 μm; paired t test; t₍₇₎ = 2.42, *p = 0.046; data from 6 rats) or >1 h of WIN incubation (3 μm; paired t test; t₍₅₎ = 3.53, *p = 0.02; 5 cells from 3 rats). Average is shown in a thick black line. C, Bar graph depicting RIM percentage increase from WIN after 15 min in WIN (white bar) or >1 h in WIN (gray bar; unpaired t test; t₍₁₁₎ =0.2, p = 0.8).

Figure 5.

Cmp101 incubation recovers CB1R inhibition of GABA release after persistent inflammation. A, WIN (3 μm) inhibition of eIPSC amplitudes from naive or CFA-treated rats. vlPAG slices were incubated in vehicle (no fill) or Cmp101 (filled bar) for >1 h. Cmp101 incubation fully recovered CB1R signaling in slices from CFA-treated rats (two-way ANOVA; main effect of Cmp101: F_(1,14) = 5.9, p = 0.029; main effect of CFA: F_(1,14) = 26.3, p = 0.0002; CFA × Cmp101 interaction: F_(1,14) = 14.63, p = 0.002; p values on graphs are Šídák post hoc test). B, DAMGO (1 μm) inhibition of eIPSC amplitude after Cmp101 (30 μm) incubation from naive or CFA-treated rats (unpaired t test; t₍₈₎ = 0.92, p = 0.4). C, Cmp101 incubation reveals eCB tone in recordings from CFA-treated rats (two-way ANOVA; main effect of Cmp101: F_(1,46) = 6.1, p = 0.02; values on graphs are from Šídák post hoc test). Experiments using either RIM or NESS are combined. Error bars represent the SEM, dots indicate individual neurons, and numbers represent the number of animals per bar.

Figure 6.

Persistent inflammation increases 2-AG activation of CB1Rs. A, Summary of DSI (+20 mV for 5 s) in tissue from naive rats (black circles; n = 21 recordings from 15 rats). DSI is blocked by NESS (0.5 μm; gray open boxes; n = 4 recordings from 3 rats). B, Proportion of patched neurons in slices from naive rats that responded to DSI (21 neurons exhibited DSI, and 29 did not). C, Summary of DSI in tissue from CFA-treated rats (red dots; n = 22 recordings from 17 rats). DSI is blocked by NESS (0.5 μm; gray open boxes; n = 9 recordings from 5 rats). D, After persistent inflammation, 22 cells exhibit DSI, while 11 do not. The proportion of neurons that produced DSI was significantly higher in slices from CFA-treated slices than naive slices (χ², p = 0.03). E, Quantification of inhibition of eIPSC amplitudes at max DSI and late DSI in vlPAG tissue from naive and CFA-treated rats (two-way repeated-measures ANOVA; main effect of DSI: F_(1,41) = 4.42, p = 0.04; main effect of treatment: F_(1,41) = 10.75, p = 0.002; DSI × treatment interaction: F_(1,41) = 4.22, p = 0.046; values on graphs from Šídák post hoc test). F, DO34 (1 μm, >1 h), an inhibitor of the 2-AG synthesis enzyme DAGLα, significantly reduces maximum DSI (two-way ANOVA; main effect of DO34, F_(1,77) = 92.19, p < 0.0001; Šídák post hoc test). G, Maximum and late DSI in slices from naive and CFA-treated animals after incubation in JZL184 (1 μm, >1 h) and JZL184 + Cmp101 (1 μm, >1 h; two-way repeated-measures ANOVA; main effect of treatment, F_(2,53) from 6.04, p = 0.004; p values on graphs from Šídák post hoc test). Dots represent individual recordings, numbers below the bar represent number of animals; error bars represent the SEM.