Elevating levels of the endocannabinoid 2-arachidonoylglycerol blunts opioid reward but not analgesia

Abstract

Converging findings have established that the endocannabinoid (eCB) system serves as a possible target for the development of new treatments for pain as a complement to opioid-based treatments. Here we show in male and female mice that enhancing levels of the eCB, 2-arachidonoylglycerol (2-AG), through pharmacological inhibition of its catabolic enzyme, monoacylglycerol lipase (MAGL), either systemically or in the ventral tegmental area (VTA) with JZL184, leads to a substantial attenuation of the rewarding effects of opioids in male and female mice using conditioned place preference and self-administration paradigms, without altering their analgesic properties. These effects are driven by CB1 receptors (CB1Rs) within the VTA as VTA CB1R conditional knockout, counteracts JZL184's effects. Conversely, pharmacologically enhancing the levels of the other eCB, anandamide (AEA), by inhibition of fatty acid amide hydrolase (FAAH) has no effect on opioid reward or analgesia. Using fiber photometry with fluorescent sensors for calcium and dopamine (DA), we find that enhancing 2-AG levels diminishes opioid reward-related nucleus accumbens (NAc) activity and DA neurotransmission. Together these findings reveal that 2-AG counteracts the rewarding properties of opioids and provides a potential adjunctive therapeutic strategy for opioid-related analgesic treatments.

Figure 1.. JZL184 attenuates morphine preference via CB1Rs.

(A) Endocannabinoid signaling pathways regulated by MAGL and FAAH enzymes that are targeted by inhibitors, JZL184 and PF-385, respectively. (B) Timeline of behavioral protocol for morphine CPP and JZL184 systemic injections pretreatment. (C) Systemic injection of JZL184 prior to each morphine conditioning session abolished morphine CPP (Two-way ANOVA, significant interaction (treatment x day), F_1,26 =19.77, P=0.0001; Bonferroni post hoc test: Veh: Test vs Pretest P<0.001***, JZL184: Test vs Pretest P>0.9999, Test: Veh vs JZL184 P<0.001†††, Veh n=7, JZL184 n=8). (D) Timeline of behavioral protocol for morphine CPP and PF-3845 systemic injections pretreatment. (E) Systemic injection of PF-3845 (Two-way ANOVA, main effect of day, F_1,32=48.49, P<0.0001; Bonferroni post hoc test: Veh: Test vs Pretest P<0.001***, PF-3845: Test vs Pretest P=0.001***, Veh n=9, PF-3845 n=9) had no effect on morphine CPP. (F) Timeline of behavioral protocol for morphine CPP and AM251 and/or JZL184 systemic injections pretreatment. (G) Systemic injections of the CB1Rs antagonist AM251 (3mg/kg) prior to JZL184 systemic exposure counteracted the JZL184 induced morphine blunted response (Stats were performed by AM251 dose; Three-way ANOVA, significant interaction ((AM251 (3mg/kg) treatment x days x JZL184 treatment), F_1,78=5.159, P=0.0259*; Bonferroni post hoc test: Veh-Veh: Test vs Pretest P<0.001***, Veh-JZL184: Test vs Pretest P>0.9999, AM251-Veh: Test vs Pretest P=0.0053**, AM251-JZL184: Test vs Pretest P=0.0025**, Test: Veh-Veh vs Veh-JZL184 P=<0.001†††) (AM251 (1mg/kg) significant interaction of AM251 x days, F_1,78=4.131, P=0.0456*, JZL184 x days, F_1,77=16.58, P=0.0001***; Bonferroni post hoc test: Veh-Veh: Test vs Pretest P<0.001***, Veh-JZL184: Test vs Pretest P>0.9999, AM251-Veh: Test vs Pretest P=0.0450*, AM251-JZL184: Test vs Pretest P<0.001***, Test: Veh-Veh vs Veh-JZL184 P=<0.001†††), Veh-Veh n=24, Veh-JZL184 n=26, AM251 (3mg/kg)-Veh n=15, AM251 (3mg/kg)-JZL184 n=17, AM251 (1mg/kg)-Veh n=15, AM251 (1mg/kg)-JZL184 n=16). Error bars ± SEM.

Figure 2.. JZL184 attenuates oxycodone preference and self-administration.

(A) Timeline of behavioral protocol for oxycodone CPP and JZL184 systemic injections pretreatment. (B) Systemic JZL184 prior to each oxycodone conditioning session attenuated oxycodone CPP (Two-way ANOVA, significant interaction (treatment x day), F_1,82 =13.31, P=0.0005; Bonferroni post hoc test: Veh: Test vs Pretest P<0.001***, JZL184: Test vs Pretest no significant, Test: Veh vs JZL184 P<0.001†††, Veh n=19, JZL184 n=24). (C) Timeline of behavioral protocol for oxycodone self-administration and JZL184 systemic injections pretreatment. (D-E). Systemic JZL184 exposure prior to oxycodone self-administration sessions, attenuated the intake of oxycodone (D; Two Way-RM ANOVA, main effect of JZL184 treatment, F_1,21 =21.43, P=0.0001***, main effect of days, F_2.962,62.20 =8.236, P=0.0001***, interaction F_9,189 =1.900, P=0.0541, E; Average of total infusions/animal (Ttest, t₍₂₁₎ = 4.63, P< 0.0001***, Veh/Saline n=12, JZL184 n=11). Error bars ± SEM.

Figure 3.. JZL184 has no effect on morphine analgesia.

(A, C) Experimental timeline of acute systemic vehicle or JZL184 pretreatment prior to the morphine dose response in the hot plate test (A), or the tail-flick test (C). (B, D) JZL184 pretreatment has no effect on morphine-induced analgesia during the hot plate test (Two-way ANOVA, main effect of dose, F_3,68= 30.65, P<0.001***, Veh n=9, JZL184 n=10), (D) or the tail flick test (Two-way ANOVA, main effect of dose, F_4,60 =22.17, P<0.001***, Veh n=7, JZL184 n=7). Error bars ± SEM.

Figure 4.. Intra-VTA JZL184 attenuates morphine preference but does not alter morphine analgesia.

(A) Experimental timeline of surgery for guide cannula placement and intra-VTA infusion of JZL184 during morphine CPP, followed by tail-flick test to measure morphine analgesia. (B) Exemplar image of guide cannula placement in the VTA. (C) Intra-VTA infusion of JZL184 attenuates morphine preference (Two-way ANOVA, significant interaction (treatment x day), F_1,54 =5.144, P=0.0274; Bonferroni post hoc test: Veh: Test vs Pretest, P=0.0051**, JZL184: Test vs Pretest no significance, P>0.999 Test: Veh vs JZL184 P=0.018*, Veh n=12, JZL184 n=17). (D) Experimental timeline for surgery, morphine CPP and morphine TFT. (E) Representative image of AAV2-Cre-GFP expression in the VTA (VTA=Ventral tegmental area). (F) Representative image of RNAscope in situ hybridization showing Cnr1 mRNA expression (magenta) and GFP-tagged cells (green) in the VTA of Cnr1 ^VTA-WT (left panel) and Cnr1 ^VTA-KO (right panel) mice (DAPI= blue). (G) Quantification of Cnr1 puncta in Cnr1^VTA-WT and Cnr1^VTA-KO mice (Ttest, t₍₇₀₎ = 4.508, P<0.001***, Cnr1^VTA-WT n=3 (42 cells in total), Cnr1^VTA-KO n=3 (25 cells in total). (H) Focal knockout of CB1Rs in the VTA counteract JZL184’s morphine blunted response (Three-way ANOVA, significant interaction (days x genotype), F_1,26 = 4.530, P<0.043*; Bonferroni post hoc test: Cnr1 ^VTA-WT Veh: Test vs Pretest, P=0.044*, Cnr1 ^VTA-WT JZL184: Test vs Pretest no significance, P>0.999, Cnr1 ^VTA-KO Veh: Test vs Pretest, P=0.020*, Cnr1 ^VTA-KO JZL184: Test vs Pretest, P=0.031*, Test: Cnr1 ^VTA-WT Veh vs Cnr1 ^VTA-WT JZL184, P=0.013†, Test: Cnr1 ^VTA-WT JZL184 vs Cnr1 ^VTA-KO JZL184, P=0.005††, Cnr1 ^VTA-WT Veh n=8, Cnr1 ^VTA-WT JZL184 n=7, Cnr1 ^VTA-KO Veh n=8, Cnr1 ^VTA-KO JZL184 n=7).

Figure 5.. JZL184 pretreatment attenuates NAc neural activity and DA dynamics time-locked to the morphine-paired chamber

(A) Brain schematic with an optic fiber implanted into the NAc expressing GCaMP6s and recording apparatus. (B) Representative image of GCaMP6s expression in the NAc (aca=anterior commissure, AcbC=NAc core, AcbSh=NAc shell). (C) Experimental timeline for surgery, morphine CPP and fiber photometry recording of calcium (GCaMP6s) on CPP preference test session on Day 6. (D) A representative trace of the mean ΔF/F (+/− SEM) photometry trace of NAc GCaMP6s fluorescence in vehicle pretreated mice time-locked to the exit from saline-paired chamber approaching the morphine-paired chamber (black) or exit from morphine chamber approaching the saline chamber (blue). (E) A representative trace of the mean ΔF/F (+/− SEM) photometry trace of NAc GCaMP6s fluorescence in JZL184 pretreated mice time-locked to the exit from saline-paired chamber approaching the morphine-paired chamber (red) or exit from morphine chamber approaching the saline chamber (grey). (F) Vehicle but not JZL184 treated mice exhibit significantly higher ΔF/F when approaching the morphine- compared to the saline-paired chamber (linear mixed effects model, **p=0.0047, Vehicle, number of approaches = 191, n = 6; p=0.974 JZL184, number of approaches = 284, n = 8). (G) Brain schematic with an optic fiber implanted into the NAc expressing dlight1.2 and recording apparatus. (H) Representative image of dLight1.2 expression in the NAc (aca=anterior commissure, AcbC=NAc core, AcbSh=NAc shell). (I) Experimental timeline for surgery, morphine CPP and fiber photometry recording of DA (dligh1.2) on CPP preference test session on Day 6. (J) A representative trace of the mean ΔF/F (+/− SEM) photometry trace of NAc dlight1.2 fluorescence in vehicle pretreated mice time-locked to entry into the morphine-paired chamber (black) or saline-paired chamber (blue). (K) A representative trace of the mean ΔF/F (+/− SEM) photometry trace of NAc dlight1.2 fluorescence in JZL184 pretreated mice time-locked to the entry into the morphine-paired (red) or saline-paired chamber (grey). (L) Vehicle but not JZL184 pretreated mice exhibit significantly higher NAc ΔF/F when entering the morphine-paired compared to the saline-paired chamber (linear mixed effects model, **p=0.007, Vehicle, number of entries = 575, n = 16; p=0.369 JZL184, number of entries = 448, n = 12). Error bars ± SEM.

Figure 6.. Morphine reward is associated with increase in NAc activity and dopamine neurotransmission that is blunted following 2-AG elevation in the VTA.

Left, Opioid reward occurs via increase of NAc activity and DA levels. Right, Elevation of 2-AG in the VTA through inhibition of MAGL with JZL184 during opioid conditioning, blunts reward, NAc activity and DA levels. Ca²⁺ = calcium, DA=dopamine, DAR = dopamine receptor, CB1= cannabinoid receptor 1, MOR= μ-opioid receptor, 2-AG= 2-Arachidonoylglycerol.