GPR55 mRNA expression in glutamate, not dopamine, neurons
To determine the cellular distributions of GPR55 in the brain, we first used RNAscope ISH assays to examine GPR55 transcript (mRNA) expression in brain DA neurons and glutamate neurons. Figure 1 shows GPR55 mRNA staining, illustrating that GPR55 is not colocalized with tyrosine hydroxylase (TH) in DA neurons in the ventral tegmental area (VTA) of the midbrain (Fig. 1A), but it is colocalized with VgluT1 mRNA in the prefrontal cortex (PFC) glutamate neurons (Fig. 1C). Genetic deletion of GPR55 almost completely blocked GPR55 mRNA signal in the VTA (Fig. 1B) and the PFC (Fig. 1D). We also examined co-localization of GPR55 and DA transporter (DAT, another DA neuronal marker) mRNAs in the VTA, but failed to detect their colocalization (Fig. S1). Figure S1-A shows the GPR55 gene structure and the deleted region in GPR55-knockout mice. Figure S2 shows a high magnification image from Fig. 1C, illustrating clear GPR55 and VgluT1 colocalization in cortical glutamate neurons. Quantitative cell counting data indicated that ~ 90% (91.65 ± 12.43%) of cortical glutamate neurons express GPR55, while in the VTA, only ~ 10% (10.42 ± 2.14%) of DA neurons show GPR55 signal.
We also examined GPR55 mRNA expression in other brain regions. We detected similar GPR55 and VgluT1 colocalization in the hippocampus and thalamus, but not in the striatum (Fig. S3).
GPR55-immunostaining is not GPR55-specific
Next, we used IHC assays to detect GPR55 protein expression in the regions where mRNA was detected. We used two commercially available GPR55 antibodies: a polyclonal anti-GPR55 antibody (Abcam), which targets the C-terminus of human GPR55, and another polyclonal anti-GPR55 antibody with an undisclosed epitope (Cayman Chemicals). Figures S4 and S5 shows the representative GPR55-immunostaining images, illustrating that GPR55-like signal was detected in the VTA, but it was not colocalized with TH-immunostaining in DA neurons when using either the Abcam GPR55 antibody (Fig. S3) or the Cayman GPR55 antibody (Fig. S4). However, the GPR55-immunostaining is not highly specific as it is still detectable in either GPR55-KO mice (Fig. S4-B) or in the presence of either of the GPR55 antibody immune peptides (Fig. S4-C; Fig. S5-B). These findings suggest that both the antibodies against the human or bovine GPR55 are not suitable to detect GPR55 receptor proteins in mice although human GPR55 show 75% and 78% homology with the rat and mouse GPR55 proteins, respectively 9.
Fluorescent cannabinoid ligand binding reveals GPR55 expression in glutamate, not DA, neurons
To further validate our RNAscope findings on GPR55 expression in glutamate versus DA neurons, we then used a fluorescent cannabinoid ligand – Tocrifluor T1117 (T1117), an analog of AM251 (a selective CB1 receptor antagonist) – to detect GPR55 expression. Because T1117 is not a selective GPR55 agonist and also has low binding affinity to the CB1 receptor 27, 28, we used CB1-KO mice to exclude its binding to the CB1 receptor in this assay. There are two types of glutamate neurons that express VgluT1 mainly in the cortex and VgluT2 mainly in the subcortical brain regions such as the hippocampus and thalamus30. Therefore, we used two different glutamatergic neuronal markers (e.g., VgluT1 and VgluT2 antibodies) to identify glutamate neurons in this study. Figure 2 shows representative T1117 binding in the VTA, PFC, and midbrain red nucleus (RN), illustrating that T1117 did not show co-localization with TH in VTA DA neurons (Fig. 2A), but showed clear T1117-VgluT1 colocalization in PFC glutamate neurons (Fig. 2B) and T1117-VgluT2 colocalization in red nucleus glutamate neurons (Fig. 2C). Notably, T1117 fluorescent signal is also detected in other non-DA neurons in the VTA (Fig. 2A) or non-glutamate neurons in the red nucleus (Fig. 2-C). Figure 3 shows the representative T1117 binding images in the hippocampus under different magnifications (20×, 40×, and 60×), indicating that T1117 and VgluT2 colocalization in the majority of VgluT2-positive glutamate neurons in the hippocampus of CB1-KO mice. We did not use GPR55-KO mice to determine the signal specificity as T1117 also binds to CB1 receptor in GPR55-KO mice and double CB1-KO and GPR55-KO mice are currently not available. Together, these ligand binding data, combined with our data from RNAscope ISH and IHC assays, support a conclusion that GPR55 is mainly expressed in cortical and subcortical glutamate neurons, but it is not expressed in midbrain DA neurons.
O-1602 elevates extracellular glutamate, not DA, in the NAc
Next we used in vivo brain microdialysis (Fig. 3A) to examine whether activation of GPR55 alters glutamate or DA release in the NAc, a critical brain region involved in cannabis action and drug abuse 2, 5. We found that O-1602, at 3 and 10 mg/kg, failed to alter extracellular DA (Fig. 3B), but produced a transient increase in extracellular glutamate levels in the NAc (Fig. 3C). An one-way RM ANOVA for the data shown in gray boxes did not reveal a O-1602 treatment main effect on extracellular DA after 3 mg/kg (Fig. 3B, F4,20=1.16, p > 0.05) or 10 mg/kg (Fig. 3B, F4,20=0.51, p > 0.05) O-1602 administration. However, the one-way RM ANOVA revealed a significant O-1602 treatment main effect in extracellular glutamate after 10 mg/kg (Fig. 3C, F2,24=4.17, p = 0.01), but not after 3 mg/kg (Fig. 3C, F4,24=1.76, p > 0.05), O-1602 administration.
O-1602 failed to alter D9-THC-induced triad effects
We have recently reported that genetic deletion of GPR55 enhanced D9-THC-induced analgesia, hypothermia, and catalepsy 22, suggesting that GPR55 activation may inhibit cannabinoid action. Therefore, we proposed that GPR55 agonists may have therapeutic effects against cannabinoid action. To test this hypothesis, we first observed the effects of O-1602 (a potent GPR55 agonist) on a high dose (30 mg/kg) of D9-THC-induced classical triad effects. We found that pretreatment with O-1602 (10, 20 mg/kg, i.p., 15 min prior to D9-THC) failed to alter 30 mg/kg D9-THC-induced analgesia, hypothermia, or catalepsy (Fig. S6) although a trend toward an increase in analgesia and hypothermia compred to the vehicle group. However, two-way RM ANOVA did not reveal a significant O-1602 treatment main effect in D9-THC-induced analgesia (Fig. S6-A, F7,2 =0.994, p > 0.05), D9-THC-induced hypothermia (Fig. S6-B, F7,2=0.441, p > 0.05), or D9-THC-induced catalepsy (Fig. S6-C, F7,2=2.31, p > 0.05).
O-1602 inhibits nicotine, not cocaine, self-administration in rats
We have recently reported that elevation of extracellular glutamate in the NAc by blockade of glial GLT-1 inhibits cocaine self-administration 31. Next, we examined whether O-1602, which transiently elevates extracellular glutamate in the NAc (Fig. 3), also inhibits intravenous cocaine self-administration in rats or mice. Figure 5 (A, B) shows that systemic administration of O-1602, at 10 mg/kg and 20 mg/kg, failed to alter cocaine self-administration under FR2 reinforcement in rats (Fig. 5A, one-way RM ANOVA, F2,14=3.69, p > 0.05) or under FR1 reinforcement in wildtype mice (Fig. 5B, one-way ANOVA, F2,20=1.40, p > 0.05), suggesting that O-1602 has no significant effect on cocaine self-administration.
We then examined the effects of O-1602 on intravenous nicotine self-administration as it was reported that systemic or intracerebroventricular administration of GPR55 agonists inhibied conditioned place preference to nicotine 25, 26. Here we chose selectively bred alcohol-preferring rats (P-rats), as P-rats displayed significantly higher vulnerability than Long-Evens (LE) rats in nicotine self-administration in our previous report 29. We found that O-1602 (3, 10, or 20 mg/kg, 15 min prior to nicotine SA session) produced a dose-dependent reduction in either the total number of nicotine infusions or the rate of nicotine self-administration (i.e., nicotine infusions/hr) (Fig. 5-C, D). This inhibitory effect was blocked by co-administration of CID 16020046 (CID), a selective GPR55 antagonist. CID alone, at 5 or 10 mg/kg, failed to alter nicotine self-administration (Fig. 5-E, F). A one-way RM ANOVA revealed a significant main effect of O-1602 treatment in the number of infusions (Fig. 5C, F4,23 =3.28, p < 0.05) and the rate of infusions (Fig. 5D, F4,23 =4.75, p < 0.01). Post-hoc individual group comparisons indicated a significant reduction in nicotine self-administration after 20 mg/kg O-1602, compared to the vehicle control group.
To determine whether O-1602 also alters motivation for nicotine taking and seeking, we observed the effects of O-1602 on nicotine self-administration under a PR reinforcement schedule. Figure 5 (E, F) shows that pretreatment with O-1602 dose-dependently inhibited PR nicotine self-administration as assessed by the number of nicotine infusions (Fig. 5E, F5,42 =12.90, p < 0.001) or PR break-point (Fig. 5F, F5,42=5.41, p < 0.001).
O-1602 inhibits nicotine self-administration in WT, not GPR55-KO, mice
To further determine whether the above action produced O-1602 is mediated by activation of GPR55, we used GPR55-KO mice in the self-administration experiment. Figure 6 (A, B) shows that GPR55-KO mice did not differ in nicotine self-administration compared to WT mice (e.g., between the vehicle control groups). However, systemic administration of O-1602 (10, 20 mg/kg, i.p.) produced a robust reduction in nicotine self-administration in WT mice as assessed by either the total number of nicotine infusions (Fig. 6A, F3,21=11.32, p < 0.001) or the rate of nicotine self-administration (Fig. 6B, F3,21=16.25, p < 0.001), but not in GPR55-KO mice (Fig. 6A, F3,18=0.71, p > 0.05; Fig. 6B, F3,18=1.06, p > 0.05). Figure 6 (C, D) shows representative nicotine self-administration (infusion) records after vehicle or O-1602 treatment in WT mice, indicating that 10 mg/kg of O-1602 significantly inhibited nicotine self-administration and altered the patterns of self-administration from a regular, evenly-distributed pattern to an irregular, extinction-like pattern, suggesting that GPR55 agonism inhibits nicotine reward.
O-1602 has no effect on oral sucrose self-administration or open-field locomotion
To determine whether the O-1602-induced reduction in nicotine self-administration was due to treatment-induced locomotor impairment, we observed the effects of O-1602 on open-field locomotion and non-drug (sucrose) self-administration. We found that systemic administration of the same doses of O-1602 neither altered sucrose self-administration (Fig. S7-A, F2,13=1.05, p > 0.05; Fig. S7-B, F2,13=1.38, p > 0.05) nor altered open-field locomotion (Fig. S7-C, main effect of O-1602 treatment, F3,21=0.25, p > 0.05; treatment × time interaction, F51,357=0.38 p > 0.05), suggesting that O-1602 selectively inhibits nicotine self-administration.
O-1602 is not rewarding or aversive by itself
Lastly, we examined whether O-1602 produces rewarding or aversive effects by itself as assessed by optical intracranial self-administration (oICSS). We have previously reported that cocaine or oxycodone produces reward-enhancing 32, 33, while cannabinoids such as D9-THC and WIN55-212-2 produces reward-attenuation (aversive) effects in oICSS in DAT-Cre mice 33. Using the same approaches (Fig. S8-A ~ D), we found that optogenetic stimulation of VTA DA neurons produced robust oICSS behavior in a stimulation frequency-dependent manner (Fig. S8-E, F). O-1602, at the same doses as used in nicotine self-administration, did not significantly alter oICSS (Fig. 6F), suggesting that GPR55 itself is not rewarding or aversive. It also suggests that O-1602 does not alter DA-dependent behavior. This is finding is consistent with our findings that GPR55 is not identified in midbrain DA neurons (Figs. 1, 2; Figs. S4 and S5) and O-1602 also failed to alter DA release in the NAc (Fig. 4). Figure 6E shows a proposed hypothesis through which O-1602 elevates extracellular glutamate that subsequently counteracts the action produced by nicotine-enhanced DA mainly in D2-expressing medium-spiny neurons (D2-MSNs).