PPARα and PPARγ are expressed in midbrain dopamine neurons and modulate dopamine- and cannabinoid-mediated behavior in mice

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate gene expression. Δ9-tetrahydrocannabinol (Δ9-THC) is a PPARg agonist and some endocannabinoids are natural activators of PPARa and PPARg. Therefore, both the receptors are putative cannabinoid receptors. However, little is known regarding their cellular distributions in the brain and functional roles in cannabinoid action. Here we first used RNAscope in situ hybridization and immunohistochemistry assays to examine the cellular distributions of PPARα and PPARγ expression in the mouse brain. We found that PPARα and PPARγ are highly expressed in ~70% midbrain dopamine (DA) neurons and in ~50% GABAergic and ~50% glutamatergic neurons in the amygdala. However, no PPARα/γ signal was detected in GABAergic neurons in the nucleus accumbens. We then used a series of behavioral assays to determine the functional roles of PPARα/γ in the CNS effects of Δ9-THC. We found that optogenetic stimulation of midbrain DA neurons was rewarding as assessed by optical intracranial self-stimulation (oICSS) in DAT-cre mice. Δ9-THC and a PPARγ (but not PPARα) agonist dose-dependently inhibited oICSS, suggesting that dopaminergic PPARγ modulates DA-dependent behavior. Surprisingly, pretreatment with PPARα or PPARγ antagonists dose-dependently attenuated the Δ9-THC-induced reduction in oICSS and anxiogenic effects. In addition, a PPARγ agonist increased, while PPARa or PPARγ antagonists decreased open-field locomotion. Pretreatment with PPARa or PPARγ antagonists potentiated Δ9-THC-induced hypoactivity and catalepsy but failed to alter Δ9-THC-induced analgesia, hypothermia and immobility. These findings provide the first anatomical and functional evidence supporting an important role of PPARa/g in DA-dependent behavior and cannabinoid action.


Introduction
In 2020, over 14,000 American adults self-reported cannabis use disorder 1 . However, recreational legalization efforts continue to progress; in the last two years alone, 5 states have passed legislation allowing non-medical use of marijuana 2 . In this social and legislative climate, a full understanding of cannabis action and the underlying neural mechanisms is critically important. Δ 9 -tetrahydrocannabinol (Δ 9 -THC) is the primary phytocannabinoid within cannabis that is responsible for its subjective effects and many of its therapeutic bene ts, which are widely believed to be mediated by activation of cannabinoid type 1 (CB1) and type 2 (CB2) receptors [3][4][5][6] . In addition to CB1 and CB2 receptors, Δ 9 -THC and other cannabinoids have high binding activity at other receptor sites such as the G protein-coupled receptor 55 (GPR55), the transient receptor potential cation channel (TRPV1), and the peroxisome proliferator-activated receptor gamma (PPARγ) and possibly alpha (PPARα) 5,7,8 . Evaluating the non-CB1 and non-CB2 receptor mechanisms underlying cannabinoid action will not only increase our understanding of cannabinoid biology but may also lead to the discovery of new interventions for treating cannabis dependence.
In this context, PPARs are of special interest due to their involvement in a number of CNS functions such as pain 9 , reward 10 , neuroin ammation 11 , and learning and memory 12 . Furthermore, the PPARγ agonist pioglitazone, an FDA-approved medication for the treatment of diabetes in humans, has been shown to be highly effective in reducing voluntary alcohol and opioid consumption and alcohol or nicotine-taking behavior in experimental animals [13][14][15][16] . However, the neural mechanisms underlying pioglitazone action are poorly understood.
PPARs are transcription factors within a subfamily of nuclear hormone receptors 17 . They are activated by lipophilic compounds and can bind directly to PPAR response elements, which are selective DNA sequences in target genes 11,18 . The PPAR family contains three isoforms: PPARα, PPARγ, and PPARβ/δ -each with distinct physiological roles 19 . Recent work has identi ed interactions between these nuclear receptors and the endocannabinoid system. For instance, the synthetic cannabinoid WIN55,212-2 promotes transcriptional activity at both PPARα and PPARγ, as do the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide [20][21][22][23][24] . As mentioned above, Δ 9 -THC binds to PPARγ, but ndings regarding Δ 9 -THC's a nity to PPARα are inconsistent. One report describes no binding a nity to PPARα 20 , while another reveals elevated transcriptional activity at PPARα in the presence of Δ 9 -THC 25 . No prior work has evaluated whether Δ 9 -THC binds to PPARβ/δ.
A small body of literature has emerged in the last two decades investigating the role of PPARs in cannabinoid activity outside of the CNS. For instance, in a neuronal cell culture model of Parkinson's disease, Δ 9 -THC is neuroprotective and this response is blocked and reinstated by a PPARγ antagonist and agonist, respectively 26 . Additionally, both the tumor suppressant effect of Δ 9 -THC against liver cancer and its vasorelaxant response in the cardiovascular system are mediated by PPARγ activation 27,28 . However, no prior work has investigated whether PPARs underlie the CNS effects of cannabinoids and very little is known regarding the phenotypes of neurons that express PPARs in the brain.
To address these knowledge gaps, we rst examined the cellular distributions of PPARα and PPARγ in multiple types of neurons in the midbrain ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala using double-staining RNAscope in situ hybridization (ISH) and immunohistochemistry (IHC) assays. Given their major distributions in midbrain dopamine (DA) neurons, we then used pharmacological approaches to manipulate PPARα and PPARγ and transgenic and optogenetic approaches to manipulate VTA DA neurons to determine the functional roles of PPARα and PPARγ in cannabinoid action and DA-dependent behavior.

Subjects
Male C57BL/6J mice (25-35 g; The Jackson Laboratory, Bar Harbor, ME) were utilized throughout the studies. Male and female DAT-Cre +/mice (25-40 g) were bred at the National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP) and underwent genotyping by Transnetyx for veri cation. All subjects were kept on a reverse light cycle (lights off at 7:00 am; on at 7:00 pm) and provided with ad lib food and water. The house room temperature was set to 21-23°C with 40-50% humidity. Experimental procedures adhered to the Guide for the Care and Use of Laboratory Animals, 8 th edition. The Animal Care and Use Committee at NIDA approved the study protocol. Chemicals Δ 9 -THC was provided by the NIDA pharmacy (Baltimore, MD). The stock solution was dissolved in ethanol at a concentration of 50 mg/ml. We diluted this solution as needed for experimental use in a 5% cremophor (Sigma-Aldrich, St. Louis, MO) saline solution. PPAR antagonists and agonists including GW9662, GW6471, pioglitazone, and GW7647 were purchased from Cayman Chemical (Ann Arbor, MI). Each compound was dissolved in a mixture of 2% DMSO, 3% tween-80 and 95% saline.

Experiment 1: RNAscope in situ hybridization
We rst performed RNAscope in situ hybridization (ISH) to examine the distribution of PPARα and PPARγ mRNA in the mesolimbic DA system and amygdala -regions associated with the affective properties of cannabinoids. In the VTA, we examined PPARα (PPARA) and PPARγ (PPARG) mRNA expression in GABAergic (GAD1 + ), glutamatergic (Slc17a6 + ) and dopaminergic (TH + ) neurons. In the NAc, we focused on PPAR expression in GABAergic (GAD1 + ) neurons, whereas in the amygdala, we looked at expression patterns in GABAergic (GAD1 + ) and glutamatergic (Slc17a6 + ) neurons. The complete RNAscope procedures are described in Supplementary Information.

Statistical Analyses
All data are presented as means ±SEM. oICSS and tetrad data were analyzed based on changes in the area under the curve (ΔAUC) to better visualize group differences. Data were converted to ΔAUC by summating the difference between each time point after drug injection and a baseline value before the injection. One-way or two-way repeated measures (RM) ANOVAs were used to analyze the data as appropriate. Signi cant effects were followed by post hoc tests using Tukey's multiple comparisons. For all tests, statistical signi cance was set to p<0.05.

Cellular distributions of PPARα and PPARγ in the VTA, NAc, amygdala
We rst examined the expression of PPARα and PPARγ in different neuronal phenotypes in the mesolimbic DA system and amygdala, which are critical brain regions involved in cannabinoid action 5 .  (Fig. S1, S2). However, in these cell types, PPARα and PPARγ mRNA expression levels were low and observed outside of DAPIlabeled nuclei, complicating cell counting analyses. As such, cell counting was not attempted on these data.
The low PPARα and PPARγ mRNA expression levels observed in DA, GABA and glutamate neurons were unexpected given previous work demonstrating a strong neuronal signal using qPCR 29 . To address this discrepancy, we utilized a different technique, double-label IHC, to measure protein expression of PPARα and PPARγ in the predominant cell types within the regions of interest. We detected strong PPARα and PPARγ immunostaining in TH + DA neurons in the VTA ( Fig. 1 -C, D) as well as in GAD67 + GABA neurons and VgluT2 + glutamate neurons in the VTA and amygdala (Fig. S3, S4). In the NAc, no PPAR immunostaining overlapped with GAD67 + GABA neurons (Fig. S5). Surprisingly, PPARα and PPARγ immunostaining was detected mainly in astrocyte-like cells in the NAc, suggesting that these may be glial receptors. Quantitative cell counting assays revealed that PPARα and PPARγ are expressed in ~ 70% of DA neurons, ~ 30% of GABA neurons and ~ 20% of glutamate neurons in the VTA ( Fig. 1 -E, F). In the amygdala, PPARα is found in ~ 60% of glutamate neurons and ~ 40% of GABA neurons, while PPARg is expressed in ~ 60% of GABA neurons and ~ 40% of glutamate neurons. In the NAc, PPARα/g and GAD67 co-expression was negligible, so no quanti cation was performed.
PPARa/g modulate DA-dependent oICSS and Δ 9 -THC action in oICSS We have recently reported that optogenetic stimulation of VTA DA neurons is rewarding as assessed by optical ICSS (oICSS) and real-time place preference 30,31 and this effect is dose-dependently attenuated by cannabinoids such as Δ 9 -THC, WIN55212,2 or AM-2201 32 . However, the receptor mechanisms underlying cannabinoid reward-attenuation in oICSS are unclear. Given that Δ 9 -THC is also a potent PPARg agonist (EC 50 = 0.3 mM) and other cannabinoids have binding a nity to PPARa 33 , we rst examined whether PPAR agonists produce similar effects as Δ 9 -THC and whether pretreatment with PPAR antagonists would block Δ 9 -THC-induced changes in oICSS in transgenic DAT-Cre mice.  Table 1. This nding that a PPARγ, but not PPARα, agonist produces a Δ 9 -THC-like effect in oICSS suggests that Δ 9 -THC may inhibit brain-stimulation reward by activation of PPARγ.
To test this hypothesis, we then determined whether the PPARα antagonist GW6471 alters Δ 9 -THCinduced changes in oICSS. We found that pretreatment with GW6471 signi cantly attenuated Δ 9 -THCinduced reduction in oICSS, with a lower dose of GW6471 being more effective in attenuation of Δ 9 -THC's action ( Fig. 2 -G, H). A two-way RM ANOVA revealed a signi cant GW6471 treatment main effect ( Fig. 2G, F 3,60 = 3.79, p < 0.05). Unexpectedly, GW6471 itself produced a dose-dependent reduction in oICSS (Fig. 2I, F 2,33 = 4.58, p < 0.05) whereas the PPARa agonist GW7647 failed to alter oICSS (Fig. 2E), suggesting that PPARα may be fully occupied and activated by endogenous ligands. Thus, the antagonist GW6471 may produce a reduction in oICSS by blockade of endogenous ligand binding to PPARa, while the agonist GW7647 may not work due to a ceiling effect caused by endogenous ligand binding.
Next, animals were pretreated with a PPARγ antagonist (GW9662). We found that GW9662 dosedependently attenuated Δ 9 -THC-induced reduction in oICSS ( Fig. 2 -J, K). Two-way RM ANOVAs over time (stimulation frequency) revealed a statistically signi cant GW9662 treatment main effect (Fig. 2J, F 3,59 = 5.80, p < 0.01). Analyzing the changes in the area under curve (DAUC) values for the data shown in  Figure 2L shows that administration of GW9662 alone failed to alter oICSS (F 2,33 = 0.04, p = 0.96). More detailed statistical analysis results are shown in supplementary Table 1. These ndings provide the rst behavioral evidence indicating that PPARa and PPARγ receptor mechanisms at least in part underlie Δ 9 -THC-induced reward attenuation.
Effects of PPAR antagonists on Δ 9 -THC-induced place aversions Next, we examined whether pretreatment with PPAR antagonists is able to block Δ 9 -THC-induced conditioned place aversion (CPA) (Fig. S6-A). Figure S6 (B, C) shows that pretreatment with either the PPARa antagonist (GW6471) or PPARγ antagonist (GW9662) failed to alter Δ 9 -THC-induced CPA, suggesting that PPARs are not critically involved in Δ 9 -THC-induced place aversion. This is consistent with our previous reports that CB1 and CB2 receptor mechanisms underlie the rewarding and aversive effects 40,41 . A two-way RM ANOVA on CPP scores in subjects administered Δ 9 -THC detected a signi cant main effect of Test (cocaine CPP) ( Figure S6 We also examined the effects of the PPAR antagonists alone in CPP. We found that the PPARα antagonist GW6471 (Fig. S6-D, F 2,21 = 1.21, p = 0.32) failed to produce either CPP or CPA, while the PPARγ antagonist GW9662, at a low dose (2 mg/kg), produced signi cant place aversion in the absence of Δ 9 -THC ( Figure  S6-E, F 1,21 = 8.95, p < 0.01), suggesting that PPARγ tonically modulates brain reward function under physiological conditions. Blockade of PPARs attenuates Δ 9 -THC-induced anxiety In addition to VTA DA neurons, PPARα and PPARγ are also expressed in 50 ~ 60% of GABA and glutamate neurons in the amygdala, a critical brain region involved in affective behavior. Therefore, we further examined the functional roles of PPARs in cannabinoid-induced anxiety. We rst examined the effects of PPAR agonists in an elevated plus maze (EPM) test. We found that systemic administration of PPARα agonist (Fig. 4A, F 2 Post hoc comparisons revealed that Δ 9 -THC-induced anxiety is statistically signi cant in the vehicle (0 mg/kg GW6471) control group. However, in subjects pretreated with 3 or 5 mg/kg GW6471 Δ 9 -THC did not produce signi cant anxiogenic effects relative to vehicle control group (Fig. 4C). Another two-way ANOVA on Δ 9 -THC-induced anxiety produced a main effect of Δ 9 -THC dose (Fig. 4D, F 1,62 = 18.93, p < 0.001), but not GW9662 dose (F 2,62 = 1.25, p = 0.29) or the interaction term (F 2,62 = 0.68, p = 0.51). Post hoc comparisons showed that subjects administered Δ 9 -THC by itself or in conjunction with 2 mg/kg GW9662 were more anxious relative to controls whereas the group given 5 mg/kg GW9662, and Δ 9 -THC did not produce signi cant anxiogenic effects compared to the vehicle controls (Fig. 4D).

Effects of Δ 9 -THC and PPAR antagonists on locomotor activity
We then examined the effects of Δ 9 -THC with or without ligands on open-eld locomotion (Fig. 5). Systemic administration of a selective PPARa agonist (GW7647) failed to alter locomotor activity (Fig. 5A, F 2 (Fig. 5B). In contrast, systemic administration of PPAR antagonists produced a signi cant reduction in open-eld locomotion (Fig. 5C, D). A two-way RM ANOVA reveal a signi cant GW6471 treatment main effect (Fig. 5C, F 2 These ndings suggest that PPARg modulates basal locomotor behavior.
We then observed the effects of PPAR antagonist pretreatment on Δ 9 -THC-induced changes in locomotion. We found that systemic administration of 3 mg/kg Δ 9 -THC produced a signi cant reduction in locomotion (Fig. 5 -E, F), consistent with our previous nding 42 . However, pretreatment with a selective PPARa antagonist (GW6471) enhanced Δ 9 -THC-induced hypoactivity (Fig. 5E), while a selective PPARg antagonist (GW9662) produced a trend toward an increase in Δ 9 -THC-induced reduction in locomotion. A two-way RM ANOVA revealed a signi cant treatment X time interaction (Fig. 5E, F 22
Additional two-way RM ANOVA results for the full-time course data (Fig. S8) are provided in the supplementary Table 4.

Discussion
The major ndings in this report include: 1) PPARa and PPARg are mainly expressed on midbrain DA neurons, GABA and glutamate neurons in the amygdala, as well as on astrocyte-like cells in the NAc. 2) Optogenetic stimulation of VTA DA neurons is rewarding, which is dose-dependently inhibited by Δ 9 -THC and a PPARg, but not PPARa, antagonist, suggesting an important role of PPARg in DA-dependent behavior. 3) PPARa and PPARg antagonism attenuated the reward-attenuating (aversive) and anxiogenic effects of Δ 9 -THC and potentiated Δ 9 -THC-induced hypoactivity and cataleptic properties, but failed to alter Δ 9 -THC-induced analgesia, hypothermia and immobility. These ndings implicate PPARa and PPARg in the VTA and amygdala in the affective pro le of cannabinoids and DA-dependent behavior.

PPARa and PPARg expression in dopamine, glutamate and GABA neurons
Prior studies using qPCR and IHC have localized PPARα to neurons, astrocytes, and microglia and PPARγ to neurons and astrocytes in both human and mouse brains and in cultured rat neurons 29,43 . However, little is known about the phenotypes of neurons or cells that express PPARa and PPARg in the mesolimbic reward system and amygdala. In the present report, we detected PPARα and PPARγ immunostaining in ~ 70% of DA neurons in the VTA, with lower but detectable levels on VTA GABA and glutamate neurons, suggesting an important role of PPARs in modulating DA-dependent behavior. This is supported by our behavioral ndings that activation of PPARg inhibited DA-dependent brain-stimulation reward as assessed by oICSS. Prior work has demonstrated that the PPARg agonist pioglitazone is effective in reducing feeding, voluntary alcohol consumption and drug self-administration [13][14][15][16] . As such, the present ndings may implicate a dopaminergic PPARg mechanism in pioglitazone's anti-reward effects.
Surprisingly, we detected PPARa and PPARg in accumbal astrocyte-like cells, but not on GABAergic medium-spiny neurons. This nding is inconsistent with previous reports in which PPARa/gimmunostaining was colocalized with primarily neuronal markers (NeuN or b-tubulin III), but not GFAP or Iba1 in the NAc and cortex 29,43 . The reasons underlying these con icting ndings are unclear. However, it is important to note that in the present work we did not employ an astrocytic marker, but assumed based on anatomical similarities in our images. Further work is needed to address this question.
It was previously reported that PPARγ transcripts are detected in both the nucleus and cytoplasm of GABA neurons in the hippocampus and amygdala 44 . Cannabinoids have biphasic anxiolytic and anxiogenic effects, which are likely mediated by GABAergic and glutamatergic neurons in the amygdala, respectively 45,46 . This inspired us to map out PPARa and PPARg expression in the amygdala and determine their preferred neuronal subtypes. PPARα was primarily expressed on glutamate neurons (57.3%) and PPARγ on GABA neurons (56.8%). These results are compatible with prior work and point to PPARs on both GABAergic and glutamatergic neurons in the amygdala as potential receptor mechanisms underlying the affective properties of cannabinoids.
We note that PPARa/g transcription levels by RNAcope ISH assays were fairly low in all three brain regions assessed and an unusual pattern of expression was observed such that individual puncta were distributed within and outside of DAPI-labeled nuclei. In previous reports, similarly low transcription levels and expression patterns have been noted in the amygdala and hippocampus 44,47 . It is not clear why mRNA levels are de cient relative to PPARa/g-immunostaining. Further study is required to address this issue.
PPARa/g activation contributes to Δ 9 -THC-induced aversion We have previously reported that cannabinoids produce a reduction in NAc DA release and DA-dependent oICSS in transgenic DAT-cre or VgluT2-cre mice 32,36,41,42 . However, the receptor mechanisms underlying cannabinoid action in oICSS have not been explored in the above studies. In the present study, we found that pretreatment with a CB1 (AM251) or CB2 (AM630) receptor antagonist signi cantly blocked or reduced Δ 9 -THC-induced reduction in oICSS, suggesting that both membrane CB1 and CB2 receptors are critically involved in cannabinoid aversion. In addition to identi cation of CB1 and CB2 receptor expression in midbrain DA neurons 35,36 , we also identi ed PPARa and PPARg in VTA DA neurons as discussed above. Furthermore, systemic administration of PPARg or pioglitazone (a selective PPARg agonist) inhibited oICSS, while pretreatment with a PPARγ antagonist signi cantly weakened the suppressive effect of Δ 9 -THC in this assay. These ndings suggest that PPARg activation may partially underlie Δ 9 -THC-induced reductions in oICSS. With PPARα, pharmacological activation failed to alter oICSS; however, pretreatment with a PPARa antagonist also reduced the suppressive effect of Δ 9 -THC, suggesting that PPARa may indirectly modulate Δ 9 -THC aversion via a non-dopaminergic mechanism. Together, these ndings suggest that multiple receptor mechanisms, including membrane CB1 and CB2 and nuclear PPARs, underlie cannabinoid or Δ 9 -THC-induced reward-attenuation or aversion (Fig. 3C).
We note that blockade of PPARα/γ failed to alter Δ 9 -THC-induced place aversion. There are several possible explanations. First, Δ 9 -THC is not a selective PPARγ agonist. It also has binding activity at CB1, CB2 and GPR55 receptors 5,7 . Thus, it is likely that Δ 9 -THC-induced place aversion is mediated by activation of multiple cannabinoid receptors and blockade of a single receptor is not su cient to prevent the establishment of Δ 9 -THC-induced place aversion. Second, the CPP/CPA test does not directly measure the acute rewarding or aversive effects of cannabinoids. Instead, it assesses reward-or aversion-associated learning and memory captured at least 24 hours after the last Δ 9 -THC administration. As such, different receptor or neural mechanisms may underlie Δ 9 -THC-induced reduction in oICSS versus place aversion. Third, CPP/CPA experiments are infamously insensitive to subtle changes in drug reward 48, 49 . In contrast, oICSS is highly sensitive to small changes in brain reward function 32 .
Last, oICSS provides a microcosm of a drug effect on a speci c phenotype of neurons in a speci c brain area, while place conditioning conveys the larger picture: the generally negative or positive associations an animal develops after repeated experiences to a drug. To summarize, both the oICSS and CPP assays are examining quantitatively and qualitatively distinct endpoints and a negative nding in a CPP test may not necessarily con ict with the positive nding in oICSS.
In prior work, both PPARγ and PPARα agonists are reported to decrease the reinforcing value of drugs of abuse including nicotine, ethanol, heroin, and methamphetamine [13][14][15][16] . However, the neural mechanisms underlying this action are poorly understood. Previous studies indicate that the PPARα agonists WY14643 and methOEA and the PPARγ agonist pioglitazone prevented nicotine-and heroin-induced increases in DA neuron ring in the VTA 13,14 . In the present study, we found that dopaminergic PPAR mechanisms may directly modulate oICSS (Fig. 3C), which may explain how PPAR agonists produce therapeutic effects against drug reward.
PPARs contribute to Δ 9 -THC-induced anxiety Another important nding in this report is that antagonism of PPARα and PPARγ attenuated Δ 9 -THCinduced anxiety, implicating these receptors in the negative affective properties of cannabinoids. This is consistent with previous work indicating that activation of PPARα via the endocannabinoid Npalmitoylethanolamine (PEA) correlated with increases in circulating cortisol in a social stress test in humans 50 . Similarly, Domi and colleagues 44 found that PPARγ knockout mice developed altered stress sensitivity and failed to display typical c-fos expression changes in the amygdala following stress exposure. As such, Δ 9 -THC may produce anxiety by activating PPARγ and PPARα in both GABA and glutamate neurons in the amygdala.
We note that PPARα/γ agonists or antagonists alone failed to alter basal anxiety levels, while PPARα or PPARγ antagonism only partially reduced Δ 9 -THC-induced anxiety, suggesting that in addition to PPARα/ γ, other receptor (such as CB1 and CB2) mechanisms are also involved in Δ 9 -THC's affective effects 5 . These ndings mirror earlier assessments in which activation of PPARs only modulated anxiety in response to lipopolysaccharide exposure or restraint stress but did not alter basal anxiety levels 44,51,52 .
In contrast, one report found that the same dose of the PPARγ antagonist (5 mg/kg GW9662) induced anxiety 44 . The reasons underlying these con icting ndings are unknown. More studies are required to further address this issue.
PPARs counteract Δ 9 -THC-induced hypoactivity and catalepsy A third important nding is that both PPARα and PPARγ modulate basal level locomotion: the agonists produced a transient increase, while the antagonists produced a robust decrease in open-eld locomotion. In agreement with these ndings, pretreatment with a PPARα antagonist, but not with a PPARγ antagonist, potentiated Δ 9 -THC-induced hypoactivity, suggesting that PPARα antagonism produced an additive or synergistic effect with Δ 9 -THC in open-eld locomotion. In addition, pretreatment with PPARα or PPARγ antagonists also potentiated Δ 9 -THC-induced catalepsy but did not alter Δ 9 -THCinduced analgesia, hypothermia, or immobility. The former nding is consistent with a previous report indicating that pretreatment with a PPARγ agonist reduced haloperidol-induced catalepsy 53 . These ndings suggest an important role of PPARα and PPARγ in modulation of locomotor behavior, but do not underlie high-dose D 9 -THC-induced tetrad effects.
In conclusion, in this study we systemically evaluated the cellular expression of PPARa and PPARg in the brain and their functional roles in the CNS effects of Δ 9 -THC. We found that PPARα and PPARγ are mainly expressed in midbrain DA neurons and in both GABA and glutamate neurons in the amygdala.
Activation of PPARg inhibits DA-dependent oICSS, while blockade of PPARa and PPARg attenuates Δ 9 -THC-induced reward-attenuation (aversion) and anxiety but potentiates Δ 9 -THC-induced hypoactivity and catalepsy. These results provide novel insights regarding the role of PPARa and PPARg in cannabis action and highlight the potential utility of PPARs as new therapeutic targets for substance use disorders.  Effects of CB1 and CB2 receptor antagonists on Δ 9 -THC-induced changes in oICSS in DAT-cre mice. A:

Declarations
The stimulation-rate response curves showing that 3 mg/kg D 9 -THC signi cantly decreased oICSS, which was blocked by AM251 and partially reduced by AM630. B: The DAUC data from the data in (A), illustrating that the reduction in oICSS by D 9 -THC was blocked by AM251 and partially reduced by activity (E), while GW9662 pretreatment did not signi cantly alter Δ 9 -THC action in locomotion (F). n = 8/group. * p<0.05, ** p<0.01, *** p<0.001, compared to the vehicle group. # p<0.05, compared to the (Vehicle + Δ 9 -THC) group (E).