Introduction

The endocannabinoid system (ECS) is importantly involved in the development of dependence to many drugs of abuse, such as cannabis, cocaine, nicotine, opiates, and alcohol [1,2,3,4,5]. The ECS consists of cannabinoid receptors, endogenous ligands, and the enzymes involved in the synthesis and degradation of endocannabinoids. Two major subtypes of cannabinoid receptors, CB1 receptor (CB1R) and CB2 receptor (CB2R), have been cloned and characterized [6]. Because CB1Rs are highly expressed in the brain, while CB2Rs were initially found in peripheral immune cells, it has been generally believed that the psychoactive effects of cannabis are mediated by activation of brain CB1Rs instead of CB2Rs [7].

However, CB2Rs are found in the brains of healthy subjects, albeit at very low levels compared to brain CB1Rs [7,35]. All mice used for experiments were heterozygous (CX3CR1eGFP/+). D1-Cre hemizygote (line FK150) and D2-Cre hemizygote (line ER44) BAC transgenic mice with C57BL/6J background were purchased from GENSAT (www.gensat.org). Homozygous RiboTag mice with C57BL/6J background expressing Cre-inducible HA-Rpl22 were purchased from Jackson Laboratory [36,37,38]. These RiboTag mice were crossed to D1-Cre or D2-Cre mouse line to generate D1-Cre-RiboTag or D2-Cre-RiboTag transgenic mice for isolation of ribosome-associated mRNAs from cell type-specific D1-MSNs or D2-MSNs.

All mice used in the present experiments were matched for age (8–14 weeks) and weight (25–35 g). They were housed individually in a climate-controlled animal colony room on a reversed light-dark cycle (lights on at 7:00 PM, lights off at 7:00 AM) with free access to food and water. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Academy of Sciences, and were approved by the Animal Care and Use Committee of the NIDA of the US National Institutes of Health and the University of Maryland School of Medicine.

Single-dose cocaine treatment

To determine whether a single dose of cocaine injection alters brain CB2 gene expression, 28 drug naïve mice (WT) were randomly divided into four dose groups (10, 20, 30 mg/kg, i.p.) or vehicle (saline) (n = 7 per group). At 1 h after cocaine injection, all mice were deeply anaesthetized with 100 mg/kg pentobarbital and transcardially perfused with saline to remove all blood from the brain. Whole brains were then rapidly removed, and the PFC and striatum tissues were dissected on dry ice for qRT-PCR assays.

Repeated cocaine or heroin treatment

To determine whether repeated cocaine or heroin administration alters brain CB2 expression, additional 20 drug naïve WT mice were divided into five groups—repeated saline vs. repeated cocaine (10, 20 mg/kg, i.p., n = 4–8 per group) or heroin (2, 8 mg/kg, s.c.) (n = 4 each group). Before the drug injection, each animal was placed in a locomotor detection chamber (Accuscan Instruments, Inc., Columbus, OH, USA) for 3 days (3 h per day) for environmental habituation. Then, each animal received 2 days of saline injection before cocaine administration. On each test day, mice were placed in the chamber for 1 h of habituation, and then removed and given either saline (0.1 mL saline, i.p.), one dose of cocaine or heroin. Animals were then placed back into the locomotor chambers for 2 h to assess the effects of cocaine or heroin on locomotion. Each mouse received one injection per day for a total of 7 successive days. Data were collected in 10 min intervals using the VersaMax data analysis system (Accuscan Instruments, Inc., Columbus, OH, USA). Total distance traveled was used to compare saline- vs. the drug (cocaine or heroin)-induced changes in locomotion. At 24 h after the last injection, all mice were deeply anaesthetized with 100 mg/kg pentobarbital and transcardially perfused with saline to remove all blood from the brain. Whole brains were then rapidly removed, and the striatum, PFC or spleen tissues were dissected on dry ice for qRT-PCR assays.

Prolonged cocaine self-administration

We also examined whether prolonged cocaine self-administration alters CB2 expression in the brain. Intravenous (i.v.) catheterization surgery and cocaine self-administration procedures were identical to those we reported previously [Data analysis

All the RT-PCR data were presented as means (±SEM). One-way analysis of variance (ANOVA) was used to analyze the difference in fold change of CB1 and CB2 mRNA among different groups. Individual group comparisons were carried out using the Student–Newman–Keuls method.

Results

Acute cocaine administration failed to alter CB2 mRNA expression

To explore whether cocaine exposure induces dynamic changes in CB2R expression, we designed two probes—one targets an exon junction of the CB2R gene (mCB2A probe) and another one targets the portion of exon 3 that is disrupted in CB2R-knockout mice (mCB2-KO probe) (Fig. 1a). In qRT-PCR analysis in WT mice, both mCB2A and mCB2-KO probes detected similar levels of CB2 mRNA signal in the striatum and spleen (Fig. 1b, c). Consistent with our previous findings [10, 16], we found that CB2 mRNA level is much higher (20–25-fold) in spleen than in striatum. In contrast, the mCB2-KO probe detected CB2 mRNA signal only in WT, not in CB2-KO mice, indicating that the detected signal is highly specific to the CB2R gene. We then used both probes to detect cocaine-induced changes in CB2 expression. We found that a single injection of cocaine at varying doses (10, 20, 30 mg/kg, i.p.) had no effect on CB2 mRNA expression in either the striatum or PFC at 1 h (Fig. 1d, e) or 3 h (data not shown) post injection.

Repeated cocaine administration increases CB2 mRNA expression in striatum and spleen

We then assessed whether repeated daily injections of cocaine similarly alter brain CB2 mRNA expression. Repeated injections of cocaine (10 or 20 mg/kg, i.p.) for 7 consecutive days produced robust locomotor sensitization (Fig. 2a), indicating circuit and behavior-level changes in response to drug exposure. At 24 h after the last cocaine injection, we measured CB1 and CB2 mRNA levels in striatum (Fig. 2b), PFC (Fig. 2c), and spleen (Fig. 2d) via qRT-PCR. We found that CB2 mRNA, as detected by mCB2-KO probe, was significantly increased in both the striatum (Fig. 2b, F2,11 = 7.03, P < 0.05) and spleen (Fig. 2d, F2,11 = 6.96, P < 0.05) in WT mice after repeated cocaine administration. In contrast, there was no change in CB2 mRNA expression in the PFC (Fig. 2c, F2,11 = 2.27, P > 0.05). Repeated cocaine injections also failed to significantly alter CB1 mRNA expression (Fig. 2b, d), with the exception of a small reduction in CB1 mRNA in the PFC following 10 mg/kg cocaine injections (Fig. 2c), suggesting that chronic cocaine treatment selectively upregulates CB2 mRNA expression in both the brain and periphery.

Fig. 2: Effects of repeated cocaine administration on CB1 and CB2 mRNA expression as measured by qRT-PCR.
figure 2

a Repeated cocaine administration produced robust locomotor sensitization at both 10 and 20 mg/kg doses. b Cocaine, at 20 mg/kg only, significantly increased CB2 mRNA in the striatum as measured with the mCB2-KO probe; c cocaine, at 10 or 20 mg/kg, failed to alter CB2 mRNA expression in the PFC. d Cocaine, at both doses, significantly increased CB2 mRNA expression in spleen as measured at 24 h after the last cocaine injection. In contrast, repeated cocaine failed to alter CB1 mRNA expression except that cocaine, at 10 mg/kg, tended to decrease CB1 mRNA expression in the PFC (c). e Repeated heroin administration also produced robust locomotor sensitization in a dose-dependent manner. f Repeated heroin-treated mice displayed a reduction in CB2 mRNA expression in the striatum. *P < 0.05, compared to saline group.

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Repeated heroin administration downregulates CB2 mRNA expression in striatum

To determine whether this effect can be generalized to other drugs of abuse, we also observed the effects of repeated heroin exposure on CB2 mRNA expression in the striatum. Figure 2e, f shows that repeated administration of heroin (2, 8 mg/kg, s.c., once daily for 7 days) produced robust locomotor sensitization in mice in a dose-dependent manner. Unexpectedly, a significant downregulation of CB2 mRNA expression was detected in the striatum as assessed by quantitative RT-PCR when measured at 24 h after the last heroin administration (Fig. 2f, F2,11 = 4.88, P < 0.05).

Cocaine self-administration upregulates CB2 mRNA expression

Molecular and circuit level changes following drug exposure can exhibit key differences depending on whether the drug is investigator administered or voluntarily consumed [45]. For this reason, we then examined whether chronic cocaine self-administration also upregulates CB2 mRNA expression in the brain. We compared these results of dynamic changes in CB2R expression following self-administration of palatable food (sucrose solution). Figure 3a shows the time courses of i.v. cocaine or oral sucrose self-administration; the majority of the animals reached the maximally allowed 50 cocaine infusions or 100 sucrose deliveries per session after ~1 week of self-administration training. At 24 h after the last cocaine self-administration, qRT-PCR analysis showed that CB2 mRNA levels were significantly increased in both the striatum and PFC (Fig. 3b, c) when compared with drug naïve mice or sucrose self-administration mice. One-way ANOVA revealed a statistically significant effect (Fig. 3b: PFC, F2,11 = 8.98, P < 0.01; ST: F2,11 = 16.35, P < 0.001; Fig. 3c: PFC, F2,13 = 7.14, P < 0.01; ST: F2,13 = 6.24, P < 0.05). In contrast, oral sucrose self-administration failed to alter brain CB2 mRNA expression when compared with drug naïve mice (P > 0.05).

Fig. 3: Effects of cocaine self-administration on CB1 and CB2 mRNA expression (by qRT-PCR).
figure 3

a Mean numbers of cocaine self-administration (infusions) or oral sucrose deliveries during the 4 weeks of self-administration training; cocaine, but not sucrose, self-administration significantly upregulated CB2 mRNA expression in both the PFC and striatum in WT mice measured with the mCB2A (b) and mCB2-KO (c) probes, respectively.  *P < 0.05, **P < 0.01, ***P < 0.001, compared to drug naïve mice or sucrose self-administration mice.

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Cellular distributions of CB2 mRNA in the striatum

To determine which type of cells displays CB2 upregulation after cocaine exposure, we then used RNAscope ISH assays to examine CB2 mRNA expression in microglia, D1-MSNs, and D2-MSNs in the NAc. In this assay, we used another mouse CB2-specific RNA probe that targets the gene-deleted region in the CB2-KO mice that we used as control in this study (Fig. 4a). We detected low-to-moderate levels of CB2 mRNA in the NAc (Fig. 4b, left panel). However, when using a microglial-specific CD11b RNA probe, we failed to detect clear microglial cells (Fig. 4b, middle panel) nor co-localization of CB2 and CD11b (Fig, 4b, right panel). However, when using Drd1 or Drd2 RNA probes, we detected clear CB2 mRNA in ~50% Drd1-labled D1-MSNs (Fig. 4c) or Drd2-labeled D2-MSNs (Fig. 4d) in the NAc.

Fig. 4: Cellular distributions of CB2 mRNA in the NAc as assessed by RNAscope in situ hybridization.
figure 4

a Diagram showing the transcript structure of the CB2A isoform and of another mCB2-specific probe (Cnr2-O2 probe) that targets the gene-deleted region in the CB2-KO mice. b CD11b mRNA is barely detectable in the NAc and no CB2 and CD11b co-localization was detected. CB2 mRNA was detected in Drd1-positive D1-MSNs (c) and Drd2-positive D2-MSNs (d).

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Repeated cocaine does not alter CB2 mRNA expression in purified microglia

Since CB2R-expressing D1-MSNs and D2-MSNs are mixedly distributed in the striatum, it is technically difficult for us to use RNAscope ISH methods to quantitatively measure CB2 mRNA density in each phenotype of cells. In addition, RNAscope ISH assays also failed to detect clear microglial cells in the NAc, which may be related to their extremely small cell sizes in normal healthy mice. Thus, to further determine which type of cells (neurons or microglia) in the NAc displays CB2 upregulation after cocaine exposure, we alternatively used three thansgenic mice and FACS technology to purify or isolate different phenotypes of cells. We first used CX3CR1eGFP/+ reporter mice to separate GFP+ microglia and GFP‒ non-microglial cells in the dorsal striatum (DST) and NAc (Fig. 5a). Surprisingly, repeated cocaine injection (20 mg/kg per day for 7 days) failed to alter CB2 mRNA expression in GFP+ microglia, but significantly increased CB2 mRNA expression in the GFP‒ non-microglial cell population (Fig. 5b, left panel, t = 2.24, P < 0.05) in the NAc. In contrast, no significant change in CB2 mRNA was observed in either GFP+ microglia or GFP‒ non-microglial cells in the DST after cocaine administration (Fig. 5b, right panels). Similarly, repeated cocaine administration did not alter CB1 mRNA expression in either cell population in the DST or NAc (Fig. 5b).

Fig. 5: Effects of repeated cocaine administration on microglial CB2 mRNA expression in the striatum.
figure 5

a Flowchart of FACS and qRT-PCR techniques for separating GFP+ microglia and GFP− non-microglial cells from CX3CR1eGFP/+ mice. b qRT-PCR results, indicating that repeated cocaine administration (20 mg/kg, i.p. for 7 days) significantly increased CB2 mRNA expression in GFP− non-microglial cells, but not in GFP+ microglia, in the NAc. There was no change in CB2 mRNA expression in either cell population in the dorsal striatum (DST).

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Repeated cocaine upregulates CB2 mRNA expression in D1-MSNs in the NAc

Given that cocaine upregulates CB2 mRNA expression in non-microglial cells within the NAc, we then sought to more clearly identify the cell population responsible for this dynamic change in CB2R expression. There are two major types of neurons in the striatum: D1- and D2-MSNs, which are differentially involved in motivated drug-taking and drug-seeking behavior [46, 47]. We then used Drd1a-Cre-RiboTag and Drd2-Cre-RiboTag mouse lines to isolate ribosome-associated RNAs from D1-MSNs and D2-MSNs, respectively. In drug naïve mice, CB2 mRNA levels in D1-MSNs or D2-MSNs were significantly lower than those in other RNA samples isolated from all the cells (Fig. 6a, left panel, F = 4.01, P < 0.05). However, when comparing D1- and D2-MSNs to one another, D2-MSNs displayed 3–4 folds higher basal levels of CB2 mRNA than D1-MSNs (Fig. 6a right two panels, t = 4.54, P < 0.05). Following repeated cocaine administration, D1-MSNs displayed significantly upregulated CB2 mRNA expression (Fig. 6b, right panel, t = 2.53, P < 0.05; left panel, t = 6.81, P < 0.05), while D2-MSNs did not (Fig. 6b, P < 0.05). CB1 mRNA expression was not altered by repeated cocaine administration in either D1-MSNs or D2-MSNs (Fig. 6c).

Fig. 6: Effects of repeated cocaine administration on CB2 mRNA expression in striatal D1-MSNs vs. D2-MSNs.
figure 6

a qRT-PCR results, indicating the relative levels of CB2 mRNA in striatal D1-MSNs, D2-MSNs, and All (unsorted) cells, prepared by FACS from D1-Cre-RiboTag and D2-Cre-RiboTag mice. D2-MSNs displayed higher basal level of CB2 mRNA than D1-MSNs when normalized to the house-keep genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or S18 protein; b repeated cocaine administration (20 mg/kg, i.p. for 7 days) selectively increased CB2 expression in D1-MSNs, not in D2-MSNs; c repeated cocaine administration failed to alter CB1R expression in the same samples. *P < 0.05, compared to “All cells” (a left panel), D1-MSNs (a right panel), or saline control mice (b).

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Discussion

In this study, we used three mouse CB2R-specific probes and multiple (4) transgenic mouse lines to examine the impact of acute vs. repeated cocaine administration on brain CB2R expression. We found that a single injection of cocaine failed to alter CB2R expression, but repeated cocaine administration or self-administration caused a significant increase in CB2 mRNA expression in the striatum and spleen tissues. Unexpectedly, this CB2 upregulation does not occur in microglial cells, but occurs mainly in D1-MSNs in the NAc. In a series of parallel studies, we did not observe significant changes in CB1 mRNA expression. These findings suggest that neuronal CB2Rs are inducible and responsive to recurring cocaine use and abuse. The present finding that NAc CB2 was upregulated in D1-MSNs, combined with our previous finding that CB2 was upregulated in VTA DA neurons [11], at least in part explain how CB2R agonists attenuate cocaine self-administration and other addiction-related behaviors in rats and mice [25, 26, 48].

The expression of functional CB2Rs in the brain, particularly in neurons, has been the subject of debate for many years [7, 49]. Concerns have been raised regarding the CB2R signal specificity when using antibodies to detect brain CB2R by immunostaining. Therefore, in the present study, we chose to detect CB2 gene expression using three custom-designed mouse CB2-specific probes (mCB2A, mCB2-KO, Cnr2-O2) based on the CB2 gene structures in WT and CB2-KO mice, combined with the use of CB2-rich spleen tissues and CB2-KO mice as positive and negative controls. These probes have been confirmed to be mouse CB2-specific as assessed by the findings in CB2-KO mice in the present study (Fig. 1) and also in our previous studies [9, 10, 40]. Using qRT-PCR assays, we detected low levels of CB2 mRNA expression in the PFC and striatum in healthy, drug naïve mice.

An important finding in the present study was that a single injection of cocaine at a wide range of doses (10, 20, 30 mg/kg) failed to alter brain CB2 mRNA expression when measured at 1 or 3 h after cocaine injection. In contrast, repeated administration of high doses of cocaine (10, 20 mg/kg) for 7 consecutive days or cocaine self-administration (for 4 weeks) significantly upregulated CB2 mRNA expression in the striatum and the spleen. In addition, cocaine self-administration, but not repeated cocaine exposure for 7 days, also upregulated CB2 mRNA expression in the PFC, suggesting that prolonged cocaine exposure is required to alter CB2 expression in the cortex. It may also suggest that voluntary consumption of cocaine or the associative learning associated with self-administration is necessary to induce CB2 upregulation in the PFC. These findings are consistent with, but expand, previous findings that brain CB2Rs can be upregulated under chronic neuroinflammation as stated above.

Another important finding is that peripheral CB2R expression appears more sensitive to cocaine exposure than that in the brain since recurrent exposure to 10 or 20 mg/kg cocaine upregulates CB2 mRNA expression in spleen, while 20 mg/kg cocaine is required to upregulate CB2 expression in the brain. Cocaine is well known to suppress immune function and cause changes in phagocytic activity of macrophages and production of immunoregulatory cytokines [50,51,52,53] and cannabinoids are known to have anti-inflammatory and immunomodulatory properties by activation of CB2Rs [54, 55]. Thus, the present finding suggests that chronic exposure to cocaine may produce immunosuppressive effects by upregulation of CB2R expression in peripheral immune cells.

An unexpected finding is that repeated administration of opioids (heroin) produced an opposite reduction in NAc CB2 mRNA expression. This is consistent with a recent report indicating that repeated morphine administration caused a significant reduction in CB2, not CB1, mRNA expression in VTA [56], but is not fully consistent with an early report that repeated administration of morphine produces conditioned place preference, which is associated with a significant reduction in CB2 mRNA expression in brain stem and a significant increase in CB2 mRNA expression in the cerebral cortex and peripheral spleen and blood immune cells in rats [57]. These findings suggest that chronic administration of opioids may also differentially alter CB2 mRNA expression in different tissues or brain regions. Given that activation of CB2Rs is protective against various insults in the brain and periphery [6, 13, 58], the present finding suggests that CB2R upregulation may be similarly protective against chronic drug-induced changes or damage in the brain and periphery.

Another unexpected finding is that recurring cocaine use did not alter microglial CB2 mRNA expression. This finding is conflicted with a well-accepted view that brain CB2 upregulation occurs mainly or exclusively in microglial cells during chronic neuroinflammation [13]. This conclusion is largely based on early antibody-based findings that CB2-like immunostaining was found in neuritic plaque-associated astrocytes and microglia in hippocampus and entorhinal cortex in post-mortem brain tissues from patients with severe Alzeimor’s Disease [59], in CD3- or CD68-positive perivascular (microglial) cells in non-human primates with simian immunodeficiency virus-induced encephalitis [60], or in activated microglia and astrocytes in the spinal cord of mice with chronic neuropathic pain [61]. This is supported by the finding that CB2R-dependent-eGFP signal was also detected in microglia in areas of intense inflammation and amyloid deposition in the brain in transgenic CB2-Cre-reporter (CB2EGFP/f/f) mice co-expressing five familial Alzheimer’s disease mutations (5XFAD) [62]. However, there are two major concerns in these studies. First, it is unknown whether those CB2 antibodies are CB2-specific, and second, the CB2-like immunostaining was seen not only in microglia, but also in astrocytes [63]. In contrast to these findings, we have previously reported that cocaine self-administration upregulates CB2R expression in VTA DA neurons, not in glial cells [11]. Systemic administration of lipopolysaccharide (LPS), a bacterial endotoxin, caused significant microglial and astrocytic proliferation in the VTA, but failed to cause CB2R upregulation in microglia or astrocytes [10]. Together, these findings suggest that CB2R upregulation may occur in different phenotypes of cells, including microglia, astrocytes, or neurons depending upon different experimental or pathological conditions, which strongly argue the current prevailing view (dogma) that brain CB2R upregulation occurs mainly or exclusively in microglia.

Perhaps the most important finding in this study is the discovery of CB2R upregulation in NAc D1-MSNs in mice after recurring cocaine use. Interestingly, D1-MSNs displayed lower basal level of CB2 mRNA expression than D2-MSNs, but displayed a higher CB2R response to repeated cocaine in the NAc, rather than in the DST. These findings suggest that neuronal CB2R upregulation in the striatum displays subregional- and cell type-specific profiles. As mentioned above, growing evidence indicates that brain CB2Rs modulate pharmacological action produced by cocaine [9, 10, 25, 26, 48], morphine [64, 65], nicotine [28], and alcohol [66] in a number of addiction- and pain-related behavioral models. However, the neural mechanisms underlying this CB2R-mediated action are poorly understood. First, it is unlikely that microglia CB2R are involved since there is little evidence supporting CB2 expression in microglia in normal healthy or in LPS-treated mice [10] or microglial CB2 upregulation in rats with cocaine self-administration history [11]. Second, as stated above, cocaine self-administration upregulates CB2 gene expression in VTA DA neurons [11], suggesting that a presynaptic CB2R mechanism in the mesolimbic DA system may be involved (Fig. 7). Lastly, in the present report, we found that chronic cocaine administration also upregulates CB2R expression in NAc D1-MSNs, suggesting that a postsynaptic CB2R mechanism may also play an important role in mediating CB2R modulation of cocaine action (Fig. 7).

Fig. 7: Schematic diagram showing the effects of cocaine on CB2R expression in the mesolimbic DA reward system.
figure 7

This system originates from DA neurons in the VTA and projects to the NAc. Acute cocaine exposure blocks DA transporter (DAT) and elevates extracellular DA level in the NAc, which subsequently activates D1-MSNs via Gs-coupled D1Rs and inhibits D2-MSNs via Gi-coupled D2Rs, producing rewarding effects. Chronic cocaine administration increases CB2R expression in VTA DA neurons as we reported previously [11] and also in NAc D1-MSNs as shown in the present study. Accordingly, activation of CB2Rs by CB2R agonists would inhibit presynaptic VTA DA neurons and postsynaptic D1-MSNs in the NAc, producing therapeutic anti-cocaine effects. VTA ventral tegmental area, NAc nucleus accumbens, (+) activation, (−) inhibition.

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It is unknown precisely how CB2Rs in D1-MSNs modulate cocaine action. Recent optogenetic studies indicate that activation of D1-MSNs in the NAc is critically associated with positive reinforcement, while activation of D2-MSNs are mostly associated with aversion [67, 68], although not all evidence supports this dichotomy [69, 70]. Accordingly, it is hypothesized that the acute rewarding effects of cocaine are most likely mediated by activation of D1-MSNs via Gs-coupled D1 receptors and inhibition of D2-MSNs via Gi-coupled D2 receptors [67, 71, 72] (Fig. 7). Since CB2Rs are Gi-coupled receptors, we further hypothesized that CB2R upregulation in VTA DA neurons or NAc D1-MSNs would increase neuronal response to endocannabinoids or CB2R agonists, producing enhanced inhibitory effects on both presynaptic DA neurons and postsynaptic D1-MSNs, which subsequently counteracts cocaine-induced increase in extracellular DA and cocaine’s action in postsynaptic D1-MSNs (Fig. 7). Thus, the present finding of CB2 upregulation in striatal D1-MSNs provides new mechanistic insight on how brain CB2Rs modulate pharmacological action produced not only by cocaine, but also by other drugs of abuse, such as opioids, nicotine and alcohol.

In conclusion, brain CB2R involvement has been recently reported in several DA-regulated CNS disorders, including substance abuse and addiction. The present findings of neuronal CB2R upregulation in NAc D1-MSNs not only challenges the well-accepted view that CB2R upregulation is mainly or exclusively expressed in activated microglia, but also provide additional evidence indicating that chronic cocaine use and abuse may upregulate CB2R expression in brain neurons and peripheral immune cells. These new findings suggest that CB2R upregulation may represent a general defense or protective mechanism in response to various insults including chronic psychostimulant abuse and addiction. Thus, brain CB2Rs may constitute a new therapeutic target in medication development for the treatment of cocaine use disorders as well as many other CNS disorders.