Cannabinoid CB1 Receptors Are Expressed in a Subset of Dopamine Neurons and Underlie Cannabinoid-Induced Aversion, Hypoactivity, and Anxiolytic Effects in Mice.

Cannabinoids modulate dopamine (DA) transmission and DA-related behavior, which has been thought to be mediated initially by activation of cannabinoid CB1 receptors (CB1Rs) on GABA neurons. However, there is no behavioral evidence supporting it. In contrast, here we report that CB1Rs are also expressed in a subset of DA neurons and functionally underlie cannabinoid action in male and female mice. RNAscope in situ hybridization (ISH) assays demonstrated CB1 mRNA in tyrosine hydroxylase (TH)-positive DA neurons in the ventral tegmental area (VTA) and glutamate decarboxylase 1 (GAD1)-positive GABA neurons. The CB1R-expressing DA neurons were located mainly in the middle portion of the VTA with the number of CB1-TH colocalization progressively decreasing from the medial to the lateral VTA. Triple-staining assays indicated CB1R mRNA colocalization with both TH and vesicular glutamate transporter 2 (VgluT2, a glutamate neuronal marker) in the medial VTA close to the midline of the brain. Optogenetic activation of this population of DA neurons was rewarding as assessed by optical intracranial self-stimulation. Δ9-tetrahydrocannabinol (Δ9-THC) or ACEA (a selective CB1R agonist) dose-dependently inhibited optical intracranial self-stimulation in DAT-Cre control mice, but not in conditional knockout mice with the CB1R gene absent in DA neurons. In addition, deletion of CB1Rs from DA neurons attenuated Δ9-THC-induced reduction in DA release in the NAc, locomotion, and anxiety. Together, these findings indicate that CB1Rs are expressed in a subset of DA neurons that corelease DA and glutamate, and functionally underlie cannabinoid modulation of DA release and DA-related behavior.SIGNIFICANCE STATEMENT Cannabinoids produce a series of psychoactive effects, such as aversion, anxiety, and locomotor inhibition in rodents. However, the cellular and receptor mechanisms underlying these actions are not fully understood. Here we report that CB1 receptors are expressed not only in GABA neurons but also in a subset of dopamine neurons, which are located mainly in the medial VTA close to the midline of the midbrain and corelease dopamine and glutamate. Optogenetic activation of these dopamine neurons is rewarding, which is dose-dependently inhibited by cannabinoids. Selective deletion of CB1 receptor from dopamine neurons blocked cannabinoid-induced aversion, hypoactivity, and anxiolytic effects. These findings demonstrate that dopaminergic CB1 receptors play an important role in mediating cannabinoid action.

New Drugs, Old Targets: Tweaking the Dopamine System to Treat Psychostimulant Use Disorders.

The abuse of illicit psychostimulants such as cocaine and methamphetamine continues to pose significant health and societal challenges. Despite considerable efforts to develop medications to treat psychostimulant use disorders, none have proven effective, leaving an underserved patient population and unanswered questions about what mechanism(s) of action should be targeted for developing pharmacotherapies. As both cocaine and methamphetamine rapidly increase dopamine (DA) levels in mesolimbic brain regions, leading to euphoria that in some can lead to addiction, targets in which this increased dopaminergic tone may be mitigated have been explored. Further, understanding and targeting mechanisms underlying relapse are fundamental to the success of discovering medications that reduce the reinforcing effects of the drug of abuse, decrease the negative reinforcement or withdrawal/negative affect that occurs during abstinence, or both. Atypical inhibitors of the DA transporter and partial agonists/antagonists at DA D3 receptors are described as two promising targets for future drug development.

Involvement of the ghrelin system in the maintenance of oxycodone self-administration: converging evidence from endocrine, pharmacologic and transgenic approaches.

Ghrelin, an orexigenic hormone, has emerged as a critical biological substrate implicated in drug reward. However, the response of the ghrelin system to opioid-motivated behaviors and the role of ghrelin in oxycodone self-administration remain to be studied. Here, we investigated the reciprocal interactions between the endogenous ghrelin system and oxycodone self-administration behaviors in rats and the role of the ghrelin system in brain stimulation reward (BSR) driven by optogenetic stimulation of midbrain reward circuits in mice. Oxycodone self-administration significantly elevated plasma ghrelin, des-acyl ghrelin and growth hormone and showed no effect on plasma LEAP2, a newly identified endogenous ghrelin receptor (GHS-R1a) antagonist. Oxycodone self-administration produced significant decreases in plasma gastric inhibitory polypeptide and insulin. Acquisition of oxycodone self-administration significantly upregulated GHS-R1a mRNA levels in dopamine neurons in the ventral tegmental area (VTA), a brain region critical in drug reward. Pretreatment with JMV2959, a selective GHS-R1a antagonist, dose-dependently reduced oxycodone self-administration and decreased the breakpoint for oxycodone under a progressive ratio reinforcement in Long-Evans rats. The inhibitory effects of JMV2959 on oxycodone self-administration is selectively mediated by GHS-R1a as JMV2959 showed a similar effect in Wistar wildtype but not in GHS-R knockout rats. JMV2959 pretreatment significantly inhibited BSR driven by selective stimulation of VTA dopamine neurons, but not by stimulation of striatal GABA neurons projecting to the VTA in mice. These findings suggest that elevation of ghrelin signaling by oxycodone or oxycodone-associated stimuli is a causal process by which oxycodone motivates oxycodone drug-taking and targeting the ghrelin system may be a viable treatment approach for opioid use disorders.

Dissecting the Role of GABA Neurons in the VTA versus SNr in Opioid Reward.

Opioid reward has traditionally been thought to be mediated by GABA-induced disinhibition of dopamine (DA) neurons in the VTA. However, direct behavioral evidence supporting this hypothesis is still lacking. In this study, we found that the µ opioid receptor (MOR) gene, Oprm1, is highly expressed in GABA neurons, with ∼50% of GABA neurons in the substantia nigra pars reticulata (SNr), ∼30% in the VTA, and ∼70% in the tail of the VTA (also called the rostromedial tegmental nucleus) in male rats. No Oprm1 mRNA was detected in midbrain DA neurons. We then found that optogenetic inhibition of VTA DA neurons reduced intravenous heroin self-administration, whereas activation of these neurons produced robust optical intracranial self-stimulation in DAT-Cre mice, supporting an important role of DA neurons in opioid reward. Unexpectedly, pharmacological blockade of MORs in the SNr was more effective than in the VTA in reducing heroin reward. Optogenetic activation of VTA GABA neurons caused place aversion and inhibited cocaine, but not heroin, self-administration, whereas optogenetic activation of SNr GABA neurons caused a robust increase in heroin self-administration with an extinction pattern, suggesting a compensatory response in drug intake due to reduced heroin reward. In addition, activation of SNr GABA neurons attenuated heroin-primed, but not cue-induced, reinstatement of drug-seeking behavior, whereas inhibition of SNr GABA neurons produced optical intracranial self-stimulation and place preference. Together, these findings suggest that MORs on GABA neurons in the SNr play more important roles in opioid reward and relapse than MORs on VTA GABA neurons.SIGNIFICANCE STATEMENT Opioid reward has long been believed to be mediated by inhibition of GABA interneurons in the VTA that subsequently leads to disinhibition of DA neurons. In this study, we found that more µ opioid receptors (MORs) are expressed in GABA neurons in the neighboring SNr than in the VTA, and that pharmacological blockade of MORs in the SNr is more effective in reducing heroin reward than blockade of MORs in the VTA. Furthermore, optogenetic activation of VTA GABA neurons inhibited cocaine, but not heroin, self-administration, whereas activation of SNr GABA neurons inhibited heroin reward and relapse. These findings suggest that opioid reward is more likely mediated by stimulation of MORs in GABA afferents from other brain regions than in VTA GABA neurons.

Dopamine, behavior, and addiction.

Addictive drugs are habit-forming. Addiction is a learned behavior; repeated exposure to addictive drugs can stamp in learning. Dopamine-depleted or dopamine-deleted animals have only unlearned reflexes; they lack learned seeking and learned avoidance. Burst-firing of dopamine neurons enables learning-long-term potentiation (LTP)-of search and avoidance responses. It sets the stage for learning that occurs between glutamatergic sensory inputs and GABAergic motor-related outputs of the striatum; this learning establishes the ability to search and avoid. Independent of burst-firing, the rate of single-spiking-or "pacemaker firing"-of dopaminergic neurons mediates motivational arousal. Motivational arousal increases during need states and its level determines the responsiveness of the animal to established predictive stimuli. Addictive drugs, while usually not serving as an external stimulus, have varying abilities to activate the dopamine system; the comparative abilities of different addictive drugs to facilitate LTP is something that might be studied in the future.

Optogenetic brain-stimulation reward: A new procedure to re-evaluate the rewarding versus aversive effects of cannabinoids in dopamine transporter-Cre mice.

Despite extensive research, the rewarding effects of cannabinoids are still debated. Here, we used a newly established animal procedure called optogenetic intracranial self-stimulation (ICSS) (oICSS) to re-examine the abuse potential of cannabinoids in mice. A specific adeno-associated viral vector carrying a channelrhodopsin gene was microinjected into the ventral tegmental area (VTA) to express light-sensitive channelrhodopsin in dopamine (DA) neurons of transgenic dopamine transporter (DAT)-Cre mice. Optogenetic stimulation of VTA DA neurons was highly reinforcing and produced a classical "sigmoidal"-shaped stimulation-response curve dependent upon the laser pulse frequency. Systemic administration of cocaine dose-dependently enhanced oICSS and shifted stimulation-response curves upward, in a way similar to previously observed effects of cocaine on electrical ICSS. In contrast, Δ9 -tetrahydrocannabinol (Δ9 -THC), but not cannabidiol, dose-dependently decreased oICSS responding and shifted oICSS curves downward. WIN55,212-2 and ACEA, two synthetic cannabinoids often used in laboratory settings, also produced dose-dependent reductions in oICSS. We then examined several new synthetic cannabinoids, which are used recreationally. XLR-11 produced a cocaine-like increase, AM-2201 produced a Δ9 -THC-like reduction, while 5F-AMB had no effect on oICSS responding. Immunohistochemistry and RNAscope in situ hybridization assays indicated that CB1 Rs are expressed mainly in VTA GABA and glutamate neurons, while CB2 Rs are expressed mainly in VTA DA neurons. Together, these findings suggest that most cannabinoids are not reward enhancing, but rather reward attenuating or aversive in mice. Activation of CB1 R and/or CB2 R in different populations of neurons in the brain may underlie the observed actions.

Newly Developed Dopamine D3 Receptor Antagonists, R-VK4-40 and R-VK4-116, Do Not Potentiate Cardiovascular Effects of Cocaine or Oxycodone in Rats.

Opioid and cocaine abuse are major public health burdens. Existing medications for opioid use disorder are limited by abuse liability and side effects, whereas no treatments are currently approved in the United States for cocaine use disorder. Dopamine D3 receptor (D3R) antagonists have shown promise in attenuating opioid and cocaine reward and mitigating relapse in preclinical models. However, translation of D3R antagonists to the clinic has been hampered by reports that the D3R antagonists GSK598,809 (5-(5-((3-((1S,5R)-1-(2-fluoro-4-(trifluoromethyl)phenyl)-3-azabicyclo[3.1.0]hexan-3-yl)propyl)thio)-4-methyl-4H-1,2,4-triazol-3-yl)-4-methyloxazole) and SB-277,011A (2-(2-((1r,4r)-4-(2-oxo-2-(quinolin-4-yl)ethyl)cyclohexyl)ethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile) have adverse cardiovascular effects in the presence of cocaine. Recently, we developed two structurally novel D3R antagonists, R-VK4-40 and R-VK4-116, which are highly selective for D3R and display translational potential for treatment of opioid use disorder. Here, we tested whether R-VK4-40 ((R)-N-(4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) and R-VK4-116 ((R)-N-(4-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) have unwanted cardiovascular effects in the presence of oxycodone, a prescription opioid, or cocaine in freely moving rats fitted with surgically implanted telemetry transmitters. We also examined cardiovascular effects of the D3R antagonist, SB-277,011A, and L-741,626 (1-((1H-indol-3-yl)methyl)-4-(4-chlorophenyl)piperidin-4-ol), a dopamine D2 receptor-selective antagonist, for comparison. Consistent with prior reports, SB-277,011A increased blood pressure, heart rate, and locomotor activity alone and in the presence of cocaine. L-741,626 increased blood pressure and heart rate. In contrast, R-VK4-40 alone dose-dependently reduced blood pressure and heart rate and attenuated oxycodone-induced increases in blood pressure and oxycodone or cocaine-induced increases in heart rate. Similarly, R-VK4-116 alone dose-dependently reduced cocaine-induced increases in blood pressure and heart rate. These results highlight the safety of new D3R antagonists and support the continued development of R-VK4-40 and R-VK4-116 for the treatment of opioid and cocaine use disorders. SIGNIFICANCE STATEMENT: Opioid and cocaine abuse are major public health challenges and new treatments that do not adversely impact the cardiovascular system are needed. Here, we show that two structurally novel dopamine D3 receptor antagonists, R-VK4-40 and R-VK4-116, do not potentiate, and may even protect against, oxycodone- or cocaine-induced changes in blood pressure and heart rate, supporting their further development for the treatment of opioid and/or cocaine use disorders.

CB2 receptor antibody signal specificity: correlations with the use of partial CB2-knockout mice and anti-rat CB2 receptor antibodies.

Cannabinoid CB1 receptors are highly expressed in the brain and functionally modulate presynaptic neurotransmitter release, while cannabinoid CB2 receptors (CB2Rs) were initially identified in the spleen and regarded as peripheral cannabinoid receptors. Recently, growing evidence indicates the presence of functional CB2Rs in the brain. However, this finding is disputed because of the specificity of CB2R antibody signals. We used two strains of currently available partial CB2-knockout (CB2-KO) mice as controls, four anti-rat or anti-mouse CB2R antibodies, and mRNA quantification to further address this issue. Western blot assays using the four antibodies detected a CB2R-like band at ~40 kD in both the brain and spleen. Notably, more bands were detected in the brain than in the spleen, and specific immune peptides blocked band detection. Immunohistochemical assays also detected CB2-like immunostaining in mouse midbrain dopamine neurons. CB2R deletion in CB2-KO mice may reduce or leave CB2R-like immunoreactivity unaltered depending on antibody epitope. Antibodies with epitopes at the receptor-deleted region detected a significant reduction in CB2R band density and immunostaining in N-terminal-deleted Deltagen and C-terminal-deleted Zimmer strain CB2-KO mice. Other antibodies with epitopes at the predicted receptor-undeleted regions detected similar band densities and immunostaining in wild-type and CB2-KO mice. Quantitative RT-PCR assays detected CB2 mRNA expression using probes that targeted upstream or downstream gene sequences but not the probe that targeted the gene-deleted sequence in Deltagen or Zimmer CB2-KO mice. These findings suggest that none of the tested four polyclonal antibodies are highly mouse CB2R-specific. Non-specific binding may be related to the expression of mutant or truncated CB2R-like proteins in partial CB2-KO mice and the use of anti-rat CB2 antibodies because the epitopes are different between rat and mouse CB2Rs.

Cannabinoid CB1 receptor neutral antagonist AM4113 inhibits heroin self-administration without depressive side effects in rats.

Cannabinoid CB1 receptors (CB1Rs) have been shown to be a promising target in medication development for the treatment of addiction. However, clinical trials with SR141716A (rimonabant, a selective CB1R antagonist/inverse agonist) for the treatment of obesity and smoking cessation failed due to unwanted side effects, such as depression, anxiety, and suicidal tendencies. Recent preclinical studies suggest that the neutral CB1R antagonist AM4113 may retain the therapeutic anti-addictive effects of SR141716A in nicotine self-administration models and possibly has fewer unwanted side effects. However, little is known about whether AM4113 is also effective for other drugs of abuse, such as opioids and psychostimulants, and whether it produces depressive side effects similar to SR141716A in experimental animals. In this study, we demonstrated that systemic administration of AM4113 (3 and 10 mg/kg) dose-dependently inhibited the self-administration of intravenous heroin but not cocaine or methamphetamine, whereas SR141716A (3 and 10 mg/kg) dose-dependently inhibited the self-administration of heroin and methamphetamine but not cocaine. In the electrical brain-stimulation reward (BSR) paradigm, SR141716A (3 and 10 mg/kg) dose-dependently increased the BSR stimulation threshold (i.e., decreased the stimulation reward), but AM4113 had no effect on BSR at the same doses, suggesting that SR141716A may produce aversive effects while AM4113 may not. Together, these findings show that neutral CB1R antagonists such as AM4113 deserve further research as a new class of CB1R-based medications for the treatment of opioid addiction without SR141716A-like aversive effects.

Working memory and salivary brain-derived neurotrophic factor as developmental predictors of cocaine seeking in male and female rats.

Poor working memory is linked to future risk-taking behaviors. Lifelong risk of habitual drug use is highest in individuals who initiate use in early adolescence. We sought to determine in rats whether juvenile traits, specifically poor working memory and low salivary brain-derived neurotrophic factor (BDNF), are related to elevated cocaine taking and relapse in adolescence and adulthood. On postnatal day (P) 20, working memory was assessed using the novel object recognition task in male and female rats. Saliva was assayed at P20 for BDNF before cocaine self-administration on P28 [0.75 or 0.25 mg/kg/infusion for 30 days under a fixed-ratio (FR) 1 to FR5 schedule] and on P94 before relapse after 30-day abstinence in adulthood. A separate cohort of P28 male rats was assayed for object discrimination and BDNF in saliva and the medial prefrontal cortex and dorsolateral striatum. Novel object discrimination correlated positively with salivary BDNF on P20 and dorsolateral striatum levels, but negatively with medial prefrontal cortex BDNF in male rats. In female rats, P20 salivary BDNF negatively correlated with object discrimination. Salivary BDNF positively correlated across age in male rats. Male rats earned more cocaine (0.75 mg/kg) at FR5 and responded more at relapse than did female rats. These elevated relapse rates in male rats were significantly associated with P20 object discrimination and salivary BDNF. Relapse after 0.75 and 0.25 mg/kg in female rats correlated only with object discrimination. In conclusion, poor working memory and low salivary BDNF in juvenile male rats may represent biomarkers for later cocaine use. Further research is needed to identify biomarkers for risk in male rats.

Neural regulation of the time course for cocaine-cue extinction consolidation in rats.

Sites within the hippocampus, amygdala and prefrontal cortex may regulate how responses maintained by cues associated with cocaine are extinguished. To test the role of various brain sites in the consolidation of cocaine-cue extinction learning, the dorsal subiculum (dSUB), rostral basolateral amygdala (rBLA) and infralimbic prefrontal cortex (IL) were manipulated in rats. Following cocaine self-administration training (cues present, cocaine available), responding was assessed during 1-h extinction tests (cues present, no cocaine available). To study extinction consolidation specifically, the protein synthesis inhibitor anisomycin or vehicle was infused bilaterally into the dSUB, rBLA or IL either immediately following or 6 h after the first two of three extinction training sessions. With manipulations made immediately after extinction sessions, infusions of anisomycin into the dSUB or the rBLA deterred extinction. Rats maintained elevated levels of cocaine seeking relative to vehicle despite the absence of cocaine delivery. Manipulations of IL had no effect. Control studies showed that bilateral protein synthesis inhibition in dSUB and rBLA 6 h after the extinction sessions ended was unable to deter extinction. Rats reduced cocaine seeking in the usual manner in the absence of cocaine delivery. Collectively, these findings suggest that the dSUB and rBLA are neural substrates important for consolidation of cocaine-cue extinction learning and have time-dependent roles. Understanding the contribution of individual neural substrates for cocaine-cue extinction consolidation may help guide treatment strategies aimed at enhancing cue exposure therapy in cocaine-dependent people.

Identification of the Risk Genes Associated With Vulnerability to Addiction: Major Findings From Transgenic Animals.

Understanding risk factors for substance use disorders (SUD) can facilitate medication development for SUD treatment. While a rich literature exists discussing environmental factors that influence SUD, fewer articles have focused on genetic factors that convey vulnerability to drug use. Methods to identify SUD risk genes include Genome-Wide Association Studies (GWAS) and transgenic approaches. GWAS have identified hundreds of gene variants or single nucleotide polymorphisms (SNPs). However, few genes identified by GWAS have been verified by clinical or preclinical studies. In contrast, significant progress has been made in transgenic approaches to identify risk genes for SUD. In this article, we review recent progress in identifying candidate genes contributing to drug use and addiction using transgenic approaches. A central hypothesis is if a particular gene variant (e.g., resulting in reduction or deletion of a protein) is associated with increases in drug self-administration or relapse to drug seeking, this gene variant may be considered a risk factor for drug use and addiction. Accordingly, we identified several candidate genes such as those that encode dopamine D2 and D3 receptors, mGluR2, M4 muscarinic acetylcholine receptors, and α5 nicotinic acetylcholine receptors, which appear to meet the risk-gene criteria when their expression is decreased. Here, we describe the role of these receptors in drug reward and addiction, and then summarize major findings from the gene-knockout mice or rats in animal models of addiction. Lastly, we briefly discuss future research directions in identifying addiction-related risk genes and in risk gene-based medication development for the treatment of addiction.

Reconceptualizing non-pharmacologic approaches to Neonatal Abstinence Syndrome (NAS) and Neonatal Opioid Withdrawal Syndrome (NOWS): A theoretical and evidence-based approach.

Discussions about non-pharmacologic interventions for Neonatal Abstinence Syndrome and Neonatal Opioid Withdrawal Syndrome (NAS/NOWS) have been minor compared with wider attention to pharmacologic treatments. Although historically under-recognized, non-pharmacologic interventions are of paramount importance for all substance-exposed infants and remain as a first line therapy for the care of infants affected by NAS. Here we examine the role of non-pharmacologic interventions for NAS/NOWS by incorporating theoretical perspectives from different disciplines that inform the importance of individualized assessment of the mother-caregiver/infant dyad and interventions that involve both individuals. NAS/NOWS is a complex, highly individualized constellation of signs/symptoms that vary widely in onset, duration, severity, expression, responses to treatment and influence on long-term outcomes. NAS/NOWS often occurs in infants with multiple prenatal/postnatal factors that can compromise neurobiological self-regulatory functioning. We propose to rethink some of the long-held assumptions, beliefs, and paradigms about non-pharmacologic care of the infant with NAS/NOWS, which is provided as non-specific or as "bundled" in current approaches. This paper is Part I of a two-part series on re-conceptualizing non-pharmacologic care for NAS/NOWS as individualized treatment of the dyad. Here, we set the foundation for a new treatment approach grounded in developmental theory and evidence-based observations of infant neurobiology and neurodevelopment. In Part II, we provide actionable, individually tailored evaluations and approaches to non-pharmacologic NAS/NOWS treatment based on measurable domains of infant neurobehavioral functioning.

Definitions of neonatal abstinence syndrome in clinical studies of mothers and infants: an expert literature review.

Neonatal abstinence syndrome (NAS) results from discontinuation of in utero exposures to opioids/substances. The rising incidence of NAS has prompted an increased need for accurate research and public health data. To examine how NAS has been defined in clinical studies of opioid-exposed mothers and infants, a review process was developed based on the RAND/UCLA Appropriateness Method, yielding 888 abstracts. Per inclusion criteria, 57 abstracts underwent full-text review. To define NAS, studies cited using modified versions of the Finnegan NAS scoring tool (n = 21; 37%), ICD-9/10 coding (n = 17; 30%), original Finnegan tool (n = 16; 28%), Eat Sleep Console (n = 3; 5%), and Lipsitz (n = 3; 5%) tools, (3 cited 2+ tools). Most studies utilized subjective NAS scoring/assessment algorithms and neonatal coding as key elements defining NAS. While most cited opioid exposure as integral to their inclusion criteria, 26% did not. These approaches highlight the need for a more refined and standardized definition of NAS.

Synaptic Zn2+ potentiates the effects of cocaine on striatal dopamine neurotransmission and behavior.

Cocaine binds to the dopamine (DA) transporter (DAT) to regulate cocaine reward and seeking behavior. Zinc (Zn2+) also binds to the DAT, but the in vivo relevance of this interaction is unknown. We found that Zn2+ concentrations in postmortem brain (caudate) tissue from humans who died of cocaine overdose were significantly lower than in control subjects. Moreover, the level of striatal Zn2+ content in these subjects negatively correlated with plasma levels of benzoylecgonine, a cocaine metabolite indicative of recent use. In mice, repeated cocaine exposure increased synaptic Zn2+ concentrations in the caudate putamen (CPu) and nucleus accumbens (NAc). Cocaine-induced increases in Zn2+ were dependent on the Zn2+ transporter 3 (ZnT3), a neuronal Zn2+ transporter localized to synaptic vesicle membranes, as ZnT3 knockout (KO) mice were insensitive to cocaine-induced increases in striatal Zn2+. ZnT3 KO mice showed significantly lower electrically evoked DA release and greater DA clearance when exposed to cocaine compared to controls. ZnT3 KO mice also displayed significant reductions in cocaine locomotor sensitization, conditioned place preference (CPP), self-administration, and reinstatement compared to control mice and were insensitive to cocaine-induced increases in striatal DAT binding. Finally, dietary Zn2+ deficiency in mice resulted in decreased striatal Zn2+ content, cocaine locomotor sensitization, CPP, and striatal DAT binding. These results indicate that cocaine increases synaptic Zn2+ release and turnover/metabolism in the striatum, and that synaptically released Zn2+ potentiates the effects of cocaine on striatal DA neurotransmission and behavior and is required for cocaine-primed reinstatement. In sum, these findings reveal new insights into cocaine's pharmacological mechanism of action and suggest that Zn2+ may serve as an environmentally derived regulator of DA neurotransmission, cocaine pharmacodynamics, and vulnerability to cocaine use disorders.

Correction: ACNP efforts toward reducing climate change.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Introduction to the Special Issue on "Informing Longitudinal Studies on the Effects of Maternal Stress and Substance Use on Child Development: Planning for the HEALthy Brain and Child Development (HBCD) Study".

The HEALthy Brain and Child Development (HBCD) study will establish a large cohort of pregnant women from regions of the country significantly affected by the opioid crisis and follow them and their children for at least 10 years. Findings from this cohort will help researchers understand normative childhood brain development as well as the long-term impact of prenatal and postnatal opioid and other drug and environmental exposures. The study will collect data on pregnancy and fetal development; infant and early childhood structural and functional brain imaging; anthropometrics; medical history; family history; biospecimens; and social, emotional, and cognitive development. Knowledge gained from this research will be critical to help predict and prevent some of the known effects of prenatal and postnatal exposure to certain drugs or environmental exposures, including risk for future substance use, mental disorders, and other behavioral and developmental problems. In this special issue, a subset of investigators that received funding for planning grants for the HBCD study provide careful guidelines and frameworks for study design, recruitment and retention of vulnerable populations, culturally sensitive practices, and biospecimen and neurodevelopmental assessment recommendations gathered in feasibility studies that will help inform the full HBCD study planned to begin recruitment in 2022.

(±)VK4-40, a novel dopamine D3 receptor partial agonist, attenuates cocaine reward and relapse in rodents.

BACKGROUND AND PURPOSE: Despite widespread abuse of cocaine, there are no approved treatments for cocaine use disorder. Chronic cocaine use is associated with up-regulated dopamine D3 receptor expression in the brain. Therefore, most D3 -based medication development has focused on D3 antagonists. However, D3 antagonists do not attenuate cocaine intake under "easy" self-administration conditions, when response requirements are low. We evaluated a novel, highly selective and metabolically stable D3 partial agonist, (±)VK4-40, for its efficacy in reducing cocaine intake and relapse to drug seeking. EXPERIMENTAL APPROACH: The impact of (±)VK4-40 on cocaine intake and relapse was evaluated using intravenous self-administration procedures under a fixed-ratio 2 reinforcement schedule and cocaine-primed reinstatement conditions in rats. Optogenetic brain-stimulation reward procedures were used to evaluate the interaction of (±)VK4-40 and cocaine in the mesolimbic dopamine system in mice. Sucrose self-administration in rats and a conditioned place preference paradigm in mice were used to evaluate the abuse potential of (±)VK4-40 alone and other unwanted effects. KEY RESULTS: (±)VK4-40 dose-dependently reduced cocaine self-administration and cocaine-primed reinstatement of drug-seeking behaviour. (±)VK4-40 also inhibited cocaine-enhanced brain-stimulation reward caused by optogenetic stimulation of dopamine neurons in the ventral tegmental area. (±)VK4-40 alone decreased brain-stimulation reward but produced neither conditioned place preference nor place aversion. This new D3 partial agonist also failed to alter oral sucrose self-administration. CONCLUSION AND IMPLICATIONS: The novel D3 partial agonist, (±)VK4-40 attenuates cocaine reward and relapse in rodents, without significant unwanted effects. These findings support further investigation of D3 partial agonists as putative treatments for cocaine use disorder.

Xie2-64, a novel CB2 receptor inverse agonist, reduces cocaine abuse-related behaviors in rodents.

Cocaine abuse remains a public health threat around the world. There are no pharmacological treatments approved for cocaine use disorder. Cannabis has received growing attention as a treatment for many conditions, including addiction. Most cannabis-based medication development has focused on cannabinoid CB1 receptor (CB1R) antagonists (and also inverse agonists) such as rimonabant, but clinical trials with rimonabant have failed due to its significant side-effects. Here we sought to determine whether a novel and selective CB2R inverse agonist, Xie2-64, has similar therapeutic potential for cocaine use disorder. Computational modeling indicated that Xie2-64 binds to CB2R in a way similar to SR144528, another well-characterized but less selective CB2R antagonist/inverse agonist, suggesting that Xie2-64 may also have CB2R antagonist profiles. Unexpectedly, systemic administration of Xie2-64 or SR144528 dose-dependently inhibited intravenous cocaine self-administration and shifted cocaine dose-response curves downward in rats and wild-type, but not in CB2R-knockout, mice. Xie2-64 also dose-dependently attenuated cocaine-enhanced brain-stimulation reward maintained by optical stimulation of ventral tegmental area dopamine (DA) neurons in DAT-Cre mice, while Xie2-64 or SR144528 alone inhibited optical brain-stimulation reward. In vivo microdialysis revealed that systemic or local administration of Xie2-64 into the nucleus accumbens reduced extracellular dopamine levels in a dose-dependent manner in rats. Together, these results suggest that Xie2-64 has significant anti-cocaine reward effects likely through a dopamine-dependent mechanism, and therefore, deserves further study as a new pharmacotherapy for cocaine use disorder.