Comparison of the Effect of Two Different Handling Conditions at Slaughter in Saliva Analytes in Pigs.

In this report, different handling conditions at slaughterhouse were studied to assess changes in salivary biomarkers. For this purpose, finishing pigs were divided into two groups, one in which handling was improved to minimize stress (Group A, n = 24, transported and stabled at the slaughterhouse at low density without mixing with unfamiliar animals throughout the whole process) and another one in which animals had a more stressful handling process (Group B, n = 24, transported and stabled at high density with unfamiliar animals). Saliva samples were taken the day before transport to the slaughterhouse at 8:00 a.m. (B0) and 12:00 a.m. (B4), and the day of slaughter just after unloading animals at the slaughterhouse at approximately 8:00 a.m. (S0) and after 4 h of lairage at approximately 12:00 a.m. (S4). Group B showed significantly higher cortisol, total esterase activity, oxytocin, adenosine deaminase and haptoglobin levels than the Group A at both S0 and S4 sampling times, and higher levels of calprotectin and creatine kinase at S4 sampling time. This report indicates that differences in the way in which the pigs are handled at the slaughterhouse can lead to changes in salivary biomarkers and opens the possibility of the use of biomarker at slaughter to monitor handling conditions.

Clinical features and outcomes of mpox in people with and without HIV: a national comparative study.

BACKGROUND: People who live with HIV (PWLH) have been one of the most affected groups during the current mpox outbreak. They are hypothesized to have a more severe clinical course than people without HIV but comparative data is scarce. We aimed to compare clinical features and outcomes of mpox in people with and without HIV in Mexico. SETTING: Country-wide study in Mexico. METHODS: We performed an observational study using nation-wide epidemiological data. We included all people with confirmed mpox diagnosed between May and November 2022 in Mexico. Clinical and sociodemographic characteristics were compared between people with and without HIV. Multivariable logistic regression models were preformed to determine the association between HIV, clinical features, and outcomes and reported with odds ratios (ORs) and 95% confidence intervals (95% CI). ORs for rare outcomes were interpreted as risk ratios. RESULTS: Among 3291 people with mpox, 59% were PWLH. PWLH had an increased risk of severe mpox (OR 2.6, 2.4-2.9) and death (OR 10.8, 9.7-11.9). They also had a higher risk of otalgia, proctitis, and urethritis. Eleven individuals died, of whom ten were PWLH. All deaths were directly attributed to mpox. CONCLUSION: People with HIV have a higher risk of severe mpox and death due to mpox.

Synthesis and preclinical evaluation of [11C]uPSEM792 for PSAM4-GlyR based chemogenetics.

Chemogenetic tools are designed to control neuronal signaling. These tools have the potential to contribute to the understanding of neuropsychiatric disorders and to the development of new treatments. One such chemogenetic technology comprises modified Pharmacologically Selective Actuator Modules (PSAMs) paired with Pharmacologically Selective Effector Molecules (PSEMs). PSAMs are receptors with ligand-binding domains that have been modified to interact only with a specific small-molecule agonist, designated a PSEM. PSAM4 is a triple mutant PSAM derived from the α7 nicotinic receptor (α7L131G,Q139L,Y217F). Although having no constitutive activity as a ligand-gated ion channel, PSAM4 has been coupled to the serotonin 5-HT3 receptor (5-HT3R) and to the glycine receptor (GlyR). Treatment with the partner PSEM to activate PSAM4-5-HT3 or PSAM4-GlyR, causes neuronal activation or silencing, respectively. A suitably designed radioligand may enable selective visualization of the expression and location of PSAMs with positron emission tomography (PET). Here, we evaluated uPSEM792, an ultrapotent PSEM for PSAM4-GlyR, as a possible lead for PET radioligand development. We labeled uPSEM792 with the positron-emitter, carbon-11 (t1/2 = 20.4 min), in high radiochemical yield by treating a protected precursor with [11C]iodomethane followed by base deprotection. PET experiments with [11C]uPSEM792 in rodents and in a monkey transduced with PSAM4-GlyR showed low peak radioactivity uptake in brain. This low uptake was probably due to high polarity of the radioligand, as evidenced by physicochemical measurements, and to the vulnerability of the radioligand to efflux transport at the blood-brain barrier. These findings can inform the design of a more effective PSAM4 based PET radioligand, based on the uPSEM792 chemotype.

In vivo photopharmacology with light-activated opioid drugs.

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using "caged" opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo, we developed photoactivatable oxymorphone (PhOX) and photoactivatable naloxone (PhNX), photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry in response to chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action.

Predictors of laboratory-confirmed mpox in people with mpox-like illness.

OBJECTIVES: We aimed to identify predictors of confirmed monkeypox (mpox) among people with mpox-like illness and to develop a multivariable model for confirmed mpox. METHODS: We performed an observational study using national epidemiologic surveillance data in Mexico from May to November 2022. People with mpox-like illness were reported to the Mexican Ministry of Health and real-time polymerase chain reaction was performed in clinical samples to confirm mpox. Sociodemographic and clinical data were collected with a case report form. We performed univariable logistic regressions to estimate the predictive capability of individual characteristics, reported with ORs and 95% CIs. Variables of interest were included in multivariable logistic regression models and Akaike information criterion was used to retain variables for the final model. Discrimination and calibration of the model were estimated in bootstrap resamples. RESULTS: A total of 5078 people were reported with mpox-like illness. Of 5078 people, 3291 (64.8%) had confirmed mpox. The strongest clinical predictors of confirmed mpox in univariable models were proctitis (OR 6.54, 5.93-7.21), inguinal adenopathy (OR 5.91, 5.36-6.52), and anogenital lesions (OR 5.45, 4.94-6.02). The final model included being a man who has sex with men (8.75, 7.37-10.38), HIV diagnosis (3.04, 2.51-3.69), inguinal adenopathy (2.24, 1.81-2.77), anogenital lesions (2.32, 1.97-2.74), and pustules (1.55, 1.32-1.81). Discrimination capability was excellent (c-statistic 0.88, 95% CI 0.87-0.89) and it was well calibrated (calibration slope 1, 95% CI 0.95-1.05). DISCUSSION: A third of people with mpox-like illness do not have mpox. Factors such as being a man who has sex with men, HIV diagnosis, inguinal adenopathy, pustules, and anogenital lesions are associated with confirmed mpox.

Expression of the excitatory opsin ChRERα can be traced longitudinally in rat and nonhuman primate brains with PET imaging.

Optogenetics is a widely used technology with potential for translational research. A critical component of such applications is the ability to track the location of the transduced opsin in vivo. To address this problem, we engineered an excitatory opsin, ChRERα (hChR2(134R)-V5-ERα-LBD), that could be visualized using positron emission tomography (PET) imaging in a noninvasive, longitudinal, and quantitative manner. ChRERα consists of the prototypical excitatory opsin channelrhodopsin-2 (ChR2) and the ligand-binding domain (LBD) of the human estrogen receptor α (ERα). ChRERα showed conserved ChR2 functionality and high affinity for [18F]16α-fluoroestradiol (FES), an FDA-approved PET radiopharmaceutical. Experiments in rats demonstrated that adeno-associated virus (AAV)-mediated expression of ChRERα enables neural circuit manipulation in vivo and that ChRERα expression could be monitored using FES-PET imaging. In vivo experiments in nonhuman primates (NHPs) confirmed that ChRERα expression could be monitored at the site of AAV injection in the primary motor cortex and in long-range neuronal terminals for up to 80 weeks. The anatomical connectivity map of the primary motor cortex identified by FES-PET imaging of ChRERα expression overlapped with a functional connectivity map identified using resting state fMRI in a separate cohort of NHPs. Overall, our results demonstrate that ChRERα expression can be mapped longitudinally in the mammalian brain using FES-PET imaging and can be used for neural circuit modulation in vivo.

In vivo photopharmacology with light-activated opioid drugs.

Traditional methods for site-specific drug delivery in the brain are slow, invasive, and difficult to interface with recordings of neural activity. Here, we demonstrate the feasibility and experimental advantages of in vivo photopharmacology using "caged" opioid drugs that are activated in the brain with light after systemic administration in an inactive form. To enable bidirectional manipulations of endogenous opioid receptors in vivo , we developed PhOX and PhNX, photoactivatable variants of the mu opioid receptor agonist oxymorphone and the antagonist naloxone. Photoactivation of PhOX in multiple brain areas produced local changes in receptor occupancy, brain metabolic activity, neuronal calcium activity, neurochemical signaling, and multiple pain- and reward-related behaviors. Combining PhOX photoactivation with optical recording of extracellular dopamine revealed adaptations in the opioid sensitivity of mesolimbic dopamine circuitry during chronic morphine administration. This work establishes a general experimental framework for using in vivo photopharmacology to study the neural basis of drug action. Highlights: A photoactivatable opioid agonist (PhOX) and antagonist (PhNX) for in vivo photopharmacology. Systemic pro-drug delivery followed by local photoactivation in the brain. In vivo photopharmacology produces behavioral changes within seconds of photostimulation. In vivo photopharmacology enables all-optical pharmacology and physiology.

Mu Opioid Receptor Activation Mediates (S)-ketamine Reinforcement in Rats: Implications for Abuse Liability.

BACKGROUND: (S)-ketamine is an NMDA receptor antagonist, but it also binds to and activates mu opioid receptors (MORs) and kappa opioid receptors in vitro. However, the extent to which these receptors contribute to (S)-ketamine's in vivo pharmacology is unknown. METHODS: We investigated the extent to which (S)-ketamine interacts with opioid receptors in rats by combining in vitro and in vivo pharmacological approaches, in vivo molecular and functional imaging, and behavioral procedures relevant to human abuse liability. RESULTS: We found that the preferential opioid receptor antagonist naltrexone decreased (S)-ketamine self-administration and (S)-ketamine-induced activation of the nucleus accumbens, a key brain reward region. A single reinforcing dose of (S)-ketamine occupied brain MORs in vivo, and repeated doses decreased MOR density and activity and decreased heroin reinforcement without producing changes in NMDA receptor or kappa opioid receptor density. CONCLUSIONS: These results suggest that (S)-ketamine's abuse liability in humans is mediated in part by brain MORs.

Effect of Selective Lesions of Nucleus Accumbens µ-Opioid Receptor-Expressing Cells on Heroin Self-Administration in Male and Female Rats: A Study with Novel Oprm1-Cre Knock-in Rats.

The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to MOR-expressing cells. After performing anatomic and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the involvement of NAc MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and FISH chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum, and dorsal hippocampus, or MOR receptor density (except dorsal striatum) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly coexpressed with Oprm1 (95%-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells. We found that the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study indicates that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in male and female rats.SIGNIFICANCE STATEMENT The brain µ-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell type-specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show that lesioning NAc MOR-expressing cells had different effects on heroin self-administration in males and females. The new Oprm1-Cre rats can be used to study the role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

6-O-(2-[18F]Fluoroethyl)-6-O-Desmethyl-Diprenorphine ([18F]FE-DPN) Preferentially Binds to Mu Opioid Receptors In Vivo.

PURPOSE: 6-O-(2-[18F]Fluoroethyl)-6-O-desmethyl-diprenorphine ([18F]FE-DPN) is regarded as a non-selective opioid receptor radiotracer. PROCEDURE: Here, we report the first characterization of [18F]FE-DPN synthesized from the novel precursor, 6-O-(2-tosyloxyethoxy)-6-O-desmethyl-3-O-trityl-diprenorphine (TE-TDDPN), using a one-pot, two-step nucleophilic radiosynthesis to image opioid receptors in rats and mice using positron emission tomography. RESULTS: We also show that [18F]FE-DPN and [3H]DPN exhibit negligible brain uptake in mu opioid receptor (MOR) knockout mice. CONCLUSIONS: Taken together with prior findings, our results suggest that [18F]FE-DPN and [3H]DPN preferentially bind to MOR in rodents in vivo.

Fluoroscopy-Guided Percutaneous Transpedicular Biopsy Versus Posterolateral Endoscopy for Infective Spondylodiskitis Diagnosis: A Comparative Study.

OBJECTIVE: We sought to determine the concordance in frequency of microbiologic isolation and species identification in specimens obtained by 2 methods. METHODS: Intervertebral disk specimens were taken simultaneously from each patient using percutaneous needle and posterolateral endoscopic biopsies. The isolates were reported in frequencies and concordance using the chi square and Cohen kappa tests. RESULTS: Thirty patients were recruited. The average age was 58.1 years, and 15 patients were women. The clinical evolution time was 7 ± 4 months. The causative organism was identified in 12 (40%) specimens obtained by fluoroscopy-guided percutaneous transpedicular biopsy and in 14 (46.6%) obtained by posterolateral endoscopy. The most common organism isolated was Staphylococcus aureus in 3 patients with the percutaneous technique and in 5 with the endoscopic one; Escherichia coli was isolated in 3 patients with each method. The kappa test showed a high degree of agreement between both methods (kappa = 0.86); the agreement in bacterial species identification was 100%. CONCLUSIONS: Fluoroscopy-guided percutaneous biopsy and endoscopic sampling have a good degree of concordance for both, frequency of organism isolation and identification in patients with infectious spondylodiskitis.

Essential role of P-glycoprotein in the mechanism of action of oliceridine.

Mu opioid receptor (MOR) agonists comprise the most effective analgesics, but their therapeutic utility is limited by adverse effects. One approach for limiting such effects has been to develop "biased" MOR agonists that show preference for activating G protein over ß-Arrestin signaling. However, the notion of biased agonism has been challenged by recent studies. Oliceridine (Olinvyk®, TRV-130, OLC) is a selective MOR agonist approved by the FDA in 2020 for pain management in controlled clinical settings. Oliceridine purportedly demonstrates diminished adverse effects compared to morphine or other MOR agonists, a profile attributed to its biased agonism. However, recent studies suggest that oliceridine does not display biased agonism but instead weak intrinsic efficacy for G protein and ß-Arrestin activation. Nevertheless, these insights have been derived from in vitro studies. To better understand oliceridine's in vivo efficacy profile, we performed a comprehensive assessment of its in vitro and in vivo pharmacology using both cultured cells and rodents. In vitro, oliceridine displayed high MOR affinity and weak intrinsic efficacy. In vivo, oliceridine showed impaired brain penetrance and rapid clearance, effects we attributed to its interaction with the P-glycoprotein (P-gp) efflux transporter. Moreover, we found that P-gp was essential for oliceridine's in vivo efficacy and adverse effect profiles. Taken together with prior studies, our results suggest that oliceridine's in vivo efficacy and adverse effect profiles are not attributed solely to its weak intrinsic efficacy or biased agonism but, to a large extent, its interaction with P-gp as well.

Adolescent nicotine administration increases nicotinic acetylcholine receptor binding and functional connectivity in specific cortico-striatal-thalamic circuits.

Nicotine exposure is associated with regional changes in brain nicotinic acetylcholine receptors subtype expression patterns as a function of dose and age at the time of exposure. Moreover, nicotine dependence is associated with changes in brain circuit functional connectivity, but the relationship between such connectivity and concomitant regional distribution changes in nicotinic acetylcholine receptor subtypes following nicotine exposure is not understood. Although smoking typically begins in adolescence, developmental changes in brain circuits and nicotinic acetylcholine receptors following chronic nicotine exposure remain minimally investigated. Here, we combined in vitro nicotinic acetylcholine receptor autoradiography with resting state functional magnetic resonance imaging to measure changes in [3H]nicotine binding and α4ß2 subtype nicotinic acetylcholine receptor binding and circuit connectivity across the brain in adolescent (postnatal Day 33) and adult (postnatal Day 68) rats exposed to 6 weeks of nicotine administration (0, 1.2 and 4.8 mg/kg/day). Chronic nicotine exposure increased nicotinic acetylcholine receptor levels and induced discrete, developmental stage changes in regional nicotinic acetylcholine receptor subtype distribution. These effects were most pronounced in striatal, thalamic and cortical regions when nicotine was administered during adolescence but not in adults. Using these regional receptor changes as seeds, resting state functional magnetic resonance imaging identified dysregulations in cortico-striatal-thalamic-cortical circuits that were also dysregulated following adolescent nicotine exposure. Thus, nicotine-induced increases in cortical, striatal and thalamic nicotinic acetylcholine receptors during adolescence modifies processing and brain circuits within cortico-striatal-thalamic-cortical loops, which are known to be crucial for multisensory integration, action selection and motor output, and may alter the developmental trajectory of the adolescent brain. This unique multimodal study significantly advances our understanding of nicotine dependence and its effects on the adolescent brain.

A tool for monitoring cell type-specific focused ultrasound neuromodulation and control of chronic epilepsy.

Focused ultrasound (FUS) is a powerful tool for noninvasive modulation of deep brain activity with promising therapeutic potential for refractory epilepsy; however, tools for examining FUS effects on specific cell types within the deep brain do not yet exist. Consequently, how cell types within heterogeneous networks can be modulated and whether parameters can be identified to bias these networks in the context of complex behaviors remains unknown. To address this, we developed a fiber Photometry Coupled focused Ultrasound System (PhoCUS) for simultaneously monitoring FUS effects on neural activity of subcortical genetically targeted cell types in freely behaving animals. We identified a parameter set that selectively increases activity of parvalbumin interneurons while suppressing excitatory neurons in the hippocampus. A net inhibitory effect localized to the hippocampus was further confirmed through whole brain metabolic imaging. Finally, these inhibitory selective parameters achieved significant spike suppression in the kainate model of chronic temporal lobe epilepsy, opening the door for future noninvasive therapies.

Effective Treatment of Patients Experiencing Primary, Acute HIV Infection Decreases Exhausted/Activated CD4+ T Cells and CD8+ T Memory Stem Cells.

Several studies have identified main changes in T- and B-lymphocyte subsets during chronic HIV infection, but few data exist on how these subsets behave during the initial phase of HIV infection. We enrolled 22 HIV-infected patients during the acute stage of infection before the initiation of antiretroviral therapy (ART). Patients had blood samples drawn previous to ART initiation (T0), and at 2 (T1) and 12 (T2) months after ART initiation. We quantified cellular HIV-DNA content in sorted naïve and effector memory CD4 T cells and identified the main subsets of T- and B-lymphocytes using an 18-parameter flow cytometry panel. We identified correlations between the patients' clinical and immunological data using PCA. Effective HIV treatment reduces integrated HIV DNA in effector memory T cells after 12 months (T2) of ART. The main changes in CD4+ T cells occurred at T2, with a reduction of activated memory, cytolytic and activated/exhausted stem cell memory T (TSCM) cells. Changes were present among CD8+ T cells since T1, with a reduction of several activated subsets, including activated/exhausted TSCM. At T2 a reduction of plasmablasts and exhausted B cells was also observed. A negative correlation was found between the total CD4+ T-cell count and IgM-negative plasmablasts. In patients initiating ART immediately following acute/early HIV infection, the fine analysis of T- and B-cell subsets has allowed us to identify and follow main modifications due to effective treatment, and to identify significant changes in CD4+ and CD8+ T memory stem cells.

Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search.

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

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.