Heterozygote Dopamine Transporter Knockout Rats Display Enhanced Cocaine Locomotion in Adolescent Females.

Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine's reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.

3-Aryl-3-arylmethoxyazetidines. A new class of high affinity ligands for monoamine transporters.

A series of 3-aryl-3-arylmethoxy-azetidines were synthesized and evaluated for binding affinities at dopamine and serotonin transporters. The 3-aryl-3-arylmethoxyazetidines were generally SERT selective with the dichloro substituted congener 7c (Ki=1.0 nM) and the tetrachloro substituted derivative 7i (Ki=1.3 nM) possessing low nanomolar affinity for the SERT. The 3-(3,4-dichlorophenyl-3-phenylmethoxyazetidine (7g) exhibited moderate affinity at both DAT and SERT transporters and suggests that substitution of the aryl rings can be used to tune the mononamine transporter affinity.

Dopamine Transporter Knockout Rats Display Epigenetic Alterations in Response to Cocaine Exposure.

(1) Background: There is an urgent need for effective treatments for cocaine use disorder (CUD), and new pharmacological approaches targeting epigenetic mechanisms appear to be promising options for the treatment of this disease. Dopamine Transporter (DAT) transgenic rats recently have been proposed as a new animal model for studying susceptibility to CUD. (2) Methods: DAT transgenic rats were treated chronically with cocaine (10 mg/kg) for 8 days, and the expression of epigenetic modulators, Lysine Demethylase 6B (KDM6B) and Bromodomain-containing protein 4 (BRD4), was examined in the prefrontal cortex (PFC). (3) Results: We show that only full knockout (KO) of DAT impacts basal levels of KDM6B in females. Additionally, cocaine altered the expression of both epigenetic markers in a sex- and genotype-dependent manner. In response to chronic cocaine, KDM6B expression was decreased in male rats with partial DAT mutation (HET), while no changes were observed in wild-type (WT) or KO rats. Indeed, while HET male rats have reduced KDM6B and BRD4 expression, HET female rats showed increased KDM6B and BRD4 expression levels, highlighting the impact of sex on epigenetic mechanisms in response to cocaine. Finally, both male and female KO rats showed increased expression of BRD4, but only KO females exhibited significantly increased KDM6B expression in response to cocaine. Additionally, the magnitude of these effects was bigger in females when compared to males for both epigenetic enzymes. (4) Conclusions: This preliminary study provides additional support that targeting KDM6B and/or BRD4 may potentially be therapeutic in treating addiction-related behaviors in a sex-dependent manner.

Further structure-activity relationship studies on 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives at monoamine transporters.

The synthesis and structure-activity relationships of 8-substituted-3-[2-(diarylmethoxyethylidenyl)]-8-azabicyclo[3.2.1]octane derivatives were investigated at the dopamine transporter (DAT), the serotonin transporter (SERT) and norepinephrine transporter (NET). The rigid ethylidenyl-8-azabicyclic[3.2.1]octane skeleton imparted modestly stereoselective binding and uptake inhibition at the DAT. Additional structure-activity studies provided a transporter affinity profile that was reminiscent of the structure-activity of GBR 12909. From these studies, the 8-cyclopropylmethyl group has been identified as a unique moiety that imparts high SERT/DAT selectivity. In this study the 8-cyclopropylmethyl derivative 22e (DAT K(i) of 4.0 nM) was among the most potent compounds of the series at the DAT and was the most DAT selective ligand of the series (SERT/DAT: 1060). Similarly, the 8-chlorobenzyl derivative 22g (DAT K(i) of 3.9 nM) was found to be highly selective for the DAT over the NET (NET/DAT: 1358).

Viral vector-mediated gene therapy for opioid use disorders.

Chronic exposure to opioids typically results in adverse consequences. Opioid use disorder (OUD) is a disease of the CNS with behavioral, psychological, neurobiological, and medical manifestations. OUD induces a variety of changes of neurotransmitters/neuropeptides in the nervous system. Existing pharmacotherapy, such as opioid maintenance therapy (OMT) is the mainstay for the treatment of OUD, however, current opioid replacement therapy is far from effective for the majority of patients. Pharmacological therapy for OUD has been challenging for many reasons including debilitating side-effects. Therefore, developing an effective, non-pharmacological approach would be a critical advancement in improving and expanding treatment for OUD. Viral vector mediated gene therapy provides a potential new approach for treating opioid abused patients. Gene therapy can supply targeting gene products directly linked to the mechanisms of OUD to restore neurotransmitter and/or neuropeptides imbalance, and avoid the off-target effects of systemic administration of drugs. The most commonly used viral vectors in rodent studies of treatment of opioid-used disorder are based on recombinant adenovirus (AV), adeno-associated virus (AAV), lentiviral (LV) vectors, and herpes simplex virus (HSV) vectors. In this review, we will focus on the recent progress of viral vector mediated gene therapy in OUD, especially morphine tolerance and withdrawal.

N-Substituted-3-alkoxy-derivatives of dextromethorphan are functional NMDA receptor antagonists in vivo: Evidence from an NMDA-induced seizure model in rats.

Interest in developing NMDA receptor antagonists with reduced side-effects for neurological and psychiatric disorders has been re-energized by the recent introduction of esketamine into clinical practice for treatment-resistant depression. Structural analogs of dextromethorphan bind with low affinity to the NMDA receptor ion channel, have functional effects in vivo, and generally display a lower propensity for side-effects than that of ketamine and other higher affinity antagonists. As such, the aim of the present study was to determine whether a series of N-substituted-3-alkoxy-substituted dextromethorphan analogs produce their anticonvulsant effects through NMDA receptor blockade. Compounds were studied against NMDA-induced seizures in rats. Compounds were administered intracerebroventricularly in order to mitigate confounds of drug metabolism that arise from systemic administration. Comparison of the anticonvulsant potencies to their affinities for NMDA, σ1, and σ2 binding sites were made in order to evaluate the contribution of these receptors to anticonvulsant efficacy. The potencies to block convulsions were positively associated with their affinities to bind to the NMDA receptor ion channel ([3H]-TCP binding) (r = 0.71, p < 0.05) but not to σ1 receptors ([3H]-SKF 10047 binding) (r = -0.31, p = 0.46) or to σ2 receptors ([3H]-DTG binding) (p = -0.38, p = 0.36). This is the first report demonstrating that these dextromethorphan analogs are functional NMDA receptor antagonists in vivo. Given their potential therapeutic utility and favorable side-effect profiles, such low affinity NMDA receptor antagonists could be considered for further development in neurological (e.g., anticonvulsant) and psychiatric (e.g., antidepressant) disorders.

Synthesis and structure-activity studies of benzyl ester meperidine and normeperidine derivatives as selective serotonin transporter ligands.

A series of benzyl esters of meperidine and normeperidine were synthesized and evaluated for binding affinity at serotonin, dopamine and norepinephrine transporters. The 4-methoxybenzyl ester 8b and 4-nitrobenzyl ester 8c in the meperidine series and 4-methoxybenzyl ester 14a in the normeperidine series exhibited low nanomolar binding affinities at the SERT (K(i) values <2nM) and high SERT selectivity (DAT/SERT >1500 and NET/SERT >1500).

Vaccination against cocaine using a modifiable dendrimer nanoparticle platform.

Pharmacological therapies for the treatment of cocaine addiction have had disappointing efficacy, and the lack of recent developments in the clinical care of cocaine-addicted patients indicates a need for novel treatment strategies. Recent studies have shown that vaccination against cocaine to elicit production of antibodies that reduce concentrations of free drug in the blood is a promising method to protect against the effects of cocaine and reduce rates of relapse. However, the poorly immunogenic nature of cocaine remains a major hurdle to active immunization. Therefore, we hypothesized that strategies to increase targeted exposure of cocaine to the immune system may produce a more effective vaccine. To specifically direct an immune response against cocaine, in the present study we have conjugated a cocaine analog to a dendrimer-based nanoparticle carrier with MHC II-binding moieties that previously has been shown to activate antigen-presenting cells necessary for antibody production. This strategy produced a rapid, prolonged, and high affinity anti-cocaine antibody response without the need for an adjuvant. Surprisingly, additional evaluation using multiple adjuvant formulations in two strains of inbred mice found adjuvants were either functionally redundant or deleterious in the vaccination against cocaine using this platform. The use of conditioned place preference in rats after administration of this vaccine provided proof of concept for the ability of this vaccine to diminish cocaine reward. Together these data demonstrate the intrinsic efficacy of an immune-targeting dendrimer-based cocaine vaccine, with a vast potential for design of future vaccines against other poorly immunogenic antigens by substitution of the conjugated cargo.

Synthesis and CB1 cannabinoid receptor affinity of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles.

A series of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles were synthesized regioselectively using click chemistry and evaluated at CB1 cannabinoid receptors. The n-propyl ester 11 (K(i)=4.6 nM) and phenyl ester 14 (K(i)=11 nM) exhibited the most potent affinity of the series.

Synthesis and monoamine transporter affinity of 3alpha-arylmethoxy-3beta-arylnortropanes.

A series of 3-arylnortrop-2-enes and 3alpha-arylmethoxy-3beta-arylnortropanes were synthesized and evaluated for binding affinity at monoamine transporters. The 3-(3,4-dichlorophenyl)nortrop-2-ene (6e) exhibited high affinity for the SERT (K(i)=0.3 nM). The 3alpha-arylmethoxy-3beta-arylnortropanes were generally SERT selective with the 3alpha-(3.4-dichlorophenylmethoxy)-3betaphenylnortrop-2-ene (7c) possessing subnanomolar potency (K(i)=0.061 nM). However, 3alpha-(3,4-dichlorophenylmethoxy)-3beta-phenylnortrop-2-ene (7b) exhibited high affinity at all three transporters [(DAT K(i)=22 nM), (SERT K(i)=6 nM) and (NET K(i)=101 nM)].

Sensitivity to cocaine conditioned reward depends on sex and age.

Human and animal laboratory studies show that females and males respond differently to drugs and that drug administration during adolescence leads to different behavioral effects than during adulthood. Adult female rats are more sensitive to the behavioral effects of cocaine than adult males, but it is not known if the same effect of sex exists during adolescence. In the present study, sensitivity to the conditioned reward of cocaine was evaluated using a conditioned place preference (CPP) paradigm where adolescent (PND 34) and adult (PND 66) male and female rats were trained and tested for the development of CPP to multiple doses of cocaine. Female rats developed CPP at lower doses than males, regardless of age. In addition, adolescent male and female rats established a CPP at lower doses of cocaine than adult male and female rats, respectively. Thus, both age and sex altered cocaine conditioned reward with the order of sensitivity being adolescent females > adult females > adolescent males > adult males. These data show that adolescents are more sensitive to the conditioned rewarding properties of cocaine than adults and that females respond to lower doses of cocaine compared to males regardless of age.

Depletion of serotonin decreases the effects of the kappa-opioid receptor agonist U-69593 on cocaine-stimulated activity.

Treatment with a kappa-opioid receptor agonist for 5 days decreases locomotor activity and reduces activity in response to a cocaine challenge 3 days later. In addition, chronic cocaine increases kappa-opioid receptor density, striatal dynorphin, and dynorphin gene expression in the striatum. The upregulation of kappa-opioid receptors after cocaine treatment occurs predominantly in brain regions that are highly innervated by serotonin. To determine if serotonin plays a role in the effects of kappa-opioid receptor agonists on cocaine-stimulated activity, parachloroamphetamine (PCA), which depleted serotonin by 53%-66%, or saline, was given prior to a five-day treatment with U-69593 or vehicle. Three days later each rat received a single injection of cocaine and locomotor activity was measured. Treatment with PCA had no effect on the ability of U-69593 alone to decrease locomotor activity. Thus, the behavioral effects of U-69593 alone were not dependent upon serotonin. In rats pretreated with saline, U-69593 treatment significantly blocked the locomotor-activating effects of cocaine. Following PCA pretreatment, however, there were no significant differences in locomotor activity in rats challenged with an injection of cocaine after treatment with U-69593 or vehicle. Thus, serotonin depletion prevented the long-lasting blockade of the locomotor-activating effects of cocaine subsequent to repeated administration of U-69593 but did not alter the effects of cocaine in rats that were treated with vehicle. Thus, the effects of PCA on U-69593 are not due to non-specific alterations in cocaine-induced locomotor activity. These findings suggest that serotonin plays an important role in mediating the effects of kappa-opioid receptor agonists on the behavioral response to cocaine.

Chronic cocaine produces decreases in N/OFQ peptide levels in select rat brain regions.

The interaction of opioids and stimulants is well established; however, the mechanisms that underlie the role that opioid receptors play in psychostimulant action are not. Nociceptin/orphaninFQ (N/OFQ), the endogenous agonist at NOP receptors, attenuates the behavioral effects of cocaine. The effects of cocaine on N/OFQ were examined in rats using immunoautoradiographic and RIA techniques. Chronic administration of cocaine decreased N/OFQ in medial regions of the caudate putamen, the nucleus accumbens shell, and the substantia nigra. These studies show that N/OFQ levels are altered by treatment with cocaine. Furthermore, the changes in N/OFQ parallel those seen for kappa-opioid receptors, suggesting that the interactions between cocaine and these systems might be similar.

Effect of MDMA (ecstasy) on activity and cocaine conditioned place preference in adult and adolescent rats.

MDMA (ecstasy) is a drug commonly used in adolescence, and many users of MDMA also use other illicit drugs. It is not known whether MDMA during adolescence alters subsequent responses to cocaine differently than in adults. This study examined the effects of MDMA in adolescent and adult rats on cocaine conditioned reward. At the start of these experiments, adolescent rats were at postnatal day (PND) 33 and adult rats at PND 60. Each rat was treated for 7 days with MDMA (2 or 5 mg/kg/day or vehicle) and locomotor activity was measured. Five days later cocaine conditioned place preference (CPP) was begun. Rats were trained for 3 days, in the morning with saline and in the afternoon with 10 mg/kg cocaine in 30 min sessions, and tested on the fourth day. MDMA stimulated activity in both age groups, but with a greater effect in the adult rats. Sensitization to the locomotor-stimulant effects of the lower dose of MDMA occurred in adult rats and in both groups to the higher dose. Cocaine did not produce a CPP in vehicle-treated adolescent rats, but a significant CPP was observed subsequent to treatment with MDMA. In contrast, cocaine-induced CPP was diminished after MDMA in adult rats. These effects were still evident 2 weeks later upon retest. Thus, under the present conditions, MDMA increased cocaine conditioned reward in adolescent and decreased it in adult rats. These findings suggest that exposure to MDMA during this critical developmental period may carry a greater risk than during adulthood and that male adolescents may be particularly vulnerable to the risk of stimulant abuse after use of MDMA.

Nicotine produces long-term increases in cocaine reinforcement in adolescent but not adult rats.

Studies have shown that many smokers begin using nicotine during adolescence, yet the influence of early nicotine use on the response to other drugs of abuse in adulthood is not fully understood. In the current study, nicotine was administered to adolescent and adult rats for seven days. Thirty days later, cocaine-induced locomotor activity and cocaine self-administration were examined when the rats pretreated as adolescents were adults. Rats exposed to nicotine during early adolescence were sensitized thirty days later to the locomotor-activating effects of cocaine and self-administered a greater number of cocaine infusions than adolescent rats pretreated with vehicle. As a result of this increased intake, the cocaine self-administration dose-response curve was shifted upward indicating an increase in cocaine reinforcement. Rats pretreated with nicotine as adults, however, did not show a difference in locomotor activity or cocaine self-administration thirty days later compared to adult rats pretreated with vehicle. These findings suggest that early exposure to nicotine has long-term consequences on cocaine use. These data further suggest that nicotine use may carry a greater risk during adolescence than adulthood and adolescents who smoke may be particularly vulnerable to stimulant use. This article is part of a Special Issue entitled SI: Adolescent plasticity.

Sexually-dimorphic alterations in cannabinoid receptor density depend upon prenatal/early postnatal history.

Recent research has demonstrated that the endogenous cannabinoid system is central to the brain's response to stress. As part of an ongoing collaboration, we sought to examine the effects of prenatal and early postnatal rearing and housing conditions on developing endocannabinoid systems. We compare brain cannabinoid receptors (CBR) in offspring of either prenatal vehicle intubated or non-treated dams (Experiment 1) or in rats derived from a vendor and shipped at weaning to a collaborating lab (Experiment 2). From postnatal day (PND) 23, all rats were either housed in isolated conditions or enriched conditions with 3 rats/cage and a variety of stimulus objects changed twice a week. All rats underwent 5days of handling as controls for a behavior study and all rats were sacrificed at approximately PND48-50 within 2hours of the last behavioral test. All brains were processed together for CB1 receptor binding using 3H CP55,940 in prefrontal cortex, striatum, amygdala and hippocampus. Conditions in the two labs were as similar as possible since the two studies were intentionally designed to be comparable and contemporary. Results show that 1) comparing offspring of non-treated dams to offspring of dams receiving daily vehicle intubations, males show decreased CB1 binding in most brain regions while females only showed alterations in the hippocampus and these were increases in the offspring of the vehicle-intubated dams. 2) When comparing offspring of non-treated dams in NY with those derived from a vendor, shipped and maintained in the collaborating lab, this latter group showed reduced CB1 binding in prefrontal cortex in males and increased binding in all four brain regions in females. Therefore, overall, both prenatal handling (intubations) and being vendor-derived, shipped and maintained in a collaborating facility reduced CB1 receptors in males and increased them in females in key limbic brain regions. Effects of environmental enrichment or isolation were minor with only the prefrontal cortex showing an increase in binding in the isolated animals that were offspring of the vehicle-intubated dams. These results support the ideas that prenatal/early postnatal conditions produce different effects in males and females and override the effects of enrichment/isolation on cannabinoid receptors. Behavioral responses to cannabinoid challenges would therefore be expected to vary depending on sex, prenatal/early postnatal history and postweaning conditions of the rats. Since exogenous cannabinoids act through the CBR, the present data may provide a molecular basis for discrepant behavioral effects reported across various labs in the literature as well as sex differences seen following stress and/or manipulation of the cannabinoid system.

Cocaine abusers have an overexpression of alpha-synuclein in dopamine neurons.

Alpha-synuclein is a presynaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease. The native protein is a major component of nigral Lewy bodies in Parkinson's disease, and full-length alpha-synuclein accumulates in Lewy neurites. Here we present evidence that alpha-synuclein levels are elevated in midbrain dopamine (DA) neurons of chronic cocaine abusers. Western blot and immunoautoradiographic studies were conducted on postmortem neuropathological specimens from cocaine users and age-matched drug-free control subjects. The results demonstrated that alpha-synuclein levels in the DA cell groups of the substantia nigra/ventral tegmental complex were elevated threefold in chronic cocaine users compared with normal age-matched subjects. The increased protein levels in chronic cocaine users were accompanied by changes in the expression of alpha-synuclein mRNA in the substantia nigra and ventral tegmental area. Although alpha-synuclein expression is prominent in the hippocampus, there was no increase in protein expression in this brain region. The levels of beta-synuclein, a possible negative regulator of alpha-synuclein, also were not affected by cocaine exposure. Alpha-synuclein protein levels were increased in the ventral tegmental area, but not the substantia nigra, in victims of excited cocaine delirium who experienced paranoia, marked agitation, and hyperthermia before death. The overexpression of alpha-synuclein may occur as a protective response to changes in DA turnover and increased oxidative stress resulting from cocaine abuse. However, the accumulation of alpha-synuclein protein with long-term cocaine abuse may put addicts at increased risk for developing the motor abnormalities of Parkinson's disease.

Synthesis and biological evaluation of meperidine analogues at monoamine transporters.

A series of aryl-substituted meperidine analogues was synthesized, and the binding affinities were determined at the DAT, SERT, and NET as well as at mu-opioid receptors. Generally the analogues exhibited increased affinity for the DAT and SERT relative to meperidine but exhibited low binding affinity for the NET. The 2-naphthyl derivative 7f was the most potent ligand at the SERT (K(i) = 0.0072 muM) and was the most selective ligand for the SERT over the DAT (DAT/SERT = 158) and mu-opioid receptors (mu/SERT = 281). The 3,4-dichlorophenyl derivative 7e was the most potent ligand at the DAT (K(i) = 0.125 muM) and was the most selective ligand for the DAT over mu-opioid receptors (mu/DAT = 16.3) but remained slightly more selective for the SERT over the DAT(DAT/SERT = 6.68). Three compounds, the 3,4-dichlorophenyl derivative 7e and the 2-naphthyl analogues 6f and 7f, were identified that were more potent at the DAT than meperidine and that exhibited well-defined biphasic dopamine uptake inhibition similar to meperidine. However, none of the analogues tested produced locomotor effects or substituted for cocaine in drug discrimination studies, suggesting that the mu-opioid effects of these analogues may contribute to the poor efficacy observed in vivo.

Differential effects of psychoactive drugs in adolescents and adults.

It is well known that most people who use psychoactive drugs started as teenagers. In spite of this, there has been little preclinical research on the effects of psychostimulants during adolescence. Recently, however, a number of laboratories have begun to focus on drug effects in adolescents as compared with adults. The data show that there are unique responses to drugs during this period of development. This review will focus on our current understanding of neurochemical and behavioral drug effects during adolescence.