Overestimation of sex differences in psychostimulant activity via comparisons of males and females from different behavioral groups.

Background: There are inconsistencies in the observation of sex differences in baseline activity and psychostimulant activity. To address this, we have developed the MISSING (Mapping Intrinsic Sex Similarities as an Integral quality of Normalized Groups) model. MISSING model proposes that sex similarities are observed when we compare similar behavioral groups of males and females, with sex differences occurring when we compare distinct groups of sexes, but this model has not been tested. Methods: To test this model, we identified within-sex groups of Sprague Dawley rats (male n = 22, female n = 23) by conducted normal mixtures clustering of baseline activity, cocaine activity (as distance traveled in cm over 90 min) and cocaine activity normalized-to-baseline activity (NBA) of all subjects. We employed 2-way ANOVA to determine the impact of within-sex heterogeneity on sex differences. We compared our cluster-based method to current median-split approaches. Results: Our new cluster-based method revealed three distinct clusters, each consisting of both males and females. We determined there were no sex differences in any of the variables when males and females from the same clusters were compared. The within-sex clusters for females were not defined by estrous phase. Median split analysis was ineffective in accurately identifying within-sex groups. Conclusions: Our results validate the MISSING model: there are no sex differences in psychostimulant activity except when we compare males and females from different behavioral groups. This has significant implications for how we proceed with research towards understanding the mechanism governing sex differences in psychostimulant activity.

Chemogenetic Inhibition of Dopamine D1-expressing Neurons in the Dorsal Striatum does not alter Methamphetamine Intake in either Male or Female Long Evans Rats.

The biochemical and molecular substrates of methamphetamine (METH) use disorder remain to be elucidated. In rodents, increased METH intake is associated with increased expression of dopamine D1 receptors (D1R) in the dorsal striatum. The present study assessed potential effects of inhibiting striatal D1R activity on METH self-administration (SA) by rats. We microinjected Cre-activated adeno-associated viruses to deliver the inhibitory DREADD construct, hM4D (Gi) - mCherry, into neurons that expressed Cre-recombinase (D1-expressing neurons) in the dorsal striatum of male and female transgenic Long Evans rats (Drd1a-iCre#3). Two weeks later, we trained rats to self-administer METH. Once this behavior was acquired, intraperitoneal injections of clozapine-N-Oxide (CNO) or its vehicle (sterile water) were given to rats before each METH SA session to determine the effect of DREADD-mediated inhibition on METH intake. After the end of the experiments, histology was performed to confirm DREADD delivery into the dorsal striatum. There were no significant effects of the inhibitory DREADD on METH SA by male or female rats. Post-mortem histological assessment revealed DREADD expression in the dorsal striatum. Our results suggest that inhibition of D1R in the dorsal striatum does not suppress METH SA. It remains to be determined if activating D1R-expressing neurons might have differential behavioral effects. Future studies will also assess if impacting D1R activity in other brain regions might influence METH SA.

A behavioral economic analysis of the effects of rimcazole on reinforcing effects of cocaine injection and food presentation in rats.

RATIONALE AND OBJECTIVES: Rimcazole, a σ-receptor antagonist with affinity for the dopamine transporter (DAT), decreases rates of cocaine self-administration at doses lower than those that affect food-reinforced responding. As response rates are multiply determined, behavioral-economic analyses were used to provide measures of the reinforcing effectiveness of cocaine and food after rimcazole treatment. Further, effects of combinations of the DAT inhibitor, methylphenidate, and σ-receptor antagonists (BD1008, BD1063) were compared to those of rimcazole to assess mechanism of rimcazole effects. METHODS: Male Sprague-Dawley rats were trained to lever press with food reinforcement (one or three 20-mg sucrose pellets) or cocaine injection (0.1 or 0.32 mg/kg) under fixed-ratio (FR) 5-response schedules. Drugs or vehicle were administered (i.p.) 5-min before sessions in which FR value was increased from 5 to 80. Economic demand functions were generated from effects of FR value (price) on intake (consumption), with the parameters of demand, consumption at no cost (Q0) and sensitivity to price (essential value, EV), derived. RESULTS: Rimcazole dose-dependently decreased Q0 and EV at both cocaine doses/injection. In contrast, rimcazole had no effect on these parameters at either food amount. Combinations of methylphenidate and the σ-receptor antagonists decreased Q0 at the lower cocaine dose/injection but had no effect on EV; these treatments were ineffective on both economic parameters at the higher cocaine dose/injection and at either food amount. CONCLUSIONS: Though the drug combinations only replicated rimcazole's effects incompletely, the present results suggest a specific decrease in the reinforcing effects of cocaine due to dual DAT σ-receptor blockade.

Molecular Adaptations in the Rat Dorsal Striatum and Hippocampus Following Abstinence-Induced Incubation of Drug Seeking After Escalated Oxycodone Self-Administration.

Repeated exposure to the opioid agonist, oxycodone, can lead to addiction. Here, we sought to identify potential neurobiological consequences of withdrawal from escalated and non-escalated oxycodone self-administration in rats. To reach these goals, we used short-access (ShA) (3 h) and long-access (LgA) (9 h) exposure to oxycodone self-administration followed by protracted forced abstinence. After 31 days of withdrawal, we quantified mRNA and protein levels of opioid receptors in the rat dorsal striatum and hippocampus. Rats in the LgA, but not the ShA, group exhibited escalation of oxycodone SA, with distinction of two behavioral phenotypes of relatively lower (LgA-L) and higher (LgA-H) oxycodone takers. Both LgA, but not ShA, phenotypes showed time-dependent increases in oxycodone seeking during the 31 days of forced abstinence. Rats from both LgA-L and LgA-H groups also exhibited decreased levels of striatal mu opioid receptor protein levels in comparison to saline and ShA rats. In contrast, mu opioid receptor mRNA expression was increased in the dorsal striatum of LgA-H rats. Moreover, hippocampal mu and kappa receptor protein levels were both increased in the LgA-H phenotype. Nevertheless, hippocampal mu receptor mRNA levels were decreased in the two LgA groups whereas kappa receptor mRNA expression was decreased in ShA and LgA oxycodone groups. Decreases in striatal mu opioid receptor protein expression in the LgA rats may serve as substrates for relapse to drug seeking because these changes occur in rats that showed incubation of oxycodone seeking.

CART peptide in the nucleus accumbens regulates psychostimulants: Correlations between psychostimulant and CART peptide effects.

In this study, we reexamined the effect of Cocaine-and-Amphetamine-Regulated-Transcript (CART) peptide on psychostimulant (PS)-induced locomotor activity (LMA) in individual rats. The Methods utilized were as previously published. The PS-induced LMA was defined as the distance traveled after PS administration (intraperitoneal), and the CART peptide effect was defined as the change in the PS-induced activity after bilateral intra-NAc administration of CART peptide. The experiments included both male and female Sprague-Dawley rats, and varying the CART peptide dose and the PS dose. While the average effect of CART peptide was to inhibit PS-induced LMA, the effect of CART peptide on individual PS-treated animals was not always inhibitory and sometimes even produced an increase or no change in PS-induced LMA. Upon further analysis, we observed a linear correlation, reported for the first time, between the magnitude of PS-induced LMA and the CART peptide effect. Because CART peptide inhibits PS-induced LMA when it is large, and increases PS-induced LMA when it is small, the peptide can be considered a homeostatic regulator of dopamine-induced LMA, which supports our earlier homeostatic hypothesis.

A Role for Sigma Receptors in Stimulant Self-Administration and Addiction.

Sigma receptors (σRs) are structurally unique proteins that function intracellularly as chaperones. Historically, σRs have been implicated as modulators of psychomotor stimulant effects and have at times been proposed as potential avenues for modifying stimulant abuse. However, the influence of ligands for σRs on the effects of stimulants, such as cocaine or methamphetamine, in various preclinical procedures related to drug abuse has been varied. The present paper reviews the effects of σR agonists and antagonists in three particularly relevant procedures: stimulant discrimination, place conditioning, and self-administration. The literature to date suggests limited σR involvement in the discriminative-stimulus effects of psychomotor stimulants, either with σR agonists substituting for the stimulant or with σR antagonists blocking stimulant effects. In contrast, studies of place conditioning suggest that administration of σR antagonists or down-regulation of σR protein can block the place conditioning induced by stimulants. Despite place conditioning results, selective σR antagonists are inactive in blocking the self-administration of stimulants. However, compounds binding to the dopamine transporter and blocking σRs can selectively decrease stimulant self-administration. Further, after self-administration of stimulants, σR agonists are self-administered, an effect not seen in subjects without that specific history. These findings suggest that stimulants induce unique changes in σR activity, and once established, the changes induced create redundant, and dopamine independent reinforcement pathways. Concomitant targeting of both dopaminergic pathways and σR proteins produces a selective antagonism of those pathways, suggesting new avenues for combination chemotherapies to specifically combat stimulant abuse.

Injection of Cocaine-Amphetamine Regulated Transcript (CART) peptide into the nucleus accumbens does not inhibit caffeine-induced locomotor activity: Implications for CART peptide mechanism.

Much evidence suggests that intra-nucleus accumbens (NAc) CART peptide (CART 55-102) injection inhibits locomotor activity (LMA) when there is an increase in the release and activity of dopamine (DA) in the NAc. However, this hypothesis has not been fully tested. One way to examine this is to determine if there is a lack of effect of intra-NAc CART peptide on LMA that does not involve increases in DA release in the NAc. Several studies have suggested that caffeine-induced LMA does not involve extracellular DA release in the NAc core. Therefore, in this study, we have examined the effect of injections of CART peptide (2.5µg) into the NAc core on the locomotor effects of caffeine in male Sprague-Dawley rats. Several LMA relevant doses of caffeine were used (0, 10, 20mg/kg i.p.), and an inverted U response curve was found as expected. We determined, in the same animals, that intra-NAc CART peptide had no effect on caffeine-induced LMA whereas it blunted cocaine-mediated LMA, as shown by other reports. We also extended a previous observation in mice by showing that at a LMA activating dose of caffeine there is no alteration of CART peptide levels in the NAc of rats. Our study supports the hypothesis that the inhibitory effects of CART peptide in the NAc may be exerted only under conditions of increased extracellular DA release and activity in this region. Our results also suggest that intra-NAc CART 55-102 does not generally inhibit increases in LMA due to all drugs, but has a more specific inhibitory effect on dopaminergic neurotransmission.

PSA-NCAM in the posterodorsal medial amygdala is necessary for the pubertal emergence of attraction to female odors in male hamsters.

During puberty, attention turns away from same-sex socialization to focus on the opposite sex. How the brain mediates this change in perception and motivation is unknown. Polysialylated neural cell adhesion molecule (PSA-NCAM) virtually disappears from most of the central nervous system after embryogenesis, but it remains elevated in discrete regions of the adult brain. One such brain area is the posterodorsal subnucleus of the medial amygdala (MePD). The MePD has been implicated in male sexual attraction, measured here as the preference to investigate female odors. We hypothesize that PSA-NCAM gates hormone-dependent plasticity necessary for the emergence of males' attraction to females. To evaluate this idea, we first measured PSA-NCAM levels across puberty in several brain regions, and identified when female odor preference normally emerges in male Syrian hamsters. We found that MePD PSA-NCAM staining peaks shortly before the surge of pubertal androgen and the emergence of preference. To test the necessity of PSA-NCAM for female odor preference, we infused endo-neuraminidase-N into the MePD to deplete it of PSAs before female odor preference normally appears. This blocked female odor preference, which suggests that PSA-NCAM facilitates behaviorally relevant, hormone-driven plasticity.

Cocaine-and-Amphetamine Regulated Transcript (CART) peptide attenuates dopamine- and cocaine-mediated locomotor activity in both male and female rats: lack of sex differences.

Cocaine-and-Amphetamine Regulated Transcript peptide (CART peptide) is known for having an inhibitory effect on dopamine (DA)- and cocaine-mediated actions and is postulated to be a homeostatic, regulatory factor in the nucleus accumbens (NAc). Some sex differences in cocaine-mediated locomotor activity (LMA) and in the expression and function of CART peptide have been reported. However, it is not known if the inhibitory effect of CART peptide on cocaine-mediated LMA is sexually dimorphic. In this study, the effects of CART 55-102 on LMA due to intra-NAc DA and i.p. cocaine were determined in male and female Sprague-Dawley rats. The results show that CART 55-102 blunted or reduced both the DA- and cocaine-induced LMA in both males and females. In conclusion, CART peptide is effective in blunting DA- and cocaine-mediated LMA in both males and females.

The inhibition of cocaine-induced locomotor activity by CART 55-102 is lost after repeated cocaine administration.

CART peptide is known for having an inhibitory effect on cocaine- and dopamine-mediated actions after acute administration of cocaine and dopamine. In this regard, it is postulated to be a homeostatic, regulatory factor on dopaminergic activity in the nucleus accumbens (NAc). However, there is no data on the effect of CART peptide after chronic administration of cocaine, and this study addresses this. It was found that CART peptide blunted cocaine-induced locomotion (LMA) after acute administration of cocaine, as expected, but it did not affect cocaine-mediated LMA after chronic administration of cocaine. The loss of CART peptide's inhibitory effect did not return for up to 9 weeks after stopping the repeated cocaine administration. It may not be surprising that homeostatic regulatory mechanisms in the NAc are lost after repeated cocaine administration, and that this may be a mechanism in the development of addiction.

µ-opioid receptors in the stimulation of mesolimbic dopamine activity by ethanol and morphine in Long-Evans rats: a delayed effect of ethanol.

RATIONALE: Naltrexone, a non-selective opioid antagonist, decreases the euphoria and positive subjective responses to alcohol in heavy drinkers. It has been proposed that the µ-opioid receptor plays a role in ethanol reinforcement through modulation of ethanol-stimulated mesolimbic dopamine release. OBJECTIVES: To investigate the ability of naltrexone and ß-funaltrexamine, an irreversible µ-opioid specific antagonist, to inhibit ethanol-stimulated and morphine-stimulated mesolimbic dopamine release, and to determine whether opioid receptors on mesolimbic neurons contribute to these mechanisms. METHODS: Ethanol-naïve male Long Evans rats were given opioid receptor antagonists either intravenously, subcutaneously, or intracranially into the ventral tegmental area (VTA), followed by intravenous administration of ethanol or morphine. We measured extracellular dopamine in vivo using microdialysis probes inserted into the nucleus accumbens shell (n = 114). RESULTS: Administration of naltrexone (intravenously) and ß-funaltrexamine (subcutaneously), as well as intracranial injection of naltrexone into the VTA did not prevent the initiation of dopamine release by intravenous ethanol administration, but prevented it from being as prolonged. In contrast, morphine-stimulated mesolimbic dopamine release was effectively suppressed. CONCLUSIONS: Our results provide novel evidence that there are two distinct mechanisms that mediate ethanol-stimulated mesolimbic dopamine release (an initial phase and a delayed phase), and that opioid receptor activation is required to maintain the delayed-phase dopamine release. Moreover, µ-opioid receptors account for this delayed-phase dopamine response, and the VTA is potentially the site of action of this mechanism. We conclude that µ-opioid receptors play different roles in the mechanisms of stimulation of mesolimbic dopamine activity by ethanol and morphine.

Intraperitoneal Administration of CART 55-102 Inhibits Psychostimulant-Induced Locomotion.

CART (cocaine and amphetamine regulated transcript) peptide functions as both a neurotransmitter and a hormone and is found both in the central nervous system (CNS) and in the periphery. CART peptide in the nucleus accumbens (NAc) has been implicated in the regulation of cocaine-dopamine-mediated locomotion and self-administration, and amphetamine-mediated locomotion and behavior. However, there are no studies on the effect of systemic administration of CART peptide on cocaine and amphetamine-mediated locomotion. In this study, we tested if the systemic administration of CART 55-102 by the intraperitoneal (ip) route has a functional effect on psychostimulant-mediated locomotion in rats as it does when given into the brain. We determined that ip CART 55-102 attenuates psychostimulant-mediated locomotion as it does when administered into the NAc and display a biphasic dose response curve.

Mu (mu) opioid receptor regulation of ethanol-induced dopamine response in the ventral striatum: evidence of genotype specific sexual dimorphic epistasis.

BACKGROUND: Ethanol stimulates the dopaminergic mesoaccumbal pathway, which is thought to play a role in ethanol reinforcement. Mu (mu)-opioid (MOP) receptors modulate accumbal dopamine activity, but it is not clear whether MOP receptors are involved in the mechanism of ethanol-stimulated accumbal dopamine release. METHODS: We investigated the role that MOP receptors play in ethanol (2.0 g/kg)-stimulated accumbal dopamine release by using MOP receptor knockout mice (C57BL/6J-129SvEv and congenic C57BL/6J genotypes) along with blockade of MOP receptors with a mu1 selective antagonist (naloxonazine). RESULTS: Both gene deletion and pharmacological antagonism of the MOP receptor decreased ethanol-stimulated accumbal dopamine release compared with controls with female mice showing a larger effect in the C57BL/6J-129SvEv genotype. However, both male and female mice showed reduced ethanol-stimulated dopamine release in the congenic MOP receptor knockout mice (C57BL/6J). No differences in the time course of dialysate ethanol concentration were found in any of the experiments. CONCLUSIONS: The data demonstrate the existence of a novel interaction between genotype and sex in the regulation of ethanol-stimulated mesolimbic dopamine release by the MOP receptor. This implies that a more complete understanding of the epistatic influences on the MOP receptor and mesolimbic dopamine function may provide more effective pharmacotherapeutic interventions in the treatment of alcoholism.

Reduced basal and ethanol stimulation of striatal extracellular dopamine concentrations in dopamine D2 receptor knockout mice.

The present study was undertaken to examine the role of the dopamine (DA) D2 receptor in the ethanol-evoked DA response in the ventral striatum. We performed microdialysis experiments using the D2 null mutant and wild-type controls and measured the effect of an intraperitoneal (i.p.) injection of either saline or ethanol (2 g/kg) on dialysate DA concentrations in the ventral striatum. Dialysate ethanol concentrations were also determined in the samples from the ventral striatum. In addition, the effects of quinpirole, a D2/D3 agonist, were examined in both the ventral and dorsal striatum. Basal dialysate concentrations of DA were significantly reduced in both the ventral and dorsal striatum of the D2 knockouts compared with wild-type controls. Ethanol administration significantly enhanced ventral striatal DA in both groups, but the increase in dialysate DA concentration was 3.5-fold higher in the wild-type controls. The time course of dialysate ethanol concentrations was similar in the two groups. Saline injection did not alter DA concentrations in either the ventral or dorsal striatum. However, quinpirole (0.3 mg/kg) administration significantly depressed striatal dialysate DA concentrations in the wild-type mice, but not in the D2 knockouts. The results suggest that the D2 receptor is necessary for normal development and regulation of striatal extracellular DA concentrations, but the mechanism for this alteration is unclear. In addition, the blunted ethanol-evoked DA response in the D2 knockouts may contribute, in part, to some of the behavioral deficits previously observed in response to ethanol.

The role of mesolimbic dopamine in the development and maintenance of ethanol reinforcement.

The neurobiological processes by which ethanol seeking and consumption are established and maintained are thought to involve areas of the brain that mediate motivated behavior, such as the mesolimbic dopamine system. The mesolimbic dopamine system is comprised of cells that originate in the ventral tegmental area (VTA) and project to several forebrain regions, including a prominent terminal area, the nucleus accumbens (NAcc). The NAcc has been subdivided into core and shell subregions. Both areas receive converging excitatory input from the cortex and amygdala and dopamine input from the VTA, with the accumbal medium spiny neuron situated to integrate the signals. Although forced ethanol administration enhances dopamine activity in the NAcc, conclusions regarding the role of mesolimbic dopamine in ethanol reinforcement cannot be made from these experiments. Behavioral experiments consistently show that pharmacological manipulations of the dopamine transmission in the NAcc alter responding for ethanol, although ethanol reinforcement is maintained after lesions of the accumbal dopamine system. Additionally, extracellular dopamine increases in the NAcc during operant self-administration of ethanol, which is consistent with a role of dopamine in ethanol reinforcement. Behavioral studies that distinguish appetitive responding from ethanol consumption show that dopamine is important in ethanol-seeking behavior, whereas neurochemical studies suggest that accumbal dopamine is also important during ethanol consumption before pharmacological effects occur. Cellular studies suggest that ethanol alters synaptic plasticity in the mesolimbic system, possibly through dopaminergic mechanisms, and this may underlie the development of ethanol reinforcement. Thus, anatomical, pharmacological, neurochemical, cellular, and behavioral studies are more clearly defining the role of mesolimbic dopamine in ethanol reinforcement.