Kevin S. Murnane

Endocrine and Neurochemical Effects of 3,4-Methylenedioxymethamphetamine and Its Stereoisomers in Rhesus Monkeys

3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative that elicits complex biological effects in humans. One plausible mechanism for this phenomenon is that racemic MDMA is composed of two stereoisomers that exhibit qualitatively different pharmacological effects. In support of this, studies have shown that R(−)-MDMA tends to have hallucinogen-like effects, whereas S(+)-MDMA tends to have psychomotor stimulant-like effects. However, relatively little is known about whether these stereoisomers engender different endocrine and neurochemical effects. In the present study, the endocrine and neurochemical effects of each stereoisomer and the racemate were assessed in four rhesus monkeys after intravenous delivery at doses (1–3 mg/kg) that approximated voluntary self-administration by rhesus monkeys and human recreational users. Specifically, fluorescence-based enzyme-linked immunosorbent assay was used to assess plasma prolactin concentrations, and in vivo microdialysis was used to assess extracellular dopamine and serotonin concentrations in the dorsal striatum. R(−)-MDMA, but not S(+)-MDMA, significantly increased plasma prolactin levels and the effects of S,R(±)-MDMA were intermediate to each of its component stereoisomers. Although S(+)-MDMA did not alter prolactin levels, it did significantly increase extracellular serotonin concentrations. In addition, S(+)-MDMA, but not R(−)-MDMA, significantly increased dopamine concentrations. Furthermore, as in the prolactin experiment, the effects of the racemate were intermediate to each of the stereoisomers. These studies demonstrate the stereoisomers of MDMA engender qualitatively different endocrine and neurochemical effects, strengthening the inference that differences in these stereoisomers might be the mechanism producing the complex biological effects of the racemic mixture of MDMA in humans.

Neuroimaging and drug taking in primates.

RationaleNeuroimaging techniques have led to significant advances in our understanding of the neurobiology of drug taking and the treatment of drug addiction in humans. Neuroimaging approaches provide a powerful translational approach that can link findings from humans and laboratory animals.ObjectiveThis review describes the utility of neuroimaging toward understanding the neurobiological basis of drug taking and documents the close concordance that can be achieved among neuroimaging, neurochemical, and behavioral endpoints.ResultsThe study of drug interactions with dopamine and serotonin transporters in vivo has identified pharmacological mechanisms of action associated with the abuse liability of stimulants. Neuroimaging has identified the extended limbic system, including the prefrontal cortex and anterior cingulate, as important neuronal circuitry that underlies drug taking. The ability to conduct within-subject longitudinal assessments of brain chemistry and neuronal function has enhanced our efforts to document long-term changes in dopamine D2 receptors, monoamine transporters, and prefrontal metabolism due to chronic drug exposure. Dysregulation of dopamine function and brain metabolic changes in areas involved in reward circuitry have been linked to drug taking behavior, cognitive impairment, and treatment response.ConclusionsExperimental designs employing neuroimaging should consider well-documented determinants of drug taking, including pharmacokinetic considerations, subject history, and environmental variables. Methodological issues to consider include limited molecular probes, lack of neurochemical specificity in brain activation studies, and the potential influence of anesthetics in animal studies. Nevertheless, these integrative approaches should have important implications for understanding drug taking behavior and the treatment of drug addiction.

Nonhuman primate positron emission tomography neuroimaging in drug abuse research.

Positron emission tomography (PET) neuroimaging in nonhuman primates has led to significant advances in our current understanding of the neurobiology and treatment of stimulant addiction in humans. PET neuroimaging has defined the in vivo biodistribution and pharmacokinetics of abused drugs and related these findings to the time course of behavioral effects associated with their addictive properties. With novel radiotracers and enhanced resolution, PET neuroimaging techniques have also characterized in vivo drug interactions with specific protein targets in the brain, including neurotransmitter receptors and transporters. In vivo determinations of cerebral blood flow and metabolism have localized brain circuits implicated in the effects of abused drugs and drug-associated stimuli. Moreover, determinations of the predisposing factors to chronic drug use and long-term neurobiological consequences of chronic drug use, such as potential neurotoxicity, have led to novel insights regarding the pathology and treatment of drug addiction. However, similar approaches clearly need to be extended to drug classes other than stimulants. Although dopaminergic systems have been extensively studied, other neurotransmitter systems known to play a critical role in the pharmacological effects of abused drugs have been largely ignored in nonhuman primate PET neuroimaging. Finally, the study of brain activation with PET neuroimaging has been replaced in humans mostly by functional magnetic resonance imaging (fMRI). There has been some success in implementing pharmacological fMRI in awake nonhuman primates. Nevertheless, the unique versatility of PET imaging will continue to complement the systems-level strengths of fMRI, especially in the context of nonhuman primate drug abuse research.

Escalation of food-maintained responding and sensitivity to the locomotor stimulant effects of cocaine in mice

Escalation of drug self-administration is a consequence of extended drug access and is thought to be specifically related to addiction, but few studies have investigated whether intake of non-drug reinforcers may also escalate with extended-access. The goal of these studies was to determine the effects of limited and extended-access to food reinforcers on behavioral and pharmacological endpoints in mice. In distinct groups, responding on a lever was maintained by liquid reinforcement, or nose-poke responses were maintained by sucrose pellets or liquid food in sessions lasting 1 h (limited-access) or 10 h (extended-access). The reinforcing strength of each food, as well as reinforcer-associated cues, was tested before and after extended-access using a progressive ratio (PR) schedule, and locomotor activity in response to novelty and increasing doses of cocaine was assessed in an open field setting in all animals after access to food reinforcers. Escalation of lever-pressing behavior reinforced by liquid food, but not nose-poke behavior reinforced by liquid food or sucrose pellets, was observed across successive extended-access sessions. A concomitant increase in the reinforcing strength of liquid food and its associated cues was apparent in mice that escalated their responding, but not in mice that did not escalate. Finally, extended reinforcer access leading to behavioral escalation was accompanied by an increased sensitivity to the psychostimulant effects of cocaine compared to limited-access. These findings indicate that behavioral escalation can develop as a consequence of extended-access to a non-drug reinforcer, although both the nature of the reinforcer (liquid versus solid food) and the topography of the operant response (lever versus nose-poke) modulate its development. These data also suggest that some of the behavioral and pharmacological corrolaries of behavioral escalation observed following extended-access to drug self-administration may not be due to drug exposure, but rather, may result from basic behavioral processes which underlie operant responding maintained by appetitive stimuli.

Development of an apparatus and methodology for conducting functional magnetic resonance imaging (fMRI) with pharmacological stimuli in conscious rhesus monkeys

Functional magnetic resonance imaging (fMRI) is a technique with significant potential to advance our understanding of multiple brain systems. However, when human subjects undergo fMRI studies they are typically conscious whereas pre-clinical fMRI studies typically utilize anesthesia, which complicates comparisons across studies. Therefore, we have developed an apparatus suitable for imaging conscious rhesus monkeys. In order to minimize subject stress and spatial motion, each subject was acclimated to the necessary procedures over several months. The effectiveness of this process was then evaluated, in fully trained subjects, by quantifying objective physiological measures. These physiological metrics were stable both within and across sessions and did not differ from when these same subjects were immobilized using standard primate handling procedures. Subject motion and blood oxygenation level dependent (BOLD) fMRI measurements were then evaluated by scanning subjects under three different conditions: the absence of stimulation, presentation of a visual stimulus, or administration of intravenous (i.v.) cocaine (0.3mg/kg). Spatial motion differed neither by condition nor along the three principal axes. In addition, maximum translational and rotational motion never exceeded one half of the voxel size (0.75 mm) or 1.5 degrees, respectively. Furthermore, the localization of changes in blood oxygenation closely matched those reported in previous studies using similar stimuli. These findings document the feasibility of fMRI data collection in conscious rhesus monkeys using these procedures and allow for the further study of the neural effects of psychoactive drugs.

Discriminative stimulus effects of psychostimulants and hallucinogens in S(+)-3,4-methylenedioxymethamphetamine (MDMA) and R(-)-MDMA trained mice.

3,4-Methylenedioxymethamphetamine (MDMA) is a substituted phenethylamine more commonly known as the drug of abuse “ecstasy.” The acute and persistent neurochemical effects of MDMA in the mice are distinct from those in other species. MDMA shares biological effects with both amphetamine-type stimulants and mescaline-type hallucinogens, which may be attributable to distinct effects of its two enantiomers, both of which are active in vivo. In this regard, among the substituted phenethylamines, R(−)-enantiomers tend to have hallucinogen-like effects, whereas S(+)-enantiomers tend to have stimulant-like effects. In the present study, mice were trained to discriminate S(+)- or R(−)-MDMA from vehicle. Drug substitution tests were then undertaken with the structurally similar phenethylamine dopamine/norepinephrine releaser S(+)-amphetamine, the structurally dissimilar tropane nonselective monoamine reuptake inhibitor cocaine, the structurally similar phenethylamine 5-hydroxytryptamine (5-HT)2A agonist 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7), and the structurally dissimilar mixed action tryptamine 5-HT2A agonist/monoamine reuptake inhibitor N,N-dipropyltryptamine (DPT). S(+)-amphetamine fully substituted in the S(+)-MDMA-treated animals but did not substitute for the R(−)-MDMA cue. 2C-T-7 fully substituted in the R(−)-MDMA-trained animals but did not substitute for the S(+)-MDMA cue. Cocaine and DPT substituted for both training drugs, but whereas cocaine was more potent in S(+)-MDMA-trained mice, DPT was more potent in R(−)-MDMA-trained mice. These data suggest that qualitative differences in the discriminative stimulus effects of each stereoisomer of MDMA exist in mice and further our understanding of the complex nature of the interoceptive effects of MDMA.

Effects of exposure to amphetamine derivatives on passive avoidance performance and the central levels of monoamines and their metabolites in mice: Correlations between behavior and neurochemistry

RationaleConsiderable evidence indicates that amphetamine derivatives can deplete brain monoaminergic neurotransmitters. However, the behavioral and cognitive consequences of neurochemical depletions induced by amphetamines are not well established.ObjectivesIn this study, mice were exposed to dosing regimens of 3,4-methylenedioxymethamphetamine (MDMA), methamphetamine (METH), or parachloroamphetamine (PCA) known to deplete the monoamine neurotransmitters dopamine and serotonin, and the effects of these dosing regimens on learning and memory were assessed.MethodsIn the same animals, we determined deficits in learning and memory via passive avoidance (PA) behavior and changes in tissue content of monoamine neurotransmitters and their primary metabolites in the striatum, frontal cortex, cingulate, hippocampus, and amygdala via ex vivo high-pressure liquid chromatography.ResultsExposure to METH and PCA impaired PA performance and resulted in significant depletions of dopamine, serotonin, and their metabolites in several brain regions. Multiple linear regression analysis revealed that the tissue concentration of dopamine in the anterior striatum was the strongest predictor of PA performance, with an additional significant contribution by the tissue concentration of the serotonin metabolite 5-hydroxyindoleacetic acid in the cingulate. In contrast to the effects of METH and PCA, exposure to MDMA did not deplete anterior striatal dopamine levels or cingulate levels of 5-hydroxyindoleacetic acid, and it did not impair PA performance.ConclusionsThese studies demonstrate that certain amphetamines impair PA performance in mice and that these impairments may be attributable to specific neurochemical depletions.

Nonhuman Primate Neuroimaging and the Neurobiology of Psychostimulant Addiction

Neuroimaging techniques have led to significant advances in our understanding of the neurobiology and treatment of drug addiction in humans. The capability to conduct parallel studies in nonhuman primates and human subjects provides a powerful translational approach to link findings in human and animal research. A significant advantage of nonhuman primate models is the ability to use drug‐naïve subjects in longitudinal designs that document the neurobiological changes that are associated with chronic drug use. Moreover, experimental therapeutics can be evaluated in subjects with well‐documented histories of drug exposure. The in vivo distribution and pharmacokinetics of drug binding in brain have been related to the time‐course of behavioral effects associated with the addictive properties of stimulants. Importantly, the characterization of drug interactions with specific protein targets in brain has identified potential targets for medication development. Neuroimaging has proven especially useful in studying the dynamic changes in neuronal function that may be associated with environmental variables. Last, neuroimaging has been used effectively in nonhuman primates to characterize both transient and long‐lasting changes in brain chemistry associated with chronic drug exposure. Although there is some evidence to suggest neurotoxicity in humans with long histories of stimulant use, parallel studies in nonhuman primates have not identified consistent long‐term changes in such neurochemical markers. Collectively, the results of these studies of nonhuman primates have enhanced our understanding of the neurobiological basis of stimulant addiction and should have a significant impact on efforts to develop medications to treat stimulant abuse.

Selective serotonin 2A receptor antagonism attenuates the effects of amphetamine on arousal and dopamine overflow in non-human primates.

The objective of the present study was to further elucidate the mechanisms involved in the wake‐promoting effects of psychomotor‐stimulants. Many previous studies have tightly linked the effects of stimulants to dopamine neurotransmission, and some studies indicate that serotonin 2A receptors modulate these effects. However, the role of dopamine in arousal is controversial, most notably because dopamine neurons do not change firing rates across arousal states. In the present study, we examined the wake‐promoting effects of the dopamine‐releaser amphetamine using non‐invasive telemetric monitoring. These effects were evaluated in rhesus monkeys as a laboratory animal model with high translational relevance for human disorders of sleep and arousal. To evaluate the role of dopamine in the wake‐promoting effects of amphetamine, we used in vivo microdialysis targeting the caudate nucleus, as this approach provides clearly interpretable measures of presynaptic dopamine release. This is beneficial in the present context because some of the inconsistencies between previous studies examining the role of dopamine in arousal may be related to differences between postsynaptic dopamine receptors. We found that amphetamine significantly and dose‐dependently increased arousal at doses that engendered higher extracellular dopamine levels. Moreover, antagonism of serotonin 2A receptors attenuated the effects of amphetamine on both wakefulness and dopamine overflow. These findings further elucidate the role of dopamine and serotonin 2A receptors in arousal, and they suggest that increased dopamine neurotransmission may be necessary for the wake‐promoting effects of amphetamine, and possibly other stimulants.