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Director's Report to the National Advisory Council on Drug Abuse
September, 2001

Research Findings

Intramural Research

Chemistry & Drug Metabolism Section, Clinical Pharmacology & Therapeutics Research Branch

Marijuana Craving Questionnaire

The purpose of this study was to develop and validate a multidimensional questionnaire on marijuana craving. Current marijuana smokers (n = 217) not seeking treatment completed a 47-item Marijuana Craving Questionnaire (MCQ) and forms assessing demographics, drug use history, marijuana quit attempts, and current mood. Exploratory and confirmatory factor analyses indicated that a four-factor solution best described the item structure. Factor subscales derived from the 17-items with significant loadings had respectable internal consistencies and were stable across settings and subgroups. The subscales exhibited low to moderate, positive intercorrelations and were significantly correlated with marijuana use history and a wide range of single-item measures of craving. Findings suggested that four specific constructs characterize craving for marijuana: 1) compulsivity, an inability to control marijuana use; 2) emotionality, use of marijuana in anticipation of relief from withdrawal or negative mood; 3) expectancy, anticipation of positive outcomes from smoking marijuana; and 4) purposefulness, intention and planning to use marijuana for positive outcomes. These data indicate that the MCQ is a valid and reliable instrument for assessing marijuana craving in individuals not seeking drug abuse treatment and that marijuana craving can be measured in the absence of withdrawal. Heishman, S.J., Singleton, E.G., and Liguori, A. Addiction, 96, pp. 1023-1034, 2001.

Brain Imaging Section, Neuroimaging Research Branch

Effect of Nicotine on Brain Activation during Performance of a Working Memory Task

Nicotine has been shown to affect cognition and behavior. Using positron emission tomography with O-15 labeled water, we measured cognitive activation during a working memory task (Two-Back task) in 11 smokers who abstained from smoking for 12 h, and 11 ex-smokers. Cerebral blood flow (CBF) was assayed twice, after the administration of placebo gum and that of 4 mg nicotine gum, respectively. Statistical parametric maps were calculated for each group (smokers and ex-smokers) and each condition (placebo and nicotine). Performance on the Two-Back task did not differ between groups in either placebo or nicotine condition. The brain regions activated by the Two-Back task were consistent with those reported in the literature. However, in the placebo condition, CBF activation in the ex-smokers predominated in the left hemisphere, and in smokers in the right hemisphere. When nicotine was administered, CBF activation was reduced in smokers but enhanced in ex-smokers. The lateralization of CBF activation as a function of nicotine dependence suggests that chronic exposure to nicotine or withdrawal from nicotine affects cognitive strategies used to perform the memory task. The lack of enhancement of CBF activation after nicotine administration in smokers, in contrast to ex-smokers, may reflect tolerance. Clarifying the effects of nicotine on brain function can improve our understanding of the mechanisms of nicotine dependence, and provide a basis for novel, rational treatment interventions. Ernst, M., Matochik, J.A., Heishman, S.J., Van Horn, J.D., Jons, P.H., Henningfield, J.E., and London, E.D. Effect of Nicotine on Brain Activation during Performance of a Working Memory Task. Proceedings of the National Academy of Sciences, USA, 98, pp. 4728-4733, 2001.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Methamphetamine Potentiates Ischemia/Reperfusion Insults after Transient Middle Cerebral Artery Ligation

Previous studies have indicated that both methamphetamine (MA) and ischemia/reperfusion injuries involve reactive oxygen species formation and activation of apoptotic mechanism. It is possible that MA may have a synergistic or additive effect with stroke-induced brain damage. The purpose of this study was to investigate if administration of MA in vivo would potentiate ischemic brain injury. Adult CD-1 mice were treated with MA or saline. Animals were later anesthetized with chloral hydrate and then placed in stereotaxic frame. A subset of animals received intracerebral administration of glial cell line -derived neurotrophic factor (GDNF). The right middle cerebral artery (MCA) and bilateral carotids were transiently occluded for 45 minutes. Regional cerebral blood flow was measured by Laser Doppler. Animals were sacrificed for tri-phenyl-tetrazolium chloride (TTC) staining and p53 mRNA Northern blot assay after 24 hours of reperfusion. Cortical and striatal GDNF levels were assayed by ELISA. Investigators found that pretreatment with MA increased ischemia-induced cerebral infarction. Ischemia or MA alone enhanced p53 mRNA expression. Moreover, MA potentiated the expression of p53 mRNA in the ischemic mouse brain. MA pretreatment decreased GDNF levels in ischemic striatum. Intracerebral administration of GDNF before ischemia reduced MA -facilitated infarction. Our data indicate that MA exacerbates ischemic insults in brain, perhaps through the inhibition of GDNF -mediated pathways, and suggest that MA may antagonize endogenous neuroprotective pathways as part of its mechanism of action. Wang,Y., Hayashi,T., Chang,C.F., Chiang,Y.H., Tsao,L.I., Su,T.P., Borlongan,C.V., and Lin,S.Z. Stroke, 32, pp. 775-782, 2001.

Contribution of the Hyperpolarization-Activated Current (Ih) to Membrane Potential and GABA Release in Hippocampal Interneurons

GABAergic interneurons provide inhibitory input to CA1 pyramidal cells of the hippocampus, and thus play a key role in regulating hippocampal activity. The excitability of these interneurons is regulated by various ion channels, including the hyperpolarization-activated current Ih. IRP investigators utilized whole-cell recording techniques in order to determine the role of Ih in regulating the baseline excitability of hippocampal interneurons and the release of GABA onto CA1 pyramidal cells. In current-clamp recordings from hippocampal stratum oriens (s.o.) interneurons at resting membrane potential (-61 1.2 mV), blockade of Ih with the selective inhibitor ZD 7288 (50 mM) resulted in a hyperpolarization of the membrane potential and a decrease in the rate of spontaneous action potentials. A small population of s.o. interneurons that did not express Ih were substantially more hyperpolarized (-73.6 5.5 mV) under baseline conditions, suggesting that Ih contributes significantly to the resting membrane potential of hippocampal interneurons. Voltage-clamp recordings from postsynaptic CA1 pyramidal cells demonstrated that blockade of Ih by ZD 7288 resulted in a significant reduction (~43%) in the frequency of spontaneous, action potential-dependent inhibitory postsynaptic currents (IPSCs). Moreover, miniature, action potential-independent IPSCs were unaffected by ZD7288, confirming the presynaptic site of action of ZD 7288. These data suggest that Ih is active at the resting membrane potential in s.o. interneurons, and thus contributes to the spontaneous activity of these cells and to the tonic inhibition of CA1 pyramidal neurons in the hippocampus. Since Ih is inhibited by both m- and d-opioid receptors, and is activated by serotonin and norepinephrine, this current may represent an important cellular target for drugs of abuse, under either normal or chronic conditions. Lupica, C.R., Bell, J.A., Hoffman, A.F., and Watson, P.L. Journal of Neurophysiology, 86, pp. 261-268, 2001.

Molecular Neuropsychiatry Section, Cellular Neurobiology Research Branch

Methamphetamine Causes Differential Regulation of Pro-Death and Anti-Death Bcl-2 Genes in the Mouse Neocortex

Bcl-2, an inner mitochondrial membrane protein, inhibits apoptotic neuronal cell death. Expression of Bcl-2 inhibits cell death by decreasing the net cellular generation of reactive oxygen species. Studies by different investigators have provided unimpeachable evidence of a role for oxygen-based free radicals in methamphetamine (METH) -induced neurotoxicity. In addition, studies conducted in this laboratory have shown that immortalized rat neuronal cells that overexpress Bcl-2 are protected against METH-induced apoptosis in vitro. Moreover, the amphetamines can cause differential changes in the expression of Bcl-X splice variants in primary cortical cell cultures. These observations suggested that METH might also cause perturbations of Bcl-2-related genes when administered to rodents. Thus, the present study was conducted to determine whether the use of METH might indeed be associated with transcriptional and translational changes in the expression of Bcl-2-related genes in the mouse brain. In this paper IRP investigators report that a toxic regimen of METH did cause significant increases in the pro-death Bcl-2 family genes BAD, BAX, and BID. Concomitantly, there were significant decreases in the anti-death genes Bcl-2 and Bcl-X(L.) These results thus support the notion that injections of toxic doses of METH trigger the activation of the programmed death pathway in the mammalian brain. Jayanthi, S., Deng, X., Bordelon, M., McCoy, M.T., and Cadet, J.L. Methamphetamine Causes Differential Regulation of Pro-Death and Anti-Death Bcl-2 Genes in the Mouse Neocortex. Jayanthi, S., Deng, X., Bordelon, M., McCoy, M.T., and Cadet, J.L. FASEB Journal, 15, pp. 1745-1752, 2001.

Marijuana Abusers are at Increased Risk for Stroke. Preliminary Evidence from Cerebrovascular Perfusion Data

IRP investigators have recorded blood flow velocity in the anterior and middle cerebral arteries by transcranial Doppler sonography in abstinent marijuana abusers (n = 16) and control subjects (n = 19) to assess the effects of prolonged marijuana use of the cerebrovascular system. The pulsatility index, a measure of cerebrovascular resistance, and systolic velocity were significantly (p < 0.005) increased in marijuana abusers compared to the control subjects. These findings suggest that cerebral perfusion observed in 18-30 year old marijuana abusers is comparable to that of normal 60 year-olds. Thus, chronic abuse of marijuana might be a risk factor for stroke. Herning, R.I., Better, W.E., Tate, K., and Cadet, J.L. Annals of the New York Academy of Science, 939, pp. 413-415, 2001.

Antiviral Medications Improve Cerebrovascular Perfusion in HIV+ Non-drug Users and HIV+ Cocaine Abusers

Antiviral medications have been useful in delaying the time course of HIV infection. Antiviral medications have also been reported to delay or reduce symptoms associated with AIDS related dementia and to improve cortical perfusion. The mechanism for this improvement is unclear. Thus, this report studies the effects of antiviral medications on cerebral blood flow velocity in HIV+ cocaine abusers, HIV+ control individuals and appropriate control individuals. Thirty-two unmedicated HIV+ individuals (28 cocaine abusers and 4 control individuals), 22 HIV+ individuals using antiviral medications (16 cocaine abusers and 6 HIV+ control individuals), 47 HIV- cocaine abusers, and 27 control HIV- subjects were studied. Blood flow velocities were determined for the anterior and middle cerebral arteries using transcranial Doppler sonography. HIV+ individuals on antiviral medications had lower pulsatility values, suggesting decreased resistance in the cerebral blood vessels, in comparison to HIV+ individuals not taking antiviral medications. HIV+ cocaine abusers and HIV+ control individuals using antiviral medications had pulsatility values similar to HIV- control subjects. Antiviral medications appear to reduce these cerebrovascular perfusion deficits in HIV+ individuals. The antiviral medications appear to have a direct neuroprotective effect in addition to their antiviral effects. The neuroprotective role of antiviral medications requires further investigation. Herning, R.I., Better, W.E., Tate, K., and Cadet, J.L. Annals of the New York Academy of Science, 939, pp. 405-412, 2001.

Fas-induced Apoptosis of Glioma Cells is Associated with Down-regulation of the hSCO1 Protein, A Subunit of Complex IV

ApoI/Fas belongs to the tumor necrosis factor receptor (TNFR) superfamily and mediates cell death in various cell types. Earlier studies from this laboratory have shown that Fas-mediated cell death of glioma cells occur, in part, through the production of reactive oxygen species (ROS). To further dissect the molecular mechanisms that are involved in Fas-induced cell death, IRP investigators compared gene expression between Fas-treated and saline-treated human neuroglioma H4 cells by using the technique of mRNA differential display. This approach led to the identification of hSCO1, a component of the inner mitochondrial membrane, which is required for the correct assembly, and catalytic function of cytochrome-c oxidase, as a Fas down-regulated gene. The decrease in hSCO1 mRNA expression was time-dependent, becoming most prominent after 4 h of Fas-treatment. Morphological changes observed by confocal microscopy revealed that after 4 h of Fas-treatment, the cells undergo membrane blebbing and early formation of apoptotic bodies. These observations are discussed in terms of their support for an important role of mitochondrial events in Fas-induced apoptosis. Jayanthi, S., Lewis, B.D., and Cadet, J.L. Brain Research Molecular Brain Research, 91, pp. 131-136, 2001.

Involvement of Free Radicals in MDMA-induced Neurotoxicity in Mice

3,4-methylenedioxymethamphetamine (MDMA or ecstasy) is a substituted amphetamine with stimulating and hallucinogenic properties. Administration of MDMA leads to the formation of metabolites responsible for its toxic effects on serotonergic neurons in rats and non-human primates and on dopaminergic neurons in mice. IRP investigators' findings indicate that overexpression of the human superoxide dismutase gene (Cu/Zn-SOD) abolishes certain effects of MDMA such as the decreased level of dopamine, DOPAC and 5-HT in the striatum, inactivation of certain antioxidant enzymes (CU/ZN-SPD, catalase or glutathione peroxidase) or peroxidation of lipids. These data are in agreement with the implication of free radicals and consequently of oxidative stress in the mode of action of MDMA. Cadet, J.L., Thiriet, N., and Subramanian, J. Annals Medicine Interne (Paris), 152, Suppl 3, pp. 57-59, 2001.

Temporal Profiling of Methamphetamine-induced Changes in Gene Expression in the Mouse Brain: Evidence from cDNA Array

Methamphetamine (METH) is a neurodegenerative drug of abuse. Its toxicity is characterized by destruction of monoaminergic terminals and by apoptosis in cortical and striatal cell bodies. Multiple factors appear to control METH neurotoxicity, including free radicals and transcription factors. Here, using cDNA arrays, IRP researchers show the temporal profile of gene expression patterns in the cortex of mice treated with this drug. Two patterns of changes were obtained from 588 genes surveyed. First, an early pattern is characterized by upregulation of transcription factors, including members of the jun family. Second, a delayed pattern includes genes related to cell death and to DNA repair. A number of trophic factors were also activated at the later timepoint. These observations suggest that METH can activate a multigene machinery that participates in the production of its toxic effects. The resulting degenerative effects of the drug are thus the result of a balance between protoxic and anti-apoptotic mechanisms triggered by its administration to these animals. These observations are of clinical relevance because of the recent identification of degenerative changes in the brains of METH abusers. Cadet, J.L., Jayanthi, S., McCoy, M.T., Vawter, M., and Ladenheim, B. Synapse, 41, pp. 40-48, 2001.

Molecular Neurobiology Section, Molecular Neurobiology Research Branch

Defining Molecular Mechanisms of Cocaine Reward

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, neither knockouts of DAT, of SERT or of NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wildtype DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place preference testing. However, mice with no DAT and either 0 or 1 SERT gene copies display no preference for places where they have previously received cocaine. The serotonin-dependence of cocaine reward in DAT KO mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide new insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anti-cocaine medication development. Sora, I., Hall, F.S., Andrews, A.M., Itokawa, M., Li, X-f., Wei, H-b., Wichems, C., Lesch, K-P., Murphy, D.L., and Uhl, G.R. Proceedings of the National Academy of Science USA, 98, pp. 5300-5305, 2001.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Laboratory

Adenosine A2A Agonist CGS 21680 Decreases the Affinity of Dopamine D2 Receptors for Dopamine in Human Striatum

Adenosine A2A receptors (A2AR) and dopamine D2 receptors (D2R) are highly concentrated in the striatum, where they are colocalized and exert reciprocal antagonistic interactions. It has been suggested that the A2R/D2R interactions might provide a therapeutic approach for basal ganglia disorders, such as Parkinson's disease, and schizophrenia. In the present work evidence is presented for the existence of an A2AR/D2R interaction in human brain by using quantitative autoradiography. The areas analyzed were the dorsal caudate nucleus and putamen. Parallel studies were performed in rat striatal sections. The A2AR agonist CGS 21680 was found to significantly increase IC50 values of competitive-inhibition curves of the D2R/D3R antagonist [125I]iodosulpiride versus dopamine both in rat striatal and human striatal brain sections. Diaz-Cabiale, Z., Hurd, Y., Guidolin, D., Finnman, U.-B., Zoli, M., Agnati, L.F., Vanderhaeghen, J.-J., Fuxe, K. and Ferre, S. NeuroReport, 9, pp. 1831-1834, 2001.

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