Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Reduction of Synapsin in the Hippocampus of Patients with Bipolar Disorder and Schizophrenia Several studies suggest that decreased expression of presynaptic proteins may be characteristic of schizophrenia. We examined one such protein, synapsin, in schizophrenia and bipolar disorder. Samples of hippocampal tissue from controls (n = 13), patients with schizophrenia (n = 16), or bipolar disorder (n = 6), and suicide victims (n = 7) were used. The membrane and cytosolic fractions were analyzed by Western immunoblotting for synapsin using an antibody that detects synapsin Ia, IIa, and IIIa proteins. Synaptophysin was also measured for comparison. Total synapsin was decreased significantly in patients with schizophrenia (P = 0.034) and in bipolar disorder (P = 0.00008) as compared to controls. The synapsin/ synaptophysin ratios were decreased in schizophrenia and bipolar disorder, and additionally in suicide victims (P = 0.014). Age, postmortem interval, percentage of protein extracted, and pH of brain were not different between groups. No changes in total synapsin or synaptophysin in the hippocampus were produced by injecting rats with either lithium or haloperidol for 30 days. Reductions in synapsin in both patients with schizophrenia (synapsin IIa and IIIa) and bipolar disorder (synapsin Ia, IIa and IIIa) imply that altered or reduced synaptic function in the hippocampus may be involved in these disorders. Vawter, M.P., Thatcher, L., Usen, N., Hyde, T.M., Kleinman, J.E., and Freed, W.J. Molecular Psychiatry, 7, pp. 571-578, 2002.

Transplantation of M213-2O Cells with Enhanced GAD(67) Expression into the Inferior Colliculus Alters Audiogenic Seizures The purpose of the present study was to examine the effects of GABA-producing cell transplants on audiogenic seizures (AGS). The M213-2O cell line was derived from fetal rat striatum and has GABAergic properties. This cell line was further modified to express human GAD(67) and produce elevated levels of GABA. The present study compares the effects of parent M213-2O cell transplants with those of GAD(67)-modified M213-2O cells in AGS-prone Long-Evans rats. Two weeks following implantation of engineered cells, latency to AGS-typical wild running was increased compared to nonimplanted subjects. Survival of the transplanted cells was confirmed by immunochemical labeling of GAD(67) and Epstein-Barr virus nuclear antigen. These findings support the use of GABA-producing cell lines to modify seizure activity. Ross, K.C., Waldman, B.C., Conejero-Goldberg, C., Freed, W., and Coleman, J.R. Experimental Neurology, 177, pp. 338-340, 2002.

Microarray Analysis of Gene Expression in the Prefrontal Cortex in Schizophrenia: A Preliminary Study Microarray studies can be used to examine expression levels for large numbers of genes simultaneously and may be applied to identify genes involved in schizophrenia. A microarray with 1127 brain-relevant genes was used to screen relative gene expression in the dorsolateral prefrontal cortex (DLPFC) from three pools of patients with schizophrenia (n=15) and three matched control pools (n=15). Pooling of tissue samples was employed as a strategy to detect changes in gene expression that are consistently found across individual cases of schizophrenia. Differences in gene expression were examined by z-ratios in addition to traditional normalized ratios. Three genes that showed consistently decreased expression in schizophrenia by both z-ratio differences and decreased normalized numerical ratios were identified. These were histidine triad nucleotide-binding protein (HINT), ubiquitin conjugating enzyme E2N (UBE2N) and glutamate receptor, ionotropic, AMPA 2 (GRIA2). Moreover, HINT gene expression was decreased to a similar degree in a prior study. In addition, a decrease in AMPA receptor expression is consistent with a decrease in glutamate synaptic function. These results are subject to limitations based on variations inherent to human subjects and tissue samples, possible effects of neuroleptic treatment, and the requirement for verification using independent techniques. Vawter, M., Crook, J., Hyde, T., Kleinman, J., Weinberger, D., Becker, K., and Freed, W. Schizophrenia Research, 58, pp. 11-20, 2002.

Molecular Neuropsychiatry Section, Cellular Neurobiology Research Branch

Analysis of Ecstasy (MDMA)-induced Transcriptional Responses in the Rat Cortex 3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a popular drug of abuse. MDMA is pharmacologically classified as an entactogen because of its affinities to classical hallucinogens and stimulants. Oral ingestion of a single dose of the drug is associated with euphoria, elevated self-confidence, and heightened sensory awareness in humans. Evidence for neurotoxicity in the human serotonin (5-HT) system has been provided. In rats, a single injection of MDMA induces hyperthermia and formation of reactive oxygen species. These effects may cause MDMA-associated, long-term 5-HT depletion, with the cortex being quite sensitive to the biochemical effects of MDMA. It has been suggested that these MDMA effects may be associated with molecular changes in this brain region. To test these ideas, IRP researchers have made use of the cDNA array analysis, which can provide a more global view of the molecular changes secondary to MDMA injections. Results show that the genes regulated by MDMA encode proteins that belong to signaling pathways, transcription regulators, or xenobiotic metabolism. Observations indicate that cortical cells respond to the acute administration of MDMA by modulating transcription of several genes that might lead to long-term changes in the brain. Thiriet, N., Ladenheim, B., McCoy, M.T., and Cadet, J.L. FASEB Journal, 16, pp. 1887-1894, 2002.

Dose-related Neurocognitive Effects of Marijuana Use Although about 7 million people in the U.S. population use marijuana at least weekly, there is a paucity of scientific data on persistent neurocognitive effects of marijuana use. In order to determine if neurocognitive deficits persist in 28-day abstinent heavy marijuana users and if these deficits are dose-related to the number of marijuana joints smoked per week IRP iunvestigators administered a battery of neurocognitive tests to 28-day abstinent heavy marijuana abusers. Results showed that as joints smoked per week increased, performance decreased on tests measuring memory, executive functioning, psychomotor speed, and manual dexterity. When dividing the group into light, middle, and heavy user groups, the heavy group performed significantly below the light group on 5 of 35 measures and the size of the effect ranged from 3.00 to 4.20 SD units. Duration of use had little effect on neurocognitive performance. The authors conclude that very heavy use of marijuana is associated with persistent decrements in neurocognitive performance even after 28 days of abstinence. It is unclear if these decrements will resolve with continued abstinence or become progressively worse with continued heavy marijuana use. Bolla, K.I., Brown, K., Eldreth, D., Tate, K., and Cadet, J.L. Neurology, 59, pp. 1337-1343, 2002.

Mice with Partial Deficiency of c-Jun Show Attenuation of Methamphetamine-induced Neuronal Apoptosis The regional distribution of c-Jun expression and of the number of apoptotic cells was compared in various brain areas after methamphetamine administration to mice. Our results showed that there was methamphetamine-induced overexpression of c-Jun in the cortex and striatum but not in the cerebellar cortex. There was an almost totally similar regional appearance of methamphetamine-induced apoptotic cells in the mouse brain; no apoptosis was present in the cerebellum. Additionally, in the neocortical area, more positive signals for c-Jun immunoreactivity were observed in the piriform cortex, an area that also showed more positive terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) signals than the frontal and parietal cortices. These observations suggested that c-Jun might be involved in methamphetamine-induced apoptosis. This idea was confirmed by using heterozygous c-Jun knockout mice that showed much less apoptosis than wild-type controls. In addition, the authors found that the majority of TUNEL-positive cells were also positive for c-Jun-like immunoreactivity in both genotypes. Moreover, methamphetamine-induced caspase-3 activity and PARP cleavage were also reduced in c-Jun heterozygous knockout mice. In contrast, methamphetamine-induced hyperthermia was essentially identical in the two genotypes. When taken together, these data support the hypothesis that c-Jun is involved in methamphetamine-induced apoptosis. Deng, X., Jayanthi, S., Ladenheim, B., Krasnova, I.N., and Cadet, J.L. Molecular Pharmacology, 62, pp. 993-1000, 2002.

cDNA Array Analysis of Gene Expression Profiles in the Striata of Wild-type and Cu/Zn Superoxide Dismutase Transgenic Mice Treated with Neurotoxic Doses of Amphetamine Amphetamine (AMPH) is a drug of abuse that causes the degeneration of striatal dopamine terminals in mammals. Superoxide radicals seem to participate in AMPH-induced damage because its toxicity is attenuated in Cu/Zn superoxide dismutase transgenic (SOD-tg) mice. To provide a detailed analysis of molecular changes associated with AMPH toxicity, IRP scientists used cDNA arrays consisting of 1176 genes to detect differential changes in gene expression in the striata of wild-type and SOD-tg mice treated with neurotoxic doses of the drug. The authors found 42 genes that showed >1.8-fold changes in at least two consecutive time points during the course of the study and were differentially affected by AMPH in the two genotypes. Specifically, more transcription factors and genes involved in responses to injury/inflammation were affected in wild-type mice after AMPH administration. Some of these stimulant-induced superoxide-dependent alterations in gene expression might affect neuronal functions and promote neuronal damage. Other changes might help to provide some degree of protection against AMPH toxicity. These results support the view that the use of global array analysis of gene expression will help to identify novel molecular mediators of AMPH-induced neurodegeneration. Krasnova, I.N., McCoy, M.T., Ladenheim, B., and Cadet, J.L. FASEB Journal, 16, pp. 1379-1388, 2002.

Molecular Neurobiology Section, Molecular Neurobiology Branch

Knockout Mice Reveal Mechanisms of Drug Reward Over the past year, IRP scientists have followed up on their seminal obeservations regarding elimination of cocaine reward in double knockout mice with deletions of dopamine and serotonin transporters; they now report that mice with combined knockouts of norepinephrine and serotonin transporters display contrastingly ENHANCED cocaine reward and that drugs that block SERT are highly rewarding in DAT knockout mice. Hall, F.S., Li, X.F., Sora, I., Xu, F., Caron, M., Lesch, K.P., Murphy, D.L., and Uhl, G.R. Neuroscience, 115, pp. 153-158, 2002.

Identifying Human Gene Variants for Substance Abuse Vulnerability Over the past year, IRP scientists have identified 42 regions of human DNA that contain variations linked to drug abuse vulnerability. Variations in sixteen of these regions are "powerful" enough that they have been identified in studies from several different laboratories; in studies of individuals addicted to several different substances; and in individuals of several different ethnic backgrounds. Over the past year, IRP scientists have reported new laboratory and analytic technologies that can identify human DNA variants linked to drug abuse vulnerability while providing the maximal possible conservation of confidentiality for these sensitive subjects. Uhl, G.R., Liu, Q.R., and Naiman, D. Trends in Genetics, 18, pp. 420-425, 2002.

Cognition and Pharmacology Section, Neuroimaging Research Branch

Cognitive Mechanisms of Nicotine on Visual Attention Nicotine is known to improve performance on a range of cognitive tasks, notably attention and to a lesser extent, working memory in both humans and animals, which could contribute to smoking maintenance by improving concentration. As such, understanding nicotine's neurobiological and cognitive mechanisms may help explain both its addictive properties and potential therapeutic applications. To this end, functional magnetic resonance imaging (fMRI) was used to determine the neural substrates of nicotine's effects on a sustained attention (rapid visual information-processing) task. Task performance activated specific bilateral frontal, parietal, thalamic, occipital and cerebellar regions previously associated with sustained attention and working memory, with additional strong task-induced activations in the anterior insula and caudate. Decreased activation in left frontal, anterior and posterior cingulate, insula and left parahippocampal regions were also seen. Along with subtle behavioral deficits, mildly abstinent smokers showed less task-induced brain activation in the parietal cortex and caudate than did non-smokers. Application of a 21 mg nicotine patch to smokers improved task performance in smokers and increased task-induced BOLD activation in attention-related areas bilaterally including the parietal cortex, thalamus and caudate, while nicotine induced a generalized increase in occipital cortex activity. The nicotine patch also prevented the decline in mood ratings that followed task performance in smokers with placebo patch. Nicotine administration further deactivated some of the brain regions deactivated by the task, suggesting that nicotine improves attention in smokers by enhancing activation in areas traditionally associated with visual attention, arousal and motor activation in order to specifically focus attentional resources on task demands. Lawrence, N.S., Ross, T.J. and Stein, E.A. Neuron, 36, pp. 539-548, 2002.

Preclinical Pharmacology Section/Behavioral Neuroscience Branch

Synergistic Interaction Between Adenosine A2A and Glutamate mGlu5 Receptors: Implications for Striatal Neuronal Function The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in GABAergic striatal neurons is intriguing. Here, IRP scientists provide in vitro and in vivo evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor/receptor interactions. Colocalization of A2AR and mGluR5 at the membrane level was demonstrated in non-permeabilized HEK-293 cells transiently co-transfected with both receptors by confocal laser microscopy. Complexes containing A2AR and mGluR5 were demonstrated by Western-blotting of immunoprecipitates of either Flag-A2AR or HA-mGluR5 in membrane preparations from co-transfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparations. In co-transfected HEK-293 cells a synergistic effect on ERK1/2 phosphorylation and c-fos expression was demonstrated upon A2AR/mGluR5 co-stimulation. No synergistic effect was observed at the second messenger level (cAMP accumulation and intracellular calcium mobilization). Accordingly, a synergistic effect on c-fos expression in striatal sections and on counteracting phencyclidine-induced motor activation was also demonstrated after the central co-administration of A2AR and mGluR5 agonists to rats with intact dopaminergic innervation. The results suggest that a functional mGluR5/A2AR interaction is required to overcome the well-known strong tonic inhibitory effect of dopamine on striatal adenosine A2AR function. Ferre, S., Karcz-Kubicha, M., Hope, B.T., Popoli, P., Burgueno, J., Casado, V., Fuxe, K., Lluis, C., Goldberg, S.R., Franco, R. and Ciruela, F. Proc. Natl. Acad. Sci. USA, 99, pp. 11940-11945, 2002.

Caffeine Induces Dopamine and Glutamate Release in the Shell of the Nucleus Accumbens An increase in the extracellular concentration of dopamine in the nucleus accumbens is believed to be one of the main mechanisms involved in the rewarding and motor-activating properties of psychostimulants, such as amphetamine and cocaine. Using in vivo microdialysis in freely-moving rats, IRP investigators demonstrated for the first time that systemic administration of behaviorally relevant doses of caffeine can preferentially increase extracellular levels of dopamine and glutamate in the shell of the nucleus accumbens. These effects could be reproduced by administration of a selective adenosine A1 receptor antagonist, but not by a selective adenosine A2A receptor antagonist. This suggests that caffeine, due to its ability to block adenosine A1 receptors, shares neurochemical properties with other psychostimulants, which could contribute to the widespread consumption of caffeine-containing beverages. Solinas, M., Ferre, S., You, Z-B., Karcz-Kubicha, M., Popoli, P. and Goldberg, S.R. Journal of Neuroscience 22, pp. 6321-6324, 2002.

Caffeine Potentiates the Discriminative-stimulus Effects of Nicotine in Rats Caffeine and nicotine are the main psychoactive ingredients of coffee and tobacco, respectively, with a high frequency of concurrent use in humans. The aim of the present study was to examine the interaction of caffeine and nicotine in rats trained to discriminate nicotine from saline. Two groups of male Sprague-Dawley rats (n=8 per group) were trained to discriminate 0.4 mg/kg nicotine, SC, from saline under a fixed ratio schedule of food presentation. One group of rats was chronically exposed to caffeine (1.0 mg/ml) dissolved in their drinking water whereas the other group was exposed to tap water. Effects of IP injections of caffeine on nicotine-lever selection were subsequently examined. In separate groups of rats exposed to the same caffeine-drinking or water-drinking regimen, effects of caffeine pretreatment on nicotine plasma levels were evaluated. Although caffeine (1.0-30.0 mg/kg) did not generalize to nicotine when administered alone, it markedly potentiated discriminative-stimulus effects of the threshold dose of nicotine (0.05 mg/kg) in both water- and caffeine-drinking rats. Nicotine plasma levels were, however, not affected by acute or chronic caffeine exposure. Caffeine appears to enhance the discriminative-stimulus effects of the threshold dose of nicotine by a pharmacodynamic rather than a pharmacokinetic interaction. This suggests that caffeine consumption may be a contributing factor in the onset, maintenance of and relapse to tobacco dependence. Gasior, M., Jaszyna, M., Munzar, P., Witkin, J.M. and Goldberg S.R. Psychopharmacology 162, pp. 385-395, 2002.

Psychobiology Section, Medications Discovery Research Branch

Intravenous Cocaine Induced-activity and Behavioral Sensitization in Norepinephrine-, but not Dopamine-transporter Knockout Mice The present studies were designed to determine the respective roles of the norepinephrine (NET) and dopamine transporters (DAT) in the stimulant effects of acute and repeated cocaine utilizing knockout (KO) mice. Mice were habituated to the test environment for sufficient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine (3-25 mg/kg) intravenously according to a within-session cumulative dose-response design. Cocaine dosing was repeated at 48-hour intervals for four sessions to assess behavioral sensitization. NET-KO mice exhibited a reduced response to acute cocaine administration compared to wild-type (WT) controls. However, comparable sensitization developed in NET-KOs and WTs. The DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to cocaine. In contrast, the DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT-KO mice, these data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine. Mead, A.N., Rocha, B.A., Donovan, D.M. and Katz, J.L. European Journal of Neuroscience, 15, pp. 514-520, 2002.

Cocaine-induced Locomotor Activity and Cocaine Discrimination in Dopamine D2 Receptor Mutant Mice Dopamine D2-like antagonists block several effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects. Because these compounds generally lack selectivity among the D2-like dopamine receptors, the specific roles of the subtypes remain unclear. Dopamine D2 receptor knockout (DA D2R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D2 dopamine receptors in the effects of cocaine. Some effects of the relatively selective DA D2-like antagonist, raclopride were also studied to further assess the role of D2 receptors. DA D2R KO, HET and WT mice were treated with cocaine (1-10 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a 2 response-key food-reinforcement (FR 20) procedure. A range of doses of cocaine (1.0 - 17 mg/kg), was administered before 15-min test-sessions. Both DA D2R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D2R WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; tested doses of 1.0 - 10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. Raclopride, at inactive to fully active doses (0.1 - 1.0 mg/kg), did not fully substitute for cocaine. Raclopride dose-dependently shifted the cocaine dose-effect curve to the right in DA D2R WT and HET mice. However, in DA D2R KO mice raclopride was inactive as an antagonist. The present data indicate an involvement of D2 dopamine receptors in the locomotor-stimulating effects and the interoceptive discriminative-stimulus effects of cocaine in WT subjects. However, the D2 receptor is not necessary for the effects, suggesting redundant dopaminergic mechanisms for the discriminative-stimulus interoceptive effects of cocaine. Chausmer, A.L., Elmer, G.I., Rubinstein, M., Low, M.J., Grandy, D.K. and Katz, J.L. Psychopharmacology, 163, pp. 54-61, 2002.

Clinical Psychopharmacology Section, Medications Development Research Branch

Salvinorin A, an Hallucinogenic Plant Extract, is a Potent Naturally Occurring Non-nitrogenous Kappa Opioid Selective Agonist Salvia divinorum, whose main active ingredient is the neoclerodane diterpene Salvinorin A, is a hallucinogenic plant in the mint family that has been used in traditional spiritual practices for its psychoactive properties by the Mazatecs of Oaxaca, Mexico. More recently, S. divinorum extracts and Salvinorin A have become more widely used in the U.S. as legal hallucinogens. IRP scientists discovered that Salvinorin A potently and selectively inhibited (3)H-bremazocine binding to cloned kappa opioid receptors. Salvinorin A had no significant activity against a battery of 50 receptors, transporters, and ion channels and showed a distinctive profile compared with the prototypic hallucinogen lysergic acid diethylamide. Functional studies demonstrated that Salvinorin A is a potent kappa opioid agonist at cloned kappa opioid receptors expressed in human embryonic kidney-293 cells and at native kappa opioid receptors expressed in guinea pig brain. Importantly, Salvinorin A had no actions at the 5-HT2A serotonin receptor, the principal molecular target responsible for the actions of classical hallucinogens. Salvinorin A thus represents, to the authors' knowledge, the first naturally occurring nonnitrogenous opioid-receptor subtype-selective agonist. Because Salvinorin A is a psychotomimetic selective for kappa opioid receptors, kappa opioid-selective antagonists may represent novel psychotherapeutic compounds for diseases manifested by perceptual distortions (e.g., schizophrenia, dementia, and bipolar disorders). Additionally, these results suggest that kappa opioid receptors play a prominent role in the modulation of human perception. Roth, B.L., Baner, K., Westkaemper, R., Siebert, D., Rice, K.C., Steinberg, S., Ernsberger, P. and Rothman, R.B., Proc. Natl. Acad. Sci USA, 99, pp. 11934-11939, 2002.

Identification of a Novel Partial Inhibitor of Dopamine Transporter Among 4-Substituted 2-phenylquinazolines Using [125I]RTI-55 to label the DA transporter (DAT), IRP scientists have consistently detected one binding site as well as one component of [3H]DA uptake. Authors report here the identification of a novel partial inhibitor of [3H]DA uptake and DAT binding (SoRI-9804). [125I]RTI-55 binding to the DAT (mouse caudate, rat caudate, HEK cells expressing the cloned DAT), the 5-HT transporter (rat brain) and [3H]DA uptake (rat caudate synaptosomes) were conducted using published procedures. 4-[(Diphenylmethyl)amino]-2-phenylquinazoline (SoRI-9804) was essentially inactive at SERT binding and resolved two DAT binding components in all 3 tissues, having high affinity (mean Ki of 465 nM) for about 40% of the binding sites and an essentially immeasurable Ki (> 100 µM) for the remaining 60% of the binding sites. The [3H]DA uptake experiments indicated that about 50% of uptake was SoRI-9804-sensitive. Saturation binding experiments showed that SoRI-9804 competitively inhibited [125I]RTI-55 binding to the SoRI-9804-sensitive binding component. Viewed collectively, the present results indicate that SoRI-9804 discriminates two components of the DA transporter. Further studies will be needed to determine the underlying mechanism of this effect and if partial inhibition of DA uptake results in any unique behavioral effects. Rothman, R.B., Dersch, C.M., Carroll, F.I. and Ananthan, S. Synapse, 43, pp. 268-274, 2002.

Persistent Antagonism of Methamphetamine-Induced Dopamine Release in Rats Pretreated with GBR12909 Decanoate Methamphetamine abuse is a serious global health problem and no effective pharmacological treatments have yet been developed for methamphetamine dependence. In animals, the addictive properties of methamphetamine are mediated via release of dopamine (DA) from nerve terminals in mesolimbic reward circuits. At the molecular level, methamphetamine promotes DA release by a nonexocytotic diffusion-exchange process involving DA transporter (DAT) proteins. IRP scientists have shown that blocking DAT activity with high-affinity DA uptake inhibitors, such as 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl) piperazine (GBR12909), can substantially reduce amphetamine-induced DA release in vivo. In the present study, the authors examined the ability of a long-acting depot formulation of GBR12909 decanoate (GBR-decanoate) to influence neurochemical actions of methamphetamine in the nucleus accumbens of rats. Rats received single injections of GBR-decanoate (480 mg/kg i.m.) and were subjected to in vivo microdialysis testing 1 and 2 weeks later. Pretreatment with GBR-decanoate produced modest elevations in basal extracellular levels of DA, but not 5-hydroxytryptamine (5-HT), at both time points. GBR-decanoate nearly eliminated the DA-releasing ability of methamphetamine (0.3 and 1.0 mg/kg i.v.) for 2 weeks, whereas methamphetamine-induced 5-HT release was unaffected. Autoradiographic analysis revealed that GBR-decanoate caused long-term decreases in DAT binding in the brain. These data suggest that GBR-decanoate, or similar agents, may be useful adjuncts in treating methamphetamine dependence. This therapeutic strategy would be especially useful for noncompliant patient populations. Baumann, M.H, Ayestas, M.A., Sharpe, L.G., Lewis, D.B., Rice, K.C. and Rothman, R.B., Journal of Pharmacology and Experimental Therapeutics, 301, pp. 1190-1197, 2002.

Medicinal Chemistry Section, Medications Discovery Research Branch

Enantioselective Synthesis of Novel Probes for the Dopamine Transporter Extensive structure-activity relationships at the dopamine transporter (DAT) have been developed around two classes of tropane-based ligands. Significant chemical modification at the 2-position in the 3-aryltropane (cocaine) class is well tolerated at the DAT, although optimal binding affinity results from the 2-substituent being in the R-configuration. In the benztropine class, a substituent need not be in the 2-position to bind to the DAT with high affinity. However, if a substituent (ex. COOMe) is placed in the 2-position it must be in the S-configuration, in order to bind. This opposing stereoselectivity suggests that these tropane-based DAT inhibitors may not access identical binding domains. In order to further investigate this hypothesis, synthesis of a series of pure S-(+)-2b-carboalkoxy-3a-[bis(4-fluorophenyl) methoxy]tropanes (>99% ee) was achieved by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high affinity binding for DAT (Ki=13-40 nM). These data support previous findings of significant divergence in structural requirements for high affinity DAT binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3a-[bis(4-fluorophenyl)methoxy] tropane series is well tolerated at the DAT but not at SERT (Ki=690-2040 nM), or muscarinic M1 receptors (Ki=133-4380 nM) resulting in highly selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic. Zou, M-F., Agoston, G.E., Belov, Y., Kopajtic, T., Katz, J.L., and Newman, A.H. Bioorganic Medicinal Chemistry Letters, 12, pp. 1249-1252, 2002.

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

Cognitive Measures in Long-term Cannabis Users The cognitive effects of long-term cannabis use are insufficiently understood. Most studies concur that cognitive deficits persist at least several days after stopping heavy cannabis use. But studies differ on whether such deficits persist long-term, or whether deficits are correlated with increasing duration of lifetime cannabis use. IRP scientists administered neuropsychological tests to 77 current heavy cannabis users who had smoked cannabis at least 5000 times in their lives, and to 87 control subjects who had smoked no more than 50 times in their lives. The heavy smokers showed deficits on memory of word lists on Days 0, 1, and 7 of a supervised abstinence period. By Day 28, however, very few significant differences were found between users and controls on any test, and the authors found no significant associations between total lifetime cannabis consumption and test performance. Although these findings may be affected by residual confounding, as in all retrospective studies, they suggest that cannabis-associated cognitive deficits are reversible and related to recent cannabis exposure, rather than irreversible and related to cumulative lifetime use. Pope, H.G. Jr., Gruber, A.J., Hudson, J.I., Huestis, M.A., and Yurgelun-Todd, D. Journal of Clinical Pharmacology, 42, pp. 41S-47S, 2002.

Urinary Elimination of Cocaine Metabolites in Chronic Cocaine Users During Cessation In an earlier study, IRP scientists showed that chronic cocaine use by active illicit users produced a longer plasma half-life than expected based on acute low-dose cocaine studies. This study reports urinary excretion patterns of cocaine metabolites as benzoylecgonine (BE) equivalents from 18 of the same individuals, housed for up to 14 days on a closed research unit. In addition, the researchers evaluated whether creatinine normalization of BE equivalents increased mean detection time and reduced mean within-subject variability. All urine voids (N=953) were individually assayed; BE equivalents were determined semi-quantitatively by FPIA (TDx®, Abbott Laboratories, Abbott Park, IL). Compared to concentration in first void after admission, BE equivalents decreased to approximately 33%, 8%, and 4% at 24, 48, and 72 hours, respectively. Mean +/- SD (range) time to first negative specimen (BE equivalents <300 ng/mL) was 43.6 +/- 17.1 (16-66) hours. BE equivalents fluctuated considerably across successive specimens; 69% of participants tested positive at least once after testing negative, and the mean time to last positive specimen was 57.5 +/- 31.6 (11-147) hours after the first specimen. Thus, mean cocaine metabolite detection times were consistent with prolonged elimination, with 63% of participants testing positive longer than the expected 48-hour window of detection after admission to the unit. Mean time to last positive after last use of cocaine, known by self-report only, was approximately 81 +/- 34 [range 34 - 162] hours. Creatinine normalization, with the cutoff of 300 ng BE equivalents/mg creatinine, increased detection time: mean time to first negative specimen was 54.8 +/- 20.7 (20-100) hours, and mean time to last positive specimen was 88.4 +/- 51.0 (35.6-235) hours. Compared to concentration in the first void after admission, BE equivalents/creatinine decreased to approximately 56%, 6%, and 5% at 24, 48, and 72 hours. However, creatinine normalization did not reduce the fluctuation of BE equivalents across successive specimens. Thus, creatinine normalized values may be useful when the goal is to maximize the probability or duration of cocaine metabolite detection, but may be less useful in determining whether an individual has used cocaine since a previous specimen collection. Preston, K.L., Epstein, D.H., Cone, E.J., Wtsadik, A.T., Huestis, M.H. and Moolchan, E.T. Journal of Analytical Toxicology, 26, pp. 393-400, 2002.

Detection Times of Methamphetamine and Amphetamine In Urine Following Oral Administration of Methamphetamine To Humans Confirmation of a workplace drug test requires urinary methamphetamine (MAMP) and amphetamine (AMP) concentrations ³500 and 200 µg/L, respectively, but cutoffs at half those values (250/100) have been proposed. IRP researchers determined the urinary excretion of MAMP after oral ingestion and examined the effect of using lower cutoffs on detection of exposure. Subjects (n=8) ingested four 10-mg doses of MAMP•HCl daily over seven days and five of them ingested four 20-mg doses four weeks later. Subjects collected all urine specimens for two weeks. After solid phase extraction, MAMP and AMP were measured by GC/PCI-MS with dual silyl derivatization. MAMP and AMP were generally detected in the first or second void (0.7-11.3 h) collected following drug administration with concentrations of 82-1,827 and 12-180 µg/L, respectively. Peak MAMP concentrations (1,871-6,004 μg/L) following single doses occurred within 1.5-60 h. MAMP >=500 µg/L was first detected in the 1st or 2nd void (1-11 h) at 524-1,871 μg/L. Lowering the MAMP cutoff to 250 μg/L changed the initial detection time little. AMP >=200 μg/L was first detected in the 2nd-13th (7-20 h) post-administration void. At a cutoff of 100 μg/L, AMP was first confirmed in the 2nd-8th void (4-13 h). Reducing the cutoff to 250/100 μg/L extended terminal MAMP detection by up to 24 h, increased total detection time by up to 34 h and increased the total number of positive specimens by 48%. At the lower cutoff, initial detection times are earlier, detection windows are longer and confirmation rates are increased. Elimination of the AMP requirement would increase detection rates and allow earlier detection. Oyler, J.M., Cone, E.J., Joseph, Jr., R.E., Moolchan, E.T., and Huestis, M.A. Clinical Chemistry, 48, pp. 1703-1714, 2002.

Pharmacokinetics and Pharmacodynamics Following Oral Codeine Administration The ease, non-invasiveness and safety of oral fluid collection have increased the use of this alternative matrix for drugs of abuse testing; however, few controlled drug administration data are available to aid in the interpretation of oral fluid results. Single 60 and 120 mg/70kg oral codeine doses were administered to 19 volunteers. Oral fluid and plasma were analyzed for free codeine, norcodeine, morphine and normorphine by SPE/GC-MS. Physiological and subjective effects were examined. Mean peak codeine concentrations were 214.2+/-27.6 and 474.3±77.0 mg/L in plasma and 638.4+/-64.4 and 1599.3+/-241.0 mg/L in oral fluid. Codeine S/P was relatively constant (approximately 4) from 1-12h. Mean t1/2 of codeine was 2.2±0.10h in plasma and 2.2+/-0.16h in oral fluid. Significant dose-related miosis and increases in sedation, psychotomimetic effect and "high" occurred after the high dose. Mean codeine oral fluid detection time was 21h with a 2.5 mg/L cutoff, longer than that of plasma (12 to 16h). Detection times with the proposed SAMHSA cutoff (40 mg/L) were only 7h. Norcodeine, but no morphine or normorphine, was quantitated in either matrix. The disposition of codeine over time was similar in plasma and oral fluid; however, due to high variability, oral fluid codeine concentrations did not reliably predict concurrent plasma concentrations. Oral fluid testing is a useful alternative matrix for monitoring codeine exposure with a detection window of 7 to 21h for single doses, depending upon cutoff concentration. These controlled drug administration data should aid the interpretation of oral fluid codeine results. Kim, I., Barnes, A.J., Oyler, J.M., Schepers, R., Joseph, Jr., R.E., Cone, E.J., Lafko, D., Moolchan, E.T., and Huestis, M.A. Clinical Chemistry, 48, pp. 1486-1497, 2002.

Drug Abuse's Smallest Victims: In Utero Drug Exposure The social and economic impact of drug use on our global population continues to increase leaving no geographical, social or cultural group untouched. The National Institute on Drug Abuse, in one of the few large surveys of maternal abuse, found that 5.5% of mothers reported taking an illicit substance during gestation. Accurate identification of in utero drug exposure has important implications for the care of the mother and child, but can raise difficult legal issues. Society discourages prenatal care with the infliction of harsh criminal penalties. Maternal drug use during pregnancy can be monitored with urine, sweat, oral fluid and/or hair testing. Detection of in utero drug exposure has traditionally been accomplished through urine testing; however, the window of detection is short, reflecting drug use for only a few days before delivery. Monitoring exposure through testing of alternative matrices, such as neonatal meconium and hair, offers advantages including non-invasive collection and detection earlier in gestation. There are many unresolved issues in monitoring in utero drug exposure that urgently require research. These can be divided into research to definitively differentiate drug exposed and non-drug exposed fetuses, determine the most efficient methods to routinely monitor women's drug use, and determine how these drug test results relate to neonatal and maternal outcomes. Research in this area is difficult and expensive to perform, but necessary to accurately assess drug effects on the fetus. By increasing our understanding of the physiological, biochemical and behavioral effects of gestational drug exposure, we may ultimately provide solutions for better drug prevention, treatment and a reduction in the number of drug-exposed children. Huestis, M.A. and Choo, R.E. Forensic Science International, 128, pp. 20-30, 2002.

Eclipse Can Deliver Crack The potential of Eclipse, a nicotine delivery device recently introduced by R.J. Reynolds Tobacco, to deliver crack cocaine was assessed in experiments utilizing a smoking machine. The quantity of cocaine delivered in smoke from crack cocaine inserted into the Eclipse cigarette was measured by gas chromatography-mass spectrometry. The Eclipse delivered 1.8 to 16.5% of the available cocaine. Eclipse could be used to deliver other drugs such as heroin, amphetamine or PCP, obviating the need to use easily identified drug paraphernalia. Steckley, S.L., Darwin, W.D., Huestis, M., Henningfield, J.E., and Pickworth, W.B. Nicotine and Tobacco Research, Suppl. pp. 189-190, 2002.

New Mass Spectrometry Technology Recently, a new atmospheric pressure ionization technique, atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI), has been introduced by Laiko and coworkers. This source has been coupled to an orthogonal time-of-flight mass spectrometer and, more recently, to an ion trap mass spectrometer (ITMS). Here, IRP investigators present the current status of work involving the development of an AP MALDI source for an ITMS. In addition, the authors present recent work from their own laboratory to demonstrate the utility of this novel configuration. Moyer, S.C., Marzilli, L.A., Laiko, V.V., Doroshenko, V.M., Woods, A.S., and Cotter, R.J. Atmospheric Pressure Matrix Assisted Laser Desorption/Ionization Mass Spectrometry (AP MALDI) On a Quadrupole Ion Trap Mass Spectrometer Int J. Mass Spec. 12299, pp. 1-18, 2002.