Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

An In Vitro Model of Human Dopaminergic Neurons Derived from Embryonic Stem Cells: MPP(+) Toxicity and GDNF Neuroprotection Human embryonic stem cells (hESCs) can proliferate indefinitely yet also differentiate in vitro, allowing normal human neurons to be generated in unlimited numbers. Here, IRP investigators describe the development of an in vitro neurotoxicity assay using human dopaminergic neurons derived from hESCs. The authors showed that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), which produces features of Parkinson's disease in humans, was toxic for hESC-derived dopaminergic neurons. Treatment with glial cell line-derived neurotrophic factor protected tyrosine hydroxylase-positive neurons against MPP(+)-induced apoptotic cell death and loss of neuronal processes as well as against the formation of intracellular reactive oxygen species. The availability of human dopaminergic neurons, derived from hESCs, therefore allows for the possibility of directly examining the unique features of human dopaminergic neurons with respect to their responses to pharmacological agents as well as environmental and chemical toxins. Zeng, X., Chen, J., Deng, X., Liu., Y., Rao, M., Cadet, J.L., and Freed, W.J. Neuropsychopharmacology, 31(12), pp. 2708-2715, 2006.

GABAergic Lineage Differentiation of AF5 Neural Progenitor Cells In Vitro AF5 neural cells derived from fetal rat mesencephalic tissue were immortalized with a truncated SV40 LT vector lacking the p53-inactivating domain to maintain long-term cultures with a p53-responsive phenotype. This study examined p53 function in producing programmed cell death in propagating AF5 neural cells after exposure to hydrogen peroxide (H2O2) and the kinase inhibitor staurosporine (STSP). Concentration-dependent exposure of AF5 cells to 0-800 mM H2O2 and STSP at 0-1000 nM revealed increasing cytotoxicity from MTS cell viability assays. Apoptosis occurred at 400 mM H2O2 as evidenced by subG1 DNA and Annexin V flow cytometry analyses and cellular immunofluorescence staining with propidium iodide, anti-Annexin V and DAPI. DNA fragmentation, caspase-3/7 activity and cytochrome c release into cytosol also confirmed H2O2-mediated apoptotic events. p53 protein levels were increased over 24 h by H2O2 in a coordinated fashion with mdm2 expression. p53 activation by H2O2 was evidenced by elevated Ser15 phosphorylation, increased luciferase p53 reporter activity and upregulation of the downstream p53 targets p21(waf1) and apoptotic proteins, bax, Noxa and PUMA. STSP exposure produced apoptosis demonstrated by DNA fragmentation, caspase-3/7 activity, cytochrome c release and over 24 h was accompanied by sustained increase in p53 and Ser15 phosphorylation, rise in p21(waf1) and bax and a transient increase in p53 reporter activity but without Annexin V binding. These findings demonstrate that AF5 cells undergo apoptosis in response to H2O2-mediated oxidative stress and signal pathway disruption by STSP that therefore would be useful in studies related to p53-dependent neuronal cell death and neurodegeneration. McNeill-Blue, C., Wetmore, B.A., Sanchez, J.F., Freed, W.J., and Merrick, B.A. Brain Res, 1112(1), pp. 1-15, 2006.

Cellular Pathobiology Unit, Development and Plasticity Section, Cellular Neurobiology Research Branch

Chronic Antidepressants Potentiate via Sigma-1 Receptors the Brain-Derived Neurotrophic Factor-Induced Signaling for Glutamate ReleaseUp-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-gamma (PLC-gamma)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. J. Biol. Chem. 277, pp. 6520-6529, 2002), IRP investigators examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T.P. Proc. Natl. Acad. Sci. U. S. A. 98, pp. 491-496, 2001), authors examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). Authors found that the BDNF-stimulated PLC-gamma activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-gamma and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release. Yagasaki, Y., Numakawa, T., Kumamaru, E., Hayashi, T., Su, T.P., and Kunugi, H. Journal of Biological Chemistry, 281(18), pp. 12941-12949, 2006.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Allosteric Modulation of Dopamine D2 Receptors by Homocysteine It has been suggested that L-DOPA-induced hyperhomocysteinemia can increase the risk of stroke, heart disease, and dementia and is an additional pathogenetic factor involved in the progression of Parkinson's disease. In Chinese hamster ovary (CHO) cells stably cotransfected with adenosine A(2A) and dopamine D2 receptors, homocysteine selectively decreased the ability of D2 receptor stimulation to internalize adenosine A(2A)-dopamine D2 receptor complexes. Radioligand-binding experiments in the same cell line demonstrated that homocysteine acts as an allosteric D2 receptor antagonist, by selectively reducing the affinity of D2 receptors for agonists but not for antagonists. Mass spectrometric analysis showed that, by means of an arginine (Arg)-thiol electrostatic interaction, homocysteine forms noncovalent complexes with the two Arg-rich epitopes of the third intracellular loop of the D2 receptor, one of them involved in A(2A)-D2 receptor heteromerization. However, homocysteine was unable to prevent or disrupt A(2A)-D2 receptor heteromerization, as demonstrated with Fluorescence Resonance Energy Transfer (FRET) experiments in stably cotransfected HEK cells. The present results could have implications for Parkinson's disease. Agnati, L.F., Ferre, S., Genedani, S., Leo, G., Guidolin, D., Filaferro, M., Carriba, P., Casado, V., Lluis, C., Franco, R., Woods, A.S., and Fuxe, K. Journal of Proteome Research, 5(11), pp. 3077-3083, 2006.

Molecular Neurobiology Research Branch

Whole Genome Association Studies of Human Addiction Vulnerability Addictions are substantially-heritable complex disorders. This work reports whole genome association studies that identify 88 genes likely to contain variants that contribute to vulnerability to addictions. Each of these genes contains clustered SNPs that display significant allele frequency differences between abusers and controls in each of two samples studied with 639,401 SNP arrays and confirmatory SNPs from each of two other abuser/control samples studied with 100k arrays. These genes are implicated in interesting functions that include "cell adhesion" processes that are important for establishing and maintaining neuronal connections of special relevance to addiction's memory-like features. Liu, Q.R., Drgon, T., Johnson, C., Walther, D., Hess, J., and Uhl, G.R. Am J Med Genet B Neuropsychiatr Genet. 2006 Nov 10; [Epub ahead of print].

Fine Mapping of Human Addiction Vulnerability Loci: Chromosome 4 GABA A Receptor Gene Cluster Strong genetic contributions to individual differences in vulnerability to addictions are well supported by classical genetic studies. Linkage and association genome scans for addiction vulnerability have provided converging evidence for several chromosomal regions which are likely to harbor allelic variants that contribute to such vulnerability. IRP scientists and others have delineated a candidate addiction-associated chromosome 4p12 "rSA3" region based on convergent data from association genome scanning studies in polysubstance abusers (Uhl and others 2001), linkage based studies in alcoholism (Long and others 1998; Reich and others 1998) and association-based studies for alcoholism and association-based studies for individual differences in electroencephalographic (EEG) spectral power phenotypes (Edenberg and others 2004; Porjesz and others 2002). The rSA3 region contains interesting candidate genes that encode the alpha2, alpha4, beta1 and gamma1 receptor subunits for the principal brain inhibitory neuron-transmitter, GABA (Covault and others 2004; Edenberg and others 2004; Lappalainen and others 2005). The authors now report assessment of single nucleotide polymorphism (SNP) genotypes in this region in three samples of substance abusers and controls. These results delineate the haplotypes and patterns of linkage disequilibrium in this region, focus attention of the GABRA2 gene and identify modest associations between GABRA2 genotypes and addiction phenotypes. These results are consistent with modest roles for GABRA2 variants in addiction vulnerabilities. Drgon, T., D'Addario, C., and Uhl, G.R. Am J Med Genet B Neuropsych Genet., 2006.

CB2 Receptors in Brain Brain expression of CB2 cannabanoid receptors has been much less well established and characterized in comparison to the expression of brain CB1 receptors. Since CB2 receptors are intensely expressed in peripheral and immune tissues, expression in brain microglia has been anticipated. This work describes expression of CB2 receptor like immunoreactivity in brain in neuronal patterns that support broader CNS roles for this receptor. These studies, and initial RT-PCR and in situ hybridization analyses of brain CB1 and CB2 mRNAs, also supported brain CB2 expression at levels much lower than those of CB1 receptors. Immunohistochemical analyses revealed abundant CB2 immunostaining in apparent neuronal and glial processes in a number of brain areas. Cerebellar Purkinje cells and hippocampal pyramidal cells revealed substantial immunoreactivity that was absent when sections were stained with preadsorbed sera. CB2 immunoreactivity was also observed in olfactory tubercle, islands of Calleja, cerebral cortex, striatum, thalamic nuclei, hippocampus, amygdale, substantia nigra, periaqueductal gray, paratrochlear nucleus, paralemniscal nucleus, red nucleus, pontine nuclei, inferior colliculus and the parvicellular portion of the medial vestibular nucleus. CB2 immunoreactivity was also present in cells of primary hippocampal cultures. Two anti-CB2 affinity purified polyclonal antibodies were raised in rabbits immunized with peptide conjugates that corresponded to amino acids 1-33 and 20-33. Western blot analyses revealed specific bands that were identified using these sera and were absent when the sera were preadsorbed with 8.3 The multifocal expression of CB2 immunoreactivity in neuronal and glial patterns in a number of brain regions suggests reevaluation of the possible roles that CB2 receptors may play in the brain. Onaivi, E.S., Ishiguro, H., Gong, J.P,, Patel, S., Perchuk, A., Meozzi, P.A., Myers, L., Mora, Z., Tagliaferro, P., Gardner, E., Brusco, A., Akinshola, B.E., Liu, Q.R., Hope, B., Iwasaki, S., Arinami, T., Teasenfitz, L., and Uhl, G.R. Ann N Y Acad Sci. 1074, pp. 514-553, August 2006.

Psychobiology Section, Medications Discovery Research Branch

The Effects of Dopamine Receptor Agonists on Food-maintained Operant Behavior Dopamine D1-like agonists have been proposed as potential treatments for cocaine abuse, however, the pharmacology of these drugs is complex and poorly understood. IRP investigators compared the effects on behavior of several D1 dopamine receptor agonists (SKF 38393, SKF 77434 and SKF 82958), both alone and in combination with the D1-receptor antagonist, SCH 23390. Each drug produced dose related reductions in the rate of a learned behavior, in order of potency: SKF 82958 > SKF 77434 > SKF 38393. Antagonism of these behavioral effects by SCH 23390 was only significant for SKF 82958; surprisingly, SCH 23390 enhanced the effects of SKF 38393. For SKF 82958, the antagonism was receptor subtype-specific, since the D2-receptor antagonist spiperone was ineffective. The non-selective serotonergic antagonist metergoline produced a significant rightward shift of the SKF 38393 dose-response function, indicating effective antagonism. Because the behavioral effects of D1-receptor agonists differed in their susceptibility to antagonism by D1-receptor antagonists, the results support the view of substantial mechanistic differences among drugs suggested to act through D1 dopamine receptors, and that the manner in which they produce their behavioral effects needs to be carefully studied with their evaluation as potential treatments for cocaine abuse. Katz, J.L., Kopajtic, T.A. and Terry, P. Behavioural Pharmacology, 17, pp. 303-309, 2006.

Medicinal Chemistry Section, Medications Discovery Research Branch

Structure-Activity Relationship Studies on a Novel Series of Benztropine Analogs for In Vivo Investigation IRP scientists have discovered that in general, benztropine-based dopamine uptake inhibitors do not demonstrate cocaine-like pharmacological activity in animal models of psychostimulant abuse and have been proposed as potential medications for the treatment of cocaine addiction. However, several (S)-2-carboalkoxy-substituted-analogues were discovered to stimulate locomotor activity and substitute in subjects trained to discriminate cocaine, suggesting a role of the 2-position substituent in mediating these cocaine-like actions. Hence, a novel a series of N- and 2-substituted-benztropine analogs were designed and synthesized to further explore the pharmacological profiles of this class of compounds. Most of these analogues demonstrated high binding affinities to the dopamine transporter (DAT; Ki=1.8-40 nM), and were selective over the other monoamine transporters and muscarinic M1 receptors. For example, when the (S)-2-carboalkoxy substituent was replaced with (S)-2-ethenyl, the resulting analogue demonstrated the highest DAT binding affinity in the series (Ki=1.81 nM) with DAT selectivity over serotonin transporters (SERT; 989-fold), norepinephrine transporters (NET; 261-fold) and muscarinic receptors (90-fold). The behavioral profiles of several analogues were evaluated and it was discovered that manipulation of the 2-, N- and 3-position substituents in the benztropine class of dopamine uptake inhibitors can result in ligands with high affinity and selectivity for the DAT, but distinctive in vivo pharmacological profiles that cannot be predicted by their effects in vitro. Zou, M.-F., Cao, J., Kopajtic, T., Desai, R. I., Katz, J. L., Newman, A. H. Journal of Medicinal Chemistry 49, pp. 6391-6399, 2006.

Design and Synthesis of a Novel Photoaffinity Ligand for the Dopamine and Serotonin Transporters Tropane-based photoaffinity ligands covalently bind to discrete points of attachment on the dopamine transporter (DAT). To further explore structure-activity relationships, a ligand in which the photoactivatable azido-group was extended from the 3-position of the tropane ring was synthesized from cocaine via a Stille or Suzuki coupling strategy. The resulting ligand, JHC 2-48, demonstrated high binding affinity for the DAT (Ki=15.1+/-2.2 nM). Moreover, this compound showed moderate binding affinity for the serotonin transporter (SERT, Ki=109+/-14 nM) suggesting the potential utility of the radioligand [125I]JHC 2-48 in both DAT and SERT protein structure studies. Newman, A.H., Cha, J.H., Cao, J., Kopajtic, T., Katz, J.L., Parnas, M.L., Vaughan, R., and Lever, R., Journal of Medicinal Chemistry, published online October 5, 2006.

Novel Analogues of the Dopamine D2 Receptor Antagonist L741,626 A series of analogues of the dopamine D2 receptor antagonist L741,626 were synthesized and evaluated for binding and function at D2 family receptor subtypes. Several analogues showed comparable binding profiles to the parent ligand, however, in general, chemical modification served to reduce D2 binding affinity and selectivity. Nevertheless, one analogue demonstrated a comparable binding profile to the parent ligand and may have utility in in vivo studies of drug addiction. Grundt, P., Husbands, S.L.J., Luedtke, R.R., Taylor, M., and Newman, A.H., Bioorganic Medicinal Chemistry Letters, published online October 29, 2006.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Amphetamine Analogs Increase Plasma Serotonin: Implications for Cardiac and Pulmonary Disease Elevations in plasma serotonin (5-HT) have been implicated in the pathogenesis of cardiac and pulmonary disease. Normally, plasma 5-HT concentrations are kept low by transporter-mediated uptake of 5-HT into platelets and by metabolism to 5-hydroxyindoleacetic acid (5-HIAA). Many abused drugs (e.g., substituted amphetamines) and prescribed medications (e.g., fluoxetine) target 5-HT transporters and could thereby influence circulating 5-HT. IRP scientists evaluated the effects of amphetamines analogs [(+/-)-fenfluramine, (+/-)-3,4-methylenedioxymethamphetamine, (+)-methamphetamine, (+)-amphetamine, phentermine] on extracellular levels (i.e., plasma levels) of 5-HT and 5-HIAA in blood from catheterized rats. Effects of the 5-HT uptake blocker fluoxetine were examined for comparison. Drugs were tested in vivo and in vitro; plasma indoles were measured using a novel microdialysis method in whole blood. Authors found that baseline dialysate levels of 5-HT are approximately 0.22 nM, and amphetamine analogs evoke large dose-dependent increases in plasma 5-HT ranging from 4 to 20 nM. The ability of drugs to elevate plasma 5-HT is positively correlated with their potency as 5-HT transporter substrates. Fluoxetine produced small, but significant, increases in plasma 5-HT. Although the drug-evoked 5-HT concentrations are below the micromolar levels required for contraction of pulmonary arteries, they approach concentrations reported to stimulate mitogenesis in pulmonary artery smooth muscle cells. Additional studies are needed to determine the effects of chronic administration of amphetamines on circulating 5-HT. Zolkowska, D., Rothman, R.B., and Baumann, M.H. J Pharmacol Exp Ther. 318, pp. 604-610, 2006.

Neuropsychopharmacology Section, Chemical Biology Research Branch

Slow-Onset Long-Duration Methylphenidate Analogs with Increased Selectivity for the Dopamine Transporter IRP scientists have previously synthesized indanamine-structure molecules that produce exceedingly slow-onset long-duration inhibition of presynaptic dopamine reuptake in the addiction-related synapses of the nucleus accumbens of the forebrain. Now, these scientists have synthesized slow-onset long-duration dopamine reuptake inhibitors of the methylphenidate chemical class. Based on a molecular modeling/superimposition model of methylphenidate with tropane-containing compounds, they hypothesized that methylphenidate analogs with the ester moiety replaced by an alkyl group should be active and have longer durations of action - potentially useful as anti-addiction medications. Many analogs were synthesized and tested using transfected human monoamine transporters. Many of the RR/SS diastereomers proved to have low nanomolar potencies in the transporter assays, and compounds with a para-Cl group had good selectivity for the dopamine transporter. In the 3,4-diCl series, RS/SR diastereomers showed significant, low nanomolar activity. In a rodent locomotor assay, one of the 4-Cl compounds (CTDP-32,476) showed a slow-onset long-duration profile that may be predictive of clinical utility as an anti-addiction medication. This same compound also showed a slow-onset, long-duration profile as assessed by in vivo brain microdialysis and by electrical brain-stimulation reward in laboratory animals - profiles that have in the past been predictive of potential clinical utility. One 4-Cl compound in the series (CTDP-32,648) met the dopamine selectivity criteria of the NIDA Cocaine Treatment Discovery Program and is being advanced by them as a possible treatment for cocaine abuse. Froimowitz, M., Gu, Y., Dakin, L.A., Kelley, C.J., Parrish, D., Deschamps, J.R., Pak, A.C., Gilbert, J.G., Peng, X.-Q., Xi, Z.-X. and Gardner, E.L., Poster, 2006. College on Problems of Drug Dependence Annual Meeting, Scottsdale, AZ, June 17-22, 2006.

Methadone Pretreatment Attenuates Heroin's Rewarding Effects and Heroin-Induced Dopamine Release in the Nucleus Accumbens: Comparison to the Effects of CTDP-31,345, a Long-Acting Dopamine Transporter Inhibitor IRP scientists have been investigating the possible utility of slow-onset long-duration monoamine transporter inhibitors as potential pharmacotherapies for psychostimulant addiction. In a conceptually analogous vein, methadone and l-_-acetyl-methadol (long-acting opiate receptor agonists), have proven to be successful treatments for opiate addiction. The long-acting dopamine transporter (DAT) inhibitors CTDP-31,345 or CTDP-32,476 have additive effects with cocaine in preclinical animal test systems. To further evaluate the potential utility of these DAT inhibitors for the treatment of psychostimulant addiction, these scientists observed the effects of methadone pretreatment on heroin self-administration and heroin-induced dopamine release in the nucleus accumbens. They found that methadone inhibited heroin self-administration in rats. In vivo microdialysis demonstrated that methadone significantly elevated extracellular dopamine, lasting for about 4 hours. However, pretreatment with methadone significantly attenuated acute heroin-induced increases in extracellular dopamine in the nucleus accumbens. Thus, methadone pretreatment appears to attenuate heroin-induced DA release, contrary to the effects of DAT inhibitors on cocaine-induced increases in nucleus accumbens dopamine. These data suggest that the analogy between methadone for opiate addiction and slow-onset long-duration DAT inhibitors for psychostimulant addiction may be more analogy than homology. Although more studies will be required to further determine the mechanisms of actions produced by methadone and DAT inhibitors, respectively, the present data raise serious conceptual concerns about the utility of slow-onset long-acting DAT inhibitors for the pharmacotherapeutic treatment of psychostimulant addiction. Peng, X.-Q., Xi, Z.-X., Li, X., Gilbert, J.G., Pak, A.C., Froimowitz, M. and Gardner, E.L., Poster, 2006. Society for Neuroscience Annual Meeting, Atlanta, GA, October 14-18, 2006.

Gamma-Vinyl GABA Inhibits Cocaine-Primed Relapse to Drug-Seeking Behavior by a Dopamine-Independent Mechanism IRP scientists have previously shown that gamma-vinyl GABA (GVG), a suicide inhibitor of GABA transaminase in the brain, shows a promising anti-addiction profile in preclinical animal models related to addiction. In the present research, these same scientists investigated whether and how GVG inhibits cocaine-triggered relapse to drug-seeking behavior in laboratory rats. Systemic administration of GVG (25-300 mg/kg i.p.) dose-dependently inhibited cocaine-triggered relapse. However, the mechanism appears to be DA-independent, because GVG pretreatment failed to block cocaine-induced increases in extracellular dopamine (DA) in the reward- and relapse-related nucleus accumbens. GVG alone also failed to alter extraellular DA. In contrast, GVG pretreatment produced an additive or synergistic increase with cocaine on extracellular glutamate, and dose-dependently elevated extracellular GABA levels. Finally, GVG-induced increase in glutamate is tetrodotoxin-dependent, while GVG-induced increases in GABA were partially blocked by blockade of type 1 GABA transporters. Together, the present data, for the first time, demonstrate that GVG inhibits cocaine-triggered relapse by a mechanism correlated to GVG-induced increases in GABA and/or glutamate, but not to a decrease in cocaine-induced increases in nucleus accumbens DA. Gardner, E.L., and Xi, Z.-X. Acta Pharmacologica Sinica, 51[suppl.1], p. 96, 2006.

S-(+)-Gamma Vinyl GABA (S-GVG) Blocks the Response to Methamphetamine in Adolescent and Adult Animals Treated with Methamphetamine and S-GVG During Adolescence IRP scientists have previously shown that gamma-vinyl GABA, a suicide inhibitor of GABA transaminase in the brain, shows a promising anti-addiction profile in preclinical animal models related to addiction. In the present research, these same scientists used small animal neuroimaging with [11C]-raclopride and [18F]-fluorodeoxyglucose positron emission tomography (PET) to examine the effects of S-GVG on methamphetamine-induced increases in brain dopamine and metabolism, respectively. Adolescent rats received baseline PET scans, then received a methamphetamine challenge followed by more PET scans. Methamphetamine significantly reduced the striatal binding of [11C]-raclopride (i.e., caused an increase in extracellular dopamine) and increased [18F]-fluorodeoxyglucose cortically, subcortically, and in the cerebellum. There were no effects of methamphetamine on [18F]-fluorodeoxyglucose uptake. However, an acute dose of S-GVG (150 mg/kg; 2.5 h prior to the methamphetamine challenge) completely abolished these increases, as it blocked the expression of methamphetamine-induced conditioned place preference. These adolescent animals were then placed on subchronic S-GVG (150 mg/kg/day) for 5 days. This subchronic S-GVG treatment blocked methamphetamine-triggered reactivation of this expression of conditioned place preference. As adults (> 90 days old), these animals received another methamphetamine-challenge during [18F]-fluorodeoxyglucose uptake. Adolescent exposure to S-GVG attenuated methamphetamine-induced changes in [18F]-fluorodeoxyglucose uptake in the adult animals. In sum, these data suggest that S-GVG may constitute an effective strategy for blocking the biochemical and behavioral effects associated with methamphetamine abuse. Dewey, S.L., Schiffer, W.K., Lee, D., Aquilina, S., Kothari, S., Mullapudi, U., Patel, V., Fowler, J., Gardner, E., Ashby, C.R. Jr., and Brodie, J.D., Poster, 2006. College on Problems of Drug Dependence Annual Meeting, Scottsdale, AZ, June 17-22, 2006.

Gabapentin has no Effect on Cocaine-Triggered Relapse to Drug-Seeking Behavior or on Cocaine-Induced Dopamine Increases in the Nucleus Accumbens of the Brain IRP scientists have previously shown that gamma-vinyl GABA shows a promising anti-addiction profile in preclinical animal models related to addiction. Since gamma-vinyl GABA is a GABAmimetic (i.e., enhances brain levels of the neurotransmitter GABA), these same scientists have now extended these studies to another GABAmimetic - gabapentin. Gabapentin's actions in the brain may involve increases in synthesis and nonvesicular release of GABA, as well as prevention of GABA catabolism. Gabapentin reportedly inhibits the subjective effects (the "high") of smoked cocaine. In the present research, IRP scientists investigated whether gabapentin attenuates intravenous cocaine self-administration, cocaine-triggered relapse to cocaine-seeking behavior, and cocaine-induced increases in the neurotransmitter dopamine in the addiction-related brain locus - the nucleus accumbens. Gabapentin failed to alter cocaine-induced reinstatement (relapse) of cocaine-seeking behavior in laboratory rats previously experienced at intravenous cocaine self-administration. In vivo brain microdialysis studies demonstrated that acute cocaine administration significantly increased extracellular dopamine in the nucleus accumbens, but that this was not altered by pretreatment with gabapentin. These findings are in stark contrast to previous findings by these same investigators that gamma-vinyl GABA significantly inhibits cocaine-triggered relapse to cocaine-seeking behavior in the laboratory rat reinstatement model. These findings strongly suggest that gamma-vinyl GABA's anti-addiction properties in preclinical animal models do not necessarily generalize to other putative GABAmimetic drugs. Li, X., Peng, X.-Q., Gilbert, J., Xi, Z.-X., and Gardner, E.L., Poster, 2006. College on Problems of Drug Dependence Annual Meeting, Scottsdale, AZ, June 17-22, 2006.

Activation of the Metabotropic Glutamate Receptor 7 (mGluR7) by AMN082 Attenuates the Rewarding Effects of Cocaine by a Dopamine-Independent Mechanism in Rats IRP scientists have begun to explore the role played by brain metabotropic glutamate receptors in the rewarding effects (the "high") produced by cocaine. In the present study, IRP scientists studied the role of the metabotropic glutamate receptor 7 (mGluR7) in drug reward and its related neurochemistry using the selective mGluR7 agonist AMN082. These scientists observed that AMN082 dose-dependently inhibited cocaine self-administration behavior under both fixed ratio and progressive-ratio reinforcement schedules, and inhibited cocaine-induced increases in locomotion. These behavioral effects appear to be DA-independent, because the same doses of AMN082 had no effect on cocaine-induced increases in extracellular dopamine in the nucleus accumbens. AMN082 alone, when administered systemically or locally into the nucleus accumbens, dose-dependently decreased extracellular GABA levels, increased extracellular glutamate levels, but had no effect on extracellular dopamine levels in the nucleus accumbens, as measured from the same in vivo brain microdialysis samples. Such agonist-induced increases in glutamate appears to be mediated by reduction in extracellular GABA levels because blockade of nucleus accumbens GABAB receptors with 2-hydroxysaclofen completely blocked AMN082-induced increases in glutamate. Further, AMN082 lowered extracellular glutamate after GABAB receptor blockade. These data, for the first time, demonstrate that activation of mGluR7 inhibits cocaine=s rewarding effects and cocaine-induced hyperactivity by a dopamine-independent mechanism. Presynaptic inhibition mediated by mGluR7 activation on the release of other neurotransmitters, such as GABA or glutamate, may underlie these effects. These findings suggest that the glutamatergic circuitry of the limbic forebrain may constitute a useful target for the development of anti-addiction medications. Li, X., Peng, X.-Q., Gilbert, J.G., Pak, A.C., Xi, Z.-X., and Gardner, E.L., Poster, 2006. Society for Neuroscience Annual Meeting, Atlanta, GA, October 14-18, 2006.

Δ9-Tetrahydrocannabinol (Δ9-THC) Inhibits Glutamate Release in the Nucleus Accumbens via a non-CB1 Receptor-Mediated Mechanism IRP scientists were the first to establish that Δ9-Tetrahydrocannabinol (Δ9-THC), the addictive constituent of marijuana, acts on the reward circuits of the brain in a manner similar to other addictive drugs. From that prior work, it is widely believed that cannabinoid addictive action is mediated by brain cannabinoid CB1 receptors and by the brain's dopamine system. In contrast, little is known about cannabinoid effects on the glutamate system, which is also proposed to be involved in drug reward and addiction. Now, IRP scientists have used in vivo microdialysis to measure brain extracellular glutamate in freely moving animals. They found that systemic or local Δ9-THC administration lowered extracellular glutamate levels in the nucleus accumbens. This reduction in glutamate appears to be CB1 receptor-independent because: 1) co-administration of Δ9-THC with SR141716A, a CB1 receptor antagonist, failed to reverse local Δ9-THC-induced inhibition of glutamate; and 2) Δ9-THC inhibited glutamate release in the nucleus accumbens in both CB1-knockout mice and littermate wild-type mice. It has been proposed that drug reward and addictive effects are mediated by dopamine-induced inhibition of nucleus accumbens medium spiny GABAergic neurons. If this is true, the presently-observed reduction in glutamatergic input to the nucleus accumbens after Δ9-THC administration might well potentiate such inhibition of the nucleus accumbens medium spiny GABAergic neurons, thus contributing to the rewarding and addictive effects of cannabinoids and Δ9-THC. Xi, Z.-X., Peng, X.-Q., Li, X., Gilbert, J., Pak, A.C., and Gardner, E.L., Poster, 2006. Society for Neuroscience Annual Meeting, Atlanta, GA, October 14-18, 2006.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Role of Brain Dopamine in Food Reward and Reinforcement The ability of food to establish and maintain response habits and conditioned preferences depends largely on the function of brain dopamine systems. While dopaminergic transmission in the nucleus accumbens appears sufficient for some forms of reward, the role of dopamine in food reward does not appear to be restricted to this region. Dopamine plays an important role in both the ability to energize feeding and to reinforce food-seeking behaviour; the role in energizing feeding is secondary to the prerequisite role in reinforcement. Dopaminergic activation is triggered by the auditory and visual as well as the tactile, olfactory, and gustatory stimuli of foods. While dopamine plays a central role in the feeding and food-seeking of normal animals, some food rewarded learning can be seen in genetically engineered dopamine-deficient mice. Wise, R.A. Philosophical Transactions of the Royal Society B-Biological Sciences, 361(1471), pp. 1149-1158, 2006.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Blockade of Adenosine A2A Receptors Prevents Protein Phosphorylation in the Striatum Induced by Cortical Stimulation Previous studies have shown that cortical stimulation selectively activates extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and immediate early gene expression in striatal GABAergic enkephalinergic neurons. In the present study, the authors demonstrate that blockade of adenosine A2A receptors with caffeine or a selective A2A receptor antagonist counteracts the striatal activation of cAMP-protein kinase A cascade (phosphorylation of the Ser845 residue of the glutamate receptor 1 subunit of the AMPA receptor) and mitogen-activated protein kinase (ERK1/2 phosphorylation) induced by the in vivo stimulation of corticostriatal afferents. The results indicate that A2A receptors strongly modulate the efficacy of glutamatergic synapses on striatal enkephalinergic neurons. Quiroz, C., Gomes, C., Pak, A. C., Riberiro, J. A., Goldberg, S. R. and Hope, B.T. Journal of Neuroscience, 26, pp. 10808-10812, 2006.

Anandamide Administration Alone and After Inhibition of Fatty Acid Amide Hydrolase (FAAH) Increases Dopamine Levels in the Nucleus Accumbens Shell Although endogenous cannabinoid systems have been implicated in the modulation of the rewarding effects of abused drugs and food, little is known about the direct effects of endogenous ligands for cannabinoid receptors on brain reward processes. Here IRP scientists show for the first time that the intravenous administration of anandamide, an endogenous ligand for cannabinoid receptors, and its longer-lasting synthetic analog methanandamide, increase the extracellular dopamine levels in the nucleus accumbens shell of awake, freely moving rats, an effect characteristic of most drugs abused by humans. Anandamide produced two distinctly different effects on dopamine levels: (1) a rapid, transient increase that was blocked by the cannabinoid CB1 receptor antagonist rimonabant, but not by the vanilloid VR1 receptor antagonist capsazepine, and was magnified and prolonged by the fatty acid amide hydrolase (FAAH) enzyme inhibitor, URB597; (2) a smaller delayed and long-lasting increase, not sensitive to CB1, VR1 or FAAH blockade. Both effects were blocked by infusing either tetrodotoxin (TTX, 1 microm) or calcium-free Ringer's solution through the microdialysis probe, demonstrating that they were dependent on the physiologic activation of dopaminergic neurotransmission. Thus, these results indicate that anandamide, through the activation of the mesolimbic dopaminergic system, participates in the signaling of brain reward processes. Solinas, M., Justinova, Z., Goldberg, S.R. and Tanda, G. Journal of Neurochemistry, 98, pp. 408-419, 2006.

Intramembrane Receptor-receptor Interactions: A Novel Principle in Molecular Medicine In 1980/81 Agnati and Fuxe introduced the concept of intramembrane receptor-receptor interactions and presented the first experimental observations for their existence in crude membrane preparations. The second step was their introduction of the receptor mosaic hypothesis of the engram in 1982. The third step was their proposal that the existence of intramembrane receptor-receptor interactions made possible the integration of synaptic (WT) and extrasynaptic (VT) signals. With the discovery of the intramembrane receptor-receptor interactions with the likely formation of receptor aggregates of multiple receptors, so called receptor mosaics, the entire decoding process becomes a branched process already at the receptor level in the surface membrane. Recent developments indicate the relevance of cooperativity in intramembrane receptor-receptor interactions namely the presence of regulated cooperativity via receptor-receptor interactions in receptor mosaics (RM) built up of the same type of receptor (homo-oligomers) or of subtypes of the same receptor (RM type1). The receptor-receptor interactions will to a large extent determine the various conformational states of the receptors and their operation will be dependent on the receptor composition (stoichiometry), the spatial organization (topography) and order of receptor activation in the RM. The biochemical and functional integrative implications of the receptor-receptor interactions are outlined and long-lived heteromeric receptor complexes with frozen RM in various nerve cell systems may play an essential role in learning, memory and retrieval processes. Intramembrane receptor-receptor interactions in the brain have given rise to novel strategies for treatment of Parkinson's disease (A2A and mGluR5 receptor antagonists), schizophrenia (A2A and mGluR5 agonists) and depression (galanin receptor antagonists). The A2A/D2, A2A/D3 and A2A/mGluR5 heteromers and heteromeric complexes with their possible participation in different types of RM are described in detail, especially in the cortico-striatal glutamate synapse and its extrasynaptic components, together with a postulated existence of A2A/D4 heteromers. Finally, the impact of intramembrane receptor-receptor interactions in molecular medicine is discussed outside the brain with focus on the endocrine, the cardiovascular and the immune systems. Ciruela, F., Ferre, S., Casado, V., Cortes, A., Cunha, R. A., Lluis, C. and Franco, R. Cellular Molecular Life Sciences, October 19, 2006, Epubmed ahead of print, PMID 17058035.

Receptor-receptor Interactions Involving Adenosine A(1) or Dopamine D (1) Receptors and Accessory Proteins The molecular basis for the known intramembrane receptor-receptor interactions among heptahelical receptors (G protein coupled receptors, GPCR) was postulated to be heteromerization based on receptor subtype specific interactions between different types of homomers of GPCR. Adenosine and dopamine receptors in the basal ganglia have been fundamental to demonstrate the existence of receptor heteromers and the functional consequences of such molecular interactions. The heterodimer is only one type of heteromeric complex and the evidence is equally compatible with the existence of higher order heteromeric complexes, where also adapter proteins such as homer proteins and scaffolding proteins can exist, assisting in the process of linking the GPCR and ion channel receptors together in a receptor mosaic that may have special integrative value and may constitute the molecular basis for learning and memory. Heteromerization of D(2) dopamine and A(2A) adenosine receptors is reviewed by Fuxe in another article in this special issue. Here, heteromerization between D(1) dopamine and A(1) adenosine receptors is reviewed. Heteromers formed by dopamine D(1) and D(2) receptors and by adenosine A(1) and A(2A) receptors also occur in striatal cells and open new perspectives to understand why two receptors with apparently opposite effects are expressed in the same neuron and in the nerve terminals. The role of accessory proteins also capable of interacting with receptor-receptor heteromers in regulating the traffic and the molecular physiology of these receptors is also discussed. Overall, the knowledge of the reason why such complex networks of receptor-receptor and receptor-protein interactions occur in striatal cells is crucial to develop new strategies to combat neurological and neuropsychiatric diseases. Franco, R., Lluis, C., Canela, E. I., Mallol, J., Agnati, L., Casado, V., Ciruela, F., Ferre, S. and Fuxe, K. Journal of Neural Transmission, October 6, 2006, Epubmed ahead of print, PMID 17024327.

Working Memory Deficits in Transgenic Rats Overexpressing Human Adenosine A(2A) Receptors in the Brain Adenosine receptors in the central nervous system have been implicated in the modulation of different behavioural patterns and cognitive functions although the specific role of A(2A) receptor (A(2A)R) subtype in learning and memory is still unclear. In the present work IRP investigators establish a novel transgenic rat strain, TGR(NSEhA2A), overexpressing adenosine A(2A)Rs mainly in the cerebral cortex, the hippocampal formation, and the cerebellum. Thereafter, the authors explore the relevance of this A(2A)Rs overexpression for learning and memory function. Animals were behaviourally assessed in several learning and memory tasks (6-arms radial tunnel maze, T-maze, object recognition, and several Morris water maze paradigms) and other tests for spontaneous motor activity (open field, hexagonal tunnel maze) and anxiety (plus maze) as modification of these behaviours may interfere with the assessment of cognitive function. Neither motor performance and emotional/anxious-like behaviours were altered by overexpression of A(2A)Rs. TGR(NSEhA2A) showed normal hippocampal-dependent learning of spatial reference memory. However, they presented working memory deficits as detected by performance of constant errors in the blind arms of the 6 arm radial tunnel maze, reduced recognition of a novel object and a lack of learning improvement over four trials on the same day which was not observed over consecutive days in a repeated acquisition paradigm in the Morris water maze. Given the interdependence between adenosinic and dopaminergic function, the present results render the novel TGR(NSEhA2A) as a putative animal model for the working memory deficits and cognitive disruptions related to overstimulation of cortical A(2A)Rs or to dopaminergic prefrontal dysfunction as seen in schizophrenic or Parkinson's disease patients. Gimenez-Llort, L., Schiffmann, S. N., Shmidt, T., Canela, L., Camon, L., Wassholm, M., Canals, M. Terasmaa, A., Fernandez-Teruel, A., Tobena, A., Popova, E., Ferre, S., Agnati, L., Ciruela, F., Martinez, E., Scheel-Kruger, J., Lluis, C., Franco, R., Fuxe, K. and Bader, M.. Neurobiology Learning Memory, July 4, 2006, Epubmed ahead of print, PMID 16824773.

Effects of Stress Modulation on Morphine-induced Conditioned Place Preferences and Plasma Corticosterone Levels in Fischer, Lewis, and Sprague-Dawley Rat Strains There is a direct relationship between hypothalamic-pituitary-adrenal axis (HPA) reactivity and susceptibility to drug use in outbred rats. Specifically, manipulations that increase or decrease HPA activity also increase or decrease drug intake, respectively. Interestingly, this relationship has not been established in the inbred Fischer (F344) and Lewis (LEW) rat strains that are often used as animal models of susceptibility to drug use. The present study investigated the effects of manipulations known to affect HPA activity on morphine-induced conditioned place preference (CPP) in male LEW, F344, and Sprague-Dawley (SD) rats. In experiment 1, animals were exposed to an injection of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and 2-h restraint stress prior to the conditioning of a morphine-induced place preference (1, 4, or 10 mg/kg subcutaneous). In experiment 2, animals were chronically exposed to corticotropin-releasing hormone type 1 receptor antagonist, antalarmin, prior to CPP training. The effects of DMCM/restraint and antalarmin on corticosterone levels were examined in experiments 3 and 4. In outbred rats, DMCM/restraint increased both HPA activity and morphine-induced CPP, while antalarmin decreased CPP and produced a slight, but nonsignificant, decrease in corticosterone levels. In the inbred rats, however, DMCM/restraint increased plasma corticosterone yet decreased place preferences in the LEW strain, and antalarmin treatment decreased plasma corticosterone but increased place preferences in the F344 strain. These data suggest that the relationship between stress and drug use may be nonmonotonic. The use of these inbred strains in genetic analysis of drug addiction may require reexamination. Grakalic, I., Schindler, C.W., Baumann, M.H., Rice, K.C. and Riley, A.L. Psychopharmacology (Berlin), October 3, 2006, Epubmed ahead of print, PMID 17016707.

Cocaine Self-administration Under Variable-dose Schedules in Squirrel Monkeys Squirrel monkeys self-administered cocaine under a variable-dose schedule, with the dose varied from injection to injection. As in earlier studies with rats, post-injection pauses varied as a monotonic function of dose, allowing a cocaine dose-effect curve to be obtained during each session. These curves were shifted by pretreatment with dopamine antagonists, demonstrating that this procedure may provide an efficient means of evaluating treatments that affect drug self-administration. However, drug intake eventually became "dysregulated" after extensive training (100-300 sessions), with relatively short pauses following all doses. Dose-sensitivity was restored by adding a 60-s timeout period after each injection, suggesting that dysregulation occurred because the monkeys developed a tendency to self-administer another injection before the previous injection had been adequately distributed. Finally, when the response requirement under the variable-dose schedule was increased from 1 to 10, both the post-injection pause and the rate of responding following the pause ("run rates") were found to vary with dose. The dose-dependency of run rates suggests that post-injection pauses reflect not only motivational factors, such as satiety, but also the direct effects of cocaine on lever pressing. Panlilio, L.V., Thorndike, E.B. and Schindler, C.W. Pharmacology Biochemistry and Behavior, 84, pp. 235-243, 2006.

Dopamine D(3) Receptor Ligands for the Treatment of Tobacco Dependence Among the 5 dopamine receptors identified, the DRD3 is located in the nucleus accumbens, ventral tegmental area and amygdala: 3 brain structures that are implicated in the motivational control of drug-seeking behaviour and drug-conditioning processes. Although it has been proposed that modulating dopamine transmission would be effective in the treatment of drug dependence, no validation has been provided in humans so far. Several highly selective DRD3 ligands have recently been evaluated in preclinical models of drug dependence. These ligands act as DRD3 antagonists in vivo and are able to decrease the motivation to take various drugs of abuse and reduce the influence of associated drug-conditioned behaviour. Of note is that these effects have been found with nicotine-seeking behaviour and nicotine relapse in rodents, suggesting a potential use of these ligands for the treatment of tobacco smokers. In contrast to nicotine replacement therapy, varenicline and bupropion (which are currently used for the treatment of smokers), DRD3 antagonists do not seem to produce nicotine-like effects in experimental animals and, therefore, may not substitute for nicotine or alleviate nicotine withdrawal symptoms in human smokers. This behavioural profile, which was also reported recently with cannabinoid CB(1) receptor antagonists, may result from effects on specific brain pathways that express DRD3 receptors and are involved in relapse and conditioning processes. These preclinical studies suggest that the clinical evaluation of DRD3 ligands should be performed with clinical trials designed specifically to evaluate the relapse phenomena. LeFoll, B., Goldberg, S.R. and Sokoloff, P. Expert Opin Investig Drugs, 16, pp. 45-57, 2007.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

The Anxiogenic Drug Yohimbine Reinstates Palatable Food-seeking in a Rat Relapse Model: A Role of CRF1 Receptors The major problem in treating excessive eating is high rates of relapse to maladaptive eating habits during diet treatments; this relapse is often induced by stress or anxiety states. Preclinical studies have not explored this clinical problem. Here, IRP researchers adapted a reinstatement model (commonly used to study relapse to abused drugs) to examine the role of stress and anxiety in relapse to palatable food seeking during dieting. Rats were placed on restricted diet (75-80% of daily standard food) and for 12 intermittent training days (9 h/d, every other day) lever-pressed for palatable food pellets (25% fat, 48% carbohydrate) under a fixed-ratio 1 (20-sec timeout) reinforcement schedule. Subsequently, the rats were given 10 daily extinction sessions during which lever-presses were not reinforced, and were then injected with yohimbine (an alpha-2 adrenoceptor antagonist that induces stress and anxiety in humans and nonhumans) or given a single food pellet to assess reinstatement of food seeking. The rats rapidly learned to lever-press for the palatable pellets and across the training days the ratio of timeout non-reinforced lever-presses to reinforced lever-presses progressively increased more than 3-fold, suggesting the development of compulsive eating-behavior. After extinction, yohimbine injections and pellet priming reliably reinstated food seeking. The corticotropin-releasing factor1 (CRF1) receptor antagonist antalarmin attenuated the reinstatement induced by yohimbine, but not pellet priming. Antalarmin also reversed yohimbine's anxiogenic effects in the social interaction test. These data suggest that CRF is involved in stress-induced relapse to palatable food seeking and that CRF1 antagonists should be considered for the treatment of maladaptive eating habits. Ghitza, U.E., Gray, S.M., Rice, K.C., Epstein, D.E. and Shaham, Y. Neuropsychopharmacology, 31, pp. 2188-2196, 2006.

Activation of Group II Metabotropic Glutamate Receptors in the Nucleus Accumbens Shell Attenuates Context-induced Relapse to Heroin Seeking Using a rat relapse model, IRP scientists previously reported that re-exposing rats to a drug-associated context, following extinction of operant responding in a different context, reinstates heroin seeking. In an initial pharmacological characterization, the authors found that the mGluR2/3 agonist LY379268, which acts centrally to reduce evoked glutamate release, attenuates context-induced reinstatement of heroin seeking when injected systemically or into the ventral tegmental area, the cell body region of the mesolimbic dopamine system. Here, the authors tested whether injections of LY379268 into the nucleus accumbens (NAc), a terminal region of the mesolimbic dopamine system, would also attenuate context-induced reinstatement of heroin seeking. Rats were trained to self-administer heroin; drug infusions were paired with a discrete tone-light cue. Subsequently, lever pressing was extinguished in the presence of the discrete cue in a context that differed from the drug self-administration context in terms of visual, auditory, tactile, and circadian cues. After extinction of responding, LY379268 was injected to different groups of rats into the NAc core or shell or into the caudate-putamen, a terminal region of the nigrastriatal dopamine system. Injections of LY379268 into the NAc shell (0.3 or 1.0 ug) dose-dependently attenuated context-induced reinstatement of heroin seeking. Injections of 1.0 ug of LY379268 into the NAc core had no effect, while a higher dose (3.0 ug) decreased this reinstatement. Injections of LY379268 (3.0 ug) 1.5 mm dorsal from the NAc core into the caudate-putamen were ineffective. Results suggest an important role of glutamate transmission in the NAc shell in context-induced reinstatement of heroin seeking. Bossert, J.M., Gray, S., Lu, L. and Shaham, Y. Neuropsychopharmacology, 31, pp. 2197-3109, 2006.