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Director's Report to the National Advisory Council on Drug Abuse - September, 2008



Research Findings - Intramural Research

Molecular Neurobiology Research Branch

Genetics of Ability to Quit Smoking
Smoking remains a major public health problem. Twin studies indicate that the ability to quit smoking is substantially heritable, with genetics that overlap modestly with the genetics of vulnerability to dependence on addictive substances. The objectives of theis study were to identify replicated genes that facilitate smokers' abilities to achieve and sustain abstinence from smoking (hereinafter referred to as quit-success genes) found in more than 2 genome-wide association (GWA) studies of successful vs. unsuccessful abstainers, and, secondarily, to nominate genes for selective involvement in smoking cessation success with bupropion hydrochloride vs. nicotine replacement therapy (NRT). The GWA results in subjects from 3 centers, with secondary analyses of NRT vs. bupropion responders were used. The study setting was 3 outpatient smoking cessation centers. European American smokers who successfully vs. unsuccessfully abstain from smoking with biochemical confirmation in a smoking cessation trial using NRT, bupropion, or placebo (N=550) served as participants. Main outcome measures were: quit-success genes, reproducibly identified by clustered nominally positive single-nucleotide polymorphisms (SNPs) in more than 2 independent samples with significant P values based on Monte Carlo simulation trials. The NRT-selective genes were nominated by clustered SNPs that display much larger t values for NRT vs. placebo comparisons. The bupropion-selective genes were nominated by bupropion-selective results. Results: Variants in quit-success genes are likely to alter cell adhesion, enzymatic, transcriptional, structural, and DNA, RNA, and/or protein-handling functions. Quit success genes are identified by clustered nominally positive SNPs from more than 2 samples and are unlikely to represent chance observations (Monte Carlo P<.0003). These genes display modest overlap with genes identified in GWA studies of dependence on addictive substances and memory. These results support polygenic genetics for success in abstaining from smoking, overlap with genetics of substance dependence and memory, and nominate gene variants for selective influences on therapeutic responses to bupropion vs. NRT. Molecular genetics should help match the types and/or intensity of antismoking treatments with the smokers most likely to benefit from them. Uhl, G.R., Liu, Q.R., Drgon, T., Johnson, C., Walther, D., Rose, J.E., David, S.P., Niaura, R., and Lerman, C. Arch. Gen. Psychiatry. 65(6), pp. 683-693, 2008.

Genetics of Vulnerability to Methamphetamine Dependence
Understanding of human methamphetamine dependence, and possibly our abilities to prevent and treat this devastating disorder can be improved by identifying genes whose allelic variants predispose to methamphetamine dependence. The objective of this study was to find "methamphetamine dependence" genes identified by each of 2 genome-wide association (GWA) studies of independent samples of methamphetamine-dependent individuals and matched controls. The study involved replicated GWA results in each of 2 case control studies and took place in Japan and Taiwan. Individuals with methamphetamine dependence and matched control subjects free from psychiatric, substance abuse, or substance dependence diagnoses (N=580) served as subjects. Main outcome measures included "Methamphetamine dependence" genes that were reproducibly identified by clusters of nominally positive single-nucleotide polymorphisms (SNPs) in both samples in ways that were unlikely to represent chance observations, based on Monte Carlo simulations that corrected for multiple comparisons, and subsets of "methamphetamine dependence" genes that were also identified by GWA studies of dependence on other addictive substances, success in quitting smoking, and memory. Results indicated that genes identified by clustered nominally positive SNPs from both samples were unlikely to represent chance observations (Monte Carlo P.00001). Variants in these "methamphetamine dependence" genes are likely to alter cell adhesion, enzymatic functions, transcription, cell structure, and DNA, RNA, and/or protein handling or modification. Cell adhesion genes CSMD1 and CDH13 displayed the largest numbers of clustered nominally positive SNPs. "Methamphetamine dependence" genes overlapped, to extents much greater than chance, with genes identified in GWAs studies of dependence on other addictive substances, success in quitting smoking, and memory (Monte Carlo P range <.04 to <.00001). These data support polygenic contributions to methamphetamine dependence from genes that include those whose variants contribute to dependence on several addictive substances, success in quitting smoking, and mnemonic processes. Uhl, G.R., Drgon. T., Liu, Q.R., Johnson, C., Walther, D., Komiyama, T., Harano, M., Sekine, Y., Inada, T., Ozaki, N., Iyo, M., Iwata, N., Yamada, M., Sora, I., Chen, C.K., Liu, H.C., Ujike, H., Lin. Arch. Gen. Psychiatry. 65(3), pp. 345-355, 2008.

Office of the Scientific Director

Exercise Stress Testing in Recently Abstinent Chronic Cocaine Abusers
Cocaine has well established acute effects on cardiac function, but less is known about effects of chronic use persisting during abstinence. IRP scientists evaluated this question by comparing the results of treadmill exercise stress testing (EST) in 28 medically screened, chronic cocaine users (abstinent for a mean of 6.2 days, range 1-32 days) with the cardiovascular effects of an intravenous cocaine challenge (25 mg or 50 mg) (9 subjects). All subjects had a clinically normal EST; all but one subject reached their predicted heart rate (i.e., was able to exercise to capacity). Only one subject had an exaggerated blood pressure response to exercise. EST produced significantly greater increases in heart rate and blood pressure than did the cocaine challenges. These findings suggest that EST may not add any additional cardiac diagnostic information in asymptomatic, recently abstinent chronic cocaine abusers who have already been medically screened as healthy, and that single low doses of cocaine may not generate substantial cardiac work (as indicated by heart rate and blood pressure) in carefully screened, otherwise healthy cocaine users. Kanneganti, P., Nelson, R.A., Boyd, S.J., Ziegelstein, R.C., and Gorelick, D.A. American Journal of Drug and Alcohol Abuse, 34, pp. 489-498, 2008.

Development and Plasticity Section, Cellular Neurobiology Research Branch

A Mechanism for the Inhibition of Neural Progenitor Cell Proliferation by Cocaine
Prenatal exposure of the developing brain to cocaine causes morphological and behavioral abnormalities, possibly caused by cocaine-induced proliferation inhibition and/or apoptosis in neural progenitor cells. IRP investigators therefore determined the molecular mechanism responsible for mediating the effect of cocaine on cell cycle regulation. Microarray analysis followed by qRT-PCR was used to screen cocaine-responsive and cell cycle-related genes in a neural progenitor cell line. Cyclin A2, among genes related to the G1-to-S cell cycle transition, was most strongly down-regulated by cocaine. Down-regulation of cyclin A was also found in cocaine-treated human primary neural and A2B5+ progenitor cells, as well as in rat fetal brains exposed to cocaine in utero. Reversing cyclin A down-regulation by gene transfer counteracted the proliferation inhibition caused by cocaine. Further, the authors found that cocaine-induced accumulation of reactive oxygen species, which involves N-oxidation of cocaine via cytochrome P450, promotes cyclin A down-regulation by causing an ER stress response, as indicated by increased phosphorylation of eIF2alpha and expression of ATF4. In the developing rat brain, the P450 inhibitor cimetidine counteracted cocaine-induced inhibition of neural progenitor cell proliferation as well as down-regulation of cyclin A. Therefore, down-regulation of cyclin A underlies cocaine-induced proliferation inhibition in neural progenitors. The down-regulation of cyclin A is initiated by N-oxidative metabolism of cocaine and consequent ER stress. Inhibition of cocaine N-oxidative metabolism by P450 inhibitors may provide a preventive strategy for counteracting the adverse effects of cocaine on fetal brain development. Lee, C.T., Chen, J., Hayashi, T., Tsai, S.Y., Sanchez, J.F., Errico, S.L., Amable, R., Su, T.P., Lowe, R.H., Huestis, M.A., Shen, J., Becker, K.G., Geller, H.M., and Freed, W.J. Public Library of Science Medicine, 5(6), pp. e117, 2008.

Benefits and Risks of Intranigral Transplantation of GABA-producing Cells Subsequent to the Establishment of Kindling-induced Seizures
IRP scientists assessed the anticonvulsant efficacy and safety of bilateral allotransplantation of genetically engineered striatal GABAergic rat cell lines into the SNr. Rats with previously-established seizures, induced by amygdala kindling, were used as a model of temporal lobe epilepsy. Three cell lines were transplanted: immortalized GABAergic cells (M213-2O) derived from embryonic rat striatum; M213-2O cells (CL4) transfected with human GAD67 cDNA to obtain higher GABA synthesis than the parent cell line; and control cells (121-1I), also derived from embryonic rat striatum, but which did not show GAD expression. A second control group received injections of medium alone. Transplantation of M213-2O cells into the SNr of kindled rats resulted in significant but transient anticonvulsant effects. Neither control cells nor medium induced anticonvulsant effects. Strong tissue reactions were, however, induced in the host brain of kindled but not of non-kindled rats, and only in animals that received grafts of genetically modified CL4 cells. These tissue reactions included graft rejection, massive infiltration of inflammatory immune cells, and gliosis. The anticonvulsant effect of M213-2O cells emphasizes the feasibility of local manipulations of seizures by intranigral transplantation of GABA-producing cells. On the other hand, kindling-induced activation of microglia in the SNr combined with immunological stimulation by CL4 cells, transfected with a human cDNA, caused graft rejection. Thus, it appears that the condition of the host brain and the production of foreign proteins by transplanted cells have to be considered in estimating the risks of rejection of transplants into the brain. Nolte, M.W., Loescher, W., Herden, C., Freed, W.J., and Gernert, M. Neurobiology of Disease, July 14, 2008, [E-pub ahead of print].

Intranigral Transplants of a GABAergic Cell Line Produce Long-term Alleviation of Established Motor Seizures
IRP researchers have previously shown that intranigral transplants of immortalized GABAergic cells decrease the number of kainic acid-induced seizures in an animal model. In the present study, recurrent spontaneous behavioral seizures were established by repeated systemic injections of this excitotoxin into male Sprague-Dawley rats. After the seizures had been established, cells were transplanted into the substantia nigra. Animals with transplants of control cells (without hGAD67 expression) or with sham transplants showed a death rate of more than 40% over the 12 weeks of observation, whereas in animals with M213-2O CL-4 transplants, the death rate was reduced to less than 20%. The M213-2O CL-4 transplants significantly reduced the percentage of animals showing behavioral seizures; animals with these transplants also showed a lower occurrence of stage V seizures than animals in the other groups. In vivo and in vitro analyses provided evidence that the GABAergic cells show sustained expression of both GAD67 and hGAD67 cDNA, as well as increased gamma-aminobutyric acid (GABA) levels in the ventral mesencephalon of transplanted animals. Therefore, transplantation of GABA-producing cells can produce long-term alleviation of behavioral seizures in an animal model. Castillo, C.G., Mendoza-Trejo, S., Aguilar, M.B., Freed, W.J., and Giordano. M. Behavioural Brain Research, May 4, 2008, [E-pub ahead of print].

Assessment of Stromal-derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by beta-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect. Vazin, T., Chen, J., Lee, C.T., Amable, R., and Freed, W.J. Stem Cells, 26(6), pp. 1517-1525, 2008.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Gene Expression Patterns in Mouse Cortical Penumbra after Focal Ischemic Brain Injury and Reperfusion
Ischemic stress in the brain causes acute and massive cell death in the targeted core area followed by a second phase of damage in the neighboring penumbra. The purpose of this study was to examine the global gene expression patterns in the penumbra, because the ischemic lesion in this region could be rescued by restoration of blood flow and other protective therapies. Adult C57Bl/6 mice were subjected to a 90-min middle cerebral artery occlusion (MCAO). Laser capture microdissection (LCM) was used for tissue dissection at 4 and 24 hr after reperfusion. Sham-operated animals were used as controls. Gene expression in the penumbra was examined by using microarray analysis and quantitative RT-PCR. In agreement with previous reports, most genes were down-regulated at 4 hr after the onset of reperfusion in the ischemic penumbra compared with controls. In contrast, at 24 hr after reperfusion, most genes were up-regulated in the ischemic penumbra. Several genes not previously reported to be associated with ischemia were found. The gene lists generated in this study will help us to understand better the spatial and temporal distribution of molecules involved in the ischemic cascade. Sarabi, A.S., Shen, H., Wang, Y., Hoffer, B.J., and Baeckman. C.M. Journal of Neuroscience Research, May 27, 2008, [E-pub ahead of print].

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Analogs of JHU75528, a PET Ligand for Imaging of Cerebral Cannabinoid Receptors (CB1): Development of Ligands with Optimized Lipophilicity and Binding Affinity
Cyano analogs of Rimonabant with high binding affinity for the cerebral cannabinoid receptor (CB1) and with optimized lipophilicity have been synthesized as potential positron emission tomography (PET) ligands. The best ligands of the series are optimal targets for the future radiolabeling with PET isotopes and in vivo evaluation as radioligands with enhanced properties for PET imaging of CB1 receptors in human subjects. Extracellular electrophysiological recordings in rodent brain slices demonstrated that JHU75528, 4, the lead compound of the new series, has functional CB antagonist properties that are consistent with its structural relationship to Rimonabant. Molecular modeling analysis revealed an important role of the binding of the cyano group with the CB1 binding pocket. Fan, H., Kotsikorou, E., Hoffman, A.F., Ravert, H.T., Holt, D., Hurst, D.P., Lupica, C.R., Reggio, P.H., Dannals, R.F., and Horti, A.G. European Journal of Medicinal Chemistry, April 18, 2008, [E-pub ahead of print].

Discovery of (-)-7-methyl-2-exo-[3'-(6-[(18)F]fluoropyridin-2-yl)-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, A Radiolabeled Antagonist for Cerebral Nicotinic Acetylcholine Receptor (alpha4beta2-nAChR) with Optimal Positron Emission Tomography Imaging Properties
Several isomers of 7-methyl-2- exo-([(18)F]fluoropyridinyl-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane have been developed as radioligands with optimized brain kinetics for PET imaging of nAChR. The binding assay demonstrated that all isomers are beta-nAChR selective ligands with Ki = 0.02-0.3 nM. The experimental lipophilicity values of all isomers were in the optimal range for the cerebral radioligands (log D 7.4= 0.67-0.99). The isomers with higher binding affinity manifested slow baboon brain kinetics, whereas the isomer with the lowest binding affinity (Ki = 0.3 nM)((-)-7-methyl-2- exo-[3'-(6-[ (18)F]fluoropyridin-2-yl)-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, [(18)F](-)- 6c) and greatest lipophilicity (log D 7.4 = 0.99) exhibited optimal brain kinetics. [(18)F](-)-6c manifests a unique combination of the optimally rapid brain kinetics, high BP and brain uptake, and favorable metabolic profile. Pharmacological studies showed that (-)-6c is an alpha4beta2-nAChR antagonist with low side effects in mice. This combination of imaging properties suggests that [(18)F]-(-)-6c is a potentially superior replacement for 2-[(18)F]fluoro-A-85380 and 6-[(18)F]fluoro-A-85380, the only available nAChR PET radioligands for humans. Gao Y., Kuwabara, H., Spivak, C.E., Xiao, Y., Kellar, K., Ravert, H.T., Kumar, A., Alexander, M., Hilton, J., Wong, D.F., Dannals, R.F., and Horti, A.G. Journal of Medicinal Chemistry, July 8, 2008, [E-pub ahead of print].

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

The Role of Phosphorylated Residues in Peptide-Peptide Noncovalent Complexes Formation
Electrospray mass spectrometry (ESI-MS) has become the tool of choice for the study of noncovalent complexes. Previous work by IRP researchers has highlighted the role of phosphorylated amino acid residues in the formation of noncovalent complexes through electrostatic interaction with arginine residues' guanidinium groups. In this study, the authors employ tandem mass spectrometry to investigate the gas-phase stability and dissociation pathways of these noncovalent complexes. The only difference in the three phosphopeptides tested is the nature of the phosphorylated amino acid residue. In addition, the absence of acidic residues and an amidated carboxyl terminus insured that the only negative charge came from the phosphate, which allowed for the comparison of the noncovalent bond between arginine residues and each of the different phosphorylated residues. Dissociation curves were generated by plotting noncovalent complex ion intensities as a function of the nominal energy given to the noncovalent complex ion before entering the collision cell. These results showed that noncovalent complexes formed with phosphorylated tyrosine were the most stable, followed by serine and threonine, which had similar stability. Jackson, S.N., Moyer, S.C., and Woods, A.S. Journal of American Society for Mass Spectrometry, July 3, 2008, [E-pub ahead of print].

How Calmodulin Interacts with the Adenosine A2A and the Dopamine D2 Receptors
Receptor heteromerization is a mechanism used by G protein-coupled receptors to diversify their properties and function. IRP researchers previously demonstrated that these interactions occur through salt bridge formation between epitopes of the involved receptors. Recent studies claim that calmodulin (CaM) binds to an Arg-rich epitope located in the amino-terminus of the dopamine D2 receptor third intracellular loop. This is the same epitope involved in adenosine A 2A-D2 receptor heteromerization, through Coulombic interaction between the Arg residues and a phosphorylated serine (pS) located in the medial segment of the C-terminus of the A 2A receptor. Mass spectrometric analysis indicates that an electrostatic interaction involving the D2 receptor Arg-rich epitope and several CaM acidic epitopes are mainly responsible for the D2 receptor-CaM binding. CaM could also form multiple noncovalent complexes by means of electrostatic interactions with an epitope localized in the proximal segment of the C-terminus of the A 2A receptor. Ca (2+) disrupted the binding of CaM to the D2 but not to the A 2A receptor epitope, and CaM disrupted the electrostatic interactions between the D2 receptor epitope and the more distal A 2A receptor epitope. A model is introduced with the possible functional implications of A 2A-D 2-CaM interactions. These in vitro findings imply a possible regulatory role for CaM in receptor heteromers formation. Woods, A.S., Marcellino, D., Jackson, S.N., Franco, R., Ferre, S., Agnati, L.F., and Fuxe, K. Journal of Proteome Research, July 1, 2008, [E-pub ahead of print].

Amazing Stability of Phosphate-Quaternary Amine Interactions
IRP scientists have previously used MALDI mass spectrometry to highlight ammonium- or guanidinium-aromatic interactions via cation-pi bonding and ammonium- or guanidinium-phosphate interactions through salt bridge formation. In the present work, the gas-phase stability and dissociation pathways of the interaction between phosphorylated peptides and compounds containing quaternary amines are demonstrated using electrospray ionization mass spectrometry. The presence of one quaternary amine in a compound is enough to form a noncovalent complex with a phosphorylated residue. However, if two quaternary amines are present in one molecule, the electrostatic interactions of the quaternary amines with the phosphate results in a "covalent-like" stability, and these bonds can withstand fragmentation by collision-induced dissociation at energies similar to those that fragment covalent bonds. Such interactions are important in accounting for physiological, pathophysiological, and pharmacological effects of many therapeutic compounds and small molecules containing quaternary amines or phosphates. Woods, A.S., Moyer, S.C., and Jackson, S.N. Journal of Proteome Research, June 26, 2008, [E-pub ahead of print].

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Identification of Dopamine D1-D3 Receptor Heteromers
Indications for a role of synergistic D1-D3 receptor interactions in the striatum. The function of dopamine D3 receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D1-D3 receptor heteromers and for an intramembrane D1-D3 receptor cross-talk in living cells and in the striatum. The formation of D1-D3 receptor heteromers was demonstrated by Fluorescence Resonance Energy Transfer (FRET) and Bioluminescence Resonance Energy Transfer (BRET) techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D1-D3 intramembrane receptor-receptor interaction was observed, by which D3 receptor stimulation enhances D1 receptor agonist affinity, indicating that the D1-D3 intramembrane receptor-receptor interaction is a biochemical characteristic of the D1-D3 receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal colocalization and heteromerization of D1 and D3 receptors. According to the synergistic D1-D3 intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D3 receptor stimulation potentiates D1 receptor-mediated behavioral effects by a different mechanism than D2 receptor stimulation. The present study shows that a main functional significance of the D3 receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors. Marcellino, D., Ferre, S., Casado, V., Cortes, A., Le Foll, B., Mazzola, C., Drago, F., Saur, O., Stark, H., Soriano, A., Barnes, C., Goldberg, S. R., Lluis, C., Fuxe, K., Franco, R. Journal of Biological Chemistry, July 25, 2008, E-pub ahead of print, PMID: 18644790.

Interactions Between Histamine H(3) and Dopamine D(2) Receptors and the Implications for Striatal Function
The striatum contains a high density of histamine H(3) receptors, but their role in striatal function is poorly understood. Previous studies have demonstrated antagonistic interactions between striatal H(3) and dopamine D(1) receptors at the biochemical level, while contradictory results have been reported about interactions between striatal H(3) and dopamine D(2) receptors. In this study, by using reserpinized mice, IRP scientists demonstrate the existence of behaviorally significant antagonistic postsynaptic interactions between H(3) and D(1) and also between H(3) and dopamine D(2) receptors. The selective H(3) receptor agonist imetit inhibited, while the H(3) receptor antagonist thioperamide potentiated locomotor activation induced by either the D(1) receptor agonist SKF 38393 or the D(2) receptor agonist quinpirole. High scores of locomotor activity were obtained with H(3) receptor blockade plus D(1) and D(2) receptor co-activation, i.e., when thioperamide was co-administered with both SKF 38393 and quinpirole. Radioligand binding experiments in striatal membrane preparations showed the existence of a strong and selective H(3)-D(2) receptor interaction at the membrane level. In agonist/antagonist competition experiments, stimulation of H(3) receptors with several H(3) receptor agonists significantly decreased the affinity of D(2) receptors for the agonist. This kind of intramembrane receptor-receptor interactions are a common biochemical property of receptor heteromers. In fact, by using Bioluminescence Resonance Energy Transfer techniques in co-transfected HEK-293 cells, H(3) (but not H(4)) receptors were found to form heteromers with D(2) receptors. This study demonstrates an important role of postsynaptic H(3) receptors in the modulation of dopaminergic transmission by means of a negative modulation of D(2) receptor function. Ferrada, C., Ferre, S., Casado, V., Cortes, A., Justinova, Z., Barnes, C., Canela, E.I., Goldberg, S.R., Leurs, R., Lluis, C., Franco, R. Neuropharmacology, May 16, 2008, E-pub ahead of print, PMID 18547596.

Blocking Cannabinoid CB1 Receptors for the Treatment of Nicotine Dependence: Insights from Pre-clinical and Clinical Studies
Tobacco use is one of the leading preventable causes of death in developed countries. Since existing medications are only partially effective in treating tobacco smokers, there is a great need for improved medications for smoking cessation. It has been recently proposed that cannabinoid CB(1) receptor antagonists represent a new class of therapeutic agents for drug dependence, and notably, nicotine dependence. Here, IRP researchers reviewed current evidence supporting the use of this class of drugs for smoking cessation treatment. Pre-clinical studies indicate that nicotine exposure produces changes in endocannabinoid content in the brain. In experimental animals, N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methylpyrazole-3-carboxamide (rimonabant, SR141716) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), two cannabinoid CB(1) receptor antagonists, block nicotine self-administration behavior, an effect that may be related to the blockade of the dopamine-releasing effects of nicotine in the brain. Rimonabant also seems efficacious in decreasing the influence of nicotine-associated stimuli over behavior, suggesting that it may act on two distinct neuronal pathways, those implicated in drug-taking behavior and those involved in relapse phenomena. The utility of rimonabant has been evaluated in several clinical trials. It seems that rimonabant is an efficacious treatment for smoking cessation, although its efficacy does not exceed that of nicotine-replacement therapy and its use may be limited by emotional side effects (nausea, anxiety and depression, mostly). Rimonabant also appears to decrease relapse rates in smokers. These findings indicate significant, but limited, utility of rimonabant for smoking cessation. Le Foll, B., Forget, B., Aubin, H.J., and Goldberg, S.R. Addiction Biology, 13, pp. 239-252, 2008.

The Endocannabinoid System in Brain Reward Processes
Food, drugs and brain stimulation can serve as strong rewarding stimuli and are all believed to activate common brain circuits that evolved in mammals to favor fitness and survival. For decades, endogenous dopaminergic and opioid systems have been considered the most important systems in mediating brain reward processes. Recent evidence suggests that the endogenous cannabinoid (endocannabinoid) system also has an important role in signalling of rewarding events. First, CB(1) receptors are found in brain areas involved in reward processes, such as the dopaminergic mesolimbic system. Second, activation of CB(1) receptors by plant-derived, synthetic or endogenous CB(1) receptor agonists stimulates dopaminergic neurotransmission, produces rewarding effects and increases rewarding effects of abused drugs and food. Third, pharmacological or genetic blockade of CB(1) receptors prevents activation of dopaminergic neurotransmission by several addictive drugs and reduces rewarding effects of food and these drugs. Fourth, brain levels of the endocannabinoids anandamide and 2-arachidonoylglycerol are altered by activation of reward processes. However, the intrinsic activity of the endocannabinoid system does not appear to play a facilitatory role in brain stimulation reward and some evidence suggests it may even oppose it. The influence of the endocannabinoid system on brain reward processes may depend on the degree of activation of the different brain areas involved and might represent a mechanism for fine-tuning dopaminergic activity. Although involvement of the various components of the endocannabinoid system may differ depending on the type of rewarding event investigated, this system appears to play a major role in modulating reward processes. Solinas, M., Goldberg, S. R., Piomelli, D.. British Journal of Pharmacology, 154, pp. 369-83, 2008.

Novel Pharmacological Targets Based on Receptor Heteromers
Studies performed in the last 10 years have provided solid evidence indicating that G-protein-coupled receptors are expressed on the plasma membrane as homo and heterodimers. The first consequence of this fact is that homo and heterodimers are the true targets of natural (hormones, neurotransmitters) and synthetic drugs. Furthermore a given receptor in a heteromer may display a different functional and/or pharmacological profile than the same receptor characterized as monomer or as homodimer. Recent evidence indicates that receptor heteromers are sensors that lead to a fine-tuning in neurotransmission or hormone regulation; mainly this is achieved by a modification of the signaling pathways activated via a given receptor when it is forming a given heteromer. Quite often antagonists display variable affinities when a given receptor is expressed with different heteromeric partners. This fact should be taken into account in the development of new drugs. Finally it should be pointed out that radioligand binding data has to be analyzed by a model that considers receptors as dimers and not as monomers. This model provides a novel approach to characterize drugs interacting with the orthosteric center (agonists/antagonists) or with allosteric centers (allosteric regulators). Franco, R., Casado, V., Cortes, A., Perez-Capote, K., Mallol, J., Canela, E., Ferre, S., Lluis, C. Brain Research Review, June 20, 2008, E-pub ahead of print, PMID: 18620000.

Future Medications for Tobacco and Cannabis Dependence
Worldwide more than 3 million deaths a year are attributable to smoking, and tobacco use is on the rise in developing countries. Consequently, smoking is one of the few causes of mortality that is increasing, with deaths projected to reach 10 million annually in 30-40 years. Cannabinoids, which are usually used in the form of marijuana, have become the most frequently used illicit drugs, but there is no pharmacological treatment for marijuana dependence. Although the dopaminergic system plays a critical role in reinforcing the effects of drugs of abuse, other neurotransmitter systems are also involved. Here IRP investigators review recent results obtained with antagonists targeting cannabinoid CB1 receptors, dopamine D3 receptors and opioid receptors, that directly or indirectly modulate dopaminergic transmission. These promising approaches warrant clinical trials in the treatment of tobacco and marijuana dependence. Le Foll, B,, Justinova, Z,, Tanda, G,, and Goldberg, S.R. Bulletin of the Academy of National Medicine, 192, pp. 45-56; discussion 56-57, 2008. PMID: 18663981.

Nicotine Psychopharmacology Unit, Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Reliability and Validity of the Tobacco Craving Questionnaire--Short Form
The Tobacco Craving Questionnaire (TCQ) is a valid and reliable, 47-item self-report instrument that assesses tobacco craving in four dimensions: emotionality, expectancy, compulsivity, and purposefulness. For use in research and clinical settings, IRP scientists constructed a 12-item version of the TCQ by selecting three items from each of the four factors that exhibited optimal within-factor reliability (Cronbach's alpha coefficient) and inter-item correlation. Smokers (n=196) completed the TCQ-Short Form (TCQ-SF) after overnight tobacco deprivation and on a separate day during ad libitum smoking. Confirmatory factor analyses indicated acceptable model fit for a 4-factor model, with congruence coefficients suggesting high to very high similarity in factor patterns and magnitude of factor loadings between the TCQ and TCQ-SF in both conditions. Scores on each factor were significantly greater after tobacco deprivation than ad libitum smoking, were associated with measures of tobacco withdrawal, and varied with degree of nicotine dependence. Cronbach's alpha coefficients and average inter-item correlations were similar in both conditions and were consistent with reliability values obtained in the initial validation of the TCQ. Test-retest correlation coefficients were also similar to those found in a previous study. These findings suggest that the TCQ-SF is as valid and reliable as the 47-item TCQ in measuring tobacco craving. Heishman, S.J., Singleton, E.G., and Pickworth, W.B. Nicotine and Tobacco Research, 10, pp. 643-651, 2008.

Chemical Biology Research Branch

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Studies of the Biogenic Amine Transporters. 12. Identification of Novel Partial Inhibitors of Amphetamine-induced Dopamine Release
Previous studies identified partial inhibitors and allosteric modulators of 5-HT ([5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-yl]carbamic acid ethyl ester [SoRI-6238], 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-piperidine [TB-1-099]) and dopamine transporters N-(Diphenylmethyl)-2-phenyl-4-quinazolinamine, [SoRI-9804]). IRP researchers report here the identification of three novel allosteric modulators of the dopamine transporter N-(2,2-Diphenylethyl)-2-phenyl-4-quinazolinamine [SoRI-20040], N-(3,3-Diphenylpropyl)-2-phenyl-4-quinazolinamine [SoRI-20041], [4-Amino-6-[(diphenylmethyl)amino]-5-nitro-2-pyridinyl]carbamic acid ethyl ester, [SoRI-2827]). Membranes were prepared from HEK cells expressing the cloned human dopamine (hDAT) transporter. [125I]RTI-55 binding and other assays followed published procedures. SoRI-20040, SoRI-20041 and SoRI-2827 partially inhibited [125I]RTI-55 binding with EC50 values ranging from ~1.4 uM to 3 uM and EMAX values decreasing as the [125I]RTI-55 concentrations increased. All three compounds decreased the [125I]RTI-55 Bmax and increased the apparent Kd in a manner well described by a sigmoid dose-response curve. In dissociation rate experiments, SoRI-20040 (10 uM) and SoRI-20041 (10 uM), but not SoRI-2827 (10 uM), slowed the dissociation of [125I]RTI-55 from hDAT by ~30%. Using rat brain synaptosomes, all three agents partially inhibited [3H]dopamine uptake with EC50 values ranging from 1.8 uM to 3.1 uM and decreased the VMAX value in a dose-dependent manner. SoRI-9804 and SoRI-20040 partially inhibited amphetamine-induced DAT-mediated release of [3H]MPP+ from rat caudate synaptosomes in a dose-dependent manner. Viewed collectively, the authors report several compounds that allosterically modulate hDAT binding and function, and identify novel partial inhibitors of amphetamine-induced dopamine release. Pariser, J.J., Partilla, J.S., Dersch, C.M., Ananthan, S., and Rothman R.B. J. Pharmacol. Exp. Ther., 326, pp. 286-295, 2008.

Chronic Fenfluramine Administration Increases Plasma Serotonin (5-HT) to Non-Toxic Levels
Large elevations in blood serotonin (5-HT) can produce valvular heart disease in humans and laboratory animals. Accordingly, one prevailing hypothesis (i.e., the "5-HT hypothesis") suggests 5-HT transporter substrates like fenfluramine increase the risk for valvular heart disease by elevating plasma 5-HT, secondary to the release of 5-HT from platelets. The main purpose of this study was to determine if chronic administration of fenfluramine increases plasma 5-HT to concentrations that are associated with the development of valvular heart disease. To the best of the authors' knowledge, this is the first study to address this issue using an in vivo microdialysis method that measures plasma 5-HT in non-hypoxic rats. The IRP scientists examined the effects of chronic (+/-)-fenfluramine and fluoxetine on plasma levels of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), in blood samples from conscious catheterized rats. Plasma indoles were measured by HPLC-ECD in dialysates of whole blood. Baseline plasma 5-HT was < 1.0 nM. Chronic fenfluramine (14-day minipump infusion) produced small increases in baseline plasma 5-HT (~2-to-4-fold), while chronic fluoxetine had no effect. Chronic fenfluramine and fluoxetine markedly decreased whole blood 5-HT, and reduced the ability of acute fenfluramine to evoke 5-HT release. Elevations in baseline plasma 5-HT produced by chronic fenfluramine are far below M levels necessary to produce valvular heart disease. Furthermore, chronic fenfluramine reduces the ability of acute fenfluramine to increase plasma 5-HT, suggesting the "5-HT hypothesis" can not explain the increased risk of valvular heart disease in patients treated with fenfluramine. Zolkowska, D., Baumann M.H., and Rothman R.B. J. Pharmacol. Exp. Ther., 324, pp. 791-797, 2008.

Drug Design and Synthesis Section, Chemical Biology Research Branch

Synthesis and Structure-Activity Relationships of a Potent mu-Agonist delta-Antagonist and an Exceedingly Potent Antinociceptive in the Enantiomeric 9-Substituted 5-(3-Hydroxyphenyl)-N-phenylethylmorphan Series
Both of the enantiomers of 5-(3-hydroxyphenyl)-N-phenylethylmorphan with C9a-methyl, C9-methylene, C9-keto and C9a- and C9b-hydroxy substituents were synthesized and pharmacologically evaluated. Three of the 10 compounds, (1R,5R,9S)-(-)-9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl-2-azabicyclo[3.3.1]nonane ((1R,5R,9S)-(-)-10),(1R,5S)-(+)-5-(3-hydroxyphenyl)-9-methylene-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S)-(+)-14), and (1R,5S,9R)-(-)-5-(3-hydroxyphenyl)-9-methyl-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S,9R)-(+)-15) had subnanomolar affinity at m-opioid receptors (Ki = 0.19, 0.19, and 0.63 nM, respectively). The (1R,5S)-(+)-14 was found to be a m-opioid agonist and a mu-, delta- and kappa-antagonist in [35S]GTP-g-S assays and was approximately 50-times more potent than morphine in a number of acute and subchronic pain assays including thermal and visceral models of nociception. The (1R,5R,9S)-(-)-10 compound with a C9-hydroxy substituent axially oriented to the piperidine ring (C9beta-hydroxy), was a mu-agonist about 500 times more potent than morphine. In the single-dose suppression assay it was greater than 1000 times more potent than morphine. It is the most potent known phenylmorphan antinociceptive. The molecular structures of these compounds were energy minimized with density functional theory at the B3LYP/6-31G* level, and then overlayed onto (1R,5R,9S)-(-)-10 using the heavy atoms in the morphan moiety as a common docking point. Based on modeling, the spatial arrangement of the protonated nitrogen atom and the 9beta-OH substituent in (1R,5R,9S)-(-)-10 may facilitate the alignment of a putative water chain enabling proton transfer to a nearby proton acceptor group in the mu-opioid receptor. Hiebel, A.-C., Lee, Y. S., Bilsky, E., Giuvelis, D., Deschamps, J.R., Aceto, M.D., May, E.L., Harris, L.S., Coop, A., Dersch, C.M., Partilla, J.S., Rothman, R B., Cheng, K., Jacobson, A.E., and Rice, K.C. J. Med. Chem., 50, pp. 3765-3776, 2007.

Drug Design and Synthesis Section, Chemical Biology Research Branch

A Novel Divergent Synthesis of ortho-Hydroxy-e and -f Oxide-Bridged 5-Phenylmorphans
5-(2-Bromo-3-methoxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-9-one was prepared in six steps from a known acetonitrile. Stereoselective reduction of the ketone furnished the corresponding - or -alcohols and their deprotonation, intramolecular cyclization, and demethylation gave ortho-hydroxy-e and -f oxide-bridged 5-phenylmorphans, respectively. This new synthetic route has the desired oxygenation pattern in place, eliminating the problematic diazonium reactions used in former syntheses. Zezula, J., Jacobson, A.E., and Rice, K.C. Heterocycles 71, pp. 881-889, 2007.

Opioid Ligands With Mixed Properties From Substituted Enantiomeric N-Phenethyl-5-Phenylmorphans. Synthesis of a micro-Agonist delta-Antagonist and delta-Inverse Agonists
Enantiomeric N-phenethyl-m-hydroxyphenylmorphans with various substituents in the ortho, meta or para positions of the aromatic ring in the phenethylamine side-chain (chloro, hydroxy, methoxy, nitro, methyl), as well as a pyridylethyl and a indolylethyl moiety on the nitrogen atom, were synthesized and their binding affinity to the mu-, delta- and kappa-opioid receptors was examined. The higher affinity ligands were further examined in the [35S]GTPgS assay to study their function and efficacy. 3-((1R,5S)-(-)-2-(4-Nitrophenethyl)-2-aza-bicyclo[3.3.1]nonan-5-yl)phenol ((-)-10m) was found to be a mu-agonist and delta-antagonist in that functional assay and was about 50 fold more potent than morphine in vivo. 3-((1R,5S)-(-)-2-(4-Chlorophenethyl)-2-aza-bicyclo[3.3.1]nonan-5-yl)phenol ((-)-10i) and several other ligands displayed inverse agonist activity at the delta-opioid receptor. The absolute configuration of all of the reported compounds was established by chemical conversion to a compound with known absolute configuration. Cheng, K., Kim, I.-J., Lee, M.J., Adah, S.A., Raymond, T.J., Bilsky, E., Aceto, M.D., May, E.L., Harris, L.S., Coop, A., Dersch, C M., Rothman, R.B., Jacobson, A.E., and Rice, K.C. Org. & Biomolec. Chem., 5, pp. 1177-1190, 2007.

A New Approach to the Synthesis of the Nonpeptide NOP Receptor Antagonist J-113397
To obtain multi-gram quantities of J-113397, a competitive antagonist of the N/OFQ-NOP receptor system, we report a new synthesis that eliminated the need for chromatographic separation. N-Benzyl protected 4-oxopiperidinecarboxylate was used as the starting material to obtain an N-benzyl intermediate that could be resolved at a relatively early stage in the synthesis. The crucial step in the synthesis was reduction of the double bond of the -enaminoester functionality of 1-benzyl-4-(3-ethyl-2-oxo-2,3-dihydro-benzoimidazol-1-yl)-1,2,5,6-tetrahydro-pyridine-3-carboxylic acid methyl ester, since Pd/C reduction gave inseparable mixtures. IRP scientits found that it could be reduced and epimerized to the desired trans diastereoisomer in a one-pot reaction by treatment with magnesium metal in methanol. Sulima, A., Folk, J., Jacobson, A.E., and Rice, K.C. Synthesis, 10, pp. 1547-1553, 2007.

Synthesis and Pharmacological Effects of the Enantiomers of the N-phenethyl Analogues of the ortho and para e- and f-oxide-Bridged Phenylmorphans
The N-phenethyl analogues of (1R*,4aR*,9aS*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2,3-c]pyridin-6-ol and 8-ol and (1R*,4aR*,9aR*)-2-phenethyl-1,3,4,9a-tetrahydro-2H-1,4a-propanobenzofuro[2.3-c]pyridin-6-ol and 8-ol, the ortho- (43) and para-hydroxy e- (20), and f-oxide-bridged 5-phenylmorphans (53 and 26) were prepared in racemic and enantiomerically pure forms from a common quaternary salt precursor. Optical resolutions were accomplished by salt formation with suitable enantiomerically pure chiral acids or by preparative HPLC on a chiral support. The N-phenethyl (-)- para-e enantiomer (1S,4aS,9aR-(-)-20) was found to be a mu-opioid agonist with morphine-like antinociceptive activity in a mouse assay. In contrast, the N-phenethyl (-)-ortho-f enantiomer (1R,4aR,9aR-(-)-53) had good affinity for the mu-opioid receptor (Ki = 7 nM) and was found to be a m-antagonist both in the [35S]GTP-g-S assay and in vivo. The molecular structures of these rigid enantiomers were energy minimized with density functional theory at the level B3LYP/6-31G* level, and then overlayed on a known potent mu-agonist. This superposition study suggests that the agonist activity of the oxide-bridged 5-phenylmorphans can be attributed to formation of a seven membered ring that is hypothesized to facilitate a proton transfer from the protonated nitrogen to a proton acceptor in the m-opioid receptor. Zezula, J., Singer, L. B., Przybyl, A.K., Hashimoto, A., Dersch, C.M., Rothman, R.B., Deschamps, J., Lee, Y.S., Jacobson, A.E., and Rice, K.C. Org. & Biomol. Chem., DOI: 10.1039/b803433h, 2008.

Behavioral Neuroscience Research Branch

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Ventral Tegmental Glutamate: A Role in Stress-, Cue-, and Cocaine-induced Reinstatement of Cocaine-seeking
Ventral tegmental dopamine neurons are activated by primary rewards and, when such rewards are predictable by reward-predicting stimuli. Glutamatergic input to the ventral tegmental area contributes to this activation: in animals trained to self-administer cocaine, cocaine-predictive cues trigger ventral tegmental glutamate release and dopaminergic activation. Mild footshock stress similarly causes glutamate release and dopaminergic activation in cocaine-trained but not cocaine-naive animals. The ability of cocaine-predictive and stress-associated cues to activate the dopamine system and to trigger cocaine craving appears to be related to changes in the ability of glutamate to activate dopaminergic neurons, changes known to be caused by experience with stress or with drugs of abuse. Wise, R. A. Neuropharmacology, 2008, [E-pub ahead of print].

Intracranial Self-administration of MDMA into the Ventral Striatum of the Rat: Differential Roles of the Nucleus Accumbens Shell, Core, and Olfactory Tubercle
Behavioral and anatomical data suggest that the ventral striatum, consisting of the nucleus accumbens and olfactory tubercle, is functionally heterogeneous. Cocaine and D-amphetamine appear to be more rewarding when administered into the medial olfactory tubercle or medial accumbens shell than into their lateral counterparts, including the accumbens core. IRP researchers sought to determine whether rats self-administer the popular recreational drug (+/-)-3,4-methylenedioxymethamphetamine (MDMA) into ventrostriatal subregions and whether the medial olfactory tubercle and medial accumbens shell mediate MDMA's positive reinforcing effects more effectively than their lateral counterparts. Rats receiving 30 mM MDMA into the medial olfactory tubercle, medial accumbens shell, or accumbens core, but not the lateral tubercle or lateral shell, showed higher self-administration rates than rats receiving vehicle. The medial shell supported more vigorous self-administration of MDMA at higher concentrations than the core or medial olfactory tubercle. In addition, intra-medial shell MDMA self-administration was disrupted by co-administration of the D1 or D2 receptor antagonists SCH 23390 (1-3 mM) or raclopride (3-10 mM). These data suggest that the ventral striatum is functionally heterogeneous. The medial accumbens shell appears to be more important than other ventrostriatal subregions in mediating the positive reinforcing effects of MDMA via both D1- and D2-type receptors. Together with previous data, our data also suggest that unidentified actions of MDMA interfere with the positive reinforcing effects of dopamine in the medial olfactory tubercle. Shin, R., Qin, M., Liu, Z-H., and Ikemoto, S. Psychopharmacology (Berl), 198(2), pp. 261-270, 2008.

Dual Role of Medial A10 Dopamine Neurons in Affective Encoding
Increasing evidence suggests that the activation of medial A10 neurons mediates positive affective encoding. However, little is known about the functions of the inhibition of midbrain dopamine neurons. Here IRP investigators show evidence suggesting that the inhibition of medial A10 neurons mediates a negative affective state, leading to negative affective encoding, whereas blunting the activation of medial A10 neurons disrupts positive affective encoding involving food reward. The authors used a microinjection procedure, in which the D(2) dopamine receptor agonist quinpirole was administered into the cell body region of the dopamine neurons, a procedure that reduces dopamine cell firing. Microinjections of quinpirole into the posteromedial ventral tegmental area, but not its more lateral counterparts, led to conditioned place aversion. Quinpirole administration to this site also decreased food intake and basal dopamine concentration in the ventromedial striatum, a major projection area of medial A10 neurons. In addition, moderate quinpirole doses that did not lead to conditioned place aversion or disrupt food intake abolished food-conditioned place preference, suggesting that blunting dopamine impulse activity in response to food reward disrupts positive affective encoding in associated external stimuli. These data support the hypothesis that activation of medial A10 dopamine neurons mediates a positive affective state, leading to positive affective encoding, while their inhibition mediates a negative affective state, leading to negative affective encoding. Together with previous findings, the authors propose that medial A10 neurons are an important component of the mechanism via which animals learn to avoid negative incentive stimuli. Liu, Z-H., Shin, R., and Ikemoto, S. Neuropsychopharmacology, 2008, E-pub ahead of print.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Context-induced Relapse to Drug Seeking: A Review
In humans, exposure to environmental contexts previously associated with drug intake often provokes relapse to drug use, but the mechanisms mediating this relapse are unknown. Based on early studies by Bouton & Bolles on context-induced 'renewal' of learned behaviors, we developed a procedure to study context-induced relapse to drug seeking. In this procedure, rats are first trained to self-administer drug in one context. Next, drug-reinforced lever responding is extinguished in a different (non-drug) context. Subsequently, context-induced reinstatement of drug seeking is assessed by re-exposing rats to the drug-associated context. Using variations of this procedure, we and others reported reliable context-induced reinstatement in rats with a history of heroin, cocaine, heroin-cocaine combination, alcohol and nicotine self-administration. Here, IRP scientists first discuss potential psychological mechanisms of context-induced reinstatement, including excitatory and inhibitory Pavlovian conditioning, and occasion setting. They then summarize results from pharmacological and neuroanatomical studies on the role of several neurotransmitter systems (dopamine, glutamate, serotonin and opioids) and brain areas (ventral tegmental area, accumbens shell, dorsal striatum, basolateral amygdala, prefrontal cortex, dorsal hippocampus and lateral hypothalamus) in context-induced reinstatement. The authors conclude by discussing the clinical implications of rat studies on context-induced reinstatement of drug seeking. Crombag, H.S., Bossert, J.M., Koya, E., and Shaham, Y. Philos Trans R Soc Lond B Biol Sci., 2008, [E-pub ahead of print].

Role of Ventral Medial Prefrontal Cortex in Incubation of Cocaine Craving
Cue-induced drug-seeking in rodents progressively increases after withdrawal from cocaine, suggesting that cue-induced cocaine craving incubates over time. Here, IRP researchers explored the role of the medial prefrontal cortex (mPFC, a brain area previously implicated in cue-induced cocaine seeking) in this incubation. They trained rats to self-administer cocaine for 10 days (6h/day, infusions were paired with a tone-light cue), and then assessed after 1 or 30 withdrawal days the effect of exposure to cocaine cues on lever presses in extinction tests. The authors found that cue-induced cocaine-seeking in the extinction tests was higher after 30 withdrawal days than after 1 day. The time-dependent increases in extinction responding were associated with large (ventral mPFC) or modest (dorsal mPFC) increases in ERK phosphorylation (a measure of ERK activity and an index of neuronal activation). After 30 withdrawal days, ventral but not dorsal injections of muscimol+baclofen (GABAa+GABAb receptor agonists that inhibit neuronal activity) decreased extinction responding. After 1 withdrawal day, ventral but not dorsal mPFC injections of bicuculline+saclofen (GABAa+GABAb receptor antagonists that increase neuronal activity) strongly increased extinction responding. Finally, muscimol+baclofen had minimal effect on extinction responding after 1 day, and in cocaine-experienced rats, ventral mPFC injections of muscimol+baclofen or bicuculline+saclofen had no effect on lever presses for an oral sucrose solution. The present results indicate that ventral mPFC neuronal activity plays an important role in the incubation of cocaine craving. Koya, E., Uejima, J.L., Wihbey, K.A., Bossert, J.M., Hope, B.T., and Shaham, Y. Neuropharmacology, 2008, [E-pub ahead of print].

In Vivo Electrophysiology Unit, Behavioral Neuroscience Research Branch

Behavioral and Temperature Effects of Delta 9-tetrahydrocannabinol in Human-relevant Doses in Rats
Marijuana smoking dramatically alters responses to various environmental stimuli. To study this phenomenon, IRP scientists assessed how delta-9-tetrahydrocannabinol (THC), a primary psychoactive ingredient of marijuana, affects locomotor and brain (nucleus accumbens or NAcc), muscle and skin temperature responses to natural arousing stimuli (one-minute tail-pinch and one-minute social interaction with another male rat) and iv cocaine (1 mg/kg) in male rats. THC was administered at three widely varying doses (0.5, 2.0 and 8.0 mg/kg, ip), and the drug-induced changes in basal values and responses to stimuli were compared to those occurring following ip vehicle injections (control). Each stimulus in control conditions caused acute locomotor activation, a prolonged increase in brain and muscle temperature (0.6-1.0 degrees C for 20-50 min) and transient decrease in skin temperature (-0.6 degrees C for 1-3 min). While THC at any dose had a tendency to decrease spontaneous locomotion as well as brain and muscle temperatures, true hypothermia and hypoactivity as well as clearly diminished locomotor and temperature responses to all stimuli were only seen following the largest dose. In this case, temperature decreases in the NAcc were stronger than in the muscle, suggesting metabolic brain inhibition as the primary cause of hypoactivity, hypothermia and hyporesponsiveness. While weaker in strength and without associated vasodilatation, this response pattern is mimicked by general anesthetics, questioning to what extent the hypothermic action of THC is specific (i.e., mediated via endogenous cannabinoid receptors) or non-specific, reflecting drug interaction with membrane lipids or other receptors. In contrast, weaker behavioral and temperature effects of THC at lower doses resemble those of diazepam, whose locomotion- and temperature-decreasing effects are evident only in activated conditions, when rats are moving and basal temperatures are elevated. Smirnov, M.S. and Kiyatkin, E.A. Brain Research, 2008, [E-pub ahead of print].

Sensory Effects of Intravenous Cocaine on Dopamine and Non-dopamine Ventral Tegmental Area Neurons
Intravenous (iv) cocaine mimics salient somato-sensory stimuli in their ability to induce rapid physiological effects, which appear to involve its action on peripherally located neural elements and fast neural transmission via somato-sensory pathways. To further clarify this mechanism, single-unit recording with fine glass electrodes was used in awake rats to examine responses of ventral tegmental area (VTA) neurons, both presumed dopamine (DA) and non-DA, to iv cocaine and tail-press, a typical somato-sensory stimulus. To exclude the contribution of DA mechanisms to the observed neuronal responses to sensory stimuli and cocaine, recordings were conducted during full DA receptor blockade (SCH23390+eticloptide). Iv cocaine (0.25 mg/kg delivered over 10 s) induced significant excitations of ~63% of long-spike (presumed DA) and ~70% of short-spike (presumed non-DA) VTA neurons. In both subgroups, neuronal excitations occurred with short latencies (4-8 s), peaked at 10-20 s (30-40% increase over baseline) and disappeared at 30-40 s after the injection onset. Most long-(67%) and short-spike (89%) VTA neurons also showed phasic responses to tail-press (5-s). All responsive long-spike cells were excited by tail-press; excitations were very rapid (peak at 1 s) and strong (100% rate increase over baseline) but brief (2-3 s). In contrast, both excitations (60%) and inhibitions (29%) were seen in short-spike cells. These responses were also rapid and transient, but excitations of short-spike units were more prolonged and sustained (10-15 s) than in long-spike cells. These data suggest that in awake animals iv cocaine, like somato-sensory stimuli, rapidly and transiently excites VTA neurons of different subtypes. Therefore, along with direct action on specific brain substrates, central effects of cocaine may occur, via an indirect mechanism, involving peripheral neural elements, visceral sensory nerves and rapid neural transmission. Via this mechanism, cocaine, like somato-sensory stimuli, can rapidly activate DA neurons and induce phasic DA release, creating the conditions for DA accumulation by a later occurring and prolonged direct inhibiting action on DA uptake. By providing a rapid neural signal and triggering transient neural activation, such a peripherally driven action might play a crucial role in the sensory effects of cocaine, thus contributing to learning and development of drug-taking behavior. Brown, P.L. and Kiyatkin, E.A. Brain Research, 1218, pp. 230-249, 2008.

Medicinal Chemistry Section, Medications Discovery Research Branch

Labeling of Dopamine Transporter Transmembrane Domain 1 with the Tropane Ligand [125I]MFZ 2-24 Implicates Proximity of Cocaine and Substrate Active Sites
The novel photoaffinity ligand N-[4- (4-azido -3-[125I]-iodophenyl) butyl] -2-carbomethoxy-3-(4-chlorophenyl) tropane ([125I]MFZ 2-24) was used to investigate the site for cocaine binding on the dopamine transporter (DAT). [125I]MFZ 2-24 irreversibly labeled both rat striatal and expressed human DAT with high affinity and appropriate pharmacological specificity. Tryptic proteolysis of [125I]MFZ 2-24 labeled DAT followed by epitope- specific immunoprecipitation demonstrated that the ligand becomes adducted almost exclusively to transmembrane domains (TMs) 1-2. Further localization of [125I]MFZ 2-24 incorporation achieved by proteolyzing labeled wild type and methionine mutant DATs with cyanogen bromide identified the sequence between residues 68-80 in TM1 as the ligand adduction site. This is in marked contrast to the previously identified attachment of the photoaffinity label [125I]RTI 82 in TM6. Because [125I]MFZ 2-24 and [125I]RTI 82 possess identical tropane pharmacophores and differ only in the placement of the reactive azido moieties, their distinct incorporation profiles identify the regions of the protein adjacent to different aspects of the cocaine molecule. These findings thus strongly support the direct interaction of cocaine on DAT with TM1 and TM6, both of which have been implicated by mutagenesis and homology to a bacterial leucine transporter as active sites for substrates. These results directly establish the proximity of TMs 1 and 6 in DAT and suggest that the mechanism of transport inhibition by cocaine involves close interactions with multiple regions of the substrate permeation pathway. Parnas, M.L., Gaffaney, J.D., Zou, M.-F., Lever, J.R., Newman, A.H., and Vaughan, R.A. Molecular Pharmacology, 73, pp. 1141-1160, 2008.

The Binding Sites for Cocaine and Dopamine in the Dopamine Transporter are Overlapping
Cocaine is a widely abused substance with psychostimulant effects attributed to inhibition of the dopamine transporter (DAT). Here, IRP investigators present molecular models for DAT binding of cocaine and its analogue CFT ((-)-2-carbomethoxy-3-(4-fluorophenyl)tropane or WIN 35,428) based on the high-resolution structure of the bacterial transporter homologue, LeuT. The authors'models suggest that the binding site for cocaine and CFT is deeply buried between transmembrane segments (TM) 1, 3, 6, and 8, and overlaps with the binding sites for the substrates, dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. The models were validated by detailed mutagenesis, and by trapping the radiolabeled cocaine analogue [3H]CFT in the transporter through cross-linking of engineered cysteines or by engineering of a Zn2+ binding site situated extracellular to the predicted common binding pocket. Summarized, the data demonstrate for the first time the molecular basis for the competitive inhibition of dopamine transport by cocaine and refutes the possibility of a dopamine-sparing cocaine antagonist. Beuming, T., Kniazeff, J., Bergmann, M.L., Shi, L., Gracia, L., Raniszewska, K., Newman, A.H., Javitch, J.A., Weinstein, H., Gether, U., and Loland, C.J. Nature Neuroscience, 11, pp. 780-789, 2008.


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