Research Findings - Intramural Research

Biomedical Informatics Section, Administrative Management Branch

Pharmacy Informatics in Controlled Substances Research
Pharmacies have become essential components in support of clinical research. Their operations become highly complex when preponderance of prescriptions is composed of controlled substances. Application of informatics will result in more efficient operations. IRP scientists present the Pharmacy Information Management System (PIMS) that includes a set of decision support systems to address the pharmacy challenges and is integrated into our electronic health record system. Lin, J.-L., Vahabzadeh, M., Mezghanni, M., Na, P.J., Leff, M., and Contoreggi, C. Proc. AMIA Annual Symposium on Biomedical and Health Informatics: From Foundations to Applications to Policy (American Medical Informatics Association-2008), pp. 1025, 2008.

Molecular Neurobiology Research Branch

Nicotine Abstinence Genotyping: Assessing the Impact on Smoking Cessation Clinical Trials
MNB workers and collaborators have used data from prior genome wide association studies and twin studies to provide the first estimate of the likely impact of application of molecular genetics to clinical trials for smoking cessation. Since this is one of the areas of addiction in which efficacies of a number of pharmacologic therapeutics have been established from well described clinical trials, this data also provides a substantial impetus for application of molecular genetics to trials for pharmacological treatments for other addictions as well. Uhl, G.R., Drgon, T., Johnson, C., and Rose, J.E. Pharmacogenomics J. Sepember 9, 2008.

Brain-derived Neurotrophic Factor and Obesity in the WAGR Syndrome
MNB investigators have aided collaborators from the NIH in elucidating clear-cut effects of human BDNF deletion on human obesity. While these comparisons of WGAR individuals who have vs. those who do not have obesity provides direct insight into a limited number of individuals, this human data and data from knockout mice both combine to provide a compelling implication of BDNF in obesity. Han, J.C., Liu, Q.R, Jones, M., Levinn, R. L., Menzie, C.M., Jefferson-George, K.S., Adler-Wailes, D.C., Sanford, E.L., Lacbawan, F L., Uhl, G.R., Rennert, O.M., and Yanovski, J.A.. N. Engl. J. Med. 359(9), pp. 918-27, 2008.

Molecular Genetics of Successful Smoking Cessation: Convergent Genome-wide Association Study Results
MNB investigators have provided the first genome wide association data that is remarkably replicated for smoking cessation. These data should provide many clues to individual differences in ability to quit use of other substances as well. 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.

OKCAM: An Ontology-based, Human-centered Knowledgebase for Cell Adhesion Molecules
MNB investigators, in collaboration with Beijing University investigators, have provided a novel database that, for the first time, enumerates the universe of human "cell adhesion" related genes. Prior MNB success in identifying GWA signals in large number of these genes implicate many of them in addiction mechanisms. Li, C.Y., Liu, Q.R, Zhang, P.W., Li, X.M., Wei, L., and Uhl, G.R. Nucleic Acids Res. 2008 Sepember 12.

Genome-wide Association for Methamphetamine Dependence: Convergent Results from 2 Samples
MNB investigators, in collaborations with Japanese JGIDA and Taiwan methamphetamine group investigators provide novel genome wide association results for methamphetamine dependence in two replicate, carefully studied samples. 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., and Lin, S.K. Arch. Gen. Psychiatry, 65(3), pp. 345-355, 2008.

Neural Protection and Regeneration Section, Molecular Neurobiology Research Branch

Bone Morphogenetic Protein-7 Reduces Toxicity Induced by High Doses of Methamphetamine in Rodents
Methamphetamine (MA) is a drug of abuse as well as a dopaminergic neurotoxin. IRP investigators have previously demonstrated that pretreatment with bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. In this study, the authors examined the neuroprotective effects of BMP7 against MA-mediated toxicity in dopaminergic neurons. Primary dopaminergic neurons, prepared from rat embryonic ventral mesencephalic tissue, were treated with MA. High doses of MA decreased tyrosine hydroxylase immunoreactivity (THir) while increasing terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling. These toxicities were significantly antagonized by BMP7. Interaction of BMP7 and MA in vivo was first examined in CD1 mice. High doses of MA (10 mg/kgx4 s.c.) significantly reduced locomotor activity and THir in striatum. I.c.v. administration of BMP7 antagonized these changes. In BMP7 +/- mice, MA suppressed locomotor activity and reduced TH immunoreactivity in nigra reticulata to a greater degree than in wild type BMP7 +/+ mice, suggesting that deficiency in BMP7 expression increases vulnerability to MA insults. Since BMP7 +/- mice also carry a LacZ-expressing reporter allele at the BMP7 locus, the expression of BMP7 was indirectly measured through the enzymatic activity of beta-galactosidase (beta-gal) in BMP7 +/- mice. High doses of MA significantly suppressed beta-gal activity in striatum, suggesting that MA may inhibit BMP7 expression at the terminals of the nigrostriatal pathway. A similar effect was also found in CD1 mice in that high doses of MA suppressed BMP7 mRNA expression in nigra. In conclusion, these data indicate that MA can cause lesioning in the nigrostriatal dopaminergic terminals and that BMP7 is protective against MA-mediated neurotoxicity in central dopaminergic neurons. Chou, J., Lu, Y., Kuo, C.C., Powers, K., Shen, H., Harvey, B.K., Hoffer, B.J., and Wang, Y. Neuroscience, 151, pp. 92-103, 2008.

Nigrostriatal Alterations in Bone Morphogenetic Protein Receptor II Dominant Negative Mice
IRP scientists previously demonstrated that exogenous application of bone morphogenetic protein 7 (BMP7) reduced 6-hydroxydopamine-mediated neurodegeneration in a rodent model of Parkinson's disease. The purpose of this study is to examine the endogenous neurotrophic properties of BMP Receptor II in dopaminergic neurons of the nigrostriatal pathway. Adult male BMPRII dominant negative (BMPRIIDN) mice and their wild type controls (WT) were placed in the activity chambers for 3 days to monitor locomotor activity. Animals were sacrificed for tyrosine hydroxylase (TH) immunostaining. A subgroup of BMPRIIDN and WT mice were injected with high doses of methamphetamine (MA) and were sacrificed for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) histochemistry at 4 days after injection. The authors found that BMPRIIDN mice had lower locomotor activity than the WT. There is a significant decrease in TH neuronal number in substantia nigra compacta, TH fiber density in the substantia nigra reticulata, and TH immunoreactivity in striatum in the BMPRIIDN mice, suggesting that deficiency in endogenous BMP signaling reduces dopaminergic innervation and motor function in the nigrostriatal pathway. Administration of MA increased TUNEL labeling in the substantia nigra in the BMPRIIDN mice. In conclusion, these data suggest that endogenous BMPs have trophic effects on nigrostriatal dopaminergic neurons. Deficiency in BMP signaling increases vulnerability to insults induced by high doses of MA. Chou, J., Harvey, B.K., Ebendal, T., Hoffer, B.J., and Wang, Y. Acta Neurochir. Suppl 101, pp. 93-98, 2008.

BMP7 Reduces Synergistic Injury Induced by Methamphetamine and Ischemia in Mouse Brain
Previous studies have indicated that methamphetamine (MA) potentiates neurodegeneration induced by ischemia in brain. IRP scientists, and others, have reported that bone morphogenetic protein 7 (BMP7) is protective against MA and ischemic brain injury. The purpose of this study was to examine whether BMP7 reduces synergistic injury induced by both MA and cerebral ischemia. Adult CD-1 mice were treated with MA (10 mg/kg x 4, each dose two hours apart) or saline. Using the quantitative real time polymerase chain reaction, the authors found that MA suppressed the expression of BMP7 mRNA in the cerebral cortex one day after injection. Ischemic and reperfusional injuries were introduced by ligation of the right middle cerebral artery for 90 min after MA injection. Animals were sacrificed for caspase 3/7 activity assay and tri-phenyl-tetrazolium chloride staining at 1 hour and 2 days after reperfusion, respectively. Cerebral infarction and caspase-3/7 activity were enhanced in the stroke animals pretreated with MA; both responses were attenuated by pretreatment with BMP7. In conclusion, these data suggest that MA facilitates cerebral infarction after ischemia possibly mediated, in part, through the suppression of BMP7. Shen, H., Luo, Y., Kuo, C.C., and Wang, Y. Neurosci. Lett., 442, pp. 15-18, 2008.

Tropism and Toxicity of Adeno-associated Viral Vector Serotypes 1, 2, 5, 6, 7, 8, and 9 in Rat Neurons and Glia in vitro
Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity. These studies facilitate experimentation of mechanistic actions of genes in models of neurodegeneration using primary neuronal cultures. Howard, D., Powers, K., Wang, Y., and Harvey, B. Virology, 372, pp. 24-34, 2008.

Molecular Neuropsychiatry Research Branch

Growth Factor Signals in Neural Cells: Coherent Patterns of Interaction Control Multiple Levels of Molecular and Phenotypic Responses
Individual neurons express receptors for several different growth factors that influence the survival, growth, neurotransmitter phenotype and other properties of the cell. While there has been considerable progress in elucidating the molecular signal transduction pathways and physiological responses of neurons and other cells to individual growth factors, little is known about if and how signals from different growth factors are integrated within a neuron. In this study, IRP investigators determined the interactive effects of nerve growth factor (NGF), insulin-like growth factor 1 (IGF-1) and epidermal growth factor (EGF) on the activation status of downstream kinase cascades and transcription factors, cell survival, and neurotransmitter production in neural cells that express receptors for all three growth factors. The authors document considerable differences in the quality and quantity of intracellular signaling and eventual phenotypic responses that are dependent on whether cells are exposed to either single or multiple growth factors. Their findings suggest that in true physiological settings where multiple growth factors are present, activation of one receptor type may result in molecular and phenotypic responses that are different from what is observed in typical experimental paradigms in which cells are exposed to only a single growth factor at a time. Martin, B., Brenneman, R., Golden, E., Walent, T., Becker, K.G,, Prabhu, V.V., Wood, W. 3rd, Ladenheim, B., Cadet, J.L., and Maudsley, S. J. Biol. Chem, E-pub 2008.

Heavy Marijuana Users Show Increased Serum Apolipoprotein C-III Levels: Evidence from Proteomic Analyses
Marijuana (MJ) is the most commonly used illicit drug in the United States. Its abuse is associated with cognitive dysfunctions and increased resistance to blood flow in the cerebral vasculature. In addition, MJ abuse is associated with increased risks of potentially serious cardiovascular disorders. In the present study, IRP scientists used the protein chip platform based on surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDITOF-MS) to test the possibility that MJ abuse might be associated with changes in serum protein levels. Indeed, MJ users showed significant increases in three protein peaks, which were identified as three isoforms of apolipoprotein (apo) C-III. Immunoprecipitation using an apoC-III antibody also validated the identification of the proteins. Marijuana-induced increases in apoC-III levels might occur through chronic stimulation of hepatic cannabinoid receptors (CB1 and/or CB2) by its active ingredient, D-9-tetrahydrocannibol (THC). Thus, chronic MJ abuse might cause increased transcription and/or translation of apoC-III in the liver with corresponding changes reflected in the plasma of these patients. In any case, because apoC-III is a cardiovascular risk factor, the increased levels observed in MJ users might explain, in part, the cardiac and cerebral abnormalities reported in these patients. Jayanthi, S., Buie, S., Moore, S., Herning, R.I., Better, W., Wilson, N.M., Contoreggi, C., and Cadet, J.L. Mol. Psychiatry E-pub 2008.

Office of the Scientific Director

Signal-Averaged Electrocardiogram in Physically Healthy, Chronic 3,4-Methylene-dioxymethamphetamine (MDMA) Users
3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) use has been associated with cardiac arrhythmias. Markers of ventricular late potentials (VLP), which may be a precursor to malignant ventricular arrhythmias, can be detected by signal-averaged electrocardiography (SA-ECG), but not by standard ECG. This study evaluated SA-ECG parameters in 21 physically healthy, recently abstinent MDMA users who also used cannabis, 18 physically healthy cannabis users, and 54 non-drug-using controls. All subjects were > 18 years old. IRP researchers analyzed three SA-ECG parameters considered markers of VLPs: duration of filtered QRS complex (fQRS), duration of low amplitude potentials during terminal 40 ms of QRS complex (LAS40), and root mean square voltage during terminal 40 ms of QRS complex (RMS40). MDMA users, cannabis users, and non-drug-using controls did not differ significantly from each other in fQRS, LAS40, or RMS40 values or in the proportion of subjects with abnormal SA-ECG parameters. There were significant gender differences among controls, but not among MDMA users. These findings suggest that chronic MDMA use or cannabis use by physically healthy adults is not associated with a high prevalence of electrophysiological risk factors for cardiac ventricular arrhythmias. Kanneganti, P.., Huestis, M.A., Kolbrich, E.A., Goodwin, R., Ziegelstein, R.C., and Gorelick, D.A. American Journal of Drug and Alcohol Abuse, 34, pp. 712-720, 2008.

Brain Mu-Opioid Receptor Binding: Relationship to Relapse to Cocaine Use After Monitored Abstinence
Cocaine users have increased regional brain mu-opioid receptor (mOR) binding which correlates with cocaine craving. The relationship of mOR binding to relapse is unknown. This study evaluated regional brain mOR binding as a predictor of relapse to cocaine use in 15 non-treatment-seeking, adult cocaine users who were housed on a closed research ward for 12 weeks of monitored abstinence, then followed for up to one year after discharge. Regional brain mOR binding was measured after one and 12 weeks using positron emission tomography (PET) with [11C]carfentanil (a selective mOR agonist). Time to first cocaine use (lapse) and to first two consecutive days of cocaine use (relapse) after discharge was based on self-report and urine toxicology. A shorter interval before relapse was associated with increased mOR binding in frontal and temporal cortical regions at one and 12 weeks of abstinence and with a lesser decrease in binding between one and 12 weeks. There were significant positive correlations between mOR binding at 12 weeks and % days of cocaine use during the first month after relapse. In multiple linear regression analysis, mOR binding contributed significantly to the prediction of time to relapse (R2 = 0.79, P < 0.001), even after accounting for clinical variables. These findings show that increased brain mOR binding in frontal and temporal cortical regions is a significant independent predictor of time to relapse to cocaine use, suggesting an important role for the brain endogenous opioid system in cocaine addiction. Gorelick, D.A., Kim, Y.-K., Bencherif, B., Boyd, S.J., Nelson, R., Copersino, M.L., Dannals, R.F., and Frost J.J. Psychopharmacology, 200, pp. 475-486, 2008.

Clinical Psychopharmacology Section, Chemical Biology Research Branch

Tolerance to 3,4-Methylenedioxymethamphetamine in Rats Exposed to Single High-Dose Binges
3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) stimulates the transporter-mediated release of monoamines, including 5-HT. High-dose exposure to MDMA causes persistent 5-HT deficits (e.g. depletion of brain 5-HT) in animals, yet the functional and clinical relevance of such deficits are poorly defined. Here IRP scientists examine functional consequences of MDMA-induced 5-HT depletions in rats. Male rats received binges of three i.p. injections of MDMA or saline, one injection every 2 h; MDMA was given at a threshold pharmacological dose (1.5 mg/kgx3, low dose) or at a fivefold higher amount (7.5 mg/kgx3, high dose). One week later, jugular catheters and intracerebral guide cannulae were implanted. Two weeks after binges, rats received acute i.v. challenge injections of 1 and 3 mg/kg MDMA. Neuroendocrine effects evoked by i.v. MDMA (prolactin and corticosterone secretion) were assessed via serial blood sampling, while neurochemical effects (5-HT and dopamine release) were assessed via microdialysis in brain. MDMA binges elevated core temperatures only in the high-dose group, with these same rats exhibiting approximately 50% loss of forebrain 5-HT 2 weeks later. Prior exposure to MDMA did not alter baseline plasma hormones or dialysate monoamines, and effects of i.v. MDMA were similar in saline and low-dose groups. By contrast, rats pretreated with high-dose MDMA displayed significant reductions in evoked hormone secretion and 5-HT release when challenged with i.v. MDMA. As tolerance developed only in rats exposed to high-dose binges, hyperthermia and 5-HT depletion are implicated in this phenomenon. These results suggest that MDMA tolerance in humans may reflect 5-HT deficits which could contribute to further dose escalation. Baumann, M.H., Clark, R.D., Franken, F.H., Rutter, J.J., and Rothman, R.B. Neuroscience, 152, pp. 773-784, 2008.

Herkinorin Analogues with Differential beta-Arrestin-2 Interactions
Salvinorin A is a psychoactive natural product that has been found to be a potent and selective kappa opioid receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such as morphine in that it mediates potent kappa opioid receptor signaling yet leads to less receptor down-regulation than observed with other kappa agonists. IRP scientists' initial chemical modifications of salvinorin A have yielded one analogue, herkinorin (1c), with high affinity at the muOR. They recently reported that 1c does not promote the recruitment of beta-arrestin-2 to the muOR or receptor internalization. Here they describe three new derivatives of 1c (3c, 3f, and 3i) with similar properties and one, benzamide 7b, that promotes recruitment of beta-arrestin-2 to the muOR and receptor internalization. When the important role mu opioid receptor regulation plays in determining physiological responsiveness to opioid narcotics is considered, mu opioids derived from salvinorin A may offer a unique template for the development of functionally selective mu opioid receptor-ligands with the ability to produce analgesia while limiting adverse side effects. Tidgewell, K., Groer, C.E., Harding, W.W., Lozama, A., Schmidt, M., Marquam, A., Hiemstra, J., Partilla, J.S., Dersch, C.M., Rothman, R.B., Bohn, L.M., and Prisinzano, T.E. J. Med. Chem., 51, pp. 2421-2431, 2008.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Effects of Chronic Caffeine Exposure on Adenosinergic Modulation of the Discriminative-stimulus Effects of Nicotine, Methamphetamine, and Cocaine in Rats
Adenosine receptors are involved in cocaine and methamphetamine discrimination and exposure to caffeine can affect behavioral effects of nicotine in rats. Here IRP researchers investigated the relative involvement of adenosine A(1) and A(2A) receptors in nicotine, cocaine, and methamphetamine discrimination, before and/or during chronic caffeine exposure. The nonselective adenosine receptor antagonist caffeine, the A(1)-receptor antagonist cyclopentyltheophylline (CPT), and the A(2A)-receptor antagonist MSX-3 were evaluated in rats trained to discriminate 0.4 mg/kg nicotine from saline under a fixed-ratio schedule of food delivery. Effects of adenosine receptor antagonists were then compared in rats discriminating nicotine, methamphetamine, or cocaine from saline during chronic caffeine exposure in their drinking water. Caffeine, CPT, and MSX-3 partially generalized to nicotine and shifted nicotine dose-response curves leftwards. During chronic caffeine exposure, however, all three ligands failed to generalize to nicotine and failed to shift nicotine dose-response curves. In previous experiments, CPT and MSX-3 partially generalized to methamphetamine and cocaine and shifted dose-response curves leftwards. In the present experiments, CPT neither generalized nor shifted dose-response curves for methamphetamine or cocaine during chronic caffeine exposure. However, MSX-3 partially generalized to both psychostimulants and shifted their dose-response curves leftwards. Caffeine partially generalized to cocaine, but not methamphetamine, and shifted both dose-response curves leftwards. The authors conclude that both adenosine A(1) and A(2A) receptors are capable of modulating the discriminative-stimulus effects of nicotine. Chronic caffeine exposure produces complete tolerance to both A(1)- and A(2A)-mediated effects in nicotine-trained rats. In contrast, chronic caffeine exposure produces tolerance to adenosine A(1)-mediated, but not A(2A)-mediated, effects in methamphetamine- and cocaine-trained rats. Justinova, Z., Ferre, S., Barnes, C., Wertheim, C.E., Pappas, L.A., Goldberg, S.R., and Le Foll, B. Psychopharmacology (Berl), August 8, 2008, Epub ahead of print, PMID: 18688601.

Looking for the Role of Cannabinoid Receptor Heteromers in Striatal Function
The introduction of two concepts, "local module" and "receptor heteromer", facilitates the understanding of the role of interactions between different neurotransmitters in the brain. In artificial cell systems, cannabinoid CB(1) receptors form receptor heteromers with dopamine D(2), adenosine A(2A) and mu opioid receptors. There is indirect but compelling evidence for the existence of the same CB(1) receptor heteromers in striatal local modules centered in the dendritic spines of striatal GABAergic efferent neurons, particularly at a postsynaptic location. Their analysis provides new clues for the role of endocannabinoids in striatal function, which cannot only be considered as retrograde signals that inhibit neurotransmitter release. Recent studies using a new method to detect heteromerization of more than two proteins, which consists of sequential BRET-FRET (SRET) analysis, has demonstrated that CB(1), D(2) and A(2A) receptors can form heterotrimers in transfected cells. It is likely that functional CB(1)-A(2A)-D(2) receptor heteromers can be found where they are highly co-expressed, in the dendritic spines of GABAergic enkephalinergic neurons. The functional properties of these multiple receptor heteromers and their role in striatal function need to be determined. Ferre, S., Goldberg, S.R., Lluis, C., and Franco, R. Neuropharm., July 19, 2008, Epub ahead of print, PMID: 18691604.

Dopamine D(2) and Adenosine A(2A) Receptors Regulate NMDA-Mediated Excitation in Accumbens Neurons through A(2A)-D(2) Receptor Heteromerization
Bursting activity of striatal medium spiny neurons results from membrane potential oscillations between a down- and an upstate that could be regulated by G-protein-coupled receptors. Among these, dopamine D(2) and adenosine A(2A) receptors are highly enriched in striatal neurons and exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. More particularly, respective involvements of common intracellular signaling cascades and A(2A)-D(2) receptor heteromerization remain unknown. Here IRP scientists show, by performing perforated-patch-clamp recordings on brain slices and loading competitive peptides, that D(2) and A(2A) receptors regulate the induction by N-methyl-D-aspartate of a depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D(2) receptor activation abolished transitions between a hyperpolarized resting potential and a depolarized plateau potential by regulating the Ca(V)1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A(2A) receptor activation had no effect per se but fully reversed the effects of D(2) receptor activation through a mechanism in which A(2A)-D(2) receptors heteromerization is strictly mandatory, demonstrating therefore a first direct physiological relevance of these heteromers. These results show that membrane potential transitions and firing patterns in striatal neurons are tightly controlled by D(2) and A(2A) receptors through specific protein-protein interactions including A(2A)-D(2) receptors heteromerization. Azdad, K., Gall, D., Woods, A.S., Ledent, C., Ferre, S., and Schiffmann, S.N. Neuropsychopharm., September 17, 2008, Epub ahead of print, PMID: 18800071.

Anandamide-Induced Behavioral Disruption through a Vanilloid-dependent Mechanism in Rats
Endocannabinoids are involved in a variety of behavioral and physiological processes that are just beginning to be understood. In the five-choice serial reaction-time task, exogenous cannabinoids have been found to alter attention, but endocannabinoids such as anandamide have not been studied. IRP scientists used this task to evaluate the effects of anandamide in rats. Since anandamide is a ligand for not only cannabinoid receptors but also transient receptor potential vanilloid 1 (TRPV1) receptors, and as recently suggested, peroxisome proliferator-activated nuclear receptor-alpha (PPARalpha), the authors also determined whether anandamide's effects in this task were mediated by each of these receptors. Whenever one of five holes was illuminated for 2 s, a food pellet was delivered if a response occurred in that hole during the light or within 2 s after the light. Anandamide increased omission errors and decreased responding during inter-trial intervals. These effects were blocked by the TRPV1 antagonist capsazepine, but not by the cannabinoid-receptor antagonist rimonabant or the PPARalpha antagonist MK886. Testing with open-field activity and food-consumption procedures in the same rats suggested that the disruption of operant responding observed in the attention task was not due to motor depression, anxiety, decreased appetite, or an inability to find and consume food pellets. The vanilloid-dependent behavioral disruption induced by anandamide was specific to the operant attention task. These effects of anandamide resemble effects of systemically administered dopamine antagonists and might reflect changes in vanilloid-mediated dopamine transmission. Panlilio, L. V., Mazzola, C., Medalie, J., Hahn, B., Justinova, Z., Drago, F., Cadet, J.L., Yasar, S., and Goldberg, S.R. Psychopharmacology (Berl.), 18 Nov 2008, Epub ahead of print, PMID: 19015836.

Diminished Iron Concentrations Increase Adenosine A(2A) Receptor Levels in Mouse Striatum and Cultured Human Neuroblastoma Cells
Brain iron insufficiency has been implicated in several neurological disorders. The dopamine system is consistently altered in studies of iron deficiency in rodent models. Changes in striatal dopamine D(2) receptors are directly proportional to the degree of iron deficiency. In light of the unknown mechanism for the iron deficiency-dopamine connection and because of the known interplay between adenosinergic and dopaminergic systems in the striatum IRP investigators examined the effects of iron deficiency on the adenosine system. They first attempted to assess whether there is a functional change in the levels of adenosine receptors in response to this low iron. Mice made iron-deficient by diet had an increase in the density of striatal adenosine A(2A) (A(2A)R) but not A(1) receptor (A(1)R) compared to mice on a normal diet. Between two inbred murine strains, which had 2-fold differences in their striatal iron concentrations under normal dietary conditions, the strain with the lower striatal iron had the highest striatal A(2A)R density. Treatment of SH-SY5Y (human neuroblastoma) cells with an iron chelator resulted in increased density of A(2A)R. In these cells, A(2A)R agonist-induced cyclic AMP production was enhanced in response to iron chelation, also demonstrating a functional upregulation of A(2A)R. A significant correlation (r(2)=0.79) was found between a primary marker of cellular iron status (transferrin receptor (TfR)) and A(2A)R protein density. In conclusion, the A(2A)R is increased across different iron-insufficient conditions. The relation between A(2A)R and cellular iron status may be an important pathway by which adenosine may alter the function of the dopaminergic system. Gulyani, S., Earley, C.J., Camandola, S., Maudsley, S., Ferre, S., Mughal, M.R., Martin, B., Cheng, A., Gleichmann, M., Jones, B.C., Allen, R.P., and Mattson, M.P. Experimental Neurology, October 28, 2008, Epubmed ahead of print, PMID: 19013457.

Fatty Acid Amide Hydrolase Inhibition Heightens Anandamide Signaling without Producing Reinforcing Effects in Primates
CB(1) cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors. However, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, IRP researchers assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys. They investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Delta(9)-tetrahydrocannabinol (THC), anandamide, or cocaine and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. They measured brain FAAH activity using an ex vivo enzyme assay. URB597 (.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597, and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine, even though, as expected, it significantly potentiated anandamide self-administration. In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. These results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse. Justinova, Z., Mangieri, R.A., Bortolato, M., Chefer, S.I., Mukhin, A.G., Clapper, J.R., King, A.R., Redhi, G.H., Yasar, S., Piomelli, D., and Goldberg, S.R. Biological Psychiatry, 64(11), pp. 930-937, 2008.

Inhibition of Anandamide Hydrolysis by Cyclohexyl Carbamic Acid 3'-carbamoyl-3-yl ester (URB597) Reverses Abuse-related Behavioral and Neurochemical Effects of Nicotine in Rats
Emerging evidence suggests that the rewarding, abuse-related effects of nicotine are modulated by the endocannabinoid system of the brain. For example, pharmacological blockade or genetic deletion of cannabinoid CB(1) receptors can reduce or eliminate many abuse-related behavioral and neurochemical effects of nicotine. Furthermore, doses of Delta(9)-tetrahydro-cannabinol and nicotine that are ineffective when given alone can induce conditioned place preference when given together. These previous studies have used systemically administered CB(1) receptor agonists and antagonists and gene deletion techniques, which affect cannabinoid CB(1) receptors throughout the brain. A more functionally selective way to alter endocannabinoid activity is to inhibit fatty acid amide hydrolase (FAAH), thereby magnifying and prolonging the effects of the endocannabinoid anandamide only when and where it is synthesized and released on demand. Here, IRP scientists combined behavioral and neurochemical approaches to evaluate whether the FAAH inhibitor URB597 (cyclohexyl carbamic acid 3'-carbamoyl-3-yl ester) could alter the abuse-related effects of nicotine in rats. They found that URB597, at a dose (0.3 mg/kg) that had no behavioral effects by itself, prevented development of nicotine-induced conditioned place preference (CPP) and acquisition of nicotine self-administration. URB597 also reduced nicotine-induced reinstatement in both CPP and self-administration models of relapse. Furthermore, in vivo microdialysis showed that URB597 reduced nicotine-induced dopamine elevations in the nucleus accumbens shell, the terminal area of the brain's mesolimbic reward system. These findings suggest that FAAH inhibition can counteract the addictive properties of nicotine and that FAAH may serve as a new target for development of medications for treatment of tobacco dependence. Scherma, M., Panlilio, L.V., Fadda, P., Fattore, L., Gamaleddin, I., Le Foll, B., Justinova, Z., Mikics, E., Haller, J., Medalie, J., Stroik, J., Barnes, C., Yasar, S., Tanda, G., Piomelli, D., Fratta, W., and Goldberg, S.R. JPET, 327(2), pp. 482-490, 2008.

Effects of Baclofen on Conditioned Rewarding and Discriminative Stimulus Effects of Nicotine in Rats
Neurochemical studies suggest that baclofen, an agonist at GABA(B) receptors, may be useful for treatment of nicotine dependence. However, its ability to selectively reduce nicotine's abuse-related behavioral effects remains in question. IRP researchers assessed effects of baclofen doses ranging from 0.1 to 3mg/kg on nicotine-induced conditioned place preferences (CPPs), nicotine discrimination, locomotor activity and food-reinforced behavior in male Sprague-Dawley rats. The high dose of baclofen (3mg/kg) totally eliminated food-reinforced responding and significantly decreased locomotor activity. Lower doses of baclofen did not have nicotine-like discriminative effects in rats trained to discriminate 0.4mg/kg nicotine from saline under a fixed-ratio 10 schedule of food delivery. Lower doses of baclofen also did not reduce discriminative stimulus effects of the training dose of nicotine and did not significantly shift the dose-response curve for nicotine discrimination. Rats treated with the high 3mg/kg dose of baclofen did not express nicotine-induced CPP. These experiments, along with previous reports that baclofen can reduce intravenous nicotine self-administration behavior, confirm the potential utility of baclofen as a tool for smoking cessation. Le Foll, B., Wetheim, C.E., and Goldberg, S.R. Neuroscience Letters, 443(3), pp. 236-240, 2008.

Sleep Deprivation Decreases Binding of [11C]Reclopride to Dopamine D2/D3 Receptors in the Human Brain
Sleep deprivation did not affect dopamine transporters (target for most wake-promoting medications) and thus dopamine increases are likely to reflect increases in dopamine cell firing and/or release rather than decreases in dopamine reuptake. Because dopamine-enhancing drugs increase wakefulness, IRP researchers postulate that dopamine increases after sleep deprivation is a mechanism by which the brain maintains arousal as the drive to sleep increases but one that is insufficient to counteract behavioral and cognitive impairment. Sleep deprivation can markedly impair human performance contributing to accidents and poor productivity. The mechanisms underlying this impairment are not well understood, but brain dopamine systems have been implicated. Here, they test whether one night of sleep deprivation changes dopamine brain activity. The authors studied 15 healthy subjects using positron emission tomography and [11C]raclopride (dopamine D2/D3 receptor radioligand) and [11C]cocaine (dopamine transporter radioligand). Subjects were tested twice: after one night of rested sleep and after one night of sleep deprivation. The specific binding of [11C]raclopride in the striatum and thalamus were significantly reduced after sleep deprivation and the magnitude of this reduction correlated with increases in fatigue (tiredness and sleepiness) and with deterioration in cognitive performance (visual attention and working memory). In contrast, sleep deprivation did not affect the specific binding of [11C]cocaine in the striatum. Because [11C]raclopride competes with endogenous dopamine for binding to D2/D3 receptors, the authors interpret the decreases in binding to reflect dopamine increases with sleep deprivation. However, they could not rule out the possibility that decreased [11C]raclopride binding reflects decreases in receptor levels or affinity. Sleep deprivation did not affect dopamine transporters (target for most wake-promoting medications) and thus dopamine increases are likely to reflect increases in dopamine cell firing and/or release rather than decreases in dopamine reuptake. Because dopamine-enhancing drugs increase wakefulness, the authors postulate that dopamine increases after sleep deprivation is a mechanism by which the brain maintains arousal as the drive to sleep increases but one that is insufficient to counteract behavioral and cognitive impairment. Volkow, N.D., Wang, G.J., Telang, F., Fowler, J.S., Logan, J., Wong, C., Ma, J., Pradhan, K., Tomasi, D., Thanos, P.K., Ferre, S., and Jayne, M. Journal of Neuroscience, August 20, 2008, 28(34), pp. 8454-8461, 2008.

Plasma Membrane Diffusion of g Protein-coupled Receptor Oligomers
G protein-coupled receptors are known to form homo-and heteromers at the plasma membrane, but the molecular properties of these oligomers are relatively unknown. Here, IRP scientists show a method that allows the diffusion of G protein-coupled receptors oligomers in the plasma membrane to be monitored in single cells by combining Bimolecular Fluorescence Complementation and Fluorescence Correlation Spectroscopy. With this approach they have measured, for the first time, the membrane diffusional characteristics of adenosine A(1) and A(2A) receptor homo-and heterodimers in Chinese Hamster Ovary cells. Interestingly, both homodimers display similar diffusion co-efficients (D) when expressed in living cells (D=5.0 and 4.8x10(-9) cm(2)/s, respectively) but the heterodimer formed by these receptors exhibit a significantly faster plasma membrane diffusion co-efficent (D=5.6x10(-9) cm(2)/s) when compared to the adenosine A(1) receptor tagged with the full-length yellow fluorescent protein (D=4.0x10(-9) cm(2)/s). Overall, these results demonstrate differences in plasma membrane diffusion between adenosine receptor homo-and heterodimers, providing new insights into the molecular plasticity of G protein-coupled receptor oligomerization. Briddon, S.J., Gandia, J., Amarol, O.B., Ferre, S., Lluis, C., Franco, R., Hill, S.J., and Ciruela, F. Biochimica Biophysica Acta, 1783(12), pp. 2262-2268, 2008.

Detection of Heteromerization of More than Two Proteins by Sequential BRET-FRET
Identification of higher-order oligomers in the plasma membrane is essential to decode the properties of molecular networks controlling intercellular communication. IRP scientists combined bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) in a technique called sequential BRET-FRET (SRET) that permits identification of heteromers formed by three different proteins. In SRET, the oxidation of a Renilla luciferase (Rluc) substrate by an Rluc fusion protein triggers acceptor excitation of a second fusion protein by BRET and subsequent FRET to a third fusion protein. The authors describe two variations of SRET that use different Rluc substrates with appropriately paired acceptor fluorescent proteins. Using SRET, they identified complexes of cannabinoid CB(1), dopamine D(2) and adenosine A(2A) receptors in living cells. SRET is an invaluable technique to identify heteromeric complexes of more than two neurotransmitter receptors, which will allow us to better understand how signals are integrated at the molecular level. Carriba, P., Navarro, G., Ciruela, F., Ferre, S., Casado, V., Agnati, L., Cortes, A., Mallol, J., Fuxe, K., Canela, E.I., Lluis, C., and Franco, R. Nature Methods, 5(8), pp. 727-733, 2008.

Potential Therapeutic Interest of Adenosine A2A Receptors in Psychiatric Disorders
The interest on targeting adenosine A(2A) receptors in the realm of psychiatric diseases first arose based on their tight physical and functional interaction with dopamine D(2) receptors. However, the role of central A(2A) receptors is now viewed as much broader than just controlling D(2) receptor function. Thus, there is currently a major interest in the ability of A(2A) receptors to control synaptic plasticity at glutamatergic synapses. This is due to a combined ability of A(2A) receptors to facilitate the release of glutamate and the activation of NMDA receptors. Therefore, A(2A) receptors are now conceived as a normalizing device promoting adequate adaptive responses in neuronal circuits, a role similar to that fulfilled, in essence, by dopamine. This makes A(2A) receptors particularly attractive targets to manage psychiatric disorders since adenosine may act as go-between glutamate and dopamine, two of the key players in mood processing. Furthermore, A(2A) receptors also control glia function and brain metabolic adaptation, two other emerging mechanisms to understand abnormal processing of mood, and A(2A) receptors are important players in controlling the demise of neurodegeneration, considered an amplificatory loop in psychiatric disorders. Current data only provide an indirect confirmation of this putative role of A(2A) receptors, based on the effects of caffeine (an antagonist of both A(1) and A(2A) receptors) in psychiatric disorders. However, the introduction of A(2A) receptor antagonists in clinics as anti-parkinsonian agents is hoped to bolster our nowledge on the role of A(2A) receptors in mood disorders in the near future. Cunha, R.A., Ferre, S., Vaugeois, J.M., and Chen, J.F. Curr. Pharm. Des., 14(15), pp. 1512-1524, 2008.

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., and Franco, R. Journal of Biological Chemistry, July 25, 2008, Epub 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 researchers 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., and Franco, R. Neuropharmacology, May 16, 2008, Epub 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 scientists 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 endocannabi-noid 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 favour 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., and Piomelli, D. British Journal of Pharmacology, 154, pp. 369-383, 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., and Lluis, C. Brain Research Review, June 20, 2008, Epub ahead of print, PMID: 18620000.

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 D 2 receptor third intracellular loop. This is the same epitope involved in adenosine A 2A-D 2 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 D 2 receptor Arg-rich epitope and several CaM acidic epitopes are mainly responsible for the D 2 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 D 2 but not to the A 2A receptor epitope, and CaM disrupted the electrostatic interactions between the D 2 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 Proteomic Research, July 1, 2008, Epub ahead of print, PMID: 18590318.

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 scientists 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.

Behavioral Neuroscience Research Branch

Acetylcholine Release in the Mesocorticolimbic Dopamine System During Cocaine Seeking: Conditioned and Unconditioned Contributions to Reward and Motivation
Microdialysis was used to assess the contribution to cocaine-seeking of cholinergic input to the mesocortico-limbic dopamine system in ventral tegmental area (VTA). VTA acetylcholine (ACh) was elevated in animals lever-pressing for IV cocaine and in cocaine-experienced and cocaine-naive animals passively receiving similar "yoked" injections. In cocaine-trained animals, the elevations comprised an initial (first hour) peak to about 160% of baseline and a subsequent plateau of 140% of baseline for the rest of the cocaine intake period. In cocaine-naive animals, yoked cocaine injections raised ACh levels to the 140% plateau but did not cause the initial 160% peak. In cocaine-trained animals that received unexpected saline (extinction conditions) rather than the expected cocaine, the initial peak was seen but the subsequent plateau was absent. VTA ACh levels played a causal role and were not just a correlate of cocaine-seeking. Blocking muscarinic input to the VTA increased cocaine intake; the increase in intake offset the decrease in cholinergic input, resulting in the same VTA dopamine levels as were seen in the absence of the ACh antagonists. Increased VTA ACh levels (resulting from 10 uM VTA neostigmine infusion) increased VTA dopamine levels and reinstated cocaine-seeking in cocaine-trained animals that had undergone extinction; these effects were strongly attenuated by local infusion of a muscarinic antagonist and weakly attenuated by a nicotinic antagonist. These findings identify two cholinergic responses to cocaine self-administration--an unconditioned response to cocaine itself and a conditioned response triggered by cocaine-predictive cues--and confirm that these cholinergic responses contribute to the control of cocaine-seeking. You, Z.-B., Wang, B., Zitzman, D., and Wise, RA. Journal of Neuroscience, 28, pp. 9021-9029, 2008.

Cocaine Serves as a Peripheral Interoceptive Conditioned Stimulus for Central Glutamate and Dopamine Release
Intravenous injections of cocaine HCl are habit-forming because, among their many actions, they elevate extracellular dopamine levels in the terminal fields of the mesocorticolimbic dopamine system. This action, thought to be very important for cocaine's strong addiction liability, is believed to have very short latency and is assumed to reflect rapid brain entry and pharmacokinetics of the drug. However, while intravenous cocaine HCl has almost immediate effects on behavior and extracellular dopamine levels, recent evidence suggests that its central pharmacological effects are not evident until 10 or more seconds after IV injection. Thus the immediate effects of a given intravenous cocaine injection on extracellular dopamine concentration and behavior appear to occur before there is sufficient time for cocaine to act centrally as a dopamine uptake inhibitor. To explore the contribution of peripheral effects of cocaine to the early activation of the dopamine system, IRP researchers used brain microdialysis to measure the effects of cocaine methiodide (MI)--a cocaine analogue that does not cross the blood brain barrier--on glutamate (excitatory) input to the dopamine cells. IP injections of cocaine MI were ineffective in cocaine-naive animals but stimulated ventral tegmental glutamate release in rats previously trained to lever-press for cocaine HCl. This peripherally triggered glutamate input was sufficient to reinstate cocaine-seeking in previously trained animals that had undergone extinction of the habit. These findings offer an explanation for short-latency behavioral responses and immediate dopamine elevations seen following cocaine injections in cocaine-experienced but not cocaine-naive animals. Wise, R.A., Wang, B., and You, Z.-B. Public Library of Science One, 3, e2846.

Medicinal Chemistry Section, Medications Discovery Research Branch

Structure-Activity Relationships for a Novel Series of Dopamine D2-like Receptor Ligands
Discovering dopamine D2-like receptor subtype-selective ligands has been a focus of significant investigation. The D2R-selective antagonist L741,626 (Ki D2R:D3R 11.2:163 nM) has previously provided a lead template for chemical modification. In the present study, a series of analogues was synthesized wherein the piperidine of L741,626 was replaced by a tropane ring that reversed the selectivity seen in the parent compound, in human hD2LR- or hD3R-transfected HEK 293 cells (Ki D2R:D3R 33.4:15.5 nM). Further exploration of both N- and aryl ring-substituted analogues resulted in the discovery of several high affinity D2R/D3R ligands with 3-benzofurylmethyl-substituents that induced high affinity not achieved in similarly N-substituted piperidine analogues, and significantly (470-fold) improved D3R binding affinity compared to the parent ligand. X-Ray crystallographic data revealed a distinctive spatial arrangement of pharmacophoric elements in the piperidinol vs tropine analogues that provide clues for the diversity in structure-activity relationships at the D2 and D3 receptor subtypes. Paul, N.M., Taylor, M., Kumar, R., Dechamps, J.R., Luedtke, R.R., and Newman, A.H. Journal of Medicinal Chemistry, 51, pp. 6095-6109, 2008.