Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

GABAergic Lineage Differentiation of AF5 Neural Progenitor Cells In Vitro IRP scientists have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage. Sanchez, J.F., Crooks, D.R., Lee, C.T., Schoen, C.J., Amable, R., Zeng, X., Florival-Victor, T., Morales, N., Truckenmiller, M.E., Smith, D.R. and Freed, W.J. Cell Tissue Research, 324, pp. 1-8, 2006.

Assessing Self-Renewal and Differentiation in Human Embryonic Stem Cell Lines Like other cell populations, undifferentiated human embryonic stem cells (hESCs) express a characteristic set of proteins and mRNA that is unique to the cells regardless of culture conditions, number of passages, and methods of propagation. IRP researchers sought to identify a small set of markers that would serve as a reliable indicator of the balance of undifferentiated and differentiated cells in hESC populations. Markers of undifferentiated cells should be rapidly downregulated as the cells differentiate to form embryoid bodies (EBs), whereas markers that are absent or low during the undifferentiated state but that are induced as hESCs differentiate could be used to assess the presence of differentiated cells in the cultures. In this paper, authors describe a list of markers that reliably distinguish undifferentiated and differentiated cells. An initial list of approximately 150 genes was generated by scanning published massively parallel signature sequencing, expressed sequence tag scan, and microarray datasets. From this list, a subset of 109 genes was selected that included 55 candidate markers of undifferentiated cells, 46 markers of hESC derivatives, four germ cell markers, and four trophoblast markers. Expression of these candidate marker genes was analyzed in undifferentiated hESCs and differentiating EB populations in four different lines by immunocytochemistry, reverse transcription-polymer-ase chain reaction (RT-PCR), microarray analysis, and quantitative RT-PCR (qPCR). Authors show that qPCR, with as few as 12 selected genes, can reliably distinguish differentiated cells from undifferentiated hESC populations. Cai, J., Chen, J., Liu, Y., Miura, T., Luo, Y., Loring, J.F., Freed, W.J., Rao, M.S., and Zeng, X. Stem Cells, 24, pp. 516-530, 2006.

Characterization of a New NIH-Registered Variant Human Embryonic Stem Cell Line, BG01V: A Tool for Human Embryonic Stem Cell Research Human embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here IRP scientists describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Authors characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination. Plaia, T.W., Josephson, R., Liu, Y., Zeng, X., Ording, C., Toumadje, A., Brimble, S.N., Sherrer, E.S., Uhl, E.W., Freed, W.J., Schulz, T.C., Maitra, A., Rao, M.S. and Auerbach, J.M. Stem Cells, 24, pp. 531-546, 2006.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Visualizing Cannabinoid Effects Using Brain Slice Imaging and Electrophysiological Approaches The use of electrophysiological recordings in brain slices is now routinely used to assess the actions of cannabinoid ligands within various central nervous system nuclei. In this chapter authors describe common protocols involving both intracellular and extracellular recording techniques in the hippocampus, where the presynaptic modulatory effects of cannabinoid receptor activation have been studied in detail. In addition to describing the basic electrophysiological setup needed for these recordings, we will address common technical problems and limitations involved in working with highly lipophilic compounds, such as the cannabinoid ligands, in brain slices. Hoffman, A.F., and Lupica, C.R. Methods in Molecular Medicine, 123, pp. 105-112, 2006.

Receptor-Independent Effects of Endocannabinoids on Ion Channels Endogenous cannabinoids (endocannabinoids), produced from membrane-bound precursors via calcium and/or G-protein dependent processes, mimic the effects of cannabinoids by activating cannabinoid CB(1) and/or CB(2) receptors. Several reports however, also indicate that endocannabinoids can produce effects that are independent of cannabinoid receptors. Thus, in pharmacologically relevant concentrations, endocannabinoids have been demonstrated to modulate the functional properties of voltage-gated ion channels including Ca(2+) channels, Na(+) channels and various types of K(+) channels, and ligand-gated ion channels such as 5-HT(3), and nicotinic ACh receptors. In addition, the functional modulations by endocannabinoids of other ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions, and neurotransmitter transporters have also been reported. These findings indicate that additional molecular targets for endocannabinoids exist and that these targets may represent important sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems. This review focuses on the results of recent studies indicating that beyond their receptor-mediated effects, endocannabinoids alter the function of ion channels directly. Oz, M. Current Pharmaceutical Design, 12, pp. 227-239, 2006.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Decoy Peptides that Bind Dynorphin Noncovalently Prevent NMDA Receptor-Mediated Neurotoxicity Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, IRP researchers show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury. Woods, A.S., Kaminski, R., Oz, M., Wang, Y., Hauser, K., Goody, R., Wang, H.Y., Jackson, S.N., Zeitz, P., Zeitz, K.P., Zolkowska, D., Schepers, R., Nold, M., Danielson, J., Graslund, A., Vukojevic, V., Bakalkin, G., Basbaum, A., and Shippenberg, T. Journal of Proteome Research, 5, pp. 1017-1023, 2006.

Phosphate Stabilization of Intermolecular Interactions Receptor heteromerization is an important phenomenon that results from the interaction of epitopes on two receptors. Previous studies have suggested the possibility of Dopamine D2-NMDA receptors' interaction. IRP scientists believe that the interaction is through an acidic epitope of the NMDA NR1 subunit (KVNSEEEEEDA) and a basic epitope of the D2 third intracellular loop (VLRRRRKRVN), which was shown to also interact with the Adenosine A2A receptor. In previous work, authors highlighted the role of certain amino acid residues, mainly two or more adjacent arginine on one peptide and two or more adjacent glutamate, or aspartate, or a phosphorylated residue on the other in the formation of noncovalent complexes (NCX) between epitopes. In the present work, they use the phosphorylated (KVNSpEEEEEDA), nonphosphorylated (KVNSEEEEEDA) and modified (KVNpSAAAAAAA) forms of the NMDA epitope that possibly interact with the D2 epitope to investigate the gas-phase stability of the NCXs as a function of the nominal energy given to the NCX ion as it enters the collision cell. In addition to theoretical calculations, the experimental data was used to calculate the stability of each electrostatic complex versus that of the dimer of KVNSpEEEEEDA. Our results demonstrate the importance of the phosphate group in stabilizing molecular interactions and that appreciably higher collision energies are required to completely dissociate any of the three different NCX ions that are formed through electrostatic interaction in comparison to the energy required to dissociate the KVNpSEEEEEDA dimer ion, which is mainly kept together by hydrogen bonding. This study emphasizes ionic bonds stability and their importance to protein structure as their potent electrostatic attractions can in the gas-phase surpass the strength of covalent bonds. Jackson, S.N., Wang, H.Y., Yergey, A., and Woods, A.S. Journal of Proteome Research, 5, pp. 122-126, 2006.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Effect of Methamphetamine Self-Administration on Tyrosine Hydroxylase and Dopamine Transporter Levels in Mesolimbic and Nigrostriatal Dopamine Pathways of the Rat Many studies have examined the effect of experimenter-delivered methamphetamine on the mesolimbic and nigrostriatal dopamine pathways. In contrast, little is known about the effect of methamphetamine self-administration on these neuronal pathways. IRP researchers studied the effect of methamphetamine self-administration on two key regulators of dopamine transmission, tyrosine hydroxylase (TH), and dopamine transporter (DAT), in components of the mesolimbic and nigrostriatal dopamine pathways. Rats self-administered methamphetamine (0.1 mg/kg per infusion, fixed-ratio-1 reinforcement schedule) or saline (control condition) for 9 h/day over 10 days. The brains of these rats were collected after 1 or 30 days of forced abstinence and the expression levels of TH and DAT were assayed by in situ, hybridization and western blot. TH mRNA and protein levels were increased in the ventral tegmental area (VTA, the cell body region of the mesolimbic dopamine system) and the substantia nigra pars compacta (SNC, the cell body region of the nigrostriatal dopamine system) after 1 day, but not 30 days, of forced abstinence from methamphetamine. In contrast, methamphetamine self-administration had no effect on TH protein levels in dopaminergic terminals located in the nucleus accumbens and caudate-putamen. In addition, methamphetamine self-administration had no effect on DAT mRNA levels in the VTA. Results suggest that extended daily access to self-administered methamphetamine results in a transient, short-lasting effect on mesolimbic and nigrostriatal dopamine neurons of the rat brain. Shepard, J.D., Chuang, D.T., Shaham, Y. and Morales, M. Psychopharmacology, 185, pp. 505-513, 2006.

Morphometric Study on Cytoskeletal Components of Neuronal and Astroglial Cells after Chronic CB1 Agonist Treatment One of the major goals for the use of digital image analysis systems in neuroanatomy is to visualize structures, cells, or other tissue components in order to compare various populations. In addition, digital image analysis allows semi-quantification of cell labeling because it is capable of measuring simultaneously the staining intensity, location, size, and shape of labeled profiles. In the present work, the morphological changes in the CB1 hippocampal area and corpus striatum induced by chronic treatment with the synthetic CB1-receptor agonist WIN55,212-2 were analyzed as an example of digital image analysis application. Twice-daily treatment for 14 d with the CB1-receptor agonist demonstrated significant changes in the expression of neuronal cytoskeletal proteins and in neuronal morphology, as evidenced by immunocytochemical and digital analysis studies. However, changes in the expression of astroglial cytoskeletal proteins were not found. Tagliaferro, P., Ramos, A.J., Onaivi, E.S., Evrard, S.G., Vega, M.D., and Brusco, A. Methods in Molecular Medicine, 123, pp. 91-104, 2006.

Molecular Neuropsychiatry Research Branch

Neurological Assessments of Marijuana Users This chapter summarizes the neurological approaches used to assess the potential long-term effects of drugs on the nervous system of drug abusers. These include the use of neuropsychological assessments, transcranial Doppler (TCD) sonography, and electroencephalographic (EEG) recordings. Neuropsychological procedures are used in an effort to provide an unbiased estimate of the individual's cognitive capacity, and included tests of language skills, attention, memory, and motor skills. TCD allows for the measurements of blood flow in the anterior cerebral and middle cerebral arteries, which supply blood to the cortex. An EEG recording was included in our assessment on marijuana abusers using a sound-attenuated, electronically shielded chamber. These neurological approaches have allowed the detection of various neurological and neurovascular deficits that are associated with the abuse of marijuana. Cadet, J.L., Bolla, K. and Herning, R. Methods of Molecular Medicine, 123, pp. 255-268, 2006.

Molecular Neurobiology Research Branch

Association Genome Scanning for Human Addiction Vulnerability Branch scientists validated pooled genome scanning using "10k" Affymetrix microarrays. They then used these arrays to provide millions of person/genotype equivalents for the DNAs that they have collected from NIDA IRP research volunteers who are dependent on illegal substances and volunteers who report no significant lifetime use of addictive substances. The genes identified using this genome scanning approach fall into several classes, and provide markers to help separate those with greater and lesser addiction vulnerabilities. Liu, Q.R., Drgon, T., Walther, D., Johnson, C., Poleskaya, O., Hess, J. and Uhl GR. Proceedings of the National Acadamy of Sciences, USA, 102, pp 11864-11869, 2005.

Fine Mapping Studies for Human Addiction Vulnerability Identify the Cell Adhesion Molecule NrCAM Branch scientists followed up on pooled genome scanning using Affymetrix microarrays and data from drug-regulated gene expression to positionally clone NrCAM as a gene whose 5' regulatory variants 1) lead to differences in levels of NrCAM expression and 2) contribute to differential vulnerability to addiction. Mouse models impressively replicated this effect: altering NrCAM expression dramatically changed place preferences for cocaine and for morphine. Ishiguro, H., Liu, Q.R., Gong, J.P., Hall, F.S., Ujike, H., Morales, M., Sakurai, T., Grumet, M. and Uhl GR. Neuropsychopharmacology, 31, pp. 572-584, 2006.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Reinforcing Effects of Nicotine are Triggered from Regions Inside and Outside the Ventral Tegmental Area Nicotine is thought to be the key substance responsible for tobacco-smoking habits and appears to trigger reinforcement via the ventral tegmental area (VTA). Recently, multiple anatomical substrates for drug reinforcement have been identified in the vicinity of the ventral midbrain. In addition to the posterior portion of the VTA, the central linear nucleus raphe and the supramammillary nucleus of the posterior hypothalamus mediate drug reinforcement. Using intracranial self-administration procedures, IRP researchers examined whether these regions mediate the reinforcing effects of nicotine. Rats learned to lever press for self-administration of nicotine into the posterior VTA, central linear nucleus, and supramammillary nucleus, suggesting a reinforcing action of nicotine in these regions. The rats did not self-administer nicotine into surrounding regions including the anterior VTA, substantia nigra, the region just dorsal to the posterior VTA, interpeduncular nucleus, or medial mammillary nucleus. The reinforcing effects of nicotine into the three brain regions were further confirmed by a two-lever discrimination procedure, in which rats learned to selectively respond between active and inactive levers. The reinforcing effects of nicotine administration into the posterior VTA, central linear nucleus, and supramammillary nucleus were blocked by coadministration of the nicotine receptor antagonist mecamylamine. The reinforcing effects of nicotine into the posterior VTA or central linear nucleus were attenuated by coadministration of the D2 receptor agonist quinpirole. These findings demonstrate that nicotine reinforcement involves multiple regions both inside and outside the VTA. Ikemoto, S., Qin, M., Liu Z-H. Journal of Neuroscience, 26, pp. 723-730, 2006.

A New Peptide Input to Learning and Addiction In this issue of Neuron, Borgland et al. report that the arousal-associated peptide orexin enhances LTP-like changes in glutamatergic excitability of ventral tegmental dopamine neurons. This parallels a similar effect of corticotropin-releasing factor and suggests a form of neuroadaptation that increases the likelihood of addiction relapse. Wise, R.A. Neuron, 49, 483-484, 2006.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

The Dopamine D3 Receptor: A Therapeutic Target for the Treatment of Neuropsychiatric Disorders The role of the D(3) receptor has remained largely elusive before the development of selective research tools, such as selective radioligands, antibodies, various highly specific pharmacological agents and knock-out mice. The data collected so far with these tools have removed some of the uncertainties regarding the functions mediated by the D(3) receptor. The D(3) receptor is an autoreceptor that controls the phasic, but not tonic activity of dopamine neurons. The D(3) receptor, via regulation of its expression by the brain-derived neurotrophic factor (BDNF), mediates sensitization to dopamine indirect agonists. This process seems responsible for side-effects of levodopa (dyskinesia) in the treatment of Parkinson's disease (PD), as well as for some aspects of conditioning to drugs of abuse. The D(3) receptor mediates behavioral abnormalities elicited by glutamate/NMDA receptor blockade, which suggests D(3) receptor-selective antagonists as novel antipsychotic drugs. These data allow us to propose novel treatment options in PD, schizophrenia and drug addiction, which are awaiting evaluation in clinical trials. Sokoloff, P., Diaz, J., Le Foll, B., Guillin, O., Leriche, L., Bezard, E. and Gross, C. CNS Neurological Disorders Drug Targets, 5, pp. 25-43, 2006.

A Comparison of Drug-seeking Behavior Maintained by d-Amphetamine, l-Deprenyl (selegiline), and d-Deprenyl under a Second-order Schedule in Squirrel Monkeys L-Deprenyl (selegiline) is used in the treatment of Parkinson's disease and has been proposed as an aid for cigarette smoking cessation and a treatment for psychostimulant abuse. L-Deprenyl is metabolized in the body to L-methamphetamine and L-amphetamine, suggesting that it may have abuse potential. The current study assessed whether L-deprenyl or its isomer would maintain drug-seeking behavior on a second-order schedule and whether L-deprenyl would alter drug-seeking behavior maintained by D-amphetamine if given as a pretreatment. Squirrel monkeys learned to respond on a second-order schedule of reinforcement, where every tenth response was followed by a brief light flash, and the first brief light flash after 30 min was paired with intravenous (i.v.) injection of D-amphetamine (0.56 mg/kg), administered over a 2-min period at the end of the session. When responding was stable, saline or different i.v. doses of D-amphetamine (0.3-1.0 mg/kg), L-deprenyl (0.1-10.0 mg/kg), and D-deprenyl (0.1-3.0 mg/kg) were substituted for 10 days each. Subsequently, monkeys were pretreated with 0.3 or 1.0 mg/kg L-deprenyl intramuscularly 30 min prior to D-amphetamine baseline sessions. D-Amphetamine maintained high rates of drug-seeking behavior on the second-order schedule. D-Deprenyl maintained high rates of drug-seeking behavior similar to D-amphetamine. L-Deprenyl maintained lower rates of responding that were not significantly above saline substitution levels. Pretreatment with L-deprenyl failed to alter drug-seeking behavior maintained by D-amphetamine. These results indicate that D-deprenyl, but not L-deprenyl, may have abuse potential. Under conditions where drug-seeking and drug-taking behaviors are actively maintained by D-amphetamine, L-deprenyl, at doses that specifically inhibit type B monoamine oxidase, may not be effective as a treatment. Yasar, S., Gaal, J., Panlilio, L.V., Justinova, Z., Molnar, S.V., Redhi, G.H. and Schindler, C.W. Psychopharmacology, 183, pp. 413-421, 2006.

The "Two-state Dimer Receptor Model": A General Model for Receptor Dimers Non-linear Scatchard plots are often found for agonist binding to G-protein-coupled receptors. Since there are clear evidences of receptor dimerization these non-linear Scatchard plots can reflect cooperativity on agonist binding to the two binding sites in the dimer. According to this, the "two-state dimer receptor model" has been recently derived. In this paper the performance of the model has been analyzed in fitting data from A1 adenosine receptors agonist binding, which is an example of receptor displaying concave downward Scatchard plots. Analysis of agonist/antagonist competition data for dopamine D1 receptors using the "two-state dimer receptor model" has also been performed. Although fitting to the "two-state dimer receptor model" is similarly good than the fitting to the "two-independent-site receptor model", the former is simpler and a discrimination test selects the "two-state dimer receptor model" as the best. This model was also very robust in fitting data of estrogen binding to the estrogen receptor, which display concave upward Scatchard plots. On the one hand, the model would predict the already demonstrated existence of estrogen receptor dimers. On the other hand, the model would predict that concave upward Scatchard plots reflect positive cooperativity, which cannot be neither predicted nor explained by assuming the existence of two different affinity states. In summary the "two-state dimer receptor model" is good for fitting data of binding to dimeric receptors displaying either linear, concave upward or concave downward Scatchard plots. Franco, R., Casado, V., Mallol, J., Ferrada, C., Ferre, S., Fuxe, K., Cortes, A., Ciruela, F., Lluis, C. and Canela, E.I. Molecular Pharmacology, February 24, 2006, Epubmed ahead of print, PMID 16501032.

Nicotine Pre-exposure Does Not Potentiate the Locomotor or Rewarding Effects of delta-9-tetrahydrocannabinol in Rats This study assessed the effects of nicotine pre-exposure on subsequent locomotor and rewarding effects of repeated Delta-9-tetrahydrocannabinol administration in Sprague-Dawley rats. Repeated administration of the same dose of Delta-9-tetrahydrocannabinol (0.01-2 mg/kg) did not produce significant tolerance or behavioral sensitization to Delta-9-tetrahydrocannabinol's locomotor effects. An unbiased place conditioning paradigm was then used to obtain a measure of the rewarding effects of Delta-9-tetrahydrocannabinol. Rats received an injection of either Delta-9-tetrahydrocannabinol (0.01-2 mg/kg) before being placed in one compartment (three trials) or saline before being placed in the other compartment (three trials) of a two-compartment apparatus. Control rats received saline injections associated with both compartments. Significant conditioned place preferences developed with 0.1 mg/kg Delta-9-tetrahydrocannabinol in control rats, but not in nicotine pre-exposed rats. Surprisingly, significant place aversions developed at higher 1 and 2 mg/kg doses of Delta-9-tetrahydrocannabinol in nicotine pre-exposed rats. To the extent that behavioral sensitization may reflect reward processes in drug dependence, the lack of behavioral sensitization on repeated Delta-9-tetrahydrocannabinol administration is consistent with the difficulties usually encountered in demonstrating rewarding or reinforcing effects of Delta-9-tetrahydrocannabinol in rats. The present findings suggest, moreover, that nicotine pre-exposure alters the qualitative nature of rewarding effects and accentuates aversive effects of Delta-9-tetrahydrocannabinol. Le Foll, B., Wiggins, M. and Goldberg, S.R. Behavioral Pharmacology, 17, pp. 195-199, 2006.,/p>

Presynaptic Control of Striatal Glutamatergic Neurotransmission by Adenosine A1-A2A Receptor Heteromers The functional role of heteromers of G-protein-coupled receptors is a matter of debate. In the present study, IRP researchers demonstrate that heteromerization of adenosine A1 receptors (A1Rs) and A2A receptors (A2ARs) allows adenosine to exert a fine-tuning modulation of glutamatergic neurotransmission. By means of coimmunoprecipitation, bioluminescence and time-resolved fluorescence resonance energy transfer techniques, authors showed the existence of A1R-A2AR heteromers in the cell surface of cotransfected cells. Immunogold detection and coimmunoprecipitation experiments indicated that A1R and A2AR are colocalized in the same striatal glutamatergic nerve terminals. Radioligand-binding experiments in cotransfected cells and rat striatum showed that a main biochemical characteristic of the A1R-A2AR heteromer is the ability of A2AR activation to reduce the affinity of the A1R for agonists. This provides a switch mechanism by which low and high concentrations of adenosine inhibit and stimulate, respectively, glutamate release. Furthermore, it is also shown that A1R-A2AR heteromers constitute a unique target for caffeine and that chronic caffeine treatment leads to modifications in the function of the A1R-A2AR heteromer that could underlie the strong tolerance to the psychomotor effects of caffeine. Ciruela, F., Casado, V., Rodrigues, R.J., Lujan, R., Burgueno, J., Canals, M., Borycz, J., Rebola, N., Goldberg, S.R., Mallol, J., Cortes, A., Canela, E.I., Lopez-Gimenez, J.F., Milligan, G., Lluis, C., Cunha, R.A., Ferre, S. and Franco, R. Journal of Neuroscience, 26, pp. 2080-2087, 2006.

Stimulation of Adenosine Receptors Selectively Activates Gene Expression in Striatal Enkephalinergic Neurons In the striatum, adenosine A(2A) and dopamine D(2) receptors exert reciprocal antagonistic interactions that modulate the function of GABAergic enkephalinergic neurons. We have previously shown that stimulation of adenosine A(1) receptors allows the stimulation of A(2A) receptors to overcome a tonic inhibitory effect of D(2) receptors and induce striatal expression of c-fos. In the present work, by studying co-localization of c-Fos immunoreactivity and preproenkephalin and preprodynorphin transcripts, IRP scientists show that co-administration of the A(1) receptor agonist CPA and the A(2A) receptor agonist CGS 21680 increases the striatal expression of c-fos in GABAergic enkephalinergic but not in GABAergic dynorphinergic neurons. Co-administration of CPA and CGS 21680 also induced a significant increase in the striatal expression of preproenkephalin. The results underscore the role of adenosine in the activation of gene expression in the GABAergic enkephalinergic neuron. Karcz-Kubica, M., Ferre, S., Diaz-Ruiz, O., Quiroz-Molina, C., Goldberg, S.R., Hope, B.T. and Morales, M. Neuropsychopharmacology, February 1, 2006, Epubmed ahead of print, PMID 16452987.

Role of Adenosine in the Control of Homosynaptic Plasticity in Striatal Excitatory Synapses Long-lasting, activity-dependent changes in synaptic efficacy at excitatory synapses are critical for experience-dependent synaptic plasticity. Synaptic plasticity at excitatory synapses is determined both presynaptically by changes in the probability of neurotransmitter release, and postsynaptically by changes in the availability of functional postsynaptic glutamate receptors. Two kinds of synaptic plasticity have been described. In homosynaptic or Hebbian plasticity, the events responsible for synaptic strengthening occur at the same synapse as is being strengthened. Homosynaptic plasticity is activity-dependent and associative, because it associates the firing of a postsynaptic neuron with that of the presynaptic neuron. Heterosynaptic plasticity, on the other hand, is activity-independent and the synaptic strength is modified as a result of the firing of a third, modulatory neuron. It has been suggested that long-term changes in synaptic strength, which are associated with gene transcription, can only be induced with the involvement of heterosynaptic plasticity. The neuromodulator adenosine plays an elaborated pre- and postsynaptic control of glutamatergic neurotransmission. This paper reviews the evidence suggesting that in some striatal excitatory synapses, adenosine can provide the heterosynaptic-like modulation essential for stabilizing homosynaptic plasticity without the need of a "third, modulatory neuron". Ferre, S., Borycz, J., Goldberg, S.R., Hope, B.T., Morales, M., Lluis, C., Franco, R., Ciruela, F. and Cunha, R. J Journal of Integrative Neuroscience, 4, pp. 445-464, 2005.

Antidepressant-like Activity and Modulation of Brain Monoaminergic Transmission by Blockade of Anandamide Hydrolysis Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, delta9-tetrahydrocannabinol, acts by binding to brain CB1 cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, IRP researchers show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB1 antagonist rimonabant; are accompanied by increased brain anandamide levels; and are maintained upon repeated URB597 administration. Unlike direct CB1 agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of delta9-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs. Proceedings of the National Adademy of Sciences USA, 102, pp. 18620-18625, 2005.

Metabolic Transformation Plays a Primary Role in the Psychostimulant-like Discriminative-stimulus Effects of Selegiline l-Deprenyl [selegiline, (R)-(-)-deprenyl] is a selective inhibitor of monoamine oxidase B (MAO-B) used in the treatment of Parkinson's disease and proposed as an antidepressant and an aid for cigarette-smoking cessation and treatment of psychostimulant abuse. Beneficial therapeutic effects of (R)-(-)-deprenyl may also result from indirect actions. Brain levels of dopamine and beta-phenylethylamine (beta-PEA), a behaviorally active endogenous trace amine, increase after (R)-(-)-deprenyl treatment due to MAO-B blockade and (R)-(-)-deprenyl is metabolized to (R)-(-)-methamphetamine and (R)-(-)-amphetamine, suggesting that (R)-(-)-deprenyl may have psychostimulant-like behavioral effects. Indeed, (R)-(-)-deprenyl produces psychostimulant-like discriminative-stimulus effects in experimental animals. Here, IRP scientists tested the hypothesis that psychostimulant-like behavioral effects of (R)-(-)-deprenyl are mainly mediated by its metabolites. Male Fisher F344 rats were trained to discriminate i.p. injection of 1.0 mg/kg (S)-(+)-methamphetamine or 10.0 mg/kg cocaine from injection of saline using two-lever choice schedules of food delivery or stimulus shock termination. When (R)-(-)-deprenyl was tested by substitution, it had (S)-(+)-methamphetamine- and cocaine-like discriminative-stimulus effects, but only at doses of 10 to 30 mg/kg, doses 10 to 20 times higher than those selective for MAO-B inhibition. Ro 16-6491 [N-(2-aminoethyl)-4-chlorobenzamide hydrochloride], a selective inhibitor of MAO-B enzyme activity without psychoactive metabolites, had no psychostimulant-like discriminative effects. In addition, blockade of (R)-(-)-deprenyl's metabolism with SKF 525A (beta-DEAE-diphenylpropylacetate hydrochloride; 50 mg/kg i.p.) reduced or eliminated (R)-(-)-deprenyl's psychostimulant-like discriminative effects. When beta-PEA synthesis was blocked by NSD 1015 (m-hydroxy-benzyl-hydrazine; 30 mg/kg i.p.), there was a modest reversal of (R)-(-)-deprenyl's psychostimulant-like discriminative effects under some conditions, indicating a facilitatory modulation of the psychostimulant-like discriminative effects of (R)-(-)-deprenyl metabolites by elevated levels of beta-PEA under certain conditions. Yasar, S., Justinova, Z., Lee, S.H., Stefanski, R., Goldberg, S.R. and Tanda, G. Journal of Pharmacology and Experimental Therapeutics, 317, pp. 387-394, 2006.

Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Assessment of Cannabis Craving Using the Marijuana Craving Questionnaire Cannabis is the most widely used illicit drug in the United States with 14.6 million current users. Cannabis-dependent individuals presenting for treatment typically report cannabis craving; however, the phenomenon has received little research attention. In the absence of a valid, multidimensional questionnaire to assess cannabis craving, IRP scientists developed the Marijuana Craving Questionnaire (MCQ). The MCQ consists of four constructs or factors that characterize cannabis craving: compulsivity, emotionality, expectancy, and purposefulness. A separate score is calculated for each factor. The MCQ can be used to measure cue-elicited craving in a research setting or natural craving in cannabis-dependent individuals presenting for treatment. Either the 47-item or 12-item version can be used, and standardized instructions for completion of the MCQ should be given. The MCQ can be administered using a paper and pencil form or a computerized version. In a research setting, the MCQ should be administered immediately after cue presentation and repeated frequently to capture the full time course. In a treatment setting, the MCQ should be administered at intake and during and at the end of treatment. Heishman, S.J. and Singleton, E.G. Methods in Molecular Medicine: Marijuana and Cannabinoid Research: Methods and Protocols, pp. 209-216. Totowa, NJ: Humana Press, 2006.

Methods for Clinical Research Involving Cannabis Administration Better scientific understanding of cannabis effects and the development of treatments for cannabis dependence require clinical studies involving cannabis administration. Cannabis can be administered by smoking a plant-derived cigarette or by oral or intravenous administration of D9-tetrahydrocannabinol (THC), the primary psychoactive chemical in cannabis. The smoked route is most commonly used outside the laboratory, but is subject to wide variation in absorbed dose. Oral synthetic THC is a legally marketed medication (dronabinol) also subject to wide pharmacokinetic variation, but offering a greater safety margin because of slower onset of action and lower potency. Intravenous THC offers precise investigator control of dose and timing. Acute adverse effects of cannabis administration include tachycardia, orthostatic hypotension, pulmonary irritation (if smoked), motor incoordination, cognitive impairment, anxiety, paranoia, and psychosis. Screening of research subjects should identify and exclude those with risk factors for such events, e.g., a history of significant cardiovascular, pulmonary, or psychiatric disorders. Monitoring of subjects during cannabis administration should include heart rate, blood pressure, and mental status. Subjects should not be discharged from research participation until reevaluation has shown that they have returned to baseline status. Gorelick, D.A. and Heishman, S.J. Methods in Molecular Medicine: Marijuana and Cannabinoid Research: Methods and Protocols, pp. 235-254. Totowa, NJ: Humana Press, 2006.

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

Smoking Rates and Topography Predict Adolescent Smoking Cessation Following Treatment with Nicotine Replacement Therapy Smoking puff topography predicts adolescent tobacco cessation outcome. IRP scientists examined the predictive value of pre-treatment smoking rates and puff topography variables for abstinence outcomes among 66 adolescents enrolled in a three-month smoking-cessation trial using nicotine replacement and cognitive-behavioral therapy. Pre-treatment variables included cigarettes per day, puff volume, puff duration, and several youth-adapted Fagerstršm-derived questionnaire scores. Outcome measures included prolonged abstinence at end of treatment and point-prevalent abstinence three months after the end of the trial. Logistic regression controlling for treatment group showed that increases in baseline cigarettes per day (OR=1.438, 95% CI 1.051-1.967) and average puff volume (OR=1.168, 95% CI 1.030-1.326) predicted continued smoking at the end of treatment. Baseline puff volume (p=0.013), but not CPD, predicted abstinence at the three-month follow-up. None of the youth-adapted Fagerstršm dependence questionnaires predicted outcome with either abstinence measure. Franken, F.H., Pickworth, W.B., Epstein, D.H. and Moolchan, E.T. Cancer Epidemiology, Biomarkers and Prevention, 15, pp. 154-157, 2006.