Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Genomic Alterations in Cultured Human Embryonic Stem Cells Cultured human embryonic stem cell (hESC) lines are an invaluable resource because they provide a uniform and stable genetic system for functional analyses and therapeutic applications. Nevertheless, these dividing cells, like other cells, probably undergo spontaneous mutation at a rate of 10(-9) per nucleotide. Because each mutant has only a few progeny, the overall biological properties of the cell culture are not altered unless a mutation provides a survival or growth advantage. Clonal evolution that leads to emergence of a dominant mutant genotype may potentially affect cellular phenotype as well. IRP investigators assessed the genomic fidelity of paired early- and late-passage hESC lines in the course of tissue culture. Relative to early-passage lines, eight of nine late-passage hESC lines had one or more genomic alterations commonly observed in human cancers, including aberrations in copy number (45%), mitochondrial DNA sequence (22%) and gene promoter methylation (90%), although the latter was essentially restricted to 2 of 14 promoters examined. The observation that hESC lines maintained in vitro develop genetic and epigenetic alterations implies that periodic monitoring of these lines will be required before they are used in in vivo applications and that some late-passage hESC lines may be unusable for therapeutic purposes. Maitra, A., Arking, D.E., Shivapurkar, N., Ikeda, M., Stastny, V., Kassauei, K., Sui, G., Cutler, D.J., Liu, Y., Brimble, S.N., Noaksson, K., Hyllner, J., Schulz, T.C., Zeng, X., Freed, W.J., Crook, J., Abraham, S., Colman, A., Sartipy, P., Matsui, S., Carpenter, M., Gazdar, A.F., Rao, M. and Chakravarti, A. Nature Genetics, 37, pp. 1099-1103, 2005.

Reactive Oxygen Species and p38 Phosphorylation Regulate the Protective Effect of Delta9-tetrahydrocannabinol in the Apoptotic Response to NMDA NMDA causes oxidative stress in neurons, and produces cell death involving elements of both necrosis and apoptosis. To examine the neuroprotective mechanism of Delta9-tetrahydrocannabinol (THC) in NMDA-induced death of AF5 cells, IRP researchers measured reactive oxygen species (ROS) formation after exposure to NMDA. ROS generation was increased by NMDA, and NMDA-induced ROS generation was significantly decreased by THC. Western blotting revealed an increase in phosphorylated p38 MAPK after NMDA treatment, which was also blocked by pretreatment with THC. The time course of ROS generation and activation of MAPK signaling pathways were similar. SB203580, a p38 inhibitor, partially blocked glutamate excitotoxicity in AF5 cells. The present data suggest that THC protects against NMDA-induced apoptosis in AF5 cells by blocking ROS generation and inhibiting the activation of p38-MAPK. Chen, J., Errico, S.L. and Freed, W.J. Neuroscience Letters, 389, pp. 99-103, 2005.

NTera2: A Model System to Study Dopaminergic Differentiation of Human Embryonic Stem Cells NTera2, a human embryonal carcinoma (EC) stem cell line, shares many characteristics with human embryonic stem cells (hESCs). To determine whether NTera2 can serve as a useful surrogate for hESCs, IRP scientists compared global gene expression between undifferentiated NTera2, multiple undifferentiated hESC cell lines, and their differentiated derivatives, and showed that NTera2 cells share multiple markers with hESCs. Similar to hESCs, NTera2 cells differentiated into TH-positive cells that express dopaminergic markers including AADC, DAT, Nurr1, TrkB, TrkC, and GFRA1 when co-cultured with PA6 cells. Flow cytometry analysis showed that tyrosine hydroxylase (TH) and neural cell adhesion molecule (NCAM) expression increased, whereas SSEA4 expression decreased as cells differentiated. Medium conditioned by PA6 cells stimulated differentiation of NTera2 cells to generate TH-positive cells that expressed dopaminergic markers. Flow cytometry selected polysialylated (PSA-NCAM) cells responded to medium conditioned by PA6 cells by differentiating into TH-positive cells and expressed dopaminergic markers. Sorted cells differentiated for 4 weeks in PA6 cell conditioned media included functional neurons that responded to neurotransmitters and exhibited electronic excitability. Therefore, NTera2 cell dopaminergic neuronal differentiation and PSA-NCAM enrichment provides a useful system for the future study of hESCs. Schwartz, C.M., Spivak, C.E., Baker, S.C., McDaniel, T.K., Loring, J.F., Nguyen, C., Chrest, F.J., Wersto, R., Arenas, E., Zeng, X., Freed, W.J., and Rao MS. Stem Cells and Development, 14, pp. 517-534, 2005.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Species and Strain Differences in the Expression of a Novel Glutamate-Modulating Cannabinoid Receptor in the Rodent Hippocampus A novel, non-CB1 cannabinoid receptor has been defined by the persistence of inhibition of glutamatergic EPSPs by the cannabinoid receptor agonist WIN55,212-2 in mice lacking the cloned CB1 receptor (CB1-/-) (Hajos et al., 2001). This novel receptor was also distinguished from CB1 by its sensitivity to the antagonist SR141716A and its insensitivity to the antagonist AM251 (Hajos & Freund, 2002). The authors have chosen to refer to this putative receptor as CBsc due to its identification on Schaffer collateral axon terminals in the hippocampus. These IRP researchers examined properties of CBsc receptors in Sprague Dawley (SD) rats and two strains of wild-type (WT) mice (C57BL/6J and CD1) used as backgrounds for two independent lines of CB1-/- mice (Ledent et al., 1999; Zimmer et al., 1999). The inhibition of synaptic glutamate release by WIN55,212-2 was observed in hippocampal slices from WT CD1 mice and SD rats but was absent in WT C57 mice. The authors also found that AM251 and SR141716A antagonized the effect of WIN55,212-2 in hippocampal slices from CD1 mice and SD rats demonstrating a lack of selectivity of these ligands for CB1 and CBsc receptors in these animals. The results indicate that the glutamate-modulating CBsc cannabinoid receptor is present in the hippocampi of CD1 mice and SD rats but not in C57BL/6J mice. Thus, the authors have identified animal models that may permit the study of cannabinoids independently of the novel CBsc receptor (C57CB1+/+), the CBsc receptor independently of the cloned CB1 receptor (CD1CB1-/-), or in the absence of both receptors (C57CB1-/-). Hoffman, A.F., Macgill, A.M., Smith, D., Oz, M. and Lupica, C.R. European Journal of Neuroscience 22, pp. 2387-2391, 2005.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

In Situ Structural Characterization of Phosphatidylcholines in Brain Tissue using MALDI-MS/MS Phosphatidylcholine (PC) is one of the most abundant classes of phospholipids and is a major component of membranes in biological systems. Recently, PCs have been detected by direct tissue analysis using MALDI-TOFMS. However, these studies did not allow for the structural characterization of PCs in tissue. In the current study, an in situ method for detection and structural analysis of PC species in brain tissue was developed using a MALDI-TOF/TOF mass spectrometer. Initial profiling of lipids in tissue is performed by MALDI-TOFMS, which allows for the assignment of PC species. However, to confirm the structure of the PC species detected in tissue, MALDI-MS/MS analysis was employed. In this work, protonated, sodiated, and potassiated PC species were detected in brain tissue using DHA matrix. MALDI-MS/MS analysis of these species yielded fragments that verified a phosphocholine head group, but did not supply any fragments that would permit the identification of acyl substituents. To obtain more structural information, lithium adducts of PC species were produced using DHA matrix dissolved in 100 mM lithium chloride. MALDI-MS/MS analysis of lithiated PC species produced fragments that allowed for the identification and positional assignment of acyl groups in PC species. Jackson, S.N., Wang, H.Y., and Woods, A.S. Journal of the American Society for Mass Spectrometry 16, pp. 2052-2056, 2005.

Localization and Analyses of Small Drug Molecules in Rat Brain Tissue Sections Traditional detection of drugs in tissue requires tissue homogenization, which precludes the mapping and localization of drugs. The use of autoradiography could compensate for such shortcomings. However, it requires expensive custom-synthesized radioactive drugs. Recent improvement in sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and MALDI-MS/MS provides an alternative approach for in situ drug detection. In this work, rat brains were collected after intracranial injection of chlorisondamine or intraperitoneal injection of cocaine and snap frozen. MALDI matrixes were applied directly to 14-mum brain cryosections and spectra acquired. The identity of the drugs was further confirmed by MS/MS. Careful matrix selection and tissue preparation allows for the successful detection of drugs and the mapping of their relative abundance across various regions of the brain. This new method is simple, safe, accurate, fast, cost-effective, and low in sample consumption and shows potential for imaging, pharmacokinetics, and toxicology applications. Wang, H.Y., Jackson, S.N., McEuen, J. and Woods, A.S. Analytical Chemistry, 77, pp. 6682-6686, 2005.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

Regulation of the Rat Brain Endothelin System by Endogenous Beta-endorphin Several lines of evidence indicate that the central endogenous opioid and endothelin (ET) system regulate each other. To explore this idea further, IRP researchers determined the effect of intracerebroventricular (i.c.v.) administration of anti-beta-endorphin IgG (rabbit) on the expression level of the opioid, corticotropin-releasing hormone and endothelin receptors, and tissue concentration of ET-1. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10mul (i.c.v.) of either control rabbit IgG (2.5 ug/mul) or anti-beta-endorphin IgG (2.5mug/mul) on days 1, 3 and 5. On day 6, animals were euthanized and caudate, cortex and hippocampus collected for Western blot analysis. Anti-beta-endorphin IgG down-regulated ET-A receptor protein expression in the caudate (51%), but had no effect on the expression of mu, delta, kappa opioid, ET-B, CRH-1 and CRH-2 receptors in any brain region. Anti-beta-endorphin IgG increased tissue ET-1 levels in the caudate by 30.3%. [(35)S]GTP-gamma-S binding assays demonstrated that anti-beta-endorphin IgG increased the efficacy of [d-Ala(2)-MePhe(4),Gly-ol(5)]enkephalin without altering its potency in caudate. Control experiments showed that there was no detectable rabbit IgG in caudate, cortex and hippocampus samples. These results suggest that beta-endorphin in the CSF coordinately regulates ET-1 levels and the ET-A receptor in rat caudate. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between opioid and ET system. Wang, X., Xu, H. and Rothman, R.B. Regulation of the Rat Brain Endothelin System by Endogenous Beta-endorphin. Peptides. [Epub ahead of print], 2005.

Medicinal Chemistry Section, Medications Discovery Research Branch

N-8-Substituted-Benztropinamine Analogs as Selective Dopamine Transporter Ligands A series of N-8-substituted benztropinamines was synthesized and evaluated for binding at the dopamine (DAT), serotonin (SERT), norepinephrine (NET) transporters, and muscarinic M1 receptors. In general, the isosteric replacement of the C-3 benzhydrol ether of benztropine by a benzhydryl amino group was well tolerated at these binding sites. However, for certain N-8 substituted derivatives, selectivity over muscarinic M1 receptor affinity was reduced. Behavioral evaluation of selected compounds in this series, in animal models of cocaine abuse, is underway. Grundt, P., Kopajtic, T., Katz, J.L. and Newman, A.H. Bioorganic Medicinal Chemistry Letters 15, pp. 5419-5423, 2005.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

The Novel mGluR2/3 Agonist LY379268 Attenuates Cue-induced Reinstatement of Heroin Seeking In humans, drug-associated stimuli can provoke heroin relapse during abstinence. In rats, cues paired with heroin self-administration reinstate heroin seeking in a relapse model. The neurobiological mechanisms involved in this reinstatement, however, are largely unknown. Here, IRP scientists determined the effect of LY379268, an mGluR2/3 agonist which decreases evoked glutamate release, on cue-induced reinstatement of heroin seeking. Systemic injections of LY379268 attenuated reinstatement of heroin seeking induced by exposure to a discrete tone-light cue that was previously paired with heroin infusions during self-administration training. In contrast, LY379268 had no effect on heroin self-administration. Results indicate that glutamate plays an important role in cue-induced reinstatement of heroin seeking and suggest that mGluR2/3 agonists should be considered for the treatment of opiate relapse. Bossert, J.M., Busch, R.F. and Gray, S.M. Neuroreport 16, pp. 1013-1016, 2005.

Differential Long-term Neuroadaptations of Glutamate Receptors in the Basolateral and Central Amygdala after Withdrawal from Cocaine Self-administration in Rats Humans and laboratory animals remain highly vulnerable to relapse to cocaine seeking after prolonged periods of withdrawal from the drug. It has been hypothesized that this persistent cocaine relapse vulnerability involves drug-induced alterations in glutamatergic synapses within the mesolimbic dopamine reward system. Previous studies have shown that cocaine self-administration induces long-lasting neuroadaptations in glutamate neurons of the ventral tegmental area and nucleus accumbens. Here, IRP investigators determined the effect of cocaine self-administration and subsequent withdrawal on glutamate receptor expression in the amygdala, a component of the mesolimbic dopamine system that is involved in cocaine seeking and craving induced by drug-associated cues. Rats were trained for 10 d to self-administer intravenous cocaine (6-h/d) or saline (a control condition) and were sacrificed after 1 or 30 withdrawal days. Basolateral and central amygdala tissues were assayed for protein expression of the AMPA receptor subunits (GluR1, GluR2) and the NMDA receptor subunits (NR1, NR2A and NR2B). In the basolateral amygdala, GluR1, but not GluR2, levels were increased on days 1 and 30, NR2A levels were increased on day 1, and NR2B levels were decreased on day 30 of withdrawal from cocaine. In the central amygdala, GluR2, but not GluR1, levels were increased on days 1 and 30, NR1 levels were increased on day 30, and NR2A or NR2B levels were not altered after withdrawal from cocaine. These results indicate that cocaine self-administration and subsequent withdrawal induces long-lasting and differential neuroadaptations in basolateral and central amygdala glutamate receptors. Lu, L., Dempsey, J., Shaham, Y. and Hope, B.T. Journal of Neurochemistry 94, pp. 161-168, 2005.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

The Supramammillary Nucleus Mediates Primary Reinforcement via GABA(A) Receptors The supramammillary nucleus (SUM), a dorsal layer of the mammillary body, has recently been implicated in positive reinforcement. The present study examined whether GABA(A) receptors in the SUM or adjacent regions are involved in primary reinforcement using intracranial self-administration procedures. Rats learned quickly to lever-press for infusions of the GABA(A) antagonist picrotoxin into the SUM. Although picrotoxin was also self-administered into the posterior hypothalamic nuclei and anterior ventral tegmental area, these regions were less responsive to lower doses of picrotoxin than the SUM. The finding that rats learned to respond selectively on the lever triggering drug infusions is consistent with picrotoxin's reinforcing effect. Coadministration of the GABA(A) agonist muscimol disrupted picrotoxin self-administration, and another GABA(A) antagonist, bicuculline, was also self-administered into the SUM; thus, the reinforcing effect of picrotoxin is mediated by GABA(A) receptors. Since rats did not self-administer the GABA(B) antagonist 2-hydroxysaclofen into the SUM, the role of GABA(B) receptors may be distinct from that of GABA(A) receptors. Pretreatment with the dopamine receptor antagonist SCH 23390 (0.05 mg/kg, i.p.) extinguished picrotoxin self-administration into the SUM, suggesting that the reinforcing effects of GABA(A) receptor blockade depend on normal dopamine transmission. In conclusion, the blockade of GABA(A) receptors in the SUM is reinforcing, and the brain 'reward' circuitry appears to be tonically inhibited via supramammillary GABA(A) receptors and more extensive than the meso-limbic dopamine system. Ikemoto, S. Neuropsychopharmacology, 30(6), pp. 1088-1095, 2005.

A Five-minute, but not a Fifteen-minute, Conditioning Trial Duration Induces Conditioned Place Preference for Cocaine Administration into the Olfactory Tubercle The establishment of conditioned place preference (CPP) with intracranial injections requires specific injection sites, drug doses, and conditioning trial durations. IRP scientists examined the role of conditioning trial duration in CPP with cocaine injections into the medial olfactory tubercle. Only those rats that had spent 5 min in the compartments showed CPP for cocaine, while rats that had been removed immediately or spent 15 min following cocaine injections did not show CPP. Effective conditioning trial durations for CPP induced by intracranial cocaine injections are apparently much shorter than those typically used for intracranial injections of other drugs of abuse. Ikemoto, S. and Donahue, K.M. Synapse 56(1), pp. 57-59, 2005.

The Functional Divide for Primary Reinforcement of D-amphetamine Lies Between the Medial and Lateral Ventral Striatum: Is the Division of the Accumbens Core, Shell, and Olfactory Tubercle Valid? When projection analyses placed the nucleus accumbens and olfactory tubercle in the striatal system, functional links between these sites began to emerge. The accumbens has been implicated in the rewarding effects of psychomotor stimulants, whereas recent work suggests that the medial accumbens shell and medial olfactory tubercle mediate the rewarding effects of cocaine. Interestingly, anatomical evidence suggests that medial portions of the shell and tubercle receive afferents from common zones in a number of regions. Here, IRP investigators report results suggesting that the current division of the ventral striatum into the accumbens core and shell and the olfactory tubercle does not reflect the functional organization for amphetamine reward. Rats quickly learned to self-administer D-amphetamine into the medial shell or medial tubercle, whereas they failed to learn to do so into the accumbens core, ventral shell, or lateral tubercle. The present results suggest that primary reinforcement of amphetamine is mediated via the medial portion of the ventral striatum. Thus, the medial shell and medial tubercle are more functionally related than the medial and ventral shell or the medial and lateral tubercle. The current core-shell-tubercle scheme should be reconsidered in light of recent anatomical data and these functional findings. Ikemoto, S., Qin, M and Liu, Z.H., Journal of Neuroscience, 25(20), pp. 5061-5065, 2005.

Cocaine Experience Establishes Control of Midbrain Glutamate and Dopamine by Corticotropin-releasing Factor: A Role in Stress-induced Relapse to Drug Seeking Footshock stress can reinstate cocaine-seeking behavior through a central action of the stress-associated neurohormone corticotropin-releasing factor (CRF). Here IRP scientists report (1) that footshock stress releases CRF in the ventral tegmental area (VTA) of the brain, (2) that, in cocaine-experienced but not in cocaine-naive rats, this CRF acquires control over local glutamate release, (3) that CRF-induced glutamate release activates the mesocorticolimbic dopamine system, and (4) that, through this circuitry, footshock stress triggers relapse to drug seeking in cocaine-experienced animals. Thus, a long-lasting cocaine-induced neuroadaptation, presumably at the level of glutamate terminals in the VTA, appears to play an important role in stress-induced relapse to drug use. Similar neuroadaptations may be important for the comorbidity between addiction and other stress-related psychiatric disorders. Wang, B., Shaham, Y, Zitzman, D, Azari, S., Wise, R.A. and You, Z.B., Journal of Neuroscience, 25(2), pp. 5389-5396, 2005.

Study of the Interaction of Chlorisondamine and Chlorisondamine Analogues with an Epitope of the Alpha-2 Neuronal Acetylcholine Nicotinic Receptor Subunit Chlorisondamine (CHL), a neuronal nicotinic ganglionic blocker, when injected in the cerebral ventricle of rats chronically blocks the increase in locomotion and rearing by subcutaneous nicotine injection. The blocking of the ion channel(s) prevents nicotine from exerting its rewarding effects on the CNS. When administered intraperitoneally, a dose 400-500 times the intracerebroventricular one is needed to cross the blood-brain barrier and to generate the same level of nicotine antagonism, resulting in severe side-effects, thus making it unlikely to be used as a therapeutic compound. Three CHL analogues, 2-(indolin-1-yl)-N,N,N-trimethylethanaminium iodide, 2-(1,3-dioxoisoindolin-2-yl)- N,N,N-trimethylethanaminium iodide, and 2-(1H-indole-3-carboxamido)- N,N,N-trimethylethanaminium iodide, were synthesized in the hope of circumventing the parent compound's shortcomings. They all share a modified indole ring, lack the four chlorines CHL carries, and have one tertiary amine and one quaternary amine. The CHL analogues form noncovalent complexes with an epitope of the alpha-2 nicotinic receptor subunit, GEREE(p)TEEEEEEEDEN, previously proposed as the possible site of CHL interaction. Complexes were analyzed using matrix-assisted laser desorption/ionization mass spectrometry for comparison with CHL. Overall, all three analogues showed better affinity than CHL for complex formation with both the nonphosphorylated and phosphorylated epitopes. Wang, H.Y., Taggi, A.E., Meinwald, J., Wise, R.A. and Woods, A.S. Journal of Proteome Research 4(2), pp. 532-539, 2005.

Brain Stimulation and Morphine Reward Deficits in Dopamine D2 Receptor-deficient Mice The rewarding effects of lateral hypothalamic brain stimulation, various natural rewards, and several drugs of abuse are attenuated by D1 or D2 dopamine receptor (D1R or D2R) antagonists. Much of the evidence for dopaminergic involvement in rewards is based on pharmacological agents with limited or "relative" selectivity for dopamine receptor subtypes. Genetically engineered animal models provide a complementary approach to pharmacological investigations. In the present study, IRP researchers explored the contribution of dopamine D2Rs to (1) brain stimulation reward (BSR) and (2) the potentiation of this behavior by morphine and amphetamine using D2R-deficient mice. Wild-type (D2Rwt), heterozygous (D2Rhet), and D2R knockout (D2Rko) mice were trained to turn a wheel for rewarding brain stimulation. Once equivalent rate-frequency curves were established, morphine-induced (0, 1.0, 3.0, and 5.6 mg/kg s.c.) and amphetamine-induced (0, 1.0, 2.0, and 4.0 mg/kg i.p.) potentiations of BSR were determined. The D2Rko mice required approximately 50% more stimulation than the D2Rwt mice did. With the equi-rewarding levels of stimulation current, amphetamine potentiated BSR equally across the three genotypes. In contrast, morphine potentiated rewarding stimulation in the D2Rwt, had no effect in the D2Rhet, and antagonized rewarding stimulation in the D2Rko mice. D2R elimination decreases, but does not eliminate, the rewarding effects of lateral hypothalamic stimulation. After compensation for this deficit, amphetamine continues to potentiate BSR, while morphine does not. Elmer, G.I., Pieper, J.O., Levy, J., Rubinstein, M., Low, M.J., Grandy, D.K. and Wise, R.A. Psychopharmacology, 182, pp. 33-44, 2005.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Nicotine as a Typical Drug of Abuse in Experimental Animals and Humans Tobacco use through cigarette smoking is the leading preventable cause of death in the developed world. Nicotine, a psychoactive component of tobacco, appears to play a major role in tobacco dependence, but reinforcing effects of nicotine often are difficult to demonstrate directly in controlled laboratory studies with animal or human subjects. The objective of the present study was to review the major findings obtained with various procedures developed to study dependence-related behavioral effects of nicotine in experimental animals and humans, i.e., drug self-administration, conditioned place preference, subjective reports of nicotine effects and nicotine discrimination, withdrawal signs, and ratings of drug withdrawal. Results showed thst nicotine can function as an effective reinforcer of drug-seeking and drug-taking behavior both in experimental animals and humans under appropriate conditions. Interruption of chronic nicotine exposure produces withdrawal symptoms that may contribute to relapse. Difficulties encountered in demonstrating reinforcing effects of nicotine under some conditions, relative to other drugs of abuse, may be due to weaker primary reinforcing effects of nicotine or to a more critical contribution of environmental stimuli to the maintenance of drug-seeking and drug-taking behavior with nicotine than with other drugs of abuse. Further experiments are also needed to delineate the role other chemical substances inhaled along with nicotine in tobacco smoke play in sustaining smoking behavior. The authors conclude that nicotine acts as a typical drug of abuse in experimental animals and humans. LeFoll, B. and Goldberg, S.R. Psychopharmacology (Berlin), October 5, 2005, Epubmed ahead of print, PMID 16205918.

A Detailed Behavioral Analysis of the Acute Motor Effects of Caffeine in the Rat: Involvement of Adenosine A1 and A2A Receptors Currently, there is no consensus on the contribution of adenosine A1 and A2A receptor blockade to motor-activating effects of caffeine. The aim of the present study was to use a detailed and continuous observational method to compare the motor effects induced by caffeine with those induced by selective A1 and A2A receptor antagonists. The behavioral repertoire induced by systemic administration of caffeine (3, 10, and 30 mg/kg), A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.2, 4.8 and 7.2 mg/kg), and A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3; 1, 3, and 10 mg/kg) was analyzed. The effects of pretreatment with the selective A1 receptor agonist N 6-cyclopentyladenosine (CPA; 0.1 mg/g) and the selective A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5¶Î-N-ethylcarboxyamidoadenosine (CGS 21680; 0.2 mg/kg) on the pattern of motor activation induced by caffeine, CPT, or MSX-3 were also examined. Results showed that the pattern of behavioral activation induced by caffeine was better mimicked by CPT than by MSX-3. Coadministration of CPT and MSX-3 gave different results depending on the dose and the type of behavioral response. CPA was more effective at decreasing the activating effects of caffeine and CPT than those of CGS 21680. On the other hand, CGS 21680 was more effective at decreasing the activating effects of MSX-3 than those of caffeine or CPT. Factor analysis revealed a complex three-dimensional behavioral profile for caffeine that was similar to the profile for CPT and was different from the profile for MSX-3. The results indicate a predominant role for A1 receptors in the motor-activating effects of acutely administered caffeine. Antoniou, K., Papadopoulou-Daifoti, Z., Hyphantis, T., Papathanasiou, G., Bekris, E., Marselos, M., Panlilio, L, MŸller, C.E., Goldberg, S.R. and Ferré, S. Psychopharmacology (Berlin), October 5, 2005, Epubmed ahead of print, PMID 16205915.

Ethanol Does Not Affect Discriminative-Stimulus Effects of Nicotine in Rats The effects of ethanol were evaluated in rats trained to discriminate 0.4 mg/kg of nicotine from saline under a fixed-ratio 10 schedule of food delivery. Ethanol (0.1-1 g/kg, i.p.) did not produce any nicotine-like discriminative effects and did not produce any shift in the dose-response curve for nicotine discrimination. Thus, the ability to discriminate nicotine's effects does not appear to be altered by ethanol administration. However, the high dose of 1 g/kg ethanol, given either alone or in combination with nicotine, markedly depressed food-maintained responding. This later effect was associated in some rats with an attenuation of the discriminative-stimulus effects of the training dose of nicotine. This suggests that previous reports of increased tobacco smoking following ethanol consumption in humans are connected, in some way, with an increase in motivation to consume nicotine that is produced by ethanol, rather than with a decrease in the subjective response to nicotine. LeFoll, B. and Goldberg, S.R. European Journal of Pharmacology, 519, pp. 96-102, 2005.

The Dopamine D3 Receptor and Drug Dependence: Effects on Reward or Beyond? Abused drugs (alcohol, heroin, cocaine, tetrahydrocannabinol and nicotine) elicit a variety of chronically relapsing disorders by interacting with brain reward systems. All of these drugs increase dopamine levels in the shell of nucleus accumbens, a structure that has been involved in their hedonic and reinforcing properties. Dopamine D3 receptors (DRD3) are predominantly expressed in the nucleus accumbens, but also in the ventral tegmental area and in the amygdala, brain structures implicated in drug dependence. Moreover, converging pharmacological, human post-mortem and genetic studies have suggested the involvement of the DRD3 in drug dependence. Based on early studies using non-selective DRD3 ligands, the DRD3 was proposed as having a direct role in the rewarding effects of psychostimulants. However, recent studies using highly selective DRD3 ligands and the DRD3-deficient mice have revealed that the DRD3 is not implicated in the direct reinforcing effects of drugs of abuse. In contrast, the DRD3 appears to be implicated in the motivation to self-administer drugs under schedules where the response requirements are high. This is consistent with a behavioral economic analysis, with the effects of DRD3 ligands revealed only in situations with high prices for drug. Drug-self administration and relapse are strongly controlled by environmental stimuli. The DRD3 strongly modulates the influence of these environmental stimuli on drug-seeking behavior. DRD3 blockade disrupts the reactivity to drug-associated stimuli in various paradigms, such as second-order schedules of drug-self administration, conditioned place preference and Pavlovian conditioning procedures. In several paradigms, the involvement of the DRD3 has been confirmed by using DRD3-deficient mice. On the contrary, reactivity to stimuli associated with natural reinforcers, such as food, appears unaffected by modulation of the DRD3. All these findings suggest that DRD3 ligands may represent a useful strategy for decreasing relapse in abstinent drug-abusers. Le Foll, B., Goldberg, S.R. and Sokoloff, P. Neuropharmacology, 49, pp. 525-541, 2005.

Existence and Theoretical Aspects of Homomeric and Heteromeric Dopamine Receptor Complexes and their Relevance for Neurological Diseases Dopamine (DA) and other receptors physically interact in the plasma membrane of basal ganglia neurons forming receptor mosaics (RMs). Two types of RMs are discussed, homomers formed only by DA-receptor (DA-R) subtypes and heteromers formed by DA-R associated with other receptors, such as A2A, A1, mGluR5, N-methyl-D-aspartate (NMDA), gamma-aminobutryic acid (GABA)-A, and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid. By being part of horizontal molecular networks, RMs tune multiple effector systems already at membrane level, such as G protein regulated inward rectifying potassium channels and dopamine transporter activity. Also, ligand-gated ion channels such as GABA-A and NMDA receptors are modulated by DA-R, e.g., in the striatal GABA output neurons through the formation of heteromeric complexes with these receptors. Thus, intramembrane DA-R-receptor interactions play an important role in the information handling in the basal ganglia. On this basis, functional implications of DA RM in physiological and pathological conditions are discussed. The effects of temperature on RM are discussed not only because receptor-decoding mechanisms are temperature sensitive, but also in view of the suggestion that possible ordering effects (i.e., changes in the entropy of a receptor complex) induced by a ligand are as a result of alterations in the receptor oligomerization (i.e., are related to rearrangements of the RM). Hence, brain temperature may have profound effects on brain integrative functions not only because its effects on the kinetics of biochemical reactions, but also for its effects on receptor geometry, building up of RM, and alterations in protein expression, as is the case of H-channels following febrile seizures. Agnati, L.F., Ferre, S., Burioni, R., Woods, A., Genedani, S., Franco, R. and Fuxe, K. Neuromolecular Medicine, 7, pp. 61-78, 2005.

Partners for Adenosine A1 Receptors G protein-coupled receptors (GPCRs) are targets for therapy in a variety of neurological diseases. Using adenosine A1 receptors (A1Rs) as paradigm of GPCRs, this review focuses on how protein-protein interactions, from monomers to heteromers, can contribute to hormone/neurotransmitter/neuromodulator regulation. The interaction of A1Rs with other membrane receptors, enzymes, and adaptor and scaffolding proteins is relevant for receptor traffic, internalization, and desensitization, and A1Rs are extremely important in driving signaling through different intracellular pathways. There is even the possibility of linking together GPCR heteromeric complexes with ion channel receptors in a receptor mosaic that might have special integrative value and might constitute the molecular basis for learning and memory. Franco, R., Ciruela, F., Casado, V., Cortes, A., Canela, E.I., Mallol, J., Agnati, L.F., Ferre, S., Fuxe, K. and Lluis, C. Journal of Molecular Neuroscience, 26, pp. 221-232, 2005.

Receptor-Receptor Interactions, Receptor Mosaics, and Basic Principles of Molecular Network Organization: Possible Implications for Drug Development The phenomenon of receptor-receptor interactions was hypothesized by Agnati and Fuxe in the 1980s, and several indirect proofs were provided in the following years by means of in vitro binding experiments and in vivo experiments in physiological and pathological animal models. This paper aims to outline some of the most important features and consequences of this phenomenon in the frame of the structural and functional aspects of molecular networks. In particular, the concepts of receptor mosaic (RM), and of horizontal and vertical molecular networks (HMNs, VMNs, respectively) are illustrated. To discuss some aspects of the functional organization of molecular networks, not only new data on protein-protein interactions but also the biochemical mechanism of cooperativity will be used. On this basis, some theoretical deductions can be drawn that allow a tentative classification of the RMs and the proposal of the extension of the concept of branching point introduced for enzymes to the possible switching role of some RMs in directing signals to various VMNs. Finally, the cooperativity phenomenon and the so-called symmetry rule will be used to introduce a proper mathematical approach that characterizes RMs as to their receptor composition, receptor topography, and order of receptor activation inside the RM. These new data on G protein-coupled receptors and molecular network organization indicate possible new approaches for drug development. Agnati, L.F., Tarakanov, A.O., Ferre, S., Fuxe, K. and Guidolin, D. Journal of Molecular Neuroscience, 26, pp. 193-208, 2005.

Dimer-based Model for Heptaspanning Membrane Receptors The existence of intramembrane receptor-receptor interactions for heptaspanning membrane receptors is now fully accepted, but a model considering dimers as the basic unit that binds to two ligand molecules is lacking. Here, IRP scientists propose a two-state-dimer model in which the ligand-induced conformational changes from one component of the dimer are communicated to the other. Our model predicts cooperativity in binding, which is relevant because the other current models fail to address this phenomenon satisfactorily. Our two-state-dimer model also predicts the variety of responses elicited by full or partial agonists, neutral antagonists and inverse agonists. This model can aid our understanding of the operation of heptaspanning receptors and receptor channels, and, potentially, be important for improving the treatment of cardiovascular, neurological and neuropsychyatric diseases. Franco, R., Casado, V., Mallol, J., Ferre, S., Fuxe, K., Cortes, A., Ciruela, F., Lluis, C. and Canela, E.I. Trends Biochemical Science 30, pp. 360-366, 2005.

Smoking Cessation Guidelines: Evidence -based Recommendations of the French Health Products Safety Agency Tobacco use is the leading preventable cause of death in developed countries. Millions of smokers are willing to stop, but few of them are able to do so. Clinicians should only use approaches that have demonstrated their efficacy in helping patients to stop smoking. This article summarizes the evidence-based major findings and clinical recommendations for the treatment of tobacco dependence of the French Health Products Safety Agency (AFSSAPS). Clinicians should enquire about the smoking status of each patient and provide information about health consequence of smoking and effective treatments available. These treatments include counseling (mainly individual or social support and behavioral and cognitive therapy) and pharmacological treatment with either nicotine replacement therapy (NRT) or bupropion LP. Pharmacological treatments should be used only for proven nicotine dependence, as assessed by the Fagerstrom test for Nicotine Dependence. The choice of pharmacologic treatment depends on the patient's preference and history and of the presence of contra-indications. The clinician should start with a single agent, but these treatments may be used in combination. Smoking behavior is a chronic problem that requires long-term management and follow-up. Access to intensive treatment combining pharmacological treatment and extensive behavioral and cognitive therapy should be available for highly dependent patients. LeFoll, B., Melihan-Cheinin, P., Rostoker, G., Lagrue, G. Working Group of AFSSAPS European Psychiatry, 20, pp. 431-441, 2005.

Intravenous Butyrylcholinesterase Administration and Plasma and Brain Levels of Cocaine and Metabolites in Rats Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. IRP researchers evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine. Carmona, G.N., Schindler, C.W., Greig, N.H., Holloway, H.W., Jufer, R.A., Cone, E.J. and Gorelick, D.A. European Journal of Pharmacology, 517, pp. 186-190, 2005.

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

Shorter Time to First Cigarette of the Day in Menthol Adolescent Cigarette Smokers Menthol smoking is thought to contribute to the addictiveness of smoking. Given the high prevalence of menthol smoking among youth, the aim of the current analysis was to examine differences in consumption and tobacco dependence, including smoking urgency among menthol and non-menthol adolescent smokers. Data for the current analysis were collected from telephone interviews with adolescent smokers applying to a cessation treatment study. Of 572 adolescent smokers (mean age=15.6+/-1.6 years; 55.1% female; 46.9% African American, 48.2% European American), 531 smoked menthol cigarettes and 41 smoked non-menthol as their usual brand. Analysis using Fisher's Exact (one-tailed) Test revealed that menthol smokers had a significantly shorter time to first (TTF) cigarette of the day compared to non-menthol smokers (smoking within the first 5 min of the day, 45% vs. 29%, respectively; p<0.04). Independent t tests revealed no significant difference in number of cigarettes per day (CPD) (mean=12.2+/-8.5 vs. 11.4+/-8.8; p<0.28) or Fagerstrom Test for Nicotine Dependence (FTND) scores (3.4+/-1.4 vs. 3.2+/-1.3; p<0.23). While preliminary, authors findings suggest greater smoking urgency among menthol compared to non-menthol adolescent cessation-treatment seekers. Further study in a broader sample of adolescent smokers is warranted to elucidate the mechanisms underlying the effects of menthol smoking for youths. Collins, C.C. and Moolchan, E.T. Addictive Behaviors November 19, 2005; [Epub ahead of print].