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Director's Report to the National Advisory Council on Drug Abuse - May, 2005

Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Truncated N-Terminal Mutants of SV40 Large T Antigen as Minimal Immortalizing Agents for CNS Cells Immortalized CNS cell lines are useful as in vitro models for innumerable purposes such as elucidating biochemical pathways, studies of effects of drugs, and ultimately, such cells may also be useful for neural transplantation. The SV40 LT oncoprotein, commonly used for immortalization, interacts with several cell cycle regulatory factors, including binding and inactivating p53 and retinoblastoma family cell-cycle regulators. In an attempt to define the minimal requirements of SV40 T antigen for immortalizing cells of CNS origin, authors constructed T155c, encoding the N-terminal 155 amino acids of LT. The p53 binding region is known to reside in the C-terminal region of LT. In a p53 temperature sensitive cell line model, T64-7B, expression of T155c and all constructs having mutations outside of the first 82 amino acids were capable of overriding cell-cycle block at the non-permissive growth temperature. Several cell lines were produced from fetal rat mesencephalic and cerebral cortical cultures using the T155c construct. The E107K construct contained a mutation in the Rb binding region, but was nonetheless capable of overcoming cell cycle block in T64-7B cell and immortalizing primary cultured cells. Cells immortalized with T155c were often highly dependent on the presence of bFGF for growth. Telomerase activity, telomere length, growth rates, and integrity of the p53 gene in cells immortalized with T155c did not change over 100 population doublings in culture, indicating that cells immortalized with T155c were generally stable during long periods of continuous culture. Freed, W.J., Zhang, P., Sanchez, J.F., Dillon-Carter, O., Coggiano, M., Errico, S.L., Lewis, B.D. and Truckenmiller, M.E. Experimental Neurology, 191, pp. S45-S59, 2005.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Independent Presynaptic and Postsynaptic Mechanisms Regulate Endocannabinoid Signaling at Multiple Synapses in the Ventral Tegmental Area Dopamine (DA) neurons in the ventral tegmental area have been implicated in psychiatric disorders and drug abuse. Understanding the mechanisms through which their activity is regulated via the modulation of afferent input is imperative to understanding their roles in these conditions. Here, IRP researchers demonstrate that endocannabinoids liberated from DA neurons activate cannabinoid CB1 receptors located on glutamatergic axons and on GABAergic terminals targeting GABA(B) receptors located on these cells. Endocannabinoid release was initiated by inhibiting either presynaptic type-III metabotropic glutamate receptors or postsynaptic calcium-activated potassium channels, two conditions that also promote enhanced DA neuron excitability and bursting. Thus, activity-dependent release of endocannabinoids may act as a regulatory feedback mechanism to inhibit synaptic inputs in response to DA neuron bursting, thereby regulating firing patterns that may fine-tune DA release from afferent terminals. Riegel, A.C. and Lupica, C.R. Journal of Neuroscience 24, pp. 11070-11078, 2004.

Effects of Saturated Long-Chain N-Acylethanolamines on Voltage-Dependent Ca2+ Fluxes in Rabbit T-Tubule Membranes The effects of saturated long-chain (C: 16-22) N-acylethanolamines and a series of saturated fatty acids with the same length of carbon chains were investigated on depolarization-induced (45)Ca(2+) fluxes mediated by voltage-dependent Ca(2+) channels in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with (45)Ca(2+) and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Arachidonoylethanolamide and docosaenoylethanolamide but not palmitoylethanolamide and stearoylethanolamide (all 10 microM) caused a significant inhibition of depolarization-induced (45)Ca(2+) fluxes and specific binding of [(3)H]Isradipine to transverse tubule membranes. On the other hand, saturated fatty acids including palmitic, stearic, arachidic, and docosanoic acids (all 10 microM) were ineffective in functional and radioligand binding experiments. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and arachidic acids were ineffective, arachidonoylethanolamide inhibited Ca(2+) effluxes and specific binding of [(3)H]Isradipine. Further studies indicated that only those fatty acids containing ethanolamine as a head group and having a chain length of more than 18 carbons were effective in inhibiting depolarization-induced Ca(2+) effluxes and specific binding of [(3)H]Isradipine. In conclusion, results indicate that depending on the chain length and the head group of fatty acid, N-acylethanolamines have differential effects on the function of voltage-dependent Ca(2+) channels and on the specific binding of [(3)H]Isradipine in skeletal muscle membranes. Oz, M., Alptekin, A., Tchugunova, Y. and Dinc, M. Archives of Biochemistry and Biophysics 434, pp. 344-351, 2005.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Direct Tissue Analysis of Phospholipids in Rat Brain using MALDI-TOFMS and MALDI-Ion Mobility-TOFMS After water, lipids are the most common biomolecules found in the brain (12%). A brief perusal of the physiology, anatomy, and pathophysiology of the brain illustrates the importance of lipids. Recent advances in mass spectrometry have allowed the direct probing of tissues. However, most studies have focused on proteins. In the present work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and MALDI-ion mobility (IM)-TOFMS were employed for direct analysis of phospholipids in rat brain tissue. Molecular ions (MH(+)) corresponding to phosphatidylcholines, phosphatidylethanolamines, and sphingomyelin, were recorded. When studying pharmacology, it has been learned that many therapeutic compounds are stored in the body's adipose tissue. MALDI-TOFMS and MALDI- IM-TOFMS were thus used to analyze rat brain tissue with chlorisondamine added directly onto the tissue slice. With both techniques, noncovalent complexes between the tissue phospholipids and chlorisondamine were detected. In addition, MALDI-IM-TOFMS of noncovalent complexes between phospholipids and chlorisondamine displayed a mobility between that of an isobaric lipid and peptide. Jackson, S.N., Wang, H.Y., Woods, A.S., Ugarov, M., Egan, T. and Schultz, J.A. Journal of American Society of Mass Spectrometry 16, pp. 133-138, 2005.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

Development of a Rationally-designed, Low-abuse Potential, Biogenic Amine Releaser that Suppresses Cocaine Self-administration Convergent lines of evidence support a dual deficit model of stimulant withdrawal, where reductions in synaptic dopamine (DA) and serotonin (5-HT) contribute to dysphoria, drug craving, and relapse. Thus, IRP scientists predicted that a non-amphetamine compound with substrate activity at DA and 5-HT transporters (i.e., a dual DA/5-HT releaser) would be an effective medication for treating stimulant addictions. Ideally, this type of medication would alleviate withdrawal symptoms, suppress cocaine self-administration, and lack side effects commonly associated with CNS stimulants. In the present work, over 350 compounds were screened in vitro for activity as substrate-type releasing agents at DA, 5-HT and norepinephrine (NE) transporters. These efforts identified PAL-287 (1-napthyl-2-aminopropane) as a non-amphetamine compound with potent substrate activity at biogenic amine transporters. In vivo microdialysis in rats demonstrated that PAL-287 (1-3 mg/kg, i.v.) increased extracellular DA and 5-HT in frontal cortex, but effects on 5-HT were somewhat greater. PAL-287 induced substantially less locomotor stimulation than (+)-amphetamine, a drug which increases only extracellular DA. Administration of high-dose (+)-methamphetamine or ()-MDMA to rats produced long-lasting depletion of cortical 5-HT, whereas PAL-287 (18 mg/kg, i.p. X 3) did not. PAL-287 displayed little or no reinforcing properties in rhesus monkeys trained to self-administer cocaine, yet PAL-287 produced a dose-dependent decrease in responding for cocaine when infused at a dose of 1.0 mg/kg/hr. Collectively, the findings reported here demonstrate that non-amphetamine monoamine releasing agents like PAL-287 might be promising candidate medications for the treatment of stimulant dependence. Rothman, R.B., Blough, B.E., Woolverton, W.L., Anderson, K.G., Negus, S.S., Mello, N.K., Roth, B.L. and Baumann, MH. Development of a Rationally-designed, Low-abuse potential, Biogenic Amine Releaser that Suppresses Cocaine Self-administration. Journal of Pharmacology and Experimental Therapeutics Fast Forward. Published on March 10, 2005 as DOI: 10.1124/jpet.104.082503.

N-substituted Piperazines Abused by Humans Mimic the Molecular Mechanism of 3,4-methylenedioxymethamphetamine (MDMA, or 'Ecstasy') 3,4-Methylenedioxy-methamphetamine (MDMA, or "Ecstasy") is an illicit drug that stimulates the release of serotonin (5-HT) and dopamine (DA) from neurons. Recent evidence reveals that drug users are ingesting piperazine analogs, like 1-benzylpiperazine (BZP, or "A2") and 1-(m-trifluoromethylphenyl)piperazine (TFMPP, or "Molly"), to mimic psychoactive effects of MDMA. In the present study, IRP scientists compared the neurochemistry of MDMA, BZP, and TFMPP in rats. The effects of MDMA, BZP, and TFMPP on transporter-mediated efflux of [3H]5-HT and [3H]MPP+ (DA transporter substrate) were determined in synaptosomes. The effects of drugs on extracellular levels of 5-HT and DA were examined using in vivo microdialysis in conscious rats. MDMA evoked transporter-mediated release of [3H]5-HT and [3H]MPP+. BZP released [3H]MPP+, whereas TFMPP was a selective releaser of [3H]5-HT. MDMA (1-3 mg/kg, i.v.) increased dialysate 5-HT and DA in a dose-related fashion, with actions on 5-HT being predominant. BZP (3-10 mg/kg, i.v.) elevated dialysate DA and 5-HT, while TFMPP (3-10 mg/kg, i.v.) elevated 5-HT. Administration of BZP plus TFMPP at a 1:1 ratio (BZP/TFMPP) produced parallel increases in dialysate 5-HT and DA; a 3 mg/kg dose of BZP/TFMPP mirrored the effects of MDMA. At a 10 mg/kg dose, BZP/TFMPP increased dialysate DA more than the summed effects of each drug alone, and some rats developed seizures. Results show that BZP/TFMPP and MDMA share the ability to evoke monoamine release, but dangerous drug-drug synergism may occur when piperazines are co-administered at high doses. Baumann, M.H., Clark, R.D., Budzynski, A.G., Partilla, J.S., Blough B.E. and Rothman, RB. N-substituted Piperazines Abused by Humans Mimic the Molecular Mechanism of 3,4-methylenedioxy-methamphetamine (MDMA, or 'Ecstasy'). Neuropsychopharmacology 30, pp. 550-560, 2005.

Evidence for Alterations in alpha2-adrenergic Receptor Sensitivity in Rats Exposed to Repeated Cocaine Administration It is well established that cocaine stimulates monoamine transmission by blocking reuptake of norepinephrine (NE), dopamine and serotonin into nerve cells, yet few investigations have addressed the effects of chronic cocaine on NE function. In the present study, IRP investigators examined the effects of repeated cocaine injections on neuroendocrine responses evoked by the alpha2-adrenergic receptor agonist, clonidine. Previous findings show that clonidine increases pituitary growth hormone (GH) secretion by a central mechanism involving postsynaptic alpha2-adrenergic receptors. Male rats previously fitted with indwelling jugular catheters received two daily injections of cocaine (15 mg/kg, i.p.) or saline for 7 days. At 42 h and 8 days after treatment, rats were challenged with clonidine (25 microg/kg, i.v.) or saline, and serial blood samples were withdrawn. Plasma GH and corticosterone levels were measured by radioimmunoassay. Prior cocaine exposure did not affect basal levels of either hormone. However, cocaine-pretreated rats displayed a significant reduction in clonidine-evoked GH secretion at 42 h, and this blunted response was still apparent 8 days later. Corticosterone responses produced by clonidine were similar regardless of pretreatment. The present data suggest that withdrawal from repeated cocaine injections may be accompanied by desensitization of postsynaptic alpha2-adrenoreceptors coupled to GH secretion. Since human patients with depression often exhibit blunted GH responses to clonidine, these findings provide evidence that cocaine withdrawal might produce depressive-like symptoms via dysregulation of NE mechanisms. Baumann, M.H., Milchanowski, A.B. and Rothman, R.B. Evidence for Alterations in alpha2-adrenergic Receptor Sensitivity in Rats Exposed to Repeated Cocaine Administration. Neuroscience, 125, pp. 683-690, 2004.

Opioid Peptide Receptor Studies. 17. Attenuation of Chronic Morphine Effects after Antisense Oligodeoxynucleotide Knock-down of RGS9 Protein in Cells Expressing the Cloned Mu Opioid Receptor RGS proteins are a recently described class of regulators that influence G-protein-mediated signaling pathways. IRP scientists have shown previously that chronic morphine results in functional uncoupling of the mu opioid receptor from its G protein in CHO cells expressing cloned human mu opioid receptors. In the present study, the authors examined the effects of morphine treatment (1 microM, 20 h) on DAMGO-stimulated high-affinity [35S]GTP-gamma-S binding and DAMGO-mediated inhibition of forskolin-stimulated cAMP accumulation in HN9.10 cells stably expressing the cloned rat mu opioid receptor, in the absence and presence of the RGS9 protein knock-down condition (confirmed by Western blot analysis). RGS9 protein expression was reduced by blocking its mRNA with an antisense oligodeoxynucleotide (AS-114). Binding surface analysis resolved two [35S]GTP-gamma-S binding sites (high affinity and low affinity sites). In sense-treated control cells, DAMGO-stimulated [35S]GTP-gamma-S binding by increasing the B(max) of the high-affinity site. In sense-treated morphine-treated cells, DAMGO-stimulated [35S]GTP-gamma-S binding by decreasing the high-affinity Kd without changing the B(max). AS-114 significantly inhibited chronic morphine-induced upregulation of adenylate cyclase activity and partially reversed chronic morphine effects as measured by DAMGO-stimulated [35S]GTP-gamma-S binding. Morphine treatment increased the EC50 (6.2-fold) for DAMGO-mediated inhibition of forskolin-stimulated cAMP activity in control cells but not in cells treated with AS-114 to knock-down RGS9. These results provide additional evidence for involvement of RGS9 protein in modulating opioid signaling, which may contribute to the development of morphine tolerance and dependence. Xu, H., Wang, X., Wang, J. and Rothman R.B. Opioid Peptide Receptor Studies. 17. Attenuation of Chronic Morphine Effects after Antisense Oligodeoxynucleotide Knock-down of RGS9 Protein in Cells Expressing the Cloned Mu Opioid Receptor. Synapse 52, pp. 209-217, 2004.

MRI Physics Unit, Neuroimaging Research Branch

Simultaneous MRI Acquisition of Blood Volume, Blood Flow and Blood Oxygenation Information during Brain Activation IRP investigators have developed a new functional MRI technique that is able to achieve concurrent acquisition of three hemodynamic images based primarily on the changes of cerebral blood volume, blood flow and blood oxygenation, respectively, associated with brain activation. The feasibility and efficacy of the new technique were assessed by brain activation experiments with visual stimulation paradigms. Experiments on healthy volunteers showed that this technique provided efficient image acquisition and thus higher contrast-to-noise ratio (CNR) per unit time, compared with conventional techniques collecting these functional images separately. In addition, it was demonstrated that the proposed technique was able to be utilized in event-related functional MRI experiments, with potential advantages of obtaining accurate transient information of the activation-induced hemodynamic responses. This new technique allows for efficient measurement of three complementary functional signals associated with brain activation, and provides a valuable tool to assist with data interpretation and functional transduction mechanisms. Yang, Y., Gu, H. and Stein, E.A. Magnetic Resonance Medicine, 52, pp. 1407-1417, 2004.

Mapping the Orientation of Intravoxel Crossing Fibers Based on the Phase Information of Diffusion Circular Spectrum IRP scientists have developed a new method to map the orientation of intravoxel crossing fibers by using the phase of the diffusion circular spectrum harmonics. In a previous paper, we demonstrated that the magnitude of the 4th order harmonic of the diffusion circular spectrum can be used to identify the existence of fiber crossings. However, the orientation of the intravoxel crossing fibers remained unknown. This study extends the previous approach so that it is able to identify the orientation of the intravoxel crossing fibers by utilizing the phase information of the circular spectrum. In general, the phase of the circular harmonic determines the rotation of the apparent diffusion coefficient (ADC) profile on the sampling circle that is spanned by the major and medium eigenvector of the diffusion tensor and thus can be used to determine the orientation of the crossing fibers. Results of simulations and in vivo experiments indicated that the estimated intravoxel crossing fibers are consistent with the orientations of the single fibers in surrounding tissues, significantly reducing the discontinuity of the fiber orientation field given by the conventional major eigenvector method. The proposed method provides important information on the white matter tracts in the fiber crossing area, and would be useful for improving accuracy in tractography. Zhan, W., Stein, E.A. and Yang, Y. NeuroImage, 23, pp. 1358-1369, 2004.

Medicinal Chemistry Section, Medications Discovery Research Branch

Novel Heterocyclic Trans Olefin Analogues of N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl}arylcarboxamides as Selective Probes with High Affinity for the Dopamine D3 Receptor Dopamine D3 receptor subtypes have been hypothesized to play a pivotal role in modulating the reinforcing and drug-seeking effects induced by cocaine. However, definitive pharmacological investigations have been hampered by the lack of highly D3 receptor selective compounds that can be used in vivo. To address this problem, the potent and D3 receptor selective antagonist NGB 2904 (9H-fluorene-2-carboxylic acid {4-[(2,3-dichlorophenyl)-piperazin-1-yl]-butyl}-amide, Ki (hD3)=2.0 nM; Ki (hD2L)=112 nM; D2/D3 selectivity ratio of 56) was chosen as a lead structure for chemical modification in an attempt to reduce its high lipophilicity (cLogD = 6.94) while optimizing D3 receptor binding affinity and D2/D3 selectivity. A series of >30 novel analogues were synthesized and their binding affinities were evaluated in competition binding assays in HEK 293 cells transfected with either D2L, D3 or D4 human dopamine receptors using the high affinity, selective D2-like receptor antagonist 125I-IABN. Structural diversity in the aryl amide end of the molecule was found to have a major influence on (sub)nanomolar D3 receptor affinity and D2/D3 selectivity, which was optimized using a more rigid trans-butenyl linker between the aryl amide and the piperazine. Several analogues demonstrated superior D3 receptor binding affinities and selectivities, as compared to the parent ligand. One compound, N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide, displayed the most promising pharmacological profile (Ki (hD3)=0.7 nM; Ki (hD2L)=93.3 nM; D2/D3 selectivity ratio of 133). In addition, this ligand inhibited quinpirole stimulation of mitogenesis at human dopamine D3 receptors transfected into Chinese hamster ovary (CHO) cells, with an EC50 of 3.0 nM. This compound was a nearly 5-times more potent antagonist at the D3 receptor than NGB 2904 (EC50=14.4 nM). Moreover, a decrease in cLogD value of ~2-orders of magnitude was determined for this novel D3 receptor preferring ligand, compared to NGB 2904. In summary, chemical modification of NGB 2904 has resulted in compounds with high affinity and selectivity for D3 receptors. The most promising candidate is currently being evaluated in animal models of cocaine abuse and will provide an important tool with which to elucidate the role of D3 receptors in drug reinforcement in vivo. Grundt, P., Carlson, E.E., Cao, J., Bennett, C.J., McElveen, E., Taylor, M., Luedtke, R.R. and Newman, A.H. Journal of Medicinal Chemistry 48, pp. 839-848, 2005.

Psychobiology Section, Medications Discovery Research Branch

Identification of a Dopamine Transporter Ligand that Blocks the Stimulant Effects of Cocaine Studies have indicated that the dopamine transporter (DAT) is the primary biological target of cocaine. Most drugs that have affinity for the DAT have behavioral effects like those of cocaine, suggesting that drugs targeting the DAT will all have abuse liability like cocaine. However, analogs of benztropine have high affinity for the DAT, and behavioral effects with varying degrees of similarity to cocaine. Among these compounds is the benztropine analog, JHW 007, which has high affinity for the DAT, but substantially reduced cocaine-like behavioral effects. The present study reports that JHW 007 antagonized the effects of cocaine in two animal models, locomotor stimulation and cocaine discrimination. JHW 007 occupied the DAT in vivo more slowly than did cocaine, and had not reached an apparent plateau up to 270 min after injection. The in vivo binding of cocaine to the DAT suggested rate of DAT occupancy as an important contributor to its behavioral effects, and the slow association with the DAT may provide an explanation for why JHW 007 is relatively devoid of cocaine-like behavioral effects. The antagonism of cocaine suggests that DAT ligands with reduced cocaine-like activity can function as cocaine antagonists and suggests JHW 007 as a lead for discovery of cocaine-abuse pharmacotherapeutics. Possibly more important, these findings indicate that the DAT is a suitable target for drug discovery efforts to identify treatments for cocaine abuse. Desai, R.I., Kopajtic, T.A., Koffarnus, K., Newman, A.H. and Katz, J.L. Identification of a Dopamine Transporter Ligand that Blocks the Stimulant Effects of Cocaine. The Journal of Neuroscience, 25, pp. 1889-1893, 2005.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Central Amygdala ERK Signaling Pathway is Critical to Incubation of Cocaine Craving Using a rat model of craving and relapse, IRP scientists previously found time-dependent increases in cocaine seeking induced by exposure to drug-associated cues over the first months of withdrawal from cocaine, suggesting that drug craving incubates over time. Here, we explored the role of amygdala extracellular signal-regulated kinases (ERK) signaling pathway in this incubation. Cocaine seeking induced by exposure to cocaine cues was substantially higher after 30 withdrawal days than after 1 day. Exposure to these cues increased ERK phosphorylation in the central, but not basolateral, amygdala after 30 days, but not 1 day, of withdrawal. After 30 days of withdrawal from cocaine, inhibition of central, but not basolateral, amygdala ERK phosphorylation decreased cocaine seeking. After 1 day of withdrawal, stimulation of central amygdala ERK phosphorylation increased cocaine seeking. Results suggest that time-dependent increases in the responsiveness of central amygdala ERK pathway to cocaine cues mediate the incubation of cocaine craving. Lu, L., Hope, B.T., Dempsey, J., Liu, S.Y., Bossert, J.M. and Shaham, Y. Nature Neuroscience 8, pp. 212-219, 2005.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Nicotine Induces Conditioned Place Preferences over a Large Range of Doses in Rats Conditioned place preference (CPP) procedures provide one measure of potential rewarding effects of abused drugs. Many attempts to induce CPP with nicotine have been unsuccessful. To assess the influence of nicotine dose and stimulus-assignment procedure on the development of nicotine-induced CPP. Initial preferences for one side of a two-compartment apparatus were first determined in Sprague-Dawley rats. In subsequent conditioning trials, the compartment paired with nicotine was the initially preferred side for half of the rats, and the initially non-preferred side for the other half. Rats received either an injection of nicotine (0.01-2 mg/kg SC) before being placed in one compartment (three trials) or saline before being placed in the other compartment (three trials). Control rats had saline injections associated with both compartments. A final test trial with no injection assessed final place preference. Significant CPP were induced by 0.1-1.4 mg/kg doses of nicotine. Nicotine-induced CPP were only apparent when nicotine was paired with the initially non-preferred side. Moreover, a very high dose of nicotine (2 mg/kg) induced conditioned place aversion when paired with the initially preferred side of the apparatus. Nicotine induced significant CPP across a wide range of doses, in accordance with its role as the primary addictive component of tobacco. Small preferences for one side of the apparatus played a major role in the development of nicotine-induced CPP. LeFoll, B. and Goldberg, S.R. Psychopharmacology (Berl), 178, pp. 481-492, 2005.

Human Cocaine-seeking Behavior and its Control by Drug-associated Stimuli in the Laboratory Second-order schedules of drug self-administration were developed to incorporate the effects of drug-related environmental stimuli into an animal model of drug abuse, making it more similar to human situations. Ironically, little is known about how human subjects behave under these schedules. In this study, human volunteers with a history of cocaine use worked on a second-order schedule in which every 100th lever response produced an auditory-visual brief stimulus (2 seconds). The first stimulus produced after 1 hour was extended to 10 seconds and paired with an intravenous injection of cocaine (25 mg). Up to three injections were allowed per session. In different phases of the experiment, presentation of the brief stimulus was discontinued and/or saline solution (placebo) was injected instead of cocaine. Injections of cocaine were found to maintain responding even when the brief stimulus was not presented. Placebo injections alone did not maintain responding. In contrast, the brief stimulus maintained high levels of responding under placebo conditions, even though self-reports indicated that subjects could clearly discriminate that they were not receiving cocaine. These results demonstrate that drug-related environmental stimuli can maintain persistent drug seeking during periods of drug unavailability. As this procedure directly measures the effects of stimuli on drug seeking, it may provide a valuable complement to indirect measures, such as self-reports of craving, that are often used with human subjects. The similarity of the response patterns in humans and animals also supports the use of second-order schedules in animals as a valid model of human drug seeking. Panlilio, L.V., Yasar, S., Nemeth-Coslett, R., Katz, J.L., Henningfield, J.E., Heishman, S. and Goldberg, S.R. Neuropsychopharmacology, 30, pp. 433-443, 2005.

Involvement of Adenosine A1 Receptors in the Discriminative-stimulus Effects of Caffeine in Rats Caffeine is a non-selective adenosine receptor antagonist in vitro, but involvement of different adenosine receptor subtypes, particularly adenosine A1 and A2A receptors, in the central effects of caffeine remains a matter of debate. The purpose of this study was to investigate the role of adenosine A1 and A2A receptors in the discriminative-stimulus effects of caffeine. Rats were trained to discriminate an injection of 30 mg/kg (i.p.) caffeine from saline. The selective A1 receptor antagonist CPT, the selective A2A receptor antagonist MSX-3 and the non-selective adenosine receptor antagonist DMPX were assessed for their ability to produce caffeine-like discriminative effects. The ability of CPT, MSX-3, the A1 receptor agonist CPA and the A2A receptor agonist CGS21680 to reduce the discriminative effects of caffeine was also tested. Radioligand binding experiments with membrane preparations from rat striatum and transfected mammalian cell lines were also performed to characterize binding affinity profiles of the different adenosine antagonists (caffeine, DMPX, CPT and MSX-3) in relation to all known adenosine receptors (A1, A2A, A2B, A3). DMPX and CPT, but not MSX-3, produced significant caffeine-like discriminative effects. MSX-3, but not CPT, markedly reduced the discriminative effects of caffeine and the caffeine-like discriminative effects of CPT. Furthermore, the A1 receptor agonist CPA, but not the A2A agonist CGS21680, reduced the discriminative effects of caffeine. Adenosine A1 receptor blockade is involved in the discriminative-stimulus effects of behaviorally relevant doses of caffeine; adenosine A2A receptor blockade does not play a central role in caffeine's discriminative effects and counteracts the A1 receptor-mediated discriminative-stimulus effects of caffeine. Solinas, M., Ferre, S., Antoniou, K., Quarta, D., Justinova, Z., Hockmeyer, J., Pappas, L., Segal, P. and Goldberg, S.R. Psychopharmacology (Berl), Feb 5, 2005. Epubmed ahead of print, PMID 15696333.

Role of Central and Peripheral Adenosine Receptors in the Cardiovascular Responses to Intraperitoneal Injections of Adenosine A1 and A2A Subtype Receptor Agonists The cardiovascular effects of the adenosine A1 receptor agonist N(6)-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of CGS 21680 were antagonized by i.p. injections of the adenosine A(2A) receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680.These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. The heart-rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery. Schindler, C.W, Karcz-Kubicha, M., Thorndike, E.B., Muller, C.E., Tella, S.R., Ferre, S. and Goldberg, S.R. British Journal of Pharmacology, Jan 24, 2005. Epubmed ahead of print, PMID 15678095.

Cannabinoid CB1 Antagonists as Promising New Medications for Drug Dependence This review examines the development of cannabinoid CB1 receptor antagonists as a new class of therapeutic agents for drug addiction. Abused drugs [alcohol, opiates, Delta(9)-tetrahydrocannabinol (THC), and psychostimulants, including nicotine] elicit a variety of chronically relapsing disorders by interacting with endogenous neural pathways in the brain. In particular, they share the common property of activating mesolimbic dopamine brain reward systems, and virtually all abused drugs elevate dopamine levels in the nucleus accumbens. Cannabinoid CB1 receptors are expressed in this brain reward circuit and modulate the dopamine-releasing effects of THC and nicotine. Rimonabant (SR141716), a CB1 receptor antagonist, blocks both the dopamine-releasing and the discriminative and rewarding effects of THC in animals. Blockade of CB1 receptor activity by genetic invalidation also decreases rewarding effects of opiates and alcohol in animals. Although CB1 receptor blockade is generally ineffective in reducing the self-administration of cocaine in rodents and primates, it reduces the reinstatement of extinguished cocaine-seeking behavior produced by cocaine-associated conditioned stimuli and cocaine-priming injections. Likewise, CB1 receptor blockade is effective in reducing nicotine-seeking behavior induced by re-exposure to nicotine-associated stimuli. Some of these findings have been recently validated in humans. In clinical trials, Rimonabant blocks the subjective effects of THC in humans and prevents relapse to smoking in ex-smokers. Findings from both clinical and preclinical studies suggest that ligands blocking CB1 receptors offer a novel approach for patients suffering from drug dependence that may be efficacious across different classes of abused drugs. LeFoll, B. and Goldberg, S.R. Journal of Pharmacology and Experimental Therapeutics, 312, pp. 875-883, 2005.

Involvement of Mu-, Delta- and Kappa-opioid Receptor Subtypes in the Discriminative-stimulus Effects of Delta-9-tetrahydrocannabinol (THC) in Rats Many behavioral effects of delta-9-tetrahydrocannabinol (THC), including its discriminative-stimulus effects, are modulated by endogenous opioid systems. To investigate opioid receptor subtypes involved in the discriminative effects of THC. Rats trained to discriminate 3 mg/kg i.p. of THC from vehicle using a two-lever, operant, drug-discrimination procedure, were tested with compounds that bind preferentially or selectively to either mu-, delta- or kappa-opioid receptors. The preferential mu-opioid receptor agonist heroin (0.3-1.0 mg/kg, i.p.), the selective delta-opioid receptor agonist SNC-80 (1-10 mg/kg, i.p.) and the selective kappa-opioid receptor agonist U50488 (1-10 mg/kg, i.p.) did not produce generalization to the discriminative effects of THC when given alone. However, heroin, but not SNC-80 or U50488, significantly shifted the dose-response curve for THC discrimination to the left. Also, the preferential mu-opioid receptor antagonist naltrexone (0.1-1 mg/kg, i.p.), the selective delta-opioid receptor antagonist, naltrindole (1-10 mg/kg, i.p.) and the kappa-opioid receptor antagonist nor-binaltorphimine (n-BNI, 5 mg/kg, s.c.), did not significantly reduce the discriminative effects of the training dose of THC. However, naltrexone, but not naltrindole or n-BNI, significantly shifted the dose-response curve for THC discrimination to the right. Finally, naltrexone, but not naltrindole or n-BNI, blocked the leftward shift in the dose-response curve for THC discrimination produced by heroin. mu- but not delta- or kappa-opioid receptors are involved in the discriminative effects of THC. Given the role that mu-opioid receptors play in THC's rewarding effects, the present findings suggest that discriminative-stimulus effects and rewarding effects of THC involve similar neural mechanisms. Solinas, M. and Goldberg, S.R. Psychopharmacology, Dec 24, 2004, Epubmed ahead of print, PMID 15619107.

Dopamine D3 Receptor Ligands Block Nicotine-induced Conditioned Place Preferences through a Mechanism that Does Not Involve Discriminative Stimulus or Antidepressant-like Effects Environmental stimuli previously paired with drug taking appear to play a critical role in nicotine dependence. Converging anatomical, pharmacological, and behavioral evidence implicates dopamine D3 receptors in the mechanisms underlying stimulus-controlled drug-seeking behavior. This study assessed the effects of BP 897, a dopamine D3 receptor partial agonist and ST 198, a dopamine D3 receptor antagonist, on nicotine-induced conditioned place preferences (CPPs), used as a measure of drug-seeking behavior, on discrimination performance under a two-lever-choice nicotine discrimination procedure and on food-maintained responding. BP 897 and ST 198 both blocked the expression of nicotine-induced CPP at doses selective for dopamine D3 receptors. They had no effect on locomotor activity in the CPP apparatus and no significant effect on nicotine discrimination performance or food-maintained responding under the discrimination procedure. Involvement of antidepressant actions in the effects of BP 897 and ST 198 on CPP is unlikely, since the authors found no effect of dopamine D3 receptor blockade with BP 897 or genetic depletion of dopamine D3 receptors in a forced-swimming test, used as a behavioral test for antidepressant activity. This suggests that dopamine D3 receptor ligands reduce the motivational effects of nicotine by a mechanism distinct from those of nicotine replacement therapy and bupropion, the two currently used aids for smoking cessation in humans. These findings support the use of dopamine D3 receptor ligands as aids for smoking cessation and indicate that their effects would be selective for those rewarding or reinforcing effects of nicotine that contribute to the maintenance of tobacco-smoking behavior, without affecting subjective responses to nicotine or producing any antidepressant-like effects. LeFoll, B., Sokoloff, P., Stark, H. and Goldberg, S.R. Neuropsychopharmacology 30, pp. 720-730, 2005.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

A Role of Ventral Tegmental Area Glutamate in Contextual Cue-induced Relapse to Heroin Seeking The environmental context previously associated with opiate use plays an important role in human relapse, but the neuronal mechanisms involved in context-induced drug relapse are not known. Using a rat relapse model, IRP researchers determined the effect of a group II metabotropic glutamate receptor agonist, LY379268, on contextual cue-induced reinstatement of heroin seeking. LY379268, which acts centrally to reduce evoked glutamate release, was injected systemically or directly into the ventral tegmental area (VTA), a brain area involved in opiate reward and conditioned drug effects. Rats were trained to self-administer intravenous heroin for 12 days; drug infusions were paired with a discrete tone-light cue. Subsequently, lever pressing was extinguished in the presence of the discrete cue in a context that differed from the drug self-administration context in terms of visual, auditory, tactile, and circadian cues. After extinction of lever responding, LY379268 was injected systemically or into the VTA, and non-reinforced responding was determined in the extinction context or the drug context. Exposure to the heroin-associated context induced robust reinstatement of drug seeking, and this effect was attenuated by systemic or intra-VTA injections of LY379268. Results indicate that glutamate transmission in the VTA plays an important role in contextual cue-induced relapse to heroin seeking. Bossert, J.M., Liu, S., Lu, L. and Shaham, Y. The Journal of Neuroscience, 24, pp. 10726-10730, 2004.

Clinical Pharmacology Section, Clinical Pharmacology and Therapeutics Research Branch

Strategies for Quitting among Non-treatment-seeking Marijuana Smokers Although marijuana is the most widely used illegal drug in the U.S., relatively few patients each year enter formal treatment for marijuana abuse. This study examined the self-reported quitting strategies used by 65 adult, non-treatment-seeking marijuana smokers who had made at least one (mean [SD] 4.9 [14.1]) serious quit attempt. Subjects rated their use and effectiveness of 13 strategies on the Marijuana Quit Questionnaire. The strategies clustered into three categories/factors, whether grouped by principal components analysis, mean helpfulness ratings, or frequency of endorsement: Change Environment, Seeking Organized/Professional Help, and Social Support. The most commonly used strategies were getting rid of all marijuana (28% of subjects), avoiding places where marijuana is used (26%), and avoiding association with marijuana users (25%). Changing one's environment was rated as most helpful, while seeking help from professionals was the least helpful. These strategies are similar to those reported by individuals trying to quit use of alcohol or tobacco. Clinicians who see marijuana users in their practice should consider incorporating these strategies into treatment plans for their marijuana-using patients. Boyd, S.J., Tashkin, D.P., Huestis, M.A., Heishman, S.J., Dermand, J.C., Simmons, M.S., and Gorelick, D.A. American Journal on Addictions, 14, pp. 35-42, 2005.


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