Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Platelet-derived Growth Factor-producing Cells Immortalized from Rat Mesencephalon with SV40 Large T Antigen Transduced by an AAV Vector Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here IRP investigators report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). AAV may be a useful vector for the transduction of oncogenes to produce cell lines. Phillips, A.W., Zhang, P., Truckenmiller, M.E., Keir, S.D., Bouvier, M., Tornatore, C., and Freed, W.J. Restorative Neurology and Neuroscience, 21, pp. 1-10, 2003.

Analysis of Microarray Data Using Z-score Transformation High-throughput cDNA microarray technology allows for the simultaneous analysis of gene expression levels for thousands of genes and, as such, rapid, relatively simple methods are needed to store, analyze, and cross-compare basic microarray data. The application of a classical method of data normalization, Z score transformation, provides a way of standardizing data across a wide range of experiments and allows the comparison of microarray data independent of the original hybridization intensities. Data normalized by Z score transformation can be used directly in the calculation of significant changes in gene expression between different samples and conditions. IRP investigators used Z scores to compare several different methods for predicting significant changes in gene expression including fold changes, Z ratios, Z and t statistical tests. The authors conclude that the Z score transformation normalization method accompanied by either Z ratios or Z tests for significance estimates offers a useful method for the basic analysis of microarray data. The results provided by these methods can be as rigorous and are no more arbitrary than other test methods, and, in addition, they have the advantage that they can be easily adapted to standard spreadsheet programs. Cheadle, C., Vawter, M.P., Freed, W.J., and Becker, K.G. Journal of Molecular Diagnostics, 5, pp. 73-81, 2003.

Cellular Pathobiology Unit, Development and Plasticity Section, Cellular Neurobiology Research Branch

Intracellular Dynamics of Sigma-1 Receptors (sigma1 binding sites) in NG108-15 Cells Sigma-1 receptors bind diverse kinds of psychoactive compounds including cocaine, and translocate upon stimulation by these compounds. However, the exact intracellular localization and dynamics of sigma-1 receptors have been unclear. IRP scientists recently found that sigma- 1 receptors specifically localize on cholesterol-enriched loci on the endoplasmic reticulum membrane that function as neutral lipid storage sites (i.e., ER lipid droplets or ER-LD) from which neutral lipids bud out to form cytosolic lipid droplets (c-LD). By combining immunocytochemistry and real-time monitoring of enhanced yellow fluorescent protein (EYFP)-tagged sigma-1 receptors (Sig-1R-EYFP) in living cells, authors characterized the sigma-1 receptor translocation in this study. (+) Pentazocine, a selective sigma-1 receptor agonist, causes a significant decrease of sigma-1 receptors in ER-LD and a diffused distribution of sigma-1 receptors over the entire endoplasmic reticulum reticular network in NG108- 15 cells. In the presence of sigma-1 receptor agonists, Sig-1R-EYFP move out from ER-LD and slide along the endoplasmic reticulum network toward nuclear envelope and the tip of neurites. Fluorescence recovery after photobleaching (FRAP) analysis demonstrates that Sig-1R- EYFP on endoplasmic reticulum reticular network are highly mobile compared to those in ER-LD. A sucrose gradient fractionation study shows that (+)pentazocine shifts sigma-1 receptors from ER-LD membranes to higher- density membranes. These results indicate that sigma-1 receptors localize on ER-LD and upon stimulation translocate on continuous endoplasmic reticulum reticular network toward peripheries of cells. Because sigma-1 receptors specifically target ER lipid storage sites and compartmentalize neutral lipids therein, these results suggest that sigma-1 receptors' dynamic translocation might affect lipid transport and distribution in neuronal cells. Hayashi, T. and Su, T.P. Journal of Pharmacology and Experimental Therapeutics, 306, pp. 726-733, 2003.

Sigma-1 Receptors (sigma1 binding sites) Form Raft- like Microdomains and Target Lipid Droplets on the Endoplasmic Reticulum: Roles in Endoplasmic Reticulum Lipid Compartmentalization and Export The brain sigma-1 receptors can bind neurosteroids and psychotropic drugs including neuroleptics and cocaine and are implicated in schizophrenia, depression and drug dependence. In this study, IRP investigators found that sigma-1 receptors specifically target lipid storage sites (lipid droplets) on the endoplasmic reticulum by forming a distinct class of lipid microdomains. Both endogenously expressing sigma-1 receptors and transfected C-terminally EYFP-tagged sigma-1 receptors (Sig-1R-EYFP) target unique "ring-like" structures associated with endoplasmic reticulum reticular networks in NG108-15 cells. The "ring- like" structures contain neutral lipids and are enlarged by the oleate treatment, indicating that they are endoplasmic reticulum-associated lipid droplets (ER-LD). Sigma-1 receptors colocalize with caveolin-2, a cholesterol-binding protein in lipid rafts on the ER-LD, but not with ADRP, a cytosolic lipid droplet (c-LD) specific protein. When the double-arginine ER retention signal on the N-terminus of sigma-1 receptors is truncated, sigma-1 receptors no longer exist on ER-LD, but predominantly target c-LD which contain ADRP. Sigma-1 receptors on ER-LD form detergent-resistant raft-like lipid microdomains, the buoyancy of which are different from those of plasma membrane lipid rafts. (+)Pentazocine causes sigma-1 receptors to disappear from the microdomains. N-terminally EYFP-tagged Sigma-1 receptors (EYFP-Sig-1R) failed to target ER-LD. EYFP-Sig-1R- transfected cells showed an unrestricted distribution of neutral lipids all over the endoplasmic reticulum network, decreases in c-LD and cholesterol in plasma membranes, and the bulbous aggregation of endoplasmic reticulum. Thus, sigma-1 receptors are unique endoplasmic reticulum proteins that regulate the compartmentalization of lipids on the endoplasmic reticulum and their export from the endoplasmic reticulum to plasma membrane and c- LD. Hayashi, T. and Su, T.P. Journal of Pharmacology and Experimental Therapeutics, 306, pp. 718-725, 2003.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Functional Tolerance and Blockade of Long-term Depression at Synapses in the Nucleus Accumbens after Chronic Cannabinoid Exposure The rewarding properties of the psychoactive constituents of marijuana, termed "cannabinoids," may reflect actions on synaptic transmission in the nucleus accumbens (NAc). Excitatory and inhibitory synapses are acutely inhibited by cannabinoids in the NAc, and endogenous cannabinoids play a critical role in the expression of long-term depression (LTD) of excitatory cortical afferents in this structure. To examine the impact of repeated cannabinoid exposure on synaptic processes in the NAc, rat brain slices were prepared for electrophysiological recordings following a 7 day treatment of the animals with vehicle solution, Delta9-tetrahydrocannabinol (THC), or the cannabinoid agonist WIN55,212-2. Extracellular glutamatergic postsynaptic potentials and whole-cell GABAergic IPSCs were concentration-dependently inhibited by WIN55,212-2 in slices from naive or vehicle-treated animals. However, the sensitivity to WIN55,212-2 was diminished in chronic agonist-treated animals. Cross-tolerance to the inhibitory effect of the mu-opioid agonist DAMGO was also observed. Endocannabinoid-mediated LTD was initiated via electrical stimulation (5 min, 10 Hz) of glutamatergic afferents to the NAc and was completely blocked by the cannabinoid receptor antagonist SR141716A in vehicle-treated animals. LTD was not observed in brain slices from rats chronically treated with Delta9-THC or WIN55,212-2. These data demonstrate that repeated exposure to cannabinoid agonists blocks synaptic plasticity in the NAc and reduces the sensitivity of GABAergic and glutamatergic synapses to both cannabinoids and opioids. Hoffman, A.F., Oz, M., Caulder, T., and Lupica, C.R. Journal of Neuroscience, 23, pp. 4815-4820, 2003.

Drugs of Abuse and Striatal Synaptic Plasticity Drug addiction can take control of the brain and behavior, activating behavioral patterns that are directed excessively and compulsively toward drug usage. Such patterns often involve the development of repetitive and nearly automatic behaviors that we call habits. The striatum, a subcortical brain region important for proper motor function as well as for the formation of behavioral habits, is a major target for drugs of abuse. Here, IRP scientists review recent studies of long-term synaptic plasticity in the striatum, emphasizing that drugs of abuse can exert pronounced influences on these processes, both in the striatum and in the dopaminergic midbrain. Synaptic plasticity in the ventral striatum appears to play a prominent role in early stages of drug use, whereas dopamine- and endocannabinoid-dependent synaptic plasticity in the dorsal striatum could contribute to the formation of persistent drug-related habits when casual drug use progresses towards compulsive drug use and addiction. Gerdeman, G.L., Partridge, J.G., Lupica, C.R., and Lovinger, D.M. Trends in Neuroscience, 26, pp. 184-192, 2003.

Beta-Funaltrexamine, a Gauge for the Recognition Site of Wildtype and Mutant H297Q mu-opioid Receptors The antagonist beta-funaltrexamine (beta-FNA), known to bind covalently to mu-opioid receptors by a two-step, doubly discriminating sequence, was used as a sensitive gauge to compare wildtype to mutant H297Q mu-opioid receptors. IRP researchers addressed whether this mutation, which enhances the intrinsic activities of alkaloid mu-receptor agents, affects both the reversible and covalent phases of beta-FNA binding. Such altered binding serves as a reporter for the dimensions and topography of the receptor's recognition site. Using the voltage-clamped Xenopus oocyte expression system with coexpressed GIRK potassium channels, we found that beta-FNA blocked the wildtype and mutant H297Q receptors both reversibly and irreversibly, indicating overall conserved tertiary structure in the mutant. The mutant H297Q receptor, however, was more resistant to both phases of blockade, indicating some disturbance of the mutant H297Q receptor recognition site. Beta-FNA acted as a partial agonist at the mutant H297Q receptor expressed in both oocytes, as measured by the activation of GIRK channels, and in COS-7 cells assayed by GTPgamma(35)S binding. Beta-FNA showed no activity at the wildtype receptor expressed in oocytes, but surprisingly induced binding of GTPgamma(35)S in transfected COS-7 cells. Thus, the topography of the mutant H297Q receptor recognition site is sufficiently conserved to allow the selective binding of beta-FNA, but the decrease in binding affinity and increase in efficacy in oocytes demonstrates clear differences from the wildtype receptor. Spivak, C.E., Beglan, C.L., Zollner, C. and Surratt. C.K. Synapse, 49, pp. 55-60, 2003.

Effects of Extracellular Sodium on mu-opioid Receptors Coupled to Potassium Channels Coexpressed in Xenopus Oocytes Wild-type or mutant H297N or H297Q of the mu-opioid receptor were co-expressed with the inwardly rectifying potassium channel GIRK1 in oocytes from Xenopus laevis. Under voltage clamp, pairs of concentration response curves were generated using the agonist normorphine in a bathing medium containing 38.5 mM sodium or an identical medium in which the sodium was replaced by an equimolar concentration of choline. The maximum currents were greater in the presence of sodium by about 30% at wild-type receptors and by about 100% at the mutant receptors. The EC(50) values tended to increase somewhat as well, though these differences reached statistical significance only for the mutant H297Q. Flame photometry detected no change in the intracellular sodium or potassium concentrations of oocytes, suggesting that the effect of sodium was solely extracellular. Thus sodium, long known for its effects on in vitro ligand binding at mu-opioid receptors, also affects overall transduction as revealed in the Xenopus oocyte model of a complete, living cell system. Oz, M. and Spivak, C.E. Pflugers Archives, 445, pp. 716-720, 2003.

Molecular Neuropsychiatry Research Branch

Frontal Cortical Tissue Composition in Abstinent Cocaine Abusers: A Magnetic Resonance Imaging Study Cocaine abusers exhibit impairment of executive cognitive functions that are mediated by the frontal cortex. This work tested for structural (i.e., tissue composition) abnormalities that may underlie such performance deficits. Research participants were cocaine abusers (n = 14) abstinent for 20 days and a non-drug-using comparison group (n = 11), who underwent magnetic resonance imaging (T1-weighted scans of the brain). Gray matter and white matter tissue densities were determined using voxel-based morphometry with small volume correction based on a priori hypotheses derived from functional imaging of the same subjects. Cocaine abusers had significantly lower gray matter tissue density than did the non drug users in 10 of 13 small volumes analyzed in the frontal cortex [bilateral anterior cingulate gyrus (infragenual and perigenual regions) and medial orbitofrontal cortex and the lateral orbitofrontal cortex and middle/dorsal cingulate gyrus in the right hemisphere]. No group differences were found in white matter density of the frontal cortex. These results extend the authors' previous findings of defective frontal cortical activation (indexed by cerebral blood flow) in cocaine abusers to include abnormalities in gray matter tissue density in the same frontal cortical regions. + Neuroimage, 19, pp. 1095-102, 2003.

Orbitofrontal Cortex Dysfunction in Abstinent Cocaine Abusers Performing a Decision-making Task Cocaine abusers demonstrate faulty decision-making as manifested by their inability to discontinue self-destructive drug-seeking behaviors. The orbitofrontal cortex (OFC) plays an important role in decision-making. In this preliminary study IRP investigators tested whether 25-day-abstinent cocaine abusers show alterations in normalized cerebral blood flow (rCBF) in the OFC using PET with (15)O during the Iowa Gambling Task (a decision-making task). This task measures the ability to weigh short-term rewards against long-term losses. A control task matched the sensorimotor aspects of the task but did not require decision-making. Cocaine abusers (N = 13) showed greater activation during performance of the Iowa Gambling Task in the right OFC and less activation in the right dorsolateral prefrontal cortex (DLPFC) and left medial prefrontal cortex (MPFC) compared to a control group (N = 13). Better Iowa Gambling Task performance was associated with greater activation in the right OFC in both groups. Also, the amount of cocaine used (grams/week) prior to the 25 days of enforced abstinence was negatively correlated with activation in the left OFC. Greater activation in the OFC in cocaine abusers compared to a control group may reflect differences in the anticipation of reward while less activation in the DLPFC and MPFC may reflect differences in planning and working memory. These findings suggest that cocaine abusers show persistent functional abnormalities in prefrontal neural networks involved in decision-making and these effects are related to cocaine abuse. Compromised decision-making could contribute to the development of addiction and undermine attempts at abstinence. Bolla, K.I., Eldreth, D.A., London, E.D., Kiehl, K.A., Mouratidis, M., Contoreggi, C., Matochik, J.A., Kurian, V., Cadet, J.L., Kimes, A.S., Funderburk, F.R. and Ernst, M. Neuroimage, 19, pp. 1085-1094, 2003.

EEG Deficits in Chronic Marijuana Abusers During Monitored Abstinence: Preliminary Findings Cognitive, cerebrovascular, and psychiatric impairments have been documented with chronic marijuana users. To better understand the nature and duration of these neurocognitive changes in marijuana abusers, IRP scientists recorded the resting EEG of 29 abstinent chronic marijuana abusers and 21 control subjects. The marijuana abusers were tested twice: the first evaluation occurred within 72 hours of admission to the inpatient research unit; the second evaluation occurred after 28 to 30 days of monitored abstinence. A three-minute period of EEG was recorded during resting eyes-closed conditions from eight electrodes (F(3), C(3), P(3), O(1), F(4), C(4), P(4), and O(2)). The artifacted EEG was converted to six frequency bands (delta, theta, alpha(1), alpha(2), beta(1), and beta(2)) using a fast Fourier transform. During early abstinence, absolute power was significantly lower (p < 0.05) for the marijuana abusers than for the control subjects for the theta and alpha(1) bands. These reductions in theta and alpha(1) power persisted for 28 days of monitored abstinence. These EEG changes, together with cerebral blood flow deficits, might underlie the cognitive alterations observed in marijuana abusers. Additional research is needed to determine how long these deficits persist during abstinence and if treatment with neuroprotective agents may reverse them. Herning, R.I., Better, W., Tate, K. and Cadet, J.L. Annals of the New York Academy of Sciences, 993, pp. 75-78, 2003.

Methylenedioxymethamphetamine (MDMA, Ecstasy) Neurotoxicity: Cellular and Molecular Mechanisms Methylenedioxymethamphetamine (MDMA, Ecstasy) is a very popular drug of abuse. This has led to new intense concerns relevant to its nefarious neuropsychiatric effects. These adverse events might be related to the neurotoxic effects of the drug. Although the mechanisms of MDMA-induced neurotoxicity remain to be fully characterized, exposure to the drug can cause acute and long-term neurotoxic effects in animals and nonhuman primates. Recent studies have also documented possible toxic effects in the developing fetus. Nevertheless, there is still much debate concerning the effects of the drug in humans and how to best extrapolate animal and nonhuman primate data to the human condition. Herein, IRP researchers review the evidence documenting the adverse effects of the drug in some animal models. The authors also discuss possible mechanisms for the development of MDMA neurotoxicity. Data supporting deleterious effects of this drug on the developing fetus are also described. Much remains to be done in order to clarify the molecular and biochemical pathways involved in the long-term neuroplastic changes associated with MDMA abuse. Lyles, J. and Cadet, J.L. Brain Research Review, 42, pp. 155-168, 2003.

Molecular Neurobiology Section, Molecular Neurobiology Branch

New Findings for Human Dopamine Transporters DAT is a principal site for cocaine reward, yet its human variants and regulation have not been well understood. In the first of these two reports, IRP scientists have described large functional effects of DAT variants found in humans, including a nearly dominant-negative effect that causes efffects on the mutant and on wildtype DAT coexpressed with mutant DAT. In the second, the authors followed up on their initial descriptions of DAT regulation by phosphorylation by elucidating specific DAT sites that are required for phosphoregulation mediated by distinct pathways. Lin, Z. and Uhl, G.R. Pharmacogenomics Journal, 3, pp. 159-168, 2003; Lin, Z., Zhang, P.W., Zhu, X., Melgari, J.M., Huff, R., Spieldoch, R.L. and Uhl, G.R. Journal of Biological Chemistry, 278, pp. 20162-20170, 2003.

Effects of Gene Variants on Reward Derived from Addictive Substances Mouse models are helping to elucidate effects of gene variants on reward elicited by addictive substances. These models can assist in understanding effects of human allelic variants in drug reward. The human BDNF locus is near markers identified in other studies of human drug abuse vulnerability. IRP scientists have also identified human allelic variants at the DAT and VMAT2 locus that influence addiction vulnerabilities. These papers describe effects of mouse models of these human allelic variants on reward elicited by stimulants and ethanol. They also describe the limitations of acute drug reward models for fully understanding effects of human allelic variants that influence vulnerability to drug dependence in humans. Hall, F.S., Sora, I. and Uhl, G.R. Neuropsychopharmacology, 28, pp. 620-628, 2003.

Gene Expression Changes with Behaviorally-Relevant Doses of Amphetamine Studying changes in gene expression provides a good window for enhancing understanding of the neuroadaptations that drug exposure causes in the brain, including those important for addiction. Little information has been available concerning the constellations of genes whose expression is changed by acute- and chronic administration of rewarding and sensitizing doses of amphetamine, although work reported after this paper was submitted describes changes that follow neurotoxic amphetamine doses. The surprisingly-widespread changes in acutely- and chronically regulated genes described here provide a powerful indication of the widespread impact that stimulant administration has on the brain. Sokolov, B.P., Polesskaya, O.O. and Uhl, G.R. Journal of Neurochemistry, 84, pp. 244-252, 2003.

Psychobiology Section, Medications Discovery Research Branch

The Validity of the Reinstatement Model of Craving and Relapse to Drug Use Several types of models used in psychopharmacology are described as conforming to definitions of formal-equivalence, correlational, and functional-equivalence models. The means of validation of each of these types of models are described. The "reinstatement" model is evaluated with regard to which type of model it is, and whether its validity has been adequately established. This assessment is conducted within the context of both preclinical and clinical research and findings. It is concluded that the model is used differently by various investigators, and thus has characteristics of each type. It is further concluded that the model possesses some face validity, as it has some similarity in form to relapse. However, the model falls short of being an established valid model when more rigorous criteria of predictive and functional validity are applied. Several areas for further research are illuminated through this analysis. Katz, J.L. and Higgins, S.T. Psychopharmacology, 168, pp. 21-30, 2003.

Medicinal Chemistry Section, Medications Discovery Research Branch

Dual Probes for the Dopamine Transporter and s1 Receptors: Novel Piperazinyl alkyl-bis-(4'-fluorophenyl)amine Analogues as Potential Cocaine-Abuse Therapeutic Agents Both dopamine uptake inhibitors and s1 receptor antagonists have been implicated as potential pharmacotherapeutics for the treatment of cocaine abuse. While the dopamine uptake inhibitors may share with cocaine neurochemical mechanisms underlying reinforcing properties, s1 antagonists have been shown to attenuate some behavioral actions and toxic side effects associated with cocaine overdose. Rimcazole, a s1 receptor antagonist that binds to the dopamine transporter (DAT; Ki =224 nM), is not behaviorally cocaine-like and attenuates some of the behavioral actions of cocaine. In order to determine the roles of both DAT and s1 receptors in the behavioral actions of rimcazole, a series of analogues was synthesized. Initial studies identified two analogues that showed high to moderate affinities for both DAT and s-1 receptors and failed to show cocaine-like discriminative stimulus (DS) effects. A second series of bis-(4'-fluorophenyl)amine analogues have now been prepared in which the most potent DAT compound, (Ki=8.5 nM) was selective over serotonin transporter (SERT/DAT=94), norepinephrine transporter (NET/DAT=63) and s1 receptor binding (s1/DAT =44). In addition, two other analogues showed superior selectivity for DAT over SERT (170- and 140-fold, respectively) and DAT over NET (219- and 190-fold, respectively) but were essentially equipotent at DAT and s1 receptors. Comparative Molecular Field Analysis studies at both DAT and s1 receptors were performed to examine structural requirements for optimal binding at these two targets as well as to assess differences between them. Behavioral evaluation of analogues with varying affinities for both DAT and s1 receptors may provide a novel approach toward designing medications for cocaine abuse. Cao, J., Kulkarni, S.S., Husbands, S.M., Bowen, W.D., Williams, W., Kopajtic, T., George, C. and Newman, A.H. Journal of Medicinal Chemistry, 46, pp. 2589-2598, 2003.

N-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butyl, butenyl and butynyl}arylcarboxamides as Novel Dopamine D3 Receptor Antagonists The dopamine D3 receptor subtype has been targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. Previous synthetic studies provided structural requirements for high affinity binding to D3 receptors, which included a 2,3-dichloro-phenylpiperazine linked to an arylamido function via a butyl chain. To reduce lipophilicity of these agents and further investigate optimal conformation, a second series of 15 novel ligands was designed that included heteroaromatic substitution and unsaturated alkyl linkers. These compounds were synthesized and evaluated for binding at rat D3 and D2 receptors stably expressed in Sf9 cells. D3 binding affinities ranged from Ki = 0.6 - 1080 nM, with a broad range of D3/D2 selectivities (2-97). The discovery of potent, selective and bioavailable D3 receptor ligands will provide essential molecular probes to elucidate the role D3 receptors play in the psychomotor stimulant and reinforcing effects of cocaine. Newman, A.H., Cao, J., Bennett, C.J., Robarge, M.J., George, C., Freeman, R. and Luedtke, R. Bioorganic Medicinal Chemistry Letters, 13, pp. 2179-2183, 2003.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

Molecular Neuroadaptations in the Accumbens and Ventral Tegmental Area During the First 90 Days of Forced Abstinence from Cocaine Self-administration in Rats Cocaine self-administration is associated with a propensity to relapse in humans and reinstatement of drug seeking in rats after prolonged withdrawal periods. These behaviors are hypothesized to be mediated by molecular neuroadaptations within the mesolimbic dopamine system. However, in most studies of drug-induced neuroadaptations, cocaine was experimenter-delivered and molecular measurements were performed after short withdrawal periods. In the present study, rats were trained to self-administer intravenous cocaine or oral sucrose (a control non-drug reward) for 10 days (6-h/day) and were sacrificed following 1, 30, or 90 days of reward withdrawal. Tissues from the accumbens and ventral tegmental area (VTA) were assayed for candidate molecular neuroadaptations, including enzyme activities of cAMP-dependent protein kinase (PKA) and adenylate cyclase (AC), and protein expression of cyclin-dependent kinase 5 (cdk5), tyrosine hydroxylase (TH) and glutamate receptor subunits (GluR1, GluR2, and NMDAR1). In the accumbens of cocaine-trained rats, GluR1 and NMDAR1 levels were increased on days 1 and 90, while GluR2 levels were increased on days 1 and 30, but not day 90; PKA activity levels were increased on days 1 and 30, but not day 90, while AC activity, TH, and cdk5 levels were unaltered. In the VTA of cocaine-trained rats, NMDAR1 levels were increased for up to 90 days, while GluR2 levels were increased only on day 1; TH and cdk5 levels were increased only on day 1, while PKA and AC activity levels were unaltered. Cocaine self-administration produces long-lasting molecular neuroadaptations in the VTA and accumbens that may underlie cocaine relapse during periods of abstinence. Lu, L., Grimm, J.W., Shaham, Y. and Hope, B.T. Journal of Neurochemistry, 85, pp. 1604--1613, 2003.

The Reinstatement Model of Drug Relapse: History, Methodology and Major Findings The reinstatement model is currently used in many laboratories to investigate mechanisms underlying relapse to drug seeking. Here IRP investigators review briefly the history of the model and describe the different procedures that have been used to study the phenomenon of reinstatement of drug seeking. The results from studies using pharmacological and neuroanatomical techniques to determine the neuronal events that mediate reinstatement of heroin, cocaine and alcohol seeking by acute priming injections of drugs, drug-associated cues and environmental stressors are summarized. In addition, several issues are discussed, including (1) the concordance between the neuronal mechanisms involved in drug-induced reinstatement and those involved in drug reward and discrimination, (2) the role of drug withdrawal states and periods in reinstatement of drug seeking, (3) the role of neuronal adaptations induced by exposure to drugs in relapse, and (4) the degree to which the rat reinstatement model provides a suitable preclinical model of relapse to drug taking. The data derived from studies using the reinstatement model suggest that: the neuronal events that mediate drug-, cue- and stress-induced reinstatement of drug seeking are not identical; that the mechanisms underlying drug-induced reinstatement are to some degree different from those mediating drug discrimination or reward; and that the duration of the withdrawal period following cocaine and heroin self-administration has a profound effect on reinstatement induced by drug cues and stress. Finally, there appears to be a good correspondence between the events that induce reinstatement in laboratory animals and those that provoke relapse in humans. Shaham, Y., Shalev, U., Lu, L., de Wit, H. and Stewart, J. Psychopharmacology, 168, pp. 3-20, 2003.

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

Reliability and Validity of the Tobacco Craving Questionnaire This study documented the initial reliability and validity of the Tobacco Craving Questionnaire (TCQ), a new multidimensional questionnaire to assess tobacco craving. Current cigarette smokers (n = 213) not attempting to reduce or quit smoking completed the 47-item TCQ and other forms assessing demographics, tobacco and other drug use history, quit attempts, and current mood. Exploratory factor analyses and structural equation modeling indicated that a four-factor solution best described the item structure. Factor subscales derived from the 17-items with significant loadings had low to high internal consistencies and inter-item correlations and exhibited low to moderate, positive intercorrelations. Factor scales were significantly correlated with single-item measures of craving, current mood, and daily cigarette smoking. Results indicated that four specific constructs characterized craving for tobacco: 1) emotionality, smoking in anticipation of relief from withdrawal or negative mood; 2) expectancy, anticipation of positive outcomes from smoking; 3) compulsivity, an inability to control tobacco use; and 4) purposefulness, intention and planning to smoke for positive outcomes. These preliminary data suggest that the TCQ is a reliable and valid instrument for assessing tobacco craving in individuals not attempting to reduce or quit smoking. Heishman, S.J., Singleton, E.G. and Moolchan, E.T. Nicotine and Tobacco Research, 5, pp. 1-10, 2003.