Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Stable Expression of hrGFP by Mouse Embryonic Stem Cells: Promoter Activity in the Undifferentiated State and During Dopaminergic Neural Differentiation Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors. Zeng, X. Chen, J., Sanchez, J.F., Coggiano, M., Dillon-Carter, O., Petersen, J., and Freed, W.J. Stem Cells, 21(6), pp. 647-653, 2003.

The Use of Microarrays to Characterize Neuropsychiatric Disorders: Postmortem Studies of Substance Abuse and Schizophrenia Neuropsychiatric disorders are generally diagnosed based on a classification of behavioral and, in some cases, specific neurological deficits. The lack of distinct quantitative and qualitative biological descriptors at the anatomical and cellular level complicates the search for and understanding of the neurobiology of these disorders. The advent of microarray technology has enabled large-scale profiling of transcriptional activity, allowing a comprehensive characterization of transcriptional patterns relating to the pathophysiology of neuropsychiatric disorders. Authors review some of the unique methodological constraints related to the use of human postmortem brain tissue in addition to the generally applicable requirements for microarray experiments. Microarray studies undertaken in neuropsychiatric disorders such as schizophrenia and substance abuse by the use of postmortem brain tissue indicate that transcriptional changes relating to synaptic function and plasticity, cytoskeletal function, energy metabolism, oligodendrocytes, and distinct intracellular signaling pathways are generally present. These have been supported by microarray studies in experimental models, and have produced multiple avenues to be explored at the functional level. The quality and specificity of information obtained from human postmortem tissue is rapidly increasing with the maturation and refinement of array-related methodologies and analysis tools, and with the use of focused cell populations. The development of experimental models of gene regulation in these disorders will serve as the initial step towards a comprehensive genome-linked analysis of the brain and associated disorders, and help characterize the integration and coordinate regulation of complex functions within the CNS. Lehrmann, E., Hyde, T.M., Vawter, M.P., Becker, K.G., Kleinman, J.E., and Freed, W.J. Current Molecular Medicine, 3(5), pp. 437-446, 2003.

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

Understanding the Molecular Mechanism of Sigma-1 Receptors: Towards a Hypothesis that Sigma-1 Receptors are Intracellular Amplifiers for Signal Transduction Although sigma receptors were discovered in 1982, the biochemical and physiological roles of sigma receptors have just begun to unveil. Sigma receptors are non-opioid, non-phencyclidine receptors that contain two subtypes: sigma-1 and sigma-2 receptors. The sigma-1 receptor has been cloned and its sequence does not resemble that of any mammalian protein. This review will be on sigma-1 receptors. Sigma-1 receptors contain 223 amino acids and reside primarily at the endoplasmic reticulum. Sigma-1 receptors exist mainly in the central nervous system, but also in the periphery. Sigma-1 receptor ligands include cocaine, (+)-benzomorphans like (+)-pentazocine and (+)N-allyl-normetazocine (or (+)-SKF-10047), and endogenous neurosteroids like progesterone and pregnenolone sulfate. Many pharmacological and physiological actions have been attributed to sigma-1 receptors. These include the regulation of IP3 receptors and calcium signaling at the endoplasmic reticulum, mobilization of cytoskeletal adaptor proteins, modulation of nerve growth factor-induced neurite sprouting, modulation of neurotransmitter release and neuronal firing, modulation of potassium channels as a regulatory subunit, alteration of psychostimulant-induced gene expression, and blockade of spreading depression. Behaviorally, sigma-1 receptors are involved in learning and memory, psychostimulant-induced sensitization, cocaine-induced conditioned place preference, and pain perception. Notably, in almost all the aforementioned biochemical and behavioral tests, sigma-1 agonists, while having no effects by themselves, caused the amplification of signal transductions incurred upon the stimulation of the glutamatergic, dopaminergic, IP3-related metabotropic, or nerve growth factor-related systems. Thus, it is hypothesized that sigma-1 receptors, at least in part, are intracellular amplifiers creating a supersensitized state for signal transduction in the biological system. Su, T.P. and Hayashi, T. Current Medical Chemistry, 10, pp. 2073-2080, 2003.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Direct Activation by Dopamine of Recombinant Human 5-HT1A Receptors: Comparison with Human 5-HT2C and 5-HT3 Receptors The effects of dopamine (DA) on the function of human 5-HT(1A) receptors expressed in Xenopus oocytes and CHO-K1 cells were investigated. In addition, the effect of DA on the activation of three different types of human 5-HT receptors (5-HT(1A), 5-HT(2C), and 5-HT(3)) were studied comparatively. In oocytes coexpressing 5-HT(1A) receptors and G-protein-activated potassium channels (GIRK1), 5-HT or DA induced currents with respective EC(50) of 4.2 nM and 11.2 microM. Maximal responses induced by DA were 85 +/- 4% of 5-HT currents and blocked by 5-HT(1A) antagonist, WAY-100635. In CHO-K1 cells expressing 5-HT(1A) receptors, 5-HT and DA inhibited the specific binding of [(3)H]-8-OH-DPAT with IC(50) of 10.2 nM and 1.4 microM, and both 5-HT and DA inhibited the forskolin-induced cAMP accumulation. In oocytes expressing 5-HT(2C) receptors, 5-HT and DA induced currents with respective EC(50) of 6.2 nM and 67.7 microM. Maximal DA responses were 42 +/- 3% of 5-HT currents and blocked by the 5-HT(2) antagonist, piperazine. In oocytes expressing 5-HT(3) receptors, 5-HT and DA induced currents with respective EC(50) of 2.1 microM and 266.3 microM. Maximal DA responses were 37 +/- 3% of 5-HT responses and blocked by 5-HT(3) antagonist LY-278584. Results indicated that the relative potency of DA increased in the order of 5-HT(3) > 5-HT(1A) > 5-HT(2C), and relative efficacy increased in the order of 5-HT(1A) > 5-HT(2C) > 5-HT(3). Results suggest that although DA activates different subtypes of human 5-HT receptors directly, the potency and efficacy of the binding site varies significantly among different receptors. Oz, M., Zhang, L., Rotondo, A., Sun, H., and Morales, M. Synapse, 50, pp. 303-313, 2003.

Diadenosine Tetraphosphate Protects Against Injuries Induced by Ischemia and 6-Hydroxydopamine in Rat Brain Diadenosine tetraphosphate (AP4A), an endogenous diadenosine polyphosphate, reduces ischemic injury in the heart. In this study, IRP investigators report the potent and protective effects of AP4A in rodent models of stroke and Parkinson's disease. AP4A, given intracerebroventricularly before middle cerebral artery (MCA) ligation, reduced cerebral infarction size and enhanced locomotor activity in adult rats. The intravenous administration of AP4A also induced protection when given early after MCA ligation. AP4A suppressed terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) induced by hypoxia/reperfusion in primary cortical cultures, and reduced both ischemia-induced translocation of mitochondrial cytochrome c and the increase in cytoplasmic caspase-3 activity In vivo. The purinergic P2/P4 antagonist di-inosine pentaphosphate or P1-receptor antagonist sulfonylphenyl theophylline, but not the P2-receptor antagonist suramin, antagonized the effect of AP4A, suggesting that the observed protection is mediated through an anti-apoptotic mechanism and the activation of P1- and P4-purinergic receptors. AP4A also afforded protection from toxicity induced by unilateral medial forebrain bundle injection of 6-hydroxydopamine (6-OHDA). One month after lesioning, vehicle-treated rats exhibited amphetamine-induced rotation. Minimal tyrosine hydroxylase immunoreactivity was detected in the lesioned nigra or striatum. No KCl-induced dopamine release was found in the lesioned striatum. All of these indices of dopaminergic degeneration were attenuated by pretreatment with AP4A. In addition, AP4A reduced TUNEL in the lesioned nigra 2 d after 6-OHDA administration. Collectively, these data suggest that AP4A is protective against neuronal injuries induced by ischemia or 6-OHDA through the inhibition of apoptosis. The authors propose that AP4A may be a potentially useful target molecule in the therapy of stroke and Parkinson's disease. Wang, Y., Chang, C.F., Morales, M., Chiang, Y.H., Harvey, B.K., Su, T.P., Tsao, L.I., Chen, S., and Thiemermann, C. Journal of Neuroscience, 23(21), pp. 7958-7965, 2003.

HSV Amplicon Delivery of Glial Cell Line-Derived Neurotrophic Factor is Neuroprotective Against Ischemic Injury Direct intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) is neuroprotective against ischemia-induced cerebral injury. Utilizing viral vectors to deliver and express therapeutic genes presents an opportunity to produce GDNF within localized regions of an evolving infarct. We investigated whether a herpes simplex virus (HSV) amplicon-based vector encoding GDNF (HSVgdnf) would protect neurons against ischemic injury. In primary cortical cultures HSVgdnf reduced oxidant-induced injury compared to the control vector HSVlac. To test protective effects In vivo, HSVgdnf or HSVlac was injected into the cerebral cortex 4 days prior to, or 3 days, after a 60-min unilateral occlusion of the middle cerebral artery. Control stroke animals developed bradykinesia and motor asymmetry; pretreatment with HSVgdnf significantly reduced such motor deficits. Animals receiving HSVlac or HSVgdnf after the ischemic insult did not exhibit any behavioral improvement. Histological analyses performed 1 month after stroke revealed a reduction in ischemic tissue loss in rats pretreated with HSVgdnf. Similarly, these animals exhibited less immunostaining for glial fibrillary acidic protein and the apoptotic marker caspase-3. Taken together, these data indicate that HSVgdnf pretreatment provides protection against cerebral ischemia and supports the utilization of the HSV amplicon for therapeutic delivery of trophic factors to the CNS. Harvey, B.K., Chang, C.F., Chiang, Y.H., Bowers, W.J., Morales, M., Hoffer, B.J., Wang, Y., and Federoff, H.J. Experimental Neurology, 183(1), pp. 47-55, 2003.

Long-term Exposure to the Active ingredient in Marijuana, D9-tetrahydrocannabinol, Causes Tolerance at the CB1 Receptor and Blocks Long-term Synaptic Plasticity in the Nucleus Accumbens Prior studies have demonstrated direct actions of cannabinoid receptor agonists in brain slices containing the nucleus accumbens. These studies demonstrated that activation of cannabinoid CB1 receptors inhibited GABA release in the nucleus accumbens entirely by a presynaptic mechanism (Hoffman and Lupica, J. Neurophysiology, 85: 72-83). The present study extends these findings by demonstrating that chronic Δ⁹-THC exposure caused marked tolerance to these presynaptic effects of cannabinoids. In these experiments, electrophysiological recordings were performed in nucleus accumbens brain slices prepared from rats following 7 days treatment with Δ⁹-THC or the synthetic cannabinoid agonist, WIN 55,212-2. Investigators found that tolerance developed to the acute inhibitory effects of WIN 55,212-2 at both glutamatergic and GABAergic synapses in the nucleus accumbens following chronic Δ⁹-THC. This finding is significant because it represents the first time that physiological tolerance to cannabinoids at defined synapses in the CNS has been described. Another consequence of long-term exposure to Δ⁹-THC was that a specific form of synaptic plasticity, known as long-term depression (LTD) of glutamatergic synaptic transmission, was completely blocked in the nucleus accumbens of rats chronically treated with Δ⁹-THC, but not in vehicle controls. Since LTD is critically dependent on endogenous cannabinoids in the nucleus accumbens (i.e. it is absent in CB1 receptor knockouts), these data suggest that chronic treatment with Δ⁹-THC can alter the sensitivity of synapses to endogenous cannabinoids, and that tolerance to Δ⁹-THC is associated with a deficit in synaptic plasticity. Hoffman, A.F., Oz, M., Caulder, T. and Lupica, C.R. The Journal of Neuroscience, 23, pp. 4815-4820, 2003.

Functional Localization of Cannabinoid Receptors and Endogenous Cannabinoid Production in Distinct Neuron Populations of the Hippocampus The mammalian hippocampus is thought to be one of the primary brain regions involved in the cognitive disrupting effects of marijuana in humans and animals. This is likely because it contains some of the highest levels of cannabinoid CB1 receptors in the brain. GABAergic interneurons represent a small fraction (<10%) of the neuronal population in the hippocampus. However, single interneurons can provide widespread input to hundreds of principal (pyramidal) cells, whose axons comprise the primary output pathway of the hippocampus. Previous work by IRP investigators demonstrated that activation of presynaptic CB1 receptors resulted in the inhibition of GABA-mediated synaptic transmission onto pyramidal neurons in the hippocampus. In order to determine whether synaptic inputs to hippocampal interneurons are also regulated by cannabinoids, whole-cell electrophysiological recordings in distinct interneuron populations were performed. In this study, authors found that, whereas glutamatergic inputs to pyramidal cells were presynaptically inhibited by the cannabinoid agonist WIN55,212-2, glutamatergic inputs to interneurons were unaffected by this agonist. In contrast, GABAergic inputs to the interneurons were inhibited by WIN55,212-2, and this effect was reversed by the CB1 receptor antagonist SR141716A. Also, using a sensitive electrophysiological bioassay for endogenous cannabinoid release, known as depolarization-induced suppression of inhibition (DSI), they found that pyramidal cells, but not interneurons, released endogenous cannabinoids. The localization of endogenous cannabinoid release to pyramidal neurons suggests that the role of these molecules may be limited to the regulation of specific synapses. This work identifies a novel neuronal pathway for consideration when examining the substrates responsible for the disruption of memory by marijuana. Hoffman, A.F., Riegel, A.C., and Lupica, C.R. European Journal of Neuroscience, 18, pp. 524-534, 2003.

MRI Physics Unit, Neuroimaging Research Branch

Circular Spectrum Mapping of Diffusion Imaging for Identifying Brain Fiber Structures Dr. Y. Yang and his colleagues in the Neuroimaging Branch developed a new imaging method to map intravoxel structures of white matter, especially fiber crossings, using circular spectrum decomposition based on high-angular resolution measurements of apparent diffusion coefficients (ADC). The basic premise of this method is to determine the ADC values voxelwise on the unit circle spanned by the major and median eigenvectors of the diffusion tensor, and then apply a 1D Fourier-transform onto this circle. The 0th, 2nd, and 4th order harmonic components of the circular spectrum provide effective indices for mean diffusivity, linear fiber, and orthogonal fiber crossing diffusion respectively. A theoretical frame work has been established for the novel diffusion imaging technique. Simulations on a digital phantom demonstrated the effectiveness of the technique to identify fiber intersections. In vivo experiments on normal subjects showed that high 4th-order components (fiber crossings) can be observed in a number of brain regions, including pons, medulla, and areas around corpus callosum. This new technique provides an innovative tool for mapping fiber crossings inside the brain. Information obtained from this technique would be used for improving fiber-tracking techniques and for better delineating neuronal pathways under normal and pathological conditions. W. Zhan, H. Gu, S. Xu, D. A. Silbersweig, E. Stern, Y. Yang, Magnetic Resonance Medicine, 49, pp. 1077-1088, 2003.

Psychobiology Section, Medications Discovery Research Branch

Behavioral Effects of Cocaine in Dopamine D5 Receptor Knockout Mice Dopamine D1-like antagonists block several effects of cocaine, including its locomotor-stimulant and discriminative-stimulus effects. Because these compounds generally lack selectivity among the dopamine D1 and D5 receptors, the specific roles of the subtypes have not been determined. Dopamine D5 receptor knockout (DA D5R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D5 dopamine receptors in the effects of cocaine. In addition, effects of the D1-like antagonist, SCH 39166 were also studied to further clarify the roles of D1 and D5 dopamine receptors in the discriminative-stimulus effects of cocaine. Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D5R WT mice. Both DA D5R KO and HET mice showed reduced levels of horizontal activity compared to WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine; doses of 1.0 - 10.0 mg/kg produced dose-related increases in the number of cocaine-appropriate responses. SCH 39166 produced a dose-dependent rightward shift in the cocaine dose-effect curve in all genotypes, with similar apparent affinities. The present data suggest an involvement of DA D5R in the locomotor stimulant effects of cocaine. In addition, the data indicate that there is little involvement of the DA D5R in the discriminative-stimulus effects of cocaine. In addition, the antagonism data suggest a role of the D1 receptor in the behavioral effects of cocaine. Elliot, E.E, Sibley, D.R. and Katz, J.L. Psychopharmacology, 169, pp. 161-168, 2003.

Clinical Psychopharmacology, Medications Discovery Research Branch

High-dose Fenfluramine Administration Decreases Serotonin Transporter Binding, but not Serotonin Transporter Protein Levels, in Rat Forebrain Administration of D-fenfluramine (D-FEN) or parachloroamphetamine (PCA) can produce long-lasting decreases in serotonin transporter (SERT) binding and tissue levels of serotonin (5-HT) in rat forebrain. These changes have been viewed as evidence for 5-HT neurotoxicity, but no studies have measured SERT protein levels. In the present study, we determined the effect of high-dose D-FEN or PCA, administered according to a "neurotoxic" dosing regimen, on the density of SERT sites using ligand binding methods and on SERT protein levels using Western blots. Rats were sacrificed 2 days and 2 weeks after administration of drug or saline. The density of SERT was determined in homogenates of caudate and whole brain minus caudate. D-FEN and PCA decreased SERT binding by 30% to 60% in both tissues and at both time points. Similarly, D-FEN and PCA administration profoundly decreased tissue 5-HT and 5-HIAA in frontal cortex. Despite the large decreases in SERT binding and depletion of tissue 5-HT that occurred with D-FEN administration, SERT protein expression, as determined by Western blot analysis, did not change in either tissue or time point. PCA administration decreased SERT protein by about 20% only at the 2 day point in the caudate. Drug treatments did not change expression of glial fibrillary acidic protein (GFAP), a hallmark indicator of neuronal damage, in whole brain minus caudate in the 2 week group. These results support the hypothesis that decreases in tissue 5-HT and SERT induced by that D-FEN- and PCA binding sites reflect neuroadapative changes, rather than neurotoxic effects. Rothman, R.B., Jayanthi, S., Wang, X., Dersch, C.M., Cadet, J.L., Prisinzano, T., Rice, K.C. and Baumann, M.H. Synapse, 50, pp. 233-239, 2003.

in vitro Characterization of Ephedrine-related Stereoisomers at Biogenic Amine Transporters and the Receptorome Reveals Selective Actions as Norepinephrine Transporter Substrates Ephedrine is a long-studied stimulant available both as a prescription and over-the-counter medication, as well as an ingredient in widely marketed herbal preparations, and is also used as a precursor for the illicit synthesis of methamphetamine. Ephedrine is related to phenylpropanolamine, a decongestant removed from the market place due to concerns that its use increased the risk of hemorrhagic stroke. Standard pharmacology texts emphasize that ephedrine is both a direct and indirect adrenergic agonist, activating adrenergic receptors both by direct agonist activity as well as by releasing norepinephrine via a carrier-mediated exchange mechanism. Chemically, ephedrine possesses two chiral centers. In the present study, IRP scientists characterized the stereoisomers of ephedrine, and the closely related compounds, pseudoephedrine, norephedrine, pseudonorephedrine (cathine), methcathinone, and cathinone, at biogenic amine transporters and a large battery of cloned human receptors (e.g. 'receptorome'). The most potent actions of ephedrine-type compounds were as substrates of the norepinephrine transporter (EC50 values of about 50 nM) followed by substrate activity at the dopamine transporter. Screening the receptorome demonstrated weak affinity at a2-adrenergic and 5-HT7 receptors (Ki values 1-10 uM) and no significant activity at b-adrenergic or a1 receptors. Viewed collectively, these data indicate that the pharmacological effects of ephedrine-like phenylpropanolamines are likely mediated by norepinephrine release and, although sharing mechanistic similarities with, differ in important respects, those of the phenylpropanonamines methcathinone and cathinone, and the phenyisopropylamines methamphetamine and amphetamine. Rothman, R. B., Vu, N., Roth, B.L., Hufeisen, V.S.J., Compton-Toth, J. and Glennon, R.A., Journal of Pharmacology and Experimental Therapeutics, 307, pp. 138-145, 2003.

(+)-Fenfluramine and its Major Metabolite, (+)-Norfenfluramine, are Potent Substrates for Norepinephrine Transporters (±)- Fenfluramine is an amphetamine analog that was once widely prescribed as an appetite suppressant. While (±)-fenfluramine is no longer clinically available, the mechanisms underlying its anorectic properties are still of interest. Upon peripheral administration, stereoisomers of (±)-fenfluramine are N-de-ethylated to form the metabolites, (+)- and (-)-norfenfluramine. It is well accepted that isomers of (±)-fenfluramine and (±)-norfenfluramine interact with serotonin (5-HT) transporters to release 5-HT from neurons. However, the effects of these drugs on other monoamine transporters are not well characterized. In this study, IRP scientists examined the interaction of stereoisomers of (±)-fenfluramine and (±)-norfenfluramine with transporters for 5-HT, norepinephrine (NE), and dopamine (DA). Results from in vitro assays confirmed these drugs are potent substrates for 5-HT transporters: (+)-fenfluramine, (-)-fenfluramine, (+)-norfenfluramine, and (-)-norfenfluramine released [3H]5-HT from synaptosomes with EC50 values of 52 nM, 147 nM, 59 nM, and 287 nM, respectively. Importantly, (+)-fenfluramine and (+)-norfenfluramine released [3H]NE with EC50 values of 302 nM and 73 nM. Results from In vivo microdialysis experiments showed that intravenous injection of (+)-norfenfluramine elevates extracellular levels of 5-HT, NE and DA in rat frontal cortex. The effects of (+)-norfenfluramine on NE and DA were antagonized by pretreatment with the NE uptake blocker, nisoxetine. In summary, administration of fenfluramines can increase synaptic levels of 5-HT, NE and DA in the cortex, and (+)-norfenfluramine likely contributes to these effects. Release of NE and DA evoked by (+)-norfenfluramine is at least partly mediated via NE transporters. Results of this study emphasize the potential involvement of noradrenergic mechanisms in the actions of fenfluramines. Rothman, R. B., Clark, R.D., Partilla, J.S. and Baumann, M.H., Journal of Pharmacology and Experimental Therapeutics, 305, pp. 1191-1199, 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., Hope, B.T. Journal of Neurochemistry, 23, pp. 742-747, 2003.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Histamine H3 Receptor Antagonists Potentiate Methamphetamine Self-administration and Methamphetamine-induced Accumbal Dopamine Release Methamphetamine administration increases brain levels of histamine and neuronal histamine attenuates several of methamphetamine's behavioral effects. The role of different subtypes of histamine receptors in this negative feedback, however, remains unclear. The aim of the present study was to evaluate the effects of two histamine H3 receptor antagonists, clobenpropit and thioperamide, on rewarding and neurochemical effects of methamphetamine utilizing three in-vivo methodologies. In rats self-administering methamphetamine intravenously under a fixed-ratio schedule, pre-session treatment with thioperamide (1.0 - 3.0 mg/kg, SC) or clobenpropit (1.0 - 3.0 mg/kg, SC) potentiated the reinforcing effects of methamphetamine, as indicated by a dose-dependent increase in responding for a low 0.03 mg/kg dose of methamphetamine, that by itself failed to maintain responding above saline substitution levels, and a decrease in responding for a higher 0.06 mg/kg training dose of methamphetamine. In contrast, neither thioperamide nor clobenpropit treatment increased responding during saline substitution. In other rats trained to discriminate IP injection of 1.0 mg/kg methamphetamine from IP injection of saline, both thioperamide and clobenpropit (0.3 - 3.0 mg/kg, SC) dose dependently increased methamphetamine-appropriate responding when administered with a low 0.3 mg/kg IP dose of methamphetamine, which by itself produced predominantly saline-appropriate responding. However, thioperamide and clobenpropit produced only saline-appropriate responding when administered with saline vehicle. Finally, thioperamide and clobenpropit potentiated methamphetamine-induced elevations in extracellular dopamine levels in the shell of the nucleus accumbens, but did not increase brain dopamine levels when given alone. These findings point to histamine H3 receptors as a new and important receptor system modulating the reinforcing, subjective and neurochemical actions of methamphetamine. Munzar, P., Tanda, G., Justinova, Z., and Goldberg, S.R. Neuropsychopharmacology http://www.acnp.org/citations/NPP12160303323/default.pdf

Involvement of Adenosine A1 and A2A Receptors in the Adenosinergic Modulation of the Discriminative-Stimulus Effects of Cocaine and Methamphetamine in Rats Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. IRP investigators have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, the authors investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the discriminative-stimulus effects of methamphetamine and cocaine. Justinova, Z., Ferré, S., Segal, P.N., Antoniou, K., Solinas, M., Pappas, L.A., Highkin, J.L., Hockemeyer, J., Munzar, P., and Goldberg, S.R. Journal of Pharmacology and Experimental Therapeutics, 307, pp. 977-986, 2003.

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

Reliability and Validity of the Tobacco Craving Questionnaire and Validation of a Craving-induction Procedure The purpose of this study was to determine the reliability and validity of the Tobacco Craving Questionnaire (TCQ) and the validity of imagery scripts to elicit self-reported tobacco craving. Current cigarette smokers (24 men, 24 women) not attempting to quit or reduce smoking engaged in active imagery of three auditory scripts that described no-, low-, and high-intensity of smoking urge. After each imagery condition, participants completed the 47-item TCQ, a Mood Form, and Visual Analog Scale (VAS) questions. Reliability of measures was demonstrated by internal consistency and unidimensionality of the four TCQ factors across imagery conditions. Criterion-related validity was demonstrated by an orderly increase in scores on the TCQ and VAS craving measures as a function of craving intensity of the imagery scripts. Increases in effect size parameters and parallel decreases in the stability of test-retest reliability for all craving measures indicated the validity of the imagery procedure. Convergent and discriminant validity were established by the craving scripts increasing self-reported craving, the no-craving (positive-affect) script increasing positive mood, the no-craving script not affecting craving, and the craving scripts not affecting positive mood. Findings further demonstrated the reliability and validity of the TCQ as a multi-factorial instrument to assess the construct of tobacco craving and suggested that the lability of craving, rather than inconsistency and instability in its measurement, was responsible for observed effects. Singleton, E.G., Anderson, L.M.and Heishman, S.J., Addiction, 98, pp. 1537-1546, 2003.