Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Dopaminergic Differentiation of Human Embryonic Stem Cells In this manuscript IRP scientists report that human embryonic stem cells (hESCs) differentiated into dopaminergic neurons when cocultured with PA6 cells. After 3 weeks of differentiation, approximately 87% of hES colonies contained tyrosine hydroxylase (TH)-positive cells, and a high percentage of the cells in most of the colonies expressed TH. Differentiation was inhibited by exposure to BMP4 or serum. TH-positive cells derived from hESCs were postmitotic, as determined by bromodeoxyurindine colabeling. Differentiated cells expressed other markers of dopaminergic neurons, including the dopamine transporter, aromatic amino acid decarboxylase, and the transcription factors associated with neuronal and dopaminergic differentiation, Sox1, Nurr1, Ptx3, and Lmx1b. Neurons that had been differentiated on PA6 cells were negative for dopamine-beta-hydroxylase, a marker of noradrenergic neurons. PA6-induced neurons were able to release dopamine and 3,4-dihydroxphe-hylacetic acid (DOPAC) but not noradrenalin when depolarized by high K(+).When transplanted into 6-hydroxydopamine-treated animals, hES-derived dopaminergic cells integrated into the rat striatum. Five weeks after transplantation, surviving TH-positive cells were present but in very small numbers compared with the high frequency of TH-positive cells seen in PA6 coculture. Larger numbers of cells positive for smooth muscle actin, but no undifferentiated ES cells, were present after transplantation. Therefore, hESCs can be used to generate human dopaminergic cells that exhibit biochemical and functional properties consistent with the expected properties of mature dopaminergic neurons. Zeng, X., Cai, J., Chen, J., Luo, Y., You, Z.B., Fotter, E., Wang, Y., Harvey, B., Miura, T., Backman, C., Chen, G.J., Rao, M.S. and Freed, W.J. Stem Cells, 22, pp. 925-940, 2004.

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

Sigma-1 Receptors at Galactosylceramide-Enriched Lipid Microdomains Regulate Oligodendrocyte Differentiation In the brain, myelin is important in regulating nerve conduction and neurotransmitter release by providing insulation at axons. Myelin is a specialized yet continuous sheet structure of differentiated oligodendrocytes (OLs) that is enriched in lipids, specifically galactosylceramides (GalCer) originated at the endoplasmic reticulum (ER). GalCer are known to affect OL differentiation. However, the mechanism whereby GalCer affect OL differentiation is not well understood. Sigma-1 receptors (Sig-1Rs), shown by IRP researchers to exist in detergent-insoluble lipid microdomains at lipid-enriched loci of ER in NG108 cells, are important in the compartmentalization/transport of ER-synthesized lipids and in cellular differentiation. In this study, authors used rat primary hippocampal cultures and found that Sig-1Rs form GalCer-enriched lipid rafts at ER lipid droplet-like structures in the entire myelin sheet of mature OLs. In rat OL progenitors (CG-4 cells), levels of lipid raft-residing Sig-1Rs and GalCer increase as cells differentiate. Sig-1Rs also increase in OLs and myelin of developing rat brains. Sig-1R, GalCer, and cholesterol are colocalized and are resistant to the Triton X-100 solubilization. Treating cells with a Sig-1R agonist or targeting Sig-1Rs at lipid rafts by overexpression of Sig-1Rs in CG-4 cells enhances differentiation, whereas reducing Sig-1Rs at lipid rafts by transfection of functionally dominant-negative Sig-1Rs attenuates differentiation. Furthermore, Sig-1R siRNA inhibits differentiation. These findings indicate that, in the brain, Sig-1Rs targeting GalCer-containing lipid microdomains are important for OL differentiation and that Sig-1Rs may play an important role in the pathogenesis of certain demyelinating diseases. Hayashi, T. and Su, T.P. Proceedings National Academy of Sciences USA, 101, pp. 14949-14954, 2004.

A Perspective on the New Mechanism of Antidepressants: Neuritogenesis Through Sigma-1 Receptors Sigma receptors were first described as one of the opiate receptor subtypes. Now it is well established that sigma receptors, existing as subtypes sigma-1 and sigma-2, are unique non-opioid receptors which are implicated in higher-ordered brain functions. Sigma-1 receptors have high to moderate affinities for (+)benzomorphans and also many psychotrophic drugs and neurosteroids. Sigma-1 receptor agonists and certain neurosteroids such as dehydroepiandrosterone sulfate (DHEA-S) have antidepressant-like effects in animal behavioral models of depression. The antidepressant-like effect induced by sigma-1 receptor agonists may involve intracellular Ca (2+) mobilization such that sigma-1 receptor agonists modulate Ca (2+) release from endoplasmic reticulum (ER) in a cytoskeletal protein-dependent manner. In addition, growth factor-induced neurite outgrowth is mediated through sigma-1 receptors, suggesting a role of antidepressants in neuroplasticity. Igmesine (JO1783), OPC-14 523 and SA4503, have recently been developed as sigma-1 agonists and are found to have antidepressant-like activity perhaps with fewer side effects. This article reviews the new potential use of sigma-1 receptor ligands in the treatment of mood disorder. Takebayashi, M., Hayashi, T., and Su, T.P. Pharmacopsychiatry, 37, pp. 208-213, 2004.

Delta Opioid Peptide (d-ala 2, d-leu 5) Enkephalin: Linking Hibernation and Neuroprotection Hibernation is a potential protective strategy for the peripheral, as well as for the central nervous system. A protein factor termed hibernation induction trigger (HIT) was found to induce hibernation in summer-active ground squirrels. Purification of HIT yielded an 88-kD peptide that is enriched in winter hibernators. Partial sequence of the 88-kD protein indicates that it may be related to the inhibitor of metalloproteinase. Using opioid receptor antagonists to elucidate the mechanisms of HIT, it was found that HIT targeted the delta opioid receptors. Indeed, delta opioid (D-Ala 2, D-Leu 5) enkephalin (DADLE) was shown to induce hibernation. Specifically, HIT and DADLE were found to prolong survival of peripheral organs, such as the lung, the heart, liver, and kidney preserved en bloc or as a single preparation. In addition, DADLE has been recently demonstrated to promote survival of neurons in the central nervous system. Exposure to DADLE dose-dependently enhanced cell viability of cultured primary rat fetal dopaminergic cells. Subsequent transplantation of these DADLE-treated dopaminergic cells into the Parkinsonian rat brain resulted in a two-fold increase in surviving grafted cells. Interestingly, delivery of DADLE alone protected against dopaminergic depletion in a rodent model of Parkinson s disease. Similarly, DADLE blocked and reversed the dopaminergic terminal damage induced by methamphetamine (METH). Such neuroprotective effects of DADLE against METH neurotoxicity was accompanied by attenuation of mRNA expressions of a tumor necrosis factor p53 and an immediate early gene c-fos. In parallel to these beneficial effects of DADLE on the dopaminergic system, DADLE also ameliorated the neuronal damage induced by ischemia-reperfusion following a transient middle cerebral artery occlusion. In vitro replication of this ischemia cell death by serum-deprivation of PC12 cells revealed that DADLE exerted neuroprotection in a naltrexone-sensitive manner. These results taken together suggest that DADLE stands as a novel therapeutic agent. In this review paper, IRP scientists present laboratory evidence supporting the use of DADLE for protection of peripheral and central nervous system. Borlongan, C.V., Wang, Y. and Su, T.P. Frontiers in Bioscience, 9, pp. 3392-3398, 2004.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Tetracycline-Inducible Expression Systems for the Generation of Transgenic Animals: A Comparison of Various Inducible Systems Carried in a Single Vector The most often used tetracycline-regulated transgenic mice system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. In this study, IRP investigators report the design of a universal and simplified regulatory gene delivery vector to facilitate the generation of conditional transgenic animals that integrate both the tetracycline regulatory and response elements in a single vector. The newly developed tetracycline reversed transactivator rtTA-M2 was used in all our constructs, based on its highly improved properties with respect to specificity, stability and inducibility. To minimize interference between the different tetracycline-inducible promoters used in this study (tetracycline-responsive element (TRE), TRE-tight, or Tk-tetO) and the rtTA-M2 transactivator, both elements were cloned in opposite directions and separated by a 5kb human p53 intron. The functionality of this system was confirmed after in vitro transfection in a mammalian cell line. Overall induction by the tetracycline-responsive element promoter was significantly higher than that induced by the newly developed TRE-tight promoter. However, the TRE-tight promoter showed a significantly tighter expression with minimal background, and still maintained high induction levels. The minimal Tk-tetO promoter showed a very weak induction capacity. This study demonstrates that this combination of elements, placed in a single vector is sufficient for delivering a functional tetracycline-inducible system to a mammalian cell line. Moreover, additional modifications to this regulatory gene delivery system, such as the introduction of specific cloning sites and selection markers, have been designed with the idea of creating a simplified and universal inducible system to facilitate the generation of conditional transgenic, knock-out, and knock-in animals. Backman, C.M., Zhang, Y., Hoffer, B.J. and Tomac, A.C. Journal Neuroscience Methods, 139, pp. 257-262, 2004.

MALDI Matrices for Biomolecular Analysis Based on Functionalized Carbon Nanomaterials When used in small molar ratios of matrix to analyte, derivatized fullerenes and single wall nanotubes are shown to be efficient matrices for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The mixing of an acidic functionalized fullerene with a solution of bioanalyte, depositing a dried droplet, and irradiating with a pulsed nitrogen laser yields protonated or cationized molecular ions. Derivatized fullerenes could offer several advantages over conventional MALDI matrices: a high analyte ionization efficiency, a small molar ratios (less than 1) of matrix/analyte, and a broader optical absorption spectrum, which should obviate specific wavelength lasers for MALDI acquisitions. The major disadvantage to the use of fullerenes is the isobaric interference between matrix and analyte ions; however, it is overcome by using MALDI-ion mobility time-of-flight (IM-oTOF) mass spectrometry to preseparate carbon cluster ions from bioanalyte ions prior to TOF mass analysis. However, an alternative to the dried droplet preparation of fullerene MALDI samples is the aerosolization of matrix-analyte solutions (or slurries) followed by impacting the aerosol onto a stainless surface. We also demonstrate that the fullerene matrices can be used to acquire spectra from rat brain tissue. Ugarov, M.V., Egan, T., Khabashesku, D.V., Schultz, J.A., Peng, H., Khabashesku, V.N., Furutani, H., Prather, K.S., Wang, H.W., Jackson, S.N. and Woods, A.S. Analytic Chemistry, 76, pp. 6734-6742, 2004.

Analysis of Phosphorylated Peptides by Ion Mobility-Mass Spectrometry An ion mobility-mass spectrometry technique for rapid screening of phosphopeptides in protein digests is described. A data set of 43 sequences (ranging in mass from 400 to 3000 m/z) of model and tryptic peptides, including serine, threonine, and tyrosine phosphorylation, was investigated, and the data support previously reported observation (Ruotolo, B.T., Verbeck, G.F., IV, Thomson, L.M., Woods, A.S., Gillig, K.J., Russell, D.H. J. Proteome Res. 303, 2001) that the drift time-m/z relationship for singly charged phosphorylated peptide ions is different from that for nonphosphorylated peptides. The data further illustrate that a combined data-dependent IM-MS/MS approach for phosphopeptide screening would have enhanced throughput over conventional MS/MS-based methodologies. Ruotolo, B.T., Gillig, K.J., Woods, A.S., Egan, T.F., Ugarov, M.V., Schultz, J.A. and Russell, D.H. Analytic Chemistry, 76, pp. 6727-6733, 2004.

Modulation of Physiological Brain Hyperthermia by Environmental Temperature and Impaired Blood Outflow in Rats To study the role of ambient temperature and brain blood outflow in modulating physiological brain hyperthermia, temperatures in two brain structures (nucleus accumbens or NAcc and hippocampus or Hippo) and a non-locomotor head muscle (musculus temporalis) were monitored in rats exposed to three arousing stimuli (placement in the cage or environmental change, 3-min social interaction with a female rat, 3-min innocuous tail-pinch) under three conditions (intact animals at 23 degrees C or control, intact animals at 29 degrees C, animals with chronically occluded jugular veins at 23 degrees C). While each stimulus in each condition induced hyperthermia, with more rapid and stronger changes in brain structures than muscle, there were significant differences between conditions. At 29 degrees C, animal placement in the cage resulted in stronger temperature increase and larger brain-muscle differentials, while basal temperatures in Hippo and muscle (but not in NAcc) were higher than control. At 29 degrees C, hyperthermia during social interaction was smaller but more prolonged, while the response to tail-pinch was similar to that seen at normal environmental temperatures. Animals with chronically occluded jugular veins had similar basal temperatures but showed much weaker hyperthermia than intact animals during each stimulus presentation; temperature increases in brain structures, however, were much stronger than in the muscle. These data suggest that the brain is able to decrease neural activation induced by environmental challenges under conditions of impaired blood outflow and restricted heat dissipation to the external environment. Kiyatkin, E.A., and Brown, P.L. Physiology and Behavior, 83, pp. 467-474, 2004.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Marijuana and Cannabinoid Regulation of Brain Reward Circuits The reward circuitry of the brain consists of neurons that synaptically connect a wide variety of nuclei. Of these brain regions, the ventral tegmental area (VTA) and the nucleus accumbens (NAc) play central roles in the processing of rewarding environmental stimuli and in drug addiction. The psychoactive properties of marijuana are mediated by the active constituent, Delta(9)-THC, interacting primarily with CB1 cannabinoid receptors in a large number of brain areas. However, it is the activation of these receptors located within the central brain reward circuits that is thought to play an important role in sustaining the self-administration of marijuana in humans, and in mediating the anxiolytic and pleasurable effects of the drug. Here IRP scientists describe the cellular circuitry of the VTA and the NAc, define the sites within these areas at which cannabinoids alter synaptic processes, and discuss the relevance of these actions to the regulation of reinforcement and reward. In addition, the authors compare the effects of Delta(9)-THC with those of other commonly abused drugs on these reward circuits, and discuss the roles that endogenous cannabinoids may play within these brain pathways, and their possible involvement in regulating ongoing brain function, independently of marijuana consumption. The authors conclude that, whereas Delta(9)-THC alters the activity of these central reward pathways in a manner that is consistent with other abused drugs, the cellular mechanism through which this occurs is likely different, relying upon the combined regulation of several afferent pathways to the VTA. Lupica, C.R., Riegel, A.C. and Hoffman, A.F. British Journal of Pharmacology, 143, pp. 227-234, 2004.

Differential Effects of Endogenous and Synthetic Cannabinoids on Alpha7-Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes The effects of endogenous and synthetic cannabinoid receptor agonists, including 2-arachidonoylglycerol (2-AG), R-methanandamide, WIN55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], and CP 55,940 [1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol], and the psychoactive constituent of marijuana, Delta9-tetrahydrocannabinol (Delta9-THC), on the function of homomeric alpha7-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. The endogenous cannabinoid receptor ligands 2-AG and the metabolically stable analog of anandamide (arachidonylethanolamide), R-methanandamide, reversibly inhibited currents evoked with ACh (100 microM) in a concentration-dependent manner (IC50 values of 168 and 183 nM, respectively). In contrast, the synthetic cannabinoid receptor agonists CP 55,940, WIN55,212-2, and the phytochemical Delta9-THC did not alter alpha7-nACh receptor function. The inhibition of alpha7-mediated currents by 2-AG was found to be non-competitive and voltage-independent. Additional experiments using endocannabinoid metabolites suggested that arachidonic acid, but not ethanolamine or glycerol, could also inhibit the alpha7-nACh receptor function. Whereas the effects of arachidonic acid were also noncompetitive and voltage-independent, its potency was much lower than 2-AG and anandamide. Results of studies with chimeric alpha7-nACh-5-hydroxytryptamine (5-HT)3 receptors comprised of the amino-terminal domain of the alpha7-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT3 receptors indicated that the site of interaction of the endocannabinoids with the alpha7-nAChR was not located on the N-terminal region of the receptor. These data indicate that cannabinoid receptor ligands that are produced in situ potently inhibit alpha7-nACh receptor function, whereas the synthetic cannabinoid ligands, and Delta9-THC, are without effect, or are relatively ineffective at inhibiting these receptors. Oz, M., Zhang, L., Ravindran, A., Morales, M. and Lupica, C.R. Journal of Pharmacology and Experimental Therapeutics, 310, pp. 1152-1160, 2004.

Differential Effects of Endogenous and Synthetic Cannabinoids on Voltage-Dependent Calcium Fluxes in Rabbit T-Tubule Membranes: Comparison with Fatty Acids The effects of cannabinoid receptor ligands including 2-arachidonoylglycerol, R-methanandamide, Delta9-THC (Delta9-tetrahydrocannabinol), WIN 55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenylcarbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one], CP 55,940 ([1alpha,2beta-(R)-5alpha]-(-)-5-(1,1-dimethyl)-2-[5-hydroxy-2-(3-hydroxypropyl) cyclohexyl-phenol]) and a series of fatty acids on depolarization-induced Ca2+ effluxes mediated by voltage-dependent Ca2+ channels were investigated comparatively in transverse tubule membrane vesicles from rabbit skeletal muscle. Vesicles were loaded with 45Ca2+ and membrane potentials were generated by establishing potassium gradients across the vesicle using the ionophore valinomycin. Endocannabinoids, 2-arachidonoylglycerol and R-methanandamide (all 10 microM), inhibited depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110 (isradipine) to transverse tubule membranes. On the other hand, synthetic cannabinoids, including CP 55,940, WIN 55,212-2, and Delta9-THC (all 10 microM), were ineffective. Additional experiments using endocannabinoid metabolites suggested that whereas ethanolamine and glycerol were ineffective, arachidonic acid inhibited Ca2+ effluxes and specific binding of [3H]PN 200-110. Further studies indicated that only those fatty acids containing two or more double bonds were effective in inhibiting depolarization-induced Ca2+ effluxes and specific binding of [3H]PN 200-110. These results indicate that endocannabinoids, but not synthetic cannabinoids, directly inhibit the function of voltage-dependent calcium channels (VDCCs) and modulate the specific binding of calcium channel ligands of the dihydropyridine (DHP) class. Oz, M., Tchugunova, Y. and Dinc, M. European Journal of Pharmacology, 502, pp. 47-58, 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.

Molecular Neuropsychiatry Section, Molecular Neuropsychiatry Research Branch

Substituted Amphetamines That Produce Long-Term Serotonin Depletion in Rat Brain ("Neurotoxicity") Do Not Decrease Serotonin Transporter Protein Expression Administration of high-dose d-fenfluramine (d-FEN) or parachloroamphetamine (PCA) produces 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 few studies have measured SERT protein levels. Thus, in the present study IRP scientists 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 d-FEN- and PCA-induced decreases in tissue 5-HT and SERT binding sites reflect neuroadaptive changes rather than neurotoxic effects. Rothman, R.B., Jayanthi, S., Cadet, J.L., Wang, X., Dersch, C.M. and Baumann, M.H. Annals of the New York Acadamy of Science, 1025, pp. 151-161, 2004.

Abnormal Brain Activity in Prefrontal Brain Regions in Abstinent Marijuana Users IRP investigators used PET (15)O and a modified version of the Stroop task to determine if 25-day abstinent heavy marijuana (MJ) users have persistent deficits in executive cognitive functioning (ECF) and brain activity. Performance on a modified version of the Stroop task and brain activity was compared between 25-day abstinent, heavy marijuana users (n = 11), and a matched comparison group (n = 11). The 25-day abstinent marijuana users showed no deficits in performance on the modified version of the Stroop task when compared to the comparison group. Despite the lack of performance differences, the marijuana users showed hypoactivity in the left perigenual anterior cingulate cortex (ACC) and the left lateral prefrontal cortex (LPFC) and hyperactivity in the hippocampus bilaterally, when compared to the comparison group. These results suggest that marijuana users display persistent metabolic alterations in brain regions responsible for ECF. It may be that marijuana users recruit an alternative neural network as a compensatory mechanism during performance on a modified version of the Stroop task. These differences in brain activity may be a common denominator in the evolution of maladaptive behaviors such as substance abuse and other neuropsychiatric disorders. Eldreth, D.A., Matochik, J., Cadet, J.L. and Bolla, K.I. Neuroimage 23, pp. 914-920, 2004.

Role of Dietary Iron Restriction in a Mouse Model of Parkinson's Disease There is a growing body of evidence suggesting that iron chelation may be a useful therapy in the treatment of Parkinson's Disease (PD). Experiments were designed to test the impact of dietary iron availability on the pathogenic process and functional outcome in a mouse model of PD. Mice were fed diets containing low (4 ppm) or adequate (48 ppm) amounts of iron for 6 weeks before the administration of MPTP, a mitochondrial toxin that damages nigrostriatal dopaminergic neurons and induces Parkinson-like symptoms. Low dietary iron increased serum total iron binding capacity (P < 0.001). Consistent with neuronal protection, iron restriction increased sphingomyelin C16:0 and decreased ceramide C16:0. However, there was a 35% decrease in striatal dopamine (DA) in iron-restricted mice. Motor behavior was also impaired in these animals. In vitro studies suggested that severe iron restriction could lead to p53-mediated neuronal apoptosis. Administration of MPTP reduced striatal DA (P < 0.01) and impaired motor behavior in iron-adequate mice. However, in iron-restricted mice, striatal dopamine levels and motor behavior were unchanged compared to saline-treated mice. Thus, while reduced iron may provide protection against PD-inducing insults such as MPTP, the role of iron in the synthesis of DA and neuronal survival should be considered, particularly in the development of iron-chelating agents to be used chronically in the clinical setting. Levenson, C.W., Cutler, R.G., Ladenheim, B., Cadet, J.L., Hare, J. and Mattson, M.P. Experimental Neurology, 190, pp. 506-514, 2004.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

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 [(3)H]5-HT and [(3)H]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 [(3)H]5-HT and [(3)H]MPP(+). BZP released [(3)H]MPP(+), whereas TFMPP was a selective releaser of [(3)H]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 coadministered at high doses. Baumann, M.H., Clark, R.D., Budzynski, A.G., Partilla, J.S., Blough, B.E. and Rothman, R.B. Neuropsychopharmacology, online publication 20 November 2004.

Intracerebroventricular Administration of Anti-endothelin-1 IgG Selectively Upregulates Endothelin-A and Kappa Opioid Receptors Endothelin (ET) type A receptor antagonists enhance morphine-induced antinociception and restore morphine analgesia in morphine tolerant rats [Peptides 23 (2002) 1837; Peptides 24 (2003) 553]. These studies suggest that the central ET and opioid systems functionally interact. To explore this idea further, IRP researchers determined the effect of i.c.v. administration of anti-ET-1 IgG (rabbit) on brain opioid receptor and ET receptor expression. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 mul (i.c.v.) of either control rabbit IgG (2.5 mug/mul) or anti-ET IgG (2.5 mug/mul) on day 1, day 3, and day 5. On day 6, animals were killed and the caudate and hippocampus collected. Anti-ET IgG had no significant effect on expression, measured by Western blots, of mu, delta or ET-B receptors, but increased kappa opioid (59%) and ET-A (33%) receptor protein expression in the caudate. [(35)S]-GTP-gamma-S binding assays demonstrated that anti-ET IgG decreased [d-Ala(2)-MePhe(4), Gly-ol(5)]enkephalin efficacy, but not potency in the caudate. Control experiments showed that there was no detectable rabbit IgG in caudate and hippocampal samples. These results suggest that ET in the CSF negatively regulates kappa opioid and ET-A receptors in certain brain regions. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between ET and the opioid receptor system. Wang, X., Xu, H. and Rothman, R.B. Neuroscience, 129, pp. 751-756, 2004.

Medicinal Chemistry Section, Medications Discovery Research Branch

Novel Azido- and Isothiocyanato- Analogues of [3-(4-phenylalkyl-piperazin-1-yl)propyl]-bis-(4-fluorophenyl)amines as Potential Irreversible Ligands for the Dopamine Transporter Potential irreversible ligands were prepared, based on a series of 3(1-piperazinyl)propyl-N,N-bis(4-fluorophenyl)amines, as molecular probes for the dopamine transporter (DAT). Both azido- and isothiocyanato-substituted phenyl alkyl analogues were synthesized and evaluated for displacement of [3H]WIN 35,428 in rat caudate-putamen tissue. All of the analogues showed moderate binding potencies at the DAT. One azido analogue was radioiodinated and used to photolabel human DAT transfected HEK 293 cell membranes. [125I]JJC 3-024 irreversibly labeled an ~80 kDa band corresponding to the DAT detected using SDS-PAGE. This radioligand provides a novel addition to the growing arsenal of structurally diverse irreversible ligands that are being used to identify binding domains on the DAT. Characterizing points of attachment of these irreversible probes to the DAT protein will ultimately help elucidate 3D- arrangement of the transmembrane domains, identify individual binding sites of the DAT inhibitors and direct future drug design. Cao, J., Lever, J.R., Kopajtic, T., Katz, J.L., Holmes, M.L., Justice, J.B. and Newman, A.H. Journal of Medicinal Chemistry, 47, pp. 6128-6136, 2004.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Unconditional Hyperactivity and Transient Reinforcing Effects of NMDA Administration into the Ventral Tegmental Area in Rats The dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens plays an important role in positive reinforcement and locomotion. Intra-VTA administration of many drugs capable of activating these neurons has been shown to be reinforcing and induce locomotion. Administration of the excitatory amino acid NMDA (N-methyl-D-aspartate) into the VTA may likewise be positively reinforcing, because it stimulates the meso-accumbens dopamine system and locomotion. Locomotor-rearing experiments were conducted to pinpoint the range of NMDA concentrations that induce significant locomotion and rearing, and to determine whether co-administration of the glycine binding site agonist d-serine would enhance the effects of NMDA administration into the VTA. Reinforcing effects of NMDA were assessed by intracranial self-administration procedures: a lever-press delivered a 75-nl infusion containing NMDA (0.1, 0.3 or 1.0 mM) plus serine into the VTA or an adjacent region, the supramammillary nucleus. Co-administration of serine slightly enhanced rearing induced by NMDA administration. Administration of NMDA at concentrations of 0.3 or 1.0 mM (500 nl) induced vigorous locomotion and rearing. NMDA (0.3 mM) was self-administered into the VTA slightly more than vehicle in the first or second sessions, yet this small reinforcing effect became irregular in subsequent sessions. The rats did not learn to self-administer NMDA into the supramammillary nucleus. Authors concluded that ventral tegmental NMDA injections, in the concentration range that induced marked unconditional hyperactivity, supported only marginal and transient self-administration. Ikemoto, S. Psychopharmacology, 172, pp. 202-210, 2004.

Mapping of Chemical Trigger Zones for Reward Addictive drugs are thought to activate brain chemistry that normally mediates more natural rewards such as food or water. Drugs activate this circuitry at synaptic junctions within the brain; identifying the junctions at which this occurs provides clues about the neurochemical and anatomical characteristics of the circuitry. One approach to identifying the junctions at which drugs interact with this circuitry is to determine if animals will lever-press for site-specific microinjections of addictive drugs. This approach has identified GABAergic, dopaminergic, glutamatergic, and cholinergic trigger zones within meso-corticolimbic circuitry important for natural reward function. Ikemoto, S. and Wise, R.A. Neuropharmacology, 47, pp. 190-201, 2004.

Rewarding Effects of AMPA Administration into the Supramammillary or Posterior Hypothalamic Nuclei But Not the Ventral Tegmental Area IRP scientists examined whether injections of the excitatory amino acid AMPA are rewarding when injected into the posterior hypothalamus and ventral tegmental area. Rats quickly learned to lever-press for infusions of AMPA into the supramammillary or posterior hypothalamic nuclei but failed to learn to lever-press for similar injections into the ventral tegmental areas. AMPA injections into the supramammillary nucleus, but not the ventral tegmental area, induced conditioned place preference. The rewarding effects of AMPA appear to be mediated by AMPA receptors, because coadministration of the AMPA antagonist CNQX blocked the rewarding effects of AMPA, and administration of the enantiomer R-AMPA did not mimic the rewarding effects. AMPA injections into the supramammillary nucleus, but not the ventral tegmental area, also increased extracellular dopamine concentrations in the nucleus accumbens. Pretreatment with the D1 dopamine antagonist SCH 23390 [R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaz epine] led to extinction of AMPA self-administration. These findings implicate posterior hypothalamic regions in reward function and suggest that reward mechanisms localized around the ventral tegmental area are more complex than has been assumed recently. Ikemoto, S., Witkin, B. M., Zangen, A. and Wise. R.A. Journal of Neuroscience, 24, pp. 5758-5765, 2004.

Cocaine-induced Fos Expression in Rat Striatum is Blocked by Chloral Hydrate or Urethane Anesthetics used in electrophysiological studies alter the effects of cocaine and amphetamine on neural activity in the striatum. However, the mechanism underlying this alteration has not been established. In the present study, IRP scientists examined the effects of anesthetics on cocaine-induced neural activity in the striatum. Authors first assayed the ability of 20 mg/kg cocaine to induce Fos expression in the striatum following pretreatment with 400 mg/kg chloral hydrate or 1.3 g/kg urethane, two of the most commonly used anesthetics for in vivo electrophysiology. Chloral hydrate blocked, while urethane strongly attenuated cocaine-induced Fos expression without affecting basal levels of expression. The investigators then examined dopaminergic and glutamatergic mechanisms for anesthetic effects on cocaine-induced Fos expression. Chloral hydrate and urethane did not attenuate basal or cocaine-induced increases of dopamine levels as assessed by microdialysis in dorsal striatum. In contrast, chloral hydrate attenuated glutamatergic neurotransmission as assessed by microdialysis in the presence of the glutamate transport blocker l-trans-pyrrolidone-2,4-dicarboxylic acid. Chloral hydrate attenuated basal levels of glutamate by 70%, while cocaine had no effect on glutamate levels. Since glutamate levels were tetrodotoxin-sensitive, the majority of glutamate measured in this assay was by synaptic release. To assess a causal role for a reduction of glutamatergic neurotransmission in anesthetic effects on cocaine-induced Fos expression, the investigators injected the glutamate receptor agonists AMPA and NMDA into the dorsal striatum of chloral hydrate-anesthetized rats. The glutamate receptor agonists partially reinstated cocaine-induced Fos expression in anesthetized rats. Authors conclude that anesthetics attenuate cocaine-induced neuronal activity by reducing glutamatergic neurotransmission. Kreuter, J.D., Mattson, B.J., Wang, B., You, Z-B. and Hope, B.T. Neuroscience, 127, pp. 233-242, 2004.

Sensitization of Psychomotor Stimulation and Conditioned Reward in Mice: Differential Modulation by Contextual Learning Incentive motivation theory ascribes a critical role to reward-associated stimuli in the generation and maintenance of goal-directed behavior. Repeated psychomotor stimulant treatment, in addition to producing sensitization to the psychomotor-activating effects, can enhance the incentive salience of reward-associated cues and increase their ability to influence behavior. In the present study, IRP scientists sought to investigate this incentive sensitization effect further by developing a model of conditioned reinforcement (CR) in the mouse and investigating the effects of a sensitizing treatment regimen of amphetamine on CR. Furthermore, these investigators assessed the role of contextual stimuli in amphetamine-induced potentiation of CR. They found that mice responded selectively on a lever resulting in the presentation of a cue previously associated with 30% condensed milk solution, indicating that the cue had attained rewarding properties. Prior treatment with amphetamine (4 x 0.5 mg/kg i.p.) resulted in psychomotor sensitization and enhanced subsequent responding for the CR. Furthermore, this enhancement of responding for the cue occurred independent of the drug-paired context, whereas the sensitized locomotor response was only observed when mice were tested in the same environment as that in which they had received previous amphetamine. These results demonstrate that the CR paradigm previously developed in the rat can be successfully adapted for use in the mouse, and suggest that behavioral sensitization to amphetamine increases the rewarding properties (incentive salience) of reward-paired cues, independent of the drug-paired context. Mead, A.N., Crombag, H.S. and Rocha, B.A. Neuropsychopharmacology, 29, pp. 249-258, 2004.

Dopamine, Learning and Motivation The hypothesis that dopamine is important for reward has been proposed in a number of forms, each of which has been challenged. Normally, reward stimuli such as food, water, lateral hypothalamic brain stimulation and several drugs of abuse become ineffective as rewards in animals given performance-sparing doses of dopamine antagonists. Dopamine release in the nucleus accumbens has been linked to the efficacy of these unconditioned reward, but dopamine release in a broader range of structures is implicated in the Ôstamping-in' of memory that attaches motivational importance to otherwise neutral environmental stimuli. Wise, R.A. Nature Review Neuroscience, 5, pp. 483-494, 2004.

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.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Adenosine Receptor-mediated Modulation of Dopamine Release in the Nucleus Accumbens Depends on Glutamate Neurotransmission and N-methyl-D-aspartate Receptor Stimulation Adenosine, by acting on adenosine A(1) and A(2A) receptors, exerts opposite modulatory roles on striatal extracellular levels of glutamate and dopamine, with activation of A(1) inhibiting and activation of A(2A) receptors stimulating glutamate and dopamine release. Adenosine-mediated modulation of striatal dopaminergic neurotransmission could be secondary to changes in glutamate neurotransmission, in view of evidence for a preferential colocalization of A(1) and A(2A) receptors in glutamatergic nerve terminals. By using in vivo microdialysis techniques, local perfusion of NMDA (3, 10 microm), the selective A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 3, 10 microm), the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 300, 1000 microm), or the non-selective A(1)-A(2A) receptor antagonist in vitro caffeine (300, 1000 microm) elicited significant increases in extracellular levels of dopamine in the shell of the nucleus accumbens (NAc). Significant glutamate release was also observed with local perfusion of CGS 21680, CPT and caffeine, but not NMDA. Co-perfusion with the competitive NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (APV; 100 microm) counteracted dopamine release induced by NMDA, CGS 21680, CPT and caffeine. Co-perfusion with the selective A(2A) receptor antagonist MSX-3 (1 microm) counteracted dopamine and glutamate release induced by CGS 21680, CPT and caffeine and did not modify dopamine release induced by NMDA. These results indicate that modulation of dopamine release in the shell of the NAc by A(1) and A(2A) receptors is mostly secondary to their opposite modulatory role on glutamatergic neurotransmission and depends on stimulation of NMDA receptors. Furthermore, these results underscore the role of A(1) vs. A(2A) receptor antagonism in the central effects of caffeine. Quarta, D., Borycz, J., Solinas, M., Patkar, K., Hockemeyer, J., Ciruela, F., Lluis, C., Woods, A.S., Goldberg S.R. and Ferre, S.J. Neurochemistry, 91, pp. 873-880, 2004.

Rimonabant, a CB1 Antagonist, Blocks Nicotine-conditioned Place Preferences The effects of Rimonabant (SR141716), an antagonist at cannabinoid CB1 receptors, were evaluated in animal models for subjective and rewarding effects of nicotine. Acute administration of 1 or 3 mg/kg SR141716 blocked expression of nicotine-induced conditioned place preferences. SR141716 (0.3-3 mg/kg) was also studied in rats trained to discriminate nicotine from saline under a fixed-ratio schedule of food delivery. In contrast to nicotine replacement therapy and bupropion, SR141716 did not have nicotine-like discriminative effects and did not alter the dose-response curve for nicotine discrimination. These findings support the proposed use of SR141716 for smoking cessation and indicate that it would selectively reduce the influence of environmental stimuli that contribute to persistent smoking behavior, without affecting subjective responses to nicotine. LeFoll, B, and Goldberg, S.R. Neuroreport, 15, pp. 2139-2143, 2004.

Combining Mass Spectrometry and Pull-down Techniques for the Study of Receptor Heteromerization. Direct epitope-epitope Electrostatic Interactions Between Adenosine A2A and Dopamine D2 Receptors Previous results from FRET and BRET experiments and computational analysis (docking simulations) have suggested that a portion of the third intracellular loop (I3) of the human dopamine D(2) receptor (D(2)R) and the C-tail from the human adenosine A(2A) receptor (A(2A)R) are involved in A(2A)R-D(2)R heteromerization. The results of the present studies, using pull-down and mass spectrometry experiments, suggest that A(2A)R-D(2)R heteromerization depends on an electrostatic interaction between an Arg-rich epitope from the I3 of the D(2)R ((217)RRRRKR(222)) and two adjacent Asp residues (DD(401-402)) or a phosphorylated Ser (S(374)) residue in the C-tail of the A(2A)R. A GST-fusion protein containing the C-terminal domain of the A(2A)R (GST-A2A(CT)) was able to pull down the whole D(2)R solubilized from D(2)R-tranfected HEK-293 cells. Second, a peptide corresponding to the Arg-rich I3 region of the D(2)R ((215)VLRRRRKRVN(224)) and bound to Sepharose was able to pull down both GST-A2A(CT) and the whole A(2A)R solubilized from A(2A)R-tranfected HEK-293 cells. Finally, mass spectometry and pull-down data showed that the Arg-rich D(2)R epitope binds to two different epitopes from the C-terminal part of the A(2A)R, containing the two adjacent Asp residues or the phosphorylated Ser residue ((388)HELKGVCPEPPGLDDPLAQDGAVGS(412) and (370)SAQEpSQGNT(378)). The present results are the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization, a new, expanding area of protein-protein interactions. Ciruela, F., Burgueno, J., Casado, V., Canals, M., Marcellino, D., Goldberg, S.R., Bader, M., Fuxe, K., Agnati, L.F., Lluis, C., Franco, R., Ferre, S. and Woods, A.S. Analytical Chemistry, 76, pp. 5354-5363, 2004.

Beta-endorphin Elevations in the Ventral Tegmental Area Regulate the Discriminative Effects of Delta-9-tetrahydrocannabinol beta-Endorphin is an endogenous opioid that produces behavioral effects similar to heroin and morphine and is released in the nucleus accumbens by cocaine, amphetamine and ethanol, suggesting a general involvement in the reinforcing effects of abused drugs. Here IRP researchers show that, in rats, Delta-9-tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis, produces large increases in extracellular levels of beta-endorphin in the ventral tegmental area and lesser increases in the shell of the nucleus accumbens. Authors then used a two-lever choice THC-discrimination procedure to investigate whether THC-induced changes in endogenous levels of beta-endorphin regulate the discriminative effects of THC. In rats that had learned to discriminate injections of THC from injections of vehicle, the opioid agonist morphine did not produce THC-like discriminative effects but markedly potentiated discrimination of THC. Conversely, the opioid antagonist naloxone reduced the discriminative effects of THC. Bilateral microinjections of beta-endorphin directly into the ventral tegmental area, but not into the shell of the nucleus accumbens, markedly potentiated the discriminative effects of ineffective threshold doses of THC but had no effect when given alone. This potentiation was blocked by naloxone. Together these results indicate that certain psychotropic effects of THC related to drug abuse liability are regulated by THC-induced elevations in extracellular beta-endorphin levels in brain areas involved in opiate reward and reinforcement processes. Solinas, M., Zangen, A., Thiriet, N. and Goldberg, S.R. European Journal of Neuroscience, 19, pp. 3183-3192, 2004.

Chlormethiazole Potentiates the Discriminative Stimulus Effects of Methamphetamine in Rats Chlormethiazole is a positive modulator of gamma-aminobutyric acid (GABA)(A) receptors used in the treatment of alcohol withdrawal seizures. It recently has been reported to attenuate seizures engendered by acute and repeated exposure to cocaine in mice and neurotoxic effects of methamphetamine in rats. The aim of the present study was to determine whether chlormethiazole could also attenuate the discriminative stimulus effects of methamphetamine, a behavior predictive of the subjective effects of methamphetamine in humans. In Sprague-Dawley rats trained to discriminate 1.0 mg/kg methamphetamine [intraperitoneally (i.p.)] from saline under a fixed-ratio schedule of food delivery, the ability of chlormethiazole (i.p.) to (1) substitute for methamphetamine, (2) antagonize effects of methamphetamine and to (3) shift the methamphetamine dose-effect function was investigated. Chlormethiazole (18 and 30 mg/kg, i.p.) partially substituted for the discriminative stimulus effects of methamphetamine when administered alone (maximum group average, 60% responses on the methamphetamine-appropriate lever). Chlormethiazole did not attenuate effects of methamphetamine when coadministered with the training dose of methamphetamine. Instead, chlormethiazole potentiated the discriminative stimulus effects of methamphetamine as demonstrated by a significant (about 2.5-fold) leftward and upward shift in the methamphetamine dose-effect function in the presence of chlormethiazole (10 mg/kg). In conclusion, the present findings suggest that there is a behavioral interaction between methamphetamine and chlormethiazole. The profile of this interaction is qualitatively different from that of methamphetamine and classical GABAergic drugs (i.e., benzodiazepines and barbiturates), suggesting the involvement of non-GABAergic mechanisms in the effects produced by chlormethiazole. Gasior, M., Witkin, J.M., Goldberg, S.R. and Munzar, P. European Journal of Pharmacology, 94, pp. 183-189, 2004.

Neuropsychopharmacology Section, Behavioral Neuroscience Research Branch

Dopamine D3 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have previously found that SB277011A, a high-potency high-selectivity dopamine D3 receptor antagonist dose-dependently inhibits nicotine-enhanced brain-stimulation reward. Now, these researchers have extended these findings to include two additional putative D3 receptor antagonists - NGB2904 and BP897. It was found that both NGB2904 and BP897 blocked (within critical dose ranges) nicotine's enhancing effect on brain reward. However, while SB277011A and NGB2904 had no significant effect on brain-stimulation reward by themselves, BP897 dose-dependently attenuated brain-stimulation reward by itself. These findings are congruent with other data suggesting that SB277011A and NGB2904 are highly selective dopamine D3 receptor antagonists, while BP897 interacts with many receptors - including, most importantly for the present results - the dopamine D2 receptor. As drug-enhanced brain reward is believed to be a neural substrate for addiction, these findings suggest that dopamine D3 receptor antagonists are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. These findings also suggest a specific utility for dopamine D3 antagonists - to assist cigarette smokers in breaking their nicotine dependence and to quit smoking. These findings further suggest that NGB2904 should be added to SB277011A as a highly selective dopamine D3 receptor antagonist with possible anti-addiction clinical utility. Campos, A.C., Xi, Z.-X., Gilbert, J., Ashby, C.R. Jr., Heidbreder, C.A., Newman, A.H. and Gardner, E.L., Poster, 2004. Society for Neuroscience Annual Meeting, San Diego, CA, October 23-27, 2004.

Dopamine D3 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have previously found that acute blockade of the dopamine D3 receptor in the rat brain (which is neuroanatomically restricted to the mesolimbic dopamine system, implicated in drug-induced reward and drug-seeking behavior) dose-dependently attenuates cocaine-enhanced brain-stimulation reward, acquisition of cocaine-induced conditioned cue preference, expression of cocaine-induced conditioned cue preference, acquisition of heroin-induced conditioned cue preference, expression of heroin-induced conditioned cue preference, cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior, and stress-triggered relapse to cocaine-seeking behavior. Now, these researchers have extended these studies, and have found that SB277011A and NGB2904, high-potency high-selectivity dopamine D3 receptor antagonists, inhibit environmental cue-induced relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. The environmental cues found capable of triggering relapse to drug-seeking behavior were sights and sounds previously associated with intravenous cocaine-taking behavior. These findings suggest that dopamine D3 receptor antagonists are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. Gilbert, J.G., Xi, Z.-X., Campos, A.C., Peng, X., Ashby, C.R. Jr., Heidbreder, C.A., Newman, A.H. and Gardner, E.L., Poster, 2004. Society for Neuroscience Annual Meeting, San Diego, CA, October 23-27, 2004.

Gamma-vinyl GABA (GVG; Vigabatrin) as a Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medication for the Treatment of Addiction IRP scientists working in collaboration with scientists at the Brookhaven National Laboratory and Saint John's University in New York have previously found that gamma-vinyl GABA (GVG; Vigabatrin), an inhibitor of GABA transaminase which pharmacologically boosts synaptic levels of the neurotransmitter gamma-aminobutyric acid (GABA) in the reward circuitry of the brain, dose-dependently attenuates cocaine-enhanced synaptic levels of the neurotransmitter dopamine in brain-reward circuits (measured by in vivo brain microdialysis and positron emission tomography), cocaine-enhanced brain-stimulation reward, cocaine-induced conditioned place preference, cocaine self-administration, cocaine-induced behavioral sensitization, nicotine-enhanced synaptic levels of the neurotransmitter dopamine in brain-reward circuits (measured by in vivo brain microdialysis and positron emission tomography), nicotine-induced conditioned place preference, and heroin-induced conditioned place preference. Now, these researchers have extended those findings, and have found that GVG - but not gabapentin - dose-dependently attenuates cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. The additional new findings with GVG suggest that GVG, but not necessarily other GABAmimetic medications, is worthy of further investigation as an anti-addiction, anti-craving, and anti-relapse medication for the treatment of drug abuse. Its effectiveness against cocaine, nicotine, and opiates suggests a broad efficacy against drug addiction. Peng, X., Xi, Z.-X., Gilbert, J., Campos, A.C., Dewey, S.L., Schiffer, W.K., Brodie, J.D., Ashby, C.R. Jr. and Gardner, E.L., Poster, 2004. Society for Neuroscience Annual Meeting, San Diego, CA, October 23-27, 2004.

Glutamate mGluR5 Receptor Antagonists as Potential Anti-Addiction, Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have found that acute blockade of the glutamate mGluR5 receptor in the rat brain (which is neuroanatomically linked to the mesolimbic dopamine system, implicated in drug-induced reward and drug-seeking behavior) dose-dependently lowers the progressive-ratio break-point (a measure of incentive motivation to self-administer drugs) during intravenous cocaine self-administration, and also attenuates cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. However, the same acute blockade of the glutamate mGluR5 receptor does not attenuate either stress-triggered or environmental cue-triggered relapse to cocaine-seeking behavior. These findings suggest that medications which produce acute blockade of the glutamate mGluR5 receptor are worthy of further investigation as potential anti-addiction, anti-craving, and anti-relapse medications for the treatment of drug abuse. Further, these findings confirm that the brain mechanisms underlying drug-triggered relapse to drug-seeking behavior is mediated by different brain mechanisms than stress-triggered or environmental cue-triggered relapse to cocaine-seeking behavior, a finding previously reported by IRP scientists. Xi, Z.-X., Gilbert, J., Campos, A.C., Peng, X., Ashby, C.R. Jr. and Gardner, E.L., Poster, 2004. Society for Neuroscience Annual Meeting, San Diego, CA, October 23-27, 2004.

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

Neonatal Abstinence Syndrome in Methadone-exposed Infants is Altered by Level of Prenatal Tobacco Exposure Maternal tobacco consumption during pregnancy has been associated with lower birth weight infants, preterm births, intrauterine growth retardation, smaller head circumference and increase in morbidity, yet few studies have examined the role tobacco has on the opiate neonatal abstinence syndrome (NAS). This study examined the effect of prenatal tobacco exposure on NAS for infants born to mothers maintained on methadone during gestation. Twenty-nine pregnant women and their newborn infants participated in this study. Tobacco exposure was based on maternal self-report with 16 women reporting cigarette consumption of 10 or less per day and 13 reporting smoking 20 cigarettes or more a day. The onset, peak, and duration of NAS were examined. Results showed that infants born to mothers who reported smoking 20 or more cigarettes per day had significantly higher NAS peak scores of 9.8 versus 4.8, and took longer to peak (113.0 h versus 37.8 h), than light smokers of 10 or fewer cigarettes per day. Investigators concluded that tobacco use in conjunction with methadone plays an important role in the timing and severity of NAS in prenatally exposed infants. Choo, R.E., Huestis, M.A., Schroeder, J.R., Shin, A.S. and Jones, H.E. Drug and Alcohol Dependence, 75, pp. 253-260, 2004.

Methamphetamine and Amphetamine Concentrations in Meconium of Neonates of Women Enrolled in the IDEAL Study of In Utero Methamphetamine Exposure The Infant Development, Environment, and Lifestyle (IDEAL) study is a multi-center, longitudinal investigation of the effects of prenatal methamphetamine exposure. Meconium, a useful matrix for identifying in utero drug exposure, was employed to identify gestational drug use. Of the 13,808 mothers screened, 1631 were consented and 176 enrolled. MA exposed mothers (n=84) were identified by self-report of gestational MA use and/or GC/MS confirmation of MA, AMP, and/or MDMA in infant meconium. Comparison participants (n=92) were matched by race, birth weight, maternal education and type of insurance, denied amphetamines use and had negative meconium results. Among the 1631 mothers, self-reported use rates were 5.2% (amphetamines), 25% (tobacco) and 5.9% (cannabis). Positive meconium screening rates were 3.6% for any amphetamine, 20% cotinine and 11.2% cannabis. For specimens that screened positive, 40.7% of amphetamines and 20.2% of cannabis specimens were confirmed. On average, 68% of the meconium from neonates whose mothers reported 3rd trimester use had detectable MA, while detection rates were ²10% for self-reported use during the 1st and/or 2nd trimesters. Mean ± SD, median and range of MA concentrations were 3674 ± 3406, 2623, 479 to 13,431 ng/g meconium and AMP 569 ± 543, 403, 30 to 2000 ng/g meconium in infants whose mothers reported 3rd trimester use. However, the highest MA (19,376 and 16,976 ng/g) and AMP (2765 ng/g) concentrations were found in offspring born to women who reported MA use only in the 1st or 1st and 2nd trimesters, raising questions about the self-report. The log transformed meconium MA concentrations significantly correlated with the frequency of MA use in the 3rd trimester (r=0.645, P=0.004), although variability prevents prediction of frequency of use for an individual mother. AMP was always detected in MA positive meconium. In 55% of the GCMS positive samples, the ratios of amphetamine to MA were 0.1 to 0.2; 14% were less than 0.1 and 18% were 0.2 to 0.3. Meconium analysis for MA is a useful adjunct to self-report for identification of MA exposure; however, the greatest sensitivity was achieved with specimens collected from offspring of women who reported use in the 3rd trimester. Further research is needed to determine if there are additional MA metabolites in meconium that could improve the identification of MA-exposed infants. Zhao, Z., Liu, J., LaGasse, L.L., Derauf, C., Grant, P., Shah, R., Arria, A., Haning, W., Smith, L.M., Lester, B. and Huestis, M.A., Poster, 2004. Joint Meeting of the Society of Forensic Toxicologists and The International Association of Forensic Toxicologists, Washington, DC, August 28-September 3, 2004.

A Validated Gas Chromatographic-Negative Chemical Ionization Mass Spectrometric Method for delta-9-tetrahydrocannabinol (THC) in Sweat A sensitive gas chromatography-negative ion chemical ionization-mass spectrometry (GC/MS-NICI) method was developed and validated for the measurement of D9-tetrahydrocannabinol (THC) in human sweat patches. THC-d0 and THC-d3 were spiked onto worn blank sweat patches (PharmChekª, PharmChem Incorporated) and extracted with 3 mL methanol/0.2 M sodium acetate buffer (pH 5.0, 3:1, v/v) on a reciprocating shaker at ambient temperature for 30 min. Two mL of extracted solution was diluted with 8 mL 0.1 M sodium acetate buffer, pH 4.5 and extracted using solid phase extraction columns (CleanScreen¨, ZSTHC020, UCT). Dried extracts were derivatized with trifluoroacetic acid (TFAA) and analyzed using an Agilent 6890 GC interfaced with an Agilent 5973 mass selective detector operated in NICI- selected ion monitoring mode. The lower limits of detection and quantification for THC in human sweat were 0.2 and 0.4 ng/patch, respectively. The standard curve was linear from 0.4 to 10 ng/patch (r2>0.995). Overall recovery of THC from blank worn patches spiked with 0.6, 4.0 and 8.0 ng THC was 44 to 46%. Assay imprecision, expressed as coefficient of variation, was less than 10.3%, for 0.6, 4.0 and 8.0 ng/patch quality control (QC) samples. Twenty-one potential interfering compounds (50 ng/patch) spiked into low QC samples (0.6 ng/patch) did not influence THC quantitation. This GC/MS-NICI assay for THC in human sweat provides adequate sensitivity and performance characteristics for analyzing THC in sweat patches and meets the requirements of the proposed Substance Abuse and Mental Health Administration's guidelines for sweat testing. Saito, T., Wtsadik, A., Scheidweiler, K., McCain, M., Fortner, N., Takeichi, S. and Huestis, M.A. Clinical Chemistry, 50, pp. 2083-2090, 2004.

Urinary Pharmacokinetics of Methamphetamine and Its Metabolite, Amphetamine Following Controlled Oral Administration to Humans Methamphetamine is widely abused for its euphoric effects. The objectives of the investigators were to characterize the urinary pharmacokinetics of methamphetamine and amphetamine after controlled methamphetamine administration to humans and to improve the interpretation of urine drug test results. Participants (n=8) received four daily 10 mg (low) oral doses of sustained release (d)-methamphetamine hydrochloride within 7 days. After 4 weeks, five participants received four daily 20 mg (high) oral doses. All urine specimens were collected during the study. Methamphetamine and amphetamine were measured by GC-MS/PCI. Maximum excretion rates ranged from 403-4919 µg/h for methamphetamine and 59-735 µg/h for amphetamine with no relationship between dose and excretion rate. The mean molar % of dose in the urine as total methamphetamine and amphetamine were 57.5 ± 21.7% (low dose) and 40.9 ± 8.5% (high dose). Mean urinary terminal elimination half-lives across doses were 23.6 ± 6.6 h for methamphetamine and 20.7 ± 7.3 h for amphetamine. Methamphetamine renal clearance across doses was 175 ± 102 mL/min. The mean amphetamine/methamphetamine % ratio based on the area under the urinary excretion-time curve increased over time from 13.4 ± 6.5 to 35.7 ± 26.6%. Slow urinary excretion results in drug accumulation and increases in detection time windows. These findings also support the presence of an active renal excretion mechanism for methamphetamine. Kim, I., Oyler, J.M., Moolchan, E.T., Cone, E.J. and Huestis, M.A. Therapeutic Drug Monitoring, 26, pp. 664-672, 2004.

Imagery-induced Tobacco Craving: Duration and Lack of Assessment Reactivity Bias Little is known about the natural history of tobacco craving, including the intensity, frequency, and duration of craving episodes during various phases of the addictive process. In this study, IRP scientists investigated the duration of imagery-induced tobacco craving and whether craving responses are biased by repeated assessment, a phenomenon known as reactivity bias. Nonabstinent smokers (n = 40) either imagined a scene describing smoking urges or rested. They then either completed the Tobacco Craving Questionnaire (TCQ) every minute for 15 minutes or completed it after imagery or rest (minute 1) and 15 minutes later. TCQ scores were greater after imagery compared with rest and remained significantly elevated at minute 15. There was no evidence that TCQ responses were affected by repeated measurement. These data suggest that imagery-induced craving can persist for at least 15 minutes and that craving responses are not biased by assessment reactivity. Heishman, S.J., Saha, S. and Singleton, E.G. Psychology of Addictive Behaviors, 18, pp. 284-288, 2004.