Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

A Perspective on Transplantation Therapy and Stem Cells for Parkinson's Disease Although the functioning of the brain is overwhelmingly complex, Parkinson's disease is one disorder that seems to be simple, straightforward, and amenable to human intervention. Mostly, a restricted population of cells (dopaminergic neurons) is lost, and it seems that the loss of striatal dopamine can, in effect, be partially replaced by administration of a drug (L-DOPA). L-DOPA simply helps the brain to produce the substance (dopamine) the lost dopamine-producing cells normally produce. These cells send projections to only a few areas, and just two of these brain regions (the caudate and putamen) seem to be responsible for almost the entire syndrome of Parkinson's disease. Therefore, if we can just find a way to restore this one simple circuit, the disease might be cured. So it appeared in 1977, and still, it seems that it might just be that simple. Freed, W.J. Cell Transplantation, 13, pp. 319-327, 2004.

Properties of Pluripotent Human Embryonic Stem Cells BG01 and BG02 Human ES (hES) cell lines have only recently been generated, and differences between human and mouse ES cells have been identified. In this manuscript IRP investigators describe the properties of two human ES cell lines, BG01 and BG02. By immunocytochemistry and reverse transcription polymerase chain reaction, undifferentiated cells expressed markers that are characteristic of ES cells, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and OCT-3/4. Both cell lines were readily maintained in an undifferentiated state and could differentiate into cells of all three germ layers, as determined by expression of beta-tubulin III neuron-specific molecule (ectoderm), cardiac troponin I (cardiomyocytes, mesoderm), and alpha-fetoprotein (endoderm). A large-scale microarray (16,659 genes) analysis identified 373 genes that were expressed at three-fold or higher levels in undifferentiated BG01 and BG02 cells as compared with pooled human RNA. Ninety-two of these genes were also highly expressed in four other hES lines (TE05, GE01, GE09, and pooled samples derived from GE01, GE09, and GE07). Included in the list are genes involved in cell signaling and development, metabolism, transcription regulation, and many hypothetical proteins. Two focused arrays designed to examine transcripts associated with stem cells and with the transforming growth factor-beta superfamily were employed to examine differentially expressed genes. Several growth factors, receptors, and components of signaling pathways that regulate embryonic development, in particular the nodal signaling pathway, were detected in both BG01 and BG02. These data provide a detailed characterization and an initial gene expression profile for the BG01 and BG02 human ES cell lines. Zeng, X., Miura, T., Luo, Y., Bhattacharya, B., Condie, B., Chen, J., Ginis, I., Lyons, I., Mejido, J., Puri, R.K., Rao, M.S., and Freed, W.J. Stem Cells, 22, pp. 292-312, 2004.

Absence of DNA Polymerase Eta Reveals Targeting of C Mutations on the Non-Transcribed Strand in Immunoglobulin Switch Regions Activation-induced cytosine deaminase preferentially deaminates C in DNA on the nontranscribed strand in vitro, which theoretically should produce a large increase in mutations of C during hypermutation of immunoglobulin genes. However, a bias for C mutations has not been observed among the mutations in variable genes. Therefore, IRP scientists examined mutations in the micro and gamma switch regions, which can form stable secondary structures, to look for C mutations. To further simplify the pattern, mutations were studied in the absence of DNA polymerase (pol) eta, which may produce substitutions of nucleotides downstream of C. DNA from lymphocytes of patients with xeroderma pigmentosum variant (XP-V) disease, whose polymerase eta is defective, had the same frequency of switching to all four gamma isotypes and hypermutation in micro-gamma switch sites (0.5% mutations per basepair) as control subjects. There were fewer mutations of A and T bases in the XP-V clones, similar to variable gene mutations from these patients, which confirms that polymerase eta produces substitutions opposite A and T. Most importantly, the absence of polymerase eta revealed an increase in C mutations on the nontranscribed strand. This data shows for the first time that C is preferentially mutated in vivo and pol eta generates hypermutation in the micro and gamma switch regions. Zeng, X., Negrete, G., Kasmer, C., and Gearhart, P.J. Journal of Experimental Medicine, 199, pp. 917-924, 2004.

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

Sigma-1 Receptor Ligands: Potential in the Treatment of Neuropsychiatric Disorders The sigma receptors are non-opioid, non-phencyclidine brain ER proteins that exist in two subtypes: sigma-1 and sigma-2. Sigma-1 receptors have been cloned and are known to bind certain sex hormones (neurosteroids) in the brain. Sigma-1 receptors regulate glutamate NMDA receptor function and the release of neurotransmitters such as dopamine, and are implicated in learning and memory, and in certain neuropsychiatric disorders. In particular, as several antipsychotics can bind to sigma-1 receptors, sigma-1 receptor ligands have been proposed as being of potential use in the treatment of schizophrenia. In clinical trials, sigma-1 receptor ligands failed to improve acute psychotic symptoms of schizophrenia but, interestingly, were shown in a few studies to attenuate negative symptomatology in schizophrenic patients. Preclinical studies, on the other hand, indicate that selective sigma-1 receptor agonists affect higher-ordered brain functions including learning and memory, cognition, and mood. These results implicate therapeutic potentials of sigma-1 agonists in depression and senile dementia. Indeed, a sigma-1 receptor agonist, igmesine, has been shown to improve depression in a clinical trial. The most distinctive feature of the action of sigma-1 receptor ligands is their "modulatory" role. In behavioral studies of depression and memory, they exert beneficial effects only when brain functions are perturbed. Given the recently accumulated preclinical and clinical data, it is time to reconstruct the concept of sigma-1 receptors and the associated pathophysiological conditions that ligands of these receptors target. This would allow clinical trials to be performed more efficiently, and the results may confirm a long-speculated possibility that sigma-1 receptor ligands represent a new class of therapeutic agents for neuropsychiatric disorders. Hayashi, T. and Su, T.P. CNS Drugs, 18, pp. 269-284, 2004.

Involvement of the Sigma1 Receptor in the Modulation of Dopaminergic Transmission by Amantadine Pharmacological effects of amantadine on dopaminergic transmission are proposed to result from an uncompetitive antagonism at glutamate N-methyl-D-aspartate (NMDA) receptors. However, the authors previous studies examining amantadine-mediated dopamine receptor regulation in the rat striatum revealed a discrepancy from a direct interference with glutamate transmission. Preliminary in vitro binding data from the literature suggested the interaction of amantadine with the sigma1 receptor. Therefore, the authors have now further characterized the pharmacological properties of amantadine and memantine at this receptor and investigated its involvement in the modulation of striatal dopaminergic transmission. Their binding studies using [3H]-(+)SKF-10,047 indicated that amantadine and memantine behave as ligands of the sigma(1) receptor in rat forebrain homogenates (Ki values of 7.44 +/- 0.82 and 2.60 +/- 0.62 microm, respectively). In NG108-15 neuroblastoma cells, both drugs (amantadine (100 microm) and memantine (10 microm)) potentiated the bradykinin-induced mobilization of intracellular Ca2+, mimicking the effect of the sigma1 receptor agonist PRE-084 (1 microm). Finally, authors previously showed that in striatal membranes from amantadine-treated rats, the functional coupling of dopamine receptors with G-proteins was enhanced. Similarly, PRE-084 dose-dependently increased the [35S]GTPgammaS binding induced by dopamine (Emax 28 and 26% of basal, 0.3 and 1 mg/kg PRE-084, respectively). By contrast, BD1047, which is without effect on its own, antagonized the effects of amantadine and PRE-084. Together, these data demonstrate that aminoadamantanes behave as sigma1 receptor agonists, and confirm an involvement of this receptor in modulating dopamine receptors exerted by therapeutically relevant concentrations of amantadine. Peeters, M., Romieu, P., Maurice, T., Su, T.P., Maloteaux, J.M., and Hermans, E. European Journal of Neuroscience, 19, pp. 2212-2220, 2004.

Sigma-1 Receptors Potentiate Epidermal Growth Factor Signaling Towards Neuritogenesis in PC12 cells: Potential Relation to Lipid Raft Reconstitution IRP investigators previously demonstrated that overexpression of sigma-1 receptors (sigma-1R) potentiated neurite sprouting caused by nerve growth factor in PC12 cells. In this study authors examined if sigma-1R may be involved in the action of epidermal growth factor (EGF). EGF is conventionally recognized as a mitogenic factor that stimulates only the proliferation of various types of cells, including PC12 cells. Authors found here that in sigma-1 receptor-overexpressing PC12 cells (sigma-1R OE cells), EGF markedly stimulates neuritogenesis without affecting cellular proliferation. EGF receptors (EGFR) are largely reduced in lipid rafts and are enriched in non-raft regions in sigma-1R OE cells. The enrichment of EGFR in the non-raft region is correlated with enhanced downstream signaling of EGFR including the phosphorylation of both EGFR and extracellular signal-regulated kinases (ERKs). Destruction of cholesterol-containing rafts by treating cells with methyl-beta-cyclodextrin also causes a reduction of EGFR in lipid rafts, a concomitant increase in the phosphorylation of both EGFR and ERK, and an increase in the EGF-induced neurite sprouting in wildtype cells. Furthermore, while overexpression of sigma-1R increases the level of lipid raft-associated cholesterol, the overexpression alters the levels of gangliosides in lipid rafts: GM1 and GM2 are decreased, whereas GD1a is increased. The authors conclude that sigma-1R cause the remodeling of lipid rafts, at least by increasing the level of lipid raft-associated cholesterol and by altering the levels of certain critical lipid raft-forming gangliosides. Sigma-1R may thus play an important role in directing EGF signaling towards neuritogenesis, perhaps by shifting EGFR from the lipid raft into non-raft regions. Takebayashi, M., Hayashi, T., and Su, T.P. Synapse, 53, pp. 90-103, 2004.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Glial Cell Line-Derived Neurotrophic Factor is Essential for Neuronal Survival in the Locus Coeruleus-Hippocampal Noradrenergic Pathway It has been shown that the noradrenergic (NE) locus coeruleus (LC)-hippocampal pathway plays an important role in learning and memory processing, and that the development of this transmitter pathway is influenced by neurotrophic factors. Although some of these factors have been discovered, the regulatory mechanisms for this developmental event have not been fully elucidated. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor influencing LC-NE neurons. IRP scientists have utilized a GDNF knockout animal model to explore its function on the LC-NE transmitter system during development, particularly with respect to target innervation. By transplanting various combinations of brainstem (including LC) and hippocampal tissues from wildtype or GDNF knockout fetuses into the brains of adult wildtype mice, the authors demonstrate that normal postnatal development of brainstem LC-NE neurons is disrupted as a result of the GDNF null mutation. Tyrosine hydroxylase immunohistochemistry revealed that brainstem grafts had markedly reduced number and size of LC neurons in transplants from knockout fetuses. NE fiber innervation into the hippocampal co-transplant from an adjacent brainstem graft was also influenced by the presence of GDNF, with a significantly more robust innervation observed in transplants from wildtype fetuses. The most successful LC/hippocampal co-grafts were generated from fetuses expressing the wildtype GDNF background, whereas the most severely affected transplants were derived from double transplants from null-mutated fetuses. These data suggest that development of the NE LC-hippocampal pathway is dependent on the presence of GDNF, most likely through a target-derived neurotrophic function. Quintero, E.M., Willis, L.M., Zaman, V., Lee, J., Boger, H.A., Tomac, A., Hoffer, B.J., Stromberg, I., and Granholm, A.C. Neuroscience, 124, pp. 137-146, 2004.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

The Solubilizing Detergents, Tween 80 and Triton X-100 Non-Competitively Inhibit Alpha(7)-Nicotinic Acetylcholine Receptor Function in Xenopus Oocytes Because many studies rely upon detergents to solubilize lipophilic agents such as cannabinoid drugs, IRP scientists examined the effect of commonly employed detergents on the function of the cloned alpha(7) subunit of the nicotinic ACh receptor. Homomeric alpha(7) receptors were expressed in Xenopus oocytes and the two-microelectrode voltage-clamp technique was used to assess their electrophysiological properties. The detergents Tween 80 and Triton X-100 reversibly inhibited ACh (100microM)-induced inward currents in a concentration-dependent manner, with IC(50) values of 610nM and 1.4microM, respectively. The effects of these detergents were independent of membrane potential, and they were not meditated by endogenous Ca(2+)-dependent Cl(-) channels, since they were unaffected by intracellularly injected BAPTA, and recorded in Ca(2+)-free bathing solution containing 2mM Ba(2+). Both detergents also decreased the maximal effect of ACh, without significantly affecting its EC(50), indicating a non-competitive interaction with the nACh alpha(7) receptors. In contrast to the effects of these detergents, authors found that cholic acid (10microM), DMSO (10microM) and Tocrisol((R)) (0.01% v/v) did not cause a significant effect on nicotinic responses. In conclusion, authors demonstrate that the detergents Tween 80 and Triton X-100 are potent inhibitors of neuronal nACh alpha(7) receptors expressed in Xenopus oocytes, and suggest that studies utilizing these detergents to solubilize lipophilic drugs be scrutinized for such effects. Oz, M., Spivak, C.E. and Lupica, C.R. Journal of Neuroscience Methods, 137, pp. 167-173, 2004.

Kinetics of Beta-Funaltrexamine Binding to Wild-Type and Mutant Micro-Opioid Receptors Expressed in Chinese Hamster Ovary Cells The two-stage reaction whereby the antagonist beta-funaltrexamine (beta-FNA) binds covalently to micro opioid receptors makes it a highly discriminating probe into the tertiary structure of the receptor's recognition pocket. To obtain a quantitative measure of how well this pocket is preserved in a mutated form of the receptor, in which His-297 is substituted with glutamine, IRP investigators employed [3H]-beta-FNA to evaluate the kinetic rate constants for both the reversible as well as the irreversible stages of its binding to wild-type and mutant H297Q micro receptors stably expressed in Chinese hamster ovary cells. The expression levels of the wild-type and mutant H297Q receptors were matched by exploiting the variation in receptor density as a function of plating day and by raising the expression level by pretreatment with naloxone. Authors found that all of the kinetic rate constants for [3H]-beta-FNA were diminished by about one-half at the mutant H297Q micro receptors with respect to wild-type receptors. By comparison, the association rate constant of [3H]-naloxone likewise decreased by one-half; however, the dissociation rate constant increased 5-fold at the mutant H297Q receptor. The authors conclude that the mutation has had only minor influence on the recognition site and that the function of position 297 is more likely as a link in the transduction chain. Spivak, C.E. and Beglan, C.L. Synapse, 52, pp. 123-135, 2004.

Molecular Neuropsychiatry Section, Molecular Neuropsychiatry Research Branch

Nicotine Dependence Criteria of the DIS and DSM-III-R: A Factor Analysis This paper reports a factor analysis of the symptoms of nicotine dependence that were determined in an assessment of 821 current cigarette-smoking research volunteers, according to the Diagnostic and Statistical Manual of Mental Disorders, 3rd edition, revised (DSM-III-R) of the American Psychiatric Association as well as an analysis of a subset who unsuccessfully attempted to quit (n=636). In the total sample, two factors with eigenvalues greater than 1 accounted for 62.7% of the variance. When the factor analysis was repeated with the subset of research volunteers who unsuccessfully attempted to quit, only one DSM-III-R nicotine dependence symptom loaded on the second factor. This finding suggests that the two-factor structure found in this and a previous factor analysis study of the nicotine dependence segment of the DSM-III-R may be an artifact of the skipout pattern of the DSM-III-R, which assumes that smokers who have not attempted to quit have not experienced withdrawal symptoms or used tobacco to avoid these symptoms. Goodness-of-fit measures suggested that the two-factor structure is a better fit than the one-factor structure for both the total population and the subset who unsuccessfully attempted to quit or cut down. The present study's sample of current smokers who had not attempted to quit (n=185) was too small to permit factor analyses. Further work with other large samples from the general population of current smokers who have unsuccessfully attempted to quit as well as those who have not attempted to quit will enhance understanding of the factor structure of the nicotine dependence segment of the DSM-III-R and clarify the effect of the skipout pattern on its factor structure. Radzius, A., Gallo, J., Gorelick, D., Cadet, J.L., Uhl, G., Henningfield, J. and Moolchan, E. Nicotine and Tobacco Research, 6, pp. 303-308, 2004.

Sex-related Differences in a Gambling Task and Its Neurological Correlates IRP scientists investigated sex-related differences in task performance and brain activity in the orbitofrontal cortex (OFC) and dorsolateral prefrontal cortex (DLPFC) during performance of a decision-making task (the Iowa Gambling Task). When men and women were examined separately, men activated extensive regions of the right lateral OFC and right DLPFC, as well as the left lateral OFC. In contrast, women activated the left medial OFC. Examining sex differences directly, men showed better task performance and greater lateralized brain activity to the right hemisphere than women. This was exemplified by greater activation in a large area of the right lateral OFC of men during their performance of the Iowa Gambling Task. In contrast, women had greater activation in the left DLPFC, left medial frontal gyrus and temporal lobe during this task. Thus, brain mechanisms engaged by men and women when solving the same decision-making task are different. These observations indicate that sex-related differences contribute to the heterogeneity observed in both normal and abnormal brain functioning. These results also provide further evidence of sexual dimorphism in neurocognitive performance and brain function. Bolla, K.I., Eldreth, D.A., Matochik, J.A. and Cadet, J.L. Cerebral Cortex, VOLUME, PAGES, 2004.

Histological Evidence Supporting a Role for the Striatal Neurokinin-1 Receptor in Methamphetamine-induced Neurotoxicity in the Mouse Brain Several studies have documented the effect of methamphetamine (METH) on the toxicity of the dopamine (DA) terminals of the striatum but only a few studies have assessed the damaging effects of METH on striatal neurons postsynaptic to the nigrostriatal DA terminals. In the present study, IRP investigators employed histological methods to study the effect of METH on DA terminals and striatal neurons. They also assessed the role of the striatal neurokinin-1 (NK-1) receptor on pre- and post-synaptic METH-induced damage. Male mice were treated with METH (10 mg/kg) four times at 2-h intervals and were sacrificed 3 days after the treatment. A number of animals received the non-peptide NK-1 receptor antagonist WIN-51,708 (10 mg/kg) 30 min before the first and fourth injections of METH. Immunocytochemical staining for tyrosine hydroxylase (TH) showed significant deficits throughout all aspects of the caudate-putamen in animals exposed to METH. Pretreatment with WIN-51,708 prevented the METH-induced loss of TH immunostaining. Sections from a separate set of mice were stained with Fluoro-Jade B (FJB), a fluorochrome that binds specifically to degenerating fibers and cell bodies of neurons. Treatment with METH shows Fluoro-Jade B positive cell bodies in the striatum and pretreatment with WIN-51,708 abolished Fluoro-Jade B staining. Moreover, double labeling with Fluoro-Jade B and glial fibrillary acidic protein (GFAP) shows reactive astrocytosis in the area adjacent to the Fluoro-Jade B-positive cells but no Fluoro-Jade B staining of the astrocytes. This observation suggests that the degenerating cells must be striatal neurons and not astrocytes. The data demonstrate that METH induces pre- and post-synaptic damage in the striatum and the damage can be prevented with pharmacological blockade of the NK-1 receptor. These findings represent a new direction in the study of the mechanism of toxicity to METH and could be useful in the treatment of some neurological disorders. Yu, J., Wang, J., Cadet, J.L. and Angulo, J.A. Brain Research, 1007, pp. 124-131, 2004.

Paroxetine Retards Disease Onset and Progression in Huntington Mutant Mice IRP scientists report that administration of paroxetine, a widely prescribed antidepressant drug that acts by inhibiting reuptake of the neurotransmitter serotonin, suppresses the neurodegenerative process and increases the survival of huntington mutant mice, an animal model of Huntington's disease (HD). Paroxetine attenuated motor dysfunction and body weight loss and improved glucose metabolism in the HD mice. Paroxetine was beneficial when treatment was initiated before or after the onset of motor dysfunction, suggesting a potential for such antidepressant drugs in the treatment of presymptomatic and symptomatic HD patients. Duan, W., Guo, Z., Jiang, H., Ladenheim, B., Xu, X., Cadet, J.L. and Mattson, M.P. Annals of Neurology, 55, pp. 590-594, 2004.

Neural Protection and Regeneration Section, Molecular Neuropsychiatry Research Branch

Neuroprotective Effects of Diadenosine Teraphosphate In Animal Models of Stroke and Parkinson's Disease Diadenosine tetraphosphate (AP4 A), 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. We 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., Su, T.P., Tsao, L.I. and Thiemermann, C. Journal of Neuroscience, 23, pp. 7958-7965, 2003.

Clinical Psychopharmacology Section, Medications Discovery Research Branch

Identification and Characterization of a Novel Allosteric Modulator (SoRI-6238) of the Serotonin Transporter In the present study, IRP scientists describe a novel agent, SoRI-6238 (ethyl 5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-ylcarbamate) that partially inhibits 5-HT transporter (SERT) binding and allosterically modulates SERT function. Membranes were prepared from rat brain. SoRI-6238 partially inhibited SERT binding to brain membranes with a plateau at about 40% of control. SoRI-6238 fully inhibited NET and DAT binding with IC50 values of 12.1 µM and 5.8 µM, respectively. The apparent Kd of [125I]RTI-55 binding to SERT increased, and then reached a plateau with increasing concentrations of SoRI-6238. SoRI-6238 fully inhibited [3H]5-HT uptake, acting to decrease the Vmax (non-competitive inhibition). In kinetic experiments, SoRI-6238 slowed the dissociation of [125I]RTI-55 from SERT and slowed the initial association rate. The authors conclude that SoRI-6238 partially inhibits SERT binding and function, most likely via an allosteric mechanism. Nandi, A., Dersch, C.M., Kulshrestha, M., Ananthan, S. and Rothman, R.B. Synapse, 53, pp. 176-183, 2004.

3,4-Methylenedioxymethamphetamine (MDMA) Administration to Rats Decreases Brain Tissue Serotonin but Not Serotonin Transporter Protein and Glial Fibrillary Acidic Protein Previous experiments conducted in this laboratory showed that administration of high-dose D-fenfluramine (D-FEN) and p-chloroamphetamine (PCA) decreased 5-HT transporter (SERT) binding and tissue 5-HT by 30% to 60% in caudate and whole brain tissue 2-days and 2-weeks after drug administration. However, protein expression as determined by Western blot analysis, did not change in either tissue or time point, except for a 30% decrease in the caudate 2-days after PCA administration. In the present study, Authors examined the effect of MDMA and 5,7-dihydroxytryptamine (5,7-DHT) on tissue 5-HT levels and the protein expression level of SERT and glial fibrillary acidic protein (GFAP), a validated neurotoxicity marker. The authors hypothesized that MDMA administration would decrease SERT expression. Two weeks after MDMA administration (7.5 mg/kg i.p., q 2hr x 3 doses) or two weeks after i.c.v. administration of 5,7,-DHT (150 µg/rat), male Sprague-Dawley rats were sacrificed, and the caudate, cortex and hippocampal tissue collected. Western blots for SERT and GFAP were generated using published methods. Tissue 5-HT levels were determined by HPLC coupled to electrochemical detection. Results indicated that MDMA treatment decreased tissue 5-HT in cortex, hippocampus and caudate by about 50%. However, MDMA treatment had no significant effect on expression level of SERT and GFAP in any brain region. In contrast, 5,7-DHT reduced tissue 5-HT by more than 90%, decreased SERT protein expression by 20% to 35%, and increased GFAP by 30% to 39%. These data suggest the MDMA treatment regimen used here does not cause degeneration of 5-HT nerve terminals. Viewed collectively with previous results from this laboratory and other published data, these data indicate that MDMA-induced persistent 5-HT depletion may occur in the absence of axotomy. Wang, X., Baumann, M.H., Xu, H. and Rothman, R.B. Synapse, 53, pp. 240-248, 2004.

Medicinal Chemistry Section, Medications Discovery Research Branch

The Effect of 6-substituted-4',4''-Difluorobenztropines on Monoamine Transporters and the Muscarinic M1 Receptor A series of racemic 6-hydroxy and carboalkoxy substituted-4',4''-difluorobenztropines was synthesized and evaluated at the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters as well as the muscarinic M1 receptor. Each of the analogs displaced [³H]WIN 35,428 from DAT with a range of affinities (K_i= 5.81 to 175 nM) and [³H]pirenzepine from muscarinic M1 receptors, with a range of affinities (K_i = 27.0-8430 nM). Binding affinities at the SERT and NET were generally low. A comparison of M1 with DAT binding affinities suggests that within this series of compounds, binding at the two sites is related, however, these hydroxy and carboalkoxy substitutions on the 6,7-bridgehead may be exploited to provide selective and potent DAT ligands. In vivo studies and the synthesis and pharmacological evaluation of enantiomerically pure derivatives is currently underway. Grundt, P., Kopajtic, T. A., Katz, J.L. and Newman, A.H. Bioorganic and Medicinal Chemistry Letters, 14, pp. 3295-3298, 2004.

Structure-Activity Relationships at Monoamine Transporters for a Series of N-Substituted-3a-(Bis[4-fluorophenyl]methoxy)tropanes: Comparative Molecular Field Analysis, Synthesis and Pharmacological Evaluation The development of structure-activity relationships (SAR) with divergent classes of monoamine transporter ligands and comparing their effects in animal models of cocaine abuse has provided insight into the complex relationship between structure, binding profiles and behavioral activity. Many 3_(diphenylmethoxy)tropane (benztropine) analogues are potent dopamine uptake inhibitors but exhibit behavioral profiles that differ from cocaine and other compounds in this class. One of the most potent and dopamine transporter (DAT)-selective N-substituted benztropine analogues (N-(4-phenyl-n-butyl)-3α-(bis[4-fluorophenyl]methoxy) tropane; 1c) is devoid of cocaine-like behaviors in rodent models but is also highly lipophilic (cLogD = 5.01), which compromises its water solubility and may adversely affect its pharmacokinetic properties. In order to further explore the SAR in this series, and ultimately design dopamine uptake inhibitors, with favorable lipophilicities for drug development, a Comparative Molecular Field Analysis (CoMFA) was performed on a set of benztropine analogs previously synthesized in this laboratory. The CoMFA field analysis on the statistically significant (r²_cv = 0.632; r²_ncv = 0.917) models provided valuable insight into the structural features required for optimal binding to the DAT, which was used to design a series of novel benztropine analogs with heteroatom substitutions at the tropane N-8. These compounds were evaluated for binding at DAT, serotonin (SERT) and norepinephrine (NET) transporters, and muscarinic M1 receptor, in rat brain. Inhibition of [³H]DA uptake in synaptosomes was also evaluated. Most of the analogues showed high DAT affinity (12-50 nM), selectivity (10-120 fold), potent inhibition of dopamine uptake and lower lipophilicities as predicted by cLogD values than the parent drug. Selected compounds are currently being evaluated in animal models of cocaine abuse. Kulkarni, S.S., Grundt, P., Kopajtic, T., Katz, J. L. and Newman, A. H. Journal of Medicinal Chemistry, 47, pp. 3388-3398, 2004.

Neurobiology of Relapse Section, Behavioral Neuroscience Research Branch

The Anxiogenic Drug Yohimbine Reinstates Methamphetamine Seeking in a Rat Model of Drug Relapse Brain noradrenaline is involved in footshock stress-induced reinstatement of drug seeking in a rat relapse model. Here, IRP scientists studied whether yohimbine, an alpha-2 adrenoceptor antagonist that increases noradrenaline release and induces anxiety-like responses in human or non-human subjects, would reinstate methamphetamine seeking in rats. In Exp. 1, the effect of yohimbine (1.25-2.5 mg/kg) on reinstatement was compared to that of intermittent footshock (5 min; 0.2-0.6 mA) in rats that were trained to lever press for intravenous methamphetamine (9-11 days) and subsequently underwent 7 days of extinction training. In Exp. 2, the effect of yohimbine on reinstatement of drug seeking was determined during early (1 day) and late (21 or 51 days) withdrawal periods. On the test days, rats were first given 3-h extinction sessions and were then tested for reinstatement induced by yohimbine. In Exp. 1, both yohimbine and footshock stress reinstated methamphetamine seeking after extinction. In Exp. 2, extinction responding was higher after 21 or 51 withdrawal days than after 1 withdrawal day. In contrast, no significant time-dependent changes in yohimbine-induced reinstatement were observed. Results indicate that the anxiogenic drug yohimbine is a potent stimulus for reinstatement of methamphetamine seeking in a rat relapse model. Shepard, J.D., Bossert, J.M., Liu, S., Shaham, Y. Biological Psychiatry, 55, pp. 1082-1089, 2004.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

On the Molecular Basis of the Receptor Mosaic Hypothesis of the Engram This paper revisits the so-called "receptor mosaic hypothesis" for memory trace formation in the light of recent findings in "functional (or interaction) proteomics." The receptor mosaic hypothesis maintains that receptors may form molecular aggregates at the plasma membrane level representing part of the computational molecular networks. Specific interactions between receptors occur as a consequence of the pattern of transmitter release from the source neurons, which release the chemical code impinging on the receptor mosaics of the target neuron. Thus, the decoding of the chemical message depends on the receptors forming the receptor mosaics and on the type of interactions among receptors and other proteins in the molecular network with novel long-term mosaics formed by their stabilization via adapter proteins formed in target neurons through the incoming neurotransmitter code. The internalized receptor heteromeric complexes or parts of them may act as transcription factors for the formation of such adapter proteins. Receptor mosaics are formed both at the pre- and postsynaptic level of the plasma membranes and this phenomenon can play a role in the Hebbian behavior of some synaptic contacts. The appropriate "matching" of the pre- with the postsynaptic receptor mosaic can be thought of as the "clamping of the synapse to the external teaching signal." According to the present hypothesis the behavior of the molecular networks at plasma membrane level to which the receptor mosaics belong can be set in a "frozen" conformation (i.e. in a frozen functional state) and this may represent a mechanism to maintain constant the input to a neuron. Thus, authors are suggesting that molecular networks at plasma membrane level may display multiple "attractors" each of which stores the memory of a specific neurotransmitter code due to a unique firing pattern. Hence, this mechanism may play a role in learning processes where the input to a neuron is likely to remain constant for a while. Agnati, L.F., Ferre, S., Leo, G., Lluis, C., Canela, E.I., Franco, R. and Fuxe, K. Cellular and Molecular Neurobiology, 24, pp. 501-516, 2004.

Striatal Plasticity at the Network Level: Focus on Adenosine A2A and D2 Interactions in Models of Parkinson's Disease Behavioral and microdialysis studies have been performed on antagonistic A(2A)/D(2) interactions in animal models of Parkinson's Disease. The behavioral analysis involved studies on locomotor activity in reserpinized mice, haloperidol-induced catalepsy in rats and rotational behavior in rats with unilateral 6-OHDA lesions of the ascending DA pathways (Ungerstedt model). Dual probe microdialysis studies were indirectly performed on the striatopallidal GABA neurons by studying extracellular glutamate levels in the striatum and globus pallidus of the awake freely moving rat. The striatum was perfused with A(2A) and/or D(2) agonists via reverse microdialysis. The results show that the A(2A) antagonists SCH58261 and KF17837 can increase locomotor activity in reserpinized mice and produce contralateral rotational behavior only after administration of subthreshold doses of l-DOPA or the D(2) like agonist quinpirole. Furthermore, antagonizing the A(2A) receptor (R) reduced haloperidol induced catalepsy. The behavioral results underline the view that A(2A) antagonists act by blocking A(2A) R in A(2A)/D(2) heterodimers where A(2A) R inhibits the D(2) R transduction and D(2) inhibits the adenylate cyclase (AC) activated by A(2A) R. The microdialysis studies show that the A(2A) agonist CGS21680 striatally coperfused with the D(2) agonist quinpirole more potently counteract the D(2) agonist (quinpirole) induced reduction of pallidal glutamate levels in the DA denervated vs the control striatum indicating an enhancement of the inhibitory A(2A)/D(2) interaction. In the DA denervated but not in the control striatum the A(2A) agonist CGS21680 could strongly increase striatal glutamate levels, indicating an increased receptor signaling in the A(2A) R located on the striatal glutamate terminals, where also D(2) like R exist, here probably as D(4). Thus, the signaling of this A(2A) R may be set free by the loss of D(4) tone on the AC activated by A(2A) in this postulated A(2A)/D(4) heteromer on the glutamate terminals. Taken together, the results indicate that the antiparkinsonian actions of A(2A) antagonists probably are produced by blockade of A(2A) R in the A(2A)/D(2) heterodimers mainly located in the striatopallidal GABA neurons. Tanganelli, S., Sandager Nielsen, K., Ferraro, L., Antonelli, T., Kehr, J., Franco, R., Ferre, S., Agnati, L. F., Fuxe, K., and Scheel-Kruger, J. Parkinsonism and Related Disorders, 10, pp. 273-280, 2004.

Adenosine A2A-dopamine D2 Receptor-receptor Heteromers. Targets for Neuro-psychiatric Disorders Emerging evidence shows that G protein-coupled receptors can form homo- and heteromers. These include adenosine A(2A) receptor-dopamine D(2) receptor heteromers, which are most probably localized in the dendritic spines of the striatopallidal GABAergic neurons, where they are in a position to modulate glutamatergic neurotransmission. The discovery of A(2A) receptor-dopamine D(2) receptor heteromers gives a frame for the well-known antagonistic interaction between both receptors, which is the bases for a new therapeutic approach for neuro-psychiatric disorders, such as Parkinson's disease and schizoprenia. The present review deals mainly with the biochemical and molecular aspects of A(2A) receptor-dopamine D(2) receptor interactions. Recent results at the molecular level show that A(2A) receptor-dopamine D(2) receptor heteromers represent the first example of epitope-epitope electrostatic interaction underlying receptor heteromerization. Most probably A(2A) receptor-D(2) receptor heteromerization is not static, but subject to a dynamic regulation, related to the phosphorylation dependence of the A(2A) receptor epitope and to the ability of the D(2) receptor epitope to bind different partners. Finding out the mechanisms involved in this dynamic regulation can have important implications for the treatment of basal ganglia disorders, schizophrenia and drug addiction. Ferre, S., Ciruela, F., Canals, M., Marcellino, D., Burgueno, J., Casado, V., Hillion, J., Torvinen, M., Fanelli, F., Benedetti, Pd. P., Goldberg, S.R., Bouvier, M., Fuxe, K., Agnati, L.F., Lluis, C., Franco, R. and Woods, A. Parkinsonism and Related Disorders, 10, pp. 265-271, 2004.

Neuropsychopharmacology Section, Behavioral Neuroscience Research Branch

Blockade of Mesolimbic Dopamine D3 Receptors Inhibits Stress-induced Reinstatement of Cocaine-seeking in Rats IRP scientists have previously reported that selective dopamine D3 receptor blockade by the novel D3 receptor antagonist SB-277011A inhibits cocaine's reinforcing action and cocaine-triggered reinstatement (relapse) of cocaine-seeking behavior. In the present study, these researchers further demonstrated that systemic injections of SB-277011A also dose-dependently inhibits stress-induced reinstatement of cocaine-seeking behavior in animals completely extinguished from their drug-taking habits. To determine the locus of action in the brain, SB-277011A was locally microinjected into the bilaterally nucleus accumbens and the dorsal striatum. SB-277011A, when administered into the nucleus accumbens, but not into the dorsal striatum, significantly blocked stress-induced reinstatement. These findings suggest that the mesolimic dopamine D3 receptor plays an important role in mediating stress-induced reinstatement, and that DA D3 receptor antagonists are worthy of further investigation as potential anti-addiction medications. Xi, Z.X., Gilbert, J., Campos, A.C., Kline, N., Ashby, C.R. Jr., Hagan, J.J., Heidbreder, C.A. and Gardner, E.L. Psychopharmacology, 2004 Apr 9 [Epub ahead of print].

The Basolateral Complex of the Amygdala Mediates the Modulation of Intracranial Self-stimulation Threshold by Drug-associated Cues Environmental cues (sights, smells, sounds) that have been previously associated with drug-taking are powerful triggers that provoke relapse to drug-seeking and drug-taking behavior. IRP scientists have previously reported that the ability of such environmental cues to trigger relapse depends upon the intact functioning of the basolateral amygdaloid nucleus in the brain. IRP scientists have also previously shown that such environmental cues alter brain-stimulation reward, and that electrical stimulation of the amygdaloid nucleus in the brain alters brain-reward functions. Now, these researchers have discovered that the ability of drug-associated environmental cues to alter brain-reward functions depends upon the intact functioning of the basolateral amygdaloid nucleus in the brain. Thus, the basolateral amygdala is necessary for cues associated with previous drug exposure to modulate reward functions within the classically-described reward circuitry of the brain. These findings have implications for understanding the brain substrates that underlie the motivation to engage in drug-taking behavior, and may help to elucidate the brain mechanisms underlying drug craving. Hayes, R.J. and Gardner, E.L. European Journal of Neuroscience, 20, pp. 273-280, 2004.

Opiate Tolerance by Heroin Self-administration: An fMRI Study in Rat Functional magnetic resonance imaging (fMRI) was employed to determine whether repeated heroin self-administration produces tolerance or sensitization in the rat brain. Twelve rats were evenly divided into saline and heroin self-administration groups. There was a progressive increase in daily heroin intake during the 8-9 days of heroin self-administration training. Within 24 hr after the last session of daily heroin self-administration, acute heroin administration induced regional blood oxygen level-dependent (BOLD) signals in the brains of both groups of rats. The positive BOLD signals appeared mainly in the cortical regions, including the prefrontal cortex, cingulate, and olfactory cortex, while the negative BOLD signals were predominantly located in subcortical regions such as caudate and putamen, nucleus accumbens, thalamus, and hypothalamus. However, the number of activated voxels was significantly fewer, and the BOLD-signal intensity was significantly less in heroin self-administration rats in regions of prefrontal cortex, nucleus accumbens, and thalamus. Application of gamma-vinyl GABA, an irreversible GABA-transaminase inhibitor, which blocks heroin-induced increase in BOLD signal in na•ve rats, failed to block opiate actions in the heroin self-administration rats. Together, these data suggest that repeated heroin self-administration produces tolerance or desensitization of opiate actions in the rat brain, which may in turn potentiate drug self-administration behavior and drug intake. Xi, Z.X., Wu, G., Stein, E.A. and Li, S.J. Magnetic Resonance in Medicine, 52, pp. 108-114, 2004.

Attenuation of fMRI Brain Response to Heroin Correlates with Reinstatement of Heroin-seeking in Rats IRP scientists further investigated the possible correlation between heroin-induced brain BOLD signal responses and heroin-triggered reinstatement (relapse) of drug-seeking behavior. Rats that had been previously heroin self-administering displayed robust reinstatement of drug-seeking behavior triggered by an acute heroin priming, whereas saline control rats did not show such a behavioral response. Regional positive or negative blood oxygen level-dependent (BOLD) signals, induced by heroin priming injection, were observed in both groups of rats during fMRI scanning. However, heroin-induced positive BOLD signals in the prefrontal cortex and parietal cortex were significantly attenuated in heroin self-administering rats. Similarly, heroin-induced negative BOLD signals in subcortical regions, such as the nucleus accumbens and hippocampus, were also significantly attenuated in both signal intensity and number of activated brain voxels in heroin self-administration rats. These data demonstrate that heroin-induced reinstatement of drug-seeking behavior coincides with a significant reduction in opiate-induced brain activity in heroin self-administration rats, suggesting a possible role of opiate tolerance in mediating reinstatement of drug-seeking behavior. Luo, F., Xi, Z.X., Wu, G., Liu, C., Gardner, E.L. and Li, S.J. Neuroimage, 22, pp. 1328-1335, 2004.

The Metabotropic Glutamate Receptor 5 Antagonist MPEP Blocks Reinstatement of Drug-seeking Triggered by Cocaine But Not By Stress or Cues Repeated exposure to cocaine has been shown to alter glutamate transmission in rat brain. Recent studies have demonstrated that mutation of the mGluR5 gene or selective mGluR5 blockade by 2-methyl-6-(phenylethynyl)pyridine (MPEP) significantly inhibits cocaine self-administration and cocaine-induced conditioned place preference. In the present study, IRP researchers investigated whether MPEP attenuates reinstatement (relapse) of cocaine-seeking behavior triggered by cocaine, stress or cocaine-associated environmental cues. Rats were allowed to self-administer cocaine until stable daily self-administration was reached. This was then followed by a once-daily extinction session until extinction criteria were met. After extinction, re-exposure to cocaine, stress or cocaine-associated environmental cues in separate groups of animals robustly reinstated the extinguished cocaine-seeking behavior. MPEP dose-dependently attenuated cocaine-induced reinstatement (maximally by 50%), but not reinstatement produced by stress or cues previously associated with cocaine self-administration. In addition, MPEP also produced a maximal 50% reduction of the 'break-point' (maximal work load to receive a cocaine infusion) under a progressive-ratio reinforcement schedule. These data suggest that mGluR5 receptors are involved in cocaine-induced reinstatement and reinforcement, and that mGluR5 antagonists may represent a promising new class of medication for the prevention of relapse to drug use. Xi, Z.X., Gilbert, J., Campos, A.C., Ashby, C.R. Jr. and Gardner, E.L. College on Problems of Drug Dependence, 66th Annual Scientific Meeting, San Juan, Puerto Rico, June 12-17, 2004.

The Dopamine D3 Receptor Antagonist SB-277011A Antagonizes THC-enhanced Brain-Stimulation Reward in Rats Marijuana is a widely used botanical with significant abuse liability, and there is no widely effective medication available to assist marijuana users in breaking the habit. Delta-9-tetrahydrocannabinol (THC), the active constituent in marijuana, stimulates the brain mesolimbic dopamine (DA) system and enhances brain stimulation reward, like other drugs with abuse potential. The mesolimbic DA system is highly enriched with DA D3 receptors, which have extraordinarily high affinity for endogenous DA. IRP researchers have previously shown that blockade of brain DA D3 receptors by SB-277011A attenuates cocaine-enhanced brain reward and cocaine-induced reinstatement of drug-seeking behavior. SB-277011A has also been previously shown to attenuate nicotine-triggered relapse to nicotine-seeking behavior in the reinstatement model. In the present study, IRP researchers further demonstrated that SB-277011A similarly inhibited THC-induced electrical brain stimulation reward, suggesting that DA D3 receptors also play an important role in mediating marijuana-enhanced brain stimulation reward, and that the D3 receptor antagonist SB-277011A may be promising in the treatment of marijuana dependence. Gilbert, J., Campos, A.C., Ashby, C.R. Jr., Heidbreder, C.A. and Gardner, E.L. International Cannabinoid Research Society, 14th Annual Meeting, Paestum, Italy, June 22 — 27, 2004.