Research Findings - Intramural Research

Development and Plasticity Section, Cellular Neurobiology Research Branch

Intranigral Transplants of Immortalized GABAergic Cells Decrease the Expression of Kainic Acid-Induced Seizures in the Rat Repeated systemic administration of low doses of kainic acid (KA) induces spontaneous convulsive seizures [Hellier et al., Epilepsy Res 31, pp. 73-84, 1998]. In this study, male Sprague-Dawley animals received intranigral transplants of a control cell line M213-2O, or a cell line transfected with human GAD67 cDNA (M213-2O CL4) [Conejero-Goldberg, C., Exp Neurol.161, pp. 453-461, 2000], or no transplant. Eight weeks after transplantation surgery, KA was administered (5 mg/kg/h) until animals reached stage V seizures as described by Racine [Racine, R.J., Electroencephalogr Clin Neurophysiol. 32, pp. 281-294, 1972]. The group transplanted with CL4 required a larger dose of KA and a longer latency to reach a stage V seizure. In addition, this group exhibited significantly fewer stage III and IV seizures. These results indicate that intranigral transplants of a GABA-producing cell line can decrease the number of kainic acid-induced seizures. Castillo, C.G., Mendoza, S., Freed, W.J., and Giordano, M. Behavioural Brain Research, 171(1), pp. 109-115, 2006.

GABAergic Lineage Differentiation of AF5 Neural Progenitor Cells In Vitro IRP scientists have previously described an immortal rat central-nervous-system progenitor cell line, AF5, which is able to exit the cell cycle and assume a differentiated state with neuronal properties. The phenotypic specification of differentiated AF5 cells, however, is not known. In the present study, when induced to differentiate by serum starvation in Neurobasal medium, AF5 cells down-regulate glial fibrillary acidic protein and up-regulate expression of beta-III-tubulin, medium-molecular-weight neurofilament protein, and neuronal growth-associated protein 43. Expression of the gamma-aminobutyric acid (GABA) lineage marker, glutamic acid decarboxylase 67 (GAD67), increases during differentiation, suggesting that AF5 cells adopt a GABAergic lineage. Time-course analysis of the GABAergic neuron specification transcription factor, Pitx2, by reverse transcription/polymerase chain reaction, has shown an increase in the Pitx2 transcript 48 h after initiation of differentiation. In differentiated AF5 cells, expression of the Pitx2 target gene products GAD65 and GABA transporter-1 increases. Cellular GABA levels in differentiated AF5 cells increase by about 26-fold, and GABA release into the medium is 150-fold higher compared with that of undifferentiated cells. Therefore, AF5 cells can be induced to differentiate to a neuronal phenotype with a GABAergic lineage. Sanchez, J.F., Crooks, D.R., Lee, C.T., Schoen, C.J., Amable, R., Zeng, X., Florival-Victor, T., Morales, N., Truckenmiller, M.E., Smith, D.R., and Freed, W.J. Cell Tissue Research, 324(1), pp. 1-8, 2006.

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

Chronic Antidepressants Potentiate via Sigma-1 Receptors the Brain-Derived Neurotrophic Factor-Induced Signaling for Glutamate ReleaseUp-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-gamma (PLC-gamma)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. J. Biol. Chem. 277, pp. 6520-6529, 2002), IRP investigators examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T.P. Proc. Natl. Acad. Sci. U. S. A. 98, pp. 491-496, 2001), authors examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). Authors found that the BDNF-stimulated PLC-gamma activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-gamma and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release. Yagasaki, Y., Numakawa, T., Kumamaru, E., Hayashi, T., Su, T.P., and Kunugi, H. Journal of Biological Chemistry, 281(18), pp. 12941-12949, 2006.

Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Visualizing Cannabinoid Effects using Brain Slice Imaging and Electrophysiological Approaches The use of electrophysiological recordings in brain slices is now routinely used to assess the actions of cannabinoid ligands within various central nervous system nuclei. In this chapter IRP scientists describe common protocols involving both intracellular and extracellular recording techniques in the hippocampus, where the presynaptic modulatory effects of cannabinoid receptor activation have been studied in detail. In addition to describing the basic electrophysiological setup needed for these recordings, authors will address common technical problems and limitations involved in working with highly lipophilic compounds, such as the cannabinoid ligands, in brain slices. Hoffman, A.F. and Lupica, C.R. Methods in Molecular Medicine, 123, pp. 105-112, 2006.

Proteomics Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Brain Tissue Lipidomics: Direct Probing Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Lipidomics is the new frontier in biomolecular structural studies. Not only are lipids the main components in membranes that define the contours of the cell and its organelles, but they are also used for storage. Lipids form stable noncovalent complexes with proteins as well as with many drugs. Lipids are a storage depot for drugs and certain types of organic molecules. To study lipid composition and distribution, complex and time-consuming techniques are used. However, recent advances in mass spectrometry, mainly matrix-assisted laser desorption/ionization (MALDI) have made it possible to directly probe tissues to study structural components, as well as for the localization of drugs. Direct tissue imaging is a powerful tool as it gives a more complete and accurate structural picture and can trace and follow where drugs localize in tissue with minimal anatomical disruption and a minimum of manipulations. Hence, we believe that in addition to its accuracy and efficiency, this new approach will lead to a better understanding of physiological processes as well as the pathophysiology of disease. Woods, A.S. and Jackson, S.N. American Association of Pharmaceutical Scientists, 8(2), pp. E391-D395, 2006.

IR-MALDI-LDI Combined with Ion Mobility Orthogonal Time-of-Flight Mass Spectrometry Most MALDI instrumentation uses UV lasers. IRP scientists have designed a MALDI-IM-oTOF-MS which employs both a Nd:YAG laser pumped optical parametric oscillator (OPOTEK, lambda = 2.8-3.2 microm at 20 Hz) to perform IR-LDI or IR-MALDI and a Nd:YLF laser (Crystalaser, lambda = 249 nm at 200 Hz) for the UV. Ion mobility (IM) gives a fast separation and analysis of biomolecules from complex mixtures in which ions of similar chemical type fall along well-defined "trend lines". Data shows that ion mobility allows multiply charged monomers and multimers to be resolved; thus, yielding pure spectra of the singly charged protein ion which are virtually devoid of chemical noise. In addition, we have demonstrated that IR-LDI produced similar results as IR-MALDI for the direct tissue analysis of phospholipids from rat brain. Woods, A.S., Ugarov, M., Jackson, S.N., Egan, T., Wang, H.Y., Murray, K.K., and Schultz, J.A. Journal of Proteome Research, 5(6), pp. 1484-1487, 2006.

Decoy Peptides that Bind Dynorphin Noncovalently Prevent NMDA Receptor-Mediated Neurotoxicity Prodynorphin-derived peptides elicit various pathological effects including neurological dysfunction and cell death. These actions are reduced by N-methyl-d-aspartate receptor (NMDAR) but not opioid receptor antagonists suggesting NMDAR-mediation. Here, IRP researchers show that a conserved epitope (KVNSEEEEEDA) of the NR1 subunit of the NMDAR binds dynorphin peptides (DYNp) noncovalently. Synthetic peptides containing this epitope form stable complexes with DYNp and prevent the potentiation of NMDAR-gated currents produced by DYNp. They attenuate DYNp-evoked cell death in spinal cord and prevent, as well as reverse, DYNp-induced paralysis and allodynia. The data reveal a novel mechanism whereby prodynorphin-derived peptides facilitate NMDAR function and produce neurotoxicity. Furthermore, they suggest that synthetic peptides that bind DYNp, thus preventing their interaction with NMDAR, may be novel therapeutic agents for the treatment of spinal cord injury. Woods, A.S., Kaminski, R., Oz, M., Wang, Y., Hauser, K., Goody, R., Wang, H.Y., Jackson, S.N., Zeitz, P., Zeitz, K.P., Zolkowska, D., Schepers, R., Nold, M., Danielson, J., Graslund, A., Vukojevic, V., Bakalkin, G., Basbaum, A., and Shippenberg, T. Journal of Proteome Research, 5(4), pp. 1017-1023, 2006.

In Vivo Electrophysiology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Stability of Substantia Nigra Pars Reticulata Neuronal Discharge Rates during Dopamine Receptor Blockade and its Possible Mechanisms It is hypothesized that substantia nigra pars reticulata neurons become overactive during a deficit of dopamine transmission. In this study, IRP scientists examined how acute dopamine receptor blockade (SCH23390 and eticlopride) affects impulse activity of substantia nigra pars reticulata neurons and their response to iontophoretic gamma-amino-n-butyric acid in awake, unrestrained rats. No changes in discharge rate were found during complete dopamine receptor blockade, but these neurons showed a diminished response to gamma-amino-n-butyric acid, suggesting gamma-amino-n-butyric acid receptor hyposensitivity. This may result from tonic increase in gamma-amino-n-butyric acid input from the striatum and globus pallidus, which are activated during dopamine receptor blockade. As substantia nigra pars reticulata neurons are autoactive and resistant to tonic increases in gamma-amino-n-butyric acid input, changes in their responsiveness to phasic gamma-amino-n-butyric acid inputs, not tonic increase discharge rate, may underlie movement disturbance following dopamine deficit. Windels, F. and Kiyatkin, E.A. NeuroReport, 17(10), pp. 1071-1075, 2006.

GABAergic Mechanisms in Regulating the Activity State of Substantia Nigra Pars Reticulata Neurons Substantia nigra reticulata is the major output structure of the basal ganglia involved in somatosensory integration and organization of movement. While previous work in vitro and in anesthetized animal preparations suggests that these neurons are autoactive and points to GABA as a primary input regulating their activity, single-unit recording coupled with iontophoresis was used in awake, unrestrained rats to further clarify the role of tonic and phasic GABA input in maintenance and fluctuations of substantia nigra reticulata neuronal activity under physiologically relevant conditions. In contrast to glutamate, which was virtually ineffective at stimulating substantia nigra reticulata neurons in awake rats, all substantia nigra reticulata neurons tested were inhibited by iontophoretic GABA and strongly excited by bicuculline, a GABA-A receptor blocker. The GABA-induced inhibition had short onset and offset latencies, a fading response pattern (a rapid decrease in rate followed by its relative restoration), and was independent of basal discharge rate. The bicuculline-induced excitation was inversely related to discharge rate and current (dose)-dependent in individual units. However, the average discharge rate during bicuculline applications at different currents increased to a similar plateau (approximately 60 impulses/s), which was about twice the mean basal rates. The excitatory effects of bicuculline were phasically inhibited or completely blocked by brief GABA applications and generally mimicked by gabazine, another selective GABA antagonist. These data as well as neuronal inhibitions induced by nipecotic acid, a selective GABA uptake inhibitor, suggest that substantia nigra reticulata neurons in awake, quietly resting conditions are under tonic, GABA-mediated inhibition. Therefore, because of inherent autoactivity and specifics of afferent inputs, substantia nigra reticulata neurons are very sensitive to phasic alterations in GABA input, which appears to be the primary factor determining fluctuations in their activity states under physiological conditions. While these cells are relatively insensitive to direct activation by glutamate, and resistant to a continuous increase in GABA input, they appear to be very sensitive to a diminished GABA input, which may release them from tonic inhibition and determine their functional hyperactivity. Windels, F. and Kiyatkin, E.A. Neuroscience, 140(4), pp. 1289-1299, 2006.

General Anesthesia as a Factor Affecting Impulse Activity and Neuronal Responses to Putative Neurotransmitters Although it is evident that general anesthesia should affect impulse activity and neurochemical responses of central neurons, there are limited studies in which these parameters were compared in both awake and anesthetized animal preparations. We used single-unit recording coupled with iontophoresis to examine impulse activity and responses of substantia nigra pars reticulata (SNr) neurons to GABA, glutamate (GLU), and dopamine (DA) in rats in awake, unrestrained conditions and during chloral hydrate anesthesia. SNr neurons in both conditions had similar organization of impulse flow, but during anesthesia, they have lower mean rates and discharge variability than in awake conditions. In individual units, discharge rate in awake, quietly resting rats was almost three-fold more variable than during anesthesia. These cells in both conditions were highly sensitive to iontophoretic GABA, but the response was stronger during anesthesia. In contrast to virtually no responses to GLU in awake conditions, most SNr neurons during anesthesia were excited by GLU; the response occurred preferentially in slow-firing units, which were atypical of awake conditions. Consistent with no postsynaptic DA receptors on SNr neurons, iontophoretic DA was ineffective in altering discharge rates in awake conditions, but often induced weak excitations during anesthesia. Although SNr neurons are autoactive, generating discharges without any excitatory input (i.e., in vitro), their impulse activity and responses to natural neurochemical inputs are strongly affected by general anesthesia. Some alterations appear to be specific to the general anesthetic used, while others probably reflect changes in the activity of afferent inputs, brain metabolism and neurotransmitter uptake that are typical to any type of general anesthesia. Therefore, an awake, freely moving animal preparation appears to be advantageous for studying impulse activity and neurochemical interactions at single-neuron level during physiologically relevant conditions. Windels, F. and Kiyatkin, E.A. Brain Research, 1086(1), pp. 104-116, 2006.

Molecular Biology Unit, Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Characterization of a Mouse Strain Expressing Cre Recombinase from the 3' Untranslated Region of the Dopamine Transporter Locus Dopamine (DA) neurotransmission has been implicated in several neurological and psychiatric disorders. The dopamine transporter (DAT) is highly expressed in dopaminergic neurons of the ventral mesencephalon and regulates neurotransmission by transporting DA back into the presynaptic terminals. To mediate restricted DNA recombination events into DA neurons using the Cre/loxP technology, IRP investigators have generated a knockin mouse expressing Cre recombinase under the transcriptional control of the endogenous DAT promoter. To minimize interference with DAT function by preservation of both DAT alleles, Cre recombinase expression was driven from the 3' untranslated region (3'UTR) of the endogenous DAT gene by means of an internal ribosomal entry sequence. Crossing this murine line with a LacZ reporter showed colocalization of DAT immunocytochemistry and beta-galactosidase staining in all regions analyzed. This knockin mouse can be used for generating tissue specific knockouts in mice carrying genes flanked by loxP sites, and will facilitate the analysis of gene function in dopaminergic neurons. Backman, C.M., Malik, N., Zhang, Y., Shan, L., Grinberg, A., Hoffer, B.J., Westphal, H., and Tomac, A.C. Genesis, 44(8), pp. 383-390, 2006.

Gene Expression Patterns for GDNF and its Receptors in the Human Putamen Affected by Parkinson's Disease: A Real-Time PCR Study Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta superfamily, is a potent trophic factor for dopaminergic neurons of the ventral midbrain, which are known to degenerate during Parkinson's disease (PD). The neuroprotective, neurorestorative, and stimulatory properties of GDNF has prompted numerous suggestions that this trophic factor may be a potential therapeutic tool to treat PD, and it has also been widely speculated that altered GDNF expression levels may be involved in the pathophysiology of the disease. In this study, IRP scientists have investigated if mRNA expression levels for GDNF and/or its receptors are altered during PD in the human putamen, a target area for dopamine neurons of the substantia nigra compacta. Expression levels were analyzed with quantitative real-time reverse transcriptase polymerase reaction (RT qPCR) in post-mortem tissues from PD patients and aged matched controls. Primer pairs specific for GDNF (isoforms I and II), and its receptor molecules, GFRalpha1 and cRET were utilized. GDNF, cRET and GFRalpha1 mRNA expression was clearly detected in the putamen of control and Parkinson's disease patients. A modest but significant upregulation of GDNF mRNA levels (Isoform I) was observed in the putamen of Parkinson's disease patients with a marked loss of nigral neurons. No significant changes were observed for the expression of cRet and GFRa1. These data suggest that the extensive loss of dopaminergic neurons in the substantia nigra, and concomitant loss of striatal dopamine, may induce compensatory changes in the expression of target derived GDNF, but not its receptor system. Backman, C.M., Shan, L., Zhang, Y.J., Hoffer, B.J., Leonard, S., Troncoso, J.C., Vonsatel, P., and Tomac A.C. Molecular and Cellular Endocrinology, 252(1-2), pp. 160-166, 2006.

Neural Protection and Regeneration Section, Molecular Neuropsychiatry Research Branch

Neuroprotective/Neuroregenerative Effects of Bone Morphogenetic Protein 7 and Adenosine A3 Receptor Agonists Authors found that bone morphogenetic protein -7 (BMP7) has neuroprotective and neuroregenerative effects in the CNS. Pretreatment with BMP-7 reduces methamphetamine-induced neuronal injury in primary dopaminergic neuronal culture. BMP-7, given after ischemic brain injury, improves locomotor activity. BMP7 treatment also enhanced immunoreactivity of BrdU in the subventricular zone, lesioned cortex, and corpus callosum. These BrdU positive cells co-labeled with nestin and NeuN. Behavioral and anatomical data suggest that BMP7 promotes neuroregeneration in stroke animals, possibly through the proliferation of new neuronal precursors after ischemia. Authors also found that selective adenosine A3 agonist reduces neurodegeneration induced by cerebral ischemia. The response was mediated through the inhibition of apoptosis. The possibility of endogeneous neuroprotection was further examined in A3R knock-out mice. After cerebral ischemia, an increase in cerebral infarction was found in the A3R knock-outs compared to the A3R wild-type controls, suggesting that A3Rs are tonically activated during ischemia. Inosine, an adenosine analog, also reduces cerebral injury and increases motor functions after brain injury through A3R receptor. Inosine did not alter basal glutamate release nor did it reduce ischemia -evoked glutamate overflow from cerebral cortex. However, inosine antagonized glutamate-induced electrophysiological excitation in cerebral cortical neurons. These data suggest that inosine inhibits glutamate post-synaptic responses and reduces cerebral infarction. Shen, H., Chen, G.J., Harvey, B.K., Bickford P.L., and Wang, Y. Stroke, 36, pp. 654-659, 2005.

Molecular Neurobiology Research Branch

Whole Genome Association for Alcohol Dependence Association genome scanning can identify markers for the allelic variants that contribute to vulnerability to complex disorders, including alcohol dependence. To improve the power and feasibility of this approach, IRP scientists validate "100k" microarray-based allelic frequency assessments in pooled DNA samples. Authors then use this approach with unrelated alcohol dependent vs control individuals sampled from pedigrees collected by the Collaborative Study on the Genetics of Alcoholism (COGA). Allele frequency differences between alcohol-dependent and control individuals are assessed in quadruplicate at 104,268 autosomal SNPs in pooled samples. One hundred thirty eight SNPs provide 1) the largest allele frequency differences between dependent vs control individuals, 2) t values > 3 for these differences and 3) clustering, so that 51 small chromosomal regions contain at least three SNPs that satisfy criteria 1 and 2 above (Monte Carlo p=0.00034). These positive SNP clusters identify interesting genes whose products are implicated in cellular signaling, gene regulation, development, "cell adhesion" and Mendelian disorders. The results converge with previous linkage and association results for alcohol and other addictive phenotypes. The data support polygenic contributions to vulnerability to alcohol dependence and support the idea that the brains of individuals who are vulnerable to alcohol dependence may differ from those who are less vulnerable. These SNPs provide new tools to aid the understanding, prevention and treatment of alcohol abuse and dependence. Johnson, C., Drgon, T., Liu, Q.R., Walther, D., Edenberg, H., Rice, J., Foroud, T., and Uhl, GR. American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 99, pp. 1-10, 2006.

Psychobiology Section, Medications Discovery Research Branch

Potential Medications for Treating Cocaine Abuse Several dopamine (DA) indirect agonists have been proposed as potential medications for treating cocaine abuse. The objective of the present study was to quantify the interactions among cocaine and DA uptake inhibitors or DA releasers in order to better understand how these drugs may be working when administered in combination. The DA uptake inhibitors GBR 12909, WIN 35,428, methylphenidate, indatraline, nomifensine and mazindol, and DA releasers methamphetamine, amphetamine, methcathinone, cathinone, fencamfamine and phentermine were examined alone and in combination with cocaine in rats trained to discriminate cocaine (10 mg/kg, i.p.) from saline injections. All of the DA indirect agonists dose-dependently substituted for cocaine, and shifted the cocaine dose-effect curve leftward. Isobolographic analysis indicated the interactions were generally additive, although both methamphetamine and d-amphetamine were quantitatively determined to be more potent than DA uptake inhibitors in shifting the cocaine dose-effect function to the left. The potential of d-amphetamine as an effective treatment for cocaine abuse, and negative clinical results with dopamine uptake inhibitors suggests that differences in shifts in dose-effect curves should be further examined with emerging clinical data as a predictive index of potential treatments for cocaine abuse. Li, S.M., Campbell, B.L., and Katz, J.L. Journal of Pharmacology and Experimental Therapeutics, 317, pp. 1088-1096, 2006.

Clinical Psychopharmacology Section, Medications Development Research Branch

Interaction of Amphetamines and Related Compounds at the Vesicular Monoamine Transporter Amphetamine-type agents interact with the vesicular monoamine transporter (VMAT2), promoting the release of intravesicular neurotransmitter and an increase in cytoplasmic neurotransmitter. Some compounds, like reserpine, "release" neurotransmitter by inhibiting the ability of VMAT2 to accumulate neurotransmitter in the vesicle, while other types of compounds can release neurotransmitter via a carrier-mediated exchange mechanism. The purpose of this study was to determine, for 42 mostly amphetamine-related compounds, their mode of interaction with the VMAT2. IRP researchers used a crude vesicular fraction prepared from rat caudate to assay VMAT2 activity. Test compounds were assessed in several assays including: a) inhibition of [(3)H]dihydrotetrabenazine binding, b) inhibition of vesicular [(3)H]dopamine uptake, and c) release of pre-loaded [(3)H]dopamine and [(3)H]tyramine. Several important findings derive from this comprehensive study. First, this work indicates that most agents are VMAT2 substrates. Two, these data strongly suggest that amphetamine-type agents deplete vesicular neurotransmitter via a carrier-mediated exchange mechanism rather than via a free-base effect, although this conclusion needs to be confirmed via direct measurement of vesicular pH. Three, these data fail to reveal differential VMAT2 interactions among agents which do and do not produce long-term 5-HT depletion. Four, the data reported revealed the presence of two pools of [(3)H]amine within the vesicle, that which is free, and that which is tightly associated with the ATP/protein complex that helps store amine. Finally, the VMAT2 assays the authors have developed should prove useful for guiding the synthesis and evaluation of novel VMAT2 agents as possible treatment agents for addictive disorders. Partilla, J.S., Dempsey, A.G., Nagpal, A.S., Blough, B.E., Baumann, M.H., and Rothman, R.B. Interaction of Amphetamines and Related Compounds at the Vesicular Monoamine Transporter. J Pharmacol Exp Ther. 2006 [Epub ahead of print].

Medicinal Chemistry Section, Medications Discovery Research Branch

High Affinity Fluorescent Probes for the Dopamine Transporter A series of novel fluorescent ligands was synthesized to identify a high affinity probe that would enable visualization of the dopamine transporter (DAT) in living cells. In this series, fluorescent tags were extended from either the N- or 2-position of a cocaine analogue, 2b-carbomethoxy-3b-(3,4-dichlorophenyl)tropane, using an ethylamino-linker. The resulting 2-substituted- and N-substituted-rhodamine-labeled ligands provided the highest DAT binding affinities expressed in COS-7 cells (Ki= 27 and 18 nM, respectively) in the series. Visualization of the DAT with these fluorescent ligands was demonstrated by confocal fluorescence laser scanning microscopy in stably transfected HEK293 cells. The most effective fluorescent probe in this series, JHC 1-064 is currently being used to visualize dopamine neurons and is enabling the dynamic visualization of DAT trafficking. Cha, J. H., Zou, M.F., Adkins, E.M., Rasmussen, S.G.F., Loland, C.J., Schoenenberger, B., Gether, U., and Newman, A.H. Journal of Medicinal Chemistry, 48, pp. 7513-7516, 2005.

Cooperative Transcription Activation by Nurr1 and Pitx3 Induces Embryonic Stem Cell Maturation to the Midbrain Dopamine Neuron Phenotype Midbrain dopamine neurons (mDNs) play a central role in the regulation of voluntary movement and their degeneration is associated with Parkinson's disease (PD). Cell replacement therapies, and in particular the use of embryonic or adult stem cell-derived dopamine neurons, offer a potential treatment strategy for PD. However, such an approach relies on the efficient generation of mature dopamine neurons that are able to respond appropriately to environmental cues and establish functional connections in the CNS. It is therefore of particular interest to determine the regulatory pathways that underlie mDN maturation from precursors. Here IRP investigators describe factors that promote mDNs maturation and identify a combination of two transcription factors, Nurr1 and PitX3, which synergistically function in this process. Nurr1 and PitX3 coordinately induce mDN maturation in both mouse and human ES cultures, and transplantation of these cells improves motor function in a toxin-based murine model of PD. One experiment in this study used the fluorescent ligand JHC 1-064 to analyze midbrain dopaminergic neurons. Martinat, C., Bacci, J.-J., Leete, T., Kim, J., Vanti, W., Newman, A.H., Cha, J.H., Gether, U., Wang, H., and Abeliovich, A. Proceedings of the National Academy of Sciences U.S.A., 103, pp. 2874-2879, 2006.

TYR95 and ILE172 in Transmembrane Segments I and III of Human Serotonin Transporters Interact to Establish High-Affinity Recognition of Antidepressants In previous studies examining the structural determinants of antidepressant and substrate recognition by serotonin transporters (SERTs), IRP scientists identified Tyr-95 in transmembrane segment 1 (TM1) of human SERT as a major determinant of binding for several antagonists, including racemic citalopram ((±)-CIT). Here authors described a separate site in hSERT TM3 (Ile-172) that impacts (±)-CIT recognition when switched to the corresponding Drosophila SERT residue (I172M). The hSERT I172M mutant displays a marked loss of inhibitor potency for multiple inhibitors such as (±)-CIT, clomipramine, RTI-55, fluoxetine, cocaine, nisoxetine, mazindol, and nomifensine, whereas recognition of substrates, including serotonin and 3,4-methylene dioxymethamphetamine, is unaffected. Selectivity for antagonist interactions is evident with this substitution because the potencies of the antidepressants tianeptine and paroxetine are unchanged. Reduced cocaine analog recognition was verified in photoaffinity labeling studies using [125I]MFZ 2-24. In contrast to the I172M substitution, other substitutions at this position significantly affected substrate recognition and/or transport activity. Additionally, the mouse mutation (mSERT I172M) exhibits similar selective changes in inhibitor potency. Unlike hSERT or mSERT, analogous substitutions in mouse dopamine transporter (V152M) or human norepinephrine transporter (V148M) result in transporters that bind substrate but are deficient in the subsequent translocation of the substrate. A double mutant hSERT Y95F/ I172M had a synergistic impact on (±)-CIT recognition (>10,000- fold decrease in (±)-CIT potency) in the context of normal serotonin recognition. The less active enantiomer (R)-CIT responded to the I172M substitution like (S)-CIT but was relatively insensitive to the Y95F substitution and did not display a synergistic loss at Y95F/ I172M.An hSERT mutant with single cysteine substitutions in TM1 and TM3 resulted in formation of a high affinity cadmium metal coordination site, suggesting proximity of these domains in the tertiary structure of SERT. These studies provided evidence for distinct binding sites coordinating SERT antagonists and revealed a close interaction between TM1 and TM3 differentially targeted by the stereoisomers of CIT. Henry, L.K., Field, J.R., Adkins, E. M., Parnas, M.L., Vaughan, R.A., Zou, M-F., and Newman, A.H., Blakely Journal of Biological Chemistry, 281, pp. 2012-2023, 2006.

Comparative Structure-Activity Relationships of Benztropine Analogues at the Dopamine Transporter and Histamine H1 Receptors Benztropine (BZT) and its analogues inhibit dopamine uptake and bind with moderate to high affinity to the dopamine transporter (DAT). However, many of these compounds, in contrast to other monoamine uptake inhibitors, lack cocaine-like behavioral effects and fail to potentiate the effects of cocaine. The BZT analogues also exhibit varied binding affinities for muscarinic M1 and histamine H1 receptors. In this study a comparative analysis was conducted of pharmacophoric features with respect to the activities of BZT analogues at the DAT and at the histamine H1 receptor. The BZT analogues showed a wide range of histamine H1 receptor (Ki =16-37600 nM) and DAT (Ki=8.5-6370 nM) binding affinities. A stereoselective histamine H1-antagonist pharmacophore, using a five-point superimposition of classical antagonists on the template, cyproheptadine, was developed. A series of superimpositions and comparisons were performed with various analogues of BZT. In general, smaller substituents were well tolerated on the aromatic rings of the diphenyl methoxy group for both the DAT and H1 receptor, however for the H1 receptor, substitution at only one of the aromatic rings was preferred. The substituents at the 2- and N-positions of the tropane ring were preferred for DAT however these groups seem to overlap receptor essential regions in the histamine H1 receptor. Molecular models at the DAT and the histamine H1 receptor provide further insight into the structural requirements for binding affinity and selectivity that can be implemented in future drug design. Kulkarni, S. S., Kopajtic, T., Katz, J. L., Newman, A. H. Bioorganic Medicinal Chemistry, 14, pp. 3625-3634, 2006.

Design and Synthesis of Noncompetitive Metabotropic Glutamate Receptor Subtype 5 Antagonists A series of diaryl amides was designed and synthesized as novel non-ethynyl mGluR5 antagonists. The systematic variation of the pharmacophoric groups led to the identification of a lead compound that demonstrated micromolar affinity for the mGluR5. Further optimization resulted in compounds with improved binding affinities and antagonist profiles, in vitro. The novel series of compounds described in this communication provide structurally distinct probes to investigate the role of mGluR5 in CNS disorders. The structure-activity relationships developed herein has led to the design of a new set of potential mGluR5 antagonists for in vivo investigation that will be reported in due course. Kulkarni, S.S., Nightingale, B., Dersch, C.M., Rothman, R.B., and Newman, A.H. Bioorganic Medicinal Chemistry Letters, 16, pp. 3371-3375, 2006.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Branch

Two Brain Sites for Cannabinoid Reward The recent findings that delta-9-tetrahydro- cannabional (THC), the active agent in marijuana and hashish, (1) is self-administered intravenously, (2) potentiates the rewarding effects of electrical brain stimulation, and (3) can establish conditioned place preferences in laboratory animals, suggest that these drugs activate biologically primitive brain reward mechanisms. Here, IRP scientists identify two chemical trigger zones for stimulant and rewarding actions of THC. Microinjections of THC into the posterior ventral tegmental area (VTA) or into the shell of the nucleus accumbens (NAS) increased locomotion, and rats learned to lever-press for injections of THC into each of these regions. Substitution of vehicle for drug or treatment with a cannabinoid CB1 receptor antagonist caused response cessation. Microinjections of THC into the posterior VTA and into the posterior shell of NAS established conditioned place preferences. Injections into the core of the NAS, the anterior VTA, or dorsal to the VTA were ineffective. These findings link the sites of rewarding action of THC to brain regions where such drugs as amphetamines, cocaine, heroin, and nicotine are also thought to have their sites of rewarding action. Zangen, A., Solinas, M., Ikemoto, S., Goldberg, S.R., and Wise, R.A. Journal of Neuroscience, 26, pp. 4901-4907, 2006.

Preclinical Pharmacology Section, Behavioral Neuroscience Research Branch

Previous Exposure to THC Alters the Reinforcing Efficacy and Anxiety Related Effects of Cocaine in Rats The hypothesis that prior cannabis exposure increases the likelihood of becoming addicted to other drugs can be evaluated by giving rats a history of tetrahydrocannabinol (THC) exposure, then allowing them to self-administer other drugs. In Experiment 1, THC pre-exposure did not alter the acquisition of cocaine self-administration or the amount of cocaine taken under a fixed-ratio 1 (FR1) schedule, with one response required for each injection. Under a progressive-ratio schedule, with the response requirement increasing exponentially with each injection, cocaine-seeking was significantly reduced in THC-exposed rats, suggesting that the regimen of THC exposure used in the present study caused cocaine to be devalued as a reinforcer. In contrast, in an earlier study that used the same regimen, a history of THC exposure did not alter the value of heroin as a reinforcer under the progressive-ratio schedule, but it increased heroin self-administration under the FR1 schedule. Experiment 2 examined how this regimen of THC pre-exposure alters the locomotor effects of cocaine and heroin. THC pre-exposure produced cross-tolerance to the motor-depressant effects of heroin; this may explain the shortened post-injection pauses exhibited by THC-exposed rats under FR1 heroin self-administration. When given cocaine, THC-exposed rats exhibited normal increases in locomotion, but they avoided the center of the open field, suggesting that this THC pre-exposure regimen enhances the anxiogenic effects of cocaine. This enhanced anxiogenic effect-which was verified in Experiment 3 using another model of anxiety, the light-dark test-may explain the reduced reinforcing value of cocaine observed in THC-exposed rats in Experiment 1. Panlilio, L.V., Solinas, M., Matthews, S.A. and Goldberg, S.R. Neuropsychopharmacology, May 31, 2006, Epubmed ahead of print, PMID 16738542.

Heteromeric Nicotinic Acetylcholine-Dopamine Autoreceptor Complexes Modulate Striatal Dopamine Release In the striatum, dopamine and acetylcholine (ACh) modulate dopamine release by acting, respectively, on dopamine D(2) autoreceptors and nicotinic ACh (nACh) heteroreceptors localized on dopaminergic nerve terminals. The possibility that functional interactions exist between striatal D(2) autoreceptors and nACh receptors was studied with in vivo microdialysis in freely moving rats. Local perfusion of nicotine in the ventral striatum (shell of the nucleus accumbens) produced a marked increase in the extracellular levels of dopamine, which was completely counteracted by co-perfusion with either the non-alpha(7) nACh receptor antagonist dihydro-beta-erythroidine or the D(2-3) receptor agonist quinpirole. Local perfusion of the D(2-3) receptor antagonist raclopride produced an increase in the extracellular levels of dopamine, which was partially, but significantly, counteracted by coperfusion with dihydro-beta-erythroidine. These findings demonstrate a potent crosstalk between G protein-coupled receptors and ligand-gated ion channels in dopaminergic nerve terminals, with the D(2) autoreceptor modulating the efficacy of non-alpha(7) nACh receptor-mediated modulation of dopamine release. Authors further demonstrate physical interactions between beta(2) subunits of non-alpha(7) nicotinic acetylcholine receptors and D(2) autoreceptors in co-immunoprecipitation experiments with membrane preparations from co-transfected mammalian cells and rat striatum. These results reveal that striatal non-alpha(7) nicotinic acetylcholine receptors form part of heteromeric dopamine autoreceptor complexes that modulate dopamine release. Quarta, D., Ciruela, F., Patkar, K., Borycz, J., Solinas, M., Lluis, C., Franco, R., Wise, R.A., Goldberg, S.R., Hope, B.T., Woods, A.S. and Ferre, S. Neuropsychopharmacology, May 17, 2006, Epubmed ahead of print, PMID 16710311.

Neuropsychopharmacology Section, Behavioral Neuroscience Research Branch

Dopamine D3 Receptor Antagonists as Potential Anti-Craving and Anti-Relapse Medications for the Treatment of Addiction IRP scientists have previously found that blockade of dopamine D3 receptors in the rat brain (which are neuroanatomically restricted to the mesolimbic dopamine system, implicated in drug-induced reward and drug-seeking behavior) by the high-potency high-selectivity dopamine D3 receptor antagonist SB277011A dose-dependently attenuates cocaine-enhanced brain-stimulation reward and cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their prior intravenous cocaine-taking behavior (see, e.g., Brain Research Reviews, 49, pp. 77-105, 2005). Now, these researchers have found that NGB2904, another high-potency high-selectivity dopamine D3 receptor antagonist, likewise dose-dependently attenuates cocaine-enhanced brain-stimulation reward and cocaine-triggered relapse to cocaine-seeking behavior in animals pharmacologically detoxified and behaviorally extinguished from their intravenous cocaine-taking behavior. These confirmatory findings with a new D3 receptor antagonist 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. Xi, Z.-X., Newman, A.H., Gilbert, J.G., Pak, A.C., Peng, X.-Q., Ashby, C.R. Jr., Gitajn, L., and Gardner, E.L. Neuropsychopharmacology, 31, pp. 1393-1405, 2006.

Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Changes in HIV Risk Behaviors Among Patients Receiving Combined Pharmacological and Behavioral Interventions for Heroin and Cocaine Dependence Cocaine use is associated with injecting and sexual HIV risk behaviors. This study was a randomized controlled trial of behavioral interventions for cocaine dependence and HIV risk behaviors among dually (cocaine and heroin) dependent outpatients. Methadone maintenance was augmented with cognitive-behavioral therapy (CBT), contingency management (CM), both (CBT+CM), or neither. The study sample (n=81) was 52% female, 70% African American, and 37.9 ± 7.0 years old. Proportions reporting HIV risk behaviors at intake were: 96.3% (78/81) injection drug use, 56.8% (46/81) sharing needles, 30.9% (25/81) unprotected sex, 28.4% (23/81) trading sex for money or drugs. Proportions who no longer reported behaviors at study exit were: 48.7% (38/78) injection drug use, 91.3% (42/46) sharing needles, 88% (22/25) unprotected sex, 91.3% (21/23) trading sex for money or drugs. Participants receiving CBT+CM were more likely to report cessation of unprotected sex relative to Control (OR=5.44, 95% CI 1.14-26.0, p=0.034) but this effect was reduced by adjusting for drug-negative urines. These results suggest broad beneficial effects of methadone maintenance augmented with behavioral interventions for reducing HIV risk behaviors. Schroeder, J.R., Epstein, D.H., Umbricht, A., and Preston, K.L. Addictive Behaviors, 31, pp. 868-879, 2006.

Adverse Events Among Patients in a Behavioral Treatment Trial for Heroin and Cocaine Dependence: Effects of Age, Race, and Gender Safety monitoring is a critical element of clinical trials evaluating treatment for substance dependence, but is complicated by participants' high levels of medical and psychiatric comorbidity. This paper describes AEs reported in a large (N = 286), 29-week outpatient study of behavioral interventions for heroin and cocaine dependence in methadone-maintained outpatients. A total of 884 AEs were reported (3.1 per patient, 0.12 per patient-week), the most common being infections (26.8%), gastrointestinal (20.5%), musculoskeletal (12.3%), and general (10%) disorders. Serious AEs were uncommon (1.6% of total). Female participants reported significantly higher rates of AEs (incidence density ratio, IDR = 1.38, p < 0.0001); lower rates of AEs were reported by African Americans (IDR = 0.73, p < 0.0001) and participants over age 40 reported lower rates of AEs (IDR = 0.84, p = 0.0095). AE incidence was not associated with the study intervention or with psychiatric comorbidity. Further work is needed to adapt AE coding systems for behavioral trials for substance dependence; the standard Medical Dictionary for Regulatory Activities, International Federation of Pharmaceutical Manufacturers Associations (MedDRA) coding system used in this report did not contain a separate category for one of the most common types of AE, dental problems. Nonetheless, the data reported here should help provide a context in which investigators and IRBs can interpret the patterns of AEs they encounter. Schroeder, J.R., Schmittner, J.P., Epstein, D.H., and Preston, K.L. Drug and Alcohol Dependence, 80, pp. 45-51, 2005.

Menstrual Cycle Length During Methadone Maintenance While the menstrual disruption of heroin has been demonstrated, there are few published data concerning methadone maintenance and menstrual function. This study was conducted to evaluate whether cycle length was more regular during methadone maintenance. A total of 191 heroin and cocaine-using women who were maintained on methadone therapy (70-100 mg/day) in two clinical trials, lasting 25-29 weeks. Start/end dates of each menses were collected weekly. Menstrual patterns were classified as regular, irregular, transient amenorrhea, persistent amenorrhea or cycle restart. Repeated-measures regression modeling determined correlates of cycle length and predictors of long cycles (> 40 days) and short cycles (< 20 days). Bleeding episodes were defined as 1 or more bleeding days, bound by at least 2 non-bleeding days. Correlates/predictors examined were body mass index, drug use, methadone dose and race. In the 133 women for whom menstrual patterns could be determined, cycle-length irregularity was common: irregular, 62 (46.7%); regular, 37 (27.8%); cycle restart, 16 (12%); persistent amenorrhea, 11 (8.3%); transient amenorrhea, seven (5.3%). Each additional week on methadone maintenance was associated with decreased risk of long (OR = 0.96, P < 0.01 and short (OR = 0.92, P < 0.01) cycles. Of 27 women with secondary amenorrhea pre-study, 16 (59%) restarted menses. Positivity for opioids or cocaine was not significantly associated with short or long cycles. Cycle length begins to normalize during methadone maintenance. Menses resumption may occur. Methadone maintenance, despite interfering with menstrual function in an absolute sense, may interfere less than illicit heroin abuse. Schmittner, J., Schroeder, J.R., Epstein, D.H., and Preston, K.L. Addiction, 100, pp. 829-836, 2005.

Nicotine Psychopharmacology Unit, Treatment Section, Clinical Pharmacology and Therapeutics Research Branch

Effect of Tobacco Craving Cues on Memory Encoding and Retrieval in Smokers Previous studies have shown that cue-elicited tobacco craving disrupted performance on cognitive tasks; however, no study has examined directly the effect of cue-elicited craving on memory encoding and retrieval. A distinction between encoding and retireval has been reported such that memory is more impaired when attention is divided at encoding than at retrieval. This study tested the hypothesis that active imagery of smoking situations would impair encoding processes, but have little effect on retrieval. Imagery scripts (cigarette craving and neutral content) were presented either before presentation of a word list (encoding trials) or before word recall (retrieval trials). A working memory task at encoding and free recall of words were assessed. Results indicated that active imagery disrupted working memory on encoding trials, but not on retrieval trials. There was a trend toward impaired working memory following craving scripts compared with neutral scripts. These data support the hypothesis that the cognitive underpinnings of encoding and retrieval processes are distinct. Heishman, S.J., Boas, Z.P., Hager, M.C., Taylor, R.C., Singleton, E.G., and Moolchan, E.T. Addictive Behaviors, 31, pp. 1116-1121, 2006.