Research Findings - Intramural Research

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

Effects of Cerebral Ischemia in Mice Deficient in Persephin

Persephin (PSP), a recently cloned member of the transforming growth factor superfamily (TGF) and glial cell line-derived neurotrophic factor (GDNF) subfamily, is distributed throughout the nervous system at extremely low levels and is thought to function as a survival factor for midbrain dopaminergic and spinal motor neurons In Vivo. Here IRP investigators report that mice lacking PSP by homologous recombination show normal development and behavior, but are hypersensitive to cerebral ischemia. A 300% increase in infarction volume was observed after middle cerebral artery occlusion. The authors found that glutamate induced Ca2+ influx, thought to be a major component of ischemic neuronal cell death, can be regulated directly by the Persephin protein (PSP) and that PSP can reduce hypoxia/reperfusion cell death In Vitro. Neuronal cell death can be prevented or markedly attenuated by administration of recombinant human PSP In Vivo before ischemia in both mouse and rat models. Taken together, these data indicate that PSP is a potent modulator of excitotoxicity in the central nervous system with pronounced neuroprotective activity. Our findings support the view that PSP signaling can exert an important control function in the context of stroke and glutamate-mediated neurotoxicity, and also suggest that future therapeutic approaches may involve this novel trophic protein. Agulnick, A.D., Haughey, N., Chang, C.-F., Zhang, Y., Backman, C., Morales, M., Mattson, M.P., Wang, Y., Westphal, H., and Hoffer, B.J., PNAS, 99, pp. 9521-9526, 2002.

Development and Plasticity Section, Cellular Neurobiology Research Branch

Analysis of Gene Expression in Schizophrenia Using DNA Microarrays

IRP investigators describe experiments performed by their group and others, in which DNA microarrays were employed to probe samples from human postmortem brain tissue to examine gene expression differences in schizophrenia. Genes for which decreases in expression were identified fell into two categories. First, decreases in the expression of genes related to presynaptic, glutamatergic, and GABAergic function have been identified. Secondly, preliminary studies have identified changes in gene expression that are suggestive of cellular stress or sub-lethal injury. The authors therefore suggest the following hypothesis. An underlying deficit in trophic, developmental, or regulatory processes in schizophrenia places stress on certain sub-populations of neurons. As a result of this cellular stress, these neurons down-regulate neurotransmitter and synaptic function. This impaired neurotransmitter and synaptic function may be directly related to the symptoms of schizophrenia. The authors suggest that an underlying defect, which has not yet been identified, may impair or stress the function of certain neurons, and is ultimately responsible for the pathophysiology of schizophrenia. Further microarray experiments with larger cohorts of subjects, and pooling of results across laboratories may lead to further insights into defects in gene expression in schizophrenia and eventually to pharmacological treatments based on these defects. Freed, W.J., Hyde, T.M., Kleinman, J.E., Becker, K.G., and Vawter, M.P. Trends in Evidence Based Neuropsychiatry, 4, pp. 48-57, 2002.

Gene Expression Profiling in Drug Abuse

Recent advances in the use of microarrays for massive parallel screening for gene expression provides an opportunity to develop an understanding of complex processes of brain function on the genomic and molecular level. Microarrays involve the use of libraries of hybridization targets, arrayed on a fixed surface, to measure gene expression for many genes simultaneously in cells or tissue samples. Both small and large scale arrays are commercially available from a number of sources. In addition, IRP investigators have developed a specialized "neuroarray" comprised of 1152 cDNAs selected for relevance to brain function. The authors used the neuroarray to examine differences in gene expression in postmortem tissue from human subjects who had abused cocaine. Subsequent animal studies of chronic cocaine administration may help to determine the degree to which brain adaptations seen in human subjects can be modeled in animal studies. Understanding the process through which drugs of abuse produce adaptations in brain function may help to identify methods for intervening to influence drug addiction. Freed, W.J., Lehrmann, E., Hyde, T.M., Kleinman, J.E., Vawter, M.P., and Becker, K. Proceedings of the ONDCP International Technology Symposium entitled "Counterdrug Research and Development: Technologies for the Next Decade", 1, pp. 119-131, 2001.

Behavioral, Hormonal and Histological Stress Markers of Anxiety-separation in Postnatal Rats are Reduced by Prepro-thyrotropin-releasing hormone 178-199

IRP scientists investigated in the present study whether systemic injections of prepro-thyrotropin-releasing-hormone 178--199 (PPTRH 178--199) in postnatal 3-days old rat pups can provide ameliorative effects in a model of anxiety-separation disorder. The pups were individually separated from their mother and placed in a novel environment. PPTRH 178--199-treated animals started exploring the novel environment in a significantly shorter time and elicited significantly less distress vocalizations than control animals. PPTRH 178--199-treated animals also had markedly lower serum adrenocorticotropic hormone and corticosterone compared to control animals. Furthermore, we observed a significant increase in PPTRH 178--199 immunoreactive cell bodies in the hypothalamus of PPTRH 178--199-treated animals compared to controls, suggesting that the peptide crossed the blood--brain barrier. PPTRH 178--199 treatment can help to reduce behavioral and hormonal disturbances associated with anxiety-separation situations. Stahl, E.E., Redei, E., Wang, Y., and Borlongan, C.E. Neuroscience Letters, 321, pp. 85-89, 2002.

T155g-immortalized Kidney Cells Produce Growth Factors and Reduce Sequelae of Cerebral Ischemia

Fetal rat kidney cells produce high levels of glial-derived neurotrophic factor (GDNF) and exert neuroprotective effects when transplanted into the brain in animal models of Parkinson's disease and stroke. The purpose of the present experiment was to produce kidney cell lines that secrete GDNF. Genes encoding two truncated N-terminal fragments of SV40 large T antigen, T155g and T155c, which does not code for small t antigen, were used. T155g was transduced into E17 cultured fetal Sprague-Dawley rat kidney cortex cells using a plasmid vector, and T155c was transduced with a plasmid and a retroviral vector. Sixteen clones were isolated from cultures transfected with the T155g-expressing plasmid. No cell lines were obtained with T155c. Four clones produced GDNF at physiological concentrations ranging from 55 to 93 pg/ml of medium. These four clones were transplanted into the ischemic core or penumbra of rats that had undergone middle cerebral artery occlusion (MCAO). Three of the four clones reduced the volume of infarction and the behavioral abnormalities normally resulting from MCAO. Blocking experiments with antibodies to GDNF and platelet-derived growth factor (PDGF) suggested that these growth factors contributed only minimally to the reduction in infarct volume and behavioral abnormality. These cell lines may be useful for intracerebral transplantation in animal models of brain injury, stroke, or Parkinson's disease. Dillon-Carter, O., Johnston, R.E., Borlongan, C.V., Truckenmiller, M.E., Coggiano M., and Freed, W.J., Cell Transplantation, 11, pp. 251-259, 2002.

AF5, a CNS Cell Line Immortalized with an N-Terminal Fragment of SV40 Large T: Growth, Differentiation, Genetic Stability and Gene Expression

Central nervous system progenitor cells that are self-renewing in culture and also differentiate under controlled conditions are potentially useful for developmental studies and for cell-based therapies. IRP investigators characterized growth and plasticity properties and gene expression in a rat mesencephalic cell line, AF5, that was immortalized with an N-terminal fragment of SV40 large T (T155g). For over 150 population doublings in culture, the growth rate of AF5 cells remained steady, the cells remained responsive to bFGF, and telomerase activity and telomere lengths were unchanged. While karyotype analyses revealed some chromosomal abnormalities, these were also unchanged over time; additionally, no mutations in p53 gene sequences were found, and wild-type p53 activation was normal. AF5 cells produced PDGF, TGFbeta1, TGFbeta2, and bFGF. Similar to primary progenitor cells, AF5 cells retained their plasticity in culture; they could be propagated in an undifferentiated state as "neurospheres" in serum-free media or as adherent cultures in serum-containing media, and they differentiated when allowed to become confluent. Adherent subconfluent actively growing cultures expressed a marker for immature neurons, nestin, while few cells expressed the mature neuronal cell marker betaIII-tubulin. Confluent cultures ceased growing, developed differentiated morphologies, contained few or no nestin-expressing cells, and acquired betaIII-tubulin expression. Global gene expression was examined using a 15,000 gene microarray, comparing exponential growth with and without bFGF stimulation, and the differentiated state. The AF5 cell line exhibited stable genetic and growth properties over extended periods of time, while retaining the ability to differentiate In Vitro. These data suggest that the AF5 cell line may be useful as an In Vitro model system for studies of neural differentiation. Truckenmiller, M.E., Vawter, M.P. Zhang, P., Conejero-Goldberg, C., Dillon-Carter, O., Morales, N., Cheadle, C., Becker, K.G., and Freed, W.J. Experimental Neurology, 175, pp. 318-337, 2002.

Psychobiology Section, Medications Discovery Research Branch

Comparison of Interactions of D1-like Agonists, SKF 81297, SKF 82958 and A-77636, with Cocaine: Locomotor Activity and Drug Discrimination Studies in Rodents

Recent data suggest that dopamine (DA) D1-like receptor full agonists may be potential pharmacotherapeutic agents for treating cocaine abuse. The structurally novel isochroman D1-like agonist, A-77636, was compared to those obtained with the better investigated benzazepine D1-like dopamine agonists, SKF 82958 and SKF 81297. Each D1-like agonist produced a dose-related decrease in the cocaine-induced stimulation of locomotor activity. Each of the D1-like agonists partially substituted for the subjective effects of cocaine, with maximal substitution approximating 49, 35, and 24% for SKF 81297, SKF 82958, and A-77636, respectively. Both SKF 82958 and SKF 81297 shifted the cocaine dose-effect curve to the left. In contrast, A-77636 either did not affect the cocaine dose-effect curve or shifted it to the right. All three agonists produced similar effects on cocaine-induced locomotor activity, however the discriminative-stimulus effects of cocaine were affected differently by the D1 agonists. These results suggest fundamental differences in the actions of these D1 agonists. Because A-77636 consistently attenuated the present effects of cocaine, it may prove more useful than the others as a pharmacotherapy to treat cocaine abuse. Chausmer, A.L. and Katz, J.L. Psychopharmacology, 159, pp. 145-153, 2002.

Synthesis and Biological Evaluation of 2-substituted 3-tolyltropane Derivatives

A series of eight 2-substituted 3-tolyltropane derivatives were synthesized and the In Vitro and In Vivo biological activities as dopamine uptake inhibitors were determined. From the In Vitro structure-activity data it is apparent that a tolyl group in the 2-position, independent of the stereochemical attachment to the tropane ring system provided compounds that exhibit high affinity binding at the DAT. Although a slight stereochemical preference in binding affinity at the DAT was observed for the 2b-(R)-alcohol over the 2b-(S)-isomer, no significant differences in behavioral effects were observed. Furthermore, despite a relatively low potency of one compound for the inhibition of dopamine uptake compared to its affinity for the DAT, its behavioral profile did not vary significantly from cocaine. These data indicate that a behavioral characterization of compounds is a critical feature of efforts to discover pharmacological treatments for cocaine abuse. Xu, L., Izenwasser, S., Katz, J.L., Kopajtic, T., Klein-Stevens, C., Zu, N., Lomenzo, S.A., Winfield. L. and Trudell, M.L. Journal of Medicinal Chemistry, 45, pp. 1203-1210, 2002.

Structure-activity Relationships at Monoamine Transporters for N-substituted Benztropines: Synthesis and Comparative Molecular Field Analysis (CoMFA)

The dopamine transporter (DAT) has been implicated as the primary molecular site of action of psychostimulant drugs of abuse. The development of structure-activity relationships of dopamine uptake inhibitors and comparing their effects models of cocaine abuse has provided insight into the complex relationships between structure, binding, and behavioral activity. Understanding the molecular interactions of the DAT with different ligands, will assist in developing effective medications for cocaine abuse. A molecular modeling study using CoMFA was performed on a set of 76 benztropine analogs. The models provided insight into the structural features for optimal binding to the DAT, which was used to design more selective compounds. The role of pharmacokinetics also entered into the design of the new compounds. Previously, potent analogs in the benztropine series were also highly lipophilic, rendering them unreasonable candidates as medications. Hence, in this study, heteroatom substitutions were used in order to retain high DAT affinity but reduced lipophilicity. A series of 10 novel N-substituted analogues were designed, synthesized and evaluated for binding at DAT, the serotonin and norepinephrine transporters, and muscarinic M1 receptors, in rat brain. Most of the analogues showed high DAT affinity (12-50 nM) and selectivity for DAT over other sites. Furthermore, reduced cLogD values suggested that decreased lipophilicity could be achieved while retaining favorable binding profiles. Future studies will further improve our understanding of the importance of physiochemical properties in the behavioral actions of these molecules. This information is essential for devising an efficacious medication strategy for cocaine addiction. Kulkarni, S.S., Kopajtic, T., Katz, J.L. and Newman, A.H. Presentation at the 2002 Gordon Conference on Medicinal Chemistry, New London, NH, August 4-9, 2002.

Intravenous Cocaine-induced Activity in A/J and C57BL/6J Mice: Behavioral Sensitization and Conditioned Activity

The purpose of this study was to develop a methodology for studying i.v. cocaine-induced activity in the mouse, which allows within-session determination of the dose-response function for a rapid characterization of activity in subjects that may be extremely valuable and in short supply (i.e. genetically engineered mice). The stimulant effects of i.v. cocaine (3-25 mg/kg) were investigated both acutely and following repeated treatments. Cocaine produced a dose-dependent increase in measures of motor activity, and repeated cocaine treatment resulted in the development of behavioral sensitization. In summary, these data extend to the i.v. route of administration previous observations on cocaine-induced activity and conditioned activity and allow an assessment of cocaine induced activity without handling of subjects. Mead, A.N., Katz, J.L. and Rocha, B.A. Neuropharmacology, 42, pp. 976-986, 2002.

Cocaine-induced Locomotor Activity and Cocaine Discrimination in Dopamine D4 Receptor Mutant Mice

The role of dopamine D4 receptors in the behavioral effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects was investigated using dopamine D4 receptor knockout (DA D4R KO) and wild-type (WT) mice. The mice were trained in daily sessions to discriminate IP injections of saline from cocaine (10 mg/kg). Responses on one of two response keys intermittently produced a food pellet; one response was reinforced in sessions following cocaine injection (10 mg/kg), and the other response was reinforced in sessions following saline injection. Each twentieth response produced a food pellet (fixed-ratio, or FR 20 schedule of reinforcement). Horizontal locomotor activity was also assessed in each genotype. Each genotype acquired the discrimination of 10 mg/kg cocaine in a comparable number of training sessions. Tested doses of 1.0 - 10.0 mg/kg of cocaine produced dose-related increases in the percentage of drug-appropriate responses. The dose-effect curve for cocaine was significantly shifted to the left approximately 2.5-fold in the DA D4R KO mice (ED50 value = 0.50 mg/kg) compared to their WT littermates (ED50 value = 2.6 mg/kg). Cocaine did not significantly alter response rates across the dose range tested. In addition, cocaine was more a potent stimulator of locomotor activity in the DA D4R KO mice compared to WT littermate mice. The present results on the stimulation of activity and interoceptive/subjective effects of cocaine are consistent with the previously reported disregulation of dopamine synthesis in DA D4R KO mice, and further suggest a role of the DA D4R in vulnerability to stimulant abuse. Katz, J.L., Chausmer, A.L., Elmer, G.I., Rubinstein, M., Low, M.J. and Grandy D.K. Presentation at the Dopamine 2002. Satellite Symposium of the IUPHAR Congress, Portland OR, July, 10-14, 2002.

Cocaine-induced Locomotor Activity and Cocaine Discrimination in Dopamine D2 Receptor Mutant Mice

Dopamine D2-like antagonists block several effects of cocaine, including its locomotor stimulant and interoceptive discriminative-stimulus effects. Because these compounds generally lack selectivity among the D2-like dopamine receptors, the specific roles of the subtypes remain unclear. Dopamine D2 receptor knockout (DA D2R KO), heterozygous (HET) and wild-type (WT) mice were used to study the role of D2 dopamine receptors in the effects of cocaine. DA D2R KO, HET and WT mice were treated with cocaine (1-10 mg/kg) or vehicle and their horizontal locomotor activity was assessed. The mice were also trained to discriminate IP injections of saline from cocaine (10 mg/kg) using a 2 response-key food-reinforcement (FR 20) procedure. Both DA D2R KO and HET mice showed reduced levels of locomotor activity compared to WT mice. Cocaine dose-dependently stimulated activity in each genotype, with the highest level of activity induced in the DA D2R WT mice, and all three genotypes acquired the discrimination of 10mg/kg cocaine. Raclopride dose-dependently shifted the cocaine dose-effect curve to the right in DA D2R WT and HET mice. However in DA D2R KO mice raclopride was inactive as an antagonist. The present data indicate an involvement of D2 dopamine receptors in the locomotor-stimulating effects and the interoceptive discriminative-stimulus effects of cocaine in WT subjects. However, the D2 receptor is not necessary for the effects, suggesting redundant dopaminergic mechanisms for the discriminative-stimulus interoceptive effects of cocaine. Chausmer, A.L., Elmer, G.I., Rubinstein, M., Low, M.J., Grandy, D.K. and Katz, J.L. Psychopharmacology, Published online: 17 July 2002 [DOI 10.1007/s00213-002-1142-y].

Medicinal Chemistry Section, Medications Discovery Research Branch

Three Generations of N-substituted Benztropine Analogues as Potential Medications for Cocaine Abuse

A series of novel dopamine uptake inhibitors, based on benztropine (BZT), have been synthesized and evaluated as potential medications for cocaine-abuse. Structure-activity relationships revealed structural features for optimal dopamine transporter (DAT) affinity and selectivity. Several first generation N-substituted 4',4"-diF-BZTs (NH, N-methyl, N-allyl, N-butyl and N-butylphenyl) were evaluated in animal models of cocaine abuse. None of these compounds was found to be as efficacious as cocaine, in stimulating locomotor activity in mice. Furthermore, only the N-methyl analogue demonstrated full generalization to the cocaine discriminative stimulus, in rats trained to discriminate 10 mg/kg cocaine from saline, at a pretreatment time of 90 min. The other N-substituted analogues were not recognized as being cocaine-like, regardless of pretreatment time. The lack of cocaine-like actions, despite potent dopamine uptake inhibition, suggested that sub-optimal pharmacokinetics, due to high lipophilicity, may be confounding the behavioral actions of these compounds. Hence, a second generation of N-substituted analogues (N-cyclopropylmethyl, N-N-indole-3-ethyl, 2'-aminoethyl, (S)-2"-amino-3"-methyl-n-butyl) were evaluated. These compounds demonstrated higher DAT binding affinities and greater selectivity compared to the first generation of analogs. These compounds also had lower lipophilicities, as measured by cLogD values. Behavioral evaluation of these compounds and comparisons to cocaine, as well as the design of a third generation of N-substituted analogues are presented. Newman, A.H., Kulkarni, S., Kopajtic, T., O'Callaghan, M. and Katz, J.L. Presentation at the Dopamine 2002 Satellite Symposium of the IUPHAR Congress, Portland OR, July, 10-14, 2002.

[3-cis-3,5-Dimethyl-1-piperazinyl) alkyl]-bis-(4'-fluorophenyl) Amine Analogs as Dual Probes for the Dopamine Transporter and Sigma1 Receptors

Rimcazole, a sigma1 receptor antagonist that binds to the DAT (Ki =224), is not behaviorally cocaine-like and attenuates some of the behavioral actions of cocaine. In order to determine the roles of both DAT and sigma1 receptors in the behavioral actions of rimcazole, a series of analogs was synthesized. Two analogs (SH 3-24 and 3-28) showed high to moderate affinities for both DAT and sigma1 receptors and failed to show cocaine-like discriminative stimulus (DS) effects. Further, a potentiation of cocaine's DS effects was not observed, unlike with other DAT inhibitors, suggesting a potential role of sigma1. Another series of bis-(4'-fluorophenyl)amine analogs were prepared in which the most potent DAT compound, JJC-2-010 (Ki = 8.5 nM) was highly selective over sigma1 receptor binding (DAT/sigma1>100). CoMFA studies at both DAT and sigma1 receptors were performed to examine structural requirements and differences. Behavioral evaluation of analogs with varying affinities for both DAT and sigma1 receptors may provide direction toward designing medications for cocaine abuse. Cao, J., Kulkarni, S.S., Bowen, W., Williams, W., Kopajtic, T., Katz, J.L. and Newman A.H. Presentation at the 2002 American Chemical Society, Boston, MA, August, 2002.

SAR comparison of (S)-2-substituted-3a-[bis (4'-fluorophenyl)methoxy] tropanes and (R)-2-substituted-3a- (3,4-dichlorophenyl)tropanes at the DAT

Extensive SAR have been developed around two classes of tropane-based dopamine transporter (DAT) ligands. Significant chemical modification at the 2-position in the cocaine class is well tolerated, although the substituent must be in the R-configuration. In the benztropine class, a substituent need not be in the 2-position to bind to the DAT with high affinity. However, if a substituent (ex. COOMe) is placed in the 2-position it must be in the S-configuration, in order to bind. This opposing enantioselectivity suggests that these tropane-based DAT inhibitors may not access identical binding domains. In order to further investigate these disparities, a series of (S)-2a-carboalkoxy-4,4'-difluorobenztropines and their identically (R)-2-substituted-3a-(3,4-dichlorophenyl) tropanes were prepared and evaluated for binding at the DAT, SERT, NET, and muscarinic receptors. SAR suggest that identical 2-position substituents on the tropane rings of these two classes of compounds confer differing binding affinities and selectivities for the DAT, which may be exploited toward the discovery of a cocaine-abuse pharmaco- therapeutic. Zou, M-.F., Kopajtic, T., Katz, J.L. and Newman, A.H. Presentation at the 2002 American Chemical Society, Boston, MA, August, 2002.

Synthesis and Evaluation of a Novel Series of 2-chloro-5- ((1-methyl-2- (S)-pyrrolidinyl) methoxy)-3-(2-(4-pyridinyl)vinyl) pyridine Analogues as Potential Positron Emission Tomography Imaging Agents for Nicotinic Acetylcholine Receptors

Reportedly, 2-[(18)F]fluoro-A-85380, 1, a promising radiotracer for imaging the nicotinic acetylcholine receptor (nAChR) by positron emission tomography (PET) in humans, exhibits slow penetration through the blood-brain barrier (BBB) due to its low lipophilicity. A ligand for nAChRs with greater lipophilicity than that of 1 would be potentially more favorable for PET imaging of nAChR due to its faster penetration through the BBB. Herein, a novel series of compounds has been developed based on the high affinity ligand for nAChRs, 2-chloro-5-((1-methyl-2-(S)-pyrrolidinyl)methoxy)-3-(2-(4-pyridinyl)vinyl)pyridine, 3b. The In Vitro binding affinities for the new series were found to be in the range of K(i) = 9-331 pM. A molecular modeling study showed differences in the comformational profiles and the electronic properties of these compounds, which provides further insight into the structure-activity relationships at nAChR. Lipophilicities of the compounds 3b-6b have been found to be substantially higher than that of 1. As a result, compounds 3b-6b might exhibit a faster penetration through the BBB than the less lipophilic 1. The N-methyl derivatives 3b and 6b demonstrated very high affinities at nAChRs (K(i) = 28 and 23 pM, respectively) and will be targets for development of (11)CH(3)-labeled derivatives as radiotracers for PET imaging of nAChRs. Brown, L.L., Kulkarni, S., Pavlova, O.A., Koren, A.O., Mukhin, A.G., Newman, A.H., and Horti, A.G. Journal of Medicinal Chemistry, 45, pp. 2841-2849, 2002.