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Director's Report to the National Advisory Council on Drug Abuse
February, 2002

Research Findings

Intramural Research

Treatment Section, Clinical Pharmacology & Therapeutics Research Branch

Shaping Cocaine Abstinence by Successive Approximation

Contingency management, a behavioral therapy in which positive behavior changes are reinforced with incentives, is one of the most effective treatments for cocaine dependence. Most commonly, patients receive an incentive each time their urine tests negative for cocaine. Though effective in many patients, this procedure sometimes fails because some patients can cut down their cocaine use, but are not immediately able to abstain long enough for their urine to test negative and earn an incentive. Investigators in the Treatment Section tested a treatment variation that reinforced patients' decreases in cocaine use prior to requiring them to be totally abstinent. Cocaine-using methadone-maintenance patients were randomized to standard contingency management (Abstinence group, n=49) or a contingency designed to increase contact with reinforcers (Shaping group, n=46). For 8 weeks, both groups earned escalating-value vouchers based on thrice-weekly urinalyses: the Abstinence group earned vouchers for cocaine-negative urine specimens only; the Shaping group earned vouchers for each urine specimen with a &Mac179;25% decrease in cocaine metabolite (first 3 weeks) and then for negative specimens only (last 5 weeks). Cocaine use was lower in the Shaping group but only in the last 5 weeks when the response requirements were identical. Thus, the shaping contingency appeared to better prepare patients for abstinence. Preston, K.L., Umbricht, A., Wong, C.J., and Epstein, D.H. Journal of Consulting and Clinical Psychology, 69, pp. 643-654, 2001.

Clinical Pharmacology Section, Clinical Pharmacology & Therapeutics Research Branch

Weight Gain and Liver Tests in Drug-Dependent Adults

This study evaluated liver function and prevalence of underweight and overweight among 264 consecutive medically screened, physically healthy drug-dependent adults admitted to the research unit of the National Institute on Drug Abuse Intramural Research Program. Liver function was assessed in terms of serum liver transaminase (ALT, AST) concentrations at admission, mid-stay, and discharge. The average length of stay on the unit was 3 weeks. At admission, the prevalence of underweight (body mass index [BMI] < 19) was 6% and of overweight [BMI > 25] 27%. Among the 204 subjects who remained on the unit at least one week, the mean weight gain was 0.14 kg/day; 69% of subjects gained weight. Heroin users had the most average weight gain; cocaine users the least. Among the 71 subjects who provided 3 blood samples, mean serum transaminase concentrations increased significantly (50-100%) from admission to mid-stay, then declined significantly (20-25%) by discharge. No adverse events were associated with the increased transaminase concentrations. There was a significant positive correlation between weight gain on the unit and increase in liver transaminase concentrations, i.e., the greater the weight gain, the greater the increase in transaminases. These findings suggest that weight gain due to refeeding can be a benign cause of abnormal liver tests in otherwise healthy drug-dependent adults. Fontaine K.R., Cheskin L.J., Carriero N.J., Jefferson L., Finley C.J., and Gorelick D.A. Journal of the American Dietetic Association, 101, pp. 1467-1469, 2001.

Brain Imaging Section, Neuroimaging Research Branch

Smoking History and Nicotine Effects on Cognitive Performance

Effects of abstinence from smoking, smoking history, and nicotine administration on visual attention (Two-Letter Search Task), verbal information processing (Logical Reasoning Task), and working memory (N-Back Tasks) were studied in 14 smokers, 15 ex-smokers, and 9 never-smokers. Subjects participated in two test sessions and received either 4 mg nicotine gum or placebo, respectively. Smokers were 12-h abstinent when they received gum. Nicotine produced significant increases in diastolic pressure and heart rate in all three groups. With respect to cognitive effects, an effect of acute nicotine administration (independent of smoking history) was observed only on the Two-Letter Search Task as reaction time was shorter after nicotine gum than after placebo in all three groups, suggesting withdrawal from or tolerance to nicotine were not significant factors. Working memory performance was related to smoking history as smokers performed most poorly and never-smokers performed best. The Logical Reasoning Task showed no effects of either acute or chronic nicotine exposure. Our determination that nicotine improves reaction time rather than accuracy of task performance, while task specific, is consistent with previous reports. Our findings further indicate that nicotine may influence focusing of attention in smokers as well as nonsmokers, and that trait-like differences in some cognitive domains, such as working memory, may be either long-term effects or etiological factors related to smoking. Ernst, M., Heishman, S.J., Spurgeon, L, and London, E.D. Neuropsychopharmacology, 25, pp. 313-319, 2001.

5-Iodo-6-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine, a Novel PET Radioligand for Nicotinic Acetylcholine Receptors: Synthesis and Initial Evaluation

Nicotinic acetylcholine receptors (nAChRs) are of growing interest due to their involvement in a variety of brain functions, including cognitive processes, and disorders (e.g., tobacco dependence and neurodegenerative disorders, such as Alzheimer’s disease). Accordingly, there has been an increasing interest in non-invasive imaging of using positron emission tomography (PET) and single photon emission computed tomography (SPECT). Radiohalogenated analogs of 3-(2(S)-azetidinylmethoxy) pyridine (A-85380) have been recognized as promising probes for in vivo imaging of brain nAChRs with PET and SPECT. To develop an 18F-labeled PET radioligand for nAChRs that retains high affinity for central alpha4beta2 nAChRs, while being superior to similar ligands with respect to safety, we synthesized 5-iodo-6-fluoro-3-(2(S)-azetidinylmethoxy) pyridine (5I-6F-A-85380) and its radiolabelled variety ([18F]). Synthesis of 5I-6F-A-85380 and its [18F]-labelled version required the synthesis of novel compounds to complete the syntheses of both compounds. In vitro characterization determined that both 5I-6F-A-85380 and 5I-6[18F]F-A-85380 had high affinities for alpha4beta2 nAChRs, Ki=15± 2 pM and Kd=17±3 pM, respectively, and in vivo studies in mice demonstrated reduced toxicity relative to closely related analogues (6F-A-85380). PET studies in Rhesus monkeys demonstrated specific accumulation of 5I-6[18F]F-A-85380 in the brain with a regional distribution consistent with alpha4beta2 nAChRs, with the highest density in the thalamus. We conclude that 5I-6[18F]F-A-85380 is a promising PET radioligand for in vivo imaging as it represents an improvement in terms of nAChR affinity, nAChR subtype selectivity and safety. Koren, A.O., Chefer, S.I., Mukhin, A.G., Pavlova, O.A., Horti, A.G., Vaupel, D.B., London, E.D. and Kimes, A.S. Journal of Labeled Compounds and Radiopharmaceuticals, 44 (S1), pp. S257-259, 2001.

Imaging studies in Humans Demonstrate that [123I]5-I-A-85380 is a Promising Single-Photon Emission Tomography (SPET) Ligand for the Study of alpha4beta2 Nicotinic Receptors in Brain Disorders

The biodistribution of radioactivity after the administration of a new tracer for alpha4beta2 nicotinic acetylcholine receptors (nAChRs), [123I]5-iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380), was studied in ten healthy human subjects. Following administration of 98±6 MBq [123I]5-I-A-85380, serial whole-body images were acquired over 24 h and corrected for attenuation. One to four brain single-photon emission tomography (SPET) images were also acquired between 2.5 and 24 h. Estimates of radiation absorbed dose were calculated using MIRDOSE 3.1 with a dynamic bladder model and a dynamic gastrointestinal tract model. The estimates of the highest absorbed dose (µGy/MBq) were for the urinary bladder wall (71 and 140), lower large intestine wall (70 and 72), and upper large intestine wall (63 and 64), with 2.4-h and 4.8-h urine voiding intervals, respectively. The whole brain activity at the time of the initial whole-body imaging at 14 min was 5.0% of the injected dose. Consistent with the known distribution of alpha4beta2 nAChRs, SPET images showed the highest activity in the thalamus. These results suggest that [123I]5-I-A-85380 is a promising SPET agent to image 42 nAChRs in humans, with acceptable dosimetry and high brain uptake. Whole-body Biodistribution, Radiation Absorbed Dose, and Brain SPET Imaging with [123I]5-I-A-85380 in Healthy Human Subjects. Fujita, M., Seibyl, J.P., Vaupel, D.B., Tamagnan, G., Early, M., Zoghbi, S.S., Baldwin, R.M., Horti, A.G., Koren, A.O., Mukhin, A.G., Khan, S., Bozkurt, A., Kimes, A.S., London, E.D., and Innis, R.B. European Journal of Nuclear Medicine, published online 4 Dec. 2001. http:/… /00695/paper/s00259-001-0695-zch110.html

Development and Plasticity Section, Cellular Neurobiology Research Branch

A Murine Dopamine Neuron-Specific cDNA Library and Microarray: Increased COXI Expression during Methamphetamine Neurotoxicity

Due to brain tissue heterogeneity, the molecular genetic profile of any neurotransmitter-specific neuronal subtype is unknown. The purpose of this study was to purify a population of dopamine neurons, construct a cDNA library, and generate an initial gene expression profile and a microarray representative of dopamine neuron transcripts. Ventral mesencephalic dopamine neurons were purified by fluorescent-activated cell sorting from embryonic day 13.5 transgenic mice harboring a 4.5-kb rat tyrosine hydroxylase promoter-lacZ fusion. Nine-hundred sixty dopamine neuron cDNA clones were sequenced and arrayed for use in studies of gene expression changes during methamphetamine neurotoxicity. A neurotoxic dose of methamphetamine produced a greater than twofold up-regulation of the mitochondrial cytochrome c oxidase polypeptide I transcript from adult mouse substantia nigra at 12 h posttreatment. This is the first work to describe a gene expression profile for a neuronal subtype and to identify gene expression changes during methamphetamine neurotoxicity. Barrett T., Xie, T., Piao, Y., Dillon-Carter, O., Kargul, G.J., Lim, M.K., Chrest, F.J., Wersto, R., Rowley, D.L., Juhaszova, M., Zhou, L., Vawter, M.P., Becker, K.G., Cheadle, C., Wood, W.H. III, McCann, U.D., Freed, W.J., Ko, M.S., Ricaurte, G.A., and Donovan, D.M. Neurobiology of Diseases, 8(5), pp. 822-833, 2001.

Application of cDNA Microarrays to Examine Gene Expression Differences in Schizophrenia

Using cDNA microarrays IRP scientists have investigated gene expression patterns in brain regions of patients with schizophrenia. A cDNA neuroarray, comprised of genes related to brain function, was used to screen pools of samples from the cerebellum and prefrontal cortex from a matched set of subjects, and middle temporal gyrus, from a separate subject cohort. Samples of cerebellum and prefrontal cortex from neuroleptic naive patients were also included. Genes that passed a 3% reproducibility criterion for differential expression in independent experiments included 21 genes for drug-treated patients and 5 genes for drug-naive patients. Of these 26 genes, 10 genes were increased and 16 were decreased. Many of the differentially expressed genes were related to synaptic signaling and proteolytic functions. A smaller number of these genes were also differentially expressed in the middle temporal gyrus. The five genes that were differentially expressed in two brain regions from separate cohorts are: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide; sialyltransferase; proteasome subunit, alpha type 1; ubiquitin carboxyl-terminal esterase L1; and solute carrier family 10, member 1. Identification of patterns of changes in gene expression may lead to a better understanding of the pathophysiology of schizophrenia disorders. Vawter, M.P., Barrett, T., Cheadle, C., Sokolov, B.P., Wood, W.H. III, Donovan, D.M., Webster, M., Freed, W.J., and Becker, K.G. Brain Research Bulletin 55(5), pp. 641-650, 2001.

Involvement of GDNF in Neuronal Protection against 6-OHDA-Induced Parkinsonism Following Intracerebral Transplantation of Fetal Kidney Tissues in Adult Rats

Exogenous application of transforming growth factors-beta (TGFbeta) family proteins, including glial cell line-derived neurotrophic factor (GDNF), neurturin, activin, and bone morphogenetic proteins, has been shown to protect neurons in many models of neurological disorders. Finding a tissue source containing a variety of these proteins may promote optimal beneficial effects for treatment of neurodegenerative diseases. Because fetal kidneys express many TGFbeta trophic factors, we transplanted these tissues directly into the substantia nigra after a unilateral 6-hydroxydopamine lesion. We found that animals that received fetal kidney tissue grafts exhibited (1) significantly reduced hemiparkinsonian asymmetrical behaviors, (2) a near normal tyrosine hydroxylase immunoreactivity in the lesioned nigra and striatum, (3) a preservation of K(+)-induced dopamine release in the lesioned striatum, and (4) high levels of GDNF protein within the grafts. In contrast, lesioned animals that received grafts of adult kidney tissues displayed significant behavioral deficits, dopaminergic depletion, reduced K(+)-mediated striatal dopamine release, and low levels of GDNF protein within the grafts. The present study suggests that fetal kidney tissue grafts can protect the nigrostriatal dopaminergic system against a neurotoxin-induced parkinsonism, possibly through the synergistic release of GDNF and several other neurotrophic factors. Borlongan, C.V., Zhou, F.C., Hayashi, T., Su, T.P., Hoffer, B.J., and Wang, Y. Neurobiology of Diseases, 8(4), pp. 636-646, 2001.

Region-specific Transcriptional Response to Chronic Nicotine in Rat Brain

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978+/-0.0035 from 0.941+/-0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10(-14)) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K(+) channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration. Konu, O., Kane, J.K., Barrett, T., Vawter, M.P., Chang, R., Ma, J.Z., Donovan, D.M., Sharp, B., Becker, K.G., and Li, M.D. Brain Research, 909, pp. 194-203, 2001.

Electrophysiological Evidence for Vasopressin v(1) Receptors on Neonatal Motoneurons, Premotor and other Ventral Horn Neurons

Prominent arginine-vasopressin (AVP) binding and AVP V(1) type receptors are expressed early in the developing rat spinal cord. IRP scientists sought to characterize their influence on neural excitability by using patch-clamp techniques to record AVP-induced responses from a population of motoneurons and interneurons in neonatal (5-18 days) rat spinal cord slices. Data were obtained from 58 thoracolumbar (T(7)-L(5)) motoneurons and 166 local interneurons. A majority (>90%) of neurons responded to bath applied AVP (10 nM to 3 &mgr;M) and (Phe(2), Orn(8))-vasotocin, a V(1) receptor agonist, but not V(2) or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near -100 mV. In interneurons, net AVP-induced inward currents displayed three patterns: decreasing membrane conductance with reversal near -100 mV, i.e., similar to that in motoneurons (24 cells); increasing conductance with reversal near -40 mV (21 cells); small reduction in conductance with no reversal within the current range tested (41 cells). A presynaptic component recorded in most neurons was evident as an increase in the frequency but not amplitude (in motoneurons) of inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs), in large part due to AVP-induced firing in inhibitory (mainly glycinergic) and excitatory (glutamatergic) neurons synapsing on the recorded cells. An increase in frequency but not amplitude of miniature IPSCs and EPSCs also indicated an AVP enhancement of neurotransmitter release from axon terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V(1) type receptors and indicate that their activation can enhance the excitability of a majority of neurons in neonatal ventral spinal cord. Oz, M., Kolaj, M., and Renaud, L.P., Journal of Neurophysiology, 86(3), pp. 1202-1210, 2001.

Characterization of Human Cleaved N-CAM and Association with Schizophrenia

The neural cell adhesion molecule (N-CAM) is a cell recognition molecule involved in cellular migration, synaptic plasticity, and CNS development. A 105- to 115-kDa isoform of N-CAM (cleaved N-CAM or cN-CAM) is increased in schizophrenia in hippocampus, prefrontal cortex, and CSF. We purified and partially characterized cN-CAM, a putative novel isoform, and confirmed that the first 9 amino acids were identical to exon 1 of N-CAM, without the signal sequence. Analysis of trypsin-digested cN-CAM fragments by matrix-assisted laser desorption ionization on a time-of-flight mass spectrometer yielded peptides that could be identified as being derived from the first 548 amino acid residues of the expected N-CAM amino acid sequence. Immunological identification with four specific N-CAM antisera directed toward cytoplasmic, secreted, variable alternative spliced exon, or GPI epitopes failed to indicate other known splice variants. Neuraminidase treatment of cN-CAM produced a minor alteration resulting in a faster migrating immunoreactive band, indicating partial glycosylation of cN-CAM. Membranous particles from cytosolic brain extract containing cN-CAM were obtained by ultracentrifugation; however, CSF contained few such particles. cN-CAM and synaptophysin were colocalized on these particles. Both cN-CAM and N-CAM 180 were present in synaptosomal preparations of human brain. Following incubation of synaptosomes or brain tissue without protease inhibitors, N-CAM 180 was degraded and cN-CAM was increased. A cN-CAM-like band was present in human fetal neuronal cultures, but not in fetal astrocyte cultures. Thus, cN-CAM represents a protease- and neuraminidase-susceptible fragment possibly derived by proteolytic cleavage of N-CAM 180. An enlargement in ventricular volume in a group of adult patients with schizophrenia over a 2-year interval was found to be correlated with CSF cN-CAM levels as measured at the time of the initial MRI scan (r = 0.53, P = 0.01). cN-CAM is associated with ventricular enlargement; thus, the release of N-CAM fragments may be part of the pathogenic mechanism of schizophrenia in vulnerable brain regions such as the hippocampus and prefrontal cortex. Alternatively, the increases in cN-CAM in schizophrenia may be a reflection of a more general abnormality in the regulation of proteolysis or of extracellular matrix stability. Vawter, M.P., Usen, N., Thatcher, L., Ladenheim, B., Zhang, P., VanderPutten, D.M., Conant, K., Herman, M.M., van Kammen, D.P., Sedvall, G., Garver, D.L., and Freed, W.J., Experimental Neurology, 172, pp. 29-46, 2001.

Cellular Neurophysiology Section, Cellular Neurobiology Research Branch

5-HT3-Receptor Subunits A and B are Co-expressed in Neurons of the Dorsal Root Ganglion

The type 3 serotonin (5-HT3) receptor is the only ligand-gated ion channel receptor for serotonin (5-HT). Many pharmacological, behavioral and electrophysiological studies indicate heterogeneous properties for this receptor. Although the basis for this heterogeneity is unknown, one possible explanation for these findings resides in the subunit composition of the receptor. Two 5-HT3-receptor subunits have been cloned: the 5-HT3-receptor subunit A (5-HT3A) and the 5-HT3-receptor subunit B (5-HT3B). Recombinant co-expression of 5-HT3A and 5-HT3B subunits produces a functional heteromeric 5-HT3A/3B receptor with pharmacological and electrophysiological properties different that those displayed by the 5-HT3A homomeric receptor. In the present report, IRP investigators used in situ hybridization histochemistry to demonstrate that the 5-HT3B subunit is expressed in rat dorsal root ganglion (DRG) neurons. Authors determined with cellular resolution that 5-HT3B subunit mRNA was expressed in 43.2%±2.8 of the total population of DRG neurons. By comparison, the 5-HT3A subunit was more widely expressed, with 70.0%±2.8 of the total population of DRG neurons expressing this subunit. Further analyses showed that most of the neurons containing mRNA for the 5-HT3B subunit (91.5%±3.4) also expressed the 5-HT3A subunit. In contrast, nearly half of the population of neurons expressing 5-HT3A subunit lacked (52.8±5.9) transcripts for the 5-HT3B subunit. These results provide the first evidence indicating that the 5-HT3B subunit of the 5-HT3 receptor is expressed in DRG, and suggest that sensory neurons have the capacity to synthesize at least two structurally different 5-HT3 receptors: a heteromeric 5-HT3A/3B receptor and a homomeric 5-HT3A receptor. Consequently, 5-HT3 receptors with different properties might be present in peripheral and central axons of the DRG. These findings open the possibility that distinct types of 5-HT3 receptors may be involved in perception and/or processing of sensory information. Furthermore, understanding the distribution, electrophysiological and pharmacological characteristics, as well as, possible participation of the different 5HT3 receptors in nociceptive perception and processing may be useful for the development of alternative drugs to opioid analgesics. Morales, M. McCollum, N., and Kirkness, E.F. 5-HT3-receptor Subunits A and B are Co-expressed in Neurons of the Dorsal Root Ganglion. Journal of Comparative Neurology, 438, pp. 163-172, 2001.

GFRa-1 mRNA in Dopaminergic and Non-dopaminergic Neurons in the Substantia Nigra and Ventral Tegmental Area

Glial cell line derived neurotrophic factor (GDNF) is a potent survival factor for several types of neurons, including dopaminergic (DAergic) neurons. GDNF binds with high affinity to the GDNF-family receptor a-1 (GFRa-1) that is highly expressed in the midbrain. Using anatomical and lesion techniques, IRP scientists demonstrated that GFRa-1 was expressed in DAergic and non-DAergic neurons in the rat midbrain. Immunohistochemical characterization of GFRa-1 expressing neurons indicated that 87%-92% of all neurons immunopositive for the DAergic marker tyrosine hydroxylase (TH) expressed GFRa-1 in the substantia nigra pars compacta (SNC). In contrast, nearly half (44%-66%) of TH containing neurons expressed GFRa-1 in the substantia nigra pars reticulata (SNR). Likewise, 50%-74% of TH-immunoreactive neurons expressed GFRa-1 in the ventral tegmental area (VTA). Depletion of GFRa-1/TH neurons was observed in the SNC following treatment with the neurotoxin 6-hydroxydopamine (6-OHDA); however, GFRa-1 expression remained in some neurons located in the SNR. The GABAergic nature of GFRa-1 expressing neurons located in the SNR which were resistant to 6-OHDA, was established by their expression of glutamic acid decarboxylase (GAD, the synthesizing enzyme for GABA). Semiquantitative analysis demonstrated rostrocaudal variability in the co-expression of GFRa-1 and GAD. Of all GAD expressing neurons, 5%-48% co-expressed GFRa-1 and GAD mRNAs in the SNR. Similar results were found in the substantia nigra pars lateralis (SNL) and VTA, where co-expression of GFRa-1 and GAD was present in 34%-52% of all GAD neurons in the SNL and 24%-45% of all GAD neurons in the VTA. Midbrain DAergic and GABAergic neurons have been previously classified according to their Ca2+ binding protein (CaBP) contents; thus, the authors also sought to investigate the proportion of midbrain GFRa-1 expressing neurons containing parvalbumin (PV), calbindin (CB) and calretinin (CR) in the midbrain. While GFRa-1 expression was found mainly in CB- and CR-immunoreactive neurons, it was rarely observed in PV immunolabeled neurons. Analysis of the proportion of GFRa-1 expressing neurons for each CaBP subpopulation indicated co-existence of GFRa-1 with CR in the VTA and all subdivisions of the SN, double labeled GFRa-1/CR neurons were distributed in the SNC (41%-67%), SNR (29%-50%), SNL (61%-90%) and VTA (40%- 62%). GFRa-1/CB neurons were also detected in the SNC (29%-42%), SNL (63%-84%) and VTA (31%-54%). This contrasts with the low proportion of GFRa-1/PV neurons (3%-16%), which were confined to the SNR. Expression of GFRa-1 in DAergic and non-DAergic neurons in the rat SN and VTA suggest that GDNF, via GFRa-1, might modulate DAergic and GABAergic functions in the nigrostriatal, mesolimbic and nigrothalamic circuits in the adult rat. Sarabi A., Hoffer, B.J., Olson, L. and Morales, M. GFRa-1 mRNA in Dopaminergic and Non-dopaminergic Neurons in the Substantia Nigra and Ventral Tegmental Area. Journal of Comparative Neurology, 441 pp. 106-117, 2001.

Molecular Neuropsychiatry Section, Cellular Neurobiology Research Branch

Amphetamine-induced Toxicity in Dopamine Terminals in CD-1 and C57BL/6J Mice: Complex Roles for Oxygen-based Species and Temperature Regulation

In order to examine differential strain susceptibility to neurotoxic effects of amphetamine and to assess the potential role of superoxide radicals in amphetamine-induced dopaminergic damage, the drug was injected into mice with different levels of copper/zinc superoxide dismutase (Cu/Zn SOD) enzyme. Administration of amphetamine (10 mg/kg, i.p., given every 2 h, a total of four times) to wild-type CD-1 and C57BL/6J mice caused significant decreases in dopamine and 3,4-dihydroxyphenylacetic acid levels, in [(125)I]RTI-121-labeled dopamine transporters as well as a significant depletion in the concentration of dopamine transporter and vesicular monoamine transporter 2 proteins. The amphetamine-induced toxic effects were less prominent in CD-1 mice, which have much higher levels of Cu/Zn SOD activity (0.69 units/mg of protein) in their striata than C57BL/6J animals (0.007 units/mg of protein). Transgenic mice on CD-1 and C57BL/6J background, which had striatal levels of Cu/Zn SOD 2.57 and 1.67 units/mg of protein, respectively, showed significant protection against all the toxic effects of amphetamine. The attenuation of toxicity observed in transgenic mice was not caused by differences in amphetamine accumulation in wild-type and mutant animals. However, CD-1-SOD transgenic mice showed marked hypothermia to amphetamine whereas C57-SOD transgenic mice did not show a consistent thermic response to the drug. The data obtained demonstrate distinctions in the neurotoxic profile of amphetamine in CD-1 and C57BL/6J mice, which show some differences in Cu/Zn SOD activity and in their thermic responses to amphetamine administration. Thus, these observations provide evidence for possible complex interactions between thermoregulation and free radical load in the long-term neurotoxic effects of this illicit drug of abuse. Krasnova, I.N., Ladenheim, B., Jayanthi, S., Oyler, J., Moran, T.H., Huestis, M.A., and Cadet J.L. Neuroscience, 107(2), pp. 265-74, 2001.

Molecular Neurotoxicological Models of Parkinsonism: Focus on Genetic Manipulation of Mice

Parkinson's disease is a neurodegenerative disorder that affects mainly the nigrostriatal dopaminergic system in humans. Several propositions have been put forward to explain the cellular and molecular pathobiology of this syndrome. Initial attempts were made through the use of various agents to manipulate the deleterious effects of toxins that destroy dopaminergic cells both in vitro and in vivo. These studies led to the idea that oxidative stress is an important factor in killing these cells. More recent attempts have made use of genetically modified mice to eliminate or over-express genes of interest. These experiments have suggested that the destruction of dopaminergic cells might be the result of the convergence of dependent and independent molecular pathways and that trigger cellular events might lead to the demise of these dopaminergic cells. Cadet, J.L. Parkinsonism Related Disorders, 8(2), pp. 85-90, 2001.

Behavioral Neuroscience Section, Behavioral Neuroscience Research Laboratory

D1 Receptors and Cocaine Reinforcement

Robert Ranaldi at the University of Mississippi and Roy Wise in the IRP have found that blockade of D1-type dopamine receptors in the ventral tegmental area attenuates the rewarding effects of intravenous cocaine. D1-type and D2-type dopamine receptors are clearly localized to different cellular elements in this region; the D-1 type receptors are localized to GABA- and glutamate-containing nerve terminals. Dopamine is thought to reach these receptors as a result of release from dopaminergic dendrites, and the effects of blockade of these receptors is among the first to suggest functional importance for dendritic dopamine release other than autoregulation of dopaminergic cell firing via the D2-type autoreceptors that are localized on dopaminergic cell bodies. Among the interesting possibilities is that modulation of GABAergic output of the ventral tegmental area--output that is "downstream" from the rewarding effect of dopamine released in nucleus accumbens--may be part of the brain circuitry of cocaine reward. Ranaldi, R., Wise, R.A. Journal of Neuroscience, 21, pp. 5841-5846, 2001.

Incubation of Cocaine Craving

Performance of a response that has been repeatedly reinforced by intravenous cocaine is the most objective and reliable indication of cocaine craving in animal models. Responding in the absence of reinforcement at various intervals after the last cocaine reinforcement gives evidence of the time-course of changes in cocaine craving following periods of cocaine intoxication. Such time-course can then be correlated with the time-course of decay of the various neuroadaptations that are caused by cocaine intoxication and that might be thought to contribute to states of cocaine craving. Jeffrey Grimm and colleagues have found evidence from this approach suggesting the cocaine craving, like learning and memory, increases as a function of the length of periods of "incubation" between the last intoxication and the first opportunity to respond again. Cocaine craving appears to continue to incubate for periods of two or more months after the last cocaine intoxication in laboratory rats. Grimm, J.W., Hope, B.T., Wise, R.A., and Shaham, Y., Nature, 412, pp. 141-142, 2001.

Addiction-prone and Addiction-resistant Rats; No Difference in Sensitivity to Cocaine Reward

Roy Wise and collaborators at Concordia University and the University of Nijmegen have found that Fischer and Lewis rats and high-responder and low-responder rats, known to be dramatically different in their likelihood to learn to self-administer intravenous cocaine, are very similar in their responsiveness to the rewarding actions of cocaine. The differences in acquisition of cocaine self-administration habits appear to result from differences in the animals' emotional responses to the testing environment rather than to any significant differences in sensitivity to the drug itself. Ranaldi, R., Bauco, P., McCormick, S., Cools, A.R., Wise, R.A. Behavioural Pharmacology, 12, pp. 527-534, 2001.

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, has not been well characterized and may prove to be useful as such an agent. The interactions of cocaine and A-77636 were compared to those obtained with the better investigated benzazepine D1-like dopamine agonists, SKF 82958 and SKF 81297. The alterations in the locomotor stimulant and discriminative-stimulus effects of cocaine by the full D1-like dopamine receptor agonists were investigated across a full range of doses in order to characterize their interactions. In Experiment 1 mice were pretreated with SKF 81297, SKF 82958 or A-77636 (1-10 mg/kg) and cocaine (5-56 mg/kg) prior to a 30-min period in which locomotor activity was assessed. Cocaine maximally stimulated activity at 20-40 mg/kg with higher and lower doses stimulating activity less. Each D1-like agonist produced a dose-related decrease in cocaine-induced locomotor activity and lowered its maximal rate. In Experiment 2, rats were trained to discriminate saline from cocaine (10 mg/kg) injections. Each of the D1-like agonists partially substituted for cocaine. In combination with cocaine, SKF 82958 and SKF 81297 shifted the cocaine dose-effect curve to the left – effects of cocaine were enhanced. In contrast, A-77636 either did not affect the cocaine dose-effect curve or shifted it to the right. In conclusion, all three D1-like agonists produced similar effects on cocaine-induced locomotor activity, however the discriminative-stimulus effects of cocaine were attenuated by A-77636. 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, 155, pp. 69-77, 2001.

Cocaine-Induced Locomotor Activity And Cocaine Discrimination in Dopamine D2 Receptor Mutant Mice

Previous studies have found that dopamine D2-like antagonists block several effects of cocaine, including its stimulation of locomotor activity and interoceptive discriminative-stimulus effects. However, given the lack of selectivity of most of these compounds among D2, D3 and D4 dopamine receptors, the specific roles of these dopamine receptors remains unclear. The role of D2 dopamine receptors in the discriminative stimulus and locomotor stimulant effects of cocaine was investigated using dopamine D2 receptor knockout (DA D2R KO), heterozygous (DA D2R HET) and wild-type (WT) mice. In addition, the role of D2 receptors was further examined in studies of the antagonism of the discriminative-stimulus effects in these mice using the relatively selective DA D2-like antagonist, raclopride. Mice were treated with cocaine (5-56 mg/kg) or vehicle and their horizontal locomotor activity was assessed for a 30-min period. The same mice were trained (FR 20) to discriminate IP injections of saline from cocaine (10 mg/kg) using a 2 response-key food-reinforcement procedure. A range of doses of cocaine (1.0 - 17 mg/kg), either alone or in combination with raclopride (0.1-1.0 mg/kg) were administered prior to a 15 min test session. Both DA D2R KO and HET mice showed reduced levels of control horizontal locomotor activity compared to WT mice. Cocaine dose-dependently stimulated locomotor activity in the WT mice, and at the highest dose studied in the DA D2R KO and HET mice, though the stimulation produced in these subjects was not to the same level as even the control level in WT mice. All three genotypes acquired the discrimination of 10 mg/kg cocaine. Once trained there was a dose-dependent generalization in each group, with doses of 1.0 to 10.0 mg/kg producing dose-related increases in the number of responses on the cocaine-appropriate key. In contrast, the D2 antagonist raclopride did not produce a proportion of cocaine-appropriate responding greater than 85% (full substitution). The doses of raclopride examined ranged from those having no effect to those decreasing response rates in WT mice. Raclopride dose dependently shifted the cocaine dose-effect curve to the right in DA D2R HET and WT 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 in subjects with an intact DA D2R system for the discriminative-stimulus interoceptive effects of cocaine. Chausmer, A.L. and Katz, J.L. Psychopharmacology, Published online: 22 September 2001.

Medicinal Chemistry and Psychobiology Sections, Medications Discovery Research Branch

[3H]MFZ 2-12: A Novel Radioligand for the Dopamine Transporter

In an effort to develop a tritiated dopamine transporter radioligand with higher affinity than the widely used [3H]WIN 35,428, we have synthesized [3H]2b-carbomethoxy-3b-[3’4’-dichlorophenyl)tropane ([3H]MFZ 2-12). Unlabeled MFZ 2-12 and the N-demethylated intermediate (MFZ 2-13) inhibited dopamine uptake by the human dopamine transporter with IC50s of 1.1 and 1.4 nM, respectively. The N-nor-intermediate (MFZ 2-13) was treated with CT-3I resulting in [3H]MFZ 2-12, S.A.= 80 Ci/mmol. [3H]MFZ 2-12 reversibly bound with a KD of 2.8 nM to human dopamine transporter expressed heterologously in EM4 cells, which is ~20-fold higher affinity than [3H]WIN 35,428. Thus at a ligand concentration of 2.5 nM, ~50% occupancy is achieved with [3H]MFZ 2-12 in contrast to ~6% by [3H]WIN35,428. Since the nonspecific binding of these two ligands is a similar fraction of the total added counts and since they are of similar specific activity, at 2.5 nM the signal and the signal to noise ratio achieved with [3H]MFZ 2-12 are approximately ten times that observed with [3H]WIN 35,428. For these reasons, [3H]MFZ 2-12 is better suited for saturation analysis, filtration assays, and use with adherent cells expressing hDAT. Newman, A. H., Zou, M-F., Ferrer, J.V. and Javitch, J.A. Bioorganic Medicinal Chemistry Letters, 11, pp. 1659-1661, 2001.

Novel Tropane-based Irreversible Ligands for the Dopamine Transporter

Novel irreversible ligands for the dopamine transporter were prepared in order to provide the required molecular tools to further characterize the binding domains at which structurally divergent dopamine transporter inhibitors bind. We previously prepared a benztropine-based photoaffinity label, [125I]GA 2-34, that covalently attached to the 1-2 transmembrane spanning region of the dopamine transporter (DAT). This was in contrast to the 4-7 transmembrane spanning region labeled by a cocaine-based photoaffinity label, [125I] RTI 82. The syntheses of the target compounds were achieved using a modification of the strategy previously developed. Evaluation of these compounds for displacing [3H]WIN 35,428 binding at DAT in rat caudate-putamen revealed that the 4’-azido-3’-iodophenyl-butyl substituent provided optimal binding affinity and was chosen to replace the N-CH3 group on RTI 82. Both the 4’-azido-3’-iodophenyl- and the 4’-isothiocyanatophenylbutyl analogs were synthesized. Both products bound to DAT with comparable potency (IC50=30 nM) to RTI 82. In addition, the isothiocyanate demonstrated wash resistant displacement of [3H]WIN 35,428 in HEK 293 cells stably transfected with hDAT. Our lead compound, MFZ 2-24, was recently radioiodinated and preliminary studies show that it binds with high affinity and in a proteolytic-resistant manner to an 80 kDa protein from rat striatum, previously identified as the DAT. Future immunological and proteolytic studies with this compound, in comparison with other irreversible ligands previously prepared in this lab and others, will allow further characterization of the binding domains on the dopamine transporter. Zou, M., Kopajtic, T., Katz, J.L., Wirtz, S., Justice, Jr., J.J. and Newman, A.H. Journal of Medicinal Chemistry, 44, pp. 4453-4461, 2001.

Design and Synthesis of Novel Ligands Selective for the Dopamine D3 Receptor Subtype

The dopamine D3 receptor subtype has been recently targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. However, definitive behavioral investigations have been hampered by the lack of highly selective D3 agonists and antagonists. In an attempt to design a novel class of D3 ligands with which to study this receptor system, a series of chemically divergent compounds that possessed various structural features that exist within several classes of reputed D3 agents was screened and compared to the recently reported NGB 2904. Based on these results, a novel series of compounds was designed that included functional moieties that were required for high affinity and selective binding to D3 receptors. All the compounds in this series included an aryl-substituted piperazine ring, varying alkyl chain linker (C3-C5) and a terminal aryl amide. The compounds were synthesized and evaluated in vitro for binding in CHO cells transfected with human D2, D3, or D4 receptor cDNAs. D3 binding affinities ranged from Ki=1.4-1460 nM. The most potent analog in this series demonstrated a D3/D2 selectivity of 64 and a D3/D4 selectivity of 1300. Structure-activity relationships for this class of ligands at D3 receptors will provide new leads toward the development of highly selective and potent molecular probes that will prove useful in the elucidation of the role D3 receptors play in the psychomotor stimulant and reinforcing properties of cocaine. Robarge, M.J., Husbands, S.M., Kieltyka, A., Brodbeck, R., Thurkauf, A. and Newman, A.H. Journal of Medicinal Chemistry, 44, pp. 3175-3186, 2001.

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