Affective Neuropharmacology Lab: Recent Papers and Abstracts

OUR RESEARCH

Abstracts of Recent Presentations

Annual Meeting of Society of Neuroscience
Miami Beach, FL
November, 1999

DIFFERENTIAL hDAT ADAPTATION INDUCED BY SUSTAINED TREATMENT WITH VARIOUS DAT INHIBTORS IN NEURONAL CULTURE
Karley Y. Little *, Lian Zhang, Huailing Zhong, Gordon Crippen Departments of Psychiatry and Pharmaceutical Chemistry, University of Michigan, and Ann Arbor VAMC
Previous experiments have demonstrated that cocaine treatment increases hDAT uptake in neuronal culture, a result complementing human brain binding studies performed with the cocaine congener [3H]WIN 35428. It appears that the increases reflect an increased fraction of DAT molecules embedded in the outer membrane. Differing binding results have been obtained using different DAT radioligands, which is likely due to differential sensitivity of some radioligands to DAT location-plasma membrane-embedded vs internalized. Also, sustained exposure with various DAT inhibitors appears to cause different DAT responses. The present experiments in hDAT-expressing neuronal culture compared the effects of treatment with an array of DAT inhibitors to those of cocaine. Wild type hDAT cDNA (inserted in the pcDNA3 vector) was expressed in a mouse neuroblastoma neuronal culture (neuro2A). Cells were then treated for 24 hours with a number of DAT inhibitors, in doses ranging from 10-9M to 10-6M. Cells were then extensively washed before assay with [3H]WIN 35428, and terminal wash samples were examined for [3H]WIN 35428 binding IC50. There were marked differences in the effects of the inhibitors. DAT binding upregulators included cocaine, WIN 35428, bupropion, and methylphenidate. Downregulators included d-amphetamine, benztropine, mazindol, and RTI-55. A 3D -QSAR model was derived which successfully predicted the effect of methylphenidate before it was assayed. Further immunofluorescent, phosphoprotein, and electrophysiological studies are underway to determine the mechanisms involved. (NIDA 09491)

[confocal]

hDAT expressing neuro2A cells

After PMA treatment, 5 mM for 30 minutes- The outer border is slightly less distinct.

Vehicle treatment

After cocaine treatment, 1mM for 24 hours-The outer membrane is clearly more intensely labeled.

Annual Meeting of Society of Neuroscience
Miami Beach, FL
November, 1999

SEROTONIN TRANSPORTER GENOTYPE EFFECTS ON OVERALL SEROTONERGIC ADAPTATION IN HUMAN ALCOHOLICS: A POST MORTEM STUDY
Lian Zhang,Umesh Patel, Huailing Zhong, Karley Y. Little Department of Psychiatry, University of Michigan, Ann Arbor VAMC
The monoamine transporters appear to be dynamically regulated, which perhaps influences synaptic signaling, as well as neuronal sodium kinetics. Previous experiments have found that a SERT promoter region polymorphism affects SERT mRNA expression and binding site density in human midbrain nuclei. It has also been found that this SERT genotype affects the SERT response in human subjects with ethanol dependence. Alcoholic subjects with the short genotype demonstrate an increase in dorsal raphe [125I]]RTI-55 binding. The present experiments further examined the status of serotonin neurons in the same alcoholic subjects. There were significant increases overall found in 5-HT1A binding ([3H]8-OH-DPAT) in the dorsal subnuclei of the dorsal raphe. There was a complementary effect of SERT genotype on 5-HT1A binding. In contrast to those alcoholic subjects with the short or mixed SERT genotype (who displayed increased SERT, but no 5-HT1A changes), alcoholic subjects with the long SERT genotype accounted for all the increased 5-HT1A receptor binding. In additon, there were significantly increased 5-HT levels in alcoholic striatum, independent of SERT genotype, or SERT or 5-HT1A binding levels. The increase in 5-HT levels were inversely related to signs of dopamine neuronal loss. The complementary effect of SERT genotype on SERT and 5-HT1A function suggests that SERT expression may have marked effects on the overall adaptation of the serotonin neuron when perturbed by ethanol exposure. Individual response to SSRI and 5-HT1A agonist treatments may be related to SERT genotype. Alcoholics appear to have increased striatal 5-HT levels, perhaps as a reaction to lost dopamine function. (NIDA 09491)

Annual Meeting of Research Society for Alcoholism
Santa Barbara, CA
June, 1999

DECREASED STRIATAL DOPAMINERGIC TERMINALS IN ALCOHOLICS: AN ACCELERATED AGEING EFFECT
Karley Y. Little, Umesh Patel, Patrick McFinton, Lian Zhang, Sid Gilman, Tim Desmond, Kirk Frey, Bader J. Cassin
Departments of Psychiatry and Neurology, University of Michigan, and Ann Arbor VAMC, Ann Arbor, MI Vesicular monoamine transporter (VMAT2) binding has been found decreased in putamen of aged alcoholics by PET. In the present experiments, striatal dopamine concentrations, as well as dopamine transporter (DAT) and VMAT2 binding site densities were examined in post mortem brain tissue from alcoholics and controls. DAT mRNA levels were also quantitated in midbrain samples. It was hypothesized that there would be a generalized age-associated dopamine cell loss. Specimens from alcoholic subjects (n=14) were matched to controls (n=17) based on age, sex, race, and post mortem interval. Dopamine and metabolite concentrations were assayed in caudate, putamen, accumbens, and frontal cortex samples, using HPLC. Striatal sections were assayed using quantitative autoradiography employing the radioligands, [3H]WIN 35428 (3nM concentration, DAT selective), and [3H]dihydrotetrabenazine, (3nM concentration, VMAT2 selective). Midbrain DAT mRNA levels were quantified using a 580bp riboprobe and in situ hybridization. Dopamine levels were significantly lower in alcoholics in each region and were inversely correlated with age. Similarly, binding to the DAT was decreased in alcoholics across the striatum, reaching statistical significance in the putamen (12% decrease, p< .05). VMAT2 binding also appeared decreased, but statistical significance was not reached. In both assays, binding strongly decreased with advancing age (DAT: r= 0.71, p<.01; VMAT2: r= 0.71, p<.01, age range 18-62 yrs). In contrast, there were no changes in DAT mRNA levels in alcoholics, nor a relationship with age. The results suggest the alcohol/age interaction effects dopamine terminals, but does not cause loss of the entire neuron.

Annual Meeting of Society of Biological Psychiatry
Washington, DC
May, 1999

ALTERED STRIATAL GENE EXPRESSION IN HUMAN COCAINE USERS DETECTED BY DD-PCR.
R. C. Thompson (1,2), P. R. McFinton (1), K. Y. Little (1,3) Departments of Psychiatry (1) and Obstetrics-Gynecology (2), University of Michigan, and Ann Arbor VAMC (3), Ann Arbor, MI, 48105
The Differential Display-Polymerase Chain Reaction (DD-PCR) technique allows tissue samples to be screened for alterations in gene expression. Novel as well as known genes can be identified with this methodology, but this approach is characteristically used to identify changes in novel proteins that would not be expected based on existing knowledge. We have utilized this technique to survey striatal samples taken at autopsy from five human cocaine users and five age-, sex-, race-, and post mortem interval- matched controls. Striatal tissue was dissected and fresh frozen on dry ice at autopsy. Approximately 150mg of tissue was used for RNA isolation. The purified RNA samples were selectively converted into pools of cDNA and subjected to PCR amplification with arbitrary primers (GeneHunter's RNAimage kit). These reactions generated radioactive DNA bands that approximated the abundance of RNA molecules in the original sample. Aliquots of reaction mixtures were examined by gel electrophoresis and differences on film identified by visual inspection. Candidate bands were excised, cloned into plasmid vectors, transformed into bacteria, grown, harvested, and characterized by DNA sequence analysis. Resulting DNA sequences were initially compared to available DNA sequence databases. Also, plasmid clones derived from each of these differentially expressed PCR bands were used to generate riboprobes for in situ hybridization. Using a single set of random primers, six mRNA species have been detected that appear altered in cocaine users as compared to control patients. The sequences for these species do not code for proteins previously associated with drug abuse. (NIDA 09491)