RSA 2007 Abstracts

University of Texas at Austin

TRANSCRIPTOME DIVERGENCE BETWEEN ADOLESCENT AND ADULT “DID” ALCOHOL CONSUMING FVBxC57BL F1 MICE: S. E. Bergeson; O. Velasquez; M. K. Mulligan; P. S. Levin; J. A. Owen. Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology, University of Texas, Austin, TX  78712.

FVBxC57BL F1 hybrid mice were recently found by Yuri Blednov to drink more alcohol than any other strain of mouse.  We tested adolescent (age 30 days) and adult (70 day old) mice of this genetic mix using several two-bottle choice paradigms and a new procedure called “DID” (Drinking In the Dark developed by J Rhodes and JC Crabbe), which allows access to alcohol only during the highest 4 hours of drinking in the light/dark cycle.  Under most conditions, both male and female adolescent mice drank more alcohol than adult mice.  The most significant blood alcohol levels were reached in both adolescent and adult F1 mice using the DID protocol, with the young animals drinking significantly more than the adult mice.  Therefore, following a 4 day repeated binge DID procedure of access to 20% alcohol, trunk blood and brain samples were taken for BEC and gene expression analysis respectively.  Age- and alcohol-related brain transcriptome differences were determined using mRNA microarray analysis.  In silico bioinformatic analysis for pathway usage revealed evidence that adolescent alcohol drinking results in significant down-regulation of translation-related genes while adult drinking showed up-regulation of kinases as a group.  Chromosomal over-representation analysis suggested age divergent chromatin remodeling.  Cluster analysis indicated that alcohol drinking in general produces a developmental left shift in brain gene expression patterns.  Follow-up specific hypotheses testing may uncover significant consequences of drinking at a young age and allow a better understanding of why early drinking leads to an increased risk for alcoholism.


ETHANOL POTENTIATES DOPAMINE UPTAKE AND INCREASES CELL SURFACE DISTRIBUTION OF STABLY EXPRESSED DOPAMINE TRANSPORTERS IN HEK-293 CELLS: D. N. Riherd; D. G. Galindo; L. R. Krause; R. D. Mayfield. Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, Austin, TX 78712. 

Drugs of abuse, including ethanol, stimulate reward-reinforcement learning through activation of dopaminergic systems. The primary protein responsible for terminating dopamine (DA) neurotransmission is the plasma membrane-bound dopamine transporter (DAT), a major target for these drugs.  DAT trafficking is critical to dopaminergic homeostasis and function of the transporter in the brain. In vitro electrophysiological and biochemical studies in Xenopus oocytes suggest that ethanol potentiates DAT function by increasing the number of functional transporters on the cell surface.  However, it is not known if this increase is due to a decrease in transporter internalization or an increase in insertion (or both) on the cell surface. Ethanol’s effects on DAT function and trafficking have not been characterized in mammalian expression systems.  The goal of this study was to examine the effects of acute ethanol exposure on transporter function and cell surface distribution in HEK-293 cells stably expressing the human DAT. Ethanol’s effects on DAT function and cell surface distribution were investigated using  [3H] DA uptake and cell surface labeling biotinylation assays.  Ethanol potentiated DAT mediated DA uptake in a time (5, 60, 120 min) and concentration (25, 50, 100mM) dependent manner.  DAT surface expression increased 40-50% after acute ethanol exposure. Uptake kinetics data showed that DA uptake velocity increased (Vmax), while DAT affinity for DA (Km) remained unchanged.  These studies suggest that ethanol-induced enhancement of transporter function occurs in DAT expressing HEK-293 cells due to an increase in DAT density on the cell surface. 

MICE DEFICIENT IN THE β2 SUBUNIT OF THE GABAA RECEPTOR EXHIBIT DIFFERENTIAL GAT1 GENE AND PROTEIN EXPRESSION: J. Sobin; I. Ponomarev; S. Park; Y. A. Blednov; R. A. Harris; R. D. Mayfield. Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712.

GABAA receptors are involved in mediating many behavioral actions of alcohol. Mice deficient in the β2 subunit of the GABAA receptor are both viable and fertile, and show no obvious phenotypical abnormalities; however, GABAA receptor expression in β2 knockout mice is reduced by more then 50% in several brain regions. Previously, we used cDNA microarrays to investigate transcriptional changes in cerebellum of β2 null mutants. Interestingly, GABA transporter (GAT1) gene expression was increased by more than 300% in these animals compared to wild type controls. GAT1 is responsible for terminating synaptic GABA currents by clearing released neurotransmitter from the synaptic cleft. We investigated the impact of these gene expression changes on GAT-mediated GABA uptake and GAT1 protein levels in cerebellum. [3H]GABA uptake in cerebellar microsacs was significantly reduced (~ 40%) in β2 deficient animals compared to wild type controls. In addition, total GAT1 protein levels in knockout mice were reduced by approximately 35%, as observed by Western blotting. The reduction in GABAA receptor expression may prompt the cell to overexpress the GAT1 gene. In addition, other post-transcriptional regulatory mechanisms may be responsible for the decrease in GAT1 protein expression in β2 deficient animals.

PROTEOMIC ANALYSIS OF NEURODEGENERATION IN THE ALCOHOLIC BRAIN: N. Etheridge; J. M. Lewohl; R. D. Mayfield; R. A. Harris; P. R. Dodd.
University of Queensland, St Lucia, Queensland, 4072, Australia.

We used a proteomics approach to identify proteins involved in novel neurodegenerative pathways by comparing susceptible and spared brain regions of alcoholics and matched controls. It is well established that neuronal loss in chronic abuse alcoholics is restricted to particular brain regions, with the superior pre-frontal cortex (SFC) notably susceptible. Proteins involved in neuron loss are likely to be concentrated in synapses in these regions. We identified these proteins by 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) applied to the synaptosomal proteome isolated from susceptible (SFC) and spared (occipital) brain regions. Approximately 1700 individual protein spots were present on the 2D-DIGE gels of each of the 24 samples. Through a multiplexed comparison of all gels, we identified proteins specifically altered by excessive alcohol consumption. Of these, 96 were differentially regulated (greater than 1.2 fold; P < 0.05, t test) between alcoholics and controls solely in the occipital region, and 33 were regulated in both the SFC and occipital regions. This result highlights the fact that, although not susceptible to neuron loss, the occipital region can still be affected by alcohol. As they are regulated in a spared region, these proteins are unlikely to be involved in neurodegenerative cascades, although they may play a part in the dendritic pruning that is widespread throughout the alcoholic cerebral cortex. There were also 49 proteins regulated exclusively in the SFC, and these have the potential to be involved in alcohol-induced neurodegeneration. Selected proteins of this group were identified by MALDI-TOF mass spectrometry, and included moieties involved in vesicle trafficking, energy transduction, and glycolysis. Alcoholism-regulated proteins in the occipital region, or in both the SFC and occipital regions, were also identified by MALDI-TOF and included G-protein a-subunits, 14-3-3 proteins and heat-shock proteins. We use this information to elucidate new signal transduction pathways implicated in neurodegeneration and to characterize the role of alcohol in this process. Financial support was provided by NIAAA under grant NIH AA12404

 

 

INIA Home   INIA Structure   Administrative Core
Genetic Animal Models Core   Gene Expression Core   Imaging Core   Neuroinformatics Core
Subgroups   Current Grants   Conference Abstracts   Publications   Links