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RSA 2005 Abstracts
INDIANA UNIVERSITY - PURDUE UNIVERSITY, INDIANAPOLIS
BOUTS OF EXCESSIVE ALCOHOL DRINKING ALTER PROTEIN EXPRESSION IN LIMBIC REGIONS OF INBRED ALCOHOL-PREFERRING (iP) RATS
M.W. Kimpel, R.L. Bell, W.N. Strother, D. Thomas, H. Coppage, N. Pedrick, F.A. Witzmann, W.J. McBride
Departments of Psychiatry & Physiology, Indiana University School of Medicine, Indianapolis, IN 46202
The objective of the present study was to determine the effects of bouts of excessive alcohol drinking on protein expression in limbic regions of iP rats. During an 8-week period 3 groups (n = 5/group) of adult female iP rats were tested: a control group given water only (W group); a group given 24-hr free-choice access to water, and 15 and 30 % ethanol (F-C group); and a group given four daily 1-hr access sessions during the dark cycle to 15 and 30% ethanol with water always available (bout-drinking [B-D] group). Both the F-C and B-D groups consumed 6.5 – 7.0 g/kg/day, but the B-D group consumed this amount in bouts of 1.2 – 2 g/kg/session. The nucleus accumbens (ACB), prefrontal cortex (PFC), amygdala (AMYG) and hippocampus (HIP) were dissected and analyzed by two-dimensional gel electrophoresis. Gels were stained with colloidal Coomaisse Brilliant Blue, scanned, and the resulting images analyzed with PDQuest software. Statistical significance (p < 0.025) was determined using an ANOVA model. Comparison of the W group with the F-C group indicated that free-choice alcohol drinking altered the expression of 20 proteins in the ACB, 15 in the PFC, 44 in the AMYG and 20 in the HIP. Moreover, comparison of the B-D group with the F-C group indicated that these two alcohol-drinking conditions produced differences in the expression of 7 proteins in the ACB, 6 in the PFC, 12 in the AMYG, and 6 in the HIP. The few proteins that have been identified thus far indicate that, in the ACB, alcohol drinking reduced the levels of several kinases involved in cellular signaling pathways and a sodium-channel associated protein, and increased the levels of a calcium-binding protein. Overall, the results of this study indicate that alcohol drinking alters the expression of proteins in the limbic system of iP rats and that, among the 4 regions assayed, the AMYG is the most sensitive to the effects of free-choice ethanol. In addition, bout drinking and free-choice drinking may produce differential effects on protein expression. (AA07611, AA07462, AA13521 [INIA Project], AA13522 [INIA Project], and INGEN®)
CHRONIC ETHANOL EXPOSURE RESULTS IN LONG-TERM ALTERATIONS IN FOS B IMMUNOREACTIVITY IN SELECT LIMBIC BRAIN REGIONS OF ALCOHOL-PREFERRING (P) RATS
W.N. Strother; C.C. Merrill; L. Lumeng; W.J. McBrideDepts Psych, Med, Indiana Univ Sch Med, Indianapolis, IN, 46202
The induction of Fos B is thought to mediate neuroadaptations to chronic drug exposure and can be used to study neuronal pathways involved in long-term neuroadaptations (Nestler et al., 2001). The objective of the present study was to determine the effects of chronic ethanol exposure on Fos B immunoreactivity (ir) in the limbic system of adult P rats. P rats received 24-hr free choice access to 10% ethanol (v/v) and water (n=10) or water only (n=10) for a period of 10 weeks. Rats were sacrificed during the light portion of the light/dark cycle with ethanol removed 4 hrs prior. Rats were transcardially perfused with 4% paraformaldehyde and post-fixed overnight. Coronal 40 µm sections were collected in phosphate buffer using a freezing microtome. Fos B-ir (Santa Cruz @ 1:1000) was detected using a modified avidin-biotin-immunoperoxidase protocol with diaminobenzidine as the chromogen. Sections were slide mounted, dehydrated and coverslipped using standard procedures. The stereological optical dissector method was used to quantitate Fos B-ir in select brain regions using Stereo Investigator software (MicroBrightField, Inc). Chronic ethanol drinking resulted in increased Fos B-ir by 20-37 % compared to control values in multiple brain regions. Significant increases were found in the nucleus accumbens core (20%) and shell (30%) subregions, the basolateral nucleus of the amygdala (30%), and the largest increase was found in the ventral tegmental area (37%). There was no significant difference between the groups in the medial prefrontal cortex and a decrease (25%) in Fos B-ir in the central nucleus of the amygdala of ethanol drinking P rats compared to control P rats. These results demonstrate that chronic ethanol drinking resulted in region-specific long-lasting alterations in Fos B protein levels. Such increases in transcription factor levels suggest long-term ethanol-induced neuroadaptations have taken place in these limbic regions. (AA07611, AA13521 [INIA])
ETHANOL SELF-ADMINISTRATION ALTERS GENE EXPRESSION IN THE NUCLEUS ACCUMBENS OF INBRED ALCOHOL-PREFERRING (iP) RATS
Z. A. Rodd, W.J. McBride, R. L. Bell; W. N. Strother; H. Aloor; J.N. McClintick; R. Jerome; W.-M. Liu; L. Lumeng; H. J. EdenbergDepts. Psychiat & Biochem, Indiana University School of Medicine, Indianapolis, IN
The objective of the current study was to examine the effects of operant oral ethanol (E) self-administration on gene expression in the nucleus accumbens (Acb) of iP rats. E-naïve iP rats were self-trained on a standard two-lever operant paradigm using daily 1 hr sessions. Rats (n = 6/group) were allowed to self-administer either water-water, E (15% v/v)- water, or saccharin (S; 0.0125%)- water. A concurrent FR5-FR1 schedule of reinforcement was used; the operant task was conducted over a 10-week period. Animals were killed 24-hr after the last operant session and the Acb was dissected; RNA was extracted and purified for microarray analysis. Affymetrix Rat Genome 230 2.0 Array GeneChips were used to analyze differences in gene expression between the animals that self-administered E-water, S-water, and water-water. Comparing across the 3 groups (with ANOVAs), there were 1604 significant differences in gene expression at the p< 0.05 level, and 359 at the p < 0.01 level. Additional t-tests were applied to detect expression differences between the groups. There were 1172 (p < 0.05) or 233 (p < 0.01) differences in expression between rats that were allowed to self-administer E-water compared to the water-water group. Comparing E-water and S-water groups, there were 1612 (p < 0.05) or 367 (p < 0.01) significant differences in gene expression. Overall, the data indicated that E self-administration altered the expression of many more genes than did S self-administration. Some genes that were differentially expressed between the E-water group and the other two groups included the NMDA receptor, metabotropic glutamate receptor, and genes involved in synaptic plasticity. These results suggest that E self-administration produces neuronal alterations that strengthen synaptic function in the Acb. [AA07611, AA13521 (INIA Project), AA13496 (INIA Project), INGEN®]
LOCAL CEREBRAL GLUCOSE UTILIZATION (LCGU) RATES IN PERI-ADOLESCENT ALCOHOL-PREFERRING (P) RATS FOLLOWING SCHEDULED ACCESS ETHANOL (E) DRINKINGH.J.K. Sable*; D. Ji; W.N. Strother; R.L. Bell; Z.A. Rodd; J.A. Schultz, L. Lumeng; W.J. McBride
Depts Psych, Med, Biochem, Indiana Univ Sch Med, Indianapolis, IN, 46202
The objective of the present study was to determine if alterations in LCGU would be observed following chronic peri-adolescent (PND 30-60) scheduled-access E drinking in P rats. Beginning on PND 30 and continuing daily until PND 60, rats were transferred to drinking cages in a separate room from 0900 to 1300 hr. During this time, half of the animals were given 15% (v/v) E and water, while the remaining half were given water only. At the conclusion of scheduled-access drinking, one-third of the animals in each group were injected i.v. with 2-[14C]-2-deoxyglucose ([14C]-2-DG) (125 µCi/kg) at 0900 hr on PND 63, 67, or 74. No E was given prior to or during the 2-DG test session. After 45 min, brains were quickly removed and frozen in isopentane (-45ŠC). Coronal sections were cut and apposed to film for two days; films were then analyzed using quantitative autoradiography. Timed arterial blood samples collected over the 2-DG session were assayed for [14C]-2-DG content and glucose. Rats given E consumed 1.9 + 0.2 g/kg/4 hr with no significant differences between groups. LCGU data were examined in several limbic regions including the medial prefrontal cortex (MPFC), nucleus accumbens core (AcbC) and shell (AcbSh), ventral tegmental area (VTA), central (CeA) and basolateral (BLA) nuclei of the amygdala, ventral pallidum (VP), lateral septum (LS), and the olfactory tubercle (OTU). Data collected from the water control animals at PND 63, 67, and 74 did not statistically differ so these groups were combined to simplify data analyses. Functional activity measured in E exposed animals on PND 67 was significantly higher than water controls and the other E exposed groups in the MPFC and BLA. Similar findings were observed in the AcbSh, with the exception that the E exposed animals tested on PND 63 were not significantly different from those tested on PND 67. These results indicate one week of E deprivation increases synaptic activity in select VTA projection areas. With only 3 days of E deprivation, functional activity in the AcbC was also elevated above water controls but returned to control levels by PND 67. Overall, these results suggest E drinking during peri-adolescence produces transient increases in functional neuronal activity that could result in long-term consequences. (AA07611, AA10256, AA13521 [INIA], AA13522 (INIA Project), INGEN®)
PATTERN OF ETHANOL DRINKING BY ADOLESCENT AND ADULT HIGH ALCOHOL DRINKING (HAD-2) RATSR.L. Bell; Z.A. Rodd; J.A. Schultz; K.M. Troy; L. Lumeng; J.M. Murphy; W.J. McBride
Depts of Psych, Med, Biochem, Indiana University School of Medicine & VAMC, & Dept Psych, Purdue School of Science, IUPUI, Indianapolis, IN 46202
Little is known about the circadian patterns of ethanol (E) consumption in HAD rats. We examined the licking pattern for E (15%) by HAD-2 rats during adolescence (ADOL), post-natal days (PNDs) 30 through 60, or adulthood (ADUL), for 30 consecutive days starting after PND 90. Male and female ADOL and male and female ADUL HAD rats increased their E intake across weeks (1st vs. 4th week: 4.0 ± 0.5 vs. 7.9 ± 0.5, 3.3 ± 0.3 vs. 5.9 ± 0.7, 4.8 ± 0.5 vs. 6.8 ± 0.6, and 5.5 ± 0.7 vs. 7.5 ± 0.8 g/kg/day, respectively). Regarding licking activity, on the E bottle, ADOL male rats displayed a substantial increase, ADOL female rats displayed a modest increase during the 2nd week which was maintained, ADUL male rats displayed a modest decrease during the 4th week, and ADUL female rats had stable licking activity across the 30 days of access, with ADOL rats displaying greater licking activity than ADUL rats. As expected, the majority of licking activity was done during the dark cycle. Average total lick activity on the E bottle for male and female ADOL and male and female ADUL HAD rats was 520 vs. 112 and 1883 vs. 469, 326 vs. 42 and 539 vs. 157, 1395 vs. 329 and 1015 vs. 277, and 300 vs. 161 and 305 vs. 164 for the dark vs. light cycle during the 1st and 4th weeks of access, respectively. These results indicate that, even though the majority of the E was consumed during the dark-cycle, the largest bout was not at the beginning, as would be expected in a limited access period, but was at the end, suggesting the animals “loaded” up before their normal sleep period. Furthermore, developmental differences in licking behavior (change across weeks) appeared to be limited, for the most part, to male HAD-2 rats. [AA07611, AA10256, AA10722, AA13522 (INIA)]
PRE- AND POSTSYNAPTIC ALTERATIONS UPON CHRONIC ALCOHOL EXPOSURE IN THE NUCLEUS ACCUMBENS
F.C. Zhou; K. Dunn; R. Bell; F. Tai; K. Cheng; Y. Sari; Z.C. Xu; P. Deng
Indiana University School of Medicine, Departments of Anatomy and Cell Biology, Nephrology, Psychiatric Research, Indianapolis, IN 46202Long-term repeated alcohol consumption elicits alcohol dependency in a subpopulation of drinkers. It is known that chronic alcohol alters brain function, it is unclear whether addictive alcohol-seeking behavior has a neuroanatomical basis. To begin to address these important questions, we are examining changes that occur at the pre- and post-synaptic level in brain regions that mediate reward in alcohol preferring P rats treated with continuous chronic alcohol (CC, 14 wks), repeated deprivation (RD, 6 wks on and twice of 2wks-on and -off), or water (Water Control, 14 wks). Our experimental end-points include examination of the vesicular glutamate transporter (vGluT) at presynaptic glutamate terminals and postsynaptic dendritic spines in median spiny neurons (MSN) in the extended amygdala. The vGluT, depending on its subtype, is clustered in the synaptic terminals of a subset of glutaminergic (GLU) innervation of specific regions of brain. We found that vGluT2 positive terminals were specifically increased in the shell of the nucleus accumbens in both the CC group and in the RD group as compared with that of WC group. Examination of Alex488 fluorescent dye-filled MSNs using two-photon laser scanning microscopy revealed that the density of spines was lower in the CC and RD groups as compared with that of WC group. However, although reduced in number, there was an increase in their size. Furthermore, larger sized spines often appeared two-headed in the RD group, and dendritic swelling was occasionally seen in both the CC and RD groups. Thus, prolonged ethanol exposure appeared to induce an increase in presynaptic GLU terminals clustered on fewer but larger sized dendritic spines. Since dendritic spines provide the post-synaptic sites for the vast majority of GLU synapses in the brain, changes in the architecture of the pre- and post-synaptic components likely reflect plastic changes and rewiring of MSN following chronic alcohol treatments. Supported by NIH U01 AA014829, AA0711, and AA13522
VALIDATION OF SELECTIVELY BRED HIGH ALCOHOL PREFERRING MICE FOR DISCOVERY OF POTENTIAL PHARMACOTHERAPIES TO REDUCE ALCOHOL DRINKING
B. Oberlin, N. GrahameIndiana University School of Medicine, Indianapolis, IN 46202
High Alcohol Preferring (HAP) mice have been selectively bred for high alcohol intake, and prefer alcohol to water during both 24-h and 30-min free-choice access (~78% preference for the alcohol tube during 30-min access). Although the HAP mice show robust ethanol consumption and preference, it has not been established that drugs used to treat human alcoholics can also reduce ethanol drinking in HAPs. Our hypothesis was that drugs showing efficacy in human alcoholism would cause a reduction in ethanol consumption in HAP mice. Furthermore, we sought to test whether putative pharmacotherapies had selective effects on ethanol intake, as opposed to causing a general reduction in consumption. Because previous studies indicated low preference for the non-alcohol containing solution, saccharin (0.1% w/v) was added to both the alcohol and water solutions to increase intake of the non-alcohol fluid. Selectivity in this study was defined as a decrease in saccharin / ethanol consumption without a corresponding decrease in saccharin / water consumption. HAP mice were individually housed with ad lib access to food and water. Tubes of either 8% ethanol or water, both with saccharin, were placed on their cages for 30 minutes during the dark part of the cycle. Drinking stabilized at about 1.38 g/kg, 63% ethanol preference, and a mean BAC of 57.5 mg/dl (range 0-143 mg/dl). To minimize floor effects, low drinkers (~14% of mice) were eliminated from the study before drugs were administered, and mice were assigned to drug groups (ns = 12-13) each week that were balanced for pre-drug alcohol intake. On test days, drug or vehicle was injected i.p. at a volume of 10 ml/kg 30 minutes prior to ethanol access. The mu-opioid receptor antagonist Naltrexone (3 & 10 mg/kg), and the NMDA antagonist Memantine (1 & 5 mg/kg) selectively decreased ethanol consumption, while the CRF1 antagonist NBI3b1996 (10 & 30 mg/kg), and the CB1 antagonist AM 251 (1, 3, 5.6, and 10 mg/kg) non-selectively decreased ethanol consumption. These findings are consistent with human and animal studies of these drugs’ effects on alcohol drinking, and validate the use of HAP mice as an animal model for testing the efficacy of novel pharmacotherapies for alcoholism. AA 13483 suppl 1 to NJG
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