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RSA 2004 Abstracts
Indiana University - Purdue University, Indianapolis
MICROARRAY ANALYSIS OF CNS LIMBIC REGIONS OF INBRED ALCOHOL-PREFERRING (iP) AND ?NONPREFERRING (iNP) RATS AND EFFECTS OF ALCOHOL DRINKING
W.N. Strother; H.J. Edenberg; L. Lumeng; T.-K. Li; W.J. McBride
Depts Psychiatry & Medicine, Indiana University School of Medicine, Indianapolis, IN 46202
Limbic regions, such as the nucleus accumbens (Acb), are sensitive to the effects of ethanol and play a large role in alcohol reward and seeking behaviors. Affymetrix microarrays were performed to measure gene expression in discrete microdissected brain regions of alcohol-na?ve male iP and iNP rats, with a focus on the Acb. There were 83 genes that significantly differed in the Acb between ethanol-na?ve iP and iNP rats (p < 0.01). There were 53 genes that had lower expression and 30 genes that had higher expression in the iP than iNP rats. Of the genes that were identified, many of the them that had higher expression in the iP were involved in regulating cell growth, cellular stress response, protein trafficking, signal transduction, synaptic function, and metabolism. Among the genes that had lower expression in the Acb of iP rats were those involved in metabolic pathways, cellular signaling systems, protein trafficking, cell death, and neurotransmission. Following chronic free-choice alcohol drinking, there were 16 genes significantly different (p ? 0.01) in their expression in the Acb of ethanol-drinking iP rats compared to alcohol-na?ve iP rats. Most were up-regulated in the ethanol-drinking iP rats. Of the identified genes, almost all of the up-regulated genes were involved in cell survival, differentiation, and/or proliferation, as well as signal transduction and immune response. Overall, the results suggest that there are innate differences in genes regulating synaptic function in the Acb between iP and iNP rats that these differences may be related to their disparate alcohol drinking behaviors. These results also indicate that alcohol drinking altered gene expression in the Acb of iP rats, and that some of these alterations may underlie the neuroadaptations associated with the rewarding effects of alcohol. (Supported by AA07611, AA13521 [INIA], INGEN)
CHRONIC ALCOHOL DRINKING ALTERS PROTEIN EXPRESSION IN LIMBIC REGIONS OF INBRED ALCOHOL-PREFERRING (iP) RATS
M. W. Kimpel, W. N. Strother, H. Coppage, N. Pedrick, L. Lumeng, F. A. Witzmann, W. J. McBride
Depts Cellular & Integrative Physiology, Psychiatry, & Medicine, Indiana Univ Sch Medicine, Indianapolis, IN
The objective of the present study was to determine the effects of chronic free-choice alcohol drinking by iP rats on protein expression in CNS regions involved in mediating drinking, development of tolerance and motor activity. Two groups of adult male iP rats (n = 5/group) were given access to water alone (alcohol-na?ve group) or a choice between 15 % ethanol (EtOH) and water (EtOH experienced). At the end of 8 weeks, rats were killed, and the brains rapidly dissected into prefrontal cortex (PFC), nucleus accumbens (ACB), hippocampus (HIP), amygdala (AMYG) and striatum (STR). Tissue samples were stored at -70o C until processed for 2-dimensional gel electrophoresis (2-DGE). Gels were stained with Coomassie Brillant Blue. The densities of individual spots were determined using an image densitometer; the resulting values were analyzed using PDQuest software. The total number of gel spots detected was 3276 (AMYG), 1662 (STR), 1230 (HIP), 2154 (ACB) and 1354 (PFC). Abundance filtering reduced the number of gel spots 30 to 50 %. Using a False Discovery Rate (FDR) analysis to reduce the number of false positives (q<0.10), only the PFC, AMYG, and ACB had significantly different values between the two groups. The number of differences detected in each region, with the relative numbers of proteins down or up regulated with EtOH drinking were: PFC, 21 (19 down/2 up), AMYG, 7 (6 down/1 up), and in the ACC, 4 (all down). Proteins that were significantly different in the PFC, AMYG, and ACB between the two groups have yet to be identified. These data suggest that EtOH experience is producing significant changes in highly abundant proteins in three limbic regions involved in regulating alcohol drinking. (AA07611, AA07462, AA13521 [INIA], INGEN)
ALCOHOL DRINKING EXPERIENCE DURING PERI-ADOLESCENCE DID NOT PRODUCE LONG-TERM EFFECTS ON LOCAL CEREBRAL GLUCOSE UTILIZATION (LCGU) RATES IN RESPONSE TO AN ETHANOL CHALLENGE IN ADULT ALCOHOL-PREFERRING (P) RATS
W.N. Strother; C.C. Merrill; L. Lumeng; T.-K. Li; W.J. McBride
Depts Psych, Med, Indiana Univ Sch Med, Indianapolis, IN, 46202.
The objective of the present study was to determine the effects of an ethanol (E) challenge on LCGU rates in animals with previous E drinking experience during adolescence. Rats received 24-hr free choice access to 15% ethanol (v/v) and water (E drinking group, n=13) or water (W) only (na?ve, n=12) from postnatal day 30-60. An average of 7.6 g/kg/day E was consumed over the 30 day free-choice period and the two E groups did not significantly differ from each other. Following 2 weeks of W only access, animals were injected i.p. with 1.0 g/kg E (10 % v/v) (W+E N=6, E+E N= 7) or saline (W+S N=6, E+S N= 6) 10 min prior to an i.v. bolus of [14C]-2-deoxyglucose ([14C]-2-DG) (125 ?Ci/kg). 2-DG experiments were conducted during the first half of the dark cycle. Timed arterial blood samples were assayed for glucose, [14C]-2-DG and E content. Brains were quickly removed, snap frozen and 20 mm coronal sections prepared. Image densities were determined using quantitative autoradiography and LCGU values calculated using the operational equation defined by Sokoloff et al. (1977). Acute E resulted in significant decreases in LCGU rates in animals with and without adolescent E drinking experience in several limbic areas, most cortical areas, and posterior regions of the hippocampus. The two E injected groups did not significantly differ from each other in any of the brain regions examined. These results suggest that the acute suppressive effect of E on neuronal activity is a robust finding, and that 24-hr free-choice E drinking experience during peri-adolescence did not produce long-term alterations in the acute effects of E on neuronal activity. (AA07611, AA10256, AA13521 [INIA])
THE ROLE OF THE EXTENDED AMYGDALA IN LOCOMOTOR SENSITIZATION TO ALCOHOL IN MICE SELECTIVELY BRED FOR ALCOHOL PREFERENCE
Behm, A. Grose, K. Keller, and N. Grahame
Indiana Univ. Schl. Med., Indianapolis, IN 46202
Previous studies have shown that High Alcohol-Preferring (HAP) line 1 mice express locomotor sensitization to ethanol whereas Low Alcohol-Preferring (LAP) line 1 mice do not. Neural pathways involved in locomotor sensitization, such as the extended amygdala, may correlate with brain regions showing increased cellular activity, such as expressed through early immediate genes like c-fos. To address this, a total of 48 HAP and LAP males were randomly assigned to one of four treatment groups and received injections of 2.75g/kg ethanol or saline every 48 hours over a period of eight days. Then 48-72 hours later mice received a test injection of either 2.0g/kg ethanol or saline. Locomotor activity was recorded for 20 minutes following each injection and mice were perfused with 4% paraformaldehyde two hours after the test injection. Brains were sectioned coronally at 40 um and immunocytochemistry for c-fos was performed on every fifth section. Fos immunostaining was quantified stereologically in the Nucleus Accumbens shell (ACB-sh, n=27), Central Nucleus of the Amygdala (CeA, n=29), and the Bed Nucleus of the Stria Terminalis (BNST, n=24) using the optical fractionator method. As expected, only HAP mice showed locomotor sensitization to ethanol. This line difference in neuroadaptation to ethanol manifested in the CeA, where only HAP mice showed tolerance to the acute effects of ethanol-induced c-fos expression. Conversely, LAP mice expressed tolerance to ethanol?s acute ability to suppress c-fos expression in the BNST, while HAP mice did not. Also, only the LAP mice showed a significant elevation of c-fos expression in the ACB-sh with ethanol pretreatment, regardless of test condition. Together, the CeA, BNST, and ACB-sh partially comprise the extended amygdala, which has been implicated in alcohol locomotor stimulation as well as alcohol preference. Therefore, these data indicate that regions of the extended amygdala, such as the CeA, show patterns of c-Fos activation consistent with their involvement in genetic differences in locomotor sensitization. These findings may help link neural mechanisms common to both high alcohol drinking and locomotor sensitization. Support provided by AA13483 to NJG and AA07462 to WMB.
REPEATED DAILY SCHEDULED ACCESS RESULTS IN BINGE-LIKE ETHANOL CONSUMPTION IN ADULT INBRED P RATS
R.L. Bell; Z.A. Rodd; C.L. Boutwell; J.M. Murphy; L. Lumeng; T.-K. Li; W.J. McBride
Depts Psych, Med, Biochem, Indiana Univ Sch Med & VAMC, & Dept Psych, Purdue Sch of Sci, IUPUI, Indianapolis, IN 46202.
It has been reported that binge-like exposure to ethanol results in alterations in the brain. We sought to establish a drinking model of binge-like access (BA), which would result in daily intakes approaching that of alcohol-preferring (P) rats given continuous access (CA) to ethanol. Adult female and male inbred P rats either had concurrent BA or concurrent CA to multiple concentrations of ethanol (15% & 30%). For the BA protocol, animals had four 1-hr access periods, starting 1-hr after dark onset and separated by 2-hrs, across the dark-cycle. To enhance intake, BA-treated animals experienced two days of deprivation after each five days of access. Across, the 6th 5-day cycle, ethanol intake (g/kg/day) of female and male BA rats (6.2 ? 0.7 and 5.5 ? 0.2, respectively) approximated that of female and male CA rats (6.4 ? 0.4 and 6.5 ? 0.6, respectively). Similarly, on the 1st day of the 6th cycle, ethanol intake (g/kg/day) of female and male BA rats (6.3 ? 0.6 and 5.8 ? 0.3, respectively) approximated that of female and male CA rats (6.8 ? 0.7 and 6.2 ? 0.4, respectively). Ethanol intakes (g/kg/hr) for the 1st, 2nd, 3rd, and 4th 1-hr access periods of the female and male inbred P rats on this day were 2.5 ? 0.3 and 2.3 ? 0.2, 1.6 ? 0.3 and 1.0 ? 0.2, 1.2 ? 0.2 and 1.2 ? 0.2, 1.3 ? 0.2 and 1.5 ? 0.2, respectively. These results indicate that inbred P rats may serve as a functional animal model of binge-like self-administration in assessing the deleterious effects of chronic alcohol binges (AA07611, AA11261, AA13522 [INIA], Indiana Genomics Initiative [INGEN]).
ETHANOL ODOR ALTERS PREPULSE INHIBITION, BUT NOT ACOUSTIC STARTLE RESPONDING, IN ETHANOL-EXPERIENCED ADULT P RATS
R.L. Bell; Z.A. Rodd; C.L. Boutwell; R.B. Stewart; N.E. Badia-Elder; J.M. Murphy; W.J. McBride
Institute of Psychiatric Research, Indiana University School of Medicine, and Department of Psychology, Purdue School of Science, IUPUI, Indianapolis, IN 46202.
It has been reported that ethanol (E) exposure alters anxiety-associated behavior, and cues associated with E self-administration enhance craving and relapse behavior. We examined whether the presence of an E (40%) odor cue during acoustic startle responding (ASR) and prepulse inhibition (PPI) testing would alter these parameters in E-experienced alcohol-preferring (P) rats. Adult female P rats had concurrent access to 15% and 30% ethanol or water alone for 30 days, which was followed by 14 days of E deprivation. The P rats were drinking approximately 7 g/kg/day at the end of the treatment period. Subsequently, they were tested for ASR using four different dB levels (70-background, 95, 105 and 115 dB) and PPI using a 90 dB prepulse preceding a 115 dB startle stimulus. Prior E experience increased ASR to the 95 and 105 dB white-noise stimuli, compared with E na?ve animals (32 ? 8 vs. 17 ? 2 and 130 ? 28 vs. 71 ? 15, for the 95 dB and 105 dB trials respectively), with no effect of E odor. Regarding PPI, there was a significant interaction between E treatment and odor treatment; such that E odor disrupted PPI in E-experienced rats compared with E-exposed rats experiencing no odor during test (67 ? 5 vs. 81 ? 4, respectively). However, E odor had no significant effect on PPI in ethanol-na?ve rats. These findings indicate that 30 days of E self-administration may sensitize neurocircuitry mediating ASR in P rats, when tested at a time not associated with overt withdrawal symptomology. Additionally, an E odor cue appears to interfere with PPI neurocircuitry in E experienced animals, which may indicate altered attentional processes. (AA07611, AA10256, AA13522 [INIA], Indiana Genomics Initiative [INGEN]).
A CONDITIONED HEART RATE RESPONSE DURING DEPRIVATION IN ETHANOL-EXPERIENCED ALCOHOL-PREFERRING (P) RATS
R.L. Bell; Z.A. Rodd; C.L. Boutwell; L. Lumeng; T.-K. Li; W.J. McBride; J.M. Murphy
Depts Psych, Med, Biochem, Indiana Univ Sch Med & VAMC, & Dept Psych, Purdue Sch of Sci, IUPUI, Indianapolis, IN 46202.
Previously we reported that ethanol (E) self-administration results in elevated heart rate (HR) in alcohol-preferring (P) rats. In the present study we examined whether prior experience with E would affect HR [beats per min (BPM)] in adult female P rats during deprivation. HR radio telemetry devices were surgically implanted in the IP cavity seven days before animals were habituated to the test procedures. After 1 week of habituation (only water was available), animals were tested for 7 consecutive weeks, with three test groups: water (W) for 8 weeks; continuous access to E (CE) for weeks 2 through 8; and an alcohol deprivation effect (ADE) group, such that rats had access to E for weeks 2 through 5 and again during week 8, but had access to water only during weeks 6 and 7. Both E groups had concurrent access to 10%, 20%, and 30% E when E was available. Once a day, animals were placed in Plexiglas test cages with water available. Ninety min later the appropriate test solution was presented to the animal. HR was averaged across the 90 min pretest (PT) and 90 min test (T) periods for each test week. E, but not water, elevated HR during the T period, such that the average HR for the CE and W groups across weeks 5 through 8 was 421 ? 8 and 377 ? 11 BPM, respectively. For the ADE group, average HR during the T period for the 5th, 6th, 7th and 8th weeks were 410 ? 7, 390 ? 5, 397 ? 4, and 417 ? 6, respectively; with E deprivation during the 6th and 7th weeks. Overall, these findings indicate that HR stimulation by E consumption can be conditioned to environmental cues in P rats, which may parallel clinical work examining cue-reactivity in alcohol-dependent individuals. (AA07611, AA13522 [INIA], Indiana Genomics Initiative [INGEN]).
SELF-INFUSION OF ETHANOL INTO THE POSTERIOR VENTRAL TEGMENTAL AREA (VTA) ALTERS GENE EXPRESSION IN VTA PROJECTION AREAS
Z.A. Rodd; R.L. Bell; W. N. Strother; H.J.K. Sable, H. Aloor; J.N. McClintick; M. Stephens; R. Jerome; W.-M. Liu; L. Lumeng; J.M. Murphy; H.J. Edenberg; W.J. McBride
Depts. Psych., Biochem., and Med. Indiana Univ. Sch. Med., Indianapolis, IN 46202.
The objective of the present study was to determine the effects of self-infusion of 100 nl of aCSF or 150 mg% ethanol (EtOH) directly into the posterior VTA for 7 consecutive sessions on changes in gene expression in the nucleus accumbens (Acb), amygdala (Ag) and frontal cortex (FC) of inbred alcohol-preferring (iP) rats. Within the Acb, there were 350 significant changes in gene expression between the control (aCSF) and EtOH group, with some values being higher (indicated by plus sign) or lower (indicated by minus sign) in the EtOH group. These included multiple genes involved in regulating cell growth (+), cell death (-), cellular regulation (+), cyctoskeletal functions (+), cellular stress (-), and metabotropic glutamate (mGlu) and GABAA receptors (-). For example, genes that code for the proteins adenylate kinase 1, acyl-coA-oxidase, palmitoyl-protein thioesterase 2, all involved in synaptic growth, were all elevated in the EtOH self-administration group (1.34, 1.46, 1.48-fold increase, respectively). The expression of mGlu receptor genes Homer 1, mGlu 1 and 4 were all significantly reduced (-3.77, -1.43, -1.56-fold decrease, respectively). Similar alterations were observed in the FC and Ag. However, the Ag differed from the Acb and FC in that gene that code for proteins associated with the mGlu and GABAA receptors were upregulated. Overall, the results indicate that self-infusion of EtOH into the posterior VTA increases the expression of genes associated with cellular growth processes, such as those involved in growth dendrites and dendritic spines, suggesting that stimulating the VTA enhances synaptic function in its projection areas. However, the mGlu and GABAA receptor changes associated with stimulation of the VTA appear to be region specific and may be a result of differences in the interactions of the VTA inputs with the local circuits within each region. [Supported by AA07611, AA13521 (INIA), AA13496 (INIA), Indiana Genomics Initiative.]
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