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RSA 2009 Abstracts
Invertebrates as a gene discovery tool for alcoholism
ORGANIZER: NIGEL S. ATKINSON
CHAIRS: NIGEL S. ATKINSON AND JON PIERCE-SHIMOMURA
RATIONALE AND CONTENT:
The focus of this symposium is the use of the invertebrate model systems Caenorhabditis elegans and Drosophila melanogaster for the identification of genes important for the response to alcohol. With all model systems, there is a trade-off between simplicity and applicability, but Caenorhabditis and Drosophila offer an attractive compromise. These animals exhibit many of the same behaviors humans do, including learning, memory, courtship, sleep, and aggression. It has also become clear that the behavioral, physiological, and cellular responses to alcohol in these animals is very similar to the responses to alcohol by mammals. Over the last decade, the powerful genetic tools specific to invertebrates have been applied to study the behavioral responses to ethanol. Because in invertebrates it is practical to screen through thousands to millions of animals, one can identify rare mutations that affect specific alcohol responses. Both Caenorhabditis and Drosophila have well-described and compact nervous systems and genes that have a surprisingly high degree of conservation with their human homologs. Furthermore most regulatory cascades important for mammalian development were originally discovered in or shown to exist in one or both of these invertebrates species. In this symposium we will discuss recent advances that capitalize on the unique genetic tools available in these two model organisms. The speakers in this session examine alcohol responsivity from different perspectives. Using either C. elegans or D. melanogaster, these speakers have identified cellular signaling pathways important for initial alcohol sensitivity and for the production of acute alcohol tolerance. The genes implicated encode highly conserved proteins such as heteromeric G proteins, NPY receptors, a Rho-type GTPase involved in the regulation of cytoskeleton dynamics, and transcription factors that regulate the expression of calcium activated potassium channels.
EVALUATING WITHDRAWAL-LIKE BEHAVIOR 8 TO 15 HOURS AFTER CESSATION OF CHRONIC 24-HR FREE-CHOICE ETHANOL DRINKING IN ALCOHOL-PREFERRING (P) RATS
R.L. Bell; S.R. Hauser; R.J. Smith; K.K. McConnell; Z.A. Rodd; W.J. McBride
Institute of Psychiatric Research, Dept. Psychiatry, Indiana University School of Medicine, Indianapolis, INChronic ethanol (E) consumption leads to withdrawal-like behavior in all species. The present study examined acoustic startle responding (ASR) and locomotor activity in a novel field (LMA) during the early acute deprivation period, following chronic E access in male P rats. Three groups of rats experienced either (1) a repeated deprivation (RD) schedule [6-weeks of E access followed by 7 cycles of 2 wk deprivation and 2 wk re-exposure], (2) continuous E access (CE) for the entire repeated deprivation schedule, or (3) remained ethanol-naïve (W) throughout. After the initial 12 hr of the 5th re-exposure, E was removed from the RD and CE rats, which were tested between 9 and 13 hr later, for 30 min, in a LMA test apparatus. E was returned following this test. After the initial 12 hr of the 6th re-exposure, E was removed from the RD and CE rats, which were tested between 8 and 15 hr later, for 35 min, in ASR chambers. The RD rats displayed a significant (p’s<.015) 1 hr, but not 24 hr, alcohol deprivation effect (ADE), relative to CE rats, during the 1st through 5th re-exposure cycles. During the 1st hr of relapse, E intakes varied across cycles between 1.7 and 2.6 g/kg/hr for the CE rats vs. 2.1 and 3.4 g/kg/hr for the RD rats. For the 1st hr of the 7th re-exposure, RD rats consumed 1.5 g/kg, whereas CE rats consumed 2.2 g/kg, with 57 mg% and 47 mg% BACs respectively. Regarding ASR, the RD rats displayed a significantly (p’s<.01) greater ASR to 95 dB and 105 dB white-noise bursts, with a trend towards significance (p=.064) at the 115 dB level, relative to W rats [RD rats: 27, 408 and 932 vs. W rats: 5, 152 and 647 in arbitrary units, for the 95, 105 and 115 dB white-noise bursts]. The CE rats only displayed a trend for significantly (p=.079) greater ASR at the 115 dB level [919], relative to W rats. Both RD and CE rats displayed higher LMA than W rats [distance (cm) 5050, 4900 and 3250; margin distance (cm) 2100, 2000 and 1450; center distance (cm) 2950, 2875 and 1850; rearing time 215, 190 and 150 as 30 min totals, respectively]. Overall, the results suggest that greater emotional/reactivity is exhibited during acute withdrawal when subjects have been exposed to repeated deprivations. (Supported in part by AA07611, AA07462, AA13522 [INIA Project])
PROTEIN EXPRESSION CHANGES IN PREFRONTAL CORTEX, CORPUS CALLOSUM, AND ANTERIOR DORSAL CEREBELLUM AFTER ETHANOL EXPOSURE IN WISTAR RATS
R.L. Bell; H.N. Ringham; N.M. Zahr; W.A. Truitt; J. Orduna; E.V. Sullivan; F.A. Witzmann; A. Pfefferbaum
Departments of Psychiatry, Physiology, Anatomy & Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202; Neuroscience Program, SRI International, Menlo Park, CA 94305
Chronic alcohol abuse is associated with regional cortical, callosal and cerebellar damage. Here, we sought to determine the effects of chronic, intermittent, heavy ethanol exposure on protein expression in these brain regions. During a 24-week period, 2 groups (n = 6-7/group) of adult male Wistar rats were exposed to escalating concentrations of ethanol vapor (EtOH) or control air (CONT) in commercially available vapor chambers. At 16 weeks the rats displayed ~ 300 mg% peak BACs, and at 24 weeks the rats displayed ~ 450 mg% peak BACs. After 24 weeks, during which the rats never experienced a protracted withdrawal period, the animals were euthanized by decapitation, the brain extracted, flash-frozen and stored, at -80 deg F, for micropunch dissection. The prefrontal cortex (PFC), corpus callosum genu (CCG) and body (CCB), cerebellar vermis (VER), and anterior dorsal cerebellum (ADC) were dissected and analyzed by two-dimensional gel electrophoresis. Gels were stained with colloidal Coomassie Brilliant Blue, scanned, and the resulting images analyzed with PDQuest software. Statistical significance (p < 0.025) was determined using an ANOVA model, which revealed that 17 protein spots in the PFC, 5 spots in the CCG, 7 spots in the CCB, 9 spots in the VER, and 8 spots in the ADC differed significantly between the EtOH and CONT groups, with no overlap in differentially expressed proteins across the brain regions. Of the 32 proteins positively identified (≥99% confidence), 17 were decreased and 15 were increased. Eight proteins, 5 increased, were associated with the cytoskeleton; 4 proteins, 2 increased, were associated with neurotransmitter synthesis or receptor activity; 9 proteins, 6 increased, were associated with cellular stress; 14 proteins, 6 increased, were associated with metabolic processes; and 10 proteins, 7 increased, were associated with the mitochondrion. Overall, the results indicate that exposure to high concentrations of ethanol alters neuronal plasticity, metabolism, and protein synthesis or transport, with the greatest number of expression changes in the PFC. (AA07611, AA07462, AA13521 [INIA Project], AA13522 [INIA Project], and INGEN®)
EXAMINING THE GENETICS OF ETHANOL REWARD USING CONSOMIC RAT LINES DERIVED FROM FHH AND BN PROGENITORS. I. CHARACTERIZING THE FHH RATS’ ETHANOL DRINKING BEHAVIOR
R.L. Bell; B.J.A. Eiler; K.K. McConnell; D. Evans; L. Lumeng; S.H. Nye
Department of Psychiatry, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202; PhysioGenix, Inc., 10437 Innovation Drive, Milwaukee, WI 53226
Identifying the causal genes for complex human disorders in rodent models by standard positional cloning methods is time consuming, laborious and expensive. To circumvent this problem for alcoholism research, we tested the feasibility of using chromosomal substitution (known as consomic) rats for studying the genetic basis of ethanol (E) reward and associated behaviors. The consomic rat panel is composed of 22 inbred strains generated by systematically substituting chromosomes from the normal (low) drinking Brown Norway (BN) rat onto the genetic background of the high E-consuming Fawn Hooded Hypertensive (FHH) rats. The present study examined the E (10%) drinking behavior of both male and female adult FHH rats, to confirm their use as a high E drinking animal model. The 2 major criteria to be tested were whether (a) FHH rats drink E in bouts, and (b) whether they achieve pharmacologically relevant BACs during continuous, free-choice, home-cage E access. Briefly, a commercial “Lickometer” test set-up recorded how often these rats licked an E or water (W) bottle spout 22 hrs (in 6 min blocks)/day during the animals’ initial 30 days of access to 10% E. As with other rat lines, female FHH rats consumed more E and water than male FHH rats (~ 5.5 g/kg/day vs. ~ 4 g/kg/day and ~ 175 ml/kg/day vs. ~ 150 mg/kg/day, respectively). At least 50% of male and female FHH rats displayed licking behavior, with 1 or more bouts/day, often more, reaching levels associated with the self-administration of 1 g/kg/6 min. Other male and female FHH rats approached this level of licking behavior. Regarding BACs, on average, female vs. male FHH rats displayed 26.4 mg% vs. 28.9 mg% and 31.8 mg% vs. 41.1 mg% after the 1st two hr and at the end of the dark cycle, respectively. These findings indicate that FHH rats will consume daily amounts of E approximating levels seen with some animal models of alcoholism, often in bouts approximating 1 g/kg/6 min, and will achieve pharmacologically relevant BACs during continuous, free-choice access to E. These results support the use of the FHH line in genetic analyses of E-associated reward and behavior (see adjacent abstract). (NIAAA AA13522 [INIA Project])
BEHAVIORAL VALIDATION OF GENE TARGETS OBTAINED FROM MICROARRAY ANALYSES.
Y.A. Blednov; I. Ponomarev; S.E. Bergeson; R.A. Harris
University of Texas, Waggoner Center for Alcohol and Addiction Research, Austin, TX 78712. Texas TECH University, Health Sciences Center, Lubbock, TX, 79430.
Analysis of mouse brain gene expression using three selected lines bred for high and low alcohol drinking, five inbred strains known to differ in voluntary alcohol consumption, and a hybrid strain with high voluntary alcohol consumption provided a number of novel candidate genes that might regulate alcohol consumption (Mulligan et al., 2006 and unpublished). We selected four genes, beta-2-microglobulin (B2m); cathepsin S (Ctss); cathepsin F (Ctsf) and interleukin 1 receptor antagonist (IL1rn) for functional validation using null mutant mice. The genes are known to be important for inflammatory and immune responses but are not related to the ‘usual suspects’ for alcohol consumption and provide a test of the idea that global gene analysis can yield novel targets. Null mutant mice were tested for ethanol intake in two tests: 24 hr two-bottle choice and two-bottle choice during limited access to ethanol (3 hrs drinking in the dark, 2B-DID). All four lines of knockout mice showed reduced ethanol consumption and preference in the 24 hr two-bottle choice test, the test which was the basis for selection of these genes. However, deletion of these genes did not consistently reduce ethanol consumption in the 2B-DID test. For example, deletion of Ctss markedly reduced ethanol preference in the 24 hr test, but not in the 3 hr DID test. The observed differences in ethanol preference and intake could not be explained by differences in taste perception (saccharin or quinine consumption). These results suggest a novel role for inflammatory signaling in regulation of alcohol consumption. We believe this is the first example of behavioral validation of alcohol effect genes obtained from microarray studies. Supported by NIH/NIAAA (AA U01 13520 -INIA Project and AA06399).
E.A. Young; T.L. Fidler; J.A. Mulgrew; A.T. Crane; P.J. Smitasin; C.L. Cunningham. Department of Behavioral Neuroscience, Oregon Health & Science University, Portland, OR 97239
INCREASING THE NUMBER OF DAYS OF PASSIVE INTRAGASTRIC (IG) ETHANOL EXPOSURE INCREASES IG ETHANOL SELF-ADMINISTRATION IN C57BL/6J AND DBA/2J MICE
TL Fidler; MS Powers; JJ Ramirez; PJ Smitasin; AT Crane; JA Mulgrew; CL Cunningham. Oregon Health & Science University, Portland, OR 97239.
We have previously shown that several days of passive ethanol exposure increases ethanol self-infusion in rats (Sprague Dawley, HAD and LAD) and in mice (C57BL/6 (B6), DBA/2J (D2), FVB/NJ and B6xFVB F1) when licks on a Kool-Aid tube are later paired with intragastric (IG) infusions of ethanol. In the current experiment, we used passive infusion parameters optimized in previous experiments, 3 infusions/day and a starting dose of 3 g/kg/infusion, to study how ethanol self-infusion is affected by the number of days of passive ethanol infusions. B6 and D2 mice were surgically implanted with IG catheters and allowed to recover. Different groups within each strain received 0, 3, 5 or 10 days of passive ethanol infusions. The 0 group received water infusions matched to the 10-day group. During subsequent self-infusion phases, mice had access (23 h/day) to one or two Kool-Aid solutions presented in drinking tubes (without ethanol). During the first 2 days of self-infusion, only one flavor was available (No-Choice) and licks produced IG ethanol infusions (20% v/v). During the next 5 days (Choice), a second flavor was also available and licks on this flavor were paired with infusions of water. As expected, in both strains, ethanol intake in the groups with passive ethanol experience exceeded that of the 0 group. Within the B6 mice, ethanol intake was highest in the 10-day group and the 3- and 5-day groups did not differ from each other. Within the D2 mice ethanol intake by the 10-day group was not different from the 5-day group but tended to be greater than the 3-day group. Consistent with our earlier work, B6 mice administered more of their ethanol in small bouts compared to the D2 mice (i.e., B6 mice sipped while D2 mice gulped). Increasing the number of days of passive ethanol exposure generally produced increases in withdrawal signs measured 7-8 h after the last passive infusion, suggesting that higher levels of ethanol self-infusion were mediated, at least in part, by higher levels of dependence or withdrawal alleviation (i.e., greater negative reinforcement by ethanol). However, it is also possible that increasing cumulative exposure to ethanol during the passive exposure phase produced greater tolerance to aversive pharmacological effects that would have otherwise produced a conditioned taste aversion to the paired Kool-Aid flavor. Supported by INIA AA13479.
D(1)-like Dopamine receptor modulation of nmda receptor-dependent long term depression in the nucleus accumbens: interactions with ethanol
Z.M. Jeanes; R.A. Morrisett. University of Texas-Austin, College of Pharmacy, Austin, TX 78712.
Alterations in mesolimbic glutamatergic synaptic plasticity are believed to underlie some of the neural adaptations that contribute to the development and/or expression of drug and ethanol dependence. Our goal in this INIA-West study is to understand how changes in N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD) in the nucleus accumbens (NAc) correlate with the induction of ethanol dependence.
We used in vitro whole-cell voltage clamp techniques to study LTD at GABAergic medium spiny neurons (MSNs) of the NAc shell. Previously, we have shown NMDAR-LTD is completely abolished by acute application of 40mM ethanol (105 ± 10%, n=7) and after in vivo chronic intermittent ethanol vapor exposure (118 ± 12%, n=8) (ACER, 31:144A, 2007). Therefore, we investigated mechanisms that may oppose the inhibitory effects of ethanol on LTD expression in the NAc shell.
Inhibition of NAc-LTD by ethanol does not increase in a concentration dependent manner. Low frequency (1Hz) stimulation in the presence of 20 and 60mM ethanol induced a modest LTD (82 ± 13% of baseline, n=7; and 83 ± 7%, n=4) respectively. However, 40mM ethanol completely blocked NAc-LTD (102 ± 9%, n=9). Previously, we reported that D(1)-like dopamine receptors activate a postsynaptic cAMP/PKA/DARPP-32 signaling cascade culminating in phosphorylation of NR1 subunits and a reduction in the sensitivity to ethanol of NMDA receptor-mediated synaptic transmission. In the presence of the D1 receptor agonist, SKF-38393 (25µM), a greater magnitude of LTD (54 ± 11%, n=8) was observed compared to control (70 ± 7%, n=12). Pre-treatment of slices with SKF-38393 (25µM) occluded inhibition of LTD by 40mM ethanol (79 ± 11%, n=5). D(1) receptor-mediated attenuation of the inhibitory actions of ethanol on NAc-LTD was completely blocked by a D(1) selective antagonist, SCH 23390.
Our prior findings indicate that adaptations occur in the NAc after in vivo chronic intermittent ethanol exposure as evident by the occlusion of LTD. In this study, we observed that D(1)-like dopamine receptor activation can reduce the inhibitory actions of 40mM ethanol on LTD in the NAc. We speculate that these interactions may contribute to the adaptations that re-wire the motivational circuitry in the ethanol-dependent brain. (Supported by U01AA16651 (RAM) and R01AA15167 (RAM)).
POTENTIATION OF CRF SENSITIVITY IN ETHANOL WITHDRAWN RATS: GENDER DIFFERENCES
M. Kang-Park; S.D. Moore. Duke University, Department of Psychiatry, VA Medical Center, Durham, NC 27705.
Dysregulation of the corticotropin releasing factor (CRF) system may underlie dependence on drugs of abuse, including ethanol. Previous work has shown that chronic ethanol treatment increases CRF levels in the central amygdala nucleus (CeA)(Menzaghi et al., 1994). Our study examined whether chronic ethanol treatment dysregulates the sensitivity of CeA neurons to CRF. We employed in vitro electrophysiological methods to measure evoked excitatory postsynaptic currents (EPSCs) in whole cell patch mode in rat brain slices. Drugs were delivered into the recording chamber by bath superfusion. Male and female rats were fed either ethanol (6~7.5 v/v%) or control calorie-adjusted liquid diet for 2 weeks, and experiments were performed one day after the last ethanol exposure. In the lateral division of CeA CRF (30-100 nM) increased the amplitude of evoked EPSCs in most neurons (52 neurons, 71 %), though in some neurons CRF had no effect (14 neurons, 19%) or depressed the amplitude of evoked EPSCs (7 neurons, 10 %). The potentiation of EPSCs was correlated with a decrease in the paired-pulse ratio, suggesting that this effect is in part mediated through increases in neurotransmitter release at presynaptic sites. NBI 27914 (a selective CRFR1 antagonist, 1 uM) did not alter this effect (n = 7), while Astressin 2B (a selective CRFR2 antagonist, 100 nM) blocked this effect (n = 5), suggesting that the EPSC enhancement is mediated by CRFR2 receptors. In control female rat slices, CRF 30 and 100 nM increased the magnitude of evoked EPSCs by 21 % (n = 15) and 32 % (n = 7), while in control male rat slices, 100 nM CRF increased the magnitude of evoked EPSCs by 21% (n = 13), which was significantly less than in females. For both male and female rats, the CRF effect was significantly greater after chronic ethanol treatment. In ethanol withdrawn female rats, CRF 30 and 100 nM increased the magnitude of evoked EPSCs by 42 % (n = 7) and 57 % (n = 7), and in the control male rats, 100 nM CRF increased the magnitude of evoked EPSCs by 32 % (n = 9), suggesting greater sensitivity in female rats. The present study suggests an involvement of the CeA-CRFR2 system in ethanol dependence that may contribute to greater sensitivity of female animals to stress and ethanol dependence.
Supported by NIAAA Integrative Neuroscience Initiative on Alcoholism (INIA-West) UO1 AA013498 and a VA Merit Review.
Tolerance to Ethanol-induced Ataxia Following Repeated Binge-like Ethanol Intake Using the DID Model
D. N. Linsenbardt, K. D. Griffin, E. M. Moore, S. L. Boehm II.
Behavioral Neuroscience, Department of Psychology, Binghamton University, Binghamton, New York 13902.A recent rodent model of binge-like alcohol (ethanol) intake referred to as ‘Drinking-in the Dark’ (DID) was developed wherein C57BL/6J (B6) mice voluntarily consume large quantities of 20% unsweetened ethanol solution when given limited access during peak arousal. Acutely, these high ethanol intakes elicit BEC’s ≥100mg/dl and lead to significant behavioral ataxia as indexed by the balance beam (Moore et al., 2007) and rotarod tests (Rhodes et al., 2005; 2007). However, the extent to which tolerance may develop following repeated DID binge drinking behavior has not been examined. To evaluate this directly, we gave 2 weeks of ethanol or water access to male B6 mice using standard DID procedures. Immediately after access on days 8 and 15 we tested animals for hind footslips on the balance beam apparatus. On the 16th day, ethanol and water consuming animals were further broken down into two ethanol challenge (i.p.) dose groups (1.5 or 1.75 g/kg) and were again tested for performance on the balance beam. Results indicated that ethanol consuming animals had significantly more footslips than water consuming controls following DID access on day 8; an effect that was no longer significant on day 15 despite similar g/kg intakes. Results of day 16 ethanol challenge indicated that ethanol consuming animals had significantly fewer footslips following i.p. injections despite no differences in ethanol pharmacokinetics., These results suggest the development of behavioral tolerance to repeated binge drinking episodes and, as there were no apparent pharmacokinetic differences, implicate some form/s of ethanol-induced neuroplasticity.
This work was funded in part by grants from NIAAA (AA015434) and the Integrative Neuroscience Initiative on Alcoholism - West.
IDENTIFICATION OF ALCOHOL-SENSITIVE SYNAPTIC PROTEIN COMPLEXES
G. Gorini; O. Ponomareva; K.S. Shores; R.A. Harris; M.D. Person; R.D. Mayfield.
Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, TX 78712
Changes in brain function induced by ethanol abuse are manifested as tolerance, physical dependence, and behavioral modifications. These conditions are likely due to alterations in gene and protein expression which are required for the cellular adaptation to persistent alcohol abuse. Recent gene expression studies from the Integrative Neuroscience Initiative on Alcoholism (INIA) consortium suggest that alcohol abuse alters the expression pattern of a number of gene families, including those involved in synaptic transmission and trafficking of synaptic proteins. Proteins encoded by these genes are important for a variety of synaptic events, including neurotransmitter vesicle transport and targeting, motor proteins involved in trafficking and targeting of synaptic proteins, and scaffolding proteins. Since cellular function of key brain signaling systems depends ultimately on interactions among proteins and other biomolecules, our goal was to identify and define novel protein complexes based on existing alcohol-responsive genes that may underlie excessive ethanol consumption. We used an interaction proteomics approach (co-immunoprecipitation, Western blotting, and mass-spectrometry) to identify protein interactions in mouse (C57BL/6J) cortical synaptosomes. Our initial studies were focused on identifying protein complexes using syntaxin-1A, synaptobrevin-2 (VAMP-2), calcium-activated potassium (BK) channel (alpha subunit), synaptosome-associated protein (SNAP-25), and dynamin-1 as bait proteins. Our results confirmed previously reported interacting partners of these synaptic proteins as well as identified a number of novel protein interactions. We will validate these findings using different antibodies, and test the effect of excessive ethanol consumption on those interacting protein complexes in future studies. (Supported by NIAAA AA016648)
FAST MICE HAVE GREATER EXPRESSION OF THE MUSCARINIC ACETYLCHOLINE SUBTYPE 5, BUT NOT SUBTYPE 4, RECEPTOR GENE COMPARED TO SLOW MICE
A.C. Scibelli; H.M. Kamens; T.J Phillips
VA Medical Center; Portland Alcohol Research Center; Oregon Health & Science University, Portland, OR 97239
Previous work in our laboratory suggests the presence of a quantitative trait locus on mouse Chromosome 2 for sensitivity to the locomotor stimulating effects of an acute treatment with 2 g/kg ethanol. Within this chromosomal region lie the genes that code for the m4 and m5 subtypes of the muscarinic acetylcholine receptor (mAChR). These experiments sought to obtain evidence that these receptor subtype genes have a role in genetic predisposition to ethanol-induced locomotor stimulation. To study this, two replicate sets of mouse lines that were selectively bred for high (FAST-1 and -2) or low (SLOW-1 and -2) sensitivity to the stimulant effects of ethanol were used. To test the hypothesis that innate differences in the expression of these genes influences sensitivity to the stimulant effects of ethanol, drug-naïve mice were used. Whole brain tissue from FAST and SLOW mice was processed for RNA extraction, cDNA generation, and gene expression analysis by qRT-PCR (quantitative reverse-transcriptase polymerase chain reaction). Hprt1 (hypoxanthine guanine phosphoribosyl transferase 1) was used as the reference gene in our expression analysis, each sample was run in triplicate, and the average crossing threshold (Ct) was determined for each gene. For each sample, the average Ct for Hprt1 was subtracted from the expression values for the mAChR gene subtypes. Relative expression was based on the ΔΔ Ct method, using the average expression of FAST-1 mice as the anchor value. Whereas there was no difference in relative expression of the m4 receptor subtype gene between FAST and SLOW mice, FAST mice showed significantly greater relative expression of the m5 receptor subtype gene compared to SLOW mice. There was no significant effect of replicate line. Expression of both genes is now being examined in specific brain regions, including the ventral tegmental area and substantia nigra for m5, the origins of the mesolimbic dopamine reward pathway, and the nucleus accumbens and striatum for m4. The greater relative expression of the m5 receptor subtype gene in FAST compared to SLOW mice may partially determine their genetically selected heightened stimulant response to ethanol.
Supported by a grant from the Department of Veterans Affairs and NIAAA (P60AA010760 and U01AA016655).
FosB Induction in Specified brain regions of c57bl6j mice Following Chronic Intermittent Ethanol Exposure and withdrawal periods
T.R. Porter, A.G. Salinas, S. Ali, R.A. Morrisett
Univ. of Texas-Austin, College of Pharmacy Austin, TX 78712FosB is a transcription factor that is induced after exposure to many drugs of abuse, including alcohol. The FosB transcript and a truncated splice variant, DFosB, accumulate in response to chronic alcohol administration and are thought to mediate long-term changes in gene transcription. This accumulation is seen in many brain regions including the Edinger Westphal nucleus (EWN) and mesolimbic dopamine system. Chronic intermittent ethanol exposure combined with withdrawal periods (CIE/WID) has been show to stably increase voluntary ethanol consumption in mice. Here we seek to determine the pattern of FosB induction in select brain regions of mice due to CIE/WID exposure.
C57BL6J mice were tested for baseline ethanol consumption by a 2-hour, 2 bottle choice drinking paradigm for five days. The animals were balanced into experimental and control groups based on ethanol consumption and body weight. Groups were exposed to 3 cycles of 16-hour ethanol vapor exposure (target blood alcohol concentration 35-45mM) or air followed by 8-hours of air exposure. Beginning one day after the third cycle, ethanol and water intake were recorded daily for 5 days. The process (baseline, vapor exposure, post chamber intake) was repeated following a rest period. One day after the final post chamber drinking session, the animals were perfused with 4% paraformaldehyde and the brains were stored in 30% sucrose until sectioning. The brains were cut into 40 uM sections and stored until immunohistochemistry processing. The free floating sections were rinsed with TBS and incubated in the following solutions (with TBS washes between steps): 1) 20% NGS in 0.4% Triton X-100/TBS (TTBS), 2) FosB antibody in 2%NGS/TTBS, 3) a biotinylated secondary antibody in 2%NGS/TTBS, 4) Vectastain ABC/0.8 mg/mL DAB in TBS. The stained sections were floated onto subbed slides and coverslipped. Images of the brain regions of interest were captured and analyzed by an observer blind to the treatment condition of each subject.
Exposure to CIE/WID resulted in a significant increase in ethanol consumption (n=8, p<0.05 t-test). Preliminary data on the EWN show a significant increase of FosB-ir in animals exposed to CIE/WID over controls (n=6, p<0.05 t-test). These data demonstrate that CIE/WID exposure-induced increases in drinking may be attributed to long term changes in gene transcription in select regions of the mouse brain.
This work was supported by NIH grant U01AA16651 to RAM.
POSTNATAL DEVELOPMENTAL EXPRESSION PROFILE OF UROCORTIN 1 IN PERIOCULOMOTOR MIDBRAIN NEURONS OF C57BL/6J MICE
A. Cservenka; A.E. Ryabinin. Oregon Health and Science University, Department of Behavioral Neuroscience, Portland, OR 97239.
Urocortin 1 (Ucn1) is an endogenous corticotropin releasing factor (CRF)-related peptide. Ucn1 has been found to be most highly expressed in the non-preganglionic Edinger-Westphal nucleus (npEW) (Vaughan et al., 1995; Weitemier et al., 2005), recently renamed the perioculomotor urocortin containing neurons (pIIIu) (May et al., 2008). Various studies indicate that these cells are involved in regulation of ethanol (EtOH) intake (see Ryabinin & Weitemier for a review, 2006). However, the development of these neurons remained unexamined. Recent studies have found that the inbred C57BL/6J (B6) strain known for high EtOH preference during adulthood, may prefer EtOH differentially during development, since infant B6 mice were found to drink less EtOH than infant DBA/2J mice, a strain that avoids EtOH as adults (Sanders, Boehm II, & Spear, 2007; Sanders, Boehm II, & Spear, 2008). The goal of the current study was to characterize the ontogenetic profile of pIIIu neurons during different stages of postnatal development, and to test whether the timing of peptide expression in pIIIu neurons may provide insight into the development of EtOH preference. B6 mice were bred, and brains of pups were collected at postnatal days (PND) 1, 4, 8, 12, 16, and 24 for sectioning in the saggital plane. Following immunohistochemical staining for Ucn1, positive Ucn1-immunoreactivity (ir) in pIIIu was only observed beginning at PND 8 with the number of cells positively stained increasing up to PND 24 and highest Ucn-ir observed in pIIIu. The absence of Ucn1-ir in pIIIu at PND 1 and 4 is in agreement with lack of EtOH preference in B6 mice at early postnatal age, and suggests that this peptide could be involved in the development of EtOH intake during ontogeny. The investigation of the developmental expression of other neuropeptides such as cocaine-and-amphetamine regulating transcript, nesfatin-1, and growth hormone secretagogue receptor, which colocalize with Ucn1 in pIIIu will be necessary to completely understand the contribution of peptides to the development of alcohol intake.
Supported by NIH grant AA 016647 (INIA Consortium Grant).
THE EFFECTS OF THE CRF RECEPTOR 1 ANTAGONIST, CP-154,526, ON ALCOHOL CONSUMPTION IN UROCORTIN 1 KNOCKOUT MICE
S. Kaur, E. Spangler, Z. Kapasova, D. M. Cote, J. Li and A.E. Ryabinin. Department of Behavioral Neuroscience, Oregon Health and Science University, School of Medicine, Portland, OR 97239
The corticotropin releasing factor (CRF) system is thought to play a role in alcoholism. This system includes four endogenous peptides: CRF, urocortin 1 (Ucn1), urocortin 2 (Ucn2) and urocortin 3 (Ucn3) and two types of receptors: CRFR1 and CRFR2. Recent studies showed that administration of the CRFR1 antagonist CP-154,526 leads to a drug-specific attenuation of increased alcohol intake in the drinking-in-the-dark (DID) procedure in C57BL/6J mice suggesting that CRFR1-mediated mechanisms are implicated in binge alcohol drinking. Two of the endogenous peptides, CRF and Ucn1, can bind CRFR1 receptors with high affinity. While CRF is thought to primarily regulate dependence–induced alcohol intake, recent studies show that Ucn1 could be involved in regulation of alcohol intake in non-dependent animals. Therefore, we hypothesized that the effect of CP-154,526 is mediated by Ucn1, and not CRF. To test this hypothesis, we investigated effects of this drug in Ucn1 knockout (KO) mice. Ucn1 KO mice were provided by Dr. W. Vale (The Salk Institute) and backcrossed onto the C57BL/6J background for 8 generations. Homozygous KO and wildtype (WT) littermates of both sexes were tested as follows. For three days, mice were injected intraperitoneally with vehicle (0.5 % carboxymethylcelluose) 3 h into the dark cycle and had their water drinking cylinders replaced (animals also had full access to water for the rest of the day). On days 4 to 6, the mice were injected as before and 30 min later were given access to 20 % (v/v) ethanol for 2 hours (water was available for the rest of the day). The volume of ethanol consumed was recorded. On day 7, mice were injected with either vehicle or 10 mg/kg CP-154,526 and 30 min later were given access to 20 % ethanol for 4 hours and the volume of ethanol consumed recorded. WT mice showed increased alcohol intakes on day 7, after 4 hours of ethanol access, but this increase was not seen in the Ucn1 KO mice. CP-154,526 attenuated the increase in drinking seen in the WT mice but had no effect in Ucn1 KO mice. These experiments confirm that Ucn1 mediates the CRFR1-dependent binge drinking seen after increased access to ethanol.
Supported by NIH grant AA01738 and INIA consortium grant AA016647.
REDUCED ETHANOL PREFERENCE AND CONSUMPTION IN COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT KNOCKOUT (CART KO) MICE
A.G. Salinas and R.A. Morrisett
The University of Texas at Austin, College of Pharmacy, Austin, Texas 78712.CART is a peptide neurotransmitter involved in addiction (for review see Rogge et al, 2008).
Several studies have characterized the role of CART in psychostimulant addiction, but surprisingly few have examined the role of CART in alcoholism. The current study utilized a CART KO mouse model to investigate what role, if any, CART plays in ethanol appetitive behaviors.
Three CART KO breeding trios were obtained from Eli Lily. The mice were crossed and their offspring were bred with wild type (WT) C57BL6J mice. The resultant heterozygote mice were crossed to produce litters with CART KO, heterozygote, and WT mice. At 8 weeks, the mice were individually housed and acclimated for 3 weeks to a reversed light schedule before beginning any experiments. A two-bottle choice paradigm was used to compare ethanol intake between WT and KO mice of both sexes. At the onset of the experiment, the mice were presented with a 3% ethanol solution and water for four days. The ethanol solution was then replaced with a 6% ethanol solution for four days, followed by 9, 12, 15, 18, & 21% ethanol solutions, each for four days. Ethanol and water consumption were measured daily. Consumption of quinine (bitter) and saccharin (sweet) solutions were assessed following the ethanol preference tests as before. Mouse body weights were measured every other day. The position of the ethanol or tastant and water bottles were alternated everyday to avoid a position preference. The amount of ethanol and tastant consumed daily was determined (in g/kg of body weight) for each animal and analyzed with a two-way ANOVA.
There was a main effect of genotype on ethanol consumption and preference in males for the 12-21% ethanol solutions (p<0.05) and 6-21% ethanol solutions for females (p<0.01) with WT males consuming 33-157% more ethanol than KOs and WT females consuming 25-82% more ethanol than KOs. There was also an effect of sex on ethanol consumption such that females consumed between 24% and 218% more ethanol than the males (p<0.01). There was no difference in saccharin consumption between groups. Female KO mice drank more quinine than WT females (p<0.05). There was a main effect of genotype on BW in males (p<0.05, with KOs weighing on average 18% more than WTs). These data demonstrate a role for CART in ethanol appetitive behaviors that cannot (in males) be attributed to differences in taste preference. This work was supported by NIH grants R01AA15167 and U01AA16651 to RAM.
EFFECT OF THE CRFR1 ANTAGONIST NBI 27914 ON WITHDRAWAL ASSOCIATED LIMITED ACCESS CONSUMPTION OF ETHANOL IN C57BL/6 MICE
C. Snelling; M.A. Tanchuck; N. Yoneyama; M.N. Strong; J. Blum; A.M. Fretwell; D.A. Finn
Department of Behavioral Neuroscience, Oregon Health & Science University, and VA Medical Research, Portland, OR 97239Over the past decade, rodent models have shown that intermittent exposures to ethanol vapor can increase subsequent self-administration of ethanol. Strong evidence from many laboratories indicate that this withdrawal associated drinking is diminished by the antagonism of corticotrophin-releasing factor (CRF) receptors, particularly CRFR1. Preliminary studies investigated the effect of NBI 27914, a non-peptide CRFR1 antagonist, on withdrawal associated ethanol intake in male C57BL/6 mice (B6). Male B6 mice were allowed 2 hr access to 15% (v/v) ethanol versus water, beginning 3 hr into their dark cycle, for a period of 14 days to establish a stable baseline. Discrete consumption matched groups were then exposed to 4 bouts of intermittent ethanol vapor exposure (3 cycles of 16 hr of ethanol vapor + 8 hr of air; multiple-withdrawal (MW) group) or air (control group), each followed by a 7 day period of limited access ethanol consumption. Following the fourth bout of intermittent vapor or air exposure, drinking in the MW and control groups was stabilized with daily handling and saline injections. Then, subjects received NBI 27914 (0.32, 0.5, 1.0, 3.2 mg/kg) in a within-subjects design. Drug doses were administered 3 days apart or more with saline injections in the interim to assure that ethanol self-administration had returned to pre-injection levels and that handling effects would be minimized. Generally, it was shown that ethanol self-administration was significantly higher in the MW group versus the control group. NBI 27914 significantly (p<0.05) decreased ethanol intake in all groups at the 1.0 and 3.2 mg/kg doses, but had a selective trend for a reduction in intake in the MW group following the 0.5 mg/kg dose. The lowest dose had no significant effect. In a very low subgroup of similarly treated mice, microinjection of NBI 27914 (0.7 µg/side) into the central nucleus of the amygdala reduced ethanol intake in the MW group. Additional studies will determine whether NBI 27914 in the 0.5-1.0 mg/kg dose range will significantly and selectively reduce ethanol self-administration in withdrawal associated drinking as well as effective doses following intra-amygdala infusion. Supported by a pilot from the INIA Consortium (grant AA013517), grant AA016981 and the Department of Veterans Affairs.
Homer-dependent kinase activation is necessary for binge alcohol drinking.
Karen K. Szumlinski
Behavioral genetic evidence indicates necessary roles for the Group1 mGluR scaffolding and signaling protein, Homer2, as well as its associated mGluR5 receptor in regulating various aspects of alcohol reward in laboratory rodents. However, the intracellular signaling molecules mediating the “anti-alcoholism” effects downstream of mGluR5/Homer activation remain unclear. To begin to address this issue, the effects of blocking PI3K and PKC epsilon activity within the nucleus accumbens (NAC) upon binge alcohol drinking were assessed in C57BL/6J (B6) mice, as well as in mice with a point mutation in mGluR5 that reduces Homer binding (FR mutant). An intra-NAC infusion of PI3K-selective doses of wortmannin dose-dependently reduced binge alcohol intake by both B6 mice and wild-type B6-129 hybrid mice, but failed to lower binge alcohol intake in FR mutants. Similarly, an intra-NAC infusion of a Tat inhibitor peptide against PKC epsilon also reduced binge alcohol drinking in B6 and WT mice, but failed to do so in FR mutants. These data provide novel in vivo evidence that mGluR5/Homer binding regulates the capacity of this receptor to signal through both its alpha q and beta-gamma subunits and, importantly, that activation of both mGluR5/Homer/PI3K and mGluR5/Homer/PKC epsilon pathways are important for the expression of a binge alcohol drinking phenotype. This work was supported by NIAAA grant AA016650 (INIA West).
Evidence that the negative regulator of Group1 mGluR function Daughter of Homer (DOH) is necessary for high-dose alcohol reward in mice
Goulding SP, Bergin M, Hu J-H, Worley PF, Szumlinski KK
The postsynaptic scaffolding protein Homer2 and its associated Group1 metabotropic glutamate receptors (mGluR1/5) are necessary and active regulators of alcohol reward in various animal models. Daughter of Homer (DOH) is a novel protein family, the members of which negatively regulate Group1 mGluR function in a Homer-dependent manner. As Homer2 deletion and Group1 mGluR blockade reduce alcohol intake under both continuous and limited alcohol access conditions, we compared the alcohol drinking phenotype of male wild-type (WT) and DOH1/2 double knock-out (KO) mice. When allowed 24-hr access to 4 sipper tubes containing 0, 3, 6 and 12% alcohol, KO mice exhibited reduced alcohol intake and greater water intake than WT mice over the course of a 2-week period. The reduction in total alcohol intake exhibited by DOH1/2 KO mice reflected reduced preference for, and consumption of, the 12% alcohol concentration. Consistent with our earlier data for Homer2 KO mice, the decrease in alcohol drinking exhibited by DOH1/2 KOs was associated with a reduction in accumbens basal extracellular glutamate levels. However, in contrast to Homer2 KO mice, but consistent with in vitro evidence that DOH inhibits mGluR function, DOH1/2 KO mice exhibited greater accumbens Group1 agonist-stimulated glutamate release than WT mice. These data provide the first evidence that, like their interacting partner Homer, DOH proteins are necessary for high-dose alcohol intake and may promote said intake by influencing accumbens extracellular glutamate levels. This work was supported by NIAAA grant AA016650 (INIA West) to KKS and NIDA grants DA 00266 and DA011742 to PFW.
INHIBITION OF ACCUMBENS PKCε IN C57BL/6J MICE ATTENUATES EXCESSIVE ALCOHOL CONSUMPTION IN TWO ANIMAL MODELS OF BINGE DRINKING
D.K. Cozzoli; C Marini; B.R. Tyrrel; R.O. Messing; K.K. Szumlinski.
Dept. Psychology and the Neuroscience Research Institute, University of California at Santa Barbara, Santa Barbara, CA, 93106-9660.Converging in vivo data support an important role for mGluR5-mediated activation of the “novel” PKC isoform PKCe in mediating the rewarding properties of alcohol. As we have shown previously that repeated bouts of binge alcohol drinking augment indices of mGluR5 activation within the nucleus accumbens (NAC), immunoblotting was conducted for changes in the expression and activational state of PKCe during short-term (24 h) withdrawal from intermittent bouts of binge alcohol drinking using the Scheduled High Alcohol Consumption (SHAC) procedure (5% alcohol for 30 min, every 3rd day for a total of 6 bouts; BECs>100 mg%). Compared to water controls, binge drinking mice exhibited an increase in the ratio of p(Ser729)PKCe:total PKCe levels within the NAC, but this increase in PKCe activation was not observed within the dorsal striatum, prefrontal cortex or hippocampus. To investigate the role for NAC PKCe activation in the maintenance of binge alcohol drinking, the effects of the local infusion of a Tat-PKCe inhibitor upon alcohol intake were assessed using both the SHAC, as well as the Drinking-in-the-Dark (DID; 20% alcohol for 2 hrs, every day; no fluid deprivation) paradigms. When compared with controls, an intra-NAC infusion of the PKCe inhibitor significantly reduced alcohol consumption in both models and these results are selective for alcohol as the inhibitor did not reduce 3% sucrose nor water intake under the same drinking schedules. These data provide novel evidence that an alcohol-induced increase in NAC PKCe signaling is important for the maintenance of binge drinking behavior and implicate NAC mGluR5 signaling through PKCe in regulating this prevalent form of excessive alcohol intake. This work was supported by NIAAA grant AA016650 (INIA West) to KKS and AA013588 to ROM
N. M. Zahr; D. Mayer; O. Hsu; S. Vinco; J. Orduna; E. V. Sullivan; A. Pfefferbaum
Psychiatry & Behavioral Sciences, Stanford University, Stanford, CA 94305, USNeuroscience Program, SRI International, Menlo Park, CA 94025, USAAlcoholics often engage in binge drinking associated with blood alcohol levels (BALs) as high as 300-500mg/dl. In vitro studies demonstrate that binge drinking can lead to neurodegeneration in corticolimbic brain regions. To investigate neurochemicalsequelae of binge alcohol ingestion, we exposed alcohol-preferring (P) rats to binge ethanol (EtOH) treatments and acquired in vivo magnetic resonance spectroscopy (MRS) data before, after 4d of EtOH exposure, and after 7d of abstinence. MRS data were acquired on a 3T MR scanner in 2h sessions per animal. Using our CT-PRESS MRS acquisition protocol, we measured brain metabolites including N-acetyl-aspartate (NAA), creatine (tCr), choline (Cho), glutamate (Glu), and myo-Inositol (mI) in a 9.8x4x4mm3 voxel in the dorsal hippocampus of 19 male P rats weighing 339±6.2g at baseline. Using baseline brain structural data to match groups, 12 rats were assigned to the EtOH group and received 25% (w/v) EtOH via oral gavage every 8h for 4d, depending on body weight and BALs, for a total average cumulative EtOH dose of 43.0±1.14g/kg. Control rats (Con) received similar doses of 5% sucrose. EtOH rats had average BALs of 271.7±10.5mg/dL, peak BALs of 434.2±17.9mg/dL, and lost 15.9% of their body weight after 4d of EtOH exposure. ANOVA indicated a group-by-time-by-metabolite interaction (p=.0001). After binge EtOH exposure, the EtOH group had lower NAAp=.004), tCr (p=.0001), and mI (p=.0003), and higher Cho (p=.0649) than the Con group. An EtOH peak was visible in the EtOH group at the post-binge scan and correlated with time between last dose and scan (r=.709, p=.0098), average BAL (r=.682, p=.0145), and peak BAL (r=.719, p=.0084). After 7d of abstinence, group differences in metabolite levels disappeared indicative of recovery. We previously performed this experiment in wild-type Wistar rats and demonstrated that average BALs of 257.8±13.2mg/dL similarly resulted in significantly decreased NAA, tCr, and mI, and increased Cho. The decline in NAA suggests that EtOH compromises neuronal integrity, while the decrease in tCr may indicate a compromise in the brain’s energy reservoir. The increase in Cho may reflect inflammation, demyelination, or abnormally high glial density. Recovery of metabolite levels in the absence of the causative agent argues for transient effects on the brain of binge EtOH exposure. (Support: AA05965, AA13521-INIA)
RNA interference targeting sodium channel â4 subunit in nucleus accumbens alters alcohol consumption
C. Adams1,2, A. Lasek3, J. Gesch3, U. Heberlein3, R. Hitzemann4, S. Edmunds4, A. Poczobutt1, B. Tabakoff5, P. Hoffman5, and W.M. Zawada1,2. 1Univ Wyoming Sch of Pharmacy, 2Neurosci Program, Laramie, WY 82070, 3Ernest Gallo Clinic Res Ctr, San Francisco, CA, 4Oregon Hlth Sci Univ, Portland, OR, 5Dept of Pharmacol, Univ of Colorado Denver.
Microarray analyses of gene expression in brains of mice bred for either high and low alcohol preference revealed that a voltage-gated sodium channel β4 subunit (Scn4b) is upregulated in alcohol preferring mice. Scn4b modulates sodium currents and neurite outgrowth. We have tested the hypothesis that reducing the expression of Scn4b in the nucleus accumbens (NAC) using a lentiviral vector encoding short hairpin RNA (shRNA) targeting Scn4b would alter alcohol consumption. A shRNA sequence found to silence the expression of Scn4b in Neuro2a cells by 80% was used to construct a lentiviral vector for silencing in vivo. Naïve male C57/B6-J mice received bilateral injections of the vector targeting Scn4b or a control vector encoding a scrambled shRNA sequence designed not to target expression of any genes into NAC. Two weeks after vector injection, all mice were entered into a protocol designed to examine alcohol preference and consumption using an unlimited access two-bottle choice test with ethanol concentrations of 6, 10, 15, 20 and 25%. Preliminary results indicate that, when tested with 10-20% ethanol, alcohol consumption was significantly higher in mice receiving the Scn4b vector than in mice injected with the control vector. Furthermore, mice injected with the Scn4b shRNA vector showed a trend toward higher alcohol preference compared to control mice. The preference for saccharin and quinine was identical in the two groups. Although these data support the notion that Scn4b modulates the development of preference for alcohol, examination of other brain areas involved in the accusition of alcohol preference is necessary for more complete understanding of the role of the Scn4b in the reward circuitry. Supported by the NIH RNAi Core of the Integrative Neuroscience Initiative on Alcoholism Consortium (U01AA016654) grant (WMZ); the NIH CoBRE grant P20 RR15640 (the Neuroscience Program at the University of Wyoming); and the Office of Research and Development at the UW.