LAD Rats Research Articles

Marinobufagenin in Urine: A Potential Marker of Predisposition to Ethanol and a Target for Spironolactone.

Previously it was demonstrated that digitalis-like cardiotonic steroid, marinobufagenin (MBG), is implicated in the development of ethanol addiction in rats. We hypothesized that (i) levels of sodium pump ligand, MBG, would be negatively correlated with the amount of ethanol consumed by rats, and (ii) that spironolactone would oppose the MBG induced ethanol-seeking behavior and blood pressure in rats. Voluntary consumption of 9% alcohol (vs. water) during 10 days period by 11 adult male Wistar rats was studied. Eight weeks after the beginning of the experiment, the animals were divided into two treatment subgroups: high alcohol drinkers (HAD, n=6, daily consumption of ethanol > 4 g/kg) and low alcohol drinkers (LAD, n=5, daily consumption of ethanol < 4 g/kg) rats. Spironolactone treatment (7 days) was started following 3-day habituation to intragastric vehicle administration. Consumption of ethanol and blood pressure were recorded daily. Urinary MBG excretion at baseline was 11.2±0.6 pmoles in HAD rats and 19.1±2.9 pmoles (p<0.05) in LAD rats, respectively. Seven days of spironolactone treatment was associated with reduction in ethanol intake (2.9 g/kg/24 hr), reduction in systolic blood pressure (5 mm Hg), and increase in sodium excretion (1 mmol/24 hr). Levels of MBG may be a predisposing factor to voluntary ethanol intake. Spironolactone, along with antihypertensive effect, decreases ethanol intake.

CIS-Acting Allele-Specific Expression Differences Induced by Alcohol and Impacted by Sex as Well as Parental Genotype of Origin.

Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high- and low-alcohol drinking (HAD and LAD) rat lines. Here, we report allele-specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. The HAD2 and LAD2 rats, which have been sequenced, were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2, and for the other 5 litters, the sire was a LAD2. From these 10 litters, 2 males and 2 females were picked from each F1 litter (N=40 total). The F1 pups were divided, balancing for sex and direction of cross, into an alcohol (15%) versus a water control group. Alcohol drinking started in the middle of adolescence (~postnatal day 35) and lasted 9weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA-sequencing and ASE analyses. Analyses revealed that adolescent ethanol (EtOH) drinking, individual EtOH drinking levels, parentage, and sex-of-animal affected ASEs of about 300genes. The identified genes included those associated with EtOH metabolism (e.g., Aldh2); neuromodulatory function (e.g., Cckbr, Slc6a7, and Slc1a1); ion channel activity (e.g., Kcnc3); and other synaptic and epigenetic functions. These data indicate that EtOH drinking differentially amplified paternal versus maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to EtOH-induced changes in cis-regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene-by-environment interactions mediating a genetic predisposition for, and/or the active development of, AUDs.

Alcohol-naïve USVs distinguish male HAD-1 from LAD-1 rat strains.

Ultrasonic vocalizations (USVs) are mediated through specific dopaminergic and cholinergic neural pathways and serve as real-time measures of positive and negative emotional status in rodents. Although most USV studies focus primarily on USV counts, each USV possesses a number of characteristics shown to reflect activity in the associated neurotransmitter system. In the present study, we recorded spontaneously emitted USVs from alcohol-naïve high alcohol drinking (HAD-1) and low alcohol drinking (LAD-1) rats. Using our recently developed WAAVES algorithm, we quantified four acoustic characteristics (mean frequency, duration, power, and bandwidth) from each 22-28kHz and 50-55kHz frequency-modulated (FM) USV. This rich USV representation allowed us to apply advanced statistical techniques to identify the USV acoustic characteristics that distinguished HAD-1 from LAD-1 rats. Linear mixed models (LMM) examined the predictability of each USV characteristic in isolation and linear discriminant analysis (LDA), and binomial logistic regression examined the predictability of linear combinations of the USV characteristics as a group. Results revealed significant differences in acoustic characteristics between HAD-1 and LAD-1 rats in both 22-28kHz and 50-55kHz FM USVs. In other words, these rats selectively bred for high- and low-alcohol consumption can be identified as HAD-1 or LAD-1 rats with high classification accuracy (approximately 92-100%) exclusively based on their emitted 22-28kHz and 50-55kHz FM USV acoustic characteristics. In addition, acoustic characteristics of 22-28kHz and 50-55kHz FM USVs emitted by alcohol-naïve HAD-1 and LAD-1 rats significantly correlate with their future alcohol consumption. Our current findings provide novel evidence that USV acoustic characteristics can be used to discriminate between alcohol-naïve HAD-1 and LAD-1 rats, and may serve as biomarkers in rodents with a predisposition for, or against, excessive alcohol intake.

Danshen injection prevents heart failure by attenuating post-infarct remodeling

Ethnopharmacological relevanceDanshen Injection (DSI) is a traditional Chinese medicine extracted from Danshen, prepared from the dried root and rhizome of Salvia miltiorrhiza Bunge. Danshen is an ancient antipyretic traditional Chinese medicine which is mostly used to improve blood circulation and dispel blood stasis. Danshen decoction or liquor-fried Danshen (with grain-based liquor) which is cool in nature is traditionally used to ‘cool the blood’ and reduce the swelling of sores and abscesses. Aim of studyThe present study aimed to examine the effect and mechanism of DSI in LAD induced heart injury. Materials and methodsOne day after LAD surgery, adult male Sprague-Dawley rats were randomized to 3 groups: MI group; DSI group (1.5ml/kg/d, intramuscular); and Valsartan group (10mg/kg/d, intragastric). Echocardiography and hemodynamic measurements (Pressure-Volume loop) were performed to evaluate cardiac function. Pathological methods (Masson, and Sirus red staining) were used to check myocardial fibrosis. Western blotting assay was used to detect the protein expression of MMP-2. RT-PCR was used to detect the gene expression of MMP-9, MPO, iNOS, Bcl-2 and Bax. ResultsDSI administration to LAD rats resulted in improved cardiac functions, hemodynamic parameters and normalized ventricular mass. Furthermore, DSI-treated group demonstrated potential regulation of myocardial collagen I and III deposition associated with MMP-2 expression. Also, DSI administration decreased gene expression of iNOS, MPO and MMP-9, and increased Bcl-2/Bax ratio. ConclusionMyocardial fibrosis, cardiac hypertrophy, hemodynamic deterioration as well as systolic and diastolic dysfunctions which characterize a failing hearts were significantly prevented by DSI. Our study may provide future directions to focus on the anti-hypertrophic mechanisms of DSI and pathological roles played by MMP-2 in myocardial hypertrophy. Meanwhile, DSI also performed the effect of anti-inflammation by the way of decreasing iNOS and MPO. The way Danshen Injection increasing Bcl-2/Bax presented the possibility that it may has the effect of inhibiting cell death.

Different Lines of Rats Selectively-Bred for High Alcohol-Drinking Demonstrate Disparate Preferences for Nicotine Self-Administration

Background. Alcohol and nicotine are commonly co- abused. The search for a common core of neural, behavioral, and genetic factors underlying addiction has been the goal of addiction research. Purpose. Genetic predisposition to high alcohol intake has been studied in rats by selectively breeding rats that have high preference for alcohol. The current experiments were conducted to determine if the level of intravenous nicotine administration for the various lines of alcohol-preferring rats differs from that for nonalcohol-preferring controls. Study design. Adult alcohol-na¨ selectively-bred alcohol-preferring male rats from four lines (P, AA, HAD-1, sP) and their control nonalcohol-preferring rats (NP, ANA, LAD-1, sNP) were trained and given access to self-administer nicotine (0.03 mg/kg/infusion). Results. The results show that the P rats self- administered significantly more nicotine than NP rats. In contrast, there were no significant differences in nicotine self-administration between the sP and sNP or the AA and ANA rats. Unexpectedly, high alcohol-drinking HAD-1 rats self-administered significantly less nicotine than low alcohol-drinking LAD-1 rats. Conclusion. This suggests that some genetic factors that underlie high-alcohol intake have more general effects in promoting high nicotine intake tendencies, while other genetic factors are more specific to only heavy drinking.

Temporal profile and mechanisms of the prompt sympathoexcitation following coronary ligation in Wistar rats.

Our aim was to assess the timing and mechanisms of the sympathoexcitation that occurs immediately after coronary ligation. We recorded thoracic sympathetic (tSNA) and phrenic activities, heart rate (HR) and perfusion pressure in Wistar rats subjected to either ligation of the left anterior descending coronary artery (LAD) or Sham operated in the working heart-brainstem preparation. Thirty minutes after LAD ligation, tSNA had increased (basal: 2.5±0.2 µV, 30 min: 3.5±0.3 µV), being even higher at 60 min (5.2±0.5 µV, P<0.01); while no change was observed in Sham animals. HR increased significantly 45 min after LAD (P<0.01). Sixty minutes after LAD ligation, there was: (i) an augmented peripheral chemoreflex – greater sympathoexcitatory response (50, 45 and 27% of increase to 25, 50 and 75 µL injections of NaCN 0.03%, respectively, when compared to Sham, P<0.01); (ii) an elevated pressor response (32±1 versus 23±1 mmHg in Sham, P<0.01) and a reduced baroreflex sympathetic gain (1.3±0.1 versus Sham 2.0±0.1%.mmHg−1, P<0.01) to phenylephrine injection; (iii) an elevated cardiac sympathetic tone (ΔHR after atenolol: −108±8 versus −82±7 bpm in Sham, P<0.05). In contrast, no changes were observed in cardiac vagal tone and bradycardic response to both baroreflex and chemoreflex between LAD and Sham groups. The immediate sympathoexcitatory response in LAD rats was dependent on an excitatory spinal sympathetic cardiocardiac reflex, whereas at 3 h an angiotensin II type 1 receptor mechanism was essential since Losartan curbed the response by 34% relative to LAD rats administered saline (P<0.05). A spinal reflex appears key to the immediate sympathoexcitatory response after coronary ligation. Therefore, the sympathoexcitatory response seems to be maintained by an angiotensinergic mechanism and concomitant augmentation of sympathoexcitatory reflexes.

Behavioral profiling of multiple pairs of rats selectively bred for high and low alcohol intake using the MCSF test

Genetic aspects of alcoholism have been modeled using rats selectively bred for extremes of alcohol preference and voluntary alcohol intake. These lines show similar alcohol drinking phenotypes but have different genetic and environmental backgrounds and may therefore display diverse behavioral traits as seen in human alcoholics. The multivariate concentric square field™ (MCSF) test is designed to provoke exploration and behaviors associated with risk assessment, risk taking and shelter seeking in a novel environment. The aim was to use the MCSF to characterize behavioral profiles in rat lines from selective breeding programs in the United States (P/NP, HAD1/LAD1, HAD2/LAD2), Italy (sP/sNP) and Finland (AA/ANA). The open field and elevated plus maze tests were used as reference tests. There were substantial differences within some of the pairs of selectively bred rat lines as well as between all alcohol-preferring rats. The most pronounced differences within the pairs of lines were between AA and ANA rats and between sP and sNP rats followed by intermediate differences between P and NP rats and minor differences comparing HAD and LAD rats. Among all preferring lines, P, HAD1 and HAD2 rats shared similar behavioral profiles, while AA and sP rats were quite different from each other and the others. No single trait appeared to form a common 'pathway' associated with a high alcohol drinking phenotype among all of the alcohol-preferring lines of rats. The marked behavioral differences found in the different alcohol-preferring lines may mimic the heterogeneity observed among human alcoholic subtypes.

Schedule-induced Polydipsia in Lines of Rats Selectively Bred for High and Low Ethanol Preference

Ethanol drinking was assessed in the P/NP, HAD1/LAD1, and HAD2/LAD2 lines of rats under environmental conditions that produce schedule-induced polydipsia. Female rats (n = 8/line), maintained at 85% of free-feeding body weights, underwent daily 1-h sessions during which 45-mg food pellets were delivered every 60 s. Water, 2, 4, 8, 16, or 32% w/v ethanol solution was available from a single bottle for 8 consecutive sessions at each concentration, with blood-ethanol levels (BELs) determined after selected sessions. P and HAD2 rats drank more water and ethanol than their non-preferring counterparts, while HAD1 and LAD1 rats did not differ. Ethanol intake and BELs were positively correlated (r = 0.75) across lines. Finally, rats were allowed 14 daily choice sessions with 8% ethanol and water concurrently available. Water intake generally exceeded ethanol intake in all lines, while P rats drank similar amounts of both fluids. These line differences indicate pleiotropic effects of genes that mediate ethanol intake and schedule-induced behaviors.

Comparison of VTA Dopamine Neuron Activity in Lines of Rats Selectively Bred to Prefer or Avoid Alcohol

Studies comparing alcohol-naive alcohol-preferring (P) and -nonpreferring (NP) rats indicate that high alcohol drinking is associated with an innate deficiency of the mesolimbic dopamine (DA) system. We previously reported that ventral tegmental area (VTA) DA neurons in alcohol-naive P rats burst fire more frequently than VTA DA neurons in alcohol-naive Wistar rats. We hypothesized that increased burst firing in P rats may represent a compensatory mechanism to maintain adequate levels of DA in terminal areas, such as the nucleus accumbens, despite the deficient mesolimbic DA system. The present study sought to extend our previous work and include NP rats and to determine whether differences in VTA DA neuron activity could be generalized to other rat lines selected for high and low alcohol preference, namely the high (HAD) and low (LAD) alcohol-drinking rats. The extracellular activity of posterior VTA DA neurons was recorded in unanesthetized alcohol-naive rats from the P/NP and HAD/LAD lines. Firing frequencies, burst activity, and the number of DA neurons encountered per electrode track were compared. Dopamine neurons in the posterior VTA of P rats had a greater percentage of action potentials in bursts and greater number of bursts compared with posterior VTA DA neurons in NP rats. There were no differences in VTA DA neuronal activity between both replicate lines of HAD and LAD rats. Burst activity of posterior VTA DA neurons distinguishes P from NP rats, but does not generalize to other lines of rats selectively bred at Indiana University for alcohol preference and nonpreference. Increased burst activity of DA neurons in the posterior VTA may be related to alcohol preference in P rats but is not necessary for high alcohol drinking.

Decreased reward during acute alcohol withdrawal in rats selectively bred for low alcohol drinking

We have previously hypothesized that increased sensitivity to the dysphoric-like or aversive effects of alcohol withdrawal following an initial exposure to alcohol might be associated with a genetic propensity to avoid alcohol. A decrease in brain reward function, as measured by an elevation in intracranial self-stimulation (ICSS) reward threshold, is one of the few methods available to model dysphoric-like or aversive effects of drug withdrawal in rats. We compared brain reward function during withdrawal following an initial exposure to alcohol in alcohol-naïve rats selectively bred for high (HAD1 line) versus low (LAD1 line) voluntary alcohol consumption. Male HAD1 ( n = 5) and LAD1 ( n = 6) rats were implanted with unilateral electrodes in the medial forebrain bundle and trained to bar press for delivery of a 100 μA current that varied in frequency from 45 to 200 Hz. Responding for ICSS was generally stable within subjects across multiple experimental sessions on a given day and across several consecutive days prior to alcohol or water administration. ICSS responding was assessed in both rat lines prior to and at 12, 14, 16, 18, 20, and 24 h following a single intragastric infusion of alcohol (4.0 g/kg body weight) or water. Rats of the LAD1 line, but not those of the HAD1 line, exhibited a decrease in brain reward function as evidenced by a decrease in bar-press responding for ICSS and an increase in ICSS stimulation threshold during alcohol withdrawal. The results suggest that rats selectively bred for low alcohol drinking may experience dysphoric-like effects during withdrawal from an initial exposure to alcohol, while rats selectively bred for high alcohol drinking may not.

EFFECTS OF CHRONIC ALCOHOL TREATMENT ON ACOUSTIC STARTLE REACTIVITY DURING WITHDRAWAL AND SUBSEQUENT ALCOHOL INTAKE IN HIGH AND LOW ALCOHOL DRINKING RATS

The purpose of the present study is to determine whether the inverse genetic association between alcohol withdrawal magnitude and genetic propensity for alcohol drinking that we have previously identified in alcohol-naive rats given alcohol acutely, would also be seen following chronic alcohol exposure. The effect of forced, chronic alcohol treatment on subsequent voluntary alcohol drinking was also examined. Male rats from the high alcohol drinking (HAD2) and low alcohol drinking (LAD2) lines received two intragastric (IG) infusions of alcohol (3.0 g/kg BW; 25% v/v) or an equal volume of water, separated by 5 h, every day for 20 consecutive days (chronic alcohol treatment). Acoustic startle reactivity was assessed at 10, 14, and 18 h after the second infusion on days 1, 5, 10, 15, and 20. After acoustic startle testing was completed, all rats received two IG infusions of 3.0 g alcohol/kg BW, separated by 5 h, and blood alcohol content was assessed at 10, 14, and 18 h after the second alcohol infusion. All rats were then given a 24 h free-choice between alcohol and water for 8 weeks. Startle magnitude to a 120 dB tone was suppressed during alcohol withdrawal in both alcohol-treated HAD2 and LAD2 rats after 5, 10, and 15 days of alcohol treatment. Forced, chronic alcohol treatment produced metabolic tolerance in both the HAD2 and LAD2 lines and significantly suppressed subsequent voluntary alcohol intake in rats of the HAD2 line. Reduced acoustic startle reactivity during alcohol withdrawal in both HAD2 and LAD2 rats is consistent with our previous findings in the HAD2 but not the LAD2 line and may reflect reduced CNS excitability during withdrawal from forced alcohol exposure. Forced alcohol exposure robustly retarded the expression of a genetic predisposition toward alcohol drinking in rats selectively bred for high alcohol intake.

Circadian activity rhythms in selectively bred ethanol-preferring and nonpreferring rats

Chronic alcohol intake is associated with dramatic disruptions in sleep and other circadian biological rhythms in both humans and experimental animals. In human alcoholics, these disruptions persist during extended abstinence and appear to promote relapse to drinking. Whereas chronic ethanol intake alters fundamental properties of the circadian pacemaker in unselected rats, nothing is known concerning circadian pacemaker function in selectively bred ethanol-preferring and nonpreferring rats, which are the most widely accepted animal models of genetic predisposition to alcoholism. The present experiments were designed to characterize free-running circadian activity (wheel-running) rhythms under both constant darkness and constant light in selectively bred ethanol-preferring (P, HAD2) and nonpreferring (NP, LAD2) rats. Differences in circadian organization between ethanol-preferring and nonpreferring animals were seen for both pairs of selected lines (P vs. NP; HAD2 vs. LAD2), but these differences were not identical in the two line pairs. For example, although P rats showed shorter free-running periods than NP rats only in constant light, HAD2 rats showed shorter free-running periods than LAD2 rats only in constant darkness. In addition, ethanol-preferring HAD2 rats showed a high rate of rhythm “splitting” that was not seen in any of the other three lines. Taken together, these results suggest that the circadian pacemakers of P and NP rats differ mainly in light sensitivity, whereas those of HAD2 and LAD2 rats differ in their intrinsic period.

Effects of Long‐Term Episodic Access to Ethanol on the Expression of an Alcohol Deprivation Effect in Low Alcohol–Consuming Rats

The alcohol-preferring (P) and -nonpreferring (NP) and high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats have been selectively bred for divergent preference for ethanol over water. In addition, both P and HAD rats display an alcohol deprivation effect (ADE). This study was undertaken to test whether the NP, LAD-1, and LAD-2 lines of rats could display an ADE as well. Adult female NP, LAD-1, and LAD-2 rats were given concurrent access to multiple concentrations of ethanol [5, 10, 15% (v/v)] and water in an ADE paradigm involving an initial 6 weeks of 24-hr access to ethanol, followed by four cycles of 2 weeks of deprivation from and 2 weeks of re-exposure to ethanol (5, 10, and 15%). A control group had continuous access to the ethanol concentrations (5, 10, and 15%) and water through the end of the fourth re-exposure period. For NP rats, a preference for the highest ethanol concentration (15%) was evident by the end of the fifth week of access (approximately 60% of total ethanol fluid intake). Contrarily, LAD rats did not display a marked preference for any one concentration of ethanol. All three lines displayed an ADE after repeated cycles of re-exposure to ethanol, with the general ranking of intake being LAD-1 > NP > LAD-2 (e.g., for the first day of reinstatement of the third re-exposure cycle, intakes were 6.5, 2.9, and 2.4 g/kg/day compared with baseline values of 3.1, 2.0, and 1.3 g/kg/day for each line, respectively). By the 13th week, rats from all three lines, with a ranking of LAD-1 > NP > LAD-2, were drinking more ethanol (3.3, 2.2, and 2.0 g/kg/day, respectively) compared with their consumption during the first week of access (approximately 1.1 g/kg/day for all three lines). These data indicate that access to multiple concentrations of ethanol and exposure to multiple deprivation cycles can partially overcome a genetic predisposition of NP, LAD-1, and LAD-2 rats for low alcohol consumption. In addition, the findings suggest that genetic control of low alcohol consumption in rats is not associated with the inability to display an ADE.

Neuropeptide Y in the Paraventricular Nucleus of the Hypothalamus Increases Ethanol Intake in High‐ and Low‐Alcohol‐Drinking Rats

The behavioral effects of neuropeptide Y (NPY) attributed to its actions in the hypothalamus are complex and include effects on feeding, sedation, and the hypothalamic-pituitary-adrenal axis. NPY infused into the paraventricular nucleus (PVN) increases ethanol intake in unselected rats. High-alcohol-drinking (HAD1) and low-alcohol-drinking (LAD1) rats differ in basal NPY levels in the PVN, and HAD1, but not LAD1, rats exhibit decreases in ethanol intake after infusion of NPY into the ventricles. This study examined whether NPY infused into the PVN alters ethanol intake in HAD1 and LAD1 rats. Female HAD1 (n = 14) and LAD1 (n = 18) rats were given 24-hr free-choice continuous access to 15% (v/v) ethanol and water for 6 weeks and then implanted bilaterally with cannulas aimed at the PVN. Two weeks later, rats received a series of microinfusions, each separated by 1 week, that included four doses of NPY (0.0, 0.25, 0.5, and 1.0 microg). Ethanol, water, and food were available ad libitum after infusions. All rats received a final microinfusion of 1.0 microg of NPY, after which ethanol and water, but no food, were made available for 2 hr. During the 2 hr after infusion, NPY yielded dose-dependent increases in both water and food consumption. With food concurrently available, the 0.25- and 1.0-microg doses of NPY did not alter baseline ethanol intake, whereas the 0.5-microg dose increased ethanol intake. Infusion of 1.0 microg of NPY in the absence of food yielded a decrease in water intake and an increase in ethanol intake relative to the same dose in the presence of food. Twenty-four hours after infusion, there were no effects of NPY on water and food intake, and increases in ethanol intake were no longer apparent. Increases in ethanol intake after infusion of NPY into the PVN may depend on NPY dose and whether food is concurrently available.

High-alcohol-drinking rats exhibit persistent freezing responses to discrete cues following Pavlovian fear conditioning

We previously reported that high-alcohol-drinking (HAD) rats exhibited selective deficits in active avoidance learning and that those deficits were partially reversed by moderate doses of ethanol under certain training conditions [Pharmacol. Biochem. Behav. 75 (2003) 89]. In that study, we hypothesized that HAD deficits resulted from exaggerated fear in the conditioning context and that the anxiolytic properties of ethanol, along with prior exposure to the conditioning apparatus, were responsible for the facilitated avoidance learning that was observed in HAD rats following moderate doses of ethanol. The current study was designed to test whether HAD rats exhibit behaviors consistent with increased fear in aversive learning contexts. We used a standard Pavlovian fear conditioning paradigm to assess behavioral freezing in HAD (HAD-1 and HAD-2) and low-alcohol-drinking (LAD; LAD-1 and LAD-2) rats. No significant differences were observed between HAD-1 and HAD-2 or between LAD-1 and LAD-2 rats, indicating that the replicate lines performed similarly in this study. Both HAD and LAD rats exhibited robust fear conditioning during training. Although no differences were observed between HAD and LAD rats during fear training, HAD rats failed to extinguish freezing behavior in response to the discrete tone conditional stimulus during subsequent fear retention tests. Thus, HAD rats demonstrated prolonged cue-elicited fear that was resistant to extinction.

Confirmation of alcohol preference quantitative trait loci in the replicate high alcohol drinking and low alcohol drinking rat lines.

Selective breeding has been employed to develop replicate high-alcohol-drinking (HAD1 and HAD2) and low-alcohol-drinking (LAD1 and LAD2) rat lines from the heterogeneous N/Nih rat. Within-family selection and a rotational breeding design were used to discourage inbreeding (Li et al., 1993). A genome screen was previously performed using 459 HAD1xLAD1 F2 progeny to identify quantitative trait loci (QTLs) on rat chromosomes 5, 10, 12 and 16 that contribute to alcohol preference and consumption in these non-inbred rat models of alcoholism. To confirm these QTLs in the replicate lines, 16 HAD2 and 16 LAD2 rats were genotyped for microsatellite markers within each of these QTL intervals. Review of the genotypic data support confirmation of the QTLs on chromosomes 5 and 10; several markers in the QTL region display different alleles in the HAD2 and LAD2 rats, suggesting linkage disequilibrium between the microsatellite markers and the QTL. Although the QTL on chromosome 12 had the highest LOD score in the HAD1 and LAD1 studies, little evidence supported confirmation of this QTL based on the genotyped markers. Further evaluation of each of these QTL regions is ongoing in a sample of HAD2xLAD2 F2 progeny currently being generated that will be used to assess the evidence of linkage in each of these QTL regions.

Effects of Neuropeptide Y on Sucrose and Ethanol Intake and on Anxiety‐Like Behavior in High Alcohol Drinking (HAD) and Low Alcohol Drinking (LAD) Rats

Background: In a previous study, neuropeptide Y (NPY) administered into the lateral ventricles decreased ethanol intake in alcohol‐preferring (P) rats but not in alcohol‐nonpreferring (NP) or unselected Wistar rats. The purpose of the present investigation is to extend these findings in selectively‐bred high‐alcohol‐drinking (HAD)1 and low‐alcohol‐drinking (LAD)1 rats by examining the effects of intracerebroventricularly administered NPY on the elevated plus maze test of anxiety and on ethanol and sucrose intake.Methods: Female HAD1 and LAD1 rats were surgically implanted with cannula into the lateral ventricle. Following recovery, a test of anxiety was conducted in which the rats (n= 12–13/group) received either artificial cerebrospinal fluid (aCSF) or NPY (10 μg) 10 min prior to a 5‐min test on an elevated plus maze. Following anxiety testing, 11 HAD and 11 LAD rats were trained to self‐administer ethanol (8% w/v), and 5 HAD and 8 LAD rats were trained to self‐administer sucrose (2.5%) during daily 2‐hr sessions. A within‐subject design was used in which the rats were pretreated once a week with aCSF, 5 μg NPY, or 10 μg NPY prior to the drinking sessions.Results: HAD and LAD rats treated with aCSF did not differ in time spent in open arms of the plus maze. NPY increased time spent on the open arms to similar degrees in both rat lines. HAD rats consumed more ethanol and sucrose than LAD rats. NPY increased sucrose intake in both rat lines. However, the same doses of NPY reduced ethanol intake in HAD but not in LAD rats.Conclusion: The plus maze results indicated that selective breeding for high and low alcohol preference in the HAD1 and LAD1 rats, respectively, did not yield differences in anxiety‐like behavior and in response to the anxiolytic effects of NPY. The increases in sucrose intake were consistent with the known orexigenic effects of NPY. The decreased ethanol intake following NPY administration in HAD rats was similar to previous observations with P rats and is consistent with the hypothesis that ethanol intake and NPY activity may be inversely related.

Moderate doses of ethanol partially reverse avoidance learning deficits in high-alcohol-drinking rats

We previously reported that ethanol-naive high-alcohol-drinking (HAD1 and HAD2) rats exhibited selective deficits in active avoidance learning, as compared to low-alcohol-drinking (LAD1 and LAD2) rats, in a signaled bar-pressing task [Alcohol. Clin. Exp. Res. 24 (2000) 1778]. In the current study, we used appetitive and aversive learning tasks to assess whether administration of ethanol influences approach and avoidance learning in HAD and LAD rats. Rats were administered 0.0, 0.5, 1.0, or 1.5 g ethanol/kg body weight during appetitive and aversive conditioning sessions. We found that ethanol impaired acquisition of the appetitive conditioned response in a dose-dependent manner in both HAD and LAD rats, with 1.5 g/kg ethanol producing the greatest deficits. Notably, moderate doses of ethanol (0.5 and 1.0 g/kg) partially reversed avoidance learning deficits in HAD rats, but only when appetitive conditioning preceded aversive conditioning. The highest dose (1.5 g/kg EtOH) abolished avoidance responding altogether in HAD rats. Avoidance responding in LAD rats was not affected by any dose of ethanol. These results are consistent with previous studies suggesting that alcohol preference may be associated with increased fear or anxiety, but the conditions under which ethanol produces a reduction of fear and anxiety in HAD rats appear to be relatively complex.

Ethanol effects on local cerebral glucose utilization in high-alcohol-drinking and low-alcohol-drinking rats

Divergent ethanol-drinking behavior in rats selectively bred for high- or low-ethanol-drinking behavior could be related to differences in the sensitivity of the CNS to ethanol. In the current study, we examined the effects of acute (i.e., single injection) ethanol administration on local cerebral glucose utilization (LCGU) within selected brain regions of high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats. Adult, male, HAD and LAD rats from replicate line 2 were injected intraperitoneally with saline, or ethanol, at doses of 0.25 g/kg or 1.0 g/kg, during their dark cycle; 10 min later, [ 14C]-2-deoxyglucose ([ 14C]-2-DG; 125 μCi/kg) was injected into the femoral vein. Timed arterial blood samples were collected over 45 min and assayed for plasma glucose, ethanol, and [ 14C]-2-DG levels. Rats were then decapitated, and their brains were quickly extracted and frozen in isopentane at –50°C. Coronal brain sections were prepared and apposed to x-ray film for 2 days, and image densities were determined by using quantitative autoradiography. Data were collected from several key limbic (nucleus accumbens, ventral tegmental area, olfactory tubercle, amygdala, hippocampus, ventral pallidum, and septum), basal ganglia, cortical (medial prefrontal, frontal, parietal, temporal, occipital, entorhinal, piriform, and cingulate), and subcortical (thalamus, habenula, and superior colliculus) structures. After administration of both low (0.25 g/kg) and moderate (1.0 g/kg) doses of ethanol, LCGU values were lower, relative to those for saline controls, in several CNS regions (lateral septum; posterior cingulate, frontal, parietal, and temporal cortices; dorsomedial striatum; and dorsomedial thalamus) of LAD but not HAD rats. These findings may indicate that certain CNS regions of LAD-2 rats are more sensitive than regions of HAD-2 rats to the effects of low-to-intermediate doses of ethanol.

Neurophysiological Profiles of Replicate Line 2 High-Alcohol-Drinking (HAD-2) and Low-Alcohol-Drinking (LAD-2) Rats

Rationale A select number of electrophysiological findings have been demonstrated to differentiate rat lines selectively bred for high and low ethanol preference. Objective In the present study, EEGs and event-related potentials (ERPs) of high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats from replicate line 2 (HAD-2 and LAD-2) were assessed to determine if their neurophysiological profiles are similar to selected lines previously evaluated. Methods Rats obtained from Indiana University were implanted with cortical and amygdalar recording electrodes. Baseline EEG and ERPs were assessed in ethanol-naïve HAD-2 and LAD-2 rats. Animals subsequently were trained to self-administer ethanol by using a sucrose-substitution procedure. Results Compared with LAD-2 rats, HAD-2 rats displayed greater parietal cortical power in the 6 to 32 Hz frequency range of the EEG. Greater parietal cortical peak frequency in the 2 to 4 Hz range and decreased frontal, parietal, and amygdalar peak frequencies in the 16 to 32 Hz frequency range were also seen. Compared with LAD-2 rats, HAD-2 rats had decreased P2 latency of ERPs recorded in the parietal cortex. HAD-2 rats also had greater frontal, parietal, and amygdalar P2 amplitudes, greater frontal and parietal cortical P1 amplitudes, and greater parietal cortical P3 amplitudes compared with LAD-2 rats. As anticipated, HAD-2 rats consumed significantly greater levels of sucrose, sucrose-ethanol, and ethanol over the course of the sucrose-substitution procedure compared with LAD-2 rats. Conclusions These data suggest that increased cortical power is associated with high ethanol preference in a number of selectively bred rat lines. However, unique electrophysiological characteristics may index alcohol preference in each line.