Inverse genetic association between alcohol preference and severity of alcohol withdrawal in two sets of rat lines selected for the same phenotype

Background: The purpose of the present study was to determine whether alcohol‐naïve rats selectively bred for alcohol preference or nonpreference differ in alcohol withdrawal severity using two sets of rat lines selectively bred for the same phenotype. Methods: Alcohol‐naïve male rats from the high alcohol drinking (HAD1) and low alcohol drinking (LAD1) rat lines and from the alcohol preferring (P) and nonpreferring (NP) rat lines received an intragastric infusion of alcohol (4.0 g/20.3 ml/kg; 25% v/v) or an equal volume of water once a day for 10 consecutive days. Alcohol withdrawal severity was assessed at using a behavioral rating scale and a radiant heat assay measured analgesia at 10, 12, 14, 16, 18, and 24 hrs following infusion of alcohol or water on days 1, 5, and 10 of treatment. Results: Data were analyzed using body weight as a co‐factor to correct for differences in body weight between the HAD1/LAD1 and P/NP lines. Acute (1 day) but not repeated alcohol treatment (5 or 10 days) produced mild behavioral signs of withdrawal in LAD1 but not in HAD1 rats. HAD1 and LAD1 rats showed alcohol‐induced analgesia after 1 and 5 days of alcohol treatment that disappeared by day 10 in both lines. Repeated alcohol treatment (5 days) produced mild behavioral signs of withdrawal in NP but not in P rats. Neither P nor NP rats showed alcohol‐induced analgesia after 1, 5, or 10 days of alcohol treatment. Conclusions: An inverse genetic association was found between alcohol preference and severity of alcohol withdrawal in two sets of rat lines selected for the same phenotype. The pattern of alcohol withdrawal that emerged over the course of the 10 days of alcohol treatment differed between the two lines selected for low alcohol drinking (LAD1 and NP), suggesting that unique sets of genes may influence alcohol withdrawal severity in the two lines.     

Further evidence of an inverse genetic relationship between innate differences in alcohol preference and alcohol withdrawal magnitude in multiple selectively bred rat lines

Background: We have previously shown that a genetic association exists between low alcohol drinking and high alcohol withdrawal magnitude after acute alcohol exposure in alcohol‐naïve rats. However, the behavioral rating scale used in this prior study was not optimal for assessing the magnitude of mild alcohol withdrawal. The present study examined whether a genetic relationship is again found between alcohol preference and alcohol withdrawal magnitude when a sensitive measure is used to index mild alcohol withdrawal in rats. Methods: Alcohol‐naïve, male rats selectively bred for alcohol preference (P, HAD1, HAD2) or nonpreference (NP, LAD1, LAD2) received a single intragastric infusion of alcohol (4.0 g/20.3 ml/kg body weight; 25% v/v) or water followed by acoustic startle testing. Results: Startle probability and magnitude was greater in water‐treated P than in water‐treated NP rats. During alcohol withdrawal, startle probability and magnitude was suppressed in P rats and elevated in NP rats relative to water‐treated controls. Startle probability and magnitude was greater in water‐treated LAD1 rats than in water‐treated HAD1 rats. During alcohol withdrawal, startle probability and magnitude was suppressed in HAD1 and elevated in LAD1 rats relative to water‐treated controls at 20 hr after acute alcohol exposure. Startle probability and magnitude did not differ between water‐treated HAD2 and water‐treated LAD2 rats. During alcohol withdrawal, there was a trend toward decreased startle probability and magnitude in HAD2 rats compared with water‐treated controls. Conclusions: The acoustic startle response to a tone stimulus is a sensitive measure of mild alcohol withdrawal in rats. Rats selectively bred for low alcohol intake showed greater alcohol withdrawal magnitude than did rats selectively bred for high alcohol intake. These results provide further evidence that an inverse genetic association exists between alcohol withdrawal magnitude and propensity toward alcohol drinking in rats.     

Acoustic startle reactivity during acute alcohol withdrawal in rats that differ in genetic predisposition toward alcohol drinking: effect of stimulus characteristics

BACKGROUND: We have previously reported an association between greater alcohol withdrawal magnitude after a single alcohol exposure and a genetic predisposition toward low alcohol drinking in rats selectively bred for differences in alcohol intake when acoustic startle reactivity to a tone stimulus was used to index acute alcohol withdrawal. The purpose of this study was to examine whether the quality of the acoustic startle stimulus (noise versus tone) is important for detecting a genetic relationship between alcohol withdrawal magnitude and alcohol drinking behavior. METHODS: Alcohol-naive male rats selectively bred for high alcohol intake [alcohol-preferring (P), high-alcohol-drinking (HAD)1, and HAD2] or low alcohol intake [alcohol-nonpreferring (NP), low-alcohol-drinking (LAD)1, and LAD2] received a single intragastric infusion of water or alcohol (4.0 g/20.3 ml/kg; 25% v/v), and acoustic startle test sessions were given at 14, 16, 18, 20, and 24 hr after infusion. Each test session consisted of a 5-min acclimation period followed by random presentation of various white noise stimuli (90, 100, 110, and 120 dB.) RESULTS: Line differences in acoustic startle magnitude under control conditions were present in all three pairs of selectively bred lines; P rats showed a greater startle magnitude relative to NP rats, whereas both LAD lines showed a greater startle magnitude relative to both HAD lines. During alcohol withdrawal, the P, HAD1, and HAD2 lines showed enhanced startle magnitude compared with their water-treated controls. No change in startle magnitude during alcohol withdrawal was found in the NP, LAD1, or LAD2 lines. CONCLUSIONS: In contrast to our prior findings, these results showed a genetic association between high alcohol drinking and a greater startle response magnitude to a noise stimulus during alcohol withdrawal. It seems that the genetic association between alcohol drinking and alcohol withdrawal, as assessed by the acoustic startle response, depends on the quality of the acoustic startle stimulus.     

Development of Alcohol Drinking Behavior in Rat Lines Selectively Bred for Divergent Alcohol Preference

A characteristic of heritable alcoholism is an early onset of alcohol abuse, which may begin at or before the age of adolescence. The objective of the present study was to determine the ontogeny of alcohol drinking behavior before and during puberty in the selectively bred alcohol-preferring (P), alcohol-nonpreferring (NP), high alcohol drinking (HAD), and low alcohol drinking (LAD) lines of rats. In addition, the effects of postweaning housing conditions (single- or pair-housed) and initiation procedure (4 days forced ethanol or free-choice) were evaluated in male and female P rats. Results indicate that high alcohol drinking in P and HAD (replicate line 2) rats, as well as low alcohol drinking behavior in NP and LAD (replicate line 2) rats, is present as early as 3 to 4 weeks of age. Ethanol intakes in juvenile P and HAD rats reached levels of ∼4 to 5 g/kg/day by 38 to 41 days of age and were comparable with levels observed in adults. Neither housing conditions nor ethanol initiation procedure significantly altered the acquisition or magnitude of alcohol intake levels in juvenile male and female P rats. These results suggest that the neural substrates underlying divergent ethanol drinking behavior in P/NP and HAD/LAD lines of rats are present early in life.     

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 microg) 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 microg NPY, or 10 microg 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.     

Low-dose stimulatory effects of ethanol during adolescence in rat lines selectively bred for high alcohol intake

Abstract : Background: The low‐dose stimulatory effect of ethanol (EtOH) in rats has been hypothesized to reflect its hedonic effects and to be associated with a genetic predisposition toward high alcohol preference. To test the hypothesis that phenotypes associated with high alcohol preference in adulthood are also present in adolescent rats at the time of onset of alcohol drinking, the current study examined the effects of EtOH on locomotor activity (LMA) during adolescence in lines of rats selectively bred for divergent alcohol intakes. Methods: Subjects were adolescent (31–40 days of age) rats from the alcohol‐preferring (P) and ‐nonpreferring (NP) lines and from the high–alcohol‐drinking (HAD) and low–alcohol‐drinking (LAD) replicate lines. On day 1, all subjects (n= 8–10/line/gender/dose) received intraperitoneal saline injections and were placed in the activity monitor for 30 min. On day 2, subjects received intraperitoneal saline or 0.25, 0.50, 0.75, 1.0, or 1.5 g EtOH/kg. Results: The LMA of male and female P rats was increased with low doses (0.25–0.75 g/kg) and decreased at the highest dose (1.5 g/kg) of EtOH. Similar effects were observed with low doses of EtOH on the LMA of HAD‐1 and HAD‐2 rats. None of the EtOH doses stimulated LMA in the NP, LAD‐1, or LAD‐2 rats, although all of the low–alcohol‐intake lines of rats showed decreased LMA at the highest dose of EtOH. Only the P rats among the high–alcohol‐consuming lines of rats showed decreased LMA at the highest dose of EtOH. Conclusion: Selective breeding for high alcohol consumption seems to be associated with increased sensitivity to the low‐dose stimulating effects of EtOH and reduced sensitivity to the high‐dose motor‐impairing effects of ethanol. The expression of these phenotypes emerges during adolescence by the age of onset of alcohol‐drinking behavior.     

A comparative study on alcohol-preferring rat lines: effects of deprivation and stress phases on voluntary alcohol intake

BACKGROUND: Voluntary alcohol intake in rats can be influenced by alcohol deprivation phases and stress. We investigated the magnitude of the effects of both deprivation and stress (forced swimming in cold water and foot-shock had been chosen as stressors distinct in their physical and psychological features) on alcohol intake and the influence of these experiences on the time course of alcohol drinking behavior. For the alcohol drinking procedure, a long-term model of alcohol self-administration originally developed for heterogeneous Wistar rats was used and was compared with different alcohol-preferring rat lines. METHODS: Adult male Alko alcohol (AA), alcohol-preferring (P), high-alcohol-drinking (HAD), and unselected Wistar rats were given ad libitum access to water, 5%, and 20% alcohol solutions for 6 months. A deprivation phase of 14 days was performed after 8 weeks of access to alcohol. After 16 weeks and 22 weeks of alcohol access, all animals were subjected to forced swimming and foot-shock, respectively, for 3 consecutive days, while alcohol intake was still being measured. RESULTS: Alcohol deprivation led to a significant increase in alcohol intake in Wistar rats and P rats. No alcohol deprivation effect was observed in HAD and AA rats; after deprivation, however, their preference for the 20% alcohol solution increased, immediately in the HAD rats and gradually over time in the AA rats. Repeated swim stress caused an increase in alcohol intake in Wistar rats but no changes in the alcohol-preferring rat lines. Foot-shock stress increased alcohol consumption in all lines of rats, but the most pronounced effects were observed in HAD and P rats. CONCLUSIONS: Wistar, HAD, P, and AA rats differentially respond to alcohol deprivation and stress, showing that the genetic background of these different rat lines profoundly affects relapse-like drinking and stress-induced drinking.     

Effects of long-term episodic access to ethanol on the expression of an alcohol deprivation effect in low alcohol-consuming rats

Background: 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. Method: 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. Results: For NP rats, a preference for the highest ethanol concentration (15%) was evident by the end of the fifth week of access (～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 (～1.1 g/kg/day for all three lines). Conclusion: 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.     

Comparison of VTA dopamine neuron activity in lines of rats selectively bred to prefer or avoid alcohol

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Bone mass and strength: phenotypic and genetic relationship to alcohol preference in P/NP and HAD/LAD rats

BACKGROUND: The association between moderate alcohol intake and elevated bone mineral density observed in several epidemiologic studies might result from common genetic pathway regulating both phenotypes. In this study, we determined whether there is a relationship between alcohol preference and high bone mass or strength and whether bone mass-regulating genes segregate during selective breeding of alcohol preferring rats. METHODS: Six different lines of male rats with high or low preference for alcohol consumption were used in this study. The high alcohol preference lines are alcohol-preferring (P), high-alcohol-drinking 1 (HAD1), and high-alcohol-drinking 2 (HAD2), and their corresponding low alcohol preference lines are alcohol-nonpreferring (NP), low-alcohol-drinking 1 (LAD1), and low-alcohol-drinking 2 (LAD2). Bone mass phenotypes were determined using dual energy x-ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and biomechanics in long bones and lumbar vertebrae from rats at 3 and 6 months of age. RESULTS: P rats had significantly higher bone mass and strength compared with NP rats, mainly due to higher cortical bone in long bones and lumbar vertebrae. HAD2 rats also had significantly higher bone mass compared with LAD2 rats, but mostly due to increased trabecular bone leading to increased strength only in lumbar vertebra. Conversely, HAD1 rats had significantly lower bone mass and strength compared with LAD1 rats in long bones. The vertebral bone mass and strength did not differ between HAD1 and LAD1 rats. CONCLUSIONS: This study demonstrated that preference for alcohol consumption had no consistent relationship with high bone mass or strength, as each alcohol-preferring rat line had their unique bone mass phenotypes. However, genes regulating bone mass and strength appear to segregate with alcohol preference genes in P and HAD rat lines, suggesting that alcohol preferring rat lines may be useful for identifying genes that regulate bone mass and structure.     

Voluntary ethanol drinking during the first three postnatal weeks in lines of rats selectively bred for divergent ethanol preference

BACKGROUND: Using a procedure first developed by Hall (1979), we examined ethanol self-administration in preweanling pups from Wistar rats and in lines of rats selectively bred for divergent ethanol preference (alcohol-preferring P, alcohol-nonpreferring NP, high-alcohol-drinking HAD-1 and -2, and low-alcohol-drinking LAD-2) to determine if factors contributing to high and low alcohol intakes are present early in development. METHODS: From postnatal days 5 to 20, nondeprived male and female rat pups received 30 min daily access to either water or a 15% (v/v) ethanol solution. In each daily session, pups were placed in a heated chamber containing Kimwipes soaked with a water or ethanol solution. Pups were weighed before and after each session, and intake levels were calculated as a percentage of body weight change. RESULTS: Similar to previous reports, Wistar pups exhibited over a 2-fold higher level of ethanol ingestion than water on postnatal days 9 through 14, with ethanol intakes approaching 3 g/kg body weight. When the drinking patterns of the selected lines were examined, only the HAD replicate lines showed a comparable preference for ethanol versus water during the preweanling period. The ethanol consumption of P, NP, and LAD lines was not consistently distinguishable from water intake levels. To reveal whether early ethanol exposure would influence later ethanol drinking behavior, a subset of HAD and LAD rat pups received free-choice ethanol access upon weaning. Although the divergent ethanol preference between high- and low-alcohol-drinking lines was evident within the first 4 days of access (>4 g/kg/day for HAD; <2 g/kg/day for LAD), preweanling ethanol exposure did not alter the acquisition or maintenance of ethanol drinking in either line. CONCLUSIONS: Overall, these results suggest that (a) the enhanced ethanol ingestion observed during the middle portion of the preweanling period is a robust phenomenon and generalizes across nonselected strains of rats, (b) selective breeding for divergent alcohol preference has arrested this age-specific effect in all but the HAD lines of rats, and (c) early ethanol exposure does not alter genetic dispositions for later high or low alcohol preference.     

Neurophysiological findings and drinking levels in high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats

BACKGROUND: Specific neurophysiological profiles, such as reduced P300 amplitude or altered spectral power in the EEG, have been associated with a risk for alcoholism in several clinical populations. In certain rodent models, high versus low alcohol consumption is associated with similar neurophysiological differences. For example, alcohol-preferring (P) rats have increased spectral power and decreased P300 amplitudes compared with alcohol-nonpreferring (NP) rats. In the present study, the neurophysiological profiles of high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats were assessed (1) to determine if their electrophysiological profiles are similar to P and NP rats and (2) to examine the relationship of these neurophysiological indices to ethanol drinking. METHODS: Ethanol-naive HAD and LAD rats were implanted with cortical and amygdalar recording electrodes. Baseline EEG and event-related potentials (ERPs) then were assessed. Subsequently, all rats were trained to self-administer ethanol by using a sucrose-substitution procedure. RESULTS: Baseline EEG and ERP (i.e., pre-ethanol exposure) were assessed based on line (HAD versus LAD) and actual ethanol consumption (high drinkers versus low drinkers). At baseline, ethanol-naive HAD rats displayed significantly greater power in the cortical EEG and decreased amygdala N1 ERP amplitude compared with ethanol-naive LAD rats. Similar EEG and ERP profiles have been observed when P and NP rats are compared. No differences in P300 between lines were observed, but high-drinking rats, independent of line, had significantly decreased P300 amplitude in the amygdala compared with low-drinking rats. CONCLUSIONS: These data suggest there are some similarities in EEG and ERP profiles of P and HAD rats compared with NP and LAD rats. Furthermore, the data suggest that decreased P300 amplitude in the amygdala is associated with increased limited access ethanol drinking.     

Comparison of Alcohol-Preferring and Nonpreferring Selectively Bred Rat Lines. I. Ethanol Initiation and Limited Access Operant Self-Administration

Several lines of alcohol-preferring and alcohol-nonpreferring rats have been developed using selective breeding based on 24-hr homecage ethanol consumption. However, it remains unclear if the selection based on two-bottle choice resulted in similar ethanol self-administration when measured using an operant procedure. In this paper, we compare our previous work using alcohol-accepting (AA) and alcohol-nonaccepting (ANA) rats with data obtained using the identical procedures in the (P) and (NP) rat lines, and both replicate lines of the high alcohol drinking (HAD1 and HAD2) and low alcohol drinking (LAD1 and LAD2) lines. All rats from each line were initiated to self-administer 10% ethanol using the sucrose fading procedure. After initiation, increasing concentrations of ethanol up to 30% ethanol were tested. The results indicated that only in the LAD1 and LAD2 lines was ethanol presentation not able to maintain lever pressing after initiation. Compared with the AA line, the P, HAD1, HAD2, and NP lines all self-administered more ethanol in the operant paradigm after initiation. The ANA line self-administered less ethanol than the AA line, but more than the LAD lines. Correlational analysis of homecage consumption with operant ethanol self-administration suggested that -62% of the genetic variance in operant self-administration resulted from genes selected for the homecage drinking. At the same time, it was clear that there were genetic influences on operant self-administration that were not selected for by homecage ethanol drinking.     

More Vasopressin mRNA in the Paraventricular Hypothalamic Nucleus of Alcohol-Preferring Rats and High Alcohol-Drinking Rats Selectively Bred for High Alcohol Preference

Both the selectively bred alcohol-preferring (P) and high alcohol-drinking (HAD) rats exhibit alcohol preference, and develop tolerance to alcohol more quickly than their counterparts, the alcohol-nonpreferring (NP) and low alcohol-drinking (LAD) rats, respectively. It has been shown that the P rats retain developed tolerance longer than do NP rats, and alcohol drinking increases concurrently with the development of tolerance. Although alcohol preference and tolerance are fundamental elements of alcoholism, the exact mechanisms underlying these two phenotypes in P and HAD rats are not well understood. Recent studies have suggested that arginine vasopressin (AVP) may be involved in modulation of alcohol tolerance. Accordingly, this study was designed to examine whether the AVP mRNA level in the hypothalamus differs in rats that have been selectively bred for alcohol preference and nonpreference. A ³⁵S-AVP antisense oligodeoxynucleotide probe was used for in situ hybridization to localize AVP mRNA in the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON), two major sites for AVP synthesis in the hypothalamus. Quantitative autoradiography demonstrated that P rats had higher levels of AVP mRNA in the PVN than NP rats. Similarly, higher levels of AVP mRNA were also found in the PVN of HAD rats, compared with LAD rats. The AVP mRNA levels in the SON were similar in the alcohol-preferring and alcohol-nonpreferring rat lines. Basal plasma AVP levels were higher in NP rats than in P rats as determined by radioimmunoassay, whereas plasma AVP levels were not significantly different between HAD and LAD rats. The results suggest that increased AVP gene expression in the PVN may contribute to alcohol preference and the development of alcohol tolerance.     

Taste Reactivity in High Alcohol Drinking and Low Alcohol Drinking Rats

High alcohol drinking (HAD) and low alcohol drinking (LAD) rats were tested, in three exposures, for taste reactivity to five concentrations of alcohol (5%, 10%, 20%, 30%, and 40%, v/v), water, and one concentration each of sucrose and quinine. Of the three reactivity exposures, one was done before a 3-week period of continuous access to water and 10% alcohol, the second test was done immediately after the consumption period, and the final reactivity test was done after 1 month of alcohol abstinence. The results showed that the groups did not differ in reactivity on the initial test. After the consumption tests (when the HAD rats consumed significantly more alcohol than the LAD rats), differences in reactivity were found: HAD rats produced significantly more ingestive responses (which promote consumption) and significantly fewer aversive responses (which facilitate fluid rejection) than LAD rats. These differences were maintained even after 1 month of alcohol abstinence. The present data replicate an earlier experiment with alcohol-preferring (P) rats and alcohol-non-preferring (NP) rats, and indicate that the selective breeding process does not produce differences in the innate perception of the taste of alcohol. However, after experience with drinking alcohol, rats selectively bred for high alcohol consumption exhibit a palatability shift reflected by high ingestive responding and little or no aversive responding. Such a shift would clearly contribute to the maintenance of high levels of alcohol consumption.     

Comparison of Alcohol-Preferring and Nonpreferring Selectively Bred Rat Lines. II. Operant Self-Administration in a Continuous-Access Situation

Several rat lines have been developed using preference/nonpreference and daily ethanol intake in the homecage as criteria for selective breeding. Using these lines, behavioral and neural factors that may underlie the genetic basis for the control of ethanol consumption have been examined. In this paper, we report data from eight of these selected lines: the Alcohol-Preferring (P) and Alcohol-Nonpreferring (NP), the Alcohol-Accepting (AA) and Alcohol-Nonaccepting (ANA), and the High Alcohol Drinking (HAD1 and HAD2) and Low Alcohol Drinking (LAD1 and LAD2) rats. All lines were tested using operant procedures and the same protocols for both the ethanol self-administration initiation and measurement of continuous-access ethanol consumption. During continuous access, the animals were housed in operant chambers with access to 10% (v/v) ethanol after responses on one lever, food pellets (45 mg) after responses on a second lever, and water in a drinking tube that was connected to a drinkometer circuit Under these procedures, both similarities and differences among the selected lines on continuous-access operant ethanol intake were observed. For example, overall total homecage ethanol drinking was similar for the AA and both HAD lines. When examined in the operant continuous-access situation, however, the AA rats displayed a different consumption pattern, compared with the HAD lines. Data suggest that the frequency of drinking bouts was a primary factor in the phenotypic homecage selection of the preferring lines that was revealed by the use of the continuous-access operant procedure. In general, data suggest that genes related to ethanol preference and intake in homecage continuous-access situations may not be identical to those related to ethanol's reinforcing function in operant continuous-access conditions. Because ethanol consumption appears to be controlled by different drinking patterns across lines, the selected lines provide for a variety of models to understand how varying genotypes can impact ethanol consumption.     

Innate Differences of Neuropeptide Y (NPY) in Hypothalamic Nuclei and Central Nucleus of the Amygdala Between Selectively Bred Rats with High and Low Alcohol Preference

BACKGROUND: Neuropeptide Y (NPY) is a neuropeptide that has been demonstrated to produce anxiolytic effects when administered centrally. To examine the hypothesis that NPY might play a role in alcohol-seeking behavior, this study took advantage of the genetic differences of the alcohol-preferring (P) rats and alcohol-nonpreferring (NP) rats, as well as the high alcohol-drinking (HAD) rats and low alcohol-drinking (LAD) rats, in voluntary alcohol consumption to examine if NPY neurons in the brains differ between these selected lines. METHODS: The NPY immunoreactivity (NPY-I) was measured using an established radioimmunohistochemical assay in discrete brain structures including the paraventricular hypothalamic nucleus (PVN), arcuate nucleus (ARC), and central nucleus of the amygdala (CeA). RESULTS: The quantitative data indicated that there was more NPY-I in the PVN and ARC of P rats than NP rats, whereas there was less NPY-I in the PVN and ARC of HAD rats than LAD rats. However, the NPY-I in the CeA was less in both the P and HAD rats than in the NP and LAD rats, respectively. Therefore, the data indicate that there are innate differences in the NPY-I in the brain between selectively bred rats with high and low alcohol preference. CONCLUSION: Because both P rats and HAD rats have high alcohol preference, the disparate finding between these two lines of rats suggests that the hypothalamic NPY neurons are probably not associated with alcohol preference. In contrast, consistent findings in the CeA of both P rats and HAD rats suggest that NPY in the CeA of P and HAD rats may contribute to the regulation of alcohol consumption. This is substantiated by a recent report showing that NPY-knockout mice drink significantly more ethanol, and transgenic mice that overexpress the NPY gene drink less alcohol, than wild-type mice. Together, the findings support the notion that NPY agonists that would enhance NPY function in the amygdala might be useful for the treatment of anxiety and alcoholism.     

Norepinephrine uptake sites in the locus coeruleus of rat lines selectively bred for high and low alcohol preference: a quantitative autoradiographic binding study using [3H]-tomoxetine

BACKGROUND: The locus coeruleus (LC) is the largest norepinephrinergic cell group in the central nervous system and contains a high density of norepinephrine (NE) uptake sites. Alcohol-preferring (AP) rats and high-alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, whereas alcohol-nonpreferring (NP) rats and low-alcohol-drinking (LAD) rats are bred for low alcohol preference. However, it is unknown whether NE uptake sites in the LC are associated with alcohol preference in AP and HAD rats when compared with their respective control rats, NP and LAD rats. This study was designed to examine this question. METHODS: Animals were decapitated and brains were removed, frozen with dry ice powder, and stored in a deep freezer. The LC tissue blocks were cut into 14 micro cryostat sections, collected on glass slides, and incubated with 0.6 nM [3H]-tomoxetine in 50 mM Tris-HCl buffer system. For nonspecific binding, 1 microM desipramine was added to the radioactive ligand. Sections were rinsed, quickly dried, and processed for quantitative autoradiography. In addition, galanin content in the LC was also studied. RESULTS: The LC possessed a high density of [3H]-tomoxetine binding sites. There were fewer tomoxetine binding sites (fmol/mg protein) in the AP rats (433.0 +/- 8.1) than in the NP rats (495.6 +/- 3.7). HAD rats (386.5 +/- 13.2) also possessed fewer tomoxetine binding sites than LAD rats (458.7 +/- 10.1). Galanin content in the LC was similar between AP and NP rats and between HAD and LAD rats. CONCLUSIONS: Because both AP rats and HAD rats were selectively bred for alcohol preference, the finding of consistently low levels of [3H]-tomoxetine binding in the LC of these two lines of rats with high alcohol preference suggests that down-regulation of NE transporters in the LC of AP and HAD rats may be associated with alcohol-seeking behavior. A possible involvement of the coerulear NE uptake sites in depression is also discussed. Galanin in the LC may not relate to alcohol preference.     

Gender and age at drinking onset affect voluntary alcohol consumption but neither the alcohol deprivation effect nor the response to stress in mice

Background: Epidemiological studies suggest that initiation of alcohol drinking at an early age is associated with an increased risk of developing an alcohol use disorder later in life. Nevertheless, relatively few studies using animal models have investigated the relationship between age of onset of drinking and ethanol drinking patterns in adulthood. Besides age at drinking onset, other factors such as gender could also affect the pattern of development of alcohol consumption. In rodents, many studies have shown that females drink more than males. However, even if it is assumed that hormonal changes occurring at puberty could explain these differences, only one study performed in rats has investigated the emergence of sex‐specific alcohol drinking patterns in adolescence and the transition from adolescence to adulthood. The aim of the present study was to compare the acquisition of voluntary alcohol consumption, relapse‐like drinking (the Alcohol Deprivation Effect—ADE) and stress‐induced alcohol drinking in male and female outbred mice that acquired alcohol consumption during adolescence or adulthood. Methods: Separate groups of naïve female and male WSC‐1 mice aged ± 28 days (adolescents) or ±70 days (adults) were given ad libitum access to water and 6% ethanol solution for 8 weeks (1st to 8th week) before undergoing a 2‐week deprivation phase (9th and 10th week). After the deprivation period, 2‐bottle preference testing (ethanol vs. water) resumed for 3 weeks (11th to 13th). During the 13th week, all animals were subjected to restraint stress for 2 consecutive days. Results: Over the entire time course of the experiment, ethanol intake and preference increased in females (both adults and adolescents). Adolescent animals (both females and males) showed a transient increase in alcohol consumption and preference compared to adults. However, by the end of continuous alcohol exposure (when all mice were adults), ethanol intake was not affected by age at drinking onset. A deprivation phase was followed by a rise in ethanol intake (ADE) that was not affected by sex or age. Finally, stress did not alter alcohol self‐administration either during or after its occurrence. Conclusions: Emergence of greater alcohol consumption in adult females does not seem to be limited to a specific developmental period (i.e., puberty). Age of voluntary drinking onset (adolescence vs. adulthood) does not affect eventual alcohol intake in adult WSC‐1 mice and does not modify the transient increase in ethanol consumption after alcohol deprivation.     

Operant Ethanol-Reinforced Behavior in P, NP, HAD, and LAD Rats Bred for High Versus Low Ethanol Preference

These studies examined the reinforcing effects of ethanol in rats selectively bred for high versus low ethanol drinking in a two-bottle choice preference task, namely the Preferring (P), Non-Preferring (NP), High Alcohol Drinking (HAD), and Low Alcohol Drinking (LAD) rats. The results substantiate findings suggesting that genetic factors are significant in determining whether ethanol will come to serve as a reinforcer. P rats exhibited high levels of responding for ethanol compared with the water vehicle, NP and HAD rats exhibited more moderate levels of responding for ethanol, and the behavior of LAD rats suggested that ethanol served only inconsistently as a reinforcer for these rats. Overall, the results suggest the existence of distinct, biologically influenced components of ethanol drinking behavior. Preference appears to measure an inherent facilitative factor allowing animals to initiate ethanol drinking. The operant chamber paradigm appears to measure factors related to whether and to what extent ethanol will serve as a positive reinforcer following conditioned exposure to the drug. Although preferring animals generally find ethanol reinforcing there seems to be little quantitative relationship between degree of preference and whether ethanol will serve as a reinforcer. Lack of preference does not seem to be predictive of lack of reinforcement. Thus, it appears that preference for ethanol and reinforcement from ethanol are somewhat overlapping, but distinct factors that contribute to ethanol drinking. These results suggest the existence of multiple components of behavior mediated by multiple mechanisms that contribute to ethanol drinking.