Nonpreferring Rat Lines Research Articles

Effects of forced alcohol drinking on alcohol–water choice in three pairs of rat lines selectively bred for differences in alcohol preference

Three pairs of Indiana University rat lines (inbred alcohol-preferring and nonpreferring rat lines [P/NPs], high- and low-alcohol-drinking rat lines [HAD/LAD1s and HAD/LAD2s]) were bred in the School of Medicine colony to drink high versus low daily amounts of a 10% vol/vol alcohol test solution (>5.0 g/kg body weight vs. <1.5 g/kg body weight), and a high versus low proportion of alcohol to water (>2:1 vs. <0.5:1) by the end of a 3-week alcohol–water choice condition. This choice phase was always preceded by four days of a forcing procedure with alcohol as the only fluid. The present study examined the contribution of the forcing procedure to the alcohol intake of animals in each pair of lines by comparing daily alcohol intake of rats housed in experimental chambers in a forced group (4 days with only alcohol solution to drink followed by 22 choice days) versus a choice group (both alcohol and water available all 26 days). As expected, under the initial alcohol exposure, high-drinking line rats drank more alcohol than low-drinking line rats, and all forced groups drank more alcohol than choice groups. At the start of the choice phase, all low-drinking line forced groups immediately dropped their alcohol intake to the level of their choice groups. In contrast, all high-drinking line forced groups maintained a high level of alcohol intake under choice, whereas all high-drinking line choice groups slowly increased average alcohol intake across the 22-day choice phase, ending near the average intake of their forced groups. However, a small subset of each high-drinking line choice animals failed to increase alcohol intake until subsequently forced with alcohol for 4 days and tested again in choice. These results indicate that the alcohol-forcing procedure used in deriving these lines resulted in the selection of more than one pathway to a high-drinking phenotype. In addition, high-drinking line animals appeared more sensitive to the differences between laboratory- and colony-testing environments than low-drinking line animals. These data suggest that these high-drinking lines may represent an unexpectedly appropriate complex model of how multiple factors may contribute to the genesis of human alcoholism.

Ethanol induces stronger dopamine release in nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol-avoiding (abstainer) rats

Several studies on the differences between ethanol-preferring versus non-preferring rat lines suggest an innate deficit in the mesolimbic dopaminergic system as an underlying factor for ethanol volition. Rats would try to overcome such deficit by engaging in a drug-seeking behaviour, when available, to drink an ethanol solution over water. Thus, in the present study we compared the effect of a single dose of ethanol (1 g/kg, i.p.) on the extracellular levels of monoamines measured by microdialysis in the shell of nucleus accumbens of University of Chile bibulous (UChB) and University of Chile Abstainer (UChA) rats, bred for 79 and 88 generations to prefer or reject ethanol, respectively. It is reported that under basal conditions extracellular dopamine levels are lower in the bibulous than in the abstainer rats, while ethanol induced a 2-fold greater increase of dopamine release in bibulous than in abstainer rats. The greater effect of ethanol in bibulous rats was not associated to differences in blood ethanol levels, since the concentration and elimination of ethanol were virtually identical in both rat lines, indicating that bibulous rats are more sensitive to the stimulation of dopamine release by ethanol than abstainer rats. No differences were observed in 5-hydroxytryptamine or metabolites measured simultaneously under basal or ethanol-stimulating conditions in bibulous and abstainer rats. Overall, the present results suggest that a low dopaminergic tone and a strong mesolimbic dopamine response to ethanol are concerted neurochemical features associated to an ethanol-seeking behaviour in rats.

Innate differences in neuropeptide Y (NPY) mRNA expression in discrete brain regions between alcohol-preferring (P) and -nonpreferring (NP) rats: a significantly low level of NPY mRNA in dentate gyrus of the hippocampus and absence of NPY mRNA in the medial habenular nucleus of P rats

The neuropeptide Y (NPY) gene in rat chromosome 4 has been shown to play an important role in alcohol-seeking behavior. NPY knockout mice drink more alcohol than wild-type mice, implicating a link between NPY deficiency and high alcohol intake. This is supported by recent studies showing that intracerebroventricular injections of NPY reduce alcohol intake in both alcohol-preferring (P) and high alcohol-drinking rats. However, it is unknown which anatomical NPY systems are involved in alcohol preference. This study was designed to investigate whether there are innate differences in NPY mRNA in cerebral cortical areas, dentate gyrus (DG) of the hippocampus and medial habenular nucleus (MHb) between P and alcohol-nonpreferring (NP) rats, as these discrete brain regions are rich in NPY mRNA. [(33)P]-labeled 28-mer oligodeoxynucleotide probe was applied for the in situ hybridization study to detect the NPY mRNA, measured using quantitative autoradiography. This study revealed an absence of NPY mRNA in the MHb of P rats. We found that NPY mRNA was significantly lower in the DG of P rats than NP rats. This innate difference of NPY mRNA expression in the DG between P and NP rats is region specific. For example, in most of the cerebral cortical areas examined, an innate difference was not seen. Our study suggests that lower NPY gene expression in the DG and MHb of P rats may be factors contributing to some of the phenotypic differences observed between the P and NP lines of rats.

Mapping of QTL influencing saccharin consumption in the selectively bred alcohol-preferring and -nonpreferring rat lines.

The inbred preferring (iP) and nonpreferring (iNP) rat strains were derived from the selectively bred alcohol-preferring (P) and alcohol-nonpreferring (NP) lines. Previously, 381 iP x iNP F2 progeny were generated to identify quantitative trait loci (QTLs) influencing alcohol consumption and preference. Saccharin consumption (ml/48 h) and saccharin intake (ml/kg/day) were also measured in the F2 sample and were significantly correlated with both alcohol consumption and preference (all r > or = .20, p < .0001), suggesting that there might be some QTLs influencing both saccharin and alcohol phenotypes. We have performed a genome screen using F2 animals with extreme saccharin or alcohol consumption to identify QTLs contributing to saccharin-related phenotypes. Lod scores greater than 2.0 were found on chromosomes 3, 16 and 18 in this sample. Additional genotyping was performed in these regions in the full sample of 381 F2 progeny to further characterize these putative QTLs. On chromosome 3, the maximum lod score in the full sample was 2.7 with saccharin consumption. This QTL appears to overlap with a QTL identified for alcohol consumption in the iP and iNP lines and has pleiotropic effects on both phenotypes. Interestingly, this region of rat chromosome 3 is syntenic with mouse chromosome 2, where a QTL influencing alcohol preference has been previously reported. The QTL on chromosome 16 has a maximum lod score of 4.0 with saccharin intake and 2.6 with saccharin consumption. The QTL on chromosome 18 has a maximum lod score of 2.7 with saccharin consumption. Taken together, these data provide the first results of a genome screen for QTLs contributing to saccharin phenotypes in the rat.

Responsivity and Development of Tolerance to the Motor Impairing Effects of Moderate Doses of Ethanol in Alcohol-Preferring (P) and -Nonpreferring (NP) Rat Lines

Background: Research comparing the alcohol-preferring (P) and -nonpreferring (NP) rat lines has detected an apparent association between ethanol preference and lower responsivity to ethanol, as well as the capacity to develop and maintain tolerance to ethanol's effects. However, past studies of tolerance to ethanol's effects generally involved relatively high doses. The present study examined recovery from functional impairment induced by moderate doses of ethanol after a single dose (responsivity) and after multiple doses (development of tolerance) in the P and NP rat lines. Method: Adult female P and NP rats were trained, for 5 consecutive days, to stay on an oscillating bar for 120 sec. Rats were then assigned to one of three groups to receive 1.0, 1.25, or 1.5 g/kg ethanol for 5 consecutive test days. Rats were tested each day at 15-min intervals, following intraperitoneal injection, until recovery to the 120 sec criterion. Results: On the first test day, NP rats took longer to recover to criterion than the P rats following the 1.0 and 1.25 g/kg doses, whereas at the 1.5 g/kg dose no line difference was evident. Trunk blood alcohol concentrations (BACs), associated with time to recovery, indicated higher values for the P than NP rat on day 1 following injection of the two lower doses. Compared to day 1, NP rats demonstrated significantly shorter times to recovery beginning on day 2 following injections of the 1.0 and 1.25 g/kg doses. However, NP rats did not show significantly different recovery times on days 2–5 compared to day 1 following injection of the 1.5 g/kg dose. The shorter recovery times at the 1.0 and 1.25 g/kg doses were associated with BACs at recovery on day 3 being equal to or greater than values obtained on day 1. In contrast, compared to day 1, P rats did not show shorter recovery times until days 3 and 5 following the 1.0 and 1.25 g/kg doses, respectively. However, P rats did demonstrate shorter recovery times on day 2 and higher BACs on day 3 compared to day 1 following the 1.5 g/kg dose. Conclusion: With regard to motor impairment, lower responsivity to moderate doses of ethanol may be a factor associated with high alcohol-seeking behavior. The present results confirm past research supporting an association between ethanol preference and low ethanol responsivity but at doses that are more reflective of those self-administered by P rats.

Responsivity and Development of Tolerance to the Motor Impairing Effects of Moderate Doses of Ethanol in Alcohol‐Preferring (P) and ‐Nonpreferring (NP) Rat Lines

Research comparing the alcohol-preferring (P) and -nonpreferring (NP) rat lines has detected an apparent association between ethanol preference and lower responsivity to ethanol, as well as the capacity to develop and maintain tolerance to ethanol's effects. However, past studies of tolerance to ethanol's effects generally involved relatively high doses. The present study examined recovery from functional impairment induced by moderate doses of ethanol after a single dose (responsivity) and after multiple doses (development of tolerance) in the P and NP rat lines. Adult female P and NP rats were trained, for 5 consecutive days, to stay on an oscillating bar for 120 sec. Rats were then assigned to one of three groups to receive 1.0, 1.25, or 1.5 g/kg ethanol for 5 consecutive test days. Rats were tested each day at 15-min intervals, following intraperitoneal injection, until recovery to the 120 sec criterion. On the first test day, NP rats took longer to recover to criterion than the P rats following the 1.0 and 1.25 g/kg doses, whereas at the 1.5 g/kg dose no line difference was evident. Trunk blood alcohol concentrations (BACs), associated with time to recovery, indicated higher values for the P than NP rat on day 1 following injection of the two lower doses. Compared to day 1, NP rats demonstrated significantly shorter times to recovery beginning on day 2 following injections of the 1.0 and 1.25 g/kg doses. However, NP rats did not show significantly different recovery times on days 2-5 compared to day 1 following injection of the 1.5 g/kg dose. The shorter recovery times at the 1.0 and 1.25 g/kg doses were associated with BACs at recovery on day 3 being equal to or greater than values obtained on day 1. In contrast, compared to day 1, P rats did not show shorter recovery times until days 3 and 5 following the 1.0 and 1.25 g/kg doses, respectively. However, P rats did demonstrate shorter recovery times on day 2 and higher BACs on day 3 compared to day 1 following the 1.5 g/kg dose. With regard to motor impairment, lower responsivity to moderate doses of ethanol may be a factor associated with high alcohol-seeking behavior. The present results confirm past research supporting an association between ethanol preference and low ethanol responsivity but at doses that are more reflective of those self-administered by P rats.

Neurotensin studies in alcohol naive, preferring and non-preferring rats

Neurotensin is a tridecapeptide, present in the central nervous system and the gastrointestinal tract in man and animals. Previous studies in mice selectively bred for differences in hypnotic sensitivity to ethanol have provided data to suggest that neurotensinergic systems may mediate differences in ethanol's actions in these animals. The present study sought to determine if brain neurotensin levels differed between two lines of rats which have been selectively bred for alcohol preferring or non-preferring behaviors. In addition, electroencephalographic and event-related potential responses to intracerebroventricular saline and neurotensin (10 or 30 μg) were evaluated between the rat lines. Similar to human subjects at high genetic risk for alcoholism, preferring rats were found to have more electroencephalographic fast frequency activity and lowered amplitude of the P3 component of the event-related potential in cortical sites under the saline condition. Overall, electrophysiological response to neurotensin, in the two rats lines, was substantially similar to what has been reported previously in outbred Wistar rats, and consisted of dose-related decreases in overall electroencephalographic spectral power concomitant with increases in amplitude and decreases in the latency of the N1 component of the event-related potential. However, differences in neurotensin responses between the preferring and non-preferring rat lines were also found. The differences in electroencephalographic high-frequency activity and in P3 amplitude seen between the rat lines under control conditions were eliminated by administration of neurotensin. In addition, preferring rats appeared to be more sensitive to neurotensin-induced increases in N1 amplitude. Brain neurotensin concentrations were also found to differ between the lines. Significantly lower concentrations of neurotensin were found in the frontal cortex of preferring rats when compared to non-preferring rats or outbred Wistars. Taken together, these studies suggest that differences in the regulation of neurotensin neurons may contribute to the expression of behavioral preference for ethanol consumption in selective rat lines. Additionally, drugs targeting the neurotensinergic system may plausibly be of utility in the treatment of alcoholism.

Housing conditions alter GABAA receptor of alcohol-preferring and -nonpreferring rats.

The effects of housing conditions on some functional properties of the GABAA benzodiazepine (BZD) receptor in the cerebral cortex were examined in the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines of rats. Compared to rats housed in pairs (P with P and NP with NP), P and NP rats housed individually had 44% (p < 0.005) and 32% (p < 0.01) lower values, respectively, for GABA-stimulated 36Cl- influx into cortical microsacs. The maximal effect (Vmax) of flunitrazepam (FNZ) to enhance GABA-stimulated 36Cl- uptake was 44% higher in individually housed P rats than pair-housed P rats (p < 0.05) and 51% higher than individually housed NP rats (p < 0.05). There was no difference between single and pair-housed NP rats for Vmax values of FNZ enhancement of GABA-stimulated 36Cl- influx. The results show housing conditions can alter some of the functional properties of the GABAA/BZD receptor in the P and NP lines of rats. The differential effect of housing conditions on FNZ enhancement of 36Cl- influx, observed between the lines, may be a result of higher levels of anxiety being produced by brief isolation in the P rat.

Comparison of alcohol-preferring (P) and nonpreferring (NP) rats on tests of anxiety and for the anxiolytic effects of ethanol

Rats of the selectively bred alcohol-preferring P and alcohol-nonpreferring NP lines were evaluated using three different behavioral measures of anxiety. Compared with NP rats, P rats (1) showed greater footshock-induced suppression of operant responding in an approach-avoidance conflict test; (2) spent less time in the open arms of an elevated plus maze; and (3) took longer in a passive avoidance test to step down from a platform to a grid floor where footshock was received 24 hours earlier. These findings indicate a greater degree of anxiety in the P than in the NP line of rats in these situations. Pretreatment with intraperitoneal (IP) ethanol (0.5–1.0 g/kg) injections produced anticonflict or anxiolytic effects in P but not in NP rats. However, the anticonflict effects of ethanol were small relative to those produced by chlordiazepoxide (CDP, 7.5 mg/kg) in both lines. The results demonstrate that selective breeding for divergent oral ethanol preference has produced associated differences between the P and NP lines of rats in behavioral tests of anxiety and in the anxiolytic effects of ethanol.

THE CONTENT AND ACTIVITY OF CYTOCHROME P450 2E1 IN LIVER MICROSOMES FROM ALCOHOL-PREFERRING AND NON-PREFERRING RATS

Constitutive levels of the ethanol-inducible cytochrome P450 (P450 2E1), as well as the extent of inducibility of this isozyme by pyrazole and 4-methylpyrazole in alcohol-preferring and non-preferring lines of rats, were investigated in order to evaluate whether the presence of this enzyme correlates with preference for ethanol. The content of P450 2E1 as detected immunochemically, as well as catalytic activity of P450 2E1 associated with the oxidation of preferred substrates such as dimethylnitrosamine and p-nitrophenol, was similar in liver microsomes from preferring and non-preferring rats. 4-Methylpyrazole was a poor inducer of P450 2E1 in both lines. Pyrazole treatment produced an identical 3- to 4-fold increase in content and catalytic activity of P450 2E1 in the two lines. The preferring and non-preferring rats do not appear to differ in their liver microsomal contents of cytochromes P450 2B1/B2 or 1A1. It appears that preference for ethanol is not associated with differences in the constitutive values or altered susceptibility to induction of P450 2E1 in the liver.

Alcohol intake is inversely related to plasma renin activity in the genetically selected alcohol-preferring and -nonpreferring lines of rats

Studies involving both animals and humans strongly suggest that alcoholism is, in part, genetically determined. One approach to studying this genetic component is to determine whether rats, genetically selected to prefer (P line) or avoid (NP line) alcohol, show differences in those physiological systems which modulate alcohol intake. It has previously been shown that alcohol intake in randomly bred stock rats is sensitive to and inversely related to manipulations which alter activity in the renin-angiotensin (R-A) system. In the present report the basal level of activity in the R-A systems of the P and NP rats as measured by plasma renin activity (PRA) was first assessed following which continuous access to alcohol (10% v/v) and water was offered for a period of five days. PRA was found to be inversely related to the amount of alcohol that was consumed. The P rats drank significantly larger amounts of alcohol than the NP rats who basically avoided the drug. The P rats had a significantly lower PRA than the NP rats. It is suggested that the genetic selection that favored different levels of alcohol consumption in the P and NP rats may have brought about this effect through differences in the activity of the renin-angiotensin systems in the two lines.

Effect of low dose ethanol on spontaneous motor activity in alcohol-preferring and -nonpreferring lines of rats.

To determine if behavioral arousal may be associated with ethanol preference, the effects of low to moderate doses of ethanol on spontaneous motor activity (SMA) were studied in the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines of rats as well as in the Maudsley Reactive (MR/N) and Nonreactive (MNR/N) strains. Alcohol-naive rats had food and water available ad lib, but food was removed 24 hr before and during activity testing. After an intraperitoneal injection of saline (5 ml) or ethanol (0.12 to 1.5 g/kg), SMA was monitored every three min for 30 min in an electronic activity monitor. The P and MR/N rats exhibited increased SMA after doses of 0.12 and 0.25 g/kg. Both the NP and MNR/N rats failed to show increased SMA at any ethanol dose. Moderate doses of ethanol, 1.0 and 1.5 g/kg, consistently depressed SMA in all lines/strains. In 24 hr-fasted rats, increased SMA occurred within 6-12 min after injection, but free-fed rats exhibited increased SMA 12-24 min after an ethanol dose of 0.25 g/kg. Free-choice drinking scores (10% ethanol (v/v) versus water) for the P, MR/N, MNR/N and NP rats were 6.6 +/- 0.5, 4.9 +/- 0.8, 2.2 +/- 0.7 and 1.4 +/- 0.3 g ethanol/kg body wt/day (mean +/- SEM), respectively. The data indicate a positive relationship between ethanol preference and ethanol-induced motor stimulation and suggest that hyperactivity may be an expression of the positive reinforcing effect of ethanol for alcohol-preferring rats.

Regional brain levels of monoamines in alcohol-preferring and -nonpreferring lines of rats

Regional brain levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA) and norepinephrine (NE) were determined in alcohol-naive rats from lines selectively bred for alcohol preference (P) and alcohol aversion (nonpreference, NP). Based on comparison by a standard t-test, the P rats had 12% lower NE in the pons-medulla, 20% higher NE and 16% lower DA content in the cerebral cortex (CX) than did the NP rats. However, the predominant finding was that the levels of 5-HT and 5-HIAA were 12–26% lower for the P than for the NP rats in the CX, hippocampus (HIP), corpus striatum (STR), thalamus (TH) and hypothalamus (HY). Regional CNS monoamines from a group of independently bred, stock Wistar rats were also compared with the NP and P groups to determine if the selectively bred rats differed widely from an unselected population. In most instances, the NP and P rats fell within the range of the stock group. When the stock group was included in an analysis of variance of the data, post-hoc differences between the NP and P groups that remained significant were the lower levels of 5-HT (and in some cases 5-HIAA) in the CX, HIP, STR, TH and HY of the P group. In the HY and HIP, the 5-HT levels of the P and NP animals diverged significantly in opposite directions from those of the stock group, possibly suggesting an involvement of these regional serotonergic systems in alcohol preference.