Low Alcohol-drinking Rats Research Articles

Regional CNS densities of serotonin and dopamine receptors in high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats

The densities of subtypes of serotonin (5-HT) and dopamine (DA) receptors were determined in the CNS of male alcohol-naive HAD and LAD lines of rats. Autoradiographic studies were undertaken to measure the densities of (a) 5-HT 1A sites labelled with 2 nM [ 3H]8-OH DPAT, (b) 5-HT 2A sites labelled with 2 nM [ 3H] ketanserin, (c) D 1 sites labelled with 1 nM [ 3H]SCH23390, and (d) D 2 sites labelled with 20 nM [ 3H]sulpiride. Membrane binding, using tissue combined from the olfactory bulb, olfactory tubercle, and nucleus accumbens, was carried out to determine K d and B max values for the binding of 0.25–8.0 nM [ 3H]7-OH DPAT to D 3 sites. Among the 14 regions measured for densities of 5-HT 1A sites, no interline differences were found in the cerebral cortical regions or in the septal nuclei; however, within the hippocampus, 15–20% lower binding of [ 3H]8-OH DPAT was observed in the posterior dorsal CA3 and dentate gyrus of the HAD line. There were no interline differences in any of the 10 regions examined for [ 3H]ketanserin binding to 5-HT 2A sites, or in the densities of D 1 and D 2 sites in the mesolimbic and nigrostriatal DA systems, except for a 35% higher density of D 2 sites in the substantia nigra pars compacta of the HAD line. There were no interline differences in the K d or B max values for [ 3H]7-OH DPAT binding to D 3 sites. Overall, these results indicate that no marked interline differences are evident in the densities of 5-HT 1A, 5-HT 2A, D 1, D 2, and D 3 receptors within the mesolimbic system that could be associated with the disparate alcohol drinking behaviors of the HAD and LAD rats.

Quantitative Autoradiography on [(35)S]TBPS Binding Sites of Gamma- Aminobutyric Acid(A) Receptors in Discrete Brain Regions of High- Alcohol-Drinking and Low-Alcohol- Drinking Rats Selectively Bred forHigh- and Low-Alcohol Preference.

It has been documented that ethanol can potentiate brain gamma-aminobutyric acid (GABA)ergic function, and there is a close link between the GABA(A) receptor complex and effects of ethanol, including reinforcement of alcohol which is a fundamental element of alcohol preference. However, it is unknown in what discrete brain regions GABA(A) receptors might be associated with alcohol preference. In the present study, [(35)S]t-butylbicyclophosphorothionate ([(35)S]TBPS) was used to localize GABA(A) receptors in high-alcohol-drinking (HAD) rats and low-alcohol-drinking (LAD) rats which were selectively bred for high and low alcohol preference, respectively. Initial qualitative observations indicated that [(35)S]TBPS binding sites were abundant in many brain areas including the cerebral cortex, hypothalamus and amygdala of HAD and LAD rats. Furthermore, the quantitative autoradiographic analysis revealed fewer [(35)S]TBPS binding sites of GABA(A) receptors in the amygdaloid complex, central medial thalamic nucleus, lateral hypothalamic nucleus and anterior hypothalamic nucleus of HAD rats than LAD rats. Collectively, this study has indicated that HAD rats selectively bred for high alcohol preference possess lower [(35)S]TBPS binding in the brain. Since lower TBPS binding has been proposed to reflect enhanced GABAergic function, as evidenced in rats with seizure or under alcohol withdrawal, the results from the present study suggest that HAD rats might have an enhanced GABAergic function. It is thus likely that enhanced GABAergic function in the brain might be related to high alcohol preference which is characteristic in HAD rats. In addition, the present result showing no difference of [(35)S]TBPS binding in the nucleus accumbens is also in agreement with a notion that [(35)S]TBPS binding may represent only a small spectrum of the GABA(A) receptor complex which is constituted of a sophisticated subunit combination whose functional compositions are still unknown. In conclusion, the present study supports the working hypothesis that GABA(A) receptors are involved in alcohol preference in HAD rats.

Comparison of Rats Selectively Bred for High and Low Ethanol Intake in a Forced-Swim-Test Model of Depression: Effects of Desipramine

This investigation examined if there is a relationship between selective breeding for high or low alcohol intake and immobility in a forced-swim-test (i.e., “behavioral despair”) model of depression. Time spent immobile in a water-filled cylinder was measured in the alcohol-preferring (P) and nonpreferring (NP) lines of rats, and in the high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) lines. Each rat was tested for 2 10-min trials administered 24 h apart, and pretreatment with saline or desipramine (10.0 or 20.0 mg desipramine/kg b.wt. IP) also was evaluated. Drug was administered immediately after Trial 1 and again 1 h before Trial 2. When tested without pretreatment in Trial 1 or with saline pretreatment in Trial 2, NP rats spent significantly more time immobile than did P rats, but no comparable line differences were found when HAD and LAD rats were tested. Desipramine pretreatment reduced the time spent immobile in rats of the 2 alcohol-nonpreferring lines (i.e., the NP and LAD rats), but had no significant effect in rats of the 2 alcohol-preferring lines (the P and HAD rats). These findings do not support the hypothesis that there is a functional relationship between high alcohol drinking and susceptibility to “behavioral despair” as measured by the forced-swim test. The results with desipramine suggest that selection for high alcohol intake may be associated with insensitivity to desipramine.

Calcitonin gene-related peptide (CGRP) content and CGRP receptor binding sites in discrete forebrain regions of alcohol-preferring vs. -nonpreferring rats, and high alcohol-drinking vs. low alcohol-drinking rats

This study showed that alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats possess fewer calcitonin gene-related peptide (CGRP) receptor binding sites than their respective controls in the central amygdaloid nucleus (CeA) which is known to be related to anxiety. Since P and HAD rats were selectively bred for high alcohol preference, and alcohol can produce anxiolytic effect, one can postulate that P and HAD rats preferentially drink alcohol in order to obtain its anxiolytic effect. This study supports a hypothesis that deficit of CGRP receptors in the CeA of P and HAD rats may contribute to alcohol preference.

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.

5-HT, dopamine, norepinephrine, and related metabolites in brain of low alcohol drinking (LAD) rats shift after chronic intra-hippocampal infusion of harman

Harman (1-methyl-beta-carboline) has been shown to induce preference for alcohol in the genetically bred, low alcohol drinking (LAD) rat. This study was undertaken in the LAD rat to determine whether monoamines and their metabolites in different regions of the brain are altered by harman infused chronically into the dorsal hippocampus. For this purpose, a cannula was implanted stereotaxically into the dorsal hippocampus. The cannula was attached to an osmotic minipump implanted subcutaneously within the intrascapular space. The pump was filled with either an artificial cerebrospinal fluid (CSF) vehicle or harman, which was delivered at a rate of 1.0 or 3.0 micrograms/h (i.e., 5.5 or 16.5 nmol/h, respectively) for a period of 14 days. Four days after surgery, a standard preference test for ethyl alcohol was given to the rats over 10 days in which concentrations were increased daily from 3%-30%. The higher concentration of harman infused into the hippocampus elevated the level of serotonin (5-HT), both ipsilateral and contralateral to the hippocampal site of infusion, as well as in the midbrain, frontal cortex, striatum and nucleus accumbens. Similarly, this treatment resulted in a rise in the levels of norepinephrine in the hippocampus and midbrain but decreases in dopamine levels in the pons. The levels of 5-hydroxyindoleacetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were diminished in the pons of rats given 3.0 micrograms/h harman, whereas both concentrations of the beta-carboline reduced the level of homovanillic acid (HVA) in the frontal cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased alcohol intake in low alcohol drinking rats after chronic infusion of the β-carboline harman into the hippocampus

Harman (1-methyl-β-carboline) has been shown to induce volitional drinking of ethyl alcohol in the rat. The purpose of this study was to examine the long-term effect of sustained delivery of harman into the dorsal hippocampus on the subsequent preference for alcohol in the genetically bred low alcohol drinking (LAD) rat. The individual pattern of preference for alcohol was first determined following a standard 3–30% alcohol self-selection test for 10 days. Thereafter, a cerebral cannula for constant infusion was implanted stereotaxically into the dorsal hippocampus. The cannula was attached to an osmotic minipump implanted subcutaneously, which was filled with either an artificial cerebrospinal fluid (CSF) vehicle or harman. Harman was delivered at a rate of 1.0 or 3.0 μg/h (i.e., 5.5 or 16.5 nmol/h, respectively) for a period of 14 days. Four days after surgery, the rats underwent a second 3–30% alcohol preference test for 10 days. Both doses of arman induced a threefold increase in the voluntary consumption of alcohol, expressed as g/kg per day. This effect of the β-carboline seems to be specific for ethanol because its intake by the LAD rats was increased significantly only when concentrations from 11% to 30% were presented. Harman also enhanced the daily intake of food in a dose-dependent manner, but did not affect body weights or the volumes of water and total fluid consumed. These results, thus, demonstrate that the long-term exposure of hippocampal neurons to harman induces a preference for high concentrations of alcohol even in a line of rats lacking such a genetic predisposition.

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.

Genetics of alcoholism: Simultaneous presentation of a chocolate drink diminishes alcohol preference in high drinking HAD rats

Through selective crossbreeding of the N/Nih heterogeneous stock of rats, two genetic lines of rats have been developed that are categorized by their preference for ethyl alcohol as high alcohol drinking (HAD) and low alcohol drinking (LAD) animals. Corresponding to other strains of rat bred for alcohol selection or rejection, they were subdivided on the basis of their intake of a solution of 10% alcohol vs. water. The present experiments were designed to determine whether the HAD-1 and LAD-1 lines are similar to the P and NP rats in their profile of alcohol consumption. Five successive three-bottle preference tests for alcohol drinking in the presence of water were undertaken in both HAD ( n = 9) and LAD ( n = 10) rats as follows: 10% alcohol for 5 days; 3–30% concentrations of alcohol increased over 11 days; the maximally preferred concentration of alcohol for 5 days; this maximally preferred concentration of alcohol plus either chocolate Slender for 5 days, or an aspartame solution for 5 days. The intake of alcohol of the LAD rats during the 10% test was 0.4 g/kg/day, whereas during the 3–30% test, the maximum intake was 1.7 g/kg/day; their maximally preferred concentrations ranged between 7% and 9% alcohol. In contrast, the intake of 10% alcohol of the HAD rats was 6.5 g/kg/day, whereas during the 3–30% test the mean daily intake was 6.6 g/kg/day; the maximally preferred solutions of the HAD rats ranged between 13 to 20%, with the mean maximum intake of 10.57 g/kg/day reached at the 20% concentration. Thus, the use of a single concentration of alcohol such as 10% to ascertain preference for alcohol for these lines of rat is not an optimal procedure. In the presence of both the chocolate drink and aspartame, the intakes of the preferred concentration of alcohol of the HAD rats declined markedly, whereas the limited drinking of the LAD rats was unaffected by either palatable fluid. These results differ with those of the P line of rats which sustained their high preference for alcohol even in the presence of the same palatable solutions. Therefore, gustatory factors associated with the sensory quality of the fluids overrode the characteristic preference of the HAD rats for a pharmacologically efficacious solution of alcohol.

Comparison of innate EEG parameters in rat lines selected for ethanol preference

Through bidirectional selective breeding, lines of rats that differ greatly in their voluntary alcohol drinking behavior have been developed — namely, the alcohol-preferring (P) and high-alcohol-drinking (HAD) lines and the alcohol-nonpreferring (NP) and low-alcohol-drinking (LAD) lines. The present experiments were designed to determine if an association exists between ethanol preference and features of the electroencephalogram (EEG) during various sleep-wake behaviors. Of the EEG parameters measured, only theta activity in the hippocampus revealed differences in the lines. However, these differences were not generally associated with ethanol preference. The peak frequency and distribution mean of hippocampal theta activity during REM sleep were significantly higher in NP rats than in P, HAD, and LAD rats. In addition, theta frequency during alert immobility tended to be higher in NP rats than in P, HAD, and LAD rats. A qualitative comparison of these data with published data from unselected rats further suggested that the NP rats are uniquely different with respect to theta frequency.

Biphasic effects of ethanol tested with drug discrimination in HAD and LAD rats

Seventh-generation selectively bred high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) rats were trained to make differential responses for ethanol (0.75 g/kg, IP) and saline vehicle, following postadministration intervals (PI) of 2 min (HAD-2 and LAD-2 animals) and 30 min (HAD-30 and LAD-30 animals). ED 50 values of 0.395 and 0.352 g/kg, respectively, for HAD-2 and LAD-2 animals and 0.269 and 0.314 g/kg, respectively, for HAD-30 and LAD-30 animals reflect the absence of any phenotypic difference for the discriminative stimulus effects of ethanol. HAD-2 animals were more responsive than LAD-2 animals to the stimulating effects of ethanol as measured by total response rates during training sessions. The differential ethanol response generalized to pentobarbital in all four groups but not to morphine, an alternative CNS depressant. The specific antagonist of 5-hydroxytryptamine 3, receptors, 3-tropanyl-3,5-dichlorobenzoate (MDL 72222), up to doses of 14.0 mg/kg failed to antagonize the discriminative effects of ethanol. Ethanol sleep times did not differ between groups.

HAD and LAD rats respond differently to stimulating effect but not discriminative effects of ethanol

The drug discrimination paradigm (DD) was used to evaluate behavioral differences of rats selectively bred for differential ethanol drinking preferences. Seventh-generation high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats were trained to discriminate between ethanol (0.5 g/kg, IP) and saline vehicle, following a 2-min presession interval (PI), using an FR-10 schedule of reinforcement. The HAD line was more responsive than the LAD line to the stimulating effect of ethanol as measured by total response rates. ED 50 values of 0.239 and 0.244 g/kg for the HAD and LAD lines, respectively, do not reflect any difference in the discriminative effects of ethanol. Response rates during DD indicated a dissociation of rate-increasing effects and discriminative performance following ethanol. In addition to differential drinking preference, these data suggest that selective breeding for the HAD and LAD animals also involves the stimulant action of ethanol but not on the discriminative effects.

Increased Number of GABAergic Terminals in the Nucleus Accumbens Is Associated with Alcohol Preference in Rats

Ethanol is known to be anxiolytic and this effect may be mediated through GABA transmission acting on the GABAA-benzodiazepine-Cl- ionophore complex. Recent studies from our laboratory have suggested that GABA transmission and the GABAA-benzodiazepine-Cl- ionophore complex might be involved in the rewarding action of ethanol in alcohol-preferring rats. We report here immunocytochemical and morphometric studies analyzing the GABAergic terminal density in the nucleus accumbens (NA), corpus striatum, nucleus tractus solitarius, and lateral septum of the selectively bred P (alcohol-preferring) and the NP (alcohol-nonpreferring) lines of rats, as well as of the high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) lines of rats. The NA was included for analysis because this structure has been implicated as an important component of the brain reward system. An increase of GABAergic terminal density was found in the NA of the P rats, when compared with the NP rats. Similarly, there were more GABAergic terminals in the NA of HAD rats than of the LAD rats. No differences between the lines were seen in the other brain regions examined. The results suggest that alcohol preference in P and HAD rats may be related to increased GABA terminals and enhanced GABAergic inhibition within the NA.

Effects of stimulation of the striatum and globus pallidus on local cerebral glucose utilization in 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.