Alcohol Drinking In Rats Research Articles

The Majchrowicz Binge Alcohol Protocol: An Intubation Technique to Study Alcohol Dependence in Rats

Binge drinking of alcohol is an important public health issue, and experimental studies are needed to understand the pathophysiological mechanisms of this problem and develop improved approaches to treatment. This unit presents a validated and widely used method to model binge alcohol drinking in rats. It consists of three daily intragastric administrations of ethanol to rats for 4 days. Ethanol is initially administered at 5 g/kg, and then each subsequent dose is determined based on the degree of intoxication the rat exhibits prior to each dose. The behavior of the animal is graded based on a well-described scale. After the fourth day, various aspects of the ethanol withdrawal syndrome can be observed over a predictable time course and additional protocols can be employed to study mechanisms of specific aspects of withdrawal and treatments to prevent them. One specific behavior observed during ethanol withdrawal and described here is sound-induced (audiogenic) convulsive seizure.

Effects of short deprivation and re-exposure intervals on the ethanol drinking behavior of selectively bred high alcohol-consuming rats

Alcoholics generally display cycles of excessive ethanol intake, abstinence and relapse behavior. Using an animal model of relapse-like drinking, the alcohol deprivation effect (ADE), our laboratory has shown that repeated 2-week cycles of ethanol deprivation and re-exposure, following an initial 6-week access period, result in a robust ADE by alcohol-preferring (P) and high alcohol-drinking (HAD-1 and HAD-2) rats. These rat lines have been selectively bred to prefer a 10% ethanol solution over water. The present study examined whether P and HAD rats would display an ADE using much shorter ethanol deprivation and re-exposure intervals. Rats were given either continuous or periodic concurrent access to multiple concentrations (10%, 20%, and 30% [vol/vol]) of ethanol. The periodic protocol involved access to ethanol for 12 days followed by four cycles of 4 days of deprivation and 4 days of re-exposure to ethanol access. High–alcohol-drinking rats displayed a robust 24-h ADE upon first re-exposure (HAD-1: ∼5 vs. 8 g/kg/day; HAD-2: ∼6 vs. 9 g/kg/day, baseline vs. re-exposure), whereas P rats (∼7 vs. 8 g/kg/day) displayed a modest, nonsignificant, increase in 24-h intake. In a separate group of rats, ethanol intake and blood alcohol concentrations after the first hour of the fourth re-exposure cycle were HAD-1: 2.0 g/kg and 97 mg%, HAD-2: 2.3 g/kg and 73 mg%, and P: 1.2 g/kg and 71 mg%; with all three lines displaying a robust first hour ADE. These findings suggest that (a) an ADE may be observed with short ethanol deprivation and re-exposure intervals in HAD rats, and (b) the genetic make-up of the P and HAD rats influences the expression of this ADE.

Dysregulation of Nociceptin/Orphanin FQ Activity in the Amygdala Is Linked to Excessive Alcohol Drinking in the Rat

Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, with an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (Marchigian Sardinian alcohol-preferring [msP]). The msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats. Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. The NOP receptor messenger RNA expression and binding was upregulated across most brain regions in msP compared with Wistar rats. However, in msP rats [(35)S]GTPgammaS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala. These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted on the basis of the anti-corticotropin releasing factor (CRF) actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.

Effects of naltrexone, duloxetine, and a corticotropin-releasing factor type 1 receptor antagonist on binge-like alcohol drinking in rats.

A 'binge' is defined by National Institute on Alcohol Abuse and Alcoholism as an excessive pattern of alcohol drinking that produces blood-alcohol levels (BALs) greater than 0.08 g% within a 2-h period and may or may not be associated with dependence. The purpose of this investigation was to explore the effects of several neuropharmacological agents in an animal model in which outbred rats voluntarily and orally self-administer pharmacologically meaningful alcohol doses that produce BALs >or=0.08 g% in daily limited access two-bottle choice and operant drinking sessions. Rats were trained to self-administer either 10% (w/v) alcohol solution sweetened with 'supersac' (3% glucose+0.125% saccharin) or supersac alone versus water in a two-bottle choice or operant situation during 30-min daily sessions. Rats were then injected systemically with multiple doses of duloxetine, naltrexone, and the corticotropin-releasing factor antagonist, MPZP, in Latin-square designs. Alcohol binge drinkers reliably consumed amounts of alcohol sufficient to produce BALs >or=0.08 g%. Duloxetine dose-dependently suppressed two-bottle choice alcohol binge drinking and operant alcohol responding as well as operant supersac responding, but did not affect two-bottle choice supersac drinking. Naltrexone-suppressed alcohol binge drinking at very low doses and suppressed supersac drinking at moderate-to-high doses. MPZP did not affect alcohol or supersac consumption. Different profiles for drugs that suppress binge-like alcohol drinking compared with dependence-induced drinking provide a heuristic foundation for future medications development.

Herbal Mixtures Consisting of Puerarin and Either Polyenylphosphatidylcholine or Curcumin Provide Comprehensive Protection Against Alcohol-Related Disorders in P Rats Receiving Free Choice Water and 15% Ethanol in Pure Water

Chronic alcohol drinking has been associated with the development of a number of abnormalities, including neuron-behavioral disorders, liver, pancreas, and heart-related diseases and inflammation and immune disorders. Because diverse mechanisms are involved in the development of these disorders, the commonly used receptor- or enzyme-specific drugs do not provide comprehensive protection against the adverse effects of alcoholism. This study describes possible therapeutic potency of puerarin (PU) from kudzu root, polyenylphosphatidylcholine from soy (SPCh), and curcumin (CU) from turmeric against alcohol's addiction-related and inflammatory-related abnormalities in alcohol-preferring P rats receiving free choice water and 15% ethanol in water. P-rats were fed once daily either the vehicle (for control) or different doses of PU, SPCh, CU, PU + SPCh, or PU + CU. The rats were divided in two groups: one received water alone, and the other free choice water and ethanol. Four rats from each group were fitted with electroencephalogram (EEG) electrodes for EEG recording. After 70 days of alcohol drinking, alcohol was withdrawn for 2 weeks, and the withdrawal symptoms were assessed. This study showed that alcohol drinking for 70 days (1) caused liver inflammation characterized by elevated tumor necrosis factor-alpha, interleukin-1beta, and matrix metalloproteinase-9 expression and (2) dysregulated lipopolysaccharide (LPS)-induced pleurisy. Alcohol withdrawal after 70 days of drinking generated severe withdrawal symptoms including seizure-type EEG activity. PU suppressed the addiction-mediated abnormalities but did not affect the inflammation-related abnormalities, while SPCh or CU suppressed only the inflammation-related abnormalities in alcohol-drinking rats subjected to LPS-induced pleurisy. A combination of PU with SPCh or CU suppressed both the addiction-related and inflammation-related abnormalities of alcohol drinking. Therefore, a mixture consisting of PU and either SPCh or CU may provide alternative therapy for alcohol-related disorders.

Repeated Light–Dark Phase Shifts Modulate Voluntary Ethanol Intake in Male and Female High Alcohol‐Drinking (HAD1) Rats

Chronic disruption of sleep and other circadian biological rhythms, such as occurs in shift work or in frequent transmeridian travel, appears to represent a significant source of allostatic load, leading to the emergence of stress-related physical and psychological illness. Recent animal experiments have shown that these negative health effects may be effectively modeled by exposure to repeated phase shifts of the daily light-dark (LD) cycle. As chronobiological disturbances are thought to promote relapse in abstinent alcoholics, and may also be associated with increased risk of subsequent alcohol abuse in nonalcoholic populations, the present experiment was designed to examine the effects of repeated LD phase shifts on voluntary ethanol intake in rats. A selectively bred, high alcohol-drinking (HAD1) rat line was utilized to increase the likelihood of excessive alcoholic-like drinking. Male and female rats of the selectively bred HAD1 rat line were maintained individually under a LD 12:12 cycle with both ethanol (10% v/v) and water available continuously. Animals in the experimental group were subjected to repeated 6-hour LD phase advances at 3 to 4 week intervals, while control rats were maintained under a stable LD cycle throughout the study. Contact-sensing drinkometers were used to monitor circadian lick patterns, and ethanol and water intakes were recorded weekly. Control males showed progressively increasing ethanol intake and ethanol preference over the course of the study, but males exposed to chronic LD phase shifts exhibited gradual decreases in ethanol drinking. In contrast, control females displayed decreasing ethanol intake and ethanol preference over the course of the experiment, while females exposed to experimental LD phase shifts exhibited a slight increase in ethanol drinking. Chronic circadian desynchrony induced by repeated LD phase shifts resulted in sex-specific modulation of voluntary ethanol intake, reducing ethanol intake in males while slightly increasing intake in females. While partially contrary to initial predictions, these results are consistent with extensive prior research showing that chronic stress may either increase or decrease ethanol intake, depending on strain, sex, stressor type, and experimental history. Thus, repeated LD phase shifts may provide a novel chronobiological model for the analysis of stress effects on alcohol intake.

Alcohol Drinking in MCH Receptor‐1‐Deficient Mice

Recently, we demonstrated that exogenous melanin-concentrating hormone (MCH) increases alcohol drinking in rats when administered into the brain. However, because the physiological relevance of this finding is unclear, we tested the hypothesis that endogenous MCH signaling enhances alcohol consumption. Alcohol intake was assessed in male and female wildtype (WT), heterozygous (HET), and homozygous MCH receptor-1-deficient (KO) mice. Mice were given 24-hour access to a series of alcohol-containing solutions. Following this, the mice were given limited (1-hour) access to 10% alcohol. Finally, mice were allowed 24-hour access to sucrose/quinine as a caloric control and a means to assess taste preference. A naïve cohort of male WT and KO mice was tested for alcohol clearance following intraperitoneal administration of 3 g/kg alcohol. Another naïve cohort of female mice was utilized to confirm that intracerebroventricular administration of MCH (5 microg) would augment alcohol drinking in mice. Exogenous MCH enhanced 10% alcohol consumption in mice (saline=0.45+/-0.08 g/kg, 5 microg MCH=0.94+/-0.20 g/kg). Male KO mice consumed more 10% alcohol (11.50+/-1.31 g/kg) than WT (6.26+/-1.23 g/kg) and HET mice (6.49+/-1.23 g/kg) during ad libitum access. However, alcohol intake was similar among genotypes during 1 hour daily access. Male KO mice tended to consume less 17.75% sucrose+1.3 mM quinine than controls (WT=10.5+/-3.6, HET=7.5+/-1.7, KO=4.4+/-0.9 g/kg). Alcohol metabolism was similar between WT and KO mice. The finding that male KO consume more alcohol than WT and HET mice, are reminiscent of the counterintuitive reports that KO mice are hyperphagic and yet eat more when administered exogenous MCH. Changes in taste preference or alcohol metabolism do not appear to be important for the increased alcohol drinking in KO mice.

Intrahippocampal allopregnanolone decreases voluntary chronic alcohol consumption in non-selected rats

We have recently shown that 0.2 μg of the neurosteroid allopregnanolone (AlloP) administered to the hippocampus induced an anxiolytic-like profile and also reduced alcohol withdrawal symptoms in voluntary and chronic alcohol-drinking rats. The aim of the present work was to study whether the administration of this dose of AlloP could affect alcohol consumption in non-selected rats that have been voluntarily ingesting high doses of alcohol for long periods of time in a limited access procedure. We used a free-choice drinking procedure that involved providing the rats with an alcoholic solution (10% ethanol) at an early age. Alcohol and control rats were assigned randomly to three groups that received an intrahippocampal (dorsal CA1) injection before the period of alcohol consumption after a long history of chronic alcohol intake. The injection groups were AlloP (0.2 μg, 1.26 μM), pregnenolone sulfate (PregS) (5 ng, 24 μM) or vehicle. Blood alcohol concentrations (BAC) were assessed before testing the effects of injections on alcohol consumption. Although AlloP did not eliminate alcohol ingestion, it significantly decreased alcohol consumption. The intrahippocampal administration of PregS, at the dose tested, did not effectively modify alcohol consumption levels. These results indicate that the positive modulation of hippocampal GABA A receptors induced by neurosteroids can be an important neurobiological target for reducing chronic alcohol consumption.

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.

NITROUS OXIDE ACUTELY SUPPRESSES ETHANOL CONSUMPTION IN HAD AND P RATS

Nitrous oxide (N2O) has been reported to reduce post-withdrawal craving in alcoholic humans, aiding in their continued abstinence. This article assessed the ability of N2O to suppress alcohol drinking in genetically selected high alcohol-drinking (HAD) and alcohol-preferring (P) rats. Although water was available ad libitum, they were accustomed to a limited access (1 h/day) choice between 10% ethanol (EtOH) and water. When drinking stabilized, the rats were exposed for 30, 60, or 120 min to a mixture of N2O and pure oxygen, timed to end 1 h before the limited access. N2O suppressed EtOH consumption at 1, but not 25 h after exposure in both HAD and P rats. This result is consistent with the efficacy of N2O for acute reduction of EtOH drinking, and supports further research into the use of N2O as an adjunct to treatment.

Effects of Intrahippocampal Nicotine and Neurosteroid Administration on Withdrawal in Voluntary and Chronic Alcohol-Drinking Rats

Previous studies have shown that 4.6 mug of nicotine administered to the hippocampus can deteriorate learning acquisition in alcohol-drinking rats. The aim of the present study was to research whether this nicotine dose can alter the alcohol withdrawal syndrome and whether the two neurosteroids, allopregnanolone (AlloP) and pregnenolone sulfate (PregS), at doses previously reported as anxiolytic and promnesic, respectively, can modulate these effects. We used a free-choice drinking procedure that involved providing the rats with an alcoholic solution (10% ethanol) at an early age. Alcohol and control rats were assigned randomly to six groups that received two consecutive intrahippocampal (dorsal CA1) injections once per week during three consecutive weeks after one hour of ethanol drinking. The first injection was nicotine (4.6 microg, 20 mM) or saline and the second injection was PregS (5 ng, 24 microM), AlloP (0.2 microg, 1.26 microM) or saline. Blood alcohol concentrations were assessed one week before the withdrawal testing. Locomotor activity and audiogenic seizures were tested during withdrawal after 110 days of voluntary ethanol consumption. Rats were injected immediately before the withdrawal testing. AlloP induced a decrease in horizontal and vertical activities, suggesting that the dose tested has sedative effects. AlloP reversed the seizures induced by ethanol withdrawal and also the spontaneous audiogenic seizures induced by the acoustic stimulation in control rats. Moreover, AlloP decreased other alcohol withdrawal signs, such as tail stiffening and body rigidity. Intrahippocampal administration of nicotine or PregS, at the doses tested, did not effectively modify the expression of audiogenic seizures induced by alcohol withdrawal. These results show that hippocampal GABAergic activity and AlloP have an important role in preventing convulsive behavior. The results also highlight the therapeutic potential of AlloP for reducing the alcohol withdrawal syndrome.

Saccharin consumption and the effect of a long-term exposure to a sweetened alcoholic solution in high- (UChB) and low- (UChA) alcohol-drinking rats

An association between saccharin consumption and alcohol intake has been observed in rodent lines genetically developed for alcohol preference or alcohol avoidance. It has also been proposed that a sweetened alcohol solution can condition rats to consume high amounts of alcohol. This work had two aims. First, to study the relationship between saccharin and alcohol intake in both high-alcohol-drinking UChB rats and low-alcohol-drinking UChA rats and, second, to determine whether a long-term exposure to a sweetened alcohol solution can increase their voluntary alcohol consumption. For the first purpose, UChB and UChA rats were tested under a free-choice paradigm between two graduated bottles, one containing a saccharin solution (0.1, 0.2, or 0.4% [wt/vol]) and the other water. For the second purpose, UChB and UChA rats that were under free choice between 10% alcohol and water, were offered a 10% alcohol solution containing 0.2% saccharin, instead of 10% alcohol for 1 month and were then returned to free choice between 10% alcohol and water. The first experiment showed that both lines have a high preference for saccharin at any concentration, but UChB rats drank twice as much saccharin solution as UChA rats and consequently they increased significantly their total daily fluid intake. This fact has been suggested to be an animal analogue of the clinical phenomenon known as “loss of control.” In the second experiment a long-term exposure to a 10% alcohol solution containing 0.2% saccharin induced a significant increase in alcohol consumption in UChB rats once saccharin was faded out, whereas the alcohol consumption in UChA rats returned to the previous low value. This result indicates that UChA rats have a genetic predisposition to avoid alcohol. In conclusion, the results reported here for UChB and UChA rats show an association between saccharin and alcohol preference, and suggest that their different genotypes are probably involved in alcohol aversion.

Intrahippocampal nicotine and neurosteroids effects on the anxiety-like behaviour in voluntary and chronic alcohol-drinking rats

Considerable evidence suggests that the anxiolytic effects of ethanol may be one of the factors that promotes alcohol consumption. The present study aimed to characterize the effects of intrahippocampal administrations of nicotine and the two neurosteroids pregnenolone sulphate (PregS) and allopregnanolone (AlloP) on anxiety-like behaviours in alcohol-drinking rats. A long-lasting free-choice drinking procedure with an early availability (from weaning) of an alcoholic solution (10% (v/v) ethanol, 3% (w/v) glucose in distilled water) was used. After 80 days of consumption, alcohol-drinking and control rats were deprived of food and assigned at random to six groups. After 100 days of consumption, each group received two consecutive intrahippocampal (dorsal CA1) injections. First injection: nicotine (4.6 μg, 20 mM) or saline; second injection: PregS (5 ng, 24 μM), AlloP (0.2 μg, 1.26 μM) or saline. Following the injections, novelty-directed activity (open field, OF), and motor coordination (80° inclined screen) were tested. Blood alcohol concentrations (BACs) were assessed. Anxiolytic-like effects of voluntary ethanol consumption and intrahippocampal AlloP administration were observed. Alcohol intake increased the novelty-induced ambulation and exploration of central areas, and decreased defecation. The high exploration levels induced by AlloP decreased significantly over sessions, indicating a rapid habituation to the environmental conditions. Motor coordination was deteriorated by ethanol consumption. These results demonstrate the effects of chronic alcohol intake and neurosteroid administration on anxiety-related behaviours, and suggest an important role of the hippocampal GABA A receptor in these behaviours.

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.

An Extract of the Chinese Herbal Root Kudzu Reduces Alcohol Drinking by Heavy Drinkers in a Naturalistic Setting

Of the available medications for treating alcohol-related problems, none are universally effective, and all have side effects that may limit their use. Extracts of kudzu containing a variety of isoflavones have been shown to reduce alcohol drinking in rats and hamsters. The present study was designed to test the efficacy of a kudzu extract in a clinical population. Male and female "heavy" alcohol drinkers were treated with either placebo or a kudzu extract for 7 days and then given an opportunity to drink their preferred brand of beer while in a naturalistic laboratory setting. Participants served as their own controls, and order of treatment exposure was counterbalanced. Drinking behavior was monitored by a digital scale that was located in the top of an end table. Kudzu treatment resulted in significant reduction in the number of beers consumed that was paralleled by an increase in the number of sips and the time to consume each beer and a decrease in the volume of each sip. These changes occurred in the absence of a significant effect on the urge to drink alcohol. There were no reported side effects of kudzu treatment. These data suggest that an extract of this leguminous plant may be a useful adjunct in reducing alcohol intake in a naturalistic setting.

The neurosteroid pregnenolone sulfate neutralized the learning impairment induced by intrahippocampal nicotine in alcohol-drinking rats

The effects of intrahippocampal administration of nicotine and the neurosteroids pregnenolone sulfate and allopregnanolone on acquiring the lever-press response and extinction in a Skinner box were examined using voluntary alcohol-drinking rats. A free-choice drinking procedure that implies early availability of the alcoholic solution (10% ethanol v/v+3% glucose w/v in distilled water) was used. Alcohol and control rats were deprived of food and assigned at random to six groups. Each group received two consecutive intrahippocampal (dorsal CA1) injections immediately after 1-h of drinking ethanol and before the free lever-press response shaping and extinction session. The groups were: saline–saline; saline–pregnenolone sulfate (5 ng, 24μM); saline–allopregnanolone (0.2μg, 1.26μM); nicotine (4.6μg, 20mM)–saline; nicotine–pregnenolone sulfate; nicotine–allopregnanolone. Blood alcohol concentrations were assessed the day before conditioning. The combination of the oral self-administration of ethanol and the intrahippocampal injection of nicotine deteriorated the ability to acquire the lever-press response. This effect was neutralized by intrahippocampal pregnenolone sulfate (negative modulator of the GABA A receptor complex), and it was not affected by intrahippocampal allopregnanolone (positive GABA receptor complex A modulator). Pregnenolone sulfate and allopregnanolone had no effects per se on lever-press acquisition, neither in alcohol-drinking rats nor in controls. Alcohol consumption facilitated operant extinction just as anxiolytics that act as positive modulators of the GABA receptor complex A receptors do, possibly reducing the anxiety or aversion related to non-reinforcement. This effect was increased by intrahippocampal nicotine.

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.

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.

Corticotropin-releasing factor gene expression is down-regulated in the central nucleus of the amygdala of alcohol-preferring rats which exhibit high anxiety: a comparison between rat lines selectively bred for high and low alcohol preference

The role of amygdaloid corticotropin-releasing factor (CRF) in alcoholism is not clear. Alcohol-preferring (P) rats and high alcohol-drinking (HAD) rats are selectively bred for high alcohol preference, and have been considered suitable animal models for studying alcoholism. The CRF neurons in the central nucleus of the amygdala (CeA) of P rats and HAD rats were studied in comparison with those of their respective counterparts, namely, alcohol-nonpreferring (NP) rats and low alcohol-drinking (LAD) rats. Specifically, CRF-immunoreactivity (ir) in the CeA and paraventricular hypothalamic nucleus (PVN) was assessed using radioimmunohistochemical (RIH) assay in alcohol-naive P/NP rats, and HAD/LAD rats. Furthermore, CRF mRNA was examined using in situ hybridization in the CeA of P/NP rats. Anxiety levels were also evaluated using an elevated plus maze. Results of the present study showed that CRF-ir was significantly lower in the CeA of P rats than NP rats. Moreover, CRF mRNA in the CeA was also much lower in P rats than NP rats. Such differences were not seen in the PVN. Interestingly, those P rats exhibited higher anxiety than NP rats. In contrary, there were no innate differences of CRF-ir in both the CeA and PVN between HAD and LAD rats whose anxiety levels were similar. This study is consistent with the literature showing CRF knockout (KO) induces alcohol drinking, and central administrations of CRF reduce alcohol intake. Collectively, the present study suggests that reduced CRF gene expression in the CeA of P rats is associated with their alcohol preference and anxiety.

Differential activation of NFκB/RelA‐p50 and NFκB/p50‐p50 in control and alcohol‐drinking rats subjected to carrageenin‐induced pleurisy

BACKGROUND: Carrageenin (CAR) injection into the pleural cavity causes local inflammation called carrageenin-induced pleurisy (CAR-IP). Inflammation onset is characterized by an activation of pro-inflammatory NFkappaB, RelA-p50, while inflammation resolution is characterized by an activation of an anti-inflammatory NFkappaB, p50-p50, that re-establishes homeostasis, an essential process for an organism's survival. Although chronic alcohol intake disrupts inflammation, the mechanism behind the development of inflammatory disorder in alcoholics is not yet known. Therefore, the aim of this investigation was to study the effects of ethanol intake on CAR-IP and NFkappaB activation in pleural fluid neutrophils in P rats. METHODS: Alcohol-preferring, P rats were given free choice of alcohol (15% ethanol) and water or water alone (for control) for 15 days. Then, each rat was injected with 0.2 ml of 2% CAR into the pleural cavity under light ether anesthesia. At different time intervals after the CAR injection, rats were anesthetized and their blood and pleural fluid samples were collected. Pleural fluid inflammatory cells were identified with Turk's or Wright-Giemsa staining. Different cell types were sorted using a fluorescence-activated cell sorter. Pleural fluid neutrophils were examined for apoptosis and activation of the two NFkappaB subspecies. RESULTS: In control rats, fluid began to accumulate in the pleural cavity 0.5 h after, which peaked 24 h after, CAR injection. Then, the values declined gradually. The increase in pleural fluid correlated with RelA-p50 activation, while the decline in pleural fluid correlated with p50-p50 activation and apoptosis in neutrophils. In alcohol-drinking rats, pleural fluid remained elevated for up to 6 days after CAR injection. Neutrophils from alcohol-drinking rats exhibited suppressed apoptosis, augmented RelA-p50 activation, and suppressed p50-p50 activation. CONCLUSIONS: Alcohol intake prolonged inflammation in P rats. An alcohol-induced upregulation of RelA-p50 activation and downregulation of p50-p50 activation may be causally related to the alcohol-induced inflammation dysregulation.