Fred O. Risinger

Alcohol drinking produces brain region-selective changes in expression of inducible transcription factors.

Mapping the effects of alcohol consumption on neural activity could provide valuable information on mechanisms of alcohols effects on behavior. The present study sought to identify effects of alcohol consumption on expression of inducible transcription factors (ITFs) in mouse brain. C57BL/6J mice were trained to consume 10% ethanol/10% sucrose solution during a 30-min limited access period. Control animals were given access to 10% sucrose solution or water. Following the final day of the procedure, animals were sacrificed and immunohistochemical analyses were performed for three ITFs (c-Fos, FosB, and Zif268). Alcohol-consuming animals had increased ITF expression in several brain areas. Specifically, c-Fos was significantly induced in the nucleus accumbens core (AcbC), the medial posteroventral portion of the central nucleus of the amygdala (CeMPV), and the Edinger-Westphal nucleus (EW). Expression of c-Fos was significantly lower in the dentate gyrus of alcohol-consuming animals vs. sucrose-consuming animals. However, it was not significantly different from the water controls. Induction of c-Fos in AcbC, CeMPV and EW was significantly related to blood alcohol concentrations (BAC). Furthermore, FosB expression in the CeMPV and the EW was also significantly higher in the alcohol-consuming animals vs. water controls. FosB expression in the EW was significantly related to BAC. The significance of these results is two-fold. First, our experiments demonstrate that ITF mapping is an effective strategy in identifying alcohol-induced changes following voluntary consumption. Second, they suggest a relationship between ITF expression in AcbC, CeMPV and EW and the level of alcohol intoxication.

Nicotine-induced conditioned place preference and conditioned place aversion in mice.

The motivational effects of nicotine were examined in mice using an unbiased place conditioning design. Swiss-Webster mice received four 15-min parings of a tactile stimulus with different doses of nicotine (0.25-2.0 mg/kg, IP). A different tactile stimulus was paired with saline injections. During conditioning, nicotine produced locomotor depression at the 2.0-mg/kg dose, with the greatest reduction in activity occurring during the latter part of each nicotine conditioning session. After four trials, nicotine produced increases in locomotor activity during the initial part of the nicotine sessions at doses 0.5 mg/kg or above. Upon testing, nicotine-induced conditioned place preference was noted in mice receiving 0.5 mg/kg nicotine. Conditioned place aversion was noted in mice receiving 2.0 mg/kg nicotine whereas doses of 0.25 and 1.0 mg/kg produced no conditioning. These results indicate that nicotine has dose-dependent rewarding and aversive effects measured in an unbiased place conditioning paradigm using mice.

Motivational properties of ethanol in mice selectively bred for ethanol-induced locomotor differences

Ethanol-induced locomotor stimulation has been proposed to be positively correlated with the rewarding effects of ethanol (Wise and Bozarth 1987). The present experiments provided a test of this hypothesis using a genetic model. Three behavioral indices of the motivational effects of ethanol (drinking, taste conditioning, place conditioning) were examined in mice from two independent FAST lines, selectively bred for sensitivity to ethanol-induced locomotor stimulation, and mice from two independent SLOW lines, selectively bred for insensitivity to ethanol-induced locomotor stimulation. In a single-bottle procedure, mice were allowed access to drinking tubes containing ethanol in a concentration (1–12% v/v) that increased over 24 consecutive days. FAST mice consumed greater amounts of ethanol solution. In a two-bottle procedure, mice were allowed access to tubes containing water or various concentrations of ethanol (2–8% v/v) over 6 days. FAST mice generally showed greater preference for ethanol solutions than SLOW mice. In a conditioned taste aversion procedure, mice received access to saccharin solution followed by injection of 2.5 g/kg ethanol (IP). SLOW mice developed aversion to the saccharin flavor more readily than FAST mice. In a series of place conditioning experiments, tactile stimuli were paired with various doses of ethanol (0.8–2.0 g/kg). During conditioning, FAST mice showed locomotor stimulation after 1.0, 1.2 and 2.0 g/kg ethanol while SLOW mice did not. During testing, mice conditioned with 1.2 g/kg and 2.0 g/kg ethanol showed conditioned place preference, but there were no line differences in magnitude of preference. These results indicate that genetic selection for sensitivity to ethanol-stimulated activity has resulted in genetic differences in ethanol drinking and ethanol-induced conditioned taste aversion but not ethanol-induced conditioned place preference. Overall, these data provide mixed support for the psychomotor stimulant theory of addiction.

Assessment of ethanol's hedonic effects in mice selectively bred for sensitivity to ethanol-induced hypothermia.

Mice selectively bred for sensitivity (COLD) or insensitivity (HOT) to the hypothermic effect of ethanol were tested in three tasks purported to assess ethanols hedonic properties: place conditioning, taste conditioning, and ethanol drinking. In the place conditioning task, distinctive tactile (floor) stimuli were differentially paired with injection of ethanol (2.25 g/kg) or saline, and preference for the tactile stimuli was assessed during a choice test without ethanol. In the taste conditioning task, fluid-deprived mice were given repeated access to saccharin followed by injection of ethanol (2.25 g/kg). In the drinking task, mice were given access on alternate days to a single drinking tube containing water or ethanol in a concentration that gradually increased from 1 to 12% (v/v) over days. HOT mice showed greater conditioned preference for ethanol-paired tactile cues, greater aversion for ethanol-paired flavor cues, and drank less ethanol at concentrations above 5% than COLD mice. HOT mice also showed higher levels of ethanol-stimulated activity than COLD mice. Control experiments indicated that the lines did not differ in initial preference for the tactile and flavor stimuli used in the conditioning tasks. Because the same line differences were seen in mice selected from two genetically independent populations, these studies offer strong evidence of genetic correlations between ethanols thermal effect and its effect on activity, place conditioning and taste conditioning. Evidence of a genetic correlation between ethanols thermal effect and ethanol drinking, however, is weaker since it is based on a line difference observed in only one of the genetic replicates. In general, these findings suggest commonality in the biological mechanisms underlying ethanols thermal effect and its effect in each behavioral task. This overall pattern of genetic correlations might indicate that these tasks measure the same motivational effect of ethanol.

Dose- and conditioning trial-dependent ethanol-induced conditioned place preference in Swiss-Webster mice

The motivational effects of ethanol were examined in Swiss-Webster mice using an unbiased place conditioning, design. Adult male Swiss-Webster mice received six 5-min pairings of a tactile stimulus with different doses of ethanol (1, 2, 3, or 4 g/kg. IP). A different tactile stimulus was paired with saline injections. A 60-min preference test was given after the first four conditioning trials and an additional 30-min preference test after the sixth conditioning trial. During conditioning, ethanol initially produced locomotor stimulation at the 2 g/kg dose and locomotor depression at the 4 g/kg dose. However, after repeated ethanol exposure, all doses produced overall increases in activity relative to saline, suggesting sensitization to ethanols stimulant effect. After four conditioning trials ethanol-induced conditioned place preference was noted in mice receiving 3 and 4 g/kg ethanol. After two additional conditioning trials all ethanol doses produced conditioned place preference. These results indicate that ethanol has dose-dependent rewarding effects measured in an unbiased place-conditioning paradigm using a standard outbred mouse strain. Further, additional place-conditioning trials enhance the development of preference at lower (1 or 2 g/kg) ethanol doses.

Genetic differences in ethanol-induced conditioned taste aversion after ethanol preexposure

The present studies examined the development of ethanol-induced conditioned taste aversion in C57BL/6J (B6) and DBA/2J (D2) mice with a history of ethanol preexposure. In Experiment 1, adult male B6 and D2 mice received four preexposure injections of either saline or 4 g/kg ethanol over an 8-day period. After preexposure, all mice were given five conditioning trials consisting of 1-h access to 0.15% w/v saccharin solution followed immediately by ethanol injections (4 g/kg, IP) on all but the last trial. Drug-naive D2 mice showed greater reductions in saccharin intake. Ethanol preexposure reduced the development of ethanol-induced taste aversion in each strain. However, B6 mice showed little taste aversion overall, hindering the characterization of genetic differences in ethanols preexposure effect. To address this problem, the parameters for taste conditioning were changed in Experiment 2 to more closely match degree of taste aversion in drug-naive mice across both strains. B6 and D2 mice received four preexposure injections of either saline, 2 g/kg ethanol, or 4 g/kg ethanol, or 4 g/kg ethanol. Subsequently, mice received five conditioning trials consisting of 1-h access to 0.2 M NaCl flavor followed by 4 g/kg ethanol (B6 mice) or 2 g/kg ethanol (D2 mice) on trials 1-4. Ethanol-naive mice of each strain developed similar levels of conditioned taste aversion. Ethanol preexposure produced greater retardation of conditioned aversion in B6 mice than in D2 mice. These results demonstrate genetic differences in the ability of ethanol preexposure to reduce the development of ethanol-induced conditioned taste aversion.

Genetic differences in ethanol-induced hyperglycemia and conditioned taste aversion

Genetic differences in the hyperglycemic response to acute ethanol exposure and ethanol-induced conditioned taste aversion were examined using inbred mice. Adult male C57BL/6J and DBA/2J mice were injected with ethanol (0-6 g/kg, I.P.) and blood glucose levels determined over 4 h. C57 mice demonstrated greater dose-dependent elevations in blood glucose compared to DBA mice. In a conditioned taste aversion procedure, water deprived mice received ethanol injections (1-4 g/kg, I.P.) immediately after access to a NaCl flavored solution. DBA mice developed aversion to the ethanol-paired flavor at a lower dose (2 g/kg) than C57 mice. These results provide further support for a possible inverse genetic relationship between sensitivity to ethanol-induced hyperglycemia and sensitivity to conditioned taste aversion.

Haloperidol does not alter expression of ethanol-induced conditioned place preference

A recent experiment (Risinger et al., Psychopharmacology, 107 (1992) 453-456) has shown that haloperidol does not prevent acquisition of ethanol-induced conditioned place preference, suggesting that dopaminergic mechanisms do not mediate the primary rewarding properties of ethanol. The present experiment examined whether haloperidol would prevent the expression of conditioned reward to ethanol-paired stimuli using the place conditioning paradigm. DBA/2J mice received four pairings of a tactile stimulus with ethanol (2 g/kg, IP). A different stimulus was paired with saline. Before preference testing, different groups received one of three doses of haloperidol (0, 0.05 or 0.1 mg/kg); ethanol was not given. Haloperidol produced a dose-dependent decrease in locomotor activity, but did not affect conditioned place preference. These results suggest that expression of ethanol-induced conditioned place preference is mediated by non-dopaminergic mechanisms.

Oral operant ethanol self-administration in 5-HT1b knockout mice

The present experiment examined oral ethanol self-administration in 5-HT1b knockout (KO) mice and 5-HT1b wide-type (WT) control mice using a continuous access operant procedure. After lever press training, adult 5-HT1b KO and 5-HT1b WT mice were placed in operant chambers on a 23 h per day basis with access to food (FR1), 10% v/v ethanol (FR4), and water from a sipper tube. KO mice displayed higher rates of responding on the ethanol-associated lever compared to WT mice. KO mice also consumed greater amounts of water. Food responding was the same in both genotypes. Following 30 sessions, ethanol concentration was altered every 5 days. Response patterns were determined using 0, 5, and 20% v/v ethanol concentrations. Ethanol responding (0, 5, 10, and 20% v/v) was also examined after the addition of 0.15% saccharin. KO mice and WT mice showed similar response rates for all ethanol concentrations. Since KO mice showed greater levels of ethanol responding only for unsweetened 10% v/v ethanol, and showed modest ethanol self-administration overall, the present results are not consistent with the notion that 5-HT1b KO have a generally greater preference for ethanol than 5-HT1b WT mice.

ITF expression in mouse brain during acquisition of alcohol self-administration

Expression of inducible transcription factors (ITFs) c-Fos and FosB was investigated during acquisition of alcohol drinking in C57BL/6J mice. A slight but statistically significant increase in c-Fos expression was found in the Edinger-Westphal nucleus (EW) of animals consuming 2% ethanol/10% sucrose for the first time. Stronger expression of c-Fos in EW was found in animals repeatedly consuming ethanol-containing solutions. These findings underscore the potential importance of EW in alcohol-related behaviors.