Motivational effects of ethanol in DARPP-32 knock-out mice.

DARPP-32 (dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa) is an important component of dopaminergic function in brain areas thought to be important for drug and alcohol addiction. The present experiments characterized the acquisition of ethanol-induced conditioned taste aversion, ethanol-induced conditioned place preference, and ethanol self-administration in DARPP-32 knock-out (KO) mice compared to wild-type (WT) controls. For taste conditioning, KO and WT mice received access to 0.2 m NaCl solution followed immediately by intraperitoneal injection of 0-4 gm/kg ethanol. Ethanol produced dose-dependent conditioned taste aversion that was the same in both genotypes. For place conditioning, KO and WT mice received eight pairings of a tactile stimulus with ethanol (2 gm/kg, i.p.), and a different stimulus with saline. Ethanol produced increases in locomotor activity during conditioning, with KO mice showing higher activity levels after ethanol compared to WT mice. WT mice, but not KO mice, acquired conditioned preference for the ethanol-paired stimulus. In the self-administration procedure, KO and WT mice were trained to lever press for access to 10% v/v ethanol. Subsequently, the mice had 23 hr/d access to food, ethanol, and water. Response patterns were determined using 0-30% v/v ethanol concentrations. WT mice displayed concentration-dependent responding for ethanol. Responding on the ethanol lever by KO mice did not change as a function of ethanol concentration. Saccharin (0.2% w/v) was subsequently added to the ethanol mixture, and responding was examined at 0, 5, 10, and 20% ethanol concentrations. Ethanol responding increased in both genotypes, although WT mice showed higher rates at all concentrations.

Attenuation of ethanol-induced conditioned taste aversion in mice sensitized to the locomotor stimulant effects of ethanol.

This study examined the effect of repeated ethanol (EtOH) injections that induced behavioral sensitization on subsequent acquisition of EtOH- and lithium chloride (LiCl)-induced conditioned taste aversion (CTA). CTA acquisition was assessed in independent groups of EtOH-sensitized and nonsensitized genetically heterogeneous female mice after injections of saline; 1, 2, or 4 g/kg EtOH; or 2 or 4 mEq/kg LiCl. Saline and 1 g/kg EtOH did not induce CTA. Four g/kg EtOH and 4 mEq/kg LiCl induced similar levels of CTA in EtOH-sensitized and nonsensitized groups. CTA induced by 2 g/kg EtOH and 2 mEq/kg LiCl was attenuated in EtOH-sensitized mice compared with nonsensitized counterparts. Thus, a sensitizing regimen of EtOH preexposure resulted in both a decrease in EtOH and LiCl aversion and an increase in EtOH locomotor sensitivity; such changes could ultimately contribute to enhanced EtOH intake and potentially to EtOH abuse.

Haloperidol reduces ethanol-induced motor activity stimulation but not conditioned place preference.

This experiment examined the impact of a dopamine receptor blocker on ethanol's rewarding effect in a place conditioning paradigm. DBA/2J mice received four pairings of a tactile stimulus with ethanol (2 g/kg, IP), haloperidol (0.1 mg/kg, IP)+ethanol, or haloperidol alone. A different stimulus was paired with saline. Ethanol produced increases in locomotor activity that were reduced by haloperidol. However, conditioned preference for the ethanol-paired stimulus was not affected by haloperidol. Haloperidol alone decreased locomotor activity during conditioning and produced a place aversion. These results indicate a dissociation of ethanol's activating and rewarding effects. Moreover, they suggest that ethanol's ability to induce conditioned place preference is mediated by nondopaminergic mechanisms.

Context-drug pairings enhance tolerance to ethanol-induced disruption of operant responding.

Much of the research implicating learning in the development of tolerance to ethanol-induced impairment has used an experimental design in which different groups receive drug either before or after an opportunity to perform an instrumental or operant task. The stronger tolerance observed in subjects who perform while intoxicated is most often attributed to the reinforced practice of a learned compensatory response. Using an experimental procedure modeled after Chen (1979), the present study examined an alternative theoretical basis for tolerance in the before-versus-after design. Specifically, the effects of Pavlovian context-drug pairings were assessed under circumstances that precluded reinforced practice of the operant response. Three groups of food-deprived rats were initially trained to barpress for sucrose on an FR15 schedule. After 30 sessions, the bar was retracted and the dipper was covered for a 3-day tolerance acquisition phase. During this phase, each group received an IP injection 15 min before and 45 min after each session. The Paired group received ethanol (1.2 g/kg) before and saline after the session, thus pairing ethanol with cues of the test chamber. The Unpaired group received saline before and ethanol after the session, while the No-Drug group always received saline. During a final test phase, all groups received ethanol (1.5 g/kg) before access to sucrose on the FR schedule. The Paired group completed the first FR15 sequence more rapidly than either control group, indicating that context-ethanol pairings enhanced tolerance to the drug's disruptive effect on the initiation of operant responding.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of flurazepam convulsive threshold by Ro 15-1788.

The influence of the benzodiazepine (BZ) receptor antagonist Ro 15-1788 on the convulsant properties of flurazepam (FLZ) was studied in rats. Animals were prepared with chronic epidural electrodes for EEG recording and respiratory rates were recorded via a rubber bulb connected to a pressure transducer. FLZ convulsive thresholds were determined by continuous IV infusion in the presence and absence of Ro 15-1788 pretreatment. All animals receiving the FLZ infusion experienced convulsions preceded by dose-dependent reduction of respiratory rate. Pretreatment with Ro 15-1788 substantially reduced the FLZ convulsive threshold, suggesting a blockade of the depressant (anticonvulsant) aspect of the FLZ effect, thus, augmenting a convulsant effect at a separate receptor. An alternate hypothesis is that Ro 15-1788 may increase the affinity or intrinsic activity of the receptor responsible for the convulsant aspect of FLZ. Ro 15-1788 did not appear to alter the respiratory rate depressant effect of FLZ, although early onset of convulsions in the pretreated animals precluded measurement of respiration at the higher dose of FLZ allowable in animals that were not pretreated with the antagonist.

Lack of operant ethanol self-administration in dopamine D2 receptor knockout mice.

RATIONALE: Dopamine D2 receptors are postulated to play an important role in modulating the reinforcing effects of abused drugs including ethanol. OBJECTIVES: This experiment examined operant ethanol self-administration in dopamine D2 receptor knockout (KO) mice and wild-type (WT) mice using a continuous access procedure. METHODS: Adult male KO and WT mice were trained in 30-min sessions to perform a lever press response for access to 10% v/v ethanol. After training, the mice were placed in test chambers on a continuous (23 h/day) basis with access to food (one lever press, i.e., FR1), 10% v/v ethanol (four lever presses, i.e., FR4), and water from a sipper tube (phase 1). After 30 consecutive sessions, response patterns were determined for 0, 5, 10, 20 and 30% v/v ethanol (phase 2). Saccharin (0.2% w/v) was subsequently added to the ethanol mixture and responding was examined for 0, 5, 10 and 20% ethanol (phase 3). RESULTS: During phase 1, WT mice displayed higher ethanol-lever responding compared to KO mice. Food lever responding and water intake was the same in both genotypes. During phase 2, WT mice displayed concentration-dependent ethanol lever responding, whereas KO mice responded at low rates regardless of ethanol concentration. WT mice also responded more for food compared to KO mice. Each genotype showed similar water intakes except at the 20% ethanol concentration, where WT mice had lower intakes. During phase 3, WT mice continued to show higher responding for all concentrations including saccharin alone. WT mice also continued to respond more for food compared to KO mice, but drank less water. In each phase, WT mice displayed episodic (bout) responding on the ethanol lever. KO mice did not respond for ethanol in bouts. CONCLUSIONS: Reduced responding in the KO mice for several reinforcers including ethanol indicates a more general role for dopamine D2 receptors in motivated responding rather than a specific role in ethanol reinforcement.

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 ethanol's 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 flavor cues, and 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 ethanol's thermal effect and its effect on activity, place conditioning and taste conditioning. Evidence of a genetic correlation between ethanol's thermal effect and ethanol drinking, however, is weaker since it is based on a line difference observed in only one of the genetic replicates. (ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Enhancement of ethanol reward by dopamine D3 receptor blockade.

This experiment examined the influence of U-99194A, a dopamine D3 receptor antagonist, on ethanol's rewarding effect in a place conditioning paradigm. Swiss-Webster mice received six pairings of a tactile stimulus with ethanol (2 g/kg, i.p.), U-99194A (20 mg/kg, i. p.) with ethanol, or U-99194A alone. A different stimulus was paired with saline. During conditioning, ethanol or ethanol/U-99194A produced similar increases in locomotor activity. U-99194A alone produced modest increases in activity on some trials. As expected, the 2 g/kg ethanol dose produced a nonsignificant trend towards conditioned place preference. However, U-99194A enhanced the acquisition of ethanol preference, whereas U-99194A alone did not produce either place preference or aversion. The results are consistent with the notion that dopamine D3 systems are important in the response to ethanol and further suggest that D3 receptor blockade increases ethanol reward.

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.

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 alcohol's 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.

Mianserin enhancement of ethanol-induced conditioned place preference.

This experiment examined the influence of mianserin, a 5-HT(2) receptor antagonist, on the rewarding effect of ethanol in a place conditioning paradigm. Swiss-Webster mice received four pairings of a tactile stimulus with drug treatment consisting of two i.p. injections separated by a 30min interval. Drug treatment groups were as follows: saline (10mg/kg) followed by ethanol (2mg/kg); mianserin (10mg/kg) followed by ethanol; mianserin followed by saline. A different stimulus was paired with two saline injections. During conditioning, ethanol produced increases in locomotor activity that were reduced by mianserin. Mianserin alone reduced activity levels. As expected, group saline-ethanol showed a nonsignificant trend towards conditioned place preference. However, mianserin enhanced the acquisition of ethanol preference, whereas mianserin alone did not produce either place preference or aversion. The results are consistent with the notion that serotonergic systems are important in the response to ethanol, and further suggest that 5-HT(2) receptor blockade increases the rewarding effects of ethanol.

Fluoxetine's effects on ethanol's rewarding, aversive and stimulus properties.

These experiments examined the influence of fluoxetine on ethanol-induced conditioned place preference, ethanol-induced conditioned taste aversion, and ethanol discrimination. In the place conditioning experiment, male Swiss-Webster mice received 4 pairings of a distinctive floor cue with 2 g/kg ethanol, 10 mg/kg fluoxetine + ethanol, or fluoxetine alone. A different floor was paired with saline. During conditioning ethanol produced locomotor stimulation. Fluoxetine + ethanol resulted in greater levels of locomotor activity during conditioning trials 2-4. Fluoxetine alone also caused increases in activity. Floor preference testing revealed conditioned place preference in groups receiving ethanol. Fluoxetine did not change the magnitude of ethanol-induced conditioned place preference nor produced place conditioning alone. In the taste conditioning procedure, mice received 1-h access to 0.2 M NaCl solution followed by injections of 0, 5 or 10 mg/kg fluoxetine and 0 or 2.5 g/kg ethanol. Ethanol produced reductions in NaCl intake. Fluoxetine (10 mg/kg) enhanced the development of ethanol-conditioned taste aversion but did not cause taste aversion alone. In the ethanol discrimination experiment, mice were trained to respond for 10% sucrose on an FR20 schedule following injections of either 1 g/kg ethanol or saline. Following acquisition, 10 mg/kg fluoxetine pretreatment enhanced ethanol-appropriate responding at a dose of ethanol (0.5 g/kg) below the training dose. These results indicate enhancement of serotonergic activity influences ethanol aversion and discrimination but not ethanol reward.

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.

Genetic differences in nicotine-induced conditioned taste aversion.

Genetic differences in nicotine-induced conditioned taste aversion were examined using inbred mice. Adult male C57BL/6J, DBA/2J, BALB/cJ and C3H/heJ mice were adapted to a 2-h per day water access regimen. Subsequently, mice received nicotine injections (0.5, 1.0 or 2.0 mg/kg) immediately after 1-h access to a NaCl flavored solution. DBA and C3H mice developed dose-dependent aversions to the nicotine-paired flavor. BALB mice showed only minor reductions in intake with no difference between the nicotine dose groups. C57BL mice did not show development of nicotine-induced conditioned taste aversion. These results demonstrate that nicotine's aversion motivational effect is strongly influenced by genotype. Further, genetic sensitivity (DBA mice) or insensitivity (C57BL mice) to nicotine-induced conditioned taste aversion was similar to reports of genetic sensitivity to ethanol's aversive effect measured in this design.

[14C]normacromerine fate in the rat.

The biological fate of [14C] normacromerine , a dimethoxylated phenethylamine derivative with putative hallucinogenic properties, was evaluated in male Sprague-Dawley rats at 100 mg/kg po. Urine was the primary elimination route accounting for 50% of administered carbon-14 after 24 h. Of this urine radioactivity, normacromerine comprised 30% at 8 h decreasing to nondetectable levels at 24h. Carbon-14 in feces represented less than 10% of the administered dose at 24 h, and 14CO2 expiration was not detected. Studies of normacromerine fate in comparison with previously studied phenethylamines may enhance evaluation of hallucinogenic potential of normacromerine .

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 ethanol's 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.

DBA/2J mice develop stronger lithium chloride-induced conditioned taste and place aversions than C57BL/6J mice.

Genetic differences in lithium-induced conditioned aversion were examined using both place- and taste-conditioning procedures. In the place-conditioning procedure, adult male C57BL/6J (B6) and DBA/2J (D2) mice were exposed to a differential conditioning procedure in which each mouse received four 30-min pairings of a distinctive floor cue immediately after IP injections of either 0.75, 1.5, or 3. 0 mEq/kg LiCl. A different floor cue was paired with saline injections. A separate group of control mice received saline injections paired with both floor types. Subsequent floor preference testing revealed greater conditioned aversion in D2 mice compared to B6 mice in groups receiving 3.0 mEq/kg LiCl. Lower LiCl doses did not produce conditioning in either strain. In a conditioned taste-aversion procedure, fluid-restricted mice received four trials in which access to 0.2 M NaCl solution was followed by IP injection of either 0.75, 1.5, 3.0, or 6.0 mEq/kg LiCl. D2 mice showed stronger conditioned taste aversion than B6 mice at all doses, suggesting that taste conditioning may be a more sensitive index of aversive drug sensitivity than place conditioning. These findings are not well explained by strain differences in general learning ability or by strain differences in stimulus salience or innate preference. Rather, these data appear more consistent with previous studies showing strain differences in lithium pharmacokinetics and in general sensitivity to aversive events.