Brain ethanol concentrations and ethanol discrimination in rats: effects of dose and time

Rationale In drug discrimination procedures, the substitution pattern for ethanol of various receptor ligands is dependent upon ethanol training dose, presumably reflecting functionally different concentrations of ethanol in the brain. The discriminative stimulus effects of ethanol are also time-dependent, although very few studies have investigated the time course of ethanol discriminations. Objectives The present study investigated the relationship between brain ethanol concentrations (BrEC), as measured by intracranial microdialysis of the nucleus accumbens, and the time course of ethanol discriminative effects. Methods Two groups of rats were trained to discriminate either 1.0 or 2.0 g/kg ethanol from water following a 30-min post-ethanol interval. Following training, the time course of the discriminative stimulus was assessed using a series of abbreviated testing trials at 20-min intervals for 5 h after the administration of various ethanol doses (0, 0.5, 1.0 and 2.0 g/kg). The rats were then fitted with microdialysis probes and the time course of BrECs were determined under conditions similar to the behavioral assessments. Results BrECs were significantly above zero at 4 min post-gavage and attained peak concentrations of 16 mmol/l, 24 mmol/l and 42 mmol/l at 9 min, 16 min and 95 min after IG administration of 0.5, 1.0 and 2.0 g/kg ethanol, respectively. BrECs were similar in ethanol-naive and ethanol-trained rats, indicating a lack of pharmacokinetic tolerance under these discrimination procedures. The discriminative stimulus effects of ethanol were dose- and time-dependent, with a threshold concentration of approximately 12 mmol/l achieved at 5 min after 1.0 g/kg ethanol gavage in rats trained to discriminate 1.0 g/kg ethanol. Acute tolerance to the discriminative stimulus effects of ethanol was evident from BrECs 2–5 h post-ethanol gavage. Conclusions Ethanol given intragastrically results in a rapid increase in BrEC, independent of ethanol exposure history. The discriminative stimulus effects of ethanol trained at 30 min post-gavage reflect a specific range of BrEC, and depend on the training dose. These data suggest that qualitatively different stimulus effects of ethanol reflect both different ranges of BrEC, as well as within dose acute tolerance to the discriminative stimulus effects.     

Environmental modification of tolerance to morphine discriminative stimulus properties in rats

The development of tolerance to the discriminative stimulus properties of morphine was examined in rats trained to discriminate saline and 3.2 mg/kg morphine under amultiple timeout 15 min, 5 min fixed-ratio 30 schedule of food delivery. Generalization gradients were generated by administering increasing doses of morphine before successive timeout periods within the experimental session. Over the course of the study, the minimal discriminable dose (MDD) of morphine under control conditions fluctuated but did not systematically increase or decrease. Acute pretreatments of 3.2–17.8 mg/kg morphine 4–24 h before a generalization test resulted in minor changes in the MDD. To examine development of tolerance, supplemental doses of morphine (17.8 mg/kg) or saline were administered twice daily while discrimination training was either suspended or continued. Tolerance was assessed by weekly generalization tests. Greater tolerance developed to the morphine stimulus when training was suspended than when training was continued. For both training conditions, response rates during generalization tests were markedly suppressed during supplemental morphine administration, and original generalization gradients were recaptured within 2 weeks after termination of supplemental morphine administration. Supplemental saline administration did not alter the discriminative or rate-altering effects of morphine under either training condition. Thus, the magnitude of tolerance to a morphine discriminative stimulus reflected an interaction of supplemental drug treatment with the training conditions imposed during that treatment.     

Reversal of overshadowing in a drug mixture discrimination in rats

Previous studies have suggested that in some circumstances, learning processes such as overshadowing may determine the effects that one drug has upon the response to another. The experiments described here examined overshadowing in rats trained to discriminate mixtures of nicotine plus midazolam in two-lever operant procedures with food reinforcement. After training for 60 sessions, midazolam (0.32 mg/kg SC) overshadowed nicotine (0.32 mg/kg SC) so that the discriminative stimulus effect of nicotine seen in control rats trained with nicotine alone was abolished (n=8–10). In the next phase of the study, the discriminative response to midazolam in one group of mixture-trained rats was devalued by means of an extinction procedure which weakened the relationship between administration of midazolam and the response that was reinforced. Dose-response determinations then showed that the devaluation procedure had indeed attenuated the response to midazolam, whereas the previously overshadowed response to nicotine was restored. Post-session injections of drugs were used to equate the pharmacological histories of the groups and the effects seen were therefore attributable to training with the drugs and not simply to repeated exposure to them. Additionally, in the control rats trained with nicotine only (with midazolam given post-session), midazolam markedly reduced response rates, whereas in the three groups of rats trained with the mixture, midazolam had little response rate-depressant effect; this observation suggests that behaviourally contingent tolerance had developed to the response rate-reducing effect of midazolam. Application of devaluation procedures in studies of the discriminative stimulus effects of single drugs with multiple effects may provide a means for manipulating the characteristics of the discriminations obtained and for identifying individual elements of the drug-produced stimulus complex.     

Inter-animal olfactory cues in operant drug discrimination procedures in rats

Olfactory cues from prior subjects in operant chambers were shown to be an effective stimulus which rodents could use to direct lever selection in a typical operant drug discrimination (DD) paradigm. Such cues persisted for very long periods of time (16h), and were deposited after very short (5 min) operant sessions. In extinction tests inter-animal olfactory cues exerted very strong stimulus control over lever selection. Furthermore, such cues were not specific to individual rodent subjects but were generalizable between subjects. Inter-animal cues directing lever selection could be abolished by cleaning operant manipulanda with a 10% alcohol solution. Reanalysis of some DD data previously reported by one of the authors (Goudie 1977) indicated that this specific earlier study (and by implication perhaps other studies) might have been confounded by inter-animal cues. In a DD study with nicotine it was found that the drug cue was antagonized by mecamylamine for all subjects except those who had a reliable olfactory cue from prior subjects to direct lever selection (subjects who possessed both an olfactory and a drug cue to direct lever selection responded in a way suggesting that the exteroceptive olfactory cue controlled behaviour rather than the interoceptive drug cue). These findings indicate that inter-animal olfactory cues could be of considerable methodological significance in DD studies. The possible significance of such cues has not previously been reported upon in detail, and in reports of many DD studies there do not appear to be explicit indications that interanimal cues have been adequately controlled.The data reported here were presented at the annual conference of the European Society for the Study of Internal Stimulus Control by Drugs and Other Means, Brighton, Sussex, England, September 1980     

Apomorphine increases ethanol discrimination

Rats were trained to discriminate between the stimulus properties of 600 mg/kg ethanol and saline in a two-lever, food-motivated operant task. Once trained, rats showed a dose-related decrease in discriminative performance with lower ethanol doses and analysis of the dose-response curve indicated an ED50 of 372 mg/kg. Pretreatment with 0.16 mg/kg apomorphine produced increased discriminative performance at each ethanol dose and the combination generated a dose-response curve parallel to ethanol administered alone with an ED50 of 232 mg/kg. This significant shift to the left of the ethanol dose-response curve after apomorphine administration is discussed in relation to dopaminergic neuronal systems and the clinical use of apomorphine alcoholics.     

Ketamine-induced locomotion in rats in an open-field

The effects of ketamine on locomotion in an open-field were determined in hooded rats. Animals were given intraperitoneal injections of saline, or of 1, 10, 50 or 100 mg ketamine/kg body weight on separate days. Open-field behavior was examined for 60 min following injection. The 50 mg/kg dose of ketamine produced an increase in locomotion which peaked approximately 30 min after injection. A cataleptic immobility produced by the 100 mg/kg dose was followed by postanesthetic locomotion. The ketamine-induced locomotion consisted largely of ambulation about the perimeter of the field and was accompanied by ataxia, but included relatively little tight circling (rotation) during the peak of activity. Comparisons with the results of past research suggests that test-chamber design influences the type of locomotion induced by ketamine.     

Effects of SR141716 and WIN 55,212-2 on tolerance to ethanol in rats using the acute and rapid procedures

Rationale Our previous findings have shown rapid cross-tolerance between ethanol and Δ⁹-tetrahydrocannabinol and that intraperitoneal (i.p.) injection of cannabinoid receptor type 1 (CB1R) antagonist SR141716 (SR) does not interfere with tolerance to either of these drugs in mice. Objectives This study investigates the effects of SR, alone or in combination with the CB receptor agonist WIN 55,212-2 (WIN), on the development of acute and rapid tolerance to the incoordinating effect of ethanol in rats. Materials and methods Male Wistar rats received SR, through i.p. (0.5–2.0 mg/kg) or intracerebroventricular (i.c.v.) injections (0.5–4.0 μg), alone or together with WIN (1.0 μg, i.c.v.), in combination with ethanol (2.7 g/kg, i.p.). Another group received WIN (1.0 μg, i.c.v.) in combination with ethanol (2.3 g/kg), and the rats were tested for motor coordination. Rapid tolerance was assessed 24 h later by administering ethanol to all animals and retesting them under the same dose regimen. Acute tolerance was evaluated for 75 min after ethanol (3.0 g/kg, i.p.) in animals treated with SR or WIN (i.c.v.). Results The reduced motor impairment on day 2 (i.e., rapid tolerance) was blocked by SR (i.p. and i.c.v.). WIN (1.0 μg, i.c.v.) facilitated rapid tolerance and also prevented the blockade of rapid tolerance by SR (1.0 μg, i.c.v.). In the acute tolerance procedure, SR did not affect the motor incoordination induced by ethanol. Conclusions The results suggest that the endocannabinoid system may contribute to the development of rapid tolerance to ethanol.     

Agmatine inhibits morphine-induced drug discrimination in rats

Our previous studies have shown that agmatine inhibited morphine-induced conditioned place preference and locomotor sensitization in rats. In the present study, we further investigated the effects of agmatine on the discriminative stimulating effects produced by morphine in rats. Agmatine, at the dose range of 10-80 mg/kg (i.g.), neither induced drug discrimination, nor substituted for morphine stimulus in rats that were previously treated with morphine, suggesting that agmatine itself has no psychomotor-stimulating potential. However, pretreatment with agmatine (40, 80 mg/kg, i.g.) significantly inhibited the acquisition, but not expression, of morphine-induced drug discrimination as assessed by the correct nose-poke response. Further, chronic administration of agmatine (40, 80 mg/kg/dayx12 days, i.g., 25 min prior to morphine) also significantly accelerated the extinction of the discrimination induced by morphine. These data suggest that agmatine inhibits the acquisition and accelerates the extinction of morphine-induced discrimination, supporting possible use of agmatine in the treatment of opioid dependence.     

Comparison of ethanol,pentobarbital, and phenobarbital using drug vs. drug discrimination training

Rats learned drug vs. drug (D vs. D) or drug vs. no drug (D vs. N) discriminations in a T-maze shock-escape task with various doses of pentobarbital, phenobarbital, or ethanol. Dose-effect curves were obtained for each drug using D vs. N training. After D vs. N training with any one of these drugs, rats made D choices during substitution tests with the other two drugs, suggesting drug interchangeability. D vs. D training also showed that pentobarbital and phenobarbital were virtually indistinguishable from one another. However, ethanol was readily discriminated from pentobarbital, showing that the two drugs differed. The results show the utility of D vs. D training as a method for studying drug differences that may be too small to detect with substitution tests.     

Differentiating the discriminative stimulus effects of gamma-hydroxybutyrate and ethanol in a three-choice drug discrimination procedure in rats

Anecdotal reports indicate that GHB produces subjective effects similar to those of ethanol. However, recent investigations comparing the discriminative stimulus effects of GHB to those of ethanol suggest that the subjective effects of these substances may differ considerably. To explore further potential differences between GHB and ethanol, 16 male Sprague–Dawley rats were trained in a three-lever drug discrimination procedure to discriminate ethanol (1.0 g/kg, experiment 1; 1.5 g/kg, experiment 2) and GHB (300 mg/kg) from vehicle. Dose–response functions determined with both training compounds revealed a clear dissociation between the discriminative stimulus effects of these drugs. As expected, the GHB precursors gamma-butyrolactone and 1,4-butanediol produced full substitution for GHB. In addition, the GABA_B receptor agonist baclofen substituted for GHB, whereas the benzodiazepine flunitrazepam and the NMDA receptor antagonist ketamine engendered greater responding on the ethanol-lever. GHB's discriminative stimulus effects were blocked by the GABA_B receptor antagonist CGP-35348 but only partially blocked by the putative GHB receptor antagonist NCS 382. These findings are consistent with previous reports of GHB's discriminative stimulus effects in two-choice drug discrimination procedures and provide additional evidence that these effects are distinct from those of ethanol.     

Effects of chlormethiazole (Heminevrin®) on drug discrimination and open-field behavior in gerbils

Chlormethiazole (CMZ, 80 mg/kg) was used as a discriminative stimulus in gerbils; i.e., the presence or absence of certain effects of the drug controlled the choice behavior (left or right turn) of the animals trained to escape electric shocks in a T-maze. Substitution tests with pentobarbital (5–25 mg/kg) and ethanol (0.5–2.5 g/kg) indicated at least a partial similarity in the stimulus effects of CMZ and the two other drugs. The CMZ stimulus was attenuated by 30 mg/kg of the analeptic bemegride (BMG). In Experiment II, gerbils were trained to discriminate CMZ (80 mg/kg) from either of two doses of ethanol (1.5 or 2.0 g/kg). The acquisition rates for the latter groups appeared some-what slower than that noted for the gerbils in Experiment I, although only one measure significantly differentiated the groups. A qualitative difference is proposed as the basis for the discrimination between CMZ and ethanol. Open-field (O-F) tests 5 min after injections of CMZ (80 mg/kg) depressed both horizontal (ambulation) and vertical (rearing) activity, effects found to be counteracted by BMG (30 mg/kg) during the initial segment of the O-F testing. However, a second O-F test carried out 60 min after injections showed that the behaviors of the gerbils treated with the combination of CMZ and BMG were now markedly depressed. The effects of the drug combination on colonic temperature of the gerbils showed similar changes over time; i.e., the mixture of BMG and CMZ resulted in a normal colonic temperature response 5 min postinjection (p.i.), after which a marked drop of temperature followed at the recordings 60 min p.i.     

Effects of single or multiple choice trials per session on drug discrimination performance

Drug discrimination procedures typically provide for multiple choice opportunities per training session. This practice allows non-drug cues (presence or absence of reinforcement) to mediate choice behavior during that portion of the session following the initial choice. In this experiment, rats were trained to discriminate 1.0 mg/kg cyclazocine from saline using a novel procedure that employed a single-choice trial per training session. Drug discrimination acquisition and generalization were compared to those of rats given discrimination training with 30 choice trials per session. The one-trial procedure yielded stable and reliable acquisition but more slowly than did the multiple trials procedure. The one-trial procedure produced longer first trial choice latencies and enhanced the tendency for subjects to respond on both choice levers during the first trial. The cyclazocine generalization functions were comparable, but the one-trial subjects more often responded on both choice levers, particularly when administered intermediate test doses of cyclazocine. Control of choice behavior by the reinforcer cue was evaluated on a mid-session cue reversal test. Multiple-trial subjects persisted in responding on the saline level following a midsession injection of cyclazocine, whereas one-trial subjects shifted to the cyclazocine-appropriate lever.     

Characterizing withdrawal in rats following repeated drug administration using an amphetamine-vehicle-haloperidol drug discrimination

Rationale: Previous research using an amphetamine (AM)-haloperidol (HA) drug- drug discrimination task has shown that predominant responding on the HA-appropriate lever occurs 24 h after a single or multiple administrations of 10 mg/kg AM. Conversely, rebound responding on the AM-appropriate lever occurs following single or multiple administrations of 1 mg/kg HA. HA-appropriate responding was also observed 24 h following a single injection of AM using a three-lever, AM-vehicle-HA discrimination task. However, a single administration of HA did not produce robust rebound responding on the AM-appropriate lever. The present studies seek to clarify the discrepancy between responding following HA in the two- and three-choice tasks. Objective: Experiment 1 examined the extent of rebound responding that could be achieved following ten daily administrations of either 10 mg/kg AM or 1 mg/kg HA. Experiment 2 explored potential differences between the two- and three-choice tasks in characterizing the post-HA cue. Methods: Animals were trained to discriminate 0.35 mg/kg AM, vehicle, and 0.033 mg/kg HA. In experiment 1, animals received ten daily injections of 10 mg/kg AM, vehicle, or 1 mg/kg HA, and were tested 24 h after the final injection, and again 8, 15, and 22 days post-treatment. In experiment 2, animals were retrained and then treated daily with either vehicle or 1.0 mg/kg HA for 10 days, and then tested 24 h after the final injection, and again 5 and 11 days post-treatment, with either all three levers or with only the AM- and HA-appropriate levers available. Results: In experiment 1, multiple injections of AM produced robust HA lever responding, which is consistent with results from previous studies that used the two-choice, AM-HA discrimination task. However, multiple injections of HA did not produce predominant responding on the AM-appropriate lever. In experiment 2, animals treated with either vehicle or HA responded predominantly on the vehicle-appropriate lever when tested with all three levers present. When tested with the vehicle lever removed, however, animals treated with vehicle responded predominantly on the HA-appropriate lever, whereas those treated with HA responded predominantly on the AM-appropriate lever. Conclusions: These results suggest that the two-choice and three-choice task used here differ in how the post-HA withdrawal cue is characterized. This finding emphasizes the importance of knowing the relative locations of the agonist-, vehicle-, and antagonist-produced cues on the interoceptive stimulus continuum established by discrimination training. Received: 3 December 1997/Final version: 16 November 1998     

Behavioral contingencies modulate tolerance to discriminative stimulus effects of morphine

Experiments examined how learning processes modulate tolerance to discriminative stimulus effects of morphine. Rats were trained to discriminate saline and 3.2 mg/kg morphine, and the doses of morphine required to mimic the training dose were determined before, during and after repeated treatment with saline or high doses of morphine (10 mg/kg, b.i.d.). In one set of experiments, training was either suspended or continued with saline and the original training dose during a 2-week treatment regimen. When training was suspended, high-dose morphine treatment increased the dose of morphine required for stimulus effects approximately 3-fold. Tolerance persisted 2 days after treatment ended, but disappeared within 7 days. In contrast, continued training with saline and 3.2 mg/kg morphine during high-dose treatment both attenuated development of tolerance and transferred control to lower doses. Transfer of control to lower doses appeared conditional upon recent termination of high-dose treatment, as it disappeared within 7 days. Treatment with saline did not change the doses of morphine required for stimulus effects under either training condition. A final experiment examined whether high-dose treatment could transfer control to higher doses of morphine. The treatment dose of 10 mg/kg morphine itself was used as the training dose during a 2-week treatment regimen. The dose of morphine required for stimulus effects increased 2- to 4-fold during treatment, but quickly returned to control values when treatment ended. These results extend previous findings that conditioning and pharmacodynamic processes jointly regulate development of tolerance to discriminative effects of morphine.     

Tolerance to the depressant effects of diazepam in the drug discrimination paradigm

Seven groups of rats (n = 35) were run in operant drug experiments. All groups were trained on a Fixed Ratio 10 schedule to discriminate diazepam from saline. Two groups (n = 7, n = 6), after extensive drug discrimination training (doses of 2.0 and 3.0 mg/kg diazepam), were submitted to generalization experiments with various doses of the training drug. Two additional groups, (n = 6, n = 8) in the initial phase of drug discrimination, were trained on intermediate and high doses of diazepam (i.e., 5.0 and 10.0 mg/kg). The development of tolerance to the depressant effects of diazepam for these two groups was compared to the low dose sophisticated rats. Of the above-mentioned groups, two groups were given tests after a waiting period in drug discrimination training. In this test the two groups were compared to an additional group (n = 8) in its initial phases of drug discrimination training. The results show that a large number of low doses (i.e., doses below 3.0 mg/kg) is not able to induce any tolerance to the depressant effects of diazepam in this particular paradigm. Intermediate doses of diazepam (i.e., 3.0 mg/kg), administered in a large number, induced some tolerance to the depressant effects, while another intermediate dose (5.0 mg/kg) and a high dose (10.0 mg/kg) rapidly induced a significant tolerance. Once developed, the tolerance persisted for 51 days.     

Acute morphine dependence: Effects observed in shock and light discrimination tasks

Alterations in shock discrimination accuracy in the rat, indicative of hyperalgesia, have been noted 1–3 days following a single injection of morphine. To establish the extent to which these withdrawal-like effects were specific to the shock discrimination paradigm, rats were trained in two separate discrimination tasks. The discriminative stimuli (S^D's) for correct lever presses were mild electric shocks of different intensities in one task and were short duration lights over the levers in the other. After achieving comparable accuracy levels in the two tasks, the animals were injected SC with 30 mg/kg morphine sulfate and performance levels assessed 1, 2, 3, and 7 days later. Shock discrimination accuracy was significantly enhanced on post-morphine day 2, while accuracy in the light position task was not significantly affected on any of the post-morphine test days. The results indicated that increased pain sensitivity, as well as other signs of dependence, can occur following acute exposure to morphine.     

Genetic differences in tolerance to ethanol: A study in UChA and UChB rats

The development of tolerance to ethanol was studied in two strains of rats, UChA (low ethanol consumer) and UChB (high ethanol consumer), by the sleeping time test. Marked tolerance (perhaps both metabolic and tissue) appeared in UCh A rats, while only a slight tissue tolerance and not a metabolic one appeared in the UChB rats, when they were offered 10% ethanol as the sole fluid during 31 days (p<0.001). In UChA, but not in the UChB rats, the chronic treatment with ethanol induced tolerance to pentobarbital as shown by the same test.     

Effects of long-term administration and withdrawal of tetrahydrocannabinols (delta 8-THC and delta 9-THC) on open-field behavior in rats

The behavior of female Wistar rats, injected daily with Δ⁸-THC (5.0 mg/kg), Δ⁹-THC (2.5 mg/kg) or vehicle was studied in an open-field test both during the two weeks' period of drug administration and after withdrawal. The behavior of rats allowed long-term acclimation to the laboratory (LTA), studied in Experiment 1, was compared with those of Experiment 2, which were subjected to short-term acclimation (STA). Throughout the injection period a depressant effect of both isomers of THC was seen on ambulation, rearing, grooming and latency. Length of acclimation period interacted with the effects of THC:s on ambulation, producing even an increased ambulation for Δ⁸-THC in the STA group. Minor indications of tolerance to Δ⁹-THC were observed in the LTA condition only. A kind of circling behavior was characteristic of both drug groups, its frequency declining with repeated injections. At drug withdrawal, most open-field measures slowly returned to control levels, whereas rate of grooming showed a manifold increase. Possible implications of the results for behavioral research with the THC:s are discussed.     

Metabolic and pharmacodynamic tolerance to ethanol in rats

The development of tolerance to ethanol was studied in rats fed nutritionally adequate liquid diets containing ethanol or sucrose for up to 5 weeks. Tolerance was shown to be due to both metabolic and pharmacodynamic factors. Tolerance began to develop after 3 days of ethanol intake, reached a plateau by 16 days and persisted for up to 22 days after stopping the ethanol intake. The rate of onset and decay of both components of tolerance was similar.