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The effect of N-methyl-D-aspartate (NMDA) antagonists [dizocilpine, (+)MK-801, and ketamine], an NMDA agonist (D-cycloserine) and of brain serotonin (5-HT) depletion with p-chlorophenylalanine (p-CPA) on acute tolerance to ethanol was examined, using the tolerance model proposed by Radlow [Psychopharmacology 114 (1994) 1-8] and Martin and Moss [Alcohol Clin Exp Res 17 (1993) 211-216]. This model is based on the concept of a linear increase of acute tolerance with time; the rate of acute tolerance development is the slope of the output function that relates blood alcohol concentrations (BACs) and intoxication. Pretreatment with NMDA antagonists inhibited the development of acute tolerance to ethanol, whereas pretreatment with D-cycloserine enhanced it. Depletion of 5-HT by p-CPA also blocked acute tolerance to ethanol. These results on acute tolerance are similar to those previously found on rapid and chronic tolerance to ethanol.

Motor impairment (tilt-plane test) test was used to assess the phenomenon of rapid tolerance and crosstolerance to benzodiazepines, barbiturates, and ethanol. The motor impairment responses to benzodiazepines (chlordiazepoxide and diazepam) and to various barbiturates (pentobarbital, phenobarbital, and barbital) were significantly reduced on day 2 in rats that had been treated on day 1 with benzodiazepines and barbiturates, respectively, compared to the control group treated with saline on day 1. Benzodiazepine treatment on day 1 resulted in rapid crosstolerance to the motor impairment effects of ethanol on day 2. Benzodiazepine treatment, however, did not result in rapid crosstolerance to the three barbiturates (pentobarbital, barbital, and phenobarbital) tested. In contrast to the lack of rapid crosstolerance to barbiturates after treatment with benzodiazepines, barbiturate treatment clearly conferred rapid crosstolerance to benzodiazepines and to ethanol. This asymmetry of rapid crosstolerance raises the possibility that benzodiazepines and barbiturates invoke tolerance by mechanisms that are not wholly identical. Therefore, tolerance to the broad range of actions of barbiturates would include crosstolerance to the effects of benzodiazepines, whereas tolerance to benzodiazepines would include only a weak or partial crosstolerance to some of the effects of barbiturates.

We have recently reported that pretreatment with NMDA receptor antagonists [(+)MK-801 and ketamine] inhibited the development of rapid tolerance to ethanol hypothermia and motor-impairment on day 2 in animals receiving ethanol on day 1, compared to the control group pretreated with saline. In these studies rats were tested at 30, 60, 90 and 120 min after ethanol on both day 1 and 2. In the present report we compared the development of rapid tolerance under 2 different conditions: (1) in groups of rats that were tested on the tilt-plane at all test times (Testing or Intoxicated Practice group), (2) in groups of rats that were not tested on the tilt-plane but were handled at all test times on day 1 (dummy testing). Rats were pretreated with ethanol or saline on day 1 and tested with ethanol on day 2 in all the above studies. Both testing (intoxicated practice) and dummy testing of animals on day 1 after pretreatment with ethanol produced rapid tolerance to ethanol on day 2. However, (+)MK-801 or ketamine pretreatment, which blocked rapid tolerance in the intoxicated practice testing paradigm, failed to block rapid tolerance in the dummy testing paradigm. Similar results were obtained for rapid tolerance and for the effect of ketamine in the hypothermia experiment. These findings suggest that NMDA antagonists block rapid tolerance in the intoxicated testing paradigm but not in the dummy testing paradigm. However, whether the two types of rapid tolerance tested in the present experiments are indeed different or interrelated remains to be further investigated.

Tolerance to ethanol and cross-tolerance to other alcohols (n-propanol, n-butanol, t-butanol, isobutanol, t-amyl alcohol, n-amyl alcohol, and benzyl alcohol) and barbiturates (pentobarbital, secobarbital, amobarbital, thiopental, barbital and phenobarbital) that differ in lipid:water partition coefficient was examined in rats after chronic pretreatment with ethanol. Tolerance and cross-tolerance were studied with three different measures (hypothermia, tilt-plane, and rotarod). Tolerance to ethanol resulted in significant cross-tolerance to alcohols with low lipid solubility (n-propanol and t-butanol), whereas no cross-tolerance was seen with alcohols of high lipid solubility (isobutanol, n-amyl alcohol, t-amyl alcohol and benzyl alcohol). Cross-tolerance to n-butanol (which has intermediate lipid solubility) appeared to be metabolic rather than functional. Tolerance to ethanol also resulted in significant cross-tolerance to barbital and phenobarbital, but not to pentobarbital, secobarbital, amobarbital or thiopental. These studies suggest that lipid solubility is an important factor in relation to specificity of cross-tolerance to alcohols and barbiturates.

In a recent study, we showed that D-cycloserine, an agonist at the glycine site of the NMDA receptor, enhances the development of rapid tolerance to ethanol. In the present study, we report that the acquisition of rapid tolerance to the motor incoordination effect of ethanol (tilt-plane test) was increased only when D-cycloserine was injected before, but not after, the intoxicated practice under ethanol. The effect of D-cycloserine on tolerance when this agonist was administered in divided doses before and after test was similar to that obtained when D-cycloserine was injected before test. Higher doses of D-cycloserine did not produce a further enhancement of rapid tolerance. Moreover, when the dose of ethanol on day 1 was large enough to induce rapid tolerance per se, D-cycloserine did not further enhance the tolerance. The enhancement of tolerance by D-cycloserine was antagonized by previous administration of ketamine. The enhancement of ethanol tolerance by D-cycloserine and the antagonism of this effect by ketamine cannot be attributed to changes in pharmacokinetics of ethanol. Taken together, these results confirm the participation of the NMDA receptor system in the development of tolerance to ethanol, and reinforce earlier findings about the involvement of learning in tolerance.

Recent studies from our laboratory have shown that NMDA antagonists ((+)MK-801 and ketamine) inhibit the development of both rapid and chronic tolerance to the motor-impairing (moving belt test) and hypothermic effects of ethanol. The present experiments were designed to determine a) the generality of this inhibition, by using a different test of motor function, the tilt-plane test, and b) the possible importance of the experimental paradigm (i.e., with and without intoxicated practice), for the effect of the NMDA antagonist on ethanol tolerance. Daily administration of ethanol 3.3 g/kg for 5 days produced the same degree of tolerance on this test, whether it was given as a single dose of 3.3 g/kg before the daily training session or as divided doses of 2.3 g/kg before and 1 g/kg immediately after the session. The inhibitory effect of a single dose of (+)MK-801 (0.25 mg/kg IP) on rapid tolerance did not last longer than 1 day. Therefore, daily administration of the NMDA antagonists was necessary to block development of chronic tolerance. Daily injection of (+)MK-801 (0.25 mg/kg IP) failed to block chronic tolerance, but inclusion of a second dose of (+)MK-801 daily, and progressive increase of this second dose during the chronic treatment period did block chronic tolerance. Unlike (+)MK-801, ketamine does not have motor-impairing effects of its own, and does not potentiate those of ethanol; it was, therefore, used in the remaining experiments. Groups of rats received ethanol (3.3 g/kg) or saline, either before a daily practice session on the tilt-plane or after it.(ABSTRACT TRUNCATED AT 250 WORDS)

We recently reported that the noncompetitive NMDA antagonists, (+)MK-801 and ketamine, block the development of rapid tolerance to ethanol. In the present article, we show that D-cycloserine (CS), an agonist at the glycine site of the NMDA receptor that enhances learning and memory, also enhances the development of rapid tolerance to ethanol. Rats were pretreated on day 1 with saline or CS, followed 30 min later by ethanol (2.3 g/kg, IP) or saline. At the end of motor impairment testing on the tilt-plane apparatus, a second injection of CS (3 mg/kg, IP, each time) or saline was given, followed 30 min later by ethanol or saline. Ethanol pretreatment alone (at this dose) did not result in rapid tolerance to ethanol on day 2. However, the group pretreated with CS and ethanol on day 1 showed significant tolerance on day 2 compared to other groups. Pretreatment with CS on day 1 did not affect the motor impairment response to the first exposure to ethanol whether this was on day 1 or day 2. In another experiment, administration of (+)MK-801 (0.25 mg/kg, IP) prior to CS abolished the rapid tolerance enhancement by CS. These findings are further evidence that the NMDA system, which requires activation by the glycine receptor, plays a major role in the development of at least some forms of ethanol tolerance.

Motor impairment (tilt-plane test) was used to investigate whether the noncompetitive N-methyl-D-aspartate (NMDA) antagonist ketamine prevents the development of chronic and acute tolerance to ethanol. Rats were treated with ethanol or saline in the presence and absence of ketamine (separate groups) for 10 days and tested for ethanol tolerance in the absence of ketamine on the fifth and tenth days. In other studies, the effect of ketamine on acute tolerance to ethanol was examined. Rats that received ethanol daily without ketamine showed significant tolerance to ethanol on days 5 and 10, but those receiving ethanol plus ketamine daily showed significantly less tolerance to ethanol. Thus, ketamine interfered with the development of chronic tolerance just as it had been found previously to prevent rapid tolerance. In contrast, ketamine failed to block acute tolerance to ethanol. These results would suggest that the phenomena of acute tolerance and chronic tolerance have differences not previously reported.

Hypothermia and motor impairment (tilt-plane) tests were used to assess the phenomenon of rapid tolerance to ethanol and cross-tolerance to various alcohols, benzodiazepines, and barbiturates that differ in lipid:water partition coefficients. The hypothermic and motor impairment responses to ethanol were significantly reduced on day 2 in rats receiving ethanol (2 doses of 2 g/kg each for the hypothermia test and 2.3 and 1.7 g/kg for the tilt-plane test) 24 and 22 h earlier compared to the control group pretreated with saline. Ethanol pretreatment resulted in rapid cross-tolerance, on both tests, to the various alcohols (n-propanol, n-butanol, and t-butanol) and the benzodiazepines (chlordiazepoxide, diazepam, oxazepam, and flurazepam) tested. Ethanol pretreatment also conferred clear rapid cross-tolerance to barbital and phenobarbital, but did not result in rapid cross-tolerance to pentobarbital, secobarbital, amobarbital, or thiopental. The results on rapid cross-tolerance on both tests seen in these studies parallel the results obtained in chronic tolerance and cross-tolerance studies reported recently. These results suggest that rapid tolerance and cross-tolerance can be used as predictors of chronic tolerance and cross-tolerance.

Chronic ethanol treatment by gastric intubation conferred tolerance to ethanol-induced motor impairment and hypnosis in four different rat strains: Fischer 344, Long-Evans, Sprague-Dawley, and Wistar. Cross-tolerance to barbital was also observed in all strains after chronic treatment with ethanol. However, chronic ethanol treatment failed to produce cross-tolerance to pentobarbital-induced motor impairment and hypnosis in any of the four strains. The demonstration of cross-tolerance to barbital and the lack of it to pentobarbital after chronic ethanol treatment confirms and extends recent observations on the specificity of the site and/or mechanism of action of sedative-hypnotic drugs that differ in lipid solubility.

Hypothermia and motor impairment (tilt-plane test) were used to assess the phenomenon of rapid cross-tolerance between ethanol and pentobarbital in rats. The hypothermic and motor-impairment responses were significantly reduced on day 2 in animals receiving ethanol on day 1, compared to the control group pretreated with saline. Ethanol pretreatment, however, did not result in rapid cross-tolerance to pentobarbital on either test. Pentobarbital pretreatment on day 1 resulted in rapid tolerance to pentobarbital on day 2. However, in contrast to the lack of rapid cross-tolerance to pentobarbital after pretreatment with ethanol, pentobarbital pretreatment clearly conferred rapid cross-tolerance to ethanol. Determination of ethanol and pentobarbital blood levels suggested that pharmacokinetic alterations did not contribute significantly to the observed rapid tolerance and cross-tolerance. The asymmetry of rapid cross-tolerance seen in these studies mimics the results obtained by us in chronic tolerance and cross-tolerance studies reported recently. These results suggest that rapid tolerance and cross-tolerance can be used as predictors of chronic tolerance and cross-tolerance.

Recently, we reported that a chronic regimen of ethanol by intubation, which produced clear tolerance to ethanol-induced hypothermia, ataxia and sleep, produced only a marginal degree of cross-tolerance to these effects of pentobarbital. The present experiments were designed to test the reverse process by examining cross-tolerance to pentobarbital after chronic pretreatment with ethanol, chronic pentobarbital treatment by gavage conferred clear cross-tolerance to both barbital- and ethanol-induced hypothermia, ataxia and sleep. In a separate experiment, cross-tolerance to barbital- and ethanol-induced hypothermia and ataxia was demonstrated over a wide range of test doses. Determination of ethanol blood levels as well as a complete time course of absorption, distribution and elimination of ethanol suggested that pharmacokinetic alterations may play a role in the development of cross-tolerance to ethanol in pentobarbital-treated subjects. The asymmetry of cross-tolerance raises the possibility that pentobarbital and ethanol invoke tolerance by mechanisms that are not wholly identical. This possibility requires further exploration. Conceivably the actions of ethanol which mediate the measured effects form a subset of a larger range of pentobarbital actions that could provide a stronger stimulus to tolerance development.

The acute administration of ethanol, pentobarbital and chlordiazepoxide impaired, in a dose-dependent manner, the performance of rats on the moving-belt and two-way shuttle-box avoidance tests. Administration of these drugs for three weeks resulted in tolerance to their motor-impairing effects. Tolerance to ethanol or pentobarbital was characterized by a parallel shift of the dose-response curve to the right. Tolerance to chlordiazepoxide, however, was of greater extent and was accompanied by an apparent flattening of the dose-response curve. Symmetrical cross-tolerance developed between ethanol and pentobarbital. On the other hand, while chlordiazepoxide treatment conferred full cross-tolerance to ethanol and pentobarbital, only partial cross-tolerance to chlordiazepoxide was observed following treatment with ethanol or pentobarbital. These results suggest that at least part of the tolerance to chlordiazepoxide depends on changes in specific benzodiazepine receptors and is independent of the tolerance associated with non-specific changes in the cell membrane.

The development of tolerance to the hypothermic effect of ethanol was examined during chronic ethanol treatment (5 g/kg PO daily) at various ambient temperatures (Ta). Tolerance to the hypothermic effect of ethanol, monitored at five-day intervals for 25 days, developed rapidly when ethanol treatment was carried out at 4 degrees C. On the other hand, rats receiving ethanol treatment at a Ta of 36 degrees C, at which they did not experience hypothermia, acquired tolerance more slowly, but achieved the same level of tolerance as other groups after 25 days of treatment. This cannot be accounted for by the repeated testing at 21 degrees C at five-day intervals, since it was also observed under a non-repeated testing condition. Once tolerance to the hypothermic effect of ethanol was acquired, termination of ethanol treatment resulted in the loss of tolerance, but mere prevention of the hypothermic effect of ethanol did not. These results suggest that tolerance still developed even though the organisms did not experience hypothermia during ethanol treatment. Therefore there appears to be a component of tolerance, that depends upon a direct cellular action of the drug, as distinct from the physiological consequences of that action. However, variation in the degree of physiological disturbance (hypothermia) during drug exposure can modulate the rate of development of this tolerance.

A chronic regimen of ethanol by intubation, which produced clear tolerance to ethanol-induced hypothermia, ataxia and narcosis, produced only a marginal degree of cross-tolerance to these effects of pentobarbital. The lack of appreciable cross-tolerance to pentobarbital-induced hypothermia and ataxia was also observed over a wide range of test doses. However, cross-tolerance to barbital was observed after chronic treatment with ethanol. Increased rate of drug biotransformation did not contribute significantly to the observed tolerance and cross-tolerance. The difference in the extent of cross-tolerance between ethanol and the two barbiturates is consistent with the hypothesis that there is a degree of specificity in the sites of action of ethanol and other sedative-hypnotic drugs.