Brain Research, 574 (1992) 251-256 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
251
BRES 17513
Differential inhibition by NMDA antagonists of rapid tolerance to, and cross-tolerance between, ethanol and chlordiazepoxide J.M. Khanna, S.J. Mihic, J. Weiner, G. Shah, P.H. Wu and H. Kalant Department of Pharmacology, University of Toronto, Toronto, Ont. (Canada) and Addiction Research Foundation of Ontario, Toronto, Ont. (Canada) (Accepted 22 October 1991)
Key words: N-Methyl-o-aspartate antagonists; Ethanol; Chlordiazepoxide; Tolerance
We have recently found that the non-competitive N-methyl-D-aspartate (NMDA) antagonists, (+)MK-801 and ketamine, block the development of rapid tolerance to ethanol. In the present report we show that they also block rapid cross-tolerance from chlordiazepoxide to ethanol as well as ethanol to chlordiazepoxide. However, NMDA antagonists fail to block the development of rapid tolerance to chlordiazepoxide. Our results suggest that NMDA antagonists may affect not only the acquisition of rapid tolerance or cross-tolerance to sedatives but also the ability to express that tolerance or cross-tolerance, depending on the drugs used. It is also possible that the phenomena of rapid tolerance and rapid cross- tolerance have basic differences not previously reported in the literature. INTRODUCTION
ment of rapid tolerance to chlordiazepoxide (C) and rapid cross-tolerance between ethanol (E) and C.
Rapid tolerance and cross-tolerance to a sedative-hypnotic agent can be demonstrated by a decreased effect following the repeated administration of the drug 8-24 h after the first dose has disappeared 6'7'12'13. This is due to a decrease in CNS sensitivity, rather than a change in drug disposition, and has been shown to be influenced by the extent of opportunity for intoxicated practice during the first drug exposure 3. This implies that learning may be a c o m p o n e n t of such tolerance, and tolerance in general has m a n y properties akin to those of learning 11. The N-methyl-D-aspartate ( N M D A ) receptor with its associated cation channel has b e e n strongly implicated in learning and memory9~ 7. Non-competitive N M D A - r e ceptor antagonists, such as (+)MK-801 and ketamine 5, have been shown to prevent long-term potentiation, which is believed to be an essential synaptic substrate of learning and m e m o r y L4. Recently, we found that the N M D A antagonist (+)MK-801 prevents the developm e n t of rapid tolerance to ethanol 14. Both ketamine and (+)MK-801 block this development of rapid tolerance as measured on the tilt-plane and hypothermia tests ( K h a n n a et al., submitted). W h e t h e r (+)MK-801 also blocks the development of rapid tolerance to other sedative agents such as the benzodiazepines or barbiturates is u n k n o w n . The present study extends our earlier work and examines the effects of (+)MK-801 on the develop-
MATERIALS AND METHODS Male Sprague-Dawley rats obtained from Charles River Canada, Ltd. (Montrdal, Qudbec) had initial body weights of 175-200 g. They were individually housed in a colony room maintained at 21 ___I°C, with lights on at 7 a.m. and off at 7 p.m. Water was available at all times. Purina Rat Chow was given ad lib for one week. Thereafter, the daily ration was restricted and individually adjusted to maintain comparable body weights in the various groups. The tilt-plane test was used as a measure of motor impairment2's. The apparatus consists of a plane which can be inclined at a fixed angular velocity through a range of 55° above the horizontal. The animal was placed on the slightly roughened surface of the plane, which was then tilted until the animal began to slide from the starting position. The test measure used was the angle at which this sliding occurred. The sliding angle was measured before and 30, 60 and 90 min after the injection of E or C. The degree of post-drug ataxia was expressed as the percentage change in the sliding angle, compared to the pre-drug value for the same animal. Maximum impairment, regardless of the time of its occurrence, was employed as the measure of E or C effect. This generally occurred about 30-60 min after injection of either E or C. Blood samples (50/~1) for E measurement were taken from the rat's tail tip immediately after the last measurement of motor impairment. Blood E was analyzed by the enzymatic method described previously1°. Two-way analysis of variance and Student's t test were used in statistical analyses.
Effects of (+)MK-801 and ketamine on the development of rapid cross-tolerance from ethanol to chlordiazepoxide (Expt. 1). Rats were randomly divided into 4 groups. On Day 1, 2 groups were injected with (+)MK-801 (0.25 mg/kg, i.p.) while the other 2 groups received saline (S). Thirty minutes later, rats from one of
Correspondence: J.M. Khanna, Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
252 the (+)MK-801 and one of the S groups were given E (2.3 g/kg, i.p.) and the other 2 groups were administered S. Before the experiment, and at successive 30 min intervals up to 90 min after E or S injection, the degree of motor impairment was assessed (tilt plane test) in all animals. Immediately after the last measurement, a second injection of E (1.7 g/kg, i.p.) or S was given. Rats were then returned to their home cages. On Day 2, an identical procedure was followed except that all animals received a challenge dose of 18.5 mg/kg i.p. C and no (+)MK-801 or S pretreatment was given. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
DAY 1
p(O.03 k.Z
Effect of (+)MK-801 and ketamine on the development of rapid tolerance to chlordiazepoxide (Expt. 3). For these studies a procedure identical to that used in the C-E cross-tolerance studies (Expt. 2) was followed except that on Day 2, C (18.5 mg/kg, i.p.) was administered to all animals before they were tested for tolerance. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
Effect of administration of ketamine on both days (Day 1 and 2) on the development of rapid tolerance to ethanol (Expt. 4). In all the studies detailed above, (+)MK-801 or ketamine was given only on Day 1, before E or C. In this study, a single dose of ketamine (1 mg/kg, i.p.) was administered on both Days 1 and 2, 30 min before E (2.3 g/kg, i.p.).
Effect of higher doses of (+)MK-801 on the development of rapid tolerance to chlordiazepoxide (Expt. 5). The experimental protocol for these studies was similar to that described above (Expt. 3) except that on Day 1, a second dose of (+)MK-801 (0.25 mg/kg, i.p.) or S was given 30 min before the second dose of C or S. In other studies, an additional dose of (+)MK-801 (0.25 mg/kg, i.p.) was also given on Day 2, before a C challenge.
Effect of ketamine and (+)MK-801 on the expression of rapid tolerance to ethanol and chlordiazepoxide (Expt. 6). Rats were randomly divided into 4 groups. On Day 1, 2 groups were injected with E (2.3 g/kg, i.p.) while the other 2 groups received S. Before the experiment, and at successive 30 min intervals up to 90 min after E or S injection, motor impairment was assessed. Immediately after the last measurement, a second injection of E (1.75 g/kg, i.p.) or S was given. Rats were then returned to their home cages. On Day 2, one set of the E- and one of the S-treated groups were given ketamine (1 mg/kg, i.p.) while the other 2 E and S-treated groups received S. Thirty minutes later, all animals received a challenge dose of E (2.3 g/kg, i.p.). A similar protocol was followed in examining the effect of (+)MK-801 (0.25 mg/kg, i.p.) on the expression of tolerance to C.
RESULTS
Effects of (+)MK-801 and ketamine on rapid cross-tolerance f r o m ethanol to chlordiazepoxide (Expt. 1). O n D a y 1, ( + ) M K - 8 0 1
(F1,24 = 12.57, P < 0 . 0 0 1 6 ) ,
40"
N.S,
a
C
w n~
/
30-
/ / / / / /
20-
Effects of (+)MK-801 and ketamine on the development of rapid cross-tolerance from chlordiazepoxide to ethanol (Expt. 2). For these studies, an experimental procedure similar to that described above was employed. C (18.5 mg/kg, i.p.) or S was given on Day I, 30 min after S or (+)MK-801 (0.25 mg/kg, i.p.) pretreatment. After the last tilt-plane test on Day 1, a second dose of C (18.5 mg/kg, i.p.) or S was given. On Day 2, E (2.3 g/kg, i.p.) was administered to all animals and tolerance was assessed on the tilt plane as described above. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
DAY 2
50.
10 0
S
E
S
CONTROL
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S-C E-C
(+)MK-801
CONTROL
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b
(+)MK-801 N.S,
d
30.
20. 10.
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KETAMINE
S--C E-C
~-C E-C
CONTROL
KETAIdlNE
Fig. 1. Effects of (+)MK-801 and ketamine on rapid cross-tolerance from E to C on the motor-impairment (tilt-plane) test. (+)MK-801 or ketamine, with E or S, was administered on the first day and rapid cross-tolerance to C-induced motor-impairment was assessed the next day (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor-impairment responses to S and E on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid cross-tolerance to C-induced motor-impairment on Day 2. S (open bars) or E (striped bars) was given on Day 1. All groups received C on Day 2. Results are presented as mean + S.E.M. of 7 animals.
but not ketamine, impaired motor performance on its own as well as enhancing the motor impairment produced by E (Fig. la,b). The results of the rapid crosstolerance test on Day 2 showed a significantly lower motor-impairing effect of C in rats injected with E 24 h earlier (E-C), compared to their respective controls (S-C) injected with S on Day 1, in both the (+)MK-801 (t12 = 2.52, P < 0.03) and ketamine (40 = 4.86, P < 0.001) experiments (Fig. lc,d). These findings indicated the development of rapid cross-tolerance to C. Maximum percent impairment, however, was almost identical when S-C vs. E - C were compared, after rats were pretreated with either (+)MK-801 or ketamine (Fig. lc,d).
253
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DAY 1
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Fig. 2. Effects of (+)MK-80I and ketamine on rapid cross-tolerance from C to E on the motor impairment (tilt plane) test. (+)MK-801 or ketamine, with C or S, was administered on Day 1 and rapid cross-tolerance to E-induced motor impairment was assessed on Day 2 (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor impairment responses to S and C on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid cross-tolerance to E-induced motor impairment. S (open bars) or C (striped bars) was given on Day 1. All groups received E on Day 2. Results are presented as mean __+S.E.M. of 7 animals.
Effects of (+)MK-801 and ketamine on rapid cross-tolerance from chlordiazepoxide to ethanol (Expt. 2). O n D a y 1, ( + ) M K - 8 0 1 (Fl,z4 = 11.95, P < 0.002), but not k e t a m i n e , slightly impaired m o t o r p e r f o r m a n c e on its own as well as enhancing C-induced m o t o r impairment (Fig. 2a,b). The results of the rapid cross-tolerance test on D a y 2 showed a significantly lower m o t o r - i m p a i r ing effect of E in rats injected with C 24 h earlier ( C - E group), c o m p a r e d to their respective controls ( S - E group) injected with S 24 h earlier in both the ( + ) M K 801 (tm = 5.27, P < 0.001) and k e t a m i n e (t12 = 4.88, P < 0.001) experiments, suggesting the d e v e l o p m e n t of rapid cross-tolerance to E (Fig. 2c,d). M a x i m u m percent i m p a i r m e n t , however, was almost identical when the
S--C C-C
S-C C-C
CONTROL KETAMINE
Fig. 3. Effects of (+)MK-801 and ketamine on rapid tolerance to C. (+)MK-801 or ketamine, with C or S, were administered on the first day and rapid tolerance to C-induced motor-impairment was assessed the next day (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor-impairment responses to S and C on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid tolerance on Day 2 to C-induced motor-impairment. S (open bars) or C (striped bars) was given on Day 1. All groups received C on Day 2. Results are presented as mean + S.E.M. of 6-7 animals.
S - E vs. C - E groups were c o m p a r e d after p r e t r e a t m e n t with either ( + ) M K - 8 0 1 or k e t a m i n e (Fig. 2c,d).
Effects of (+)MK-801 and ketamine on rapid tolerance to chlordiazepoxide (Expt. 3). A d m i n i s t r a t i o n of ( + ) M K - 8 0 1 (F1,22 = 9.52, P < 0.0054), but not ketamine, on D a y 1 impaired m o t o r performance and enhanced the m o t o r i m p a i r m e n t produced by C (Fig. 3a,b). Rats injected with C on both days ( C - C groups) showed significantly less m o t o r imp a i r m e n t by C on D a y 2 when c o m p a r e d to rats which received S on D a y 1 ( S - C groups) in both the ( + ) M K 801 (h0 = 3.31, P < 0.01) and k e t a m i n e (ho = 6.8, P < 0.001) experiments (Fig. 3c,d). A d m i n i s t r a t i o n of ( + ) M K - 8 0 1 or k e t a m i n e 30 min
254
DAY 1
DAY 2
DAY 2
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Fig. 4. Effects of dosing with ketamine on both Days 1 and 2, on the development of rapid tolerance to E. On Day 1, rats were pretreated with S or ketamine and then given S or E. On Day 2, all animals received E and those which had been given ketamine on the first day, received it again on the second day. No rapid tolerance to E was seen after ketamine pretreatment, even if ketamine was given on both days. S (open bars) or E (striped bars) was given on Day 1. Results are presented as mean _+ S.E.M. of 7 animals.
Effect of administration of ketamine on both Day 1 and Day 2 on the development of rapid tolerance to ethanol (Expt. 4). In o r d e r to examine whether state-dependent learning played a role in the b l o c k a d e of rapid tolerance by the N M D A antagonists, k e t a m i n e was given on both days and rapid tolerance to E was examined (Fig. 4). O n D a y 2, tolerance was clearly seen in the control group (rio = 7.92, P < 0.0001) but not in the group which received k e t a m i n e on both t r e a t m e n t days. These results were not different from those of Expt. 2 in which ketamine was given only on D a y 1; i.e. k e t a m i n e blocked rapid tolerance to E.
Effect of higher doses of ( + )MK-801 on the development of rapid tolerance to chlordiazepoxide (Expt. 5). The administration of 2 doses (0.25 g/kg, i.p. each) of ( + ) M K - 8 0 1 on D a y 1 (see Methods) did not affect the d e v e l o p m e n t of rapid tolerance to C on D a y 2 (flz =
C
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s-c o-c
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prior to C on D a y 1 had no effect on d e v e l o p m e n t of rapid tolerance to C on D a y 2. A two-way A N O V A showed neither a significant effect of p r e t r e a t m e n t (F1,22 = 0.22, P > 0.65 and F1,22 = 1.90, P > 0.19 for ( + ) M K 801 and ketamine, respectively) nor a significant pretreatment x t r e a t m e n t interaction (F~,22 = 0.71, P > 0.41 and F1,22 = 0.39, P > 0.40 for ( + ) M K - 8 0 1 and ketamine, respectively). T h e r e was, however, a significant effect of C t r e a t m e n t (F1,22 = 55.6, P < 0.0001 and/'1,22 = 28.2, P < 0.0001 for the ( + ) M K - 8 0 1 and ketamine experiments, respectively.
S
i
30' 20
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C
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(+)MK-B01
S-,-C C-C CONTROL
S-C C-C (+)MK-801
Fig. 5. a: Effect of doubling the Day 1 (+)MK-801 dose on the development of rapid tolerance to C. On Day 1, rats were pretreated with S or (+)MK-801 (2 doses of 0.25 mg/kg i.p.) and then given S or C. On Day 2, all animals received C. (+)MK-801 was not able to prevent rapid tolerance to C. S (open bars) or C (striped bars) was given on Day 1. Results are presented as mean + S.E.M. of 7 animals, b: Effect of doubling the Day 1 dose of (+)MK-801, and adding a Day 2 dose, on the development of rapid tolerance to C. On Day 1, rats were pretreated with S or (+)MK801 (2 doses of 0.25 mg/kg i.p.) and then given S or C. On Day 2, all animals received C and those which had received (+)MK-801 on Day 1 received a single 0.25 mg/kg dose on Day 2. (+)MK-801 was not able to prevent C rapid tolerance. S (open bars) or C (striped bars) was given on Day 1. Results are presented as mean + S.E.M. of 20-21 animals.
3.45, P < 0.005 comparing S - C vs. C - C in the Control group; t12 = 2.21, P < 0.05 in the ( + ) M K - 8 0 1 group) (Fig. 5a). The difference in rapid tolerance d e v e l o p m e n t between the S - C and C - C groups was the same in those that received S as in those receiving 2 doses of ( + ) M K 801 on D a y 1. In the next series of experiments, we further increased the dose of ( + ) M K - 8 0 1 and gave 2 doses of ( + ) M K - 8 0 1 on D a y 1 and an additional dose on D a y 2. The results from 3 separate experiments were p o o l e d and are shown in Fig. 5b. O n D a y 2, rapid tolerance de-
255
Effect of ( +)MK-80I and ketamine on the expression of rapid tolerance to chlordiazepoxide and ethanol (Expt.
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Rapid tolerance to C was seen on Day 2 in both the control group (t12 = 6.04, P < 0.001) and the group given (+)MK-801 only on Day 2 (t12 = 2.33, P < 0.05) (Fig. 6a). Similarly, rapid tolerance to E was seen on Day 2 in both the control group (t12 = 5.43, P < 0.001) and the group pretreated with ketamine only on Day 2 (t12 = 4.93, P < 0.001) (Fig. 6b). This suggests that NMDA antagonists do not affect the expression of rapid tolerance to C or E. (+)MK-801 or ketamine administration did not affect blood ethanol concentrations in any of the experiments conducted. It is therefore unlikely that pharmacokinetic factors are playing a significant role in the findings detailed above. DISCUSSION
p(O.O01
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- 301
6).
$ - E E-E
CONTROL KETAMINE
Fig. 6. Effects of (+)MK-801 or ketamine on the expression of rapid tolerance to C or E. a: Two groups of rats were pretreated with S and then given either S or C on Day 1. On Day 2, 1 of the 2 groups was once again pretreated with S while the other received (+)MK-801; all animals received C. (+)MK-801 was not able to prevent the expression of C rapid tolerance. Open bars, S (vehicle), and striped bars, C (treatment dose), given on the first day. b: Two groups of rats were pretreated with S and then given either S or E on Day 1. On Day 2, 1 of the 2 groups was once again pretreated with S while the other received ketamine; all animals received E. Ketamine was not able to prevent the expression of E rapid tolerance. Open bars, S (vehicle), and striped bars, E (treatment dose), given on the first day. Results are presented as mean + S.E.M. of 7 animals.
velopment was again seen in the groups receiving C-C, in either the presence or absence of (+)MK-801. A twoway ANOVA showed an effect of C treatment (F1,78 = 31.48, P < 0.0001) and as well as a significant effect of (+)MK-801 pretreatment (F1,78 = 20.61, P < 0.0001). There was, however, no significant pretreatment x treatment interaction (F1,7s = 3.53, P < 0.064), suggesting that (+)MK-801 is not able to block tolerance to C as clearly as it does tolerance to E.
(+)MK-801 and ketamine failed to block rapid tolerance to C as assessed on the tilt plane (Figs. 3 and 5), in clear contrast to their ability to block rapid tolerance to E 14 (Expt. 4), as well as cross-tolerance from E to C and vice versa (Figs. 1 and 2). It could be argued that the failure to see rapid tolerance to E on the second day is a phenomenon related to state-dependent learning. The effects of the NMDA antagonist + E, as perceived by the rat on Day 1, may be different from the effects of E alone on Day 2, resulting in a failure to display rapid tolerance. However, when ketamine was administered before E on both days, still no tolerance was seen (Fig. 4). Further, when ketamine was given only on Day 2 (Fig. 6b), it did not block the expression of rapid tolerance to E, suggesting that ketamine blocks the acquisition of rapid tolerance to E. The lack of cross-tolerance in the E - C experiment (Fig. 1) is also likely due to the NMDA antagonists blocking the acquisition of rapid tolerance to E after the first dose; this would explain why no rapid cross-tolerance to C was seen on the second day. However, the fact that rapid cross-tolerance from C to E (Fig. 2) was blocked by the NMDA antagonists, whereas rapid tolerance to C itself was not, argues against the idea of NMDA antagonists retarding the acquisition of rapid tolerance in general. It does not appear that NMDA antagonists block expression of rapid tolerance to either E or C, since (+)MK-801 and ketamine, when administered only on Day 2, were not able to prevent the expression of either E or C rapid tolerance (Fig. 6). However, we have not ruled out the possibility that expression of cross-tolerance from C to E is blocked by NMDA antagonists.
256 It is possible that the dose of N M D A antagonists required to block rapid tolerance to C m a y be higher than the dose required to block rapid tolerance and rapid cross-tolerance to E. H o w e v e r , doubling the D a y 1 ( + ) M K - 8 0 1 dose (Fig. 5a) or even doubling the D a y 1 (+)MK-801 dose plus giving an additional dose on D a y 2 had no significant effect on tolerance to C (Fig. 5b). Therefore, the dose of the N M D A antagonist does not a p p e a r to be a factor in the failure of N M D A antagonists to prevent rapid tolerance to C. It should also be noted that ( + ) M K - 8 0 1 , but not ketamine, usually produced some m o t o r incoordination on its own and enhanced the effects of E and C in an additive fashion. ( + ) M K - 8 0 1 has also been r e p o r t e d to enhance the hypnotic effects of ethanol in mice TM. Thus, if anything, ( + ) M K - 8 0 1 should have increased the stimulus for rapid tolerance and cross-tolerance d e v e l o p m e n t in rats concurrently receiving ( + ) M K - 8 0 1 + E (or C) on D a y 1. Since ethanol, like ( + ) M K - 8 0 1 , inhibits N M D A - a c t i vated ion currents 16, some sort of interaction at the level of the N M D A - r e c e p t o r might explain why tolerance to E, but not to C, is inhibited by (+)MK-801. To our knowledge, no one has r e p o r t e d any effects of benzodiazepines on N M D A - s t i m u l a t e d cation currents. A specific interaction b e t w e e n ( + ) M K - 8 0 1 and E would also explain the ability of ( + ) M K - 8 0 1 to prevent cross-tolerance from E to C; ( + ) M K - 8 0 1 could be blocking the acquisition of tolerance to E and consequently, indirectly, the expression of cross-tolerance to C. H o w e v e r , this
antagonism of tolerance d e v e l o p m e n t to E could not explain why ( + ) M K - 8 0 1 also blocks cross-tolerance from C to E (Fig. 2), while not affecting tolerance to C (Fig. 3), especially since the expression of tolerance to E is not blocked by N M D A antagonists (Fig. 6). In conclusion, our results suggest a number of possibilities: (1) that N M D A antagonists affect not only the acquisition of rapid tolerance or cross-tolerance to sedatives but possibly also the ability to express that tolerance, depending on the drugs used; (2) that there is a basic difference between the p h e n o m e n a of rapid tolerance and rapid cross-tolerance that has previously not been r e p o r t e d in the literature; or (3) that there is an incomplete overlap in the mechanisms of action of E and C and in the ways rapid tolerance and cross-tolerance develop to them. Some support for this third possibility m a y be p r o v i d e d by the observation that the changes in d o s e - r e s p o n s e curves during the d e v e l o p m e n t of tolerance to E are not identical to those in tolerance to C xS. F u r t h e r studies characterizing the effects of N M D A antagonists on the d e v e l o p m e n t of rapid tolerance and cross-tolerance to sedatives are in progress.
REFERENCES
and post-synaptic antagonism, Brain Research, 334 (1985) 348353. 10 Hawkins, R.D., Kalant, H. and Khanna, J.M., Effect of chronic intake of ethanol on rate of ethanol metabolism, Can. J. Physiol. Pharmacol., 44 (1966) 241-257. 11 Kalant, H., LeBlanc, A.E. and Gibbins, R.J., Tolerance to, and dependence on, some nonopiate psychotropic drugs, Pharmacol. Rev., 23 (1971) 135-191. 12 Khanna, J.M., Kalant, H., Shah, G. and Weiner, J., Rapid tolerance as an index of chronic tolerance, Pharmacol. Biochem. Behav., 38 (1991) 427-432. 13 Khanna, J.M., Kalant, H. and Shah, G., Tolerance to ethanol and cross-tolerance to other alcohols, barbiturates and benzodiazepines, International Society for Biomedical Research on Alcoholism, Abstract 317, Toronto, Canada, 1990. 14 Khanna, J.M., Wu, P.H., Weiner, J. and Kalant, H., NMDA antagonist inhibits rapid tolerance to ethanol, Brain Res. Bull., 26 (1991) 643-645. 15 IA, A.D., J.M. Khanna, H. Kalant and Grossi, E, Tolerance to and cross-tolerance among ethanol, pentobarbital and chlordiazepoxide, Pharmacol. Biochem. Behav., 24 (1986) 93-98. 16 Lovinger, D.M., White, G. and Weight EE, Ethanol inhibits NMDA-activated ion current in hippocampal neurons, Science, 243 (1989) 1721-1724. 17 Morris, R.G.M., Anderson, E., Lynch, G.S. and Baudry, M., Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate antagonist, APS, Nature, 319 (1986) 774-776. 18 Wilson, W.R., Bosy, T.Z. and Ruth, J.A., NMDA agonists and antagonists alter the hypnotic response to ethanol in LS and SS mice, Alcohol, 7 (1990) 389-395.
1 Abraham, W.C. and Mason, S.E., Effects of the NMDA receptor/channel antagonists CPP and MK801 on hippocampal field potentials and long-term potentiation in anesthesized rats, Brain Research, 462 (1988) 40-46. 2 Arvola, A., Sammaiisto, L. and Wallgren, H., A test for level of alcohol intoxication in the rat, J. Stud. Alcohol, 19 (1958) 563-572. 3 Bitran, M. and Kaiant, H., Learning factor in rapid tolerance to ethanol-induced motor impairment, Pharmacol. Biochem. Behav., 39 (1991) 917-922. 4 Coan, E.J., Saywood, W. and Collingridge, G.L., MK-801 blocks NMDA receptor-mediated synaptic transmission and long term potentiation in rat hippocampai slices, Neurosci. Lett., 80 (1987) 111-114. 5 Collingfidge, G.L. and Lester, R.A.J., Excitatory amino acid receptors in the vertebrate central nervous system, Pharmacol. Rev., 40 (1989) 143-210. 6 Crabbe, J.C., Rigter, H., Uijlen, J. and Strijbos, C., Rapid development of tolerance to the hypothermic effect of ethanol in mice, J. Pharmacol. Exp. Ther., 208 (1979) 129-133. 7 File, S.E., Mabbutt, P.S. and Hitchcott, EK., Characterisation of the phenomenon of 'one trial tolerance' to the anxiolytic effect of chlordiazepoxide in the elevated plus-maze, Psychopharmacology, 102 (1990) 98-101. 8 Gougos, A., Khanna, J.M., IA, A.D. and Kalant, H., Tolerance to ethanol and cross-tolerance to pentobarbital and barbital, Pharmacol. Biochem. Behav., 24 (1986) 801-807. 9 Harris, E.W. and Cotman, C.W., Effects of synaptic antagonists on perforant path paired-pulse plasticity: differentiation of pre-
Acknowledgments: This work was supported by NIAAA Grant AA08212-02 and the Addiction Research Foundation of Ontario. The authors would like to thank Mrs. Anita Chau for technical assistance. The views expressed in this paper are those of the authors, and do not necessarily represent the policy of the Addiction Research Foundation.
251
BRES 17513
Differential inhibition by NMDA antagonists of rapid tolerance to, and cross-tolerance between, ethanol and chlordiazepoxide J.M. Khanna, S.J. Mihic, J. Weiner, G. Shah, P.H. Wu and H. Kalant Department of Pharmacology, University of Toronto, Toronto, Ont. (Canada) and Addiction Research Foundation of Ontario, Toronto, Ont. (Canada) (Accepted 22 October 1991)
Key words: N-Methyl-o-aspartate antagonists; Ethanol; Chlordiazepoxide; Tolerance
We have recently found that the non-competitive N-methyl-D-aspartate (NMDA) antagonists, (+)MK-801 and ketamine, block the development of rapid tolerance to ethanol. In the present report we show that they also block rapid cross-tolerance from chlordiazepoxide to ethanol as well as ethanol to chlordiazepoxide. However, NMDA antagonists fail to block the development of rapid tolerance to chlordiazepoxide. Our results suggest that NMDA antagonists may affect not only the acquisition of rapid tolerance or cross-tolerance to sedatives but also the ability to express that tolerance or cross-tolerance, depending on the drugs used. It is also possible that the phenomena of rapid tolerance and rapid cross- tolerance have basic differences not previously reported in the literature. INTRODUCTION
ment of rapid tolerance to chlordiazepoxide (C) and rapid cross-tolerance between ethanol (E) and C.
Rapid tolerance and cross-tolerance to a sedative-hypnotic agent can be demonstrated by a decreased effect following the repeated administration of the drug 8-24 h after the first dose has disappeared 6'7'12'13. This is due to a decrease in CNS sensitivity, rather than a change in drug disposition, and has been shown to be influenced by the extent of opportunity for intoxicated practice during the first drug exposure 3. This implies that learning may be a c o m p o n e n t of such tolerance, and tolerance in general has m a n y properties akin to those of learning 11. The N-methyl-D-aspartate ( N M D A ) receptor with its associated cation channel has b e e n strongly implicated in learning and memory9~ 7. Non-competitive N M D A - r e ceptor antagonists, such as (+)MK-801 and ketamine 5, have been shown to prevent long-term potentiation, which is believed to be an essential synaptic substrate of learning and m e m o r y L4. Recently, we found that the N M D A antagonist (+)MK-801 prevents the developm e n t of rapid tolerance to ethanol 14. Both ketamine and (+)MK-801 block this development of rapid tolerance as measured on the tilt-plane and hypothermia tests ( K h a n n a et al., submitted). W h e t h e r (+)MK-801 also blocks the development of rapid tolerance to other sedative agents such as the benzodiazepines or barbiturates is u n k n o w n . The present study extends our earlier work and examines the effects of (+)MK-801 on the develop-
MATERIALS AND METHODS Male Sprague-Dawley rats obtained from Charles River Canada, Ltd. (Montrdal, Qudbec) had initial body weights of 175-200 g. They were individually housed in a colony room maintained at 21 ___I°C, with lights on at 7 a.m. and off at 7 p.m. Water was available at all times. Purina Rat Chow was given ad lib for one week. Thereafter, the daily ration was restricted and individually adjusted to maintain comparable body weights in the various groups. The tilt-plane test was used as a measure of motor impairment2's. The apparatus consists of a plane which can be inclined at a fixed angular velocity through a range of 55° above the horizontal. The animal was placed on the slightly roughened surface of the plane, which was then tilted until the animal began to slide from the starting position. The test measure used was the angle at which this sliding occurred. The sliding angle was measured before and 30, 60 and 90 min after the injection of E or C. The degree of post-drug ataxia was expressed as the percentage change in the sliding angle, compared to the pre-drug value for the same animal. Maximum impairment, regardless of the time of its occurrence, was employed as the measure of E or C effect. This generally occurred about 30-60 min after injection of either E or C. Blood samples (50/~1) for E measurement were taken from the rat's tail tip immediately after the last measurement of motor impairment. Blood E was analyzed by the enzymatic method described previously1°. Two-way analysis of variance and Student's t test were used in statistical analyses.
Effects of (+)MK-801 and ketamine on the development of rapid cross-tolerance from ethanol to chlordiazepoxide (Expt. 1). Rats were randomly divided into 4 groups. On Day 1, 2 groups were injected with (+)MK-801 (0.25 mg/kg, i.p.) while the other 2 groups received saline (S). Thirty minutes later, rats from one of
Correspondence: J.M. Khanna, Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada
252 the (+)MK-801 and one of the S groups were given E (2.3 g/kg, i.p.) and the other 2 groups were administered S. Before the experiment, and at successive 30 min intervals up to 90 min after E or S injection, the degree of motor impairment was assessed (tilt plane test) in all animals. Immediately after the last measurement, a second injection of E (1.7 g/kg, i.p.) or S was given. Rats were then returned to their home cages. On Day 2, an identical procedure was followed except that all animals received a challenge dose of 18.5 mg/kg i.p. C and no (+)MK-801 or S pretreatment was given. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
DAY 1
p(O.03 k.Z
Effect of (+)MK-801 and ketamine on the development of rapid tolerance to chlordiazepoxide (Expt. 3). For these studies a procedure identical to that used in the C-E cross-tolerance studies (Expt. 2) was followed except that on Day 2, C (18.5 mg/kg, i.p.) was administered to all animals before they were tested for tolerance. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
Effect of administration of ketamine on both days (Day 1 and 2) on the development of rapid tolerance to ethanol (Expt. 4). In all the studies detailed above, (+)MK-801 or ketamine was given only on Day 1, before E or C. In this study, a single dose of ketamine (1 mg/kg, i.p.) was administered on both Days 1 and 2, 30 min before E (2.3 g/kg, i.p.).
Effect of higher doses of (+)MK-801 on the development of rapid tolerance to chlordiazepoxide (Expt. 5). The experimental protocol for these studies was similar to that described above (Expt. 3) except that on Day 1, a second dose of (+)MK-801 (0.25 mg/kg, i.p.) or S was given 30 min before the second dose of C or S. In other studies, an additional dose of (+)MK-801 (0.25 mg/kg, i.p.) was also given on Day 2, before a C challenge.
Effect of ketamine and (+)MK-801 on the expression of rapid tolerance to ethanol and chlordiazepoxide (Expt. 6). Rats were randomly divided into 4 groups. On Day 1, 2 groups were injected with E (2.3 g/kg, i.p.) while the other 2 groups received S. Before the experiment, and at successive 30 min intervals up to 90 min after E or S injection, motor impairment was assessed. Immediately after the last measurement, a second injection of E (1.75 g/kg, i.p.) or S was given. Rats were then returned to their home cages. On Day 2, one set of the E- and one of the S-treated groups were given ketamine (1 mg/kg, i.p.) while the other 2 E and S-treated groups received S. Thirty minutes later, all animals received a challenge dose of E (2.3 g/kg, i.p.). A similar protocol was followed in examining the effect of (+)MK-801 (0.25 mg/kg, i.p.) on the expression of tolerance to C.
RESULTS
Effects of (+)MK-801 and ketamine on rapid cross-tolerance f r o m ethanol to chlordiazepoxide (Expt. 1). O n D a y 1, ( + ) M K - 8 0 1
(F1,24 = 12.57, P < 0 . 0 0 1 6 ) ,
40"
N.S,
a
C
w n~
/
30-
/ / / / / /
20-
Effects of (+)MK-801 and ketamine on the development of rapid cross-tolerance from chlordiazepoxide to ethanol (Expt. 2). For these studies, an experimental procedure similar to that described above was employed. C (18.5 mg/kg, i.p.) or S was given on Day I, 30 min after S or (+)MK-801 (0.25 mg/kg, i.p.) pretreatment. After the last tilt-plane test on Day 1, a second dose of C (18.5 mg/kg, i.p.) or S was given. On Day 2, E (2.3 g/kg, i.p.) was administered to all animals and tolerance was assessed on the tilt plane as described above. This experiment was repeated with ketamine (1 mg/kg, i.p.) instead of (+)MK-801 on a new batch of animals.
DAY 2
50.
10 0
S
E
S
CONTROL
E
S-C E-C
(+)MK-801
CONTROL
50F-Z
W
S--C E-C
p(O,O01
40
b
(+)MK-801 N.S,
d
30.
20. 10.
s
E
CONTROL
S
E
KETAMINE
S--C E-C
~-C E-C
CONTROL
KETAIdlNE
Fig. 1. Effects of (+)MK-801 and ketamine on rapid cross-tolerance from E to C on the motor-impairment (tilt-plane) test. (+)MK-801 or ketamine, with E or S, was administered on the first day and rapid cross-tolerance to C-induced motor-impairment was assessed the next day (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor-impairment responses to S and E on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid cross-tolerance to C-induced motor-impairment on Day 2. S (open bars) or E (striped bars) was given on Day 1. All groups received C on Day 2. Results are presented as mean + S.E.M. of 7 animals.
but not ketamine, impaired motor performance on its own as well as enhancing the motor impairment produced by E (Fig. la,b). The results of the rapid crosstolerance test on Day 2 showed a significantly lower motor-impairing effect of C in rats injected with E 24 h earlier (E-C), compared to their respective controls (S-C) injected with S on Day 1, in both the (+)MK-801 (t12 = 2.52, P < 0.03) and ketamine (40 = 4.86, P < 0.001) experiments (Fig. lc,d). These findings indicated the development of rapid cross-tolerance to C. Maximum percent impairment, however, was almost identical when S-C vs. E - C were compared, after rats were pretreated with either (+)MK-801 or ketamine (Fig. lc,d).
253
DAY 2
DAY 1
DAY 2
DAY 1
5050,
p(O.O01
iz- 40 I.d "5
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.< 30I:L
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a
L
2010. 0
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s
c
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p(O.O01
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40
i
b
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(+)MK-801
P(O.O01
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30/ / '/
20-
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~
CONTROL (÷)MK-801
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CONTROL CONTROL (+)MK-801
P(O.Ol
20--~ 10-
/ s
c
s
c
S-E C-E
S-E C-£
CONTROL
KETAMINE
S
C
S
C
CONTROL KErAMINE
CONTROL
KETAM[NE
Fig. 2. Effects of (+)MK-80I and ketamine on rapid cross-tolerance from C to E on the motor impairment (tilt plane) test. (+)MK-801 or ketamine, with C or S, was administered on Day 1 and rapid cross-tolerance to E-induced motor impairment was assessed on Day 2 (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor impairment responses to S and C on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid cross-tolerance to E-induced motor impairment. S (open bars) or C (striped bars) was given on Day 1. All groups received E on Day 2. Results are presented as mean __+S.E.M. of 7 animals.
Effects of (+)MK-801 and ketamine on rapid cross-tolerance from chlordiazepoxide to ethanol (Expt. 2). O n D a y 1, ( + ) M K - 8 0 1 (Fl,z4 = 11.95, P < 0.002), but not k e t a m i n e , slightly impaired m o t o r p e r f o r m a n c e on its own as well as enhancing C-induced m o t o r impairment (Fig. 2a,b). The results of the rapid cross-tolerance test on D a y 2 showed a significantly lower m o t o r - i m p a i r ing effect of E in rats injected with C 24 h earlier ( C - E group), c o m p a r e d to their respective controls ( S - E group) injected with S 24 h earlier in both the ( + ) M K 801 (tm = 5.27, P < 0.001) and k e t a m i n e (t12 = 4.88, P < 0.001) experiments, suggesting the d e v e l o p m e n t of rapid cross-tolerance to E (Fig. 2c,d). M a x i m u m percent i m p a i r m e n t , however, was almost identical when the
S--C C-C
S-C C-C
CONTROL KETAMINE
Fig. 3. Effects of (+)MK-801 and ketamine on rapid tolerance to C. (+)MK-801 or ketamine, with C or S, were administered on the first day and rapid tolerance to C-induced motor-impairment was assessed the next day (see Materials and Methods for details), a and b: Effect of (+)MK-801 and ketamine on the acute motor-impairment responses to S and C on Day 1. c and d: Effect of (+)MK-801 and ketamine on rapid tolerance on Day 2 to C-induced motor-impairment. S (open bars) or C (striped bars) was given on Day 1. All groups received C on Day 2. Results are presented as mean + S.E.M. of 6-7 animals.
S - E vs. C - E groups were c o m p a r e d after p r e t r e a t m e n t with either ( + ) M K - 8 0 1 or k e t a m i n e (Fig. 2c,d).
Effects of (+)MK-801 and ketamine on rapid tolerance to chlordiazepoxide (Expt. 3). A d m i n i s t r a t i o n of ( + ) M K - 8 0 1 (F1,22 = 9.52, P < 0.0054), but not ketamine, on D a y 1 impaired m o t o r performance and enhanced the m o t o r i m p a i r m e n t produced by C (Fig. 3a,b). Rats injected with C on both days ( C - C groups) showed significantly less m o t o r imp a i r m e n t by C on D a y 2 when c o m p a r e d to rats which received S on D a y 1 ( S - C groups) in both the ( + ) M K 801 (h0 = 3.31, P < 0.01) and k e t a m i n e (ho = 6.8, P < 0.001) experiments (Fig. 3c,d). A d m i n i s t r a t i o n of ( + ) M K - 8 0 1 or k e t a m i n e 30 min
254
DAY 1
DAY 2
DAY 2
50-
50p(O.O001
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/ / / / /
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z;
DAY 1
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Fig. 4. Effects of dosing with ketamine on both Days 1 and 2, on the development of rapid tolerance to E. On Day 1, rats were pretreated with S or ketamine and then given S or E. On Day 2, all animals received E and those which had been given ketamine on the first day, received it again on the second day. No rapid tolerance to E was seen after ketamine pretreatment, even if ketamine was given on both days. S (open bars) or E (striped bars) was given on Day 1. Results are presented as mean _+ S.E.M. of 7 animals.
Effect of administration of ketamine on both Day 1 and Day 2 on the development of rapid tolerance to ethanol (Expt. 4). In o r d e r to examine whether state-dependent learning played a role in the b l o c k a d e of rapid tolerance by the N M D A antagonists, k e t a m i n e was given on both days and rapid tolerance to E was examined (Fig. 4). O n D a y 2, tolerance was clearly seen in the control group (rio = 7.92, P < 0.0001) but not in the group which received k e t a m i n e on both t r e a t m e n t days. These results were not different from those of Expt. 2 in which ketamine was given only on D a y 1; i.e. k e t a m i n e blocked rapid tolerance to E.
Effect of higher doses of ( + )MK-801 on the development of rapid tolerance to chlordiazepoxide (Expt. 5). The administration of 2 doses (0.25 g/kg, i.p. each) of ( + ) M K - 8 0 1 on D a y 1 (see Methods) did not affect the d e v e l o p m e n t of rapid tolerance to C on D a y 2 (flz =
C
$
C
CONTROL (+)MK-B01 DAY 1
s-c o-c
s-c c-c
CONTROL (+)MK-B01 DAY 2
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prior to C on D a y 1 had no effect on d e v e l o p m e n t of rapid tolerance to C on D a y 2. A two-way A N O V A showed neither a significant effect of p r e t r e a t m e n t (F1,22 = 0.22, P > 0.65 and F1,22 = 1.90, P > 0.19 for ( + ) M K 801 and ketamine, respectively) nor a significant pretreatment x t r e a t m e n t interaction (F~,22 = 0.71, P > 0.41 and F1,22 = 0.39, P > 0.40 for ( + ) M K - 8 0 1 and ketamine, respectively). T h e r e was, however, a significant effect of C t r e a t m e n t (F1,22 = 55.6, P < 0.0001 and/'1,22 = 28.2, P < 0.0001 for the ( + ) M K - 8 0 1 and ketamine experiments, respectively.
S
i
30' 20
10
0
S
C
CONTROL
S
C
(+)MK-B01
S-,-C C-C CONTROL
S-C C-C (+)MK-801
Fig. 5. a: Effect of doubling the Day 1 (+)MK-801 dose on the development of rapid tolerance to C. On Day 1, rats were pretreated with S or (+)MK-801 (2 doses of 0.25 mg/kg i.p.) and then given S or C. On Day 2, all animals received C. (+)MK-801 was not able to prevent rapid tolerance to C. S (open bars) or C (striped bars) was given on Day 1. Results are presented as mean + S.E.M. of 7 animals, b: Effect of doubling the Day 1 dose of (+)MK-801, and adding a Day 2 dose, on the development of rapid tolerance to C. On Day 1, rats were pretreated with S or (+)MK801 (2 doses of 0.25 mg/kg i.p.) and then given S or C. On Day 2, all animals received C and those which had received (+)MK-801 on Day 1 received a single 0.25 mg/kg dose on Day 2. (+)MK-801 was not able to prevent C rapid tolerance. S (open bars) or C (striped bars) was given on Day 1. Results are presented as mean + S.E.M. of 20-21 animals.
3.45, P < 0.005 comparing S - C vs. C - C in the Control group; t12 = 2.21, P < 0.05 in the ( + ) M K - 8 0 1 group) (Fig. 5a). The difference in rapid tolerance d e v e l o p m e n t between the S - C and C - C groups was the same in those that received S as in those receiving 2 doses of ( + ) M K 801 on D a y 1. In the next series of experiments, we further increased the dose of ( + ) M K - 8 0 1 and gave 2 doses of ( + ) M K - 8 0 1 on D a y 1 and an additional dose on D a y 2. The results from 3 separate experiments were p o o l e d and are shown in Fig. 5b. O n D a y 2, rapid tolerance de-
255
Effect of ( +)MK-80I and ketamine on the expression of rapid tolerance to chlordiazepoxide and ethanol (Expt.
DAY 2
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DAY 2 p(O.O01
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Rapid tolerance to C was seen on Day 2 in both the control group (t12 = 6.04, P < 0.001) and the group given (+)MK-801 only on Day 2 (t12 = 2.33, P < 0.05) (Fig. 6a). Similarly, rapid tolerance to E was seen on Day 2 in both the control group (t12 = 5.43, P < 0.001) and the group pretreated with ketamine only on Day 2 (t12 = 4.93, P < 0.001) (Fig. 6b). This suggests that NMDA antagonists do not affect the expression of rapid tolerance to C or E. (+)MK-801 or ketamine administration did not affect blood ethanol concentrations in any of the experiments conducted. It is therefore unlikely that pharmacokinetic factors are playing a significant role in the findings detailed above. DISCUSSION
p(O.O01
40
- 301
6).
$ - E E-E
CONTROL KETAMINE
Fig. 6. Effects of (+)MK-801 or ketamine on the expression of rapid tolerance to C or E. a: Two groups of rats were pretreated with S and then given either S or C on Day 1. On Day 2, 1 of the 2 groups was once again pretreated with S while the other received (+)MK-801; all animals received C. (+)MK-801 was not able to prevent the expression of C rapid tolerance. Open bars, S (vehicle), and striped bars, C (treatment dose), given on the first day. b: Two groups of rats were pretreated with S and then given either S or E on Day 1. On Day 2, 1 of the 2 groups was once again pretreated with S while the other received ketamine; all animals received E. Ketamine was not able to prevent the expression of E rapid tolerance. Open bars, S (vehicle), and striped bars, E (treatment dose), given on the first day. Results are presented as mean + S.E.M. of 7 animals.
velopment was again seen in the groups receiving C-C, in either the presence or absence of (+)MK-801. A twoway ANOVA showed an effect of C treatment (F1,78 = 31.48, P < 0.0001) and as well as a significant effect of (+)MK-801 pretreatment (F1,78 = 20.61, P < 0.0001). There was, however, no significant pretreatment x treatment interaction (F1,7s = 3.53, P < 0.064), suggesting that (+)MK-801 is not able to block tolerance to C as clearly as it does tolerance to E.
(+)MK-801 and ketamine failed to block rapid tolerance to C as assessed on the tilt plane (Figs. 3 and 5), in clear contrast to their ability to block rapid tolerance to E 14 (Expt. 4), as well as cross-tolerance from E to C and vice versa (Figs. 1 and 2). It could be argued that the failure to see rapid tolerance to E on the second day is a phenomenon related to state-dependent learning. The effects of the NMDA antagonist + E, as perceived by the rat on Day 1, may be different from the effects of E alone on Day 2, resulting in a failure to display rapid tolerance. However, when ketamine was administered before E on both days, still no tolerance was seen (Fig. 4). Further, when ketamine was given only on Day 2 (Fig. 6b), it did not block the expression of rapid tolerance to E, suggesting that ketamine blocks the acquisition of rapid tolerance to E. The lack of cross-tolerance in the E - C experiment (Fig. 1) is also likely due to the NMDA antagonists blocking the acquisition of rapid tolerance to E after the first dose; this would explain why no rapid cross-tolerance to C was seen on the second day. However, the fact that rapid cross-tolerance from C to E (Fig. 2) was blocked by the NMDA antagonists, whereas rapid tolerance to C itself was not, argues against the idea of NMDA antagonists retarding the acquisition of rapid tolerance in general. It does not appear that NMDA antagonists block expression of rapid tolerance to either E or C, since (+)MK-801 and ketamine, when administered only on Day 2, were not able to prevent the expression of either E or C rapid tolerance (Fig. 6). However, we have not ruled out the possibility that expression of cross-tolerance from C to E is blocked by NMDA antagonists.
256 It is possible that the dose of N M D A antagonists required to block rapid tolerance to C m a y be higher than the dose required to block rapid tolerance and rapid cross-tolerance to E. H o w e v e r , doubling the D a y 1 ( + ) M K - 8 0 1 dose (Fig. 5a) or even doubling the D a y 1 (+)MK-801 dose plus giving an additional dose on D a y 2 had no significant effect on tolerance to C (Fig. 5b). Therefore, the dose of the N M D A antagonist does not a p p e a r to be a factor in the failure of N M D A antagonists to prevent rapid tolerance to C. It should also be noted that ( + ) M K - 8 0 1 , but not ketamine, usually produced some m o t o r incoordination on its own and enhanced the effects of E and C in an additive fashion. ( + ) M K - 8 0 1 has also been r e p o r t e d to enhance the hypnotic effects of ethanol in mice TM. Thus, if anything, ( + ) M K - 8 0 1 should have increased the stimulus for rapid tolerance and cross-tolerance d e v e l o p m e n t in rats concurrently receiving ( + ) M K - 8 0 1 + E (or C) on D a y 1. Since ethanol, like ( + ) M K - 8 0 1 , inhibits N M D A - a c t i vated ion currents 16, some sort of interaction at the level of the N M D A - r e c e p t o r might explain why tolerance to E, but not to C, is inhibited by (+)MK-801. To our knowledge, no one has r e p o r t e d any effects of benzodiazepines on N M D A - s t i m u l a t e d cation currents. A specific interaction b e t w e e n ( + ) M K - 8 0 1 and E would also explain the ability of ( + ) M K - 8 0 1 to prevent cross-tolerance from E to C; ( + ) M K - 8 0 1 could be blocking the acquisition of tolerance to E and consequently, indirectly, the expression of cross-tolerance to C. H o w e v e r , this
antagonism of tolerance d e v e l o p m e n t to E could not explain why ( + ) M K - 8 0 1 also blocks cross-tolerance from C to E (Fig. 2), while not affecting tolerance to C (Fig. 3), especially since the expression of tolerance to E is not blocked by N M D A antagonists (Fig. 6). In conclusion, our results suggest a number of possibilities: (1) that N M D A antagonists affect not only the acquisition of rapid tolerance or cross-tolerance to sedatives but possibly also the ability to express that tolerance, depending on the drugs used; (2) that there is a basic difference between the p h e n o m e n a of rapid tolerance and rapid cross-tolerance that has previously not been r e p o r t e d in the literature; or (3) that there is an incomplete overlap in the mechanisms of action of E and C and in the ways rapid tolerance and cross-tolerance develop to them. Some support for this third possibility m a y be p r o v i d e d by the observation that the changes in d o s e - r e s p o n s e curves during the d e v e l o p m e n t of tolerance to E are not identical to those in tolerance to C xS. F u r t h e r studies characterizing the effects of N M D A antagonists on the d e v e l o p m e n t of rapid tolerance and cross-tolerance to sedatives are in progress.
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Acknowledgments: This work was supported by NIAAA Grant AA08212-02 and the Addiction Research Foundation of Ontario. The authors would like to thank Mrs. Anita Chau for technical assistance. The views expressed in this paper are those of the authors, and do not necessarily represent the policy of the Addiction Research Foundation.
