Brain Research, 562 (1991) 164-168 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 A DONL~ 000689939124870T
164
BRES 24870
Repeated administration of MK-801 produces sensitization to its own locomotor stimulant effects but blocks sensitization to amphetamine Marina
E. Wolf and Mohamad
R. Khansa
Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, MI 48207 (U.S.A.)
(Accepted 9 July 1991) Key words: Amphetamine; Sensitization; Locomotion; MK-801; Glutamate; N-Methyl-D-aspartate
Repeated amphetamine administration produced behavioral sensitization to subsequent amphetamine challenge. The development of sensitization was blocked by coadministration of the N-methyl-D-aspartate (NMDA) antagonist MK-801. Conditioned locomotion, as revealed by saline challenge, was also blocked by MK-801, suggesting that behavioral sensitization and conditioned locomotion may share a requirement for NMDA receptor stimulation. Repeated MK-801 administration produced behavioral sensitization to MK-801 but not amphetamine challenge, suggesting that MK-801 itself produces sensitization through a different mechanism than amphetamine. Repeated administration of amphetamine (AMPH) and other psychomotor stimulants results in an augmentation of their locomotor activating effects, a phenomenon known as behavioral sensitization. Dopamine (DA) neurons projecting from the A10 region to the nucleus accumbens are thought to play an important role in the locomotor activating effects of psychomotor stimulants and in the development of sensitization t2. Behavioral sensitization resembles long-term potentiation (LTP), a well-characterized form of synaptic plasticity in the CNS, in that both involve persistent changes in neuronal responsiveness and both share certain properties with associative learning. It is well established that stimulation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors is required for certain forms of LTP in the hippocampus 9. It was recently demonstrated that behavioral sensitization to AMPH and cocaine may also require N M D A receptor stimulation since it is blocked by MK-8015"6. MK-801 is a non-competitive NMDA receptor antagonist which acts as an agonist at the phencyclidine (PCP) receptor located in the NMDA ion channel complex 18. The purpose of the present study was to investigate further the role of N M D A receptor stimulation in behavioral sensitization to AMPH and to examine the effects of acute and repeated administration of MK-801 on locomotor activity. Male Harlan Sprague-Dawley rats were used in these studies. They were housed, two/cage, with constant temperature, humidity and a 12-h light/dark cycle. Locomo-
tion was monitored in 12 photobeam chambers (San Diego Instruments) located in a separate testing room within the animal colony. Each chamber was 50 x 30 cm, with 3 photocells (separated by 10.5 cm) located lengthwise, 3.5 cm above the floor. Standard polyethylene rat cages were set inside each chamber. One rat was placed in each chamber and its activity was measured automatically with a personal computer using SDI software which provides user-defined intervals of total photobeam interruptions as well as ambulation counts. Ambulation is defined as breaking of consecutive photobeams and separates true horizontal locomotion from repetitive interruptions of the same photobeam produced by stereotyped movements. Both measurements are helpful because sensitization can sometimes be manifest as a shift from horizontal locomotion to stereotyped behavior. In other words, a dose of amphetamine which produces horizontal locomotion in a naive rat may become sufficient to elicit stereotypy after the rat is sensitized. On test days, rats were habituated to the photobeam chambers for 30 min prior to AMPH injection. Activity was measured for 2 h following AMPH injection. The following paradigm was used to examine behavioral sensitization after repeated AMPH or MK-801 treatment. Rats were randomly assigned to 4 groups: repeated saline (SAL), repeated AMPH, repeated MK801, or repeated AMPH + MK-801. On the initial test day, all groups received saline. On day 2, the AMPH and AMPH + MK-801 groups received 0.5 mg/kg AMPH
Correspondence: M.E. Wolf, Department of Psychiatry, Lafayette Clinic, 951 E. Lafayette, Detroit, MI 48207, U.S.A.
165 TABLE I
Effect of acute drug administration on locomotor activity Data are presented as the m e a n s ± S.E.M. n = 6 rats per group. Asterisks indicate significant (P < 0.05) differences from SAL group.
Drug treatment
Ambulation counts
Total counts
SAL A M P H (1.0 mg/kg) MK-801 (0.25 mg/kg) A M P H + MK-801
104 190 172 192
417 783 851 958
± -+ -+
17 23 40 45
± 50 +-- 53* ± 66* - 53*
(i.p.) and the SAL and MK-801 groups received saline. On the third day, the treatment regimen began. The SAL group received daily injections of saline on days 3-5 and 8-12. The A M P H group received daily injections of A M P H (1 mg/kg, i.p.). The MK-801 group received daily injections of MK-801 (0.25 mg/kg, i.p.). The A M P H + MK-801 group received daily injections of MK-801 (0.25 mg/kg) followed 30 min later by A M P H (1 mg/kg). No injections were made on days 6-7 and 13-14. Rats were tested for locomotion on days 1, 2, 3, 5, 8 and 12. On days 15 and 16, all groups were tested with saline and 0.5 mg/kg A M P H , respectively. A lower dose of A M P H was used on test days (0.5 mg/kg) than treatment days (1.0 mg/kg) to avoid the induction of stereotypy in AMPH-sensitized rats. On day 19, all groups were tested with 0.25 mg/kg MK-801. Data from 2 experiments were pooled (n = 6 rats per group in each experiment) and analyzed by one-way analysis of variance. Post hoc analyses were performed using a Newman-Keuls' test. This treatment regimen allows several important comparisons to be made, 3 of which will be focused upon in this study: (1) between-groups on day 15 following saline challenge (a measure of conditioned locomotion resuiting from pairing of A M P H injection with placement in the locomotor activity chamber; see below), (2) between-groups on day 16 following A M P H challenge (AMPH sensitization), (3) between-groups on day 19 following MK-801 challenge (MK-801 sensitization). Locomotor activity in response to saline, A M P H , MK-801, and A M P H + MK-801 on the first treatment day is shown in Table I. MK-801 produced almost as great a stimulation of locomotor activity as AMPH. The locomotor stimulant effects of MK-801 have been reported previously 3"7"16. The combination of A M P H and MK-801 produced approximately the same increase in locomotion as either drug administered alone. This lack of additivity could reflect either a common mechanism for stimulating locomotion or an attenuation of the acute locomotor stimulatory effects of A M P H by MK-801.
Studies are in progress to address this issue. Fig. 1 shows the results obtained when rats in all of the repeated treatment groups were challenged on day 16 with 0.5 mg/kg AMPH. The A M P H group exhibited significantly greater ambulation and total counts than the SAL group, indicating the development of robust behavioral sensitization to AMPH. In other experiments, the behavioral sensitization produced by this paradigm was shown to persist for 1 month (SAL group ambulation counts = 152 +-- 24; A M P H group ambulation counts = 230 +- 19; significantly different, P < 0.05). MK-801, when administered repeatedly with AMPH, appeared to block the development of behavioral sensitization to A M P H since the A M P H + MK-801 group did not exhibit a significantly different response to A M P H challenge than the SAL group. The MK-801 group also did not differ significantly from the SAL group, suggesting that repeated MK-801 treatment does not sensitize rats to AMPH. In light of the locomotor stimulant effects of MK-801 presented in Table I and described by other investigators 3'7'16, it is not entirely surprising that repeated administration of MK-801 was found to produce behavioral sensitization. These results are shown in Fig. 2, which illustrates the response of the 4 repeated treatment groups to challenge with MK-801 on day 19. The MK-801 and A M P H + MK-801 groups exhibited a robust increase in ambulation and total counts as compared to both the SAL and A M P H groups. These resuits suggest that repeated treatment with MK-801 produces behavioral sensitization to subsequent challenge with MK-801 and that A M P H does not block the
[] II
SAL/AMPH MK 801 /AMPH
I ~ AM~H/AMPH 1000 I ~ AMPH + MK801/AMPH I
800
f-
600
o u 3~
400
._>
200 0 Ambulation
Totat counts
Fig. 1. Rats treated repeatedly with SAL, A M P H , MK-801 or A M P H + MK-801 were challenged with 0.5 mg/kg A M P H (day 16). Data are presented as the m e a n ± S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the S A L group. T h e A M P H group also differed significantly from the MK-801 and A M P H + MK-801 groups.
166 development of behavioral sensitization to MK-801. These data also demonstrate that A M P H and MK-801 do not cross-sensitize since the repeated A M P H group exhibits sensitization to A M P H challenge (Fig. 1) but not MK-801 challenge (Fig. 2). It should be noted that the MK-801 challenge was performed 3 days after the AMPH challenge. However, we have shown that the AMPH sensitization produced by our treatment regimen persists for at least 1 month (see above). Thus, the repeated AMPH rats were still sensitized to A M P H at the time they were challenged with MK-801. It is well known that the behavioral activating effects of A M P H and other psychostimulants are controlled by environmental stimuli and that repeated pairing of psychostimulant administration with a particular set of environmental stimuli can result in the production of locomotion by exposure to the stimuli alone ~5. This is known as conditioned locomotion, and is studied by administering repeated injections of a psychostimulant and then measuring locomotion on a subsequent test day when animals are placed in the testing environment without drug. To determine whether conditioned locomotion had occurred in the present study, all rats were placed in photobeam chambers and challenged with saline on day 15 (Fig. 3). When challenged with saline, the A M P H group exhibited significantly greater ambulation and total counts than the SAL group. This suggests that repeated pairing of A M P H administration with the environmental stimuli of placement in the photobeam chamber had resulted in conditioned locomotion; in other words, the conditioned stimulus (placement in the chamber) became capable of eliciting the same response as the unconditioned stimulus (AMPH). Interestingly, the
AMPH + MK-801 group did not differ from the SAL group in response to saline challenge, suggesting that MK-801 blocks conditioned locomotion as well as behavioral sensitization. When challenged with saline, the MK-801 group exhibited a small, but statistically significant, increase in ambulation and total counts as compared to the SAL group (Fig. 3). This is consistent with the fact that MK801 itself produced an increase in locomotor activity during the treatment days as compared to the SAL group and would therefore be expected to produce conditioned locomotion. In summary, the present study demonstrates that MK801, when administered repeatedly with AMPH, blocks the development of behavioral sensitization to A M P H in rats. These results are consistent with a previous study in which 0.25 mg/kg MK-801 blocked the development of sensitization to the locomotor stimulant effects of A M P H (4 mg/kg, i.p.) and cocaine (20 mg/kg, s.c.) in mice 5. This study differed from the present one in that it utilized higher doses of A M P H and mice rather than rats, and was performed by placing 3 mice in each activity chamber and measuring their aggregate locomotor activity. MK-801 has also been reported to block sensitization of mice and rats to stereotyped behavior and convulsions produced by higher doses of AMPH and cocaine 5,~. The mechanism by which MK-801 blocks sensitization is unknown. Recent studies have shown that MK-801 does not prevent metamphetamine-stimulated D A release from striatal slices ], consistent with the fact that MK-801 is a poor inhibitor of D A uptake in vitro 14 and does not interact with the DA transporter in in vivo
I--'7 SAL/SAL
F-'I SAL/MK 801
I 1500
MKS01
IMK 801
AMPH /MK 801 R ~ A M P H + MK801 /MKS01
"~
500
1200
I
MKs01/SAL
I~ I~
AMPH/SAL AMPH + MK801 /SAL
400
~
bq 8O
\\\ 300
900 -
"+-
u
""" \\%
0--
0
Ambulation
Total counts
Fig. 2. Rats treated repeatedly with SAL, A M P H , MKo801 or A M P H + MK-801 were challenged with 0.25 mg/kg MK-801 (day 19). Data are presented as the m e a n -+ S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the SAL and A M P H groups.
i Ambulation
\\\ Total counts
Fig. 3. Rats treated repeatedly with SAL, A M P H , MK-801 or A M P H + MK-801 were challenged with S A L (day 15). Data are presented as the m e a n -+ S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the S A L and A M P H + MK-801 groups.
167 binding experiments 1°. These results suggest that MK801 does not interfere with AMPH-induced DA release, but may act instead to modify either the effect of D A on its target cells or other neuronal events subsequen t to D A receptor activation. In addition, the present results show for the first time that MK-801 blocks the development of conditioned locomotion. The relationship between conditioned locomotion and behavioral sensitization is complex. While behavioral sensitization can, under certain circumstances, come under the control of conditioned stimuli 15, it is apparent that conditioning alone cannot account for the phenomenon of behavioral sensitization (see ref. 12 for review). Thus, the present findings do not imply that MK-801 prevents behavioral sensitization by virtue of its ability to block conditioning. Rather, these findings suggest that two related phenomena (behavioral sensitization to AMPH and the control of its expression by conditioning) may share a common requirement for stimulation of NMDA receptors. Another novel finding is that repeated administration of MK-801 results in behavioral sensitization to a subsequent challenge with MK-801. Since rats sensitized to MK-801 did not exhibit cross-sensitization to AMPH, and vice versa, it is likely that AMPH sensitization and MK-801 sensitization occur via different mechanisms. Sensitization to MK-801 has been found to persist for at least 3 weeks after the final treatment day. Thus, when the repeated SAL group was first challenged with MK801, they exhibited ambulation counts of 120 -+ 24 and total counts of 460 _+ 35. The repeated MK-801 group was first challenged with MK-801 one week after the final treatment day (results shown in Fig. 2). When they were challenged again with MK-801 2 weeks later, they exhibited approximately the same degree of sensitization as shown in Fig. 2 (ambulation counts of 453 -+ 72 and total counts of 1336 +- 153; significantly different from repeated SAL, P < 0.05).
In conclusion, the present findings, along with previous reports 5'6, suggest that the development of behavioral sensitization requires stimulation of NMDA receptors since it is blocked by the N M D A receptor antagonist MK-801. MK-801 has also been shown to block other types of changes in the intensity of neuronal responses resulting from repeated presentation of stimuli, such as the development of morphine tolerance and dependence 17, the priming effect of long-term D i agonist administration in neonatal 6-hydroxydopamine-lesioned rats 2, and the priming effect of D A agonists on D 1 agonist-induced turning in unilaterally 6-hydroxydopaminelesioned rats ll. These results may suggest a common role for NMDA receptors in mediating long-term changes in the responsiveness of dopaminergic and other neurotransmitter systems. It should be noted that this conclusion rests upon data obtained exclusively with MK-801, a non-competitive NMDA antagonist. Non-competitive NMDA antagonists act by occupying the phencyclidine (PCP) binding site within the NMDA receptor ion channel s. In general, non-competitive and competitive NMDA antagonists exert similar effects 13'16. However, this is not always the case. For example, many effects of PCP on dopaminergic neurons appear to reflect its interaction with the D A transporter rather than the NMDA receptor 4. MK-801 does not appear to share this property of PCP l°a4. However, the hypothesized role of NMDA receptors in behavioral sensitization would be strengthened by the demonstration that competitive NMDA antagonists share the ability of non-competitive antagonists to block its induction.
1 Bowyer, J.E, Scallet, A.C., Holson, R.R., Lipe, G.W., Slikker Jr., W. and Ali, S.E, Interactions of MK-801 with glutamate-, glutamine- and metamphetamine-evoked release of [3H]dopamine from striatal slices, J. Pharmacol. Exp. Ther., 257 (1991) 262-270. 2 Criswell, H.E., Mueller, R.A. and Breese, G.R., Long-term D~-dopamine receptor sensitization in neonatal 6-OHDA-lesioned rats is blocked by an NMDA antagonist, Brain Research, 512 (1990) 284-290. 3 Hiramatsu, M., Cho, A.K. and Nabeshima, T., Comparison of the behavioral and biochemical effects of the NMDA receptor antagonists, MK-801 and phencyclidine, Eur. J. Pharmacol., 166 (1989) 359-366. 4 Johnson, K.M. and Jones, S.M., Neuropharmacology of phencyclidine: basic mechanisms and therapeutic potential, Annu. Rev. Pharmacol. Toxicol., 30 (1990) 707-750. 5 Karler, R., Calder, L.D., Chaudhry, I.A. and Turkanis, S.A.,
Blockade of 'reverse tolerance' to cocaine and amphetamine by MK-801, Life Sci., 45 (1989) 599-606. 6 Kader, R., Chaudhry, I.A., Calder, L.D. and Turkanis, S.A., Amphetamine sensitization and the excitatory amino acids, Brain Research, 537 (1990) 76-82. 7 Koek, W., Woods, J.H. and Winger, G.D., MK-801, a proposed noncompetitive antagonist of excitatory amino acid neurotransmission, produces phencyclidine-like behavioral effects in pigeons, rats and rhesus monkeys, J. Pharmacol. Exp. Ther., 245 (1988) 969-974. 8 Lodge, D. and Johnson, K.M., Noncompetitive excitatory amino acid receptor antagonists, Trends Pharmacol. Sci., 11
We are grateful to Dr. Francis J. White for advice on experimental design and for helpful discussions. Supported by the National Alliance for Research on Schizophrenia and Depression and the Pharmaceutical Manufacturers Association Foundation.
(1990) 81-86. 9 Madison, D.V., Malenka, R.C. and Nicoll, R.A., Mechanisms underlying long-term potentiation of synaptic transmission, Annu. Rev. Neurosci., 14 (1991) 379-397. 10 Maurice, T., Vignon, J., Kamenka, J.-M. and Chicheportiche,
168
11
12
13
14
R., Differential interaction of phencyclidine-like drugs with the dopamine uptake complex in vivo, J. Neurochem., 56 (1991) 553-559. Morelli, M. and Di Chiara, G., Stereospecific blockade of N-methyI-D-aspartate transmission by MK-801 prevents priming of SKF 38393-induced turning, Psychopharmacology, 101 (1990) 287-288. Robinson, T.E. and Becker, J.B., Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev., 11 (1986) 157-198. Snell, L.D. and Johnson, K.M., Phencyclidine: behavioral correlates of NMDA antagonism. In D. Lodge (Ed.), Excitatory Amino Acids in Health and Disease, Wiley, Chichester, 1988, pp. 261-273. Snell, L.D., Yi, S.-J. and Johnson, K.M. Comparison of the effects of MK-801 and phencyclidine on catecholamine uptake and NMDA-induced norepinephrine release, Eur. J. Pharma-
col., 145 (1988) 223-226. 15 Stewart, J. and Vezina, P., Conditioning and behavioral sensitization. In P.W. Kalivas and C.D. Barnes (Eds.), Sensitization in the Nervous System, Telford Press, Caldwell, NJ, 1988, pp. 207-224. i6 Tricklebank, M.D., Singh, L., Oles, R.J., Preston, C. and lversen, S.D., The behavioural effects of MK-801: a comparison with antagonists acting non-competitively and competitively at the NMDA receptor, Eur. J. Pharmacol., 167 (1989) 127135. 17 Trujillo, K.A. and Akil, H., Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801, Science, 251 (1991) 85-87. 18 Wong, E.H.F., Kemp, J.A., Priestly, T., Knight, A.R., Woodruff, G.N. and lversen, L.L., The anticonsulvant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. ScL U.S.A., 83 (1986) 7104-7108.
164
BRES 24870
Repeated administration of MK-801 produces sensitization to its own locomotor stimulant effects but blocks sensitization to amphetamine Marina
E. Wolf and Mohamad
R. Khansa
Cellular and Clinical Neurobiology Program, Department of Psychiatry, Wayne State University School of Medicine, Detroit, MI 48207 (U.S.A.)
(Accepted 9 July 1991) Key words: Amphetamine; Sensitization; Locomotion; MK-801; Glutamate; N-Methyl-D-aspartate
Repeated amphetamine administration produced behavioral sensitization to subsequent amphetamine challenge. The development of sensitization was blocked by coadministration of the N-methyl-D-aspartate (NMDA) antagonist MK-801. Conditioned locomotion, as revealed by saline challenge, was also blocked by MK-801, suggesting that behavioral sensitization and conditioned locomotion may share a requirement for NMDA receptor stimulation. Repeated MK-801 administration produced behavioral sensitization to MK-801 but not amphetamine challenge, suggesting that MK-801 itself produces sensitization through a different mechanism than amphetamine. Repeated administration of amphetamine (AMPH) and other psychomotor stimulants results in an augmentation of their locomotor activating effects, a phenomenon known as behavioral sensitization. Dopamine (DA) neurons projecting from the A10 region to the nucleus accumbens are thought to play an important role in the locomotor activating effects of psychomotor stimulants and in the development of sensitization t2. Behavioral sensitization resembles long-term potentiation (LTP), a well-characterized form of synaptic plasticity in the CNS, in that both involve persistent changes in neuronal responsiveness and both share certain properties with associative learning. It is well established that stimulation of the N-methyl-D-aspartate (NMDA) class of glutamate receptors is required for certain forms of LTP in the hippocampus 9. It was recently demonstrated that behavioral sensitization to AMPH and cocaine may also require N M D A receptor stimulation since it is blocked by MK-8015"6. MK-801 is a non-competitive NMDA receptor antagonist which acts as an agonist at the phencyclidine (PCP) receptor located in the NMDA ion channel complex 18. The purpose of the present study was to investigate further the role of N M D A receptor stimulation in behavioral sensitization to AMPH and to examine the effects of acute and repeated administration of MK-801 on locomotor activity. Male Harlan Sprague-Dawley rats were used in these studies. They were housed, two/cage, with constant temperature, humidity and a 12-h light/dark cycle. Locomo-
tion was monitored in 12 photobeam chambers (San Diego Instruments) located in a separate testing room within the animal colony. Each chamber was 50 x 30 cm, with 3 photocells (separated by 10.5 cm) located lengthwise, 3.5 cm above the floor. Standard polyethylene rat cages were set inside each chamber. One rat was placed in each chamber and its activity was measured automatically with a personal computer using SDI software which provides user-defined intervals of total photobeam interruptions as well as ambulation counts. Ambulation is defined as breaking of consecutive photobeams and separates true horizontal locomotion from repetitive interruptions of the same photobeam produced by stereotyped movements. Both measurements are helpful because sensitization can sometimes be manifest as a shift from horizontal locomotion to stereotyped behavior. In other words, a dose of amphetamine which produces horizontal locomotion in a naive rat may become sufficient to elicit stereotypy after the rat is sensitized. On test days, rats were habituated to the photobeam chambers for 30 min prior to AMPH injection. Activity was measured for 2 h following AMPH injection. The following paradigm was used to examine behavioral sensitization after repeated AMPH or MK-801 treatment. Rats were randomly assigned to 4 groups: repeated saline (SAL), repeated AMPH, repeated MK801, or repeated AMPH + MK-801. On the initial test day, all groups received saline. On day 2, the AMPH and AMPH + MK-801 groups received 0.5 mg/kg AMPH
Correspondence: M.E. Wolf, Department of Psychiatry, Lafayette Clinic, 951 E. Lafayette, Detroit, MI 48207, U.S.A.
165 TABLE I
Effect of acute drug administration on locomotor activity Data are presented as the m e a n s ± S.E.M. n = 6 rats per group. Asterisks indicate significant (P < 0.05) differences from SAL group.
Drug treatment
Ambulation counts
Total counts
SAL A M P H (1.0 mg/kg) MK-801 (0.25 mg/kg) A M P H + MK-801
104 190 172 192
417 783 851 958
± -+ -+
17 23 40 45
± 50 +-- 53* ± 66* - 53*
(i.p.) and the SAL and MK-801 groups received saline. On the third day, the treatment regimen began. The SAL group received daily injections of saline on days 3-5 and 8-12. The A M P H group received daily injections of A M P H (1 mg/kg, i.p.). The MK-801 group received daily injections of MK-801 (0.25 mg/kg, i.p.). The A M P H + MK-801 group received daily injections of MK-801 (0.25 mg/kg) followed 30 min later by A M P H (1 mg/kg). No injections were made on days 6-7 and 13-14. Rats were tested for locomotion on days 1, 2, 3, 5, 8 and 12. On days 15 and 16, all groups were tested with saline and 0.5 mg/kg A M P H , respectively. A lower dose of A M P H was used on test days (0.5 mg/kg) than treatment days (1.0 mg/kg) to avoid the induction of stereotypy in AMPH-sensitized rats. On day 19, all groups were tested with 0.25 mg/kg MK-801. Data from 2 experiments were pooled (n = 6 rats per group in each experiment) and analyzed by one-way analysis of variance. Post hoc analyses were performed using a Newman-Keuls' test. This treatment regimen allows several important comparisons to be made, 3 of which will be focused upon in this study: (1) between-groups on day 15 following saline challenge (a measure of conditioned locomotion resuiting from pairing of A M P H injection with placement in the locomotor activity chamber; see below), (2) between-groups on day 16 following A M P H challenge (AMPH sensitization), (3) between-groups on day 19 following MK-801 challenge (MK-801 sensitization). Locomotor activity in response to saline, A M P H , MK-801, and A M P H + MK-801 on the first treatment day is shown in Table I. MK-801 produced almost as great a stimulation of locomotor activity as AMPH. The locomotor stimulant effects of MK-801 have been reported previously 3"7"16. The combination of A M P H and MK-801 produced approximately the same increase in locomotion as either drug administered alone. This lack of additivity could reflect either a common mechanism for stimulating locomotion or an attenuation of the acute locomotor stimulatory effects of A M P H by MK-801.
Studies are in progress to address this issue. Fig. 1 shows the results obtained when rats in all of the repeated treatment groups were challenged on day 16 with 0.5 mg/kg AMPH. The A M P H group exhibited significantly greater ambulation and total counts than the SAL group, indicating the development of robust behavioral sensitization to AMPH. In other experiments, the behavioral sensitization produced by this paradigm was shown to persist for 1 month (SAL group ambulation counts = 152 +-- 24; A M P H group ambulation counts = 230 +- 19; significantly different, P < 0.05). MK-801, when administered repeatedly with AMPH, appeared to block the development of behavioral sensitization to A M P H since the A M P H + MK-801 group did not exhibit a significantly different response to A M P H challenge than the SAL group. The MK-801 group also did not differ significantly from the SAL group, suggesting that repeated MK-801 treatment does not sensitize rats to AMPH. In light of the locomotor stimulant effects of MK-801 presented in Table I and described by other investigators 3'7'16, it is not entirely surprising that repeated administration of MK-801 was found to produce behavioral sensitization. These results are shown in Fig. 2, which illustrates the response of the 4 repeated treatment groups to challenge with MK-801 on day 19. The MK-801 and A M P H + MK-801 groups exhibited a robust increase in ambulation and total counts as compared to both the SAL and A M P H groups. These resuits suggest that repeated treatment with MK-801 produces behavioral sensitization to subsequent challenge with MK-801 and that A M P H does not block the
[] II
SAL/AMPH MK 801 /AMPH
I ~ AM~H/AMPH 1000 I ~ AMPH + MK801/AMPH I
800
f-
600
o u 3~
400
._>
200 0 Ambulation
Totat counts
Fig. 1. Rats treated repeatedly with SAL, A M P H , MK-801 or A M P H + MK-801 were challenged with 0.5 mg/kg A M P H (day 16). Data are presented as the m e a n ± S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the S A L group. T h e A M P H group also differed significantly from the MK-801 and A M P H + MK-801 groups.
166 development of behavioral sensitization to MK-801. These data also demonstrate that A M P H and MK-801 do not cross-sensitize since the repeated A M P H group exhibits sensitization to A M P H challenge (Fig. 1) but not MK-801 challenge (Fig. 2). It should be noted that the MK-801 challenge was performed 3 days after the AMPH challenge. However, we have shown that the AMPH sensitization produced by our treatment regimen persists for at least 1 month (see above). Thus, the repeated AMPH rats were still sensitized to A M P H at the time they were challenged with MK-801. It is well known that the behavioral activating effects of A M P H and other psychostimulants are controlled by environmental stimuli and that repeated pairing of psychostimulant administration with a particular set of environmental stimuli can result in the production of locomotion by exposure to the stimuli alone ~5. This is known as conditioned locomotion, and is studied by administering repeated injections of a psychostimulant and then measuring locomotion on a subsequent test day when animals are placed in the testing environment without drug. To determine whether conditioned locomotion had occurred in the present study, all rats were placed in photobeam chambers and challenged with saline on day 15 (Fig. 3). When challenged with saline, the A M P H group exhibited significantly greater ambulation and total counts than the SAL group. This suggests that repeated pairing of A M P H administration with the environmental stimuli of placement in the photobeam chamber had resulted in conditioned locomotion; in other words, the conditioned stimulus (placement in the chamber) became capable of eliciting the same response as the unconditioned stimulus (AMPH). Interestingly, the
AMPH + MK-801 group did not differ from the SAL group in response to saline challenge, suggesting that MK-801 blocks conditioned locomotion as well as behavioral sensitization. When challenged with saline, the MK-801 group exhibited a small, but statistically significant, increase in ambulation and total counts as compared to the SAL group (Fig. 3). This is consistent with the fact that MK801 itself produced an increase in locomotor activity during the treatment days as compared to the SAL group and would therefore be expected to produce conditioned locomotion. In summary, the present study demonstrates that MK801, when administered repeatedly with AMPH, blocks the development of behavioral sensitization to A M P H in rats. These results are consistent with a previous study in which 0.25 mg/kg MK-801 blocked the development of sensitization to the locomotor stimulant effects of A M P H (4 mg/kg, i.p.) and cocaine (20 mg/kg, s.c.) in mice 5. This study differed from the present one in that it utilized higher doses of A M P H and mice rather than rats, and was performed by placing 3 mice in each activity chamber and measuring their aggregate locomotor activity. MK-801 has also been reported to block sensitization of mice and rats to stereotyped behavior and convulsions produced by higher doses of AMPH and cocaine 5,~. The mechanism by which MK-801 blocks sensitization is unknown. Recent studies have shown that MK-801 does not prevent metamphetamine-stimulated D A release from striatal slices ], consistent with the fact that MK-801 is a poor inhibitor of D A uptake in vitro 14 and does not interact with the DA transporter in in vivo
I--'7 SAL/SAL
F-'I SAL/MK 801
I 1500
MKS01
IMK 801
AMPH /MK 801 R ~ A M P H + MK801 /MKS01
"~
500
1200
I
MKs01/SAL
I~ I~
AMPH/SAL AMPH + MK801 /SAL
400
~
bq 8O
\\\ 300
900 -
"+-
u
""" \\%
0--
0
Ambulation
Total counts
Fig. 2. Rats treated repeatedly with SAL, A M P H , MKo801 or A M P H + MK-801 were challenged with 0.25 mg/kg MK-801 (day 19). Data are presented as the m e a n -+ S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the SAL and A M P H groups.
i Ambulation
\\\ Total counts
Fig. 3. Rats treated repeatedly with SAL, A M P H , MK-801 or A M P H + MK-801 were challenged with S A L (day 15). Data are presented as the m e a n -+ S.E.M. for 12 rats per group. Asterisks indicate significant (P < 0.05) differences from the S A L and A M P H + MK-801 groups.
167 binding experiments 1°. These results suggest that MK801 does not interfere with AMPH-induced DA release, but may act instead to modify either the effect of D A on its target cells or other neuronal events subsequen t to D A receptor activation. In addition, the present results show for the first time that MK-801 blocks the development of conditioned locomotion. The relationship between conditioned locomotion and behavioral sensitization is complex. While behavioral sensitization can, under certain circumstances, come under the control of conditioned stimuli 15, it is apparent that conditioning alone cannot account for the phenomenon of behavioral sensitization (see ref. 12 for review). Thus, the present findings do not imply that MK-801 prevents behavioral sensitization by virtue of its ability to block conditioning. Rather, these findings suggest that two related phenomena (behavioral sensitization to AMPH and the control of its expression by conditioning) may share a common requirement for stimulation of NMDA receptors. Another novel finding is that repeated administration of MK-801 results in behavioral sensitization to a subsequent challenge with MK-801. Since rats sensitized to MK-801 did not exhibit cross-sensitization to AMPH, and vice versa, it is likely that AMPH sensitization and MK-801 sensitization occur via different mechanisms. Sensitization to MK-801 has been found to persist for at least 3 weeks after the final treatment day. Thus, when the repeated SAL group was first challenged with MK801, they exhibited ambulation counts of 120 -+ 24 and total counts of 460 _+ 35. The repeated MK-801 group was first challenged with MK-801 one week after the final treatment day (results shown in Fig. 2). When they were challenged again with MK-801 2 weeks later, they exhibited approximately the same degree of sensitization as shown in Fig. 2 (ambulation counts of 453 -+ 72 and total counts of 1336 +- 153; significantly different from repeated SAL, P < 0.05).
In conclusion, the present findings, along with previous reports 5'6, suggest that the development of behavioral sensitization requires stimulation of NMDA receptors since it is blocked by the N M D A receptor antagonist MK-801. MK-801 has also been shown to block other types of changes in the intensity of neuronal responses resulting from repeated presentation of stimuli, such as the development of morphine tolerance and dependence 17, the priming effect of long-term D i agonist administration in neonatal 6-hydroxydopamine-lesioned rats 2, and the priming effect of D A agonists on D 1 agonist-induced turning in unilaterally 6-hydroxydopaminelesioned rats ll. These results may suggest a common role for NMDA receptors in mediating long-term changes in the responsiveness of dopaminergic and other neurotransmitter systems. It should be noted that this conclusion rests upon data obtained exclusively with MK-801, a non-competitive NMDA antagonist. Non-competitive NMDA antagonists act by occupying the phencyclidine (PCP) binding site within the NMDA receptor ion channel s. In general, non-competitive and competitive NMDA antagonists exert similar effects 13'16. However, this is not always the case. For example, many effects of PCP on dopaminergic neurons appear to reflect its interaction with the D A transporter rather than the NMDA receptor 4. MK-801 does not appear to share this property of PCP l°a4. However, the hypothesized role of NMDA receptors in behavioral sensitization would be strengthened by the demonstration that competitive NMDA antagonists share the ability of non-competitive antagonists to block its induction.
1 Bowyer, J.E, Scallet, A.C., Holson, R.R., Lipe, G.W., Slikker Jr., W. and Ali, S.E, Interactions of MK-801 with glutamate-, glutamine- and metamphetamine-evoked release of [3H]dopamine from striatal slices, J. Pharmacol. Exp. Ther., 257 (1991) 262-270. 2 Criswell, H.E., Mueller, R.A. and Breese, G.R., Long-term D~-dopamine receptor sensitization in neonatal 6-OHDA-lesioned rats is blocked by an NMDA antagonist, Brain Research, 512 (1990) 284-290. 3 Hiramatsu, M., Cho, A.K. and Nabeshima, T., Comparison of the behavioral and biochemical effects of the NMDA receptor antagonists, MK-801 and phencyclidine, Eur. J. Pharmacol., 166 (1989) 359-366. 4 Johnson, K.M. and Jones, S.M., Neuropharmacology of phencyclidine: basic mechanisms and therapeutic potential, Annu. Rev. Pharmacol. Toxicol., 30 (1990) 707-750. 5 Karler, R., Calder, L.D., Chaudhry, I.A. and Turkanis, S.A.,
Blockade of 'reverse tolerance' to cocaine and amphetamine by MK-801, Life Sci., 45 (1989) 599-606. 6 Kader, R., Chaudhry, I.A., Calder, L.D. and Turkanis, S.A., Amphetamine sensitization and the excitatory amino acids, Brain Research, 537 (1990) 76-82. 7 Koek, W., Woods, J.H. and Winger, G.D., MK-801, a proposed noncompetitive antagonist of excitatory amino acid neurotransmission, produces phencyclidine-like behavioral effects in pigeons, rats and rhesus monkeys, J. Pharmacol. Exp. Ther., 245 (1988) 969-974. 8 Lodge, D. and Johnson, K.M., Noncompetitive excitatory amino acid receptor antagonists, Trends Pharmacol. Sci., 11
We are grateful to Dr. Francis J. White for advice on experimental design and for helpful discussions. Supported by the National Alliance for Research on Schizophrenia and Depression and the Pharmaceutical Manufacturers Association Foundation.
(1990) 81-86. 9 Madison, D.V., Malenka, R.C. and Nicoll, R.A., Mechanisms underlying long-term potentiation of synaptic transmission, Annu. Rev. Neurosci., 14 (1991) 379-397. 10 Maurice, T., Vignon, J., Kamenka, J.-M. and Chicheportiche,
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R., Differential interaction of phencyclidine-like drugs with the dopamine uptake complex in vivo, J. Neurochem., 56 (1991) 553-559. Morelli, M. and Di Chiara, G., Stereospecific blockade of N-methyI-D-aspartate transmission by MK-801 prevents priming of SKF 38393-induced turning, Psychopharmacology, 101 (1990) 287-288. Robinson, T.E. and Becker, J.B., Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis, Brain Res. Rev., 11 (1986) 157-198. Snell, L.D. and Johnson, K.M., Phencyclidine: behavioral correlates of NMDA antagonism. In D. Lodge (Ed.), Excitatory Amino Acids in Health and Disease, Wiley, Chichester, 1988, pp. 261-273. Snell, L.D., Yi, S.-J. and Johnson, K.M. Comparison of the effects of MK-801 and phencyclidine on catecholamine uptake and NMDA-induced norepinephrine release, Eur. J. Pharma-
col., 145 (1988) 223-226. 15 Stewart, J. and Vezina, P., Conditioning and behavioral sensitization. In P.W. Kalivas and C.D. Barnes (Eds.), Sensitization in the Nervous System, Telford Press, Caldwell, NJ, 1988, pp. 207-224. i6 Tricklebank, M.D., Singh, L., Oles, R.J., Preston, C. and lversen, S.D., The behavioural effects of MK-801: a comparison with antagonists acting non-competitively and competitively at the NMDA receptor, Eur. J. Pharmacol., 167 (1989) 127135. 17 Trujillo, K.A. and Akil, H., Inhibition of morphine tolerance and dependence by the NMDA receptor antagonist MK-801, Science, 251 (1991) 85-87. 18 Wong, E.H.F., Kemp, J.A., Priestly, T., Knight, A.R., Woodruff, G.N. and lversen, L.L., The anticonsulvant MK-801 is a potent N-methyl-D-aspartate antagonist, Proc. Natl. Acad. ScL U.S.A., 83 (1986) 7104-7108.
