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Apomorphine induces latent rotation in lesioned rats, amphetamine does not
Life Sciences, Vol. 42, pp. 2397-2401 Printed in the U.S.A.
Pergamon Press
APOMORPHINE INDUCES LATENT ROTATION IN LESIONED RATS, AMPHETAMINE DOES NOT Peter B. Silverman Department of Psychiatry and Behavioral Sciences - MSI University of Texas Medical School at Houston Houston, TX 77030 (Received in final form April 6, 1988) Summary Rats with unilateral 6-hydroxydopamine (6HDA) lesions of one nigrostriatal pathway were used to compare the efficacy of apomorphine (Apo) and (+)-amphetamine (Amph) at inducing latent rotational behavior. Rats treated with Apo rotated contralaterally acutely after drug treatment and subsequently, weeks after drug treatment, exhibited rapid contralateral circling when re-introduced to the rotation environment. Amph treated animals, on the other hand, rotated ipsilaterally acutely after drug treatment and exhibited no latent drug effect. Rotational (circling) behavior in unilaterally lesioned rodents has proven to be a popular model for investigation of central dopamine (DA) function. Several years ago we described a striking behavioral consequence of Apo administration in rats with unilateral nigrostriatal lesions (i). When returned to the bowl in which even a single prior Apo treatment had been given, the animals burst into contralateral rotation which lasted 2-3 minutes. Lesioned animals that had no drug experience never exhibited this behavior. This behavior appeared to correspond with the "paradoxical" rotation which Ungerstedt had described earlier but had not attributed to drug treatment (2,3). The behavior did not develop fully until two weeks after treatment, but once developed was observable for at least a year after drug treatment, so long as the undrugged tests were conducted at spaced intervals. The behavior seemed to be learned, but had strange characteristics, such as the long latency to develop and an unusual dose-response effect. Recently, Burunat et al. (4) extended our work and concluded that the behavior they observed after repeated Apo administrations was strictly Pavlovian conditioning (associative learning). The work presented here shows that while the latent rotation after Apo treatment may well be associative learning, it remains unusual in that Amph does not produce an analogous ipsilaterally-directed latent rotation. Methods Male Sprague-Dawley rats (Simonsen, Gilroy, CA) weighing 180-240 g m w e r e anesthetized with 40 mg/kgpentobarbital and mounted in a stereotaxic device. A lesion was produced by infusion of 4 ~i of saline containing 0.1% ascorbate and 2 ~g/pl (base wt) 6-hydroxydopamine HBr. Stereotaxic coordinates, with incisor bar at zero, were 1.4 L and 4.2 P from bregma, 8 mm ventral from the
0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
2398
Latent Rotation
Vol. 42, No. 23, 1988
skull surface. The 6HDA solution was administered at 0.33 ~i/min via syringe pump, i0 ~i syringe, PE I0 tubing and 27 gauge needle. Observations of rotational behavior were made in 26 cm diameter clear plastic bowls. Complete 360 ° turns were counted in three minute observation periods inanediately prior to injection, immediately after injection, and at intervals thereafter. After Apo treatment, observations were made beginning at 0, 15, 30 and 45 minutes after injection. After Amph treatment, observations were made (with an exception noted below) beginning 0, 15, 30, 45, 60, 90 and 120 minutes after injection. In both cases, acute rotation had slowed considerably by the time of the last observation. Subsequent observations of undrugged behavior consisted solely of three minute observation periods beginning immediately after the animal was placed in the bowl. During the intervals between drug tests and undrugged observations, rats remained undisturbed in their home cages with ad lib food and water throughout. Drugs tested were (+)-amphetamine sulfate (Sigma) and apomorphine HCI (Merck). These compounds were administered i.p. and s.c., respectively, in 1 ml/kg saline. Statistical evaluation of undrugged rotation scores within each group was made by a repeated measures analysis of variance followed by a Dunnett's procedure comparing each test subsequent to drug treatment with the pre-drug score obtained prior to any treatment. Results The results, summarized in Table I, show that all groups of animals exhibited a slight ipsilateral bias prior to any drug treatment. Group 1 animals (n = 8) were treated with a single dose of 0.05 mg/kg Apo two weeks after having been lesioned. They exhibited the typical latent (undrugged) rotational response two weeks later when placed in the rotation bowls for a three minute observation period. Group 2 animals (n = 6) were injected with 0.05 mg/kg Apo on three consecutive days two weeks after being lesioned. When placed, undrugged, in the rotation bowl four weeks after being lesioned for a three minute test they exhibited rapid contralateral rotation. Group 3 animals (n = 9) were injected with 2 mg/kg Amph on three consecutive days two weeks after being lesioned. Despite exhibiting good ipsilateral rotation when under the acute influence of the drug, they showed essentially no change in the magnitude of their undrugged response at four weeks after being lesioned. Group 4 animals (n = 7) were treated with 0.25 mg/kg Apo at 4, 6 and 8 weeks after being lesioned. After their first Apo treatment and prior to their second, their undrugged response had shifted from slightly ipsilateral to moderately contralateral. This undrugged contralateral rotation became more pronounced after subsequent treatments. Group 5 animals (n = 5) were injected with 2 mg/kg amphetamine at 4, 6, 8 and ii weeks after lesion. Despite good ipsilateral rotational response to these treatments, there was essentially no change in their undrugged response upon subsequent exposure to the bowl.
Vol. 42, No. 23, 1988
Latent Rotation
2399
Group 6 animals (n = 4) were injected with 5 mg/kg amphetamine at 4, 6 and 8 weeks after lesion. This group exhibited rapid ipsilateral rotation which lasted over two hours. Nonetheless, this group showed essentially no change in their subsequent undrugged response to the rotation environment when tested at ii weeks after lesion. A smaller (0.5 mg/kg) dose was then administered to this group, with three minute observations made up to one hour later, to see if it would induce latent rotation. No such effect was apparent when they were tested at 16 weeks post-lesion.
TABLE I Acute and Latent Rotational Responses to Apo and Amph*
Group (n)
Pre-drug response a
Treatment (mg/kg)
Acute response b (wks post-lesion)
1 (8)
-2 ± 1
Apo (.05)
25 ± 3 (2)
36 ± 7 (4) d
2 (6)
-4 ± 1
Apo (.05) APO (.05) APO (.05) None
26 ± 2 (2) 26 ± 4 (2+1 day) 25 ± 4 (2+2 day) --
4 1 55 57
± ± ± f
2 1 7 6
(2+1 day) (2+ 2 day) (4)~ (8) a
3 (8)
-4 ± 1
Amph (2) Amph (2) Amph (2) None
-20 ± 2 (2) -21 ± 3 (2+1 day) -21 ± 4 (2+2 day) --
-5 -2 -4 -3
± ± ± ±
1 0 1 1
(2+1 day) (2+2 day) (4) (8)
4 (7)
-2 ± 1
Apo (.25) Apo (.25) Apo (.25) None
9 18 28 42
± ± ± ±
2 5 5 8
(6). (8) a. (II)~ (16) a
5 (5)
-2 ± 0
Amph Amph Amph Amph
(2) (2) (2) (2)
-19 -19 -31 -33
± ± ± ±
4 2 6 4
(4) (6) (8) (ii)
-4 -6 -5 -3
± ± ± ±
1 1 1 1
(6) (8) (ii) (16)
6 (4)
-2 ± 0
Amph Amph ;~ph Amph
(5) (5) (5) (.5)
-48 -49 -54 - S
± ± ± i
8 3 8 1
(4) (6) (8) (ii)
-2 -2 -3 -2
± ± ± ±
1 0 1 1
(6) (8) (ii) (16)
30 ± 5 (4) 35 ± 3 (6) 52 ± 4 (8) --
Latent response c (wks post-lesion)
*Scores are mean ± SEM net rotations (rounded to whole numbers) in 3 minute observation periods with contralateral turns expressed positively and ipsilateral negatively. arotation prior to any drug treatment bpeak observed rotation acutely after drug treatment undrugged rotation subsequent to indicated treatment(s) < .01 different from pre-drug response for the same group
2400
Latent Rotation
Vol. 42, No. 23, 1988
Discussion Ungerstedt (2,3) described an unusual behavior in rats that had been nigrostriatally lesioned with 6HDA. Some time after being lesioned, the undrugged animals would show "explosive" contralateral rotation at the beginning of experimental sessions. Ungerstedt termed this strange behavior "paradoxical" rotation and attributed it in part to stress from handling. We subsequently demonstrated (i) that paradoxical rotation was not a consequence of lesion alone, but the result of drug treatment. Lesioned, drug naive animals never demonstrated the behavior, but those that had received even a single Apo treatment did so months after the treatment. The behavior seemed to be a demonstration of associative learning, exhibiting considerable specificity for the environment in which the drug had previously been administered, but was peculiar in several respects. Smaller doses (0.05 mg/kg) of Apo had significantly greater long-term effects than larger doses (0.25 and 1.0 mg/kg) despite the fact that the acute behavior was of doseresponsive duration. Thus, under a single-treatment condition with Apo, there seamed to be no practice effect (although there was a practice effect with spaced, repeated doses). There was also conspicuous absence of a recency effect; rats rotated more two weeks after their single drug treatment than did those tested at earlier post-treatment intervals. Finally, the behavior was undiminished even a year after treatment and, anthropomorphically, did not appear to be voluntary. We suggested that the behavior might reflect a permanent biochemical change in addition to, or as the basis of, the associative learning and cautioned that use of Apo to "screen" lesions resulted in a changed experimental animal. This was recently confirmed by the finding that lesioned rats rotated in response to the D-I agonist, SKF 38393, when they had previously received one treatment with Apo or the D-2 agonist, LY 171555, but not if they were drug naive (5). It is interesting to speculate on whether previously reported "nonspecific" supersensitivity of denervated striata (i.e., supersensitivity to norepinephrine and serotonin) was a result of lesion or the lesion plus Apo screening (6). Burunat et al (4), using repeated Apo treatments, have replicated our findings of a persistent, latent rotational effect and have concluded that the effect is purely associative learning. The data presented here show that, under conditions in which Apo is highly effective in inducing the latent rotational effect, Amph is not. In experiments utilizing repeated injections of cocaine, which induce ipsilateral rotation, we've never seen a latent ipsilateral effect comparable to the latent contralateral effect of Apo. This was so despite the fact that cocaine did induce a highly significant increase in rotational response to subsequent cocaine treatment (7,8). That is, cocaine sensitization was clear, but no undrugged rotation was induced. LSD, on the other hand, potently stimulates contralateral rotation acutely (9), and it too induced a long term latent contralateral rotation after one or more doses (i0). The latent rotational effect thus seems to be a property of direct, but not indirect, DA agonists. There being no a priori reason that association of the rotation environment with contralateral turning should be preferred to association of the environment with ipsilateral turning, failure of Amph and cocaine to induce the behavior argues either that denervation supersensitivity facilitates associative learning or that associative learning is an incomplete explanation of the behavior. It is plausible that the behavior results from associative learning plus a direct agonist-induced increase in sensitivity to agents including amines released as a result of
Vol. 42, No. 23, 1988
Latent Rotation
2401
stress. Consistent with this explanation is the observation that the behavior is extinguished by daily undrugged testing (and thus handling) only to reappear after a period free of handling. Acknowledgment This research was supported in part by NIDA grant DA 04423. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0.
P.B. SILVERMAN and B.T. HO, Nature 294 475 (1981). U. UNGERSTEDT, Acta Physiol. Scand. Suppl. 3&7 49 (1971). U. UNGERSTEDT, Pharmac. Ther. B. ~ 37 (1976). E. BURUNAT, M.D. DIAZ-PALAREA, R. CASTRO and M. RODRIGUEZ, Life Sciences 41 309 (1987). M. MORELLI, S. FENU and G. DI CHIARA, Life Sciences 40 245 (1987). B. COSTALL, R.J. NAYLOR and C. PYCOCK, Eur. J. Pharmacol. 35 275 (1976). P.B. SILVERMAN and B.T. HO, The Pharmacologist -20 172 (1978). -f P.B. SILVERMAN, Neurosci. Abst. 13 1719 (1987). L. PIERI, M. PIERI and W. HAEFELY-? Nature 252 586' (1974). P.B. SILVERMAN, Eur. J. Pharmacol. 147 309 (1988).
Pergamon Press
APOMORPHINE INDUCES LATENT ROTATION IN LESIONED RATS, AMPHETAMINE DOES NOT Peter B. Silverman Department of Psychiatry and Behavioral Sciences - MSI University of Texas Medical School at Houston Houston, TX 77030 (Received in final form April 6, 1988) Summary Rats with unilateral 6-hydroxydopamine (6HDA) lesions of one nigrostriatal pathway were used to compare the efficacy of apomorphine (Apo) and (+)-amphetamine (Amph) at inducing latent rotational behavior. Rats treated with Apo rotated contralaterally acutely after drug treatment and subsequently, weeks after drug treatment, exhibited rapid contralateral circling when re-introduced to the rotation environment. Amph treated animals, on the other hand, rotated ipsilaterally acutely after drug treatment and exhibited no latent drug effect. Rotational (circling) behavior in unilaterally lesioned rodents has proven to be a popular model for investigation of central dopamine (DA) function. Several years ago we described a striking behavioral consequence of Apo administration in rats with unilateral nigrostriatal lesions (i). When returned to the bowl in which even a single prior Apo treatment had been given, the animals burst into contralateral rotation which lasted 2-3 minutes. Lesioned animals that had no drug experience never exhibited this behavior. This behavior appeared to correspond with the "paradoxical" rotation which Ungerstedt had described earlier but had not attributed to drug treatment (2,3). The behavior did not develop fully until two weeks after treatment, but once developed was observable for at least a year after drug treatment, so long as the undrugged tests were conducted at spaced intervals. The behavior seemed to be learned, but had strange characteristics, such as the long latency to develop and an unusual dose-response effect. Recently, Burunat et al. (4) extended our work and concluded that the behavior they observed after repeated Apo administrations was strictly Pavlovian conditioning (associative learning). The work presented here shows that while the latent rotation after Apo treatment may well be associative learning, it remains unusual in that Amph does not produce an analogous ipsilaterally-directed latent rotation. Methods Male Sprague-Dawley rats (Simonsen, Gilroy, CA) weighing 180-240 g m w e r e anesthetized with 40 mg/kgpentobarbital and mounted in a stereotaxic device. A lesion was produced by infusion of 4 ~i of saline containing 0.1% ascorbate and 2 ~g/pl (base wt) 6-hydroxydopamine HBr. Stereotaxic coordinates, with incisor bar at zero, were 1.4 L and 4.2 P from bregma, 8 mm ventral from the
0024-3025/88 $3.00 + .00 Copyright (c) 1988 Pergamon Press plc
2398
Latent Rotation
Vol. 42, No. 23, 1988
skull surface. The 6HDA solution was administered at 0.33 ~i/min via syringe pump, i0 ~i syringe, PE I0 tubing and 27 gauge needle. Observations of rotational behavior were made in 26 cm diameter clear plastic bowls. Complete 360 ° turns were counted in three minute observation periods inanediately prior to injection, immediately after injection, and at intervals thereafter. After Apo treatment, observations were made beginning at 0, 15, 30 and 45 minutes after injection. After Amph treatment, observations were made (with an exception noted below) beginning 0, 15, 30, 45, 60, 90 and 120 minutes after injection. In both cases, acute rotation had slowed considerably by the time of the last observation. Subsequent observations of undrugged behavior consisted solely of three minute observation periods beginning immediately after the animal was placed in the bowl. During the intervals between drug tests and undrugged observations, rats remained undisturbed in their home cages with ad lib food and water throughout. Drugs tested were (+)-amphetamine sulfate (Sigma) and apomorphine HCI (Merck). These compounds were administered i.p. and s.c., respectively, in 1 ml/kg saline. Statistical evaluation of undrugged rotation scores within each group was made by a repeated measures analysis of variance followed by a Dunnett's procedure comparing each test subsequent to drug treatment with the pre-drug score obtained prior to any treatment. Results The results, summarized in Table I, show that all groups of animals exhibited a slight ipsilateral bias prior to any drug treatment. Group 1 animals (n = 8) were treated with a single dose of 0.05 mg/kg Apo two weeks after having been lesioned. They exhibited the typical latent (undrugged) rotational response two weeks later when placed in the rotation bowls for a three minute observation period. Group 2 animals (n = 6) were injected with 0.05 mg/kg Apo on three consecutive days two weeks after being lesioned. When placed, undrugged, in the rotation bowl four weeks after being lesioned for a three minute test they exhibited rapid contralateral rotation. Group 3 animals (n = 9) were injected with 2 mg/kg Amph on three consecutive days two weeks after being lesioned. Despite exhibiting good ipsilateral rotation when under the acute influence of the drug, they showed essentially no change in the magnitude of their undrugged response at four weeks after being lesioned. Group 4 animals (n = 7) were treated with 0.25 mg/kg Apo at 4, 6 and 8 weeks after being lesioned. After their first Apo treatment and prior to their second, their undrugged response had shifted from slightly ipsilateral to moderately contralateral. This undrugged contralateral rotation became more pronounced after subsequent treatments. Group 5 animals (n = 5) were injected with 2 mg/kg amphetamine at 4, 6, 8 and ii weeks after lesion. Despite good ipsilateral rotational response to these treatments, there was essentially no change in their undrugged response upon subsequent exposure to the bowl.
Vol. 42, No. 23, 1988
Latent Rotation
2399
Group 6 animals (n = 4) were injected with 5 mg/kg amphetamine at 4, 6 and 8 weeks after lesion. This group exhibited rapid ipsilateral rotation which lasted over two hours. Nonetheless, this group showed essentially no change in their subsequent undrugged response to the rotation environment when tested at ii weeks after lesion. A smaller (0.5 mg/kg) dose was then administered to this group, with three minute observations made up to one hour later, to see if it would induce latent rotation. No such effect was apparent when they were tested at 16 weeks post-lesion.
TABLE I Acute and Latent Rotational Responses to Apo and Amph*
Group (n)
Pre-drug response a
Treatment (mg/kg)
Acute response b (wks post-lesion)
1 (8)
-2 ± 1
Apo (.05)
25 ± 3 (2)
36 ± 7 (4) d
2 (6)
-4 ± 1
Apo (.05) APO (.05) APO (.05) None
26 ± 2 (2) 26 ± 4 (2+1 day) 25 ± 4 (2+2 day) --
4 1 55 57
± ± ± f
2 1 7 6
(2+1 day) (2+ 2 day) (4)~ (8) a
3 (8)
-4 ± 1
Amph (2) Amph (2) Amph (2) None
-20 ± 2 (2) -21 ± 3 (2+1 day) -21 ± 4 (2+2 day) --
-5 -2 -4 -3
± ± ± ±
1 0 1 1
(2+1 day) (2+2 day) (4) (8)
4 (7)
-2 ± 1
Apo (.25) Apo (.25) Apo (.25) None
9 18 28 42
± ± ± ±
2 5 5 8
(6). (8) a. (II)~ (16) a
5 (5)
-2 ± 0
Amph Amph Amph Amph
(2) (2) (2) (2)
-19 -19 -31 -33
± ± ± ±
4 2 6 4
(4) (6) (8) (ii)
-4 -6 -5 -3
± ± ± ±
1 1 1 1
(6) (8) (ii) (16)
6 (4)
-2 ± 0
Amph Amph ;~ph Amph
(5) (5) (5) (.5)
-48 -49 -54 - S
± ± ± i
8 3 8 1
(4) (6) (8) (ii)
-2 -2 -3 -2
± ± ± ±
1 0 1 1
(6) (8) (ii) (16)
30 ± 5 (4) 35 ± 3 (6) 52 ± 4 (8) --
Latent response c (wks post-lesion)
*Scores are mean ± SEM net rotations (rounded to whole numbers) in 3 minute observation periods with contralateral turns expressed positively and ipsilateral negatively. arotation prior to any drug treatment bpeak observed rotation acutely after drug treatment undrugged rotation subsequent to indicated treatment(s) < .01 different from pre-drug response for the same group
2400
Latent Rotation
Vol. 42, No. 23, 1988
Discussion Ungerstedt (2,3) described an unusual behavior in rats that had been nigrostriatally lesioned with 6HDA. Some time after being lesioned, the undrugged animals would show "explosive" contralateral rotation at the beginning of experimental sessions. Ungerstedt termed this strange behavior "paradoxical" rotation and attributed it in part to stress from handling. We subsequently demonstrated (i) that paradoxical rotation was not a consequence of lesion alone, but the result of drug treatment. Lesioned, drug naive animals never demonstrated the behavior, but those that had received even a single Apo treatment did so months after the treatment. The behavior seemed to be a demonstration of associative learning, exhibiting considerable specificity for the environment in which the drug had previously been administered, but was peculiar in several respects. Smaller doses (0.05 mg/kg) of Apo had significantly greater long-term effects than larger doses (0.25 and 1.0 mg/kg) despite the fact that the acute behavior was of doseresponsive duration. Thus, under a single-treatment condition with Apo, there seamed to be no practice effect (although there was a practice effect with spaced, repeated doses). There was also conspicuous absence of a recency effect; rats rotated more two weeks after their single drug treatment than did those tested at earlier post-treatment intervals. Finally, the behavior was undiminished even a year after treatment and, anthropomorphically, did not appear to be voluntary. We suggested that the behavior might reflect a permanent biochemical change in addition to, or as the basis of, the associative learning and cautioned that use of Apo to "screen" lesions resulted in a changed experimental animal. This was recently confirmed by the finding that lesioned rats rotated in response to the D-I agonist, SKF 38393, when they had previously received one treatment with Apo or the D-2 agonist, LY 171555, but not if they were drug naive (5). It is interesting to speculate on whether previously reported "nonspecific" supersensitivity of denervated striata (i.e., supersensitivity to norepinephrine and serotonin) was a result of lesion or the lesion plus Apo screening (6). Burunat et al (4), using repeated Apo treatments, have replicated our findings of a persistent, latent rotational effect and have concluded that the effect is purely associative learning. The data presented here show that, under conditions in which Apo is highly effective in inducing the latent rotational effect, Amph is not. In experiments utilizing repeated injections of cocaine, which induce ipsilateral rotation, we've never seen a latent ipsilateral effect comparable to the latent contralateral effect of Apo. This was so despite the fact that cocaine did induce a highly significant increase in rotational response to subsequent cocaine treatment (7,8). That is, cocaine sensitization was clear, but no undrugged rotation was induced. LSD, on the other hand, potently stimulates contralateral rotation acutely (9), and it too induced a long term latent contralateral rotation after one or more doses (i0). The latent rotational effect thus seems to be a property of direct, but not indirect, DA agonists. There being no a priori reason that association of the rotation environment with contralateral turning should be preferred to association of the environment with ipsilateral turning, failure of Amph and cocaine to induce the behavior argues either that denervation supersensitivity facilitates associative learning or that associative learning is an incomplete explanation of the behavior. It is plausible that the behavior results from associative learning plus a direct agonist-induced increase in sensitivity to agents including amines released as a result of
Vol. 42, No. 23, 1988
Latent Rotation
2401
stress. Consistent with this explanation is the observation that the behavior is extinguished by daily undrugged testing (and thus handling) only to reappear after a period free of handling. Acknowledgment This research was supported in part by NIDA grant DA 04423. References i. 2. 3. 4. 5. 6. 7. 8. 9. I0.
P.B. SILVERMAN and B.T. HO, Nature 294 475 (1981). U. UNGERSTEDT, Acta Physiol. Scand. Suppl. 3&7 49 (1971). U. UNGERSTEDT, Pharmac. Ther. B. ~ 37 (1976). E. BURUNAT, M.D. DIAZ-PALAREA, R. CASTRO and M. RODRIGUEZ, Life Sciences 41 309 (1987). M. MORELLI, S. FENU and G. DI CHIARA, Life Sciences 40 245 (1987). B. COSTALL, R.J. NAYLOR and C. PYCOCK, Eur. J. Pharmacol. 35 275 (1976). P.B. SILVERMAN and B.T. HO, The Pharmacologist -20 172 (1978). -f P.B. SILVERMAN, Neurosci. Abst. 13 1719 (1987). L. PIERI, M. PIERI and W. HAEFELY-? Nature 252 586' (1974). P.B. SILVERMAN, Eur. J. Pharmacol. 147 309 (1988).