Conditioned response to apomorphine in nigro-striatal system-lesioned rats: The origin of undrugged rotational response

Life Sciences, Vol. 41, pp. 1861-1866 Printed in the U.S.A.

Pergamon Journals

CONDITIONED RESPONSE TO APOMORPHINE IN NIGRO-STRIATAL SYSTEM-LESIONED RATS: THE ORIGIN OF UNDRUGGED ROTATIONAL RESPONSE

Enrique Burunat, Ph.D. (I), Rafael Castro (2), Maria D. Diaz-Palarea (2) and Manuel Rodriguez (2) (i) Dept. of Psychobiology, (2) Dept. of Physiology University of La Laguna, Canary Islands, Spain (Received in final form August 13, 1987)

Summary Development and time-course characteristics of Early rotational response (ER) to apomorphine in 6-hydroxydopaminelesioned rats (i) is explored. We show here how this ER can be considered a conditioned response that arises when the drug is repeatedly administered, according to a classical conditioning paradigm. In this way, the ER to apomorphine can be considered a non-pharmacological, conditioned, placebo response, the drug action being the unconditioned stimulus (UCS). In our model, the un~ed rotational response elicited by saline injections two weeks after drug treatment (2) can be considered as the conditioned response (CR) to the conditioned stimulus, the CS being the environment associated with the drug treatment. This CR had not previously been identified during the drug treatment. Thus, we studied the acquisition of the ER, nonexistent after the first injection of apomorphine (2). Furthermore, we distinguish between this ER and the later, strictly pharmacological rotational response (LR) to apomorphine. Finally, we related this ER to the u n d r ~ d , paradoxical response to saline. In conclussion, we demonstrate the paradigm of pharmacological conditioning using this animal model of Parkinson's disease, supported by our own results and those of Silverman and Ho (1981). In this paper, we report how the rotational model of Parkinson's disease could be considered an excellent animal model to investigate the origins and characteristics of pharmacological conditioning after repeated dopaminergic compound administration. We try to show how pharmacological conditioning, which arises when a drug is repeatedly administered, is a strictly Pavlovian phenomenon wherein the UCR (unconditioned response) is a pharmacological response to a drug. Using this animal model of Parkinson's disease, the conditioned response (CR) in a pharmacological conditioning process can be identified and defined. Materials

and Methods

Experiments were carried out on male Sprague-Dawley rats (Panlab,Barcelona) (200-250 g). Animals were housed under normal laboratory conditions of 22+i°C on a standard light-dark schedule (12:12 with 3.00-15.00 h. light on) and free access to standard diet and water, except during the behavioral measurements. The rats were tested for circling behavior in individual cages (lenght, width and height, 25 cm.) with grid bars on the floor and illuminated with white light (50 W lamp located 15 cm. under the cage floor). The cage walls were filled with sound attenuating foam. The behavioral tests were made over 8-10 h. of the light period, at room temperature (22+i°C). The total number of full-body turns 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.

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(complete rotations) was observed every minute at different intervals after apomorphine injections. The rotational response in the first minute after injection is referred to as Early Response (ER). The sum of rotations observed at minute i0, 20, 30, 40, 50, 60, 70 and 80, is referred to as Late Respom,se (LR). Circling behavior was video-tape recorded. As apomorphine induces contralateral circling behavior in rats only when more than 90~ of the nigrostriatal system has been destroyed (3), rats were unilaterally lesioned with 6-hydroxydopamine according to the previously described method (4). Apomorphine hydrochloride (Sigma, St. Louis) was dissolved in a 0.9~ saline solution and immediately injected i.p. (i ml total volume per rat). 6-Hydroxydopamine (6-OHDA) (Sigma, St. Louis) was prepared for injection by dissolving it in a 0.9% saline solution and 0.3 ug/ul of ascorhic acid to retard oxidation. Statistical analysis comprised analysis of variance (ANOVA) followed by Scheff~ test for unpaired data or by two-tailed Student's paired test comparisons. In a]l the following experiments, lesioned rats were repeatedly injected with apomorphine (i mg/kg i.p.). Injections began two weeks after lesion, when postsynaptic striatal supersentivity is developed (i). In Experiment i, apomorphine was injected with a two day interval (Group I, 15 injections, dashed lines) or a two hour interval (Group II, 4 injections, continuous line) between injections. Immediately after injection, the rats (n=8 per group) were placed in the experimental cages and circling behavior was evaluated. We studied: (A) the appearance and development of ER, and (B) the development of LR. In EXperiment 2 we studied the effect of a three week d r ~ - f r e e period after apomorphine treatment on: (A) the ER and (B) the LR in rats from Experiment I. In Experiment 3 we studied the effect of an eight apomorphine injection, with a ten times smaller dose (0.I mg/kg) than that previously administered (i mg/kg, seven injections, every other day, n=8) on: (A) the ER and (B) the LR. In Experiment 4 we studied the relation between ER to the drug (seven injections, every other day, n=8) and undr~EEed behavior to saline, two weeks after dru E treatment had finished. In Experiment 5 we studied the development of ER (11 doses, every other day, n=8 per group) in rats adapted to the experimental environment (three times, 80 minutes each, after a saline injection). The control group only received the drug treatment. Results Experiment I: The LR (Figure IB): The group injected every other day showed an increased LR after successive injections. The two hour interval group showed, however, a decreased LR after the fourth injection (" p.05 15th vs. 7th doses). Experiment 2: Three weeks after the drug treatment had finished, independent of dosage interval, a new apomorphine injection elicited a similar LR than that elicited after the initial injection (Figure IB: p>.05 3-week after vs. ist dose). However, in both groups the ER remained increased (Figure IA). Experiment 3 (Figure 2): Seven apomorphine injections (I mg/kg,i.p.) every other day) e~cited an increased ER and LR. Two days after the seventh apomorphine injection, when both ER and LR were stable, the injection of a ten times smaller dose (0.i mg/kg, 8th injection) elicited a smaller LR (Figure 2B:

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• p<.Ol) but a similar ER (Figure 2_A).

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Faq~erlmemt 4 (Figure 3): Two weeks after the last drug injection administered every other day, the injection of saline solution (0.9%) in the same volume and environment where the drug was repeatedly administered, elicited a placebo, undrugged, paradoxical response lasting two or three minutes after injection. The number of turns in the first minute after injection was significantly the same than the last ER. The time course of this undrugEed response ( ~ in Figure 3) can be compared with the first minutes of circling behavior after the first and seventh apomorphine doses (I mg/kg i.p.). In the first drug dose, the rotations began two minutes after injection. In the seventh dose, the rotational behavior begad immediately after the animals were placed in the experimental cages. UndrugEed response began immediately after the animals were placed in the cages and dissapeared in two minutes, exactly when the rotational behavior elicited by the first apomorphine began.

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Experiment 5 (Figure 4): The repeated exposure of the rats to the experimental environment with saline injections (three times, every other day), blocked ER development in animals treated with ii apomorphine doses beginning two days after this '&daptation"period ( * p~.O01 adapted vs. non-adapted group ).

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Discussion The present results can be analyzed from two different viewpoints. First of all, we have here the first characterization of a pharmacological conditioned response where the several components of the conditioning phenomenon are defined exactly: a) The conditioned response (CR) has very exact, temporal, limits. In our model, the CR is the Early rotational response to apomorphine. b) The CR is clearly differentiated from the unconditioned, pharmacological response (UCR); in our model, this is the Late response to the drug. c) The CR appears and increases with the conditioning -with the repeated drug administration, d) The conditioned stimulus (CS) is the environment associated with the drug injection, e) The sole presentation of the CS elicits the CR -undrugged rotations after saline injection, f) The unconditioned stimulus (UCS) is the action of the drug on the central dopaminergic receptors -and it elicits the UCR, the Late response, g) The CS precedes the UCb. h) The CR precedes the UCR. i) The CS-UCS association can be perturbed by the previous exposure to the CS in the absence of the UCS -"latent inhibition". In this way, we can define Pharmacological condltioni~E as the result of a Pavlovian conditioning occurring when a drug is repeatedly administered. We suggest the utility of using CNS-active drugs as UCSs for studying very basic aspects of learning and memory in Mammalians. This is because the neurochemical action of these UCS is much better understood than for other UCSs used in Pavlovian conditioning designs. Pharmacological treatments of neurological and psychiatric illness could be specially affected by this

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approach, as we have shown using this animal model of Parkinson's disease. Currently there is considerable evidence that the behavioral and/or physiological effects of many CNS-active drugs are modified by learning phenomena (see, for example, the demonstrative works of Siegel et al (5), Beninger et al (6,7) and Post et al (8); and for a general review, ref. 9). The modification of the therapeutic effectiveness of these drugs during extended treatments (i0), as well as the placebo phenomenon (ii), could both be related to these learning phenomena. We formally propose that they be named Phaz~macological conditioning. It would be that variety of Pavlovian conditioning wherein the UCSs are the action of CNS-active drugs upon central structures, and the UCRs, the behavioral or physiological responses elicited by these drugs. The CSs would be the stimuli (i.e., physical environment, handling, injection, etc.) associated with the drug effects. The CRs would be similar responses (but shorter) than the UCR, but after the mere exposure to the CSs. In this paper we have reported a CR that develops after apomorphine treatment in an animal model of Parkinson's disease. The influence of learning processes on the modification of drug effects are usually demonstrated by two methods: through the appearance of a behavioral or physiological response to a placebo in the context previously associated with a drug, or through the modification of the expected effect of a drug by a CR previously elicited by another drug (12). Here, we have shown that the CR in Pharmacological conditioning, using this animal model, may be differentiated from the strictly pharmacological UCR. Such differentiation had not been previously reported. The CR in Pharmacological conditioning may be manipulated using different Pavlovian conditioning designs. We have shown the effect of a latent inhibition (LI) procedure on the development of this CR. So, the usual "adaptation period" of animals to the experimental environment prior to the evaluation of drugs action may be considered as LI training. But this "adaptation period" is not employed when these drugs are administered to humans. Therefore, we propose that Pharmacological conditioning as a specific paradigm of learning could have important consequences for both, the evaluation of the therapeutic effectiveness of new CNS-active drugs, and for the pharmacological management of neurological and psychiatric illness. Secondly, we show that the u n d r ~ e d (2) or paradoxical (13) response in nigrostriatal system-lesioned and apomorphine pretreated rats, is the result of Pharmacological conditioning. It has previously been reported that undrugged response has very peculiar characteristics. Silverman and Ho (2) stablished an inverse relationship between a sole apomorphine dose and the intensity of the subsequent rotational behavior after saline. Furthermore, this undruE~ed response only appeared two weeks after drug treatment. Our previous study showed that the undruE~ed behavior can be considered a CR in a Pharmacological conditioning: The Early response developed after repeated apomorphine injections will be, two weeks after the treatment has finished, the undrup~ed response. This rotational CR to saline shows some of the usual characteristics of CRs: it may be extinguished with the repeated administration of saline; the extinction is faster with shorter administration intervals, and there is a partial spontaneous recovery of this CR. Furthermore, we have reported in this previous paper (14) that this CR has similar values to the last ER for six months after the drug treatment. So, the extraordinary persistence of the CR in Pharmacological conditioning may be considered as another peculiar characteristic of this paradigm of learning. Some of these characteristics are explained in a Pavlovian context, such as the latency period required for the appearance of the undruE=j~ed response: in Pavlovian conditioning, the same CR may be elicited by similar stimuli than the original CS, but after a generalization period has elapsed.

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Generalization implies that the specific characteristics of the original CS have been forgotten. We have identified the CR, which develops during drug treatment, and it is the Early response to apomorphine. But, as in the Silverman and Ho (1981) work, the CR to saline is only elicited two weeks after the last drug injection. The only differences between the apomorphine and saline administration procedures are the periferic side effects caused by the drug. According to the results obtained in our laboratory (submitted), we suggest that the 2-week latency period required for the appearance of the undrugged response is the period in which the animal forgets the periferic sideeffects of apomorphine. The inverse dose-response reported by Silverman and Ho (2) is also in agreement with the generalization hypothesis: A higher dose will cause more intense periferic side-effects and, consequently, the generalization period will be longer than for a lower dose. So, two weeks after the drug administration, the undrugged response will be less in the higher dose-pretreated group, which experienced more intense periferic side-effects. Many other CNS-active drugs cause undesirable periferic side-effects. Therefore, a specific characteristic of Pharmacological conditioning is the importance of the generalization phenomena as well as the persistence of this conditioning. The importance of the periferic side-effects of apomorphine may explain, as in Aversive conditioning, the fact that a sole dose causes the conditioning and the fact of the extraordinary persistence of the conditioning. In short, we have here identified and differentiated the CR from the UCR after repeated apomorphine administration in the rotational model of Parkinson's disease, wherein this behavioral response is considered an antiparkinsonian effect. This CR may be elicited by saline injections in the context previously associated with the drug. So, the undrugged or paradoxical response to saline may be characterized as a placebo, antiparkinsonian CR after Pharmacological conditioning. Pharmacological conditioning may explain some modifications of the therapeutic effectiveness of CNS-active drugs after repeated administration. Furthermore, Pharmacological conditioning could be considered the main cause of placebo phenomenon (15,16). The peculiar characteristics of Pharmacological conditioning that we have commented on presumably underly the little understood placebo phenomenon. References

i. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.

U. UNGERSTEDT, Acta physiol. Scand. Suppl. 367 69-93 (1971). P.B. SILVERMAN and B.T. HO, Nature 294 475'477 (1981). F. HEFTI, E. MELAMED and R.J.WURTMAN, Brain Res. 195 123-137 (1980). R. CASTRO et al, Psychopharmacol. 85 333-339 (1985--~. S. SIEGEL et al, Science 216 436-437 (1982). R.J. BENINGER and B.L. HAHN, Science 220 1304-1306 (1983). R.J. BENINGER and R.S. HERZ, Life Sci. 38 1425-1431 (1986). R.M. POST et al, Life Sci. 28 755-760 (1981). S. SIEGEL and J. MACRAE, Trends Neuro-Sci. 7 140-143 (1984). E. MELAMED, Arch. Neurol. 43 402-404 (1986)? L. WHITE, B. TURSKY and G.E. SCHWARTZ (Eds~: Placebo: Theory, Research and mechanisms. Guilford Press (London) 1985. R.E. HINSON and M. RHIJNSBURGER, Life Sci. 34 2633-2640 (1984). U. UNGERSTEDT, Pharmacol. Ther. (B) 2 37-40"-[1976). E. BURUNAT et al, Life Sci. 41 309-353 (1987). E. BURUNAT et al, European J. Pharmacol. (in press). E. BURUNAT et al, Functional Neurol. (in press).