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Behavioral effects of d -amphetamine in developing cats
150
Developmental Brain Research, 22 (1985) 150-155 Elsevier
BRD 60090
Behavioral effects of D-amphetamine in developing cats M. S. LEVINE, J. H. HANNIGAN, R. S. FISHER, S. HOWARD-BUTCHER, C. D. HULL and N. A. BUCHWALD Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted May 14th, 1985) Key words: amphetamine - - development - - cat - - dopamine - - stereotypy - - locomotion
The development of the behavioral effects of amphetamine was assessed in kittens of 1-53 days of age. Amphetamine-induced increases in locomotion occurred when animals were beyond 35 days of age. Stereotypic behavior was induced at all ages tested but the predominant type of stereotypy was age-related. From 1 to 14 days amphetamine induced licking. Pendular head movements occurred when animals were under 35 days. At 14 days of age darting, a response consisting of rapid pacing and turning began to occur. Tracking, a series of horizontal and vertical head movements also began to occur after 14 days. The adult response of vertical and horizontal head movements became most prominent after 35 days. This report is part of a continuing series of studies assessing behavioral d e v e l o p m e n t in neonatal kittens and its alteration by pharmacological t r e a t m e n t and/or experimental brain d a m a g e 17A9-21. In the current study the behavioral alterations p r o d u c e d by a single dose of D - a m p h e t a m i n e have been assessed in kittens of different ages. Differential effects of amphetamine during d e v e l o p m e n t are of particular interest because this c o m p o u n d had been used in the treatment of hyperkinetic disorders and so-called 'minimal brain dysfunction' in children 1,27. A l t h o u g h its use has been discontinued for the most part, amphetamine-like c o m p o u n d s such as m e t h y l p h e n i d a t e remain in use 10. A m p h e t a m i n e is a widely used drug of abuse that has been implicated in the etiology of certain varieties of psychoses as well 5. Recent evidence has also indicated that a m p h e t a m i n e administration p r o d u c e d long-term neurochemical and neurophysiological changes 22.25. A d u l t animals respond to acute a m p h e t a m i n e administration with consistent, dose-dependent changes in locomotion and in a variety of other behaviors that b e c o m e stereotypic 10. Developing animals often respond differently 2-4,7,15. F o r example in rats, locomotion is increased by low doses ( 0 . 5 - 4 . 0 mg/kg) when animals below 25 days of age or above
40 days are tested. In contrast, rats of 3 0 - 3 5 days old ('periadolescence') are not behaviorally sensitive to the a m p h e t a m i n e . A t higher doses ( 6 . 0 - 1 6 . 0 mg/kg), both younger and adult rats, but not the 'periadolescent' rats show r e d u c e d l o c o m o t i o n and increased stereotypies2-4,7,t5. A d u l t cats r e s p o n d to a m p h e t a m i n e with changes in locomotion and stereotypic head m o v e m e n t s 12,19. Although there are a n u m b e r of studies on the effects of a m p h e t a m i n e in the adult cat 12,19, there have been, to our knowledge, no consistent observations in the developing kitten. Thus, the present experiment was designed to provide systematic information on the d e v e l o p m e n t of behavioral responses to amphetamine in kittens. The behavior of kittens was monitored after injections of D - a m p h e t a m i n e to determine the types of responses e v o k e d and the agerelated changes in responses during the early developmental period. A total of 43 kittens of both sexes (2l males and 23 females) o b t a i n e d from 11 litters were used. There were no consistent gender differences in response to a m p h e t a m i n e or saline at any age tested and data obtained from males and females were pooled. All animals were born and r e a r e d in the cat breeding colony of the Mental R e t a r d a t i o n Research C e n t e r at
Correspondence: M. S. Levine, Mental Retardation Research Center, Room 58-258A, UCLA School of Medicine, 760 Westwood Plaza, Los Angeles, CA 90024, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
151 U C L A and ranged in age from 1 to 53 days. For all age measurements the day of birth was considered day 1. Each kitten was tested only once and animals were tested individually. Whenever possible two animals from the same litter were tested, one with amphetamine and one with saline. Thus, age ranges and average age of testing for amphetamine-treated and saline-treated groups were approximately the same. Animals were divided into the following age groups according to the age at which they were tested: 1-14 days (15 kittens, 8 amphetamine-treated, mean age 11 days and 7 saline-treated, mean age 9 days); 15-35 days (17 kittens, 9 amphetamine-treated, mean age 27 days and 8 saline-treated, mean age 29 days); and 36-53 days (11 kittens, 7 amphetamine-treated, mean age 47 days and 4 saline-treated, mean age 44 days). There were several reasons for dividing animals into these age categories. First, our previous work showed that marked changes in locomotor activity are occurring over each of these time periods TM. From 1-14 days kittens basically locomote by crawling. From 15-35 days they begin to support their weight and walk. Locomotor activity increases during this time. From 36-60 days locomotor activity remains at levels reached between 15-35 days. Amphetamine treatment in the present experiment produced different effects on locomotion depending upon age. Second, amphetamine appeared to produce different types of stereotypies during each of these age periods. For testing behavioral response to amphetamine, each animal was first placed into a 60 x 60 x 47 cm plywood 'open field' chamber in which the floor was gridded into 36 10 x 10 cm squares by photocells to determine locomotor activity~8. Interruption of each light beam during locomotion produced a single count. The number of photocell counts were recorded every 3 min. After 15 min of baseline testing the kittens received an injection of either D-amphetamine sulfate (2 mg/kg i.p.) or an equivalent volume of saline vehicle. The dose chosen was based on our previously published research on adult cats 19 which shows that doses of 1-2 mg/kg (i.p.) induced marked stereotypies lasting for more than 2 h. The injection was followed immediately by a second test in the open field for 15 min. After this test the kitten was placed in a sound-attenuating chamber equipped with a one-way mirror through which the animal's be-
havior was monitored in detail for 30 min. The behaviors scored included licking, various head movements and patterns of body movement and position. Behaviors were recorded every 3 min and rated on a 4-point scale as follows: 0, behavior did not occur; 1, behavior occurred for less than 30 s during the 3 min rating period; 2, behavior occurred for more than 30 s but less than 3 rain; 3, behavior lasted the entire 3 min to the exclusion of all other behaviors. Following this test locomotor activity was assessed again for 15 min in the open field and then behavior was monitored once again in detail for an additional 30 min in the sound attenuating chamber. Behavibr was monitored periodically thereafter for another 30 min-1 h but was not rated. This sequence of testing made it possible to assess changes quantitatively in locomotor activity and other behaviors influenced by amphetamine. Segments of the session were videotaped for subsequent reanalysis and reliability estimation. lnterrater reliability correlation coefficients exceeded 0.90. Locomotor activity and behavioral rating scores were averaged for each test period for all animals. Differences in locomotor activity scores between groups receiving amphetamine and saline at different ages were assessed with appropriate 3 way analyses of variance and post hoc tests 30. Differences in rating scores were assessed with non-parametric tests2S. Temperature in the activity and observation chambers was maintained between 24-26 °C by infrared heat lamps mounted above each chamber. Animals were tested from 10.00-15.00 h. When two animals from a single litter were tested on the same day order of testing of amphetamine-treated and saline-treated animals was counter-balanced so that one-half of the amphetamine-treated group was tested first and onehalf of the saline-treated group tested first. There were marked age-related changes in the behavioral effects of D-amphetamine. These were characterized by differential alterations in both .locomotor activity and drug-induced stereotypies. Locomotor activity. Increases in locomotion in adult cats following amphetamine administration are dose-dependent 19. Increasing doses (up to 2 mg/kg) produce increased locomotion. At higher dosages locomotion then decreases. The dose used in the present study (2 mg/kg) produces increases in both locomotion and head movement stereotypies in adult
152 MEAN 1-14 DAYS z
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which the kitten remained immobile but slowly and repeatedly moved its head from side to side. This behavior occurred more frequently 60-90 min postinjection than at 15-45 min. The increases in the frequency of occurrence of this behavior were statistically significant at both 1-14 and 15-35 days of age (P < 0.05). A second behavior that occurred frequently was stereotypic licking of the forepaws and/or floor of the observation chamber. This behavior was slightly more prevalent 60-90 min than 15-45 min postinjection. The increase in the frequency of occurrence of this behavior was significantly different from that of the saline-treated group at 1-14 days only (P < 0.05). In the 15-35-day-old kittens, a transition in the types of behavior induced by amphetamine began to occur. Amphetamine still induced pendular head movements. The occurrence of AGE (DAYS)
cats. In kittens 1-14- and 15-35-days-old, amphetamine administration produced no change in locomotor activity relative to kittens in the same age group receiving saline injections (Fig. 1). In both saline-and amphetamine-treated 1-14 day groups locomotor activity was low. At 15-35 days there were no significant differences between saline- and amphetaminetreated groups. Overall, activity levels were significantly higher compared to those in the younger group (P < 0.05). In 36-53-day-old kittens, amphetamine produced a marked increase in locomotor activity. After the amphetamine injection there was a marked and statistically significant increase in locomotor activity during both postinjection sessions (P < 0.01). This increase was greatest during the second postinjection test (45-60 min). There was no statistically significant difference in baseline activity levels between the saline-treated kittens in the 15-35- and 36-53-day groups. Stereotypic behavior. In adult cats the major stereotypy produced by amphetamine is a dose-dependent increase in horizontal and vertical head movement12j 9. During this behavior the cat remains stationary and moves its head from side to side or up and down, as if tracking an object in space 12,19. In the youngest group of kittens tested (1-14 days), amphetamine produced two consistent behavioral patterns that were different from its effects in adult cats (Fig. 2). The first was pendular head movement in
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Fig. 2. Mean rating scores for stereotypic behaviors at 15-45 and 60-90 rain postinjection for each age group. Range above or below each point refers to S.E.M.
153 licking decreased markedly. Two new behaviors, 'darting' and 'tracking', began to occur. Darting was a locomotor behavior in which the kitten paced back and forth for a few steps, turned sharply and then repeated the behavior. Tracking is a behavior that is frequently seen after amphetamine injections in adult cats. When this behavior occurs the animal appears to be fixated on a non-existent object. The eyes and head change position constantly and both vertical and horizontal head movements occur although the latter predominate. In kittens older than 35 days the frequency of both darting and tracking increased markedly. In contrast the frequency of pendular head movements decreased. Increases in frequency in both darting and tracking were statistically signifiAGE (DAYS) 1-14
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Fig. 3. Mean rating scores for other types of behaviors induced at 15-45 and 60-90 postinjection for each age group. Range above or below each point refers to S.E.M.
cant at 36-53 days (P < 0.05). Only the increase in the frequency of darting at 60-90 min postinjection was significant at 15-35 days. Although we systematically rated behaviors and quantified motor activity for only about 2 h postinjection, we observed that most amphetamine-induced behaviors continued to occur for 3-4 h postinjection in all age groups. During this latter 1 h the frequency and intensity of amphetamine-induced behavior decreased. While this report emphasizes the occurrence of increased locomotion and age-related stereotypies other behaviors were also induced by amphetamine (Fig. 3). For example, amphetamine-treated animals occasionally engaged in circling or climbing. Such behaviors tended to be idiosyncratic and did not occur consistently across animals. Indeed saline-treated animals sometimes exhibited these behaviors as frequently as amphetamine-treated animals. Most saline-treated kittens at all ages were inactive during a large part of the observation test. They remained immobile and, if old enough to have already opened their eyes, would close their eyes. This inactivity and/or sleep occurred less frequently in amphetamine-treated animals especially in the 36-53 day group (P < 0.05). The development of amphetamine-induced autonomic responses was also observed. In 1-14 day kittens piloerection and salivation occurred frequently and in kittens over 15 days pupillary dilation was observed as well. The major findings of this study indicate that amphetamine treatment in early postnatal periods in kittens produces markedly different effects than similar treatment in older kittens and adult cats. For the first 5 postnatal weeks kittens did not display increased locomotion in response to amphetamine. After this time kittens became hyperactive after injections of 2 mg/kg of the drug. Stereotypic behavior was induced by amphetamine at all ages tested. However, the nature of the stereotypy evoked was qualitatively different from the adult-like response at each age. The youngest animals (1-14 days) engaged in stereotypic licking. This behavior did not persist after 2 weeks of age and is rarely observed in adult cats. Up to 5 weeks of age, the predominant stereotypy was a pendular head movement. From 15-35 days a transitional behavior, 'darting', began to occur. Darting and the adult response 'tracking' became prominent after
154 35 days of age. There are a number of mechanisms that may underly the developmental behavioral changes induced by the actions of amphetamine. These mechanisms are probably not independent and there is evidence that they all contribute to the effects of the compound. First, the patterns of behavioral response to amphetamine in any age animal reflects the specific movement capabilities of the animal at that age. Locomotion in the cat develops extensively between 2 and 3 weeks of age is. Kittens in early postnatal periods move about by crawling for short distances 18,29. Thus, it might be expected that amphetamine would not markedly increase locomotion before 4-5 postnatal weeks. Similarly, most visual behaviors do not develop until about 1 month of age or later in the cat 29 although eye opening occurs between the first and second postnatal weeklS,29 Thus, increases in the occurrence of tracking would not be expected until after this time. The present data allow a limited interpretation of the development of sensitivity to amphetamine in the kitten. In the adult, there is a biphasic dose-response curve where locomotion is elevated at lower but not higher doses jg. Stereotypies increase roughly monotonically with increasing dose of amphetamine, behaviorally competing with locomotion. For the kittens tested with the single dose of amphetamine, the absence of elevated locomotion in the two younger age groups could represent either relative insensitivity of this behavior to amphetamine in animals of this age, or a sensitivity high enough so that only stereotypies but not locomotion are affected. In preliminary data, in which different dosages of amphetamine were used, we found that below 1 month of age locomotor activity was rarely increased, whereas the occurrence of amphetamine-induced stereotypies was dose-dependent. Thus, it appears that cats have to mature beyond 30 days before amphetamine markedly increases locomotion. The development of the responses to amphetamine reflects the maturation of dopamine systems in the brain. In the mature nervous system, amphetamine acts primarily by promoting the release and blocking of reuptake and subsequently increasing the synthesis of dopamine 11. A major site of action of amphetamine on dopamine is believed to be the basal ganglia and its associated structures 11. Basal ganglia
dopamine concentration and tyrosine hydroxylase activity develop slowly over the first 40-60 days postnatally in the rat 14,24. In the cat, development of neostriatal dopamine and tyrosine hydroxylase occurs even more slowly over the first 3-5 postnatal months 6. Adult levels are reached at about 4 months of age 6. Thus, in early postnatal periods the ability of amphetamine to produce alterations in dopamine release, re-uptake and synthesis is severely limited by the immaturity of the dopamine system. The functional status of basal ganglia neurons matures over the first two postnatal months and this maturation affects the ability of amphetamine to alter neuronal activityS,16,23. The electrophysiological responses of caudate and substantia nigra neurons to activation of their afferents is markedly different in kittens below one month of age than in older animals. Amphetamine is capable of altering electrophysiological response in substantia nigra neurons in young kittens9. The type of alteration induced in kittens, however, was different from the alteration produced in adult cats. The behavioral response to amphetamine in the kitten may be different from that of the adult because of the differential state of younger dopamine systems. It is not merely the case that the animals become gradually more responsive to the amphetamine. Rather, the induction of stereotypic licking in the youngest group suggests that the dopamine systems in these animals may be organized to respond differently than the dopamine systems in adult cats. It is also possible that the differences may reflect maturation of separate components of dopamine systems at different times (e.g. mesolimbic vs nigrostriatal) 26. Different dopamine systems have been hypothesized to separately mediate locomotor and stereotypic responses to amphetamine 13. Finally, the systems upon which amphetamine acts are not developing in isolation. Areas other than the basal ganglia and neurotransmitter systems other than the dopamine system are concurrently maturing. Locomotion and stereotypies are also influenced by manipulation of these other systems and the role of amphetamine in behavior must be considered in the context of these interactions. Supported by USPHS Grants HD-05958, D A 3107 and HD-07032.
155 1 Arnold, L. E., Wender, P. H., McCloskey, K. and Snyder, S. H., Levoamphetamine and dextroamphetamine: comparative efficiacy in the hyperkinetic syndrome, Arch. Gen. Psychiatry, 27 (1972) 816-822. 2 Bauer, R. H., The effects of L-, D- and para-hydroxy-amphetamine on locomotor activity and wall clinging in rats of different ages, Pharmacol. Biochem. Behav., 13 (1980) 155-165. 3 Bauer, R. H. and Duncan, D. L., Differential effect of D-amphetamine in mature and immature rats, Physiol. Psychol., 3 (1975) 312-316. 4 Bauer, R. H. and Evey, L., Differential effects of L-amphetamine on ontogeny of active avoidance, intertrial response, and locomotor activity, Psychopharmacology, 75 (1981) 299-304. 5 Bell, D. S., The experimental reproduction of amphetamine psychosis, Arch. Gen. Psychiatry, 29 (1973) 35-40. 6 Buchwald, N. A., Hull, C. D., Levine, M. S. and Villablanca, J. R., Developmental assessment of intact and brain lesioned kittens. In M. A. B. Brazier and F. Coceani (Eds.), Brain Dysfunction in Infantile Febrile Convulsions, Raven, New York, 1976, pp. 179-186. 7 Campbell, B. A. and Randall, P. J., Paradoxical effects of amphetamine on preweaning and postweaning rats, Science, 195 (1977) 882-891. 8 Fisher, R. S., Levine, M. S., Hull, C. D. and Buchwald, N. A., Postnatal ontogeny of evoked neuronal response in substantia nigra of the cat, Dev. Brain Res., 3 (1982) 443-462. 9 Fisher, R. S. Levine, M. S., Hull, C. D. and Buchwald, N. A., Amphetamine alters evoked response of nigral neurons in kittens and adult cats, Brain Research, 237 (1982) 415-427. 10 Groves, P. M. and Rebec, G. V., Biochemistry and behavior: some central actions of amphetamine and antipsychotic drugs, Anna. Rev. Psychol., 27 (1976) 91-127. 11 Groves, P. M. and Tepper, J. M., Neuronal mechanism of actions of amphetamine. In I. Creese (Ed.), Stimulants: Neurochemical, Behavioral and Clinical Perspectives, Raven, New York, 1983, pp. 81-129. 12 Jacobs, B. L., Heym, J. and Trulson, M. E., Cats develop tolerance to D-amphetamine's effect upon locomotion and stereotyped behaviors, Eur. J. Pharmacol., 69 (1981) 353-356. 13 Kelly, P. D., Seviour, P. W. and Iversen, S. D., Amphetamine and apomorphine response in the rat following 6-hydroxydopamine lesions of the nucleus accumbens septi and corpus striatum, Brain Research, 94 (1975) 507-522. 14 Lanier, L. P., Dunn, A. J. and Van Hartesveldt, C., Development of neurotransmitters and their function in the brain. In S. E. Ehrenpreis and I. J. Kopin (Eds.), Reviews of Neuroscience, Vol. 2, Raven, New York, 1976, pp. 195-256. 15 Lanier, L. P. and Isaacson, R. L., Early developmental changes in the locomotor response to amphetamine and their relation to hippocampal function, Brain Research, 126 (1977) 567-575.
16 Levine, M. S., Cherubini, E., Novack, G. D., Hull, C. D. and Buchwald, N. A., Development of responses of globus pallidus and entopeduncular nucleus neurons to stimulation of caudate nucleus and precruciate cortex, Exp. Neurol., 66 (1976) 479-492. 17 Levine, M. S., Clausen, M. L. and Butcher, S. H., Behavioral effects of phencyclidine in the developing cat, Neuropharmacology, 20 (1981) 743-745. 18 Levine, M. S. and Hull, C. D. and Buchwald, N. A., Development of motor activity in kittens, Dev. Psychobiol., 13 (1980) 357-371. 19 Levine, M. S., Hull, C. D. and Buchwald, N. A., Effects of neonatal destruction of the medial forebrain bundle in the cat: long-term disturbances in learning ability, response to amphetamine challenge and reactivity to auditory stimuli, Exp. Neurol., 81 (1983) 359-375. 20 Levine, M. S., Hull, C. D., Fisher, R. S., Buchwald, N. A. and Heller, A., Effects of neonatal destruction of the medial forebrain bundle in the cat: long-term neurochemical, locomotor, and regulatory deficits, Exp. Neurol., 81 (1983) 340-350. 21 Levine, M. S., Hull, C. D., Buchwald, N. A. and Villablanca, J. R., Effects of caudate nuclei or frontal cortical ablations in kittens: motor activity and visual discrimination performance in n~onatal and juvenile kittens, Exp. Neurol., 62 (1978) 555-569. 22 Levine, M. S., Hull, C. D., Garcia-Rill, E., Erinoff, L., Buchwald, N. A. and Heller, A., Long-term decreases in spontaneous firing of caudate neurons induced by amphetamine in cats, Brain Research, 194 (1980) 263-268. 23 Morris, R., Levine, M. S., Cherubini, E., Buchwald, N. A. and Hull, C. D., Intracellular analysis of the development of responses of caudate neurons to stimulation of cortex thalamus and substantia nigra in the kitten, Brain Research, 73 (1979) 471-487. 24 Porcher, W. and Heller, A., Regional development of Gatecholamine biosynthesis in rat brain, J. Neurochem., 19 (1972) 1917-1930. 25 Seiden, L. S., Fischman, M. W. and Schuster, C. R., Longterm methamphetamine induced changes in brain catecholamines in tolerant rhesus monkeys, Drug Alcohol Depend., 1 (1975/76) 215-219. 26 Shalamby, I. A., Dendel, P. S. and Spear, L. P., Differential functional ontogeny of dopamine presynaptic receptor regulation, Dev. Brain Res., 1 (1981) 434-439. 27 Shaywitz, B. A., Yager, R. D. and Kopper, J. H., Selective brain dopamine depletion in developing rats: an experimental model for minimal brain dysfunction, Science, 191 (1976) 305-308. 28 Siegel, S., Nonparametric Statistics for the Behavioral Sciences, McGraw-Hill, New York, 1956. 29 Villablanca, J. R. and Olmstead, C. E., Neurological development in kittens, Dev. Psychobiol., 12 (1979) 101-127. 30 Winer, B. J., Statistical Principles in Experimental Design, McGraw-Hill, San Francisco, 1971.
Developmental Brain Research, 22 (1985) 150-155 Elsevier
BRD 60090
Behavioral effects of D-amphetamine in developing cats M. S. LEVINE, J. H. HANNIGAN, R. S. FISHER, S. HOWARD-BUTCHER, C. D. HULL and N. A. BUCHWALD Mental Retardation Research Center, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A.) (Accepted May 14th, 1985) Key words: amphetamine - - development - - cat - - dopamine - - stereotypy - - locomotion
The development of the behavioral effects of amphetamine was assessed in kittens of 1-53 days of age. Amphetamine-induced increases in locomotion occurred when animals were beyond 35 days of age. Stereotypic behavior was induced at all ages tested but the predominant type of stereotypy was age-related. From 1 to 14 days amphetamine induced licking. Pendular head movements occurred when animals were under 35 days. At 14 days of age darting, a response consisting of rapid pacing and turning began to occur. Tracking, a series of horizontal and vertical head movements also began to occur after 14 days. The adult response of vertical and horizontal head movements became most prominent after 35 days. This report is part of a continuing series of studies assessing behavioral d e v e l o p m e n t in neonatal kittens and its alteration by pharmacological t r e a t m e n t and/or experimental brain d a m a g e 17A9-21. In the current study the behavioral alterations p r o d u c e d by a single dose of D - a m p h e t a m i n e have been assessed in kittens of different ages. Differential effects of amphetamine during d e v e l o p m e n t are of particular interest because this c o m p o u n d had been used in the treatment of hyperkinetic disorders and so-called 'minimal brain dysfunction' in children 1,27. A l t h o u g h its use has been discontinued for the most part, amphetamine-like c o m p o u n d s such as m e t h y l p h e n i d a t e remain in use 10. A m p h e t a m i n e is a widely used drug of abuse that has been implicated in the etiology of certain varieties of psychoses as well 5. Recent evidence has also indicated that a m p h e t a m i n e administration p r o d u c e d long-term neurochemical and neurophysiological changes 22.25. A d u l t animals respond to acute a m p h e t a m i n e administration with consistent, dose-dependent changes in locomotion and in a variety of other behaviors that b e c o m e stereotypic 10. Developing animals often respond differently 2-4,7,15. F o r example in rats, locomotion is increased by low doses ( 0 . 5 - 4 . 0 mg/kg) when animals below 25 days of age or above
40 days are tested. In contrast, rats of 3 0 - 3 5 days old ('periadolescence') are not behaviorally sensitive to the a m p h e t a m i n e . A t higher doses ( 6 . 0 - 1 6 . 0 mg/kg), both younger and adult rats, but not the 'periadolescent' rats show r e d u c e d l o c o m o t i o n and increased stereotypies2-4,7,t5. A d u l t cats r e s p o n d to a m p h e t a m i n e with changes in locomotion and stereotypic head m o v e m e n t s 12,19. Although there are a n u m b e r of studies on the effects of a m p h e t a m i n e in the adult cat 12,19, there have been, to our knowledge, no consistent observations in the developing kitten. Thus, the present experiment was designed to provide systematic information on the d e v e l o p m e n t of behavioral responses to amphetamine in kittens. The behavior of kittens was monitored after injections of D - a m p h e t a m i n e to determine the types of responses e v o k e d and the agerelated changes in responses during the early developmental period. A total of 43 kittens of both sexes (2l males and 23 females) o b t a i n e d from 11 litters were used. There were no consistent gender differences in response to a m p h e t a m i n e or saline at any age tested and data obtained from males and females were pooled. All animals were born and r e a r e d in the cat breeding colony of the Mental R e t a r d a t i o n Research C e n t e r at
Correspondence: M. S. Levine, Mental Retardation Research Center, Room 58-258A, UCLA School of Medicine, 760 Westwood Plaza, Los Angeles, CA 90024, U.S.A. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
151 U C L A and ranged in age from 1 to 53 days. For all age measurements the day of birth was considered day 1. Each kitten was tested only once and animals were tested individually. Whenever possible two animals from the same litter were tested, one with amphetamine and one with saline. Thus, age ranges and average age of testing for amphetamine-treated and saline-treated groups were approximately the same. Animals were divided into the following age groups according to the age at which they were tested: 1-14 days (15 kittens, 8 amphetamine-treated, mean age 11 days and 7 saline-treated, mean age 9 days); 15-35 days (17 kittens, 9 amphetamine-treated, mean age 27 days and 8 saline-treated, mean age 29 days); and 36-53 days (11 kittens, 7 amphetamine-treated, mean age 47 days and 4 saline-treated, mean age 44 days). There were several reasons for dividing animals into these age categories. First, our previous work showed that marked changes in locomotor activity are occurring over each of these time periods TM. From 1-14 days kittens basically locomote by crawling. From 15-35 days they begin to support their weight and walk. Locomotor activity increases during this time. From 36-60 days locomotor activity remains at levels reached between 15-35 days. Amphetamine treatment in the present experiment produced different effects on locomotion depending upon age. Second, amphetamine appeared to produce different types of stereotypies during each of these age periods. For testing behavioral response to amphetamine, each animal was first placed into a 60 x 60 x 47 cm plywood 'open field' chamber in which the floor was gridded into 36 10 x 10 cm squares by photocells to determine locomotor activity~8. Interruption of each light beam during locomotion produced a single count. The number of photocell counts were recorded every 3 min. After 15 min of baseline testing the kittens received an injection of either D-amphetamine sulfate (2 mg/kg i.p.) or an equivalent volume of saline vehicle. The dose chosen was based on our previously published research on adult cats 19 which shows that doses of 1-2 mg/kg (i.p.) induced marked stereotypies lasting for more than 2 h. The injection was followed immediately by a second test in the open field for 15 min. After this test the kitten was placed in a sound-attenuating chamber equipped with a one-way mirror through which the animal's be-
havior was monitored in detail for 30 min. The behaviors scored included licking, various head movements and patterns of body movement and position. Behaviors were recorded every 3 min and rated on a 4-point scale as follows: 0, behavior did not occur; 1, behavior occurred for less than 30 s during the 3 min rating period; 2, behavior occurred for more than 30 s but less than 3 rain; 3, behavior lasted the entire 3 min to the exclusion of all other behaviors. Following this test locomotor activity was assessed again for 15 min in the open field and then behavior was monitored once again in detail for an additional 30 min in the sound attenuating chamber. Behavibr was monitored periodically thereafter for another 30 min-1 h but was not rated. This sequence of testing made it possible to assess changes quantitatively in locomotor activity and other behaviors influenced by amphetamine. Segments of the session were videotaped for subsequent reanalysis and reliability estimation. lnterrater reliability correlation coefficients exceeded 0.90. Locomotor activity and behavioral rating scores were averaged for each test period for all animals. Differences in locomotor activity scores between groups receiving amphetamine and saline at different ages were assessed with appropriate 3 way analyses of variance and post hoc tests 30. Differences in rating scores were assessed with non-parametric tests2S. Temperature in the activity and observation chambers was maintained between 24-26 °C by infrared heat lamps mounted above each chamber. Animals were tested from 10.00-15.00 h. When two animals from a single litter were tested on the same day order of testing of amphetamine-treated and saline-treated animals was counter-balanced so that one-half of the amphetamine-treated group was tested first and onehalf of the saline-treated group tested first. There were marked age-related changes in the behavioral effects of D-amphetamine. These were characterized by differential alterations in both .locomotor activity and drug-induced stereotypies. Locomotor activity. Increases in locomotion in adult cats following amphetamine administration are dose-dependent 19. Increasing doses (up to 2 mg/kg) produce increased locomotion. At higher dosages locomotion then decreases. The dose used in the present study (2 mg/kg) produces increases in both locomotion and head movement stereotypies in adult
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which the kitten remained immobile but slowly and repeatedly moved its head from side to side. This behavior occurred more frequently 60-90 min postinjection than at 15-45 min. The increases in the frequency of occurrence of this behavior were statistically significant at both 1-14 and 15-35 days of age (P < 0.05). A second behavior that occurred frequently was stereotypic licking of the forepaws and/or floor of the observation chamber. This behavior was slightly more prevalent 60-90 min than 15-45 min postinjection. The increase in the frequency of occurrence of this behavior was significantly different from that of the saline-treated group at 1-14 days only (P < 0.05). In the 15-35-day-old kittens, a transition in the types of behavior induced by amphetamine began to occur. Amphetamine still induced pendular head movements. The occurrence of AGE (DAYS)
cats. In kittens 1-14- and 15-35-days-old, amphetamine administration produced no change in locomotor activity relative to kittens in the same age group receiving saline injections (Fig. 1). In both saline-and amphetamine-treated 1-14 day groups locomotor activity was low. At 15-35 days there were no significant differences between saline- and amphetaminetreated groups. Overall, activity levels were significantly higher compared to those in the younger group (P < 0.05). In 36-53-day-old kittens, amphetamine produced a marked increase in locomotor activity. After the amphetamine injection there was a marked and statistically significant increase in locomotor activity during both postinjection sessions (P < 0.01). This increase was greatest during the second postinjection test (45-60 min). There was no statistically significant difference in baseline activity levels between the saline-treated kittens in the 15-35- and 36-53-day groups. Stereotypic behavior. In adult cats the major stereotypy produced by amphetamine is a dose-dependent increase in horizontal and vertical head movement12j 9. During this behavior the cat remains stationary and moves its head from side to side or up and down, as if tracking an object in space 12,19. In the youngest group of kittens tested (1-14 days), amphetamine produced two consistent behavioral patterns that were different from its effects in adult cats (Fig. 2). The first was pendular head movement in
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153 licking decreased markedly. Two new behaviors, 'darting' and 'tracking', began to occur. Darting was a locomotor behavior in which the kitten paced back and forth for a few steps, turned sharply and then repeated the behavior. Tracking is a behavior that is frequently seen after amphetamine injections in adult cats. When this behavior occurs the animal appears to be fixated on a non-existent object. The eyes and head change position constantly and both vertical and horizontal head movements occur although the latter predominate. In kittens older than 35 days the frequency of both darting and tracking increased markedly. In contrast the frequency of pendular head movements decreased. Increases in frequency in both darting and tracking were statistically signifiAGE (DAYS) 1-14
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cant at 36-53 days (P < 0.05). Only the increase in the frequency of darting at 60-90 min postinjection was significant at 15-35 days. Although we systematically rated behaviors and quantified motor activity for only about 2 h postinjection, we observed that most amphetamine-induced behaviors continued to occur for 3-4 h postinjection in all age groups. During this latter 1 h the frequency and intensity of amphetamine-induced behavior decreased. While this report emphasizes the occurrence of increased locomotion and age-related stereotypies other behaviors were also induced by amphetamine (Fig. 3). For example, amphetamine-treated animals occasionally engaged in circling or climbing. Such behaviors tended to be idiosyncratic and did not occur consistently across animals. Indeed saline-treated animals sometimes exhibited these behaviors as frequently as amphetamine-treated animals. Most saline-treated kittens at all ages were inactive during a large part of the observation test. They remained immobile and, if old enough to have already opened their eyes, would close their eyes. This inactivity and/or sleep occurred less frequently in amphetamine-treated animals especially in the 36-53 day group (P < 0.05). The development of amphetamine-induced autonomic responses was also observed. In 1-14 day kittens piloerection and salivation occurred frequently and in kittens over 15 days pupillary dilation was observed as well. The major findings of this study indicate that amphetamine treatment in early postnatal periods in kittens produces markedly different effects than similar treatment in older kittens and adult cats. For the first 5 postnatal weeks kittens did not display increased locomotion in response to amphetamine. After this time kittens became hyperactive after injections of 2 mg/kg of the drug. Stereotypic behavior was induced by amphetamine at all ages tested. However, the nature of the stereotypy evoked was qualitatively different from the adult-like response at each age. The youngest animals (1-14 days) engaged in stereotypic licking. This behavior did not persist after 2 weeks of age and is rarely observed in adult cats. Up to 5 weeks of age, the predominant stereotypy was a pendular head movement. From 15-35 days a transitional behavior, 'darting', began to occur. Darting and the adult response 'tracking' became prominent after
154 35 days of age. There are a number of mechanisms that may underly the developmental behavioral changes induced by the actions of amphetamine. These mechanisms are probably not independent and there is evidence that they all contribute to the effects of the compound. First, the patterns of behavioral response to amphetamine in any age animal reflects the specific movement capabilities of the animal at that age. Locomotion in the cat develops extensively between 2 and 3 weeks of age is. Kittens in early postnatal periods move about by crawling for short distances 18,29. Thus, it might be expected that amphetamine would not markedly increase locomotion before 4-5 postnatal weeks. Similarly, most visual behaviors do not develop until about 1 month of age or later in the cat 29 although eye opening occurs between the first and second postnatal weeklS,29 Thus, increases in the occurrence of tracking would not be expected until after this time. The present data allow a limited interpretation of the development of sensitivity to amphetamine in the kitten. In the adult, there is a biphasic dose-response curve where locomotion is elevated at lower but not higher doses jg. Stereotypies increase roughly monotonically with increasing dose of amphetamine, behaviorally competing with locomotion. For the kittens tested with the single dose of amphetamine, the absence of elevated locomotion in the two younger age groups could represent either relative insensitivity of this behavior to amphetamine in animals of this age, or a sensitivity high enough so that only stereotypies but not locomotion are affected. In preliminary data, in which different dosages of amphetamine were used, we found that below 1 month of age locomotor activity was rarely increased, whereas the occurrence of amphetamine-induced stereotypies was dose-dependent. Thus, it appears that cats have to mature beyond 30 days before amphetamine markedly increases locomotion. The development of the responses to amphetamine reflects the maturation of dopamine systems in the brain. In the mature nervous system, amphetamine acts primarily by promoting the release and blocking of reuptake and subsequently increasing the synthesis of dopamine 11. A major site of action of amphetamine on dopamine is believed to be the basal ganglia and its associated structures 11. Basal ganglia
dopamine concentration and tyrosine hydroxylase activity develop slowly over the first 40-60 days postnatally in the rat 14,24. In the cat, development of neostriatal dopamine and tyrosine hydroxylase occurs even more slowly over the first 3-5 postnatal months 6. Adult levels are reached at about 4 months of age 6. Thus, in early postnatal periods the ability of amphetamine to produce alterations in dopamine release, re-uptake and synthesis is severely limited by the immaturity of the dopamine system. The functional status of basal ganglia neurons matures over the first two postnatal months and this maturation affects the ability of amphetamine to alter neuronal activityS,16,23. The electrophysiological responses of caudate and substantia nigra neurons to activation of their afferents is markedly different in kittens below one month of age than in older animals. Amphetamine is capable of altering electrophysiological response in substantia nigra neurons in young kittens9. The type of alteration induced in kittens, however, was different from the alteration produced in adult cats. The behavioral response to amphetamine in the kitten may be different from that of the adult because of the differential state of younger dopamine systems. It is not merely the case that the animals become gradually more responsive to the amphetamine. Rather, the induction of stereotypic licking in the youngest group suggests that the dopamine systems in these animals may be organized to respond differently than the dopamine systems in adult cats. It is also possible that the differences may reflect maturation of separate components of dopamine systems at different times (e.g. mesolimbic vs nigrostriatal) 26. Different dopamine systems have been hypothesized to separately mediate locomotor and stereotypic responses to amphetamine 13. Finally, the systems upon which amphetamine acts are not developing in isolation. Areas other than the basal ganglia and neurotransmitter systems other than the dopamine system are concurrently maturing. Locomotion and stereotypies are also influenced by manipulation of these other systems and the role of amphetamine in behavior must be considered in the context of these interactions. Supported by USPHS Grants HD-05958, D A 3107 and HD-07032.
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