378
Brain Research, 135 (1977) 378-382 Elsevier/North-Holland Biomedical Press
Clonidine sensitivity in the developing rat
DANIEL K. REINSTEIN and ROBERT L. iSAACSON
Department of Psychology, University of Florida, Gainesville, Fla. 32611 (U.S.A.) (Accepted June 22nd, 1977)
Pharmacological manipulations of catecholamine (CA) systems during development may help to clarify the relationship between the maturation of neural systems and behavior. The present study employs pharmacological manipulation of a catecholamine (CA) system in order to examine the relation of that system to behavior over the course of development. Previously, Kellogg and Lundborg5 were able to separate components of the L-DOPA-induced behavioral response in the developing rat. LDOPA produced different response patterns in 4- and 7-day-old rats relative to patterns found after drug administration in 14- and 21-day-old animals. Using pharmacological agents such as apomorphine (l mg/kg) and clonidine (2 mg/kg) which more specifically affect dopamine (DA) and norepinephrine (NE) systems, respectively, drug-specific reactions were observed. Changes in the character of the drug-induced behavioral reactions were observed over development. For example, apomorphine produced forward crawling at 7 days but much less of this type of activity at 14 days after birth. Clonidine produced more intense crawling at these respective ages than apomorphine as well as other behavioral signs such as ataxia and decreases in head raising. At day 21, apomorphine induced intense grooming and digging, while clonidine markedly depressed all motor activity. In a subsequent study Kellogg and Lundborg4 found evidence that changes in the response to L-DOPA and ~he response to CA receptor stimulants may be due to differential functional rates of DA and NE development. It was suggested that at least portions of the NE systems mature earlier than DA systems, a finding supported by histochemicalS and behavioral TM evidence. There is evidence to suggest that regional concentrations of catecholamines may approach adult levels of functioning at different periodse. The present study is an attempt to specifically examine the development of behavioral responsivity of the NE systems using the adrenergic agonist, clonidine. Clonidine acts as an adrenergic receptor stimulating agent whose cardiovascular and behavioral effects are primarily mediated by central a-adrenergic receptors1. Cionidine has recently been found to additionally activate histamine H~-rec~ptors in rat cerebral cortexTM. In the current study, examination of the behavioral effects of clonidine were
379 carried out over a longer period than that studied by previous workers and, in addition, dose-response relationships were determined. Pilot work in our laboratory also indicated that the depression of motor activity described in the 21-day-old rat given clonidine resembles cataleptic states observed with certain classes of DA blocking drugs, e.g. haloperidol 3. Therefore, special attention was given to this class of behaviors. Approximately equal numbers of laboratory-bred male and female offspring of Long-Evans hooded rats were used in the experimental groups. The litters were culled to 9-11 pups/mother at birth. Pups were weaned at 25 days and housed in colony cages. Animals were individually tested in square chambers measuring 25 cm on a side and 15 cm high. Four such chambers were situated close to each other during testing and the behavior of animals in all 4 chambers were monitored by a single observer. Ambient temperature was maintained between 30 and 31 °C. Testing was done under red light and began 3-5 h after the onset of the light portion of the light-dark cycle. Each litter was run in groups of 4 subjects over two testing sessions 'centered on' specific postnatal days. Testing termed as being done on day 7, for example, was done on days 6 and 7 or days 7 and 8. Groups were tested so as to center on postnatal days 7, 14, 21, 28 and 35 in this manner. Four animals were placed in the testing compartments 5-10 rain prior to injection. Drug doses of clonidine at 2 mg/kg, mg/kg and 0.5 mg/kg dissolved in physiological saline and control injections of saline were given interperitoneally to the respective animals in volumes proportional to each animal's body weight. Behavioral observations were recorded beginning 10 rain after injection and continued for 2.5 h. A time-sampling technique was used. Each animal was observed at half-minute intervals and the behavior exhibited recorded. Categories of forward locomotion, paddling, and wall climbing were combined for a quantitative measure of activity. Sitting and lying behaviors were used as an index of inactivity. On postnatal days 21, 28 and 35, animals were also tested for catalepsy 30 min after injection. This was done by placing cylindrical objects 7.5 mm in diameter under both hind limbs and at least o~e front limb. A positive test for catalepsy required that the animal remain on the objects at least one rain. The amount of time spent on the objects was taken as a quantitative measure of catalepsy. When not cataleptic, animals were tested for 'waxy flexibility' at approximately 5-min intervals during the session. "Waxy flexibility" was characterizea by an animal's maintainance of an experimenter-initiated abducted-limb posture. The drug-dose groups were made up of 8 animals on every testing day except on postnatal days 7 and 14, when the saline groups had 7 and 6 animals per group, respectively. As shown in Fig. 1, on postnatal day 7, all doses of clonidine produced a doserelated increase in activity. A one-tailed Wilcoxin signed ranks test for matched pair comparisons was used in the analysis of same week dose-response measures for litter mates. All dose levels produced responses that were significantly different from each other at P -~ 0.05. Again, on day 14 dose-dependent increases of activity were found. In addition ~o the dose-related differences in activation observed on days 7 and 14, the scores for each dose on day 7 were significantly greater than the same dose scores on
380
m,%
I~i.ily w
*]i
I
!1
g
I.s
g
klm
7
1~
21
28
3S
hstaatal Oal Fig. I. Activity measures of animals treated with clonidme or saline at different postnatal days. Positive counts on the vertical axis correspond to activity observed during the time period sampled at 30-sec intervals.
postnatal day 14. This indicates a reduced effect of the drug on postnatal day 14. Comparisons between weeks for each dose level were made using two-tailed MannWhitney U-tests. On days 21 and 28, all doses ofclonidine produced significant decreases in motor activity. Dose dependency was not observed. On day 35 t~ere were significant differences between the drug and saline-injected animals only for the 1 mg/kg dose of clonidine. Drug-induced catalepsy was first observed on days 21 and 28 (see Fig. 2). On day
Catalepsy m
=.,)
T
-=~
'i
i.
L
I.s
n.,®
T
~Salme
TT "V
7
14
21
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35
Postnatal h l Fig. 2. Catalepsy measures of animals treated with cionidineor saline at differentpostnatal days.
381 21, the clonidine-induced catalepsy lasted approximately I h, and, on day 28, the effect lasted slightly under 50 min. On day 35, catalepsy was observed in only half the animals and, when observed, it lasted less than 15 min. These cataleptic effects observed on day 35 were significantly less than those observed on days 21 and 28 for both the high and middle doses. In addition to the quantitative measures of activity and catalepsy presented, the behavioral response to clonidine took various other forms. After the drug injections on day 7, the animals engaged in intense forward crawling which was accompanied by bouts of wall climbing. Head raising and body tremor were also observed. Movement was often initiated by a single clonic jerk. In the early portion of the testing session, movements were accompanied by repeated vocalizations. The animals were markedly ataxic and most animals had difficulty in ribhting. Those that could not maintain an upright posture would still maintain limb movements appropriate for locomotion while lying on their sides for long periods of time. On day 14, the same basic pattern emerged a,~ on day 7. Although wall climbing and forward crawling were seen, vocalizations were not heard. All animals were much less ataxic than those treated earlier. Bouts of activity alternated with periods of immobility. Tests for catalepsy at this time, however, were negative. A slight piloerection of the fur was noted. On day 21, activation was severely depressed following the drug and the animals were cataleptic. The animals evidenced piloerection, were exophthalmic and had rigid tails. After the animals had passed through the cataleptic phase, they often reacted violently to the experimenter's attempts to test for waxy flexibility. The hyperreactive response to virtually every stimulus often took the form of a nociceptively-induced rage response. This was followed by a period in which a test for waxy flexibility was positive. The same pattern of reaction to the drug was observed on clay 2~. On day 35, the cataleptic response to clonidine was seen less frequently. When present in an animal, the response followed the same basic pattern observed on days 21 and 28, but was of much shorter duration. The animals that did show the catalepti,~ i'~ponse at this age resumed normal posturing and activity more rapidly than those animals showing the reaction at the earlier ages. In summary, clonidine is observed to produce dose-dependent hyperactivity in 7day-old rats and to a lesser extent in rats of 14 days of age. A dramatic shift occurs at day 21 when clonidine induces catalepsy. This effect was also found in animals tested on postnatal day 28 but declined markedly in frequency of occurrence by day 35. Clonidine produces a depression in motor activity in the adult rat but not catalepsy, though some evidence of abnormal postural attitudes have been reported 9. These changes in the behavioral response to clonidine may be related to the development of interacting neural systems or regional changes in a-adrenergic receptor sites over development. Noradrenergic systems in brain stem and cortex e that develop early in life may be mediating the hyperactive response to clonidine at 7 and 14 days. Later developing noradrenergic systems in the diencephalon e may exert influences directly or indirectly on striatum, where antagonistic interactions of DA and NE have been
382 p,~stulatede as mediators of the cataleptic response to neuroleptics. Another indirect influence of noradrenergic systems on striatum may be through locus coeruleus input to hippocampus. Postnatal development of portions of the hippocampus between days 20 and 30 has been correlated with mo0ification of the behavioral response to amphetamine7. Similar dynamic interaction~ may be responsible for changes in the behavioral response to clonidine. Changes in receptor sensitivity to NE have been observed in hypothalamus as a function of age. The appearance of morphologically immature chubby spines of hypothalamic dendrites has been associated with a membrane rich specific CA-binding protein TM. Increased ratios of morphologically mature to immature dendrites in the 4-10-week-old kitten have been correlated with decreases in stimulus hypersensitivity it. It is possible that regional changes in receptor sensitivity are associated with corresponding changes in the clonidine response. Both hyperactivity as seen at 7 and 14 days and catalepsy at 21 and 28 days may reflect regional hypersensitive responses to cionidine which become modified as the respective systems attain adult levels of functioning. Recent findings suggesting central histamine He-receptor effects of clonidine TM must also be considered. It is possible that clonidine-induced behavioral responses may be correlated with its effects on different transmitter systems. Functional development of these systems may be occurring at different rates. The examination of regional effects of clonidine may serve to clarify the relation of developing brain systems to the changes in clonidine-induced behaviors over development. Support was provided by NIH Grant RR-07-021-11 to the University of Florida.
1 And~n, N. E., Corrodi, H., Fuxe, K., H6kfelt, B., H6kfelt, T., Rydin, C. and Svensson, T., Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci., 9 (1970) 513-523. 2 Baez, L. A., Eskridge, N. K. and Schein, R., Postnatal development of dopaminergi¢ and cholinergic catalepsy in the rat, Europ. J. Pharmacoi., 36 (1976) 155-162. 3 Janssen, P. A. S., The pharmacology of haloperidol, Int. J. Neuropsychiat., 3 (1967) 10-18. 4 Kellogg, C. and Lundborg, P., Inhibition of catecholamine synthesis during ontogenic development, Brain Research, 61 (1973) 321-329. 5 Kellogg, C. and Lundborg, P., Ontogenic variations in responses to L-DOPA and mono~mine receptor-stimulating agents, Psychopharmacologia, 23 (1972) 187-200. 6 Kellogg, C. and Wennerstrom, G., An ontogenic study on the effect of catecholamine receptorstimulating agents on the turnover of noradrenaline and dopamine in the brain, Brain Research, 79 (1974) 451-464. 7 Lanier, L. P. and lsaacson, R. L., Early developmental changes in the locomotor response to amphetamine and their relation to hippocampal function, Brain Research, 126 (1977) 567-575. 8 Loizou, L. A., ~he postnatal ontogeny of monoamine-containing neurones in the central nervous system of the albino rat, Brain Research, 40 (1972) 395-418. 9 Morpurgo, C., Aggressive behavior induced by large doses of 2-(2,6-dichlorphenylamino)-2imidazoline hydrochloride (ST 155) in mice, Europ. J. Pharmacol., 3 (1968) 374-377. 10 Torda, C., Why babies cry, J. Amer. med. Worn. Ass., 31 (1976) 271-284. 11 Torda, C., Bioelectric measurements on adult and newborn hypothalamic dendrites, 3". Neurosci. Res., 2 (1976) 21. 12 Sastry, B. S. R. and Phyllis, J. W., Evidence that clonidine can activate histamine H~-receptors in rat cerebral cortex, Neuropharmacology, 16 (1977) 223-225. 13 Van Hartesveldt, C. and Lindquist, D., Behavioral effects of unilateral basal ganglia lesions in neonatal rat, Develop. Psychobiol., (1977) in press.
Brain Research, 135 (1977) 378-382 Elsevier/North-Holland Biomedical Press
Clonidine sensitivity in the developing rat
DANIEL K. REINSTEIN and ROBERT L. iSAACSON
Department of Psychology, University of Florida, Gainesville, Fla. 32611 (U.S.A.) (Accepted June 22nd, 1977)
Pharmacological manipulations of catecholamine (CA) systems during development may help to clarify the relationship between the maturation of neural systems and behavior. The present study employs pharmacological manipulation of a catecholamine (CA) system in order to examine the relation of that system to behavior over the course of development. Previously, Kellogg and Lundborg5 were able to separate components of the L-DOPA-induced behavioral response in the developing rat. LDOPA produced different response patterns in 4- and 7-day-old rats relative to patterns found after drug administration in 14- and 21-day-old animals. Using pharmacological agents such as apomorphine (l mg/kg) and clonidine (2 mg/kg) which more specifically affect dopamine (DA) and norepinephrine (NE) systems, respectively, drug-specific reactions were observed. Changes in the character of the drug-induced behavioral reactions were observed over development. For example, apomorphine produced forward crawling at 7 days but much less of this type of activity at 14 days after birth. Clonidine produced more intense crawling at these respective ages than apomorphine as well as other behavioral signs such as ataxia and decreases in head raising. At day 21, apomorphine induced intense grooming and digging, while clonidine markedly depressed all motor activity. In a subsequent study Kellogg and Lundborg4 found evidence that changes in the response to L-DOPA and ~he response to CA receptor stimulants may be due to differential functional rates of DA and NE development. It was suggested that at least portions of the NE systems mature earlier than DA systems, a finding supported by histochemicalS and behavioral TM evidence. There is evidence to suggest that regional concentrations of catecholamines may approach adult levels of functioning at different periodse. The present study is an attempt to specifically examine the development of behavioral responsivity of the NE systems using the adrenergic agonist, clonidine. Clonidine acts as an adrenergic receptor stimulating agent whose cardiovascular and behavioral effects are primarily mediated by central a-adrenergic receptors1. Cionidine has recently been found to additionally activate histamine H~-rec~ptors in rat cerebral cortexTM. In the current study, examination of the behavioral effects of clonidine were
379 carried out over a longer period than that studied by previous workers and, in addition, dose-response relationships were determined. Pilot work in our laboratory also indicated that the depression of motor activity described in the 21-day-old rat given clonidine resembles cataleptic states observed with certain classes of DA blocking drugs, e.g. haloperidol 3. Therefore, special attention was given to this class of behaviors. Approximately equal numbers of laboratory-bred male and female offspring of Long-Evans hooded rats were used in the experimental groups. The litters were culled to 9-11 pups/mother at birth. Pups were weaned at 25 days and housed in colony cages. Animals were individually tested in square chambers measuring 25 cm on a side and 15 cm high. Four such chambers were situated close to each other during testing and the behavior of animals in all 4 chambers were monitored by a single observer. Ambient temperature was maintained between 30 and 31 °C. Testing was done under red light and began 3-5 h after the onset of the light portion of the light-dark cycle. Each litter was run in groups of 4 subjects over two testing sessions 'centered on' specific postnatal days. Testing termed as being done on day 7, for example, was done on days 6 and 7 or days 7 and 8. Groups were tested so as to center on postnatal days 7, 14, 21, 28 and 35 in this manner. Four animals were placed in the testing compartments 5-10 rain prior to injection. Drug doses of clonidine at 2 mg/kg, mg/kg and 0.5 mg/kg dissolved in physiological saline and control injections of saline were given interperitoneally to the respective animals in volumes proportional to each animal's body weight. Behavioral observations were recorded beginning 10 rain after injection and continued for 2.5 h. A time-sampling technique was used. Each animal was observed at half-minute intervals and the behavior exhibited recorded. Categories of forward locomotion, paddling, and wall climbing were combined for a quantitative measure of activity. Sitting and lying behaviors were used as an index of inactivity. On postnatal days 21, 28 and 35, animals were also tested for catalepsy 30 min after injection. This was done by placing cylindrical objects 7.5 mm in diameter under both hind limbs and at least o~e front limb. A positive test for catalepsy required that the animal remain on the objects at least one rain. The amount of time spent on the objects was taken as a quantitative measure of catalepsy. When not cataleptic, animals were tested for 'waxy flexibility' at approximately 5-min intervals during the session. "Waxy flexibility" was characterizea by an animal's maintainance of an experimenter-initiated abducted-limb posture. The drug-dose groups were made up of 8 animals on every testing day except on postnatal days 7 and 14, when the saline groups had 7 and 6 animals per group, respectively. As shown in Fig. 1, on postnatal day 7, all doses of clonidine produced a doserelated increase in activity. A one-tailed Wilcoxin signed ranks test for matched pair comparisons was used in the analysis of same week dose-response measures for litter mates. All dose levels produced responses that were significantly different from each other at P -~ 0.05. Again, on day 14 dose-dependent increases of activity were found. In addition ~o the dose-related differences in activation observed on days 7 and 14, the scores for each dose on day 7 were significantly greater than the same dose scores on
380
m,%
I~i.ily w
*]i
I
!1
g
I.s
g
klm
7
1~
21
28
3S
hstaatal Oal Fig. I. Activity measures of animals treated with clonidme or saline at different postnatal days. Positive counts on the vertical axis correspond to activity observed during the time period sampled at 30-sec intervals.
postnatal day 14. This indicates a reduced effect of the drug on postnatal day 14. Comparisons between weeks for each dose level were made using two-tailed MannWhitney U-tests. On days 21 and 28, all doses ofclonidine produced significant decreases in motor activity. Dose dependency was not observed. On day 35 t~ere were significant differences between the drug and saline-injected animals only for the 1 mg/kg dose of clonidine. Drug-induced catalepsy was first observed on days 21 and 28 (see Fig. 2). On day
Catalepsy m
=.,)
T
-=~
'i
i.
L
I.s
n.,®
T
~Salme
TT "V
7
14
21
2g
35
Postnatal h l Fig. 2. Catalepsy measures of animals treated with cionidineor saline at differentpostnatal days.
381 21, the clonidine-induced catalepsy lasted approximately I h, and, on day 28, the effect lasted slightly under 50 min. On day 35, catalepsy was observed in only half the animals and, when observed, it lasted less than 15 min. These cataleptic effects observed on day 35 were significantly less than those observed on days 21 and 28 for both the high and middle doses. In addition to the quantitative measures of activity and catalepsy presented, the behavioral response to clonidine took various other forms. After the drug injections on day 7, the animals engaged in intense forward crawling which was accompanied by bouts of wall climbing. Head raising and body tremor were also observed. Movement was often initiated by a single clonic jerk. In the early portion of the testing session, movements were accompanied by repeated vocalizations. The animals were markedly ataxic and most animals had difficulty in ribhting. Those that could not maintain an upright posture would still maintain limb movements appropriate for locomotion while lying on their sides for long periods of time. On day 14, the same basic pattern emerged a,~ on day 7. Although wall climbing and forward crawling were seen, vocalizations were not heard. All animals were much less ataxic than those treated earlier. Bouts of activity alternated with periods of immobility. Tests for catalepsy at this time, however, were negative. A slight piloerection of the fur was noted. On day 21, activation was severely depressed following the drug and the animals were cataleptic. The animals evidenced piloerection, were exophthalmic and had rigid tails. After the animals had passed through the cataleptic phase, they often reacted violently to the experimenter's attempts to test for waxy flexibility. The hyperreactive response to virtually every stimulus often took the form of a nociceptively-induced rage response. This was followed by a period in which a test for waxy flexibility was positive. The same pattern of reaction to the drug was observed on clay 2~. On day 35, the cataleptic response to clonidine was seen less frequently. When present in an animal, the response followed the same basic pattern observed on days 21 and 28, but was of much shorter duration. The animals that did show the catalepti,~ i'~ponse at this age resumed normal posturing and activity more rapidly than those animals showing the reaction at the earlier ages. In summary, clonidine is observed to produce dose-dependent hyperactivity in 7day-old rats and to a lesser extent in rats of 14 days of age. A dramatic shift occurs at day 21 when clonidine induces catalepsy. This effect was also found in animals tested on postnatal day 28 but declined markedly in frequency of occurrence by day 35. Clonidine produces a depression in motor activity in the adult rat but not catalepsy, though some evidence of abnormal postural attitudes have been reported 9. These changes in the behavioral response to clonidine may be related to the development of interacting neural systems or regional changes in a-adrenergic receptor sites over development. Noradrenergic systems in brain stem and cortex e that develop early in life may be mediating the hyperactive response to clonidine at 7 and 14 days. Later developing noradrenergic systems in the diencephalon e may exert influences directly or indirectly on striatum, where antagonistic interactions of DA and NE have been
382 p,~stulatede as mediators of the cataleptic response to neuroleptics. Another indirect influence of noradrenergic systems on striatum may be through locus coeruleus input to hippocampus. Postnatal development of portions of the hippocampus between days 20 and 30 has been correlated with mo0ification of the behavioral response to amphetamine7. Similar dynamic interaction~ may be responsible for changes in the behavioral response to clonidine. Changes in receptor sensitivity to NE have been observed in hypothalamus as a function of age. The appearance of morphologically immature chubby spines of hypothalamic dendrites has been associated with a membrane rich specific CA-binding protein TM. Increased ratios of morphologically mature to immature dendrites in the 4-10-week-old kitten have been correlated with decreases in stimulus hypersensitivity it. It is possible that regional changes in receptor sensitivity are associated with corresponding changes in the clonidine response. Both hyperactivity as seen at 7 and 14 days and catalepsy at 21 and 28 days may reflect regional hypersensitive responses to cionidine which become modified as the respective systems attain adult levels of functioning. Recent findings suggesting central histamine He-receptor effects of clonidine TM must also be considered. It is possible that clonidine-induced behavioral responses may be correlated with its effects on different transmitter systems. Functional development of these systems may be occurring at different rates. The examination of regional effects of clonidine may serve to clarify the relation of developing brain systems to the changes in clonidine-induced behaviors over development. Support was provided by NIH Grant RR-07-021-11 to the University of Florida.
1 And~n, N. E., Corrodi, H., Fuxe, K., H6kfelt, B., H6kfelt, T., Rydin, C. and Svensson, T., Evidence for a central noradrenaline receptor stimulation by clonidine, Life Sci., 9 (1970) 513-523. 2 Baez, L. A., Eskridge, N. K. and Schein, R., Postnatal development of dopaminergi¢ and cholinergic catalepsy in the rat, Europ. J. Pharmacoi., 36 (1976) 155-162. 3 Janssen, P. A. S., The pharmacology of haloperidol, Int. J. Neuropsychiat., 3 (1967) 10-18. 4 Kellogg, C. and Lundborg, P., Inhibition of catecholamine synthesis during ontogenic development, Brain Research, 61 (1973) 321-329. 5 Kellogg, C. and Lundborg, P., Ontogenic variations in responses to L-DOPA and mono~mine receptor-stimulating agents, Psychopharmacologia, 23 (1972) 187-200. 6 Kellogg, C. and Wennerstrom, G., An ontogenic study on the effect of catecholamine receptorstimulating agents on the turnover of noradrenaline and dopamine in the brain, Brain Research, 79 (1974) 451-464. 7 Lanier, L. P. and lsaacson, R. L., Early developmental changes in the locomotor response to amphetamine and their relation to hippocampal function, Brain Research, 126 (1977) 567-575. 8 Loizou, L. A., ~he postnatal ontogeny of monoamine-containing neurones in the central nervous system of the albino rat, Brain Research, 40 (1972) 395-418. 9 Morpurgo, C., Aggressive behavior induced by large doses of 2-(2,6-dichlorphenylamino)-2imidazoline hydrochloride (ST 155) in mice, Europ. J. Pharmacol., 3 (1968) 374-377. 10 Torda, C., Why babies cry, J. Amer. med. Worn. Ass., 31 (1976) 271-284. 11 Torda, C., Bioelectric measurements on adult and newborn hypothalamic dendrites, 3". Neurosci. Res., 2 (1976) 21. 12 Sastry, B. S. R. and Phyllis, J. W., Evidence that clonidine can activate histamine H~-receptors in rat cerebral cortex, Neuropharmacology, 16 (1977) 223-225. 13 Van Hartesveldt, C. and Lindquist, D., Behavioral effects of unilateral basal ganglia lesions in neonatal rat, Develop. Psychobiol., (1977) in press.
