Journal of the Neurological Seienees, 1976, 2 8 : 7 7 - 8 2
77
'.(~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
IN VIVO REVERSAL OF T H Y R O X I N E I N D U C T I O N OF DNA SYNTHESIS BY D I B U T Y R Y L CYCLIC AMP IN D E V E L O P I N G RAT CEREBELLUM
M O R T O N E. WEICHSEL, Jr. and JAMES E. T R O S K O
Department Of Human Development, Michigan State University, East Lansing, Mich. (U.S.A.) (Received 21 August, 1975)
SUMMARY
Thyroxine, dibutyryl cyclic AMP, and a combination of both drugs were administered daily from birth to 2, 2 and 3 pups, respectively from each of 5 litters of Sprague-Dawley rats. Body weight, brain weight, cerebellar weight, and cerebellar DNA were measured in each animal at age 5 days and compared with values from a pair of controls from each litter. Cerebellar weight and DNA content were affected more severely than body weight in cyclic AM P-treated animals, with cerebellar DNA reduced significantly to 8 8 ~ of control values. Cerebellar DNA was significantly elevated to 117~, of control values in thyroxine-treated animals. This augmentation of cerebellar DNA synthesis by thyroxine was negated by administration of dibutyryl cyclic AMP 10 min prior to the thyroxine injection. These results support an hypothesis that the enhancement of cerebellar cell division by thyroxine involves an increase in the ratio of intracellular cyclic guanosine monophosphate to cyclic adenosine monophosphate. The reversal of the thyroxine-induced increase in cerebellar DNA synthesis by a prior injection of dibutyryl cyclic AMP suggests that the early stimulation of cell division by thyroxine may be mediated by cyclic AMP, and that the intracellular balance between cerebellar cyclic AMP and cyclic G M P was distorted by in vivo elevation of intracellular cyclic AMP levels.
1NTRODUCTION
The relationships between hormones and the developing nervous system have recently become of increasing interest. The rat cerebellum has been used as a model This research was supported by National Institutes of Health Grants H D 07275-01 and H D 09277-01, United States Public Health Service. J. E. Trosko is a recipient of a PHS Career
Development Award (1 K4 CA 24, 085q33). Reprint requests to: M. E. Weichsel, Jr., M.D., Department of Pediatrics, Harbor General Hospital, 1000 W. Carson Street, Torrance, Calif. 90509, U.S.A.
78 for the study of cell division during brain development because of its multi-fold increase in DNA (cell number) during the first 3 postnatal weeks with the peak rate of cell replication occurring near the middle of that period. Cortisol administration at birth in the rat has been shown to suppress severely cerebellar cell division (Cotterrell, Bal~tzs and Johnson 1972). Furthermore, in neonatal hypothyroidism, cerebellar DNA content is below control values during the first part of cerebellar ontogenesis with a gradual return towards normal values occurring over a prolonged period of time (Gourdon, CIos, Coste, Dainat and Legrand 1973). In contrast to the effects on cerebellar DNA biosynthesis in these two conditions, Weichsel (1974) has recently noted a significant increase in cerebellar DNA biosynthesis by the second day of life in the cerebella of rat pups injected with thyroxine daily from birth. This increase above normal values lasts through 6 days of age, following which DNA synthesis is prematurely terminated. This finding suggests that a critical relationship exists between thyroxine and the regulation of cerebellar DNA biosynthesis. The mechanisms underlying the regulatory processes that control cell division tinder the influences of hormonal stimuli are poorly understood, although it is known that intracellular concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) influence these processes (Clarkson and Baserga 1974). Goldberg, Haddox, Estensen, Lopez aod Hadden (1975) have proposed a "dualism" theory by which cellular processes that are "bidirectionally controlled" may be related to the ratio of the intracellular concentration of cGMP to cAMP, with the concentrations varying with the direction of the process. Under this hypothesis, the early acceleration of cerebellar DNA biosynthesis reported by Weichsel (1974) should be accompanied by an increase in the ratio of these two nucleotides. The majority of studies supporting the Goldberg hypothesis for cellular growth regulation have been conducted with in vitro systems. We initiated the present study in order to determine whether elevation of cerebellar cAMP in vivo might retard rat cerebellar cell division and reverse or counteract potential intracellular changes in cyclic nucleotide relationships secondary to the early thyroxine-mediated enhancement of cell division METHODS
Five litters of Sprague-Dawley rats born within a 12-hr period were used in this study. All pups were the products of a second pregnancy. Litters were reduced to a total of 9 pups regardless of sex. The day of birth was considered day 0. All injections were administered daily during the same 2 hr period. Two pups randomly selected from each litter were treated with 0.4 #g thyroxine per g body weight subcutaneously in the right flank. Two pups received dibutyryl cAMP, 0.t2 mg per g body weight, subcutaneously in the left flank, and 3 pups received the dosage of dibutyryl cAMP subcutaneously in the left flank followed 10 rain later by 0.4 #g thyroxine per g body weight subcutaneously in the right flank. The remaining 2 pups from each litter were used as controls and injected with 0.1 ml of saline. Dams were fed Wayne lab blox rat chow and all animals were housed at 22
77
'.(~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
IN VIVO REVERSAL OF T H Y R O X I N E I N D U C T I O N OF DNA SYNTHESIS BY D I B U T Y R Y L CYCLIC AMP IN D E V E L O P I N G RAT CEREBELLUM
M O R T O N E. WEICHSEL, Jr. and JAMES E. T R O S K O
Department Of Human Development, Michigan State University, East Lansing, Mich. (U.S.A.) (Received 21 August, 1975)
SUMMARY
Thyroxine, dibutyryl cyclic AMP, and a combination of both drugs were administered daily from birth to 2, 2 and 3 pups, respectively from each of 5 litters of Sprague-Dawley rats. Body weight, brain weight, cerebellar weight, and cerebellar DNA were measured in each animal at age 5 days and compared with values from a pair of controls from each litter. Cerebellar weight and DNA content were affected more severely than body weight in cyclic AM P-treated animals, with cerebellar DNA reduced significantly to 8 8 ~ of control values. Cerebellar DNA was significantly elevated to 117~, of control values in thyroxine-treated animals. This augmentation of cerebellar DNA synthesis by thyroxine was negated by administration of dibutyryl cyclic AMP 10 min prior to the thyroxine injection. These results support an hypothesis that the enhancement of cerebellar cell division by thyroxine involves an increase in the ratio of intracellular cyclic guanosine monophosphate to cyclic adenosine monophosphate. The reversal of the thyroxine-induced increase in cerebellar DNA synthesis by a prior injection of dibutyryl cyclic AMP suggests that the early stimulation of cell division by thyroxine may be mediated by cyclic AMP, and that the intracellular balance between cerebellar cyclic AMP and cyclic G M P was distorted by in vivo elevation of intracellular cyclic AMP levels.
1NTRODUCTION
The relationships between hormones and the developing nervous system have recently become of increasing interest. The rat cerebellum has been used as a model This research was supported by National Institutes of Health Grants H D 07275-01 and H D 09277-01, United States Public Health Service. J. E. Trosko is a recipient of a PHS Career
Development Award (1 K4 CA 24, 085q33). Reprint requests to: M. E. Weichsel, Jr., M.D., Department of Pediatrics, Harbor General Hospital, 1000 W. Carson Street, Torrance, Calif. 90509, U.S.A.
78 for the study of cell division during brain development because of its multi-fold increase in DNA (cell number) during the first 3 postnatal weeks with the peak rate of cell replication occurring near the middle of that period. Cortisol administration at birth in the rat has been shown to suppress severely cerebellar cell division (Cotterrell, Bal~tzs and Johnson 1972). Furthermore, in neonatal hypothyroidism, cerebellar DNA content is below control values during the first part of cerebellar ontogenesis with a gradual return towards normal values occurring over a prolonged period of time (Gourdon, CIos, Coste, Dainat and Legrand 1973). In contrast to the effects on cerebellar DNA biosynthesis in these two conditions, Weichsel (1974) has recently noted a significant increase in cerebellar DNA biosynthesis by the second day of life in the cerebella of rat pups injected with thyroxine daily from birth. This increase above normal values lasts through 6 days of age, following which DNA synthesis is prematurely terminated. This finding suggests that a critical relationship exists between thyroxine and the regulation of cerebellar DNA biosynthesis. The mechanisms underlying the regulatory processes that control cell division tinder the influences of hormonal stimuli are poorly understood, although it is known that intracellular concentrations of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) influence these processes (Clarkson and Baserga 1974). Goldberg, Haddox, Estensen, Lopez aod Hadden (1975) have proposed a "dualism" theory by which cellular processes that are "bidirectionally controlled" may be related to the ratio of the intracellular concentration of cGMP to cAMP, with the concentrations varying with the direction of the process. Under this hypothesis, the early acceleration of cerebellar DNA biosynthesis reported by Weichsel (1974) should be accompanied by an increase in the ratio of these two nucleotides. The majority of studies supporting the Goldberg hypothesis for cellular growth regulation have been conducted with in vitro systems. We initiated the present study in order to determine whether elevation of cerebellar cAMP in vivo might retard rat cerebellar cell division and reverse or counteract potential intracellular changes in cyclic nucleotide relationships secondary to the early thyroxine-mediated enhancement of cell division METHODS
Five litters of Sprague-Dawley rats born within a 12-hr period were used in this study. All pups were the products of a second pregnancy. Litters were reduced to a total of 9 pups regardless of sex. The day of birth was considered day 0. All injections were administered daily during the same 2 hr period. Two pups randomly selected from each litter were treated with 0.4 #g thyroxine per g body weight subcutaneously in the right flank. Two pups received dibutyryl cAMP, 0.t2 mg per g body weight, subcutaneously in the left flank, and 3 pups received the dosage of dibutyryl cAMP subcutaneously in the left flank followed 10 rain later by 0.4 #g thyroxine per g body weight subcutaneously in the right flank. The remaining 2 pups from each litter were used as controls and injected with 0.1 ml of saline. Dams were fed Wayne lab blox rat chow and all animals were housed at 22
