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Effects of prenatal administration of psychotropic drugs to rats on brain butytylcholinesterase activity at birth
460
SHORT COMMUNICATIONS
Effects of prenatal administration of psychotropic drugs to rats on brain butytylcholinesterase activity at birth Studies have shown that prenatal administration of CNS drugs to rats can lead to functional21 and behavioralg-ll,2z,23 changes in the offspring. Little is known, however, about the biochemical factors underlying such changes. The present study was designed to investigate the effects of prenatal administration of chlorpromazine or amphetamine on brain biochemical parameters of the offspring at birth. Sprague-Dawley pregnant rats were used. On the 12th, 13th, 14th, and 15th days of gestation 5 rats were given subcutaneously 1 mg/kg amphetamine sulfate; 4 rats, 3 mg/kg chlorpromazine HCI, and 4 rats, 0.9 ~ NaC1, which served as controls. These days were chosen because this period is critical for organogenesis in the rat 16. Chlorpromazine was prepared immediately prior to treatment and was kept in a bottle wrapped with aluminum foil to avoid deterioration as a result of exposure to light 13. At birth, 20-21 days, 6 rats from each group were sacrificed and the brains used for biochemical determinations to be described below. The remaining offspring were used to study behavioral changes during development (unpublished experiments). The brain was rapidly removed, dissected free of grossly visible blood vessels over ice, blotted free of moisture, and weighed. The following CNS areas were separated: the entire spinal cord (removed using the 'toothpaste method' of Pomerat 18); diencephalon-midbrain; and cerebral hemispheres. Acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) activities were determined colorimetrically by means of a Beckman DU spectrophotometer, using the rate of hydrolysis of the substrates acetylthiocholine (AcTCh) and butyrylthiocholine (BuTCh) according to the method of Ellman et al. 4. For determination of BuChE, the selective AChE inhibitor, 1,5-bis-(4-trimethyl-ammoniumphenyl)pentan-3-one di-iodide (Wellcome Laboratories, 62c47), was used 1. An aliquot from the same sample used for enzyme analysis was utilized for protein determination by the Lowry method 14. Extraction of RNA and DNA was done according to the method of Schneiderag, as modified by Geel and Timiras 6. Determinations of RNA from portions of the nucleic acid extract were made according to the orcinol procedure of Ceriotti 3 as modified by Geel and Timiras 6. The method used for DNA analysis was the diphenylamine procedure described by Burton z and modified by Geel and Timiras 6. To determine whether the parameters measured in control and drugtreated offspring differed significantly in their means, the t test for nonpaired data was applied 5. Previous studies~5 have shown that AChE reaches adult levels in the spinal cord between 18 days of gestation and 2 days after birth, and in the cerebral cortex, around 45 days after birth. The data of the present study on AChE activity are in agreement with the previous findings. The high DNA content in newborn animals found in this study (Table I) and in a previous study17, as compared to lower values found in rats at 22 and 90 days of age ~0, gives further evidence of the immaturity of the cerebral hemispheres at birth. Brain Research, 21 (1970) 460-463
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Brain Research, 21 (1970) 460-463
462
SHORT COMMUNICATIONS
Prenatal treatment with chlorpromazine or amphetamine affected only BuChE activity in the diencephalon-midbrain and the cerebral hemispheres, the least mature of the CNS structures studied (Table I). Whether the drugs directly affected the fetal CNS or induced maternal alterations of hormonal influences on the fetus is not evident from these data, although it has been shown that m a n y psychoactive drugs cross the placental barrier 7. BuChE is thought to be primarily localized in the neuroglia 8. The decrease in BuChE activity observed in the cerebral hemispheres of rats whose mothers were treated either with chlorpromazine or amphetamine can be interpreted to reflect either a lower number of neuroglia as compared to controls, or a lower metabolic activity in these cells. Changes in glial cells have been reported to take place under various experimental conditions. For example, RNA, proteins and respiratory enzyme activities increase in neurons and decrease in glial cells as a function of rotatory stimulation in rats vz. During sleep in rabbits, respiratory enzyme activity is high in the neurons and low in the glial cells, whereas during wakefulness this relationship is reversed 12. Treating rabbits with phenylcyclopropylamine, a monoamine oxidase inhibitor, increases neuronal and decreases glial R N A and cytochrome oxidase activities lz. The implication from the present data, that the glial cells may be sensitive to chlorpromazine and amphetamine, is therefore very likely. These two drugs, which have generally opposite CNS effects and mechanisms of action in the adult, had the same biochemical effect on the developing organism. This investigation was supported by a U.S. Public Health Service Research G r a n t (R01 MH-15931-02). Dr. Vernadakis has a Research Scientist Development Award (K02 MH-42479-01) from the National Institute of Mental Health, National Institutes of Health. Dr. Clark held a Psychology Research Associateship with the Veterans Administration. Chlorpromazine HC1 and amphetamine sulfate were made available through the generosity of Smith, Kline and French Laboratories. The acetylcholinesterase inhibitor, 62c47, was kindly supplied by Burroughs Wellcome and Company Laboratories. The able technical assistance of Mrs. Judith Astrin is gratefully acknowledged. Departments of Psychiatry and Pharmacology, University of Colorado Medical Center, and Veterans Administration Hospital, Denver, Colo. 80220 (U.S.A.)
ANTONIA VERNADAKIS CAROL V. H. CLARK
1 BAYLISS,B. J., AND TODRICK, A., The use of a selective acetylcholinesterase inhibitor in the estima-
tion of pseudo-cholinesterase activity in rat brain, Biochem. J., 62 (1956) 62-67. 2 BURTON,K., A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA, Biochem. J., 62 (1956) 315-323. 3 CERIOTTI,G., Determinations of nucleic acids in animal tissues, J. biol. Chem., 214 (1955) 59-70. 4 ELLMAN,G. L., COURTNEY,K. D., ANDRES,V., AND FEATHERSTONE,R. M., m new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 FISHER,R. A., Statistical Methods for Research Workers, Hafner, New York, 1950. 6 GEEL,S., ANDTIMIRAS,P. S., The influence of neonatal hypothyroidism and of thyroxine on the Brain Research, 21 (1970) 460-463
SHORT COMMUNICATIONS
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ribonucleic acid and deoxyribonucleic acid concentrations of rat cerebral cortex, Brain Research, 4 (1967) 135-142. GHETTI, B., GLIOZZI, E., AND CASSANO,G. B., Placental transfer and foetal distribution of psychoactive and hypnotic drugs. In A. CERLETT~AND F. J. BOV~ (Eds.), The Present Status of Psychotropic Drugs, Excerpta Medica Found., Amsterdam, 1969, pp. 349-352. GIACOBINI, E., Metabolic relations between glia and neurons studied in single cells. In M. M. COHEN AND R. S. SNIDER (Eds.), Morphological and Biochemical Correlates of Neural Activit.v, Harper and Row, New York, 1964, pp. 15-38. HOFFELD, D. R., AND WEBSTER, R. L., Effect of injection of tranquilizing drugs during pregnancy on offspring, Nature (Lond.), 205 (1965) 1030-1072. HOrFELD, D. R., WEBSTER, R. L., AND MCNEW, J., Adverse effects on offspring of tranquilizing drugs during pregnancy, Nature (Lond.), 215 (1967) 182-183. HOFFELD, D. R., MCNEW, J., AND WEBSTER, R. L., Effect of tranquilizing drugs during pregnancy on activity of offspring, Nature (Lond.), 218 (1968) 357-358. HYD~N, H., R N A in brain cells. In G. C. QUARTON, T. MELNICHUK AND F. O. SCHMITT (Eds.), The Neurosciences, Rockefeller Univ. Press, New York, 1967, pp. 248-266. JARVIK, M. E., Drugs used in the treatment of psychiatric disorders. In L. S. GOODMAN AND A. GILMAN (Eds.), The Pharmacological Basis of Therapeutics, MacMillan, New York, 1965, pp. 159214. LowRY, O. H., ROSEBROUGH, N. J,, FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. MALETTA, G. J., VERNADAKIS, A., AND TIMIRAS, P. S., Acetylcholinesterase activity and protein content of brain and spinal cord after prenatal X-irradiation, J. Neurochem., 14 (1967) 647-752. NICHOLAS, J. S., Experimental methods and rat embroys. In J. A. GRrVFITH, JR. AND E. J. FARRIS (Eds.), The Rat in Laboratory Investigation, Lippincott, Philadelphia, 1949, pp. 51-57. PETROPOULOS, E. A., VERNADAKIS, A., AND TIMIRAS, P. S,, Nucleic acid content in developing rat brain after prenatal and/or neonatal exposure to high altitude, Fed. Proc., 28 (1969) 1001-1005. POMERAT, C. M., DRAGER, G. A., AND PAINTER, J. T., Effect of some barbiturates on tissues in vitro, Proc. Soc. exp. Biol. Med., 63 (1946) 322-325. SCHNEIDER, W. C., Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid, J. biol. Chem., 161 (1945) 293-303. VERNADAKIS, A., VALCANA, T., CURRY, J. J., MALETTA, C. J., HUDSON, D., AND TIMIRAS, P. S., Alterations in growth of brain and other organs after electroshock in rats, Exp. Neurol., 17 (1967) 505-516. VERNADAKIS,A., ANDWOODBURY,D. M., The developing animal as a model, Epilepsia (Amst.), 10 (1969) 163-178. WERBOFF,J., AND DEMBICKI, E. L., Toxic effects of tranquilizers administered to gravid rats, J. Neuropsychiat., 4 0962) 87-91. WERBOFE, J., AND KESNER, R., Learning deficits of offspring after administration of tranquilizing drugs to the mothers, Nature (Lond.), 197 (1963) 106-107.
(Accepted May 22nd, 1970)
Brain Research, 21 (1970) 460-463
SHORT COMMUNICATIONS
Effects of prenatal administration of psychotropic drugs to rats on brain butytylcholinesterase activity at birth Studies have shown that prenatal administration of CNS drugs to rats can lead to functional21 and behavioralg-ll,2z,23 changes in the offspring. Little is known, however, about the biochemical factors underlying such changes. The present study was designed to investigate the effects of prenatal administration of chlorpromazine or amphetamine on brain biochemical parameters of the offspring at birth. Sprague-Dawley pregnant rats were used. On the 12th, 13th, 14th, and 15th days of gestation 5 rats were given subcutaneously 1 mg/kg amphetamine sulfate; 4 rats, 3 mg/kg chlorpromazine HCI, and 4 rats, 0.9 ~ NaC1, which served as controls. These days were chosen because this period is critical for organogenesis in the rat 16. Chlorpromazine was prepared immediately prior to treatment and was kept in a bottle wrapped with aluminum foil to avoid deterioration as a result of exposure to light 13. At birth, 20-21 days, 6 rats from each group were sacrificed and the brains used for biochemical determinations to be described below. The remaining offspring were used to study behavioral changes during development (unpublished experiments). The brain was rapidly removed, dissected free of grossly visible blood vessels over ice, blotted free of moisture, and weighed. The following CNS areas were separated: the entire spinal cord (removed using the 'toothpaste method' of Pomerat 18); diencephalon-midbrain; and cerebral hemispheres. Acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) activities were determined colorimetrically by means of a Beckman DU spectrophotometer, using the rate of hydrolysis of the substrates acetylthiocholine (AcTCh) and butyrylthiocholine (BuTCh) according to the method of Ellman et al. 4. For determination of BuChE, the selective AChE inhibitor, 1,5-bis-(4-trimethyl-ammoniumphenyl)pentan-3-one di-iodide (Wellcome Laboratories, 62c47), was used 1. An aliquot from the same sample used for enzyme analysis was utilized for protein determination by the Lowry method 14. Extraction of RNA and DNA was done according to the method of Schneiderag, as modified by Geel and Timiras 6. Determinations of RNA from portions of the nucleic acid extract were made according to the orcinol procedure of Ceriotti 3 as modified by Geel and Timiras 6. The method used for DNA analysis was the diphenylamine procedure described by Burton z and modified by Geel and Timiras 6. To determine whether the parameters measured in control and drugtreated offspring differed significantly in their means, the t test for nonpaired data was applied 5. Previous studies~5 have shown that AChE reaches adult levels in the spinal cord between 18 days of gestation and 2 days after birth, and in the cerebral cortex, around 45 days after birth. The data of the present study on AChE activity are in agreement with the previous findings. The high DNA content in newborn animals found in this study (Table I) and in a previous study17, as compared to lower values found in rats at 22 and 90 days of age ~0, gives further evidence of the immaturity of the cerebral hemispheres at birth. Brain Research, 21 (1970) 460-463
461
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Brain Research, 21 (1970) 460-463
462
SHORT COMMUNICATIONS
Prenatal treatment with chlorpromazine or amphetamine affected only BuChE activity in the diencephalon-midbrain and the cerebral hemispheres, the least mature of the CNS structures studied (Table I). Whether the drugs directly affected the fetal CNS or induced maternal alterations of hormonal influences on the fetus is not evident from these data, although it has been shown that m a n y psychoactive drugs cross the placental barrier 7. BuChE is thought to be primarily localized in the neuroglia 8. The decrease in BuChE activity observed in the cerebral hemispheres of rats whose mothers were treated either with chlorpromazine or amphetamine can be interpreted to reflect either a lower number of neuroglia as compared to controls, or a lower metabolic activity in these cells. Changes in glial cells have been reported to take place under various experimental conditions. For example, RNA, proteins and respiratory enzyme activities increase in neurons and decrease in glial cells as a function of rotatory stimulation in rats vz. During sleep in rabbits, respiratory enzyme activity is high in the neurons and low in the glial cells, whereas during wakefulness this relationship is reversed 12. Treating rabbits with phenylcyclopropylamine, a monoamine oxidase inhibitor, increases neuronal and decreases glial R N A and cytochrome oxidase activities lz. The implication from the present data, that the glial cells may be sensitive to chlorpromazine and amphetamine, is therefore very likely. These two drugs, which have generally opposite CNS effects and mechanisms of action in the adult, had the same biochemical effect on the developing organism. This investigation was supported by a U.S. Public Health Service Research G r a n t (R01 MH-15931-02). Dr. Vernadakis has a Research Scientist Development Award (K02 MH-42479-01) from the National Institute of Mental Health, National Institutes of Health. Dr. Clark held a Psychology Research Associateship with the Veterans Administration. Chlorpromazine HC1 and amphetamine sulfate were made available through the generosity of Smith, Kline and French Laboratories. The acetylcholinesterase inhibitor, 62c47, was kindly supplied by Burroughs Wellcome and Company Laboratories. The able technical assistance of Mrs. Judith Astrin is gratefully acknowledged. Departments of Psychiatry and Pharmacology, University of Colorado Medical Center, and Veterans Administration Hospital, Denver, Colo. 80220 (U.S.A.)
ANTONIA VERNADAKIS CAROL V. H. CLARK
1 BAYLISS,B. J., AND TODRICK, A., The use of a selective acetylcholinesterase inhibitor in the estima-
tion of pseudo-cholinesterase activity in rat brain, Biochem. J., 62 (1956) 62-67. 2 BURTON,K., A study of the conditions and mechanisms of the diphenylamine reaction for the colorimetric estimation of DNA, Biochem. J., 62 (1956) 315-323. 3 CERIOTTI,G., Determinations of nucleic acids in animal tissues, J. biol. Chem., 214 (1955) 59-70. 4 ELLMAN,G. L., COURTNEY,K. D., ANDRES,V., AND FEATHERSTONE,R. M., m new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol., 7 (1961) 88-95. 5 FISHER,R. A., Statistical Methods for Research Workers, Hafner, New York, 1950. 6 GEEL,S., ANDTIMIRAS,P. S., The influence of neonatal hypothyroidism and of thyroxine on the Brain Research, 21 (1970) 460-463
SHORT COMMUNICATIONS
7
8
9 10 11 12 13
14 15 16 17 18 19 20
21 22 23
463
ribonucleic acid and deoxyribonucleic acid concentrations of rat cerebral cortex, Brain Research, 4 (1967) 135-142. GHETTI, B., GLIOZZI, E., AND CASSANO,G. B., Placental transfer and foetal distribution of psychoactive and hypnotic drugs. In A. CERLETT~AND F. J. BOV~ (Eds.), The Present Status of Psychotropic Drugs, Excerpta Medica Found., Amsterdam, 1969, pp. 349-352. GIACOBINI, E., Metabolic relations between glia and neurons studied in single cells. In M. M. COHEN AND R. S. SNIDER (Eds.), Morphological and Biochemical Correlates of Neural Activit.v, Harper and Row, New York, 1964, pp. 15-38. HOFFELD, D. R., AND WEBSTER, R. L., Effect of injection of tranquilizing drugs during pregnancy on offspring, Nature (Lond.), 205 (1965) 1030-1072. HOrFELD, D. R., WEBSTER, R. L., AND MCNEW, J., Adverse effects on offspring of tranquilizing drugs during pregnancy, Nature (Lond.), 215 (1967) 182-183. HOFFELD, D. R., MCNEW, J., AND WEBSTER, R. L., Effect of tranquilizing drugs during pregnancy on activity of offspring, Nature (Lond.), 218 (1968) 357-358. HYD~N, H., R N A in brain cells. In G. C. QUARTON, T. MELNICHUK AND F. O. SCHMITT (Eds.), The Neurosciences, Rockefeller Univ. Press, New York, 1967, pp. 248-266. JARVIK, M. E., Drugs used in the treatment of psychiatric disorders. In L. S. GOODMAN AND A. GILMAN (Eds.), The Pharmacological Basis of Therapeutics, MacMillan, New York, 1965, pp. 159214. LowRY, O. H., ROSEBROUGH, N. J,, FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. MALETTA, G. J., VERNADAKIS, A., AND TIMIRAS, P. S., Acetylcholinesterase activity and protein content of brain and spinal cord after prenatal X-irradiation, J. Neurochem., 14 (1967) 647-752. NICHOLAS, J. S., Experimental methods and rat embroys. In J. A. GRrVFITH, JR. AND E. J. FARRIS (Eds.), The Rat in Laboratory Investigation, Lippincott, Philadelphia, 1949, pp. 51-57. PETROPOULOS, E. A., VERNADAKIS, A., AND TIMIRAS, P. S,, Nucleic acid content in developing rat brain after prenatal and/or neonatal exposure to high altitude, Fed. Proc., 28 (1969) 1001-1005. POMERAT, C. M., DRAGER, G. A., AND PAINTER, J. T., Effect of some barbiturates on tissues in vitro, Proc. Soc. exp. Biol. Med., 63 (1946) 322-325. SCHNEIDER, W. C., Phosphorus compounds in animal tissues. I. Extraction and estimation of desoxypentose nucleic acid and of pentose nucleic acid, J. biol. Chem., 161 (1945) 293-303. VERNADAKIS, A., VALCANA, T., CURRY, J. J., MALETTA, C. J., HUDSON, D., AND TIMIRAS, P. S., Alterations in growth of brain and other organs after electroshock in rats, Exp. Neurol., 17 (1967) 505-516. VERNADAKIS,A., ANDWOODBURY,D. M., The developing animal as a model, Epilepsia (Amst.), 10 (1969) 163-178. WERBOFF,J., AND DEMBICKI, E. L., Toxic effects of tranquilizers administered to gravid rats, J. Neuropsychiat., 4 0962) 87-91. WERBOFE, J., AND KESNER, R., Learning deficits of offspring after administration of tranquilizing drugs to the mothers, Nature (Lond.), 197 (1963) 106-107.
(Accepted May 22nd, 1970)
Brain Research, 21 (1970) 460-463