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Changes in free amino acids of the rat hypothalamus around the critical period
Brain Research, 104 (1976) 363-366
363
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Changes in free amino acids of the rat hypothalamus around the critical period
HAJIME MORISHITA, SHUNICHIRO KUROIWA, MICHIO TOMIOKA, KAZUHIKO HIGUCHI, HIROSHI MITANI, NORIO NAGAMACHI, MASARU KAWAMOTO, TERUYASU OZASA AND HARUO ADACHI
Department of Obstetrics and Gynecology, School of Medicine, Tokushima University, 50, 2-chome, Kuramoto-cho, Tokushima 770 (Japan) (Accepted December 1st, 1975)
From the results of both stimulation and ablation studies, it is now well established that the central nervous system, especially the hypothalamus, is of importance in the regulation of hypophyseal secretion of gonadotropic hormones6, l°,z°. Our aim is to investigate whether the cyclic changes of the hypothalamic regulation of gonadotropin secretion during the estrous cycle are accompanied by cyclic changes in the metabolism of the hypothalamus. Cyclic events related to ovulation in the rat generally show marked changes on the day of proestrus. These events can usually be fitted into a framework around the critical period during which the nervous system triggers the release of an ovulatory surge of gonadotropin from the pituitary gland. The present experiments were performed to investigate the changes in the concentrations of free amino acids of the rat hypothalamus around the critical period. Experiments were carried out on adult female Wistar rats, weighing 200-250 g. They were kept on a light schedule of 12 h light and 12 h dark (light from 22:00 to 10:00) for 30 days before the start of taking vaginal smears. Under our laboratory conditions, the critical period is around 5:00 to 8:00 (ref. 16). Each of the rats finally selected had shown at least 3 consecutive 4-day estrous cycles before the cycle when it was used. The animals were decapitated without anesthesia. After excision of the upper cranial bones, the whole brain was removed as rapidly as possible. The entire hypothalamus was dissected out in a block, limited anteriorly by the anterior margin of the optic chiasma, laterally by the lateral fissures and posteriorly by the posterior margin of the mammillary body. The block was 2.5 mm deep from the basal surface of the hypothalamus. The hypothalamus was then divided into 3 portions by frontal sections, i.e. the anterior, middle and posterior hypothalamus as described previously 16. Each determination was made on a pool from 15 rats. Each pooled sample was extracted according to the procedure described by Anderson et al. 1. The concentrations of free amino acids were measured with the Yanagimoto amino acid analyzer. The free amino acids estimated were as follows: threonine, serine, glutamic acid plus
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365 glutamine (Glx), glycine, alanine and leucine. The protein content was estimated by the method of Lowry et al. 15. Data were analyzed using the Student's t-test. The concentrations of free amino acids in the anterior, middle and posterior hypothalamus around the critical period are summarized in Table I. The concentrations of alanine and leucine showed no changes in the anterior, middle and posterior hypothalamus during each phase. There were no differences with regard to the concentrations of threonine and serine in the anterior and posterior hypothalamus during each phase. The mean values of the concentrations of threonine and serine in the middle hypothalamus during the critical period were slightly higher than those in the other stages, but these differences were not significant on statistical examination (P > 0.05). The concentrations of Glx and glycine in the anterior hypothalamus showed no changes during each phase but the concentrations of Glx and glycine in the middle hypothalamus during the critical period were significantly higher than those in the other phases (P < 0.05). The ratio of the middle hypothalamus to the anterior hypothalamus for either Glx or glycine during the critical period was significantly higher than in the other phases (P < 0.05). Since the early studies showing that the free amino acid composition of brain is not obviously related to the composition of brain protein 11, much experimental evidence has accumulated to support the idea that the free amino acids play a special role in the functioning of the nervous system. In this experiment significant increases were observed in glycine and Glx of the middle hypothalamus during the critical period. Much experimental evidence suggesting the view that glycine is an inhibitory synaptic transmitter in the mammalian spinal cord has accumulated2,a,7,1L Glutamate has been found to be an excitatory transmitter candidate in the spinal cord3,a,9 and cerebral cortexS, 12-14. But little is known about the function of glycine and glutamic acid in the hypothalamus. Singh and Malhotra17, la reported that chlorpromazine and reserpine cause decreases in the concentrations of glycine and Glx, especially in the monkey hypothalamus. These drugs are considered ovulation-blocking agents. When chlorpromazine and reserpine were injected before the critical period, ovulation failed to occur that night, whereas rats treated after the critical period did ovulatea. It is well known that the middle hypothalamus exerts its regulatory influence on the ovulatory mechanism6,2°. These reports suggest that the high concentrations of glycine and Glx in the middle hypothalamus during the critical period might be related to the release of gonadotropin releasing factor (GRF). However, further studies are required to substantiate the relationship between glycine and Glx concentrations and GRF releasing mechanisms. The authors are grateful to Dr. Y. Kuroda and Mr. H. Miyai for their valuable collaboration.
1 ANDERSON,H. L., BENEVENGA,N. J., AND HARPER, A. E., Associations among food and protein intake, serine dehydratase, and plasma amino acids, .4met. J. Physiol., 214 (1968) 1008-1013. 2 APRISON, M. H., AND WERMAN, R., The distribution of glycine in cat spinal cord and roots, Life Sci., 4 (1965) 2075-2083.
366 3 APRISON, M. H., AND WERMAN, R., A combined neurochemical and neurophysiological approach to identification of central nervous system transmitters. In S. EHRENPREIS AND O. C. SOLNITZKY (Eds.), Neuroseienee Research, Vol. l, Academic Press, New York, 1968, pp. 143-174. 4 BARRACLOUGH,C. A., AND SAWYER, C. H., Blockade of the release of pituitary ovulating hormone in the rat by chlorpromazine and reserpine: possible mechanisms of action, Endocrinology, 61 (1957) 341-351. 5 CRAWEORD, J. M., AND CURTIS, D. R., The excitation and depression of mammalian cortical neurones by amino acids, Brit. J. Pharmacol., 23 (1964) 313-329. 6 CRITCHLOW, V., Ovulation induced by hypothalamic stimulation in the anesthetized rat, Amer. J~ Physiol., 195 (1958) 171-174. 7 CURTIS, D. R., HOSLI, L., AND JOHNSTON, G. A. R., Inhibition of spinal neurones by glycine, Nature (Lond.), 215 (1967) 1502-1503. 8 CURTIS, D. R., PHILLIS, J. W., AND WATK1NS, J. C., Chemical excitation of spinal neurones, Nature (Lond.), 183 (1959) 611-612. 9 GRAHAM, L. T., JR., SHANK, R. P., WERMAN, R., AND APRISON, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord: glutamic acid, aspartic acid, ),-aminobutyric acid, glycine, and glutamine, J. Neurochem., 14 (1967) 465472. 10 HILLARP, N. A., Studies on the localization of hypothalamic centres controlling the gonadotrophic function of the hypophysis, Acta endocr. (Kbh.), 2 (1949) I 1-23. 11 KNAUFF, H. G., MAYER, G., UND MARX, D., Ober die Aminos~iurezusammensetzung der Gehirnproteine, Hoppe-Seyler' s Z. Physiol. Chem., 326 (1961) 78-88. 12 KRNJEVI~, K., Actions of drugs on single neurones in the cerebral cortex, Brit. reed. Bull., 21 (1965) 10-14. 13 KRNJEVIC, K., AND PHILLIS, J. W., Iontophoretic studies of neurones in the mammalian cerebral cortex, J. PhysioL (Lond.), 165 (1963) 274-304. 14 LEGGE, K. F., RANDIt~, M., AND STRAUGHAN, D. W., The pharmacology of neurones in the pyriform cortex, Brit. J. Pharmacol., 26 (1966) 87-107. 15 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biok Chem., 193 (1951) 265-275. 16 MORISHITA, n . , NAGAMACHI, N., KAWAMOTO, M., YOSHIDA, J., OZASA, T., AND ADACHI, H., Cyclic change in hypothalamic lactic dehydrogenase activity in the adult female rat, Acta endocr. (Kbh.}, 71 (1972) 226-232. 17 SINGH, S. I., AND MALHOTRA, C. L., Amino acid content of monkey brain. IlI. Effects of reserpine on some amino acids of certain regions of monkey brain, J. Neurochem., I I (1964) 865-872. 18 SINGH, S. I., AND MALHOTRA, C. L., Amino acid content of monkey brain. IV. Effects of chlorpromazine on some amino acids of certain regions of monkey brain, J. Neurochem., 14 0967) 135-140. 19 TEN BRUGGENCATE, G., AND ENGBERG, I., Analysis of glycine actions on spinal interneurones by intracellular recording, Brain Research, I l (1968) 446-450. 20 TERASAWA,E., AND SAWYER, C. H., Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophyseal function, Endocrinology, 85 (1969) 143-149.
363
© Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
Changes in free amino acids of the rat hypothalamus around the critical period
HAJIME MORISHITA, SHUNICHIRO KUROIWA, MICHIO TOMIOKA, KAZUHIKO HIGUCHI, HIROSHI MITANI, NORIO NAGAMACHI, MASARU KAWAMOTO, TERUYASU OZASA AND HARUO ADACHI
Department of Obstetrics and Gynecology, School of Medicine, Tokushima University, 50, 2-chome, Kuramoto-cho, Tokushima 770 (Japan) (Accepted December 1st, 1975)
From the results of both stimulation and ablation studies, it is now well established that the central nervous system, especially the hypothalamus, is of importance in the regulation of hypophyseal secretion of gonadotropic hormones6, l°,z°. Our aim is to investigate whether the cyclic changes of the hypothalamic regulation of gonadotropin secretion during the estrous cycle are accompanied by cyclic changes in the metabolism of the hypothalamus. Cyclic events related to ovulation in the rat generally show marked changes on the day of proestrus. These events can usually be fitted into a framework around the critical period during which the nervous system triggers the release of an ovulatory surge of gonadotropin from the pituitary gland. The present experiments were performed to investigate the changes in the concentrations of free amino acids of the rat hypothalamus around the critical period. Experiments were carried out on adult female Wistar rats, weighing 200-250 g. They were kept on a light schedule of 12 h light and 12 h dark (light from 22:00 to 10:00) for 30 days before the start of taking vaginal smears. Under our laboratory conditions, the critical period is around 5:00 to 8:00 (ref. 16). Each of the rats finally selected had shown at least 3 consecutive 4-day estrous cycles before the cycle when it was used. The animals were decapitated without anesthesia. After excision of the upper cranial bones, the whole brain was removed as rapidly as possible. The entire hypothalamus was dissected out in a block, limited anteriorly by the anterior margin of the optic chiasma, laterally by the lateral fissures and posteriorly by the posterior margin of the mammillary body. The block was 2.5 mm deep from the basal surface of the hypothalamus. The hypothalamus was then divided into 3 portions by frontal sections, i.e. the anterior, middle and posterior hypothalamus as described previously 16. Each determination was made on a pool from 15 rats. Each pooled sample was extracted according to the procedure described by Anderson et al. 1. The concentrations of free amino acids were measured with the Yanagimoto amino acid analyzer. The free amino acids estimated were as follows: threonine, serine, glutamic acid plus
364
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365 glutamine (Glx), glycine, alanine and leucine. The protein content was estimated by the method of Lowry et al. 15. Data were analyzed using the Student's t-test. The concentrations of free amino acids in the anterior, middle and posterior hypothalamus around the critical period are summarized in Table I. The concentrations of alanine and leucine showed no changes in the anterior, middle and posterior hypothalamus during each phase. There were no differences with regard to the concentrations of threonine and serine in the anterior and posterior hypothalamus during each phase. The mean values of the concentrations of threonine and serine in the middle hypothalamus during the critical period were slightly higher than those in the other stages, but these differences were not significant on statistical examination (P > 0.05). The concentrations of Glx and glycine in the anterior hypothalamus showed no changes during each phase but the concentrations of Glx and glycine in the middle hypothalamus during the critical period were significantly higher than those in the other phases (P < 0.05). The ratio of the middle hypothalamus to the anterior hypothalamus for either Glx or glycine during the critical period was significantly higher than in the other phases (P < 0.05). Since the early studies showing that the free amino acid composition of brain is not obviously related to the composition of brain protein 11, much experimental evidence has accumulated to support the idea that the free amino acids play a special role in the functioning of the nervous system. In this experiment significant increases were observed in glycine and Glx of the middle hypothalamus during the critical period. Much experimental evidence suggesting the view that glycine is an inhibitory synaptic transmitter in the mammalian spinal cord has accumulated2,a,7,1L Glutamate has been found to be an excitatory transmitter candidate in the spinal cord3,a,9 and cerebral cortexS, 12-14. But little is known about the function of glycine and glutamic acid in the hypothalamus. Singh and Malhotra17, la reported that chlorpromazine and reserpine cause decreases in the concentrations of glycine and Glx, especially in the monkey hypothalamus. These drugs are considered ovulation-blocking agents. When chlorpromazine and reserpine were injected before the critical period, ovulation failed to occur that night, whereas rats treated after the critical period did ovulatea. It is well known that the middle hypothalamus exerts its regulatory influence on the ovulatory mechanism6,2°. These reports suggest that the high concentrations of glycine and Glx in the middle hypothalamus during the critical period might be related to the release of gonadotropin releasing factor (GRF). However, further studies are required to substantiate the relationship between glycine and Glx concentrations and GRF releasing mechanisms. The authors are grateful to Dr. Y. Kuroda and Mr. H. Miyai for their valuable collaboration.
1 ANDERSON,H. L., BENEVENGA,N. J., AND HARPER, A. E., Associations among food and protein intake, serine dehydratase, and plasma amino acids, .4met. J. Physiol., 214 (1968) 1008-1013. 2 APRISON, M. H., AND WERMAN, R., The distribution of glycine in cat spinal cord and roots, Life Sci., 4 (1965) 2075-2083.
366 3 APRISON, M. H., AND WERMAN, R., A combined neurochemical and neurophysiological approach to identification of central nervous system transmitters. In S. EHRENPREIS AND O. C. SOLNITZKY (Eds.), Neuroseienee Research, Vol. l, Academic Press, New York, 1968, pp. 143-174. 4 BARRACLOUGH,C. A., AND SAWYER, C. H., Blockade of the release of pituitary ovulating hormone in the rat by chlorpromazine and reserpine: possible mechanisms of action, Endocrinology, 61 (1957) 341-351. 5 CRAWEORD, J. M., AND CURTIS, D. R., The excitation and depression of mammalian cortical neurones by amino acids, Brit. J. Pharmacol., 23 (1964) 313-329. 6 CRITCHLOW, V., Ovulation induced by hypothalamic stimulation in the anesthetized rat, Amer. J~ Physiol., 195 (1958) 171-174. 7 CURTIS, D. R., HOSLI, L., AND JOHNSTON, G. A. R., Inhibition of spinal neurones by glycine, Nature (Lond.), 215 (1967) 1502-1503. 8 CURTIS, D. R., PHILLIS, J. W., AND WATK1NS, J. C., Chemical excitation of spinal neurones, Nature (Lond.), 183 (1959) 611-612. 9 GRAHAM, L. T., JR., SHANK, R. P., WERMAN, R., AND APRISON, M. H., Distribution of some synaptic transmitter suspects in cat spinal cord: glutamic acid, aspartic acid, ),-aminobutyric acid, glycine, and glutamine, J. Neurochem., 14 (1967) 465472. 10 HILLARP, N. A., Studies on the localization of hypothalamic centres controlling the gonadotrophic function of the hypophysis, Acta endocr. (Kbh.), 2 (1949) I 1-23. 11 KNAUFF, H. G., MAYER, G., UND MARX, D., Ober die Aminos~iurezusammensetzung der Gehirnproteine, Hoppe-Seyler' s Z. Physiol. Chem., 326 (1961) 78-88. 12 KRNJEVI~, K., Actions of drugs on single neurones in the cerebral cortex, Brit. reed. Bull., 21 (1965) 10-14. 13 KRNJEVIC, K., AND PHILLIS, J. W., Iontophoretic studies of neurones in the mammalian cerebral cortex, J. PhysioL (Lond.), 165 (1963) 274-304. 14 LEGGE, K. F., RANDIt~, M., AND STRAUGHAN, D. W., The pharmacology of neurones in the pyriform cortex, Brit. J. Pharmacol., 26 (1966) 87-107. 15 LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., Protein measurement with the Folin phenol reagent, J. biok Chem., 193 (1951) 265-275. 16 MORISHITA, n . , NAGAMACHI, N., KAWAMOTO, M., YOSHIDA, J., OZASA, T., AND ADACHI, H., Cyclic change in hypothalamic lactic dehydrogenase activity in the adult female rat, Acta endocr. (Kbh.}, 71 (1972) 226-232. 17 SINGH, S. I., AND MALHOTRA, C. L., Amino acid content of monkey brain. IlI. Effects of reserpine on some amino acids of certain regions of monkey brain, J. Neurochem., I I (1964) 865-872. 18 SINGH, S. I., AND MALHOTRA, C. L., Amino acid content of monkey brain. IV. Effects of chlorpromazine on some amino acids of certain regions of monkey brain, J. Neurochem., 14 0967) 135-140. 19 TEN BRUGGENCATE, G., AND ENGBERG, I., Analysis of glycine actions on spinal interneurones by intracellular recording, Brain Research, I l (1968) 446-450. 20 TERASAWA,E., AND SAWYER, C. H., Changes in electrical activity in the rat hypothalamus related to electrochemical stimulation of adenohypophyseal function, Endocrinology, 85 (1969) 143-149.