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Glucocorticoids and hippocampal enzyme activity
172
Brain Research, 166 (1979) 172-175 ((~)Elsevier/North-Holland Biomedical Press
Glucocorticoids and hippocampal enzyme activity
JERROLD S. MEYER*, VICTORIA N. LUINE, RADA I. KHYLCHEVSKAYA** and BRUCE S. McEWEN*** The Rockefeller University, New York, N. Y. 10021 (U.S.A.)
(Accepted December 14th, 1978)
The hippocampus is generally presumed to be a major glucocorticoid target site in the rat brain since it displays a high level of cytosolic binding of corticosterone in vitro 1 and pronounced uptake of this hormone in vivo 14. Autoradiographic experiments have ascertained that much of the hippocampal corticosterone uptake occurs in the nuclei of pyramidal and dentate granule neuronsL Such localization studies, however, have been accompanied by only a few recent experiments investigating the biochemical actions of glucocorticoids in this region of the brain. In one instance, Miller et al. 16 found that adrenalectomy increased the rate of GABA transport into hippocampal synaptosomes while Lee et al. 7 also reported changes in the synthesis of an unidentified hippocampal protein following exposure to corticosterone. These studies did not examine the influence of corticosterone on any hippocampal enzymes, but glucocorticoids are known to exert many of their peripheral metabolic effects via enzyme induction tg. They also control the activities of several enzymes in neural tissues other than the hippocampuslT,ls,20, 21. With these facts in mind, we investigated the possible adrenocortical regulation of a number of key metabolic and neurotransmitter-related enzymes in the rat hippocampus. The results were largely negative, thereby suggesting that other biochemical mechanisms are possibly of greater importance in mediating glucocorticoid action in this target area. Male Sprague-Dawley derived (Charles River, CD strain) rats of 200-300 g were used as subjects. They were group housed under a 14:10 light-dark cycle (lights on at 0500 h) and given lab chow and tap water (or 0.85 ~ saline following adrenalectomy) ad libitum. In most experiments, rats were bilaterally adrenalectomized and then, starting one week later, given a week of replacement therapy or placebo. Replacement was accomplished by either a 100 mg corticosterone pellet implanted subcutaneously 15 or by daily s.c. injections of 0.5 mg corticosterone hemisuccinate. Controls received a * Present address: Department of Psychology, University of Massachusetts, Amherst, Mass. 01035, U.S.A. ** Present address: Institute of General Genetics, U.S,S.R. Academy of Sciences, Moscow 117312, U.S,S.R. *** To whom reprint requests should be sent.
173 cholesterol pellet or saline injections respectively. In one experiment, 3-week adrenalectomized rats were compared to sham-operated controls. At the indicated times, subjects were decapitated and sera collected for corticosterone determination by fluorometry 4 or radioimmunoassay 6. The hippocampus from each animal was dissected over ice and usually homogenized in ice-cold 0.1 M Tris'HC1 buffer, pH 8.0. The buffer was supplemented with 30/~M reduced glutathione and 20 #M pyridoxal phosphate when ~,-aminobutyrate aminotransferase was being assayed. In the case of glutamate decarboxylase, samples were homogenized in 0.1 M phosphate buffer, pH 6.4 supplemented with 1 mM aminoethylisothiouronium bromide (AET). The following enzymes were assayed in crude homogenates by monitoring the appearance or disappearance of nicotinamide adenine dinucleotide (NADH) or NADPH) cofactor in a Farrand filter fluorometer: malate dehydrogenase (E.C. 1.1.1.40, MDH), lactate dehydrogenase (EC 1.1.1.27, LDH), isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), glucose 6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH), 6-phosphogluconate dehydrogenase (EC 1.1.1.44, 6PGDH), glutamate dehydrogenase (EC 1.4.1.2, GDH), and glycerolphosphate dehydrogenase (EC 1.1.1.8, GPDH) (see Lowry and Passonneau 9 for the general method of enzyme analysis by pyridine nucleotide fluorescence). Other enzymes were assayed fluorometrically with the aid of auxiliary enzymes which utilize pyridine nucleotide cofactors: hexokinase (EC 2.7.1.1, HK), pyruvate kinase (EC 2.7.1.40, PK), glutaminase (EC 3.5.1.2, GLN-ASE), y-aminobutyrate aminotransferase (EC 2.6.1.19, GABA-T), and aspartate aminotransferase (EC 2.6.1.1, ASP-T). The remaining enzymes were measured by radioisotopic methods: glutamate decarboxylase (EC 4.1.1.15, GAD) 22, monoamine oxidase (EC 1.4.3.4, MAO) 11, choline acetyltransferase (EC 2.3.1.6, CAT) 11, and acetylcholinesterase (EC 3.1.1.7, ACE) 5. Substrates and cofactors for all enzymes were present at concentrations which gave maximal in vitro activity. GDH, GAD, GABA-T and CAT were activated prior to assay by treating homogenates with Triton X-100 at a final concentration of 0.1 ~. Homogenate protein concentrations were determined by the method of Lowry et al. 1°. The results are presented in Table I. It is apparent that adrenalectomy and corticosterone replacement affected only the activity of GPDH, an enzyme already known to be regulated by glucocorticoids in other areas of the rat brain 2. Since G P D H appears to be a glial-specific enzyme s, its induction sheds no light on the metabolic function of the neuronal corticosterone uptake system in the hippocampus. However, the presence of this positive effect does provide a standard of comparison for the remaining negative results and establishes the efficacy of our experimental paradigm. Serum corticosterone concentrations at the time of sacrifice ranged from 6-15 #g/100 ml in the injected animals and from 10-25 #g/100 ml in those which received pellet implants. These levels which are in the normal afternoon to evening range for male rats in our lab lz, should have been sufficient to induce any enzymes under physiological control by the adrenal cortex. Of course, many hippocampal enzymes which were not investigated could be modulated by glucocorticoids. However, it seems more likely that the enzyme induction model of estradiol action in the hypothalamus
174 TABLE I Enzyme activities (l~molproduct/g protein~h) in crude hippocampal homogenatesjkom adrenalectomized male rats with or without corticosterone replacement Values are expressed as the mean zk S.E.M. Number of subjects is shown in parentheses. Enzyme
Group ADX
MDH LDH ICDH SDH G6PDH 6PGDH GDH GPDH HK PK GLN-ASE GABA-T ASP-T GAD MAO CAT ACE
153 8329 273 124 98 136 1200 283 2698 2272 695 580 11730 202 45 14 1386
* ADX vs. sham-ADX.
ADX ± CORT ~_ 5 ± 563 ± 14 zk 4 i 4 ± 6 zL- 130 -5:17 ± 134 ± 77 ± 65 ± 25 ± 400 ± 6 :-L 2 ± 1 ± 48
(11) (10) (11) (11) (10) (11) (7) (6) (11) (10) (6) (6) (7) (6) (6) (6) (7)
153 8964 303 122 101 142 1130 481 2823 2528 735 550 12360 212 45 15 1347
-4- 7 (8) ± 649 (8) ± 12 (8) ± 4 (8) ± 8 (8) ± 8 (8) ± 100 (7) ± 49 (6)** ± 172 (8) ± 135 (7) ± 60 (6) ± 45 (6) ± 980 (7) i 6 (6) ± 2 (6) ± 0.5 (6) ± 60 (7)*
** P < 0.01 by t-test.
(see M c E w e n , K r e y a n d Luine 12) does n o t generally apply to adrenal steroids in their target area, the h i p p o c a m p u s . Alternative m e c h a n i s m s c o u l d include the previously cited effects of n e u r o t r a n s m i t t e r u p t a k e a n d p r o t e i n synthesis, the regulation of p r o t e i n p h o s p h o r y l a t i o n , or direct influences o n m e m b r a n e permeability. This research was s u p p o r t e d by N I H G r a n t NS 07080 a n d by a n I n s t i t u t i o n a l G r a n t R F 70095 f r o m the Rockefeller F o u n d a t i o n . J.M. is recipient of U S P H S p o s t d o c t o r a l fellowship NS 05040. We would like to t h a n k B. S. S t e p h e n s o n for expert technical assistance.
1 De Kloet, R., Wallach, G. and McEwen, B. S., Differences in corticosterone and dexamethasone binding in rat brain and pituitary, Endocrinology, 96 (1975) 598-609. 2 De Vellis, J. and Inglish, D., Hormonal control of glycerolphosphate dehydrogenase in the rat brain, J. Neurochem., 15 (1968) 1061-1070. 3 Gerlach, J. L. and McEwen, B. S., Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone, Science, 175 (1972) 1133-1136. 4 Guillemin, R., Clayton, G. W., Smith, J. D. and Lipscomb, H. S., Measurement of free corticosteroid in rat plasma: physiological validation of a method, Endocrinology, 63 (1958) 349-358. 5 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Analyt. Biochem., 64 (1975) 229-238. 6 Krey, L. C., Lu, K.-H., Butler, W. R., Hotchkiss, J., Piva, F. and Knob±l, E., Surgical disconnection of the medial basal hypothalamus and pituitary function in the rhesus monkey. II. GH and cortisol secretion, Endocrinology, 96 (1975) 1088-1093.
1.7.5 7 Lee, K. S., Etgen, A. M. and Lynch, G. S., Corticosterone modification of the synthesis of specific proteins in the central nervous system, Neurosci. Abstr., 3 (1977) 350. 8 Leveille, P. J., DeVellis, J. and Maxwell, D. S., Immunocytochemical localization of glycerol-3phosphate dehydrogenase in rat brain: are oligudendrocytes target cells for glucocorticoids? Neurosci. Abstr., 3 (1977) 333. 9 Lowry, O. H. and Passonneau, J. V., A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972. 10 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. 11 Luine, V. N., Khylchevskaya, R. I. and McEwen, B. S., Effect of gonadal steroids on activities of monoamine oxidase and choline acetylase in rat brain, Brain Research, 86 (1975) 293-306. 12 McEwen, B. S., Krey, L. C. and Luine, V. N., Steroid hormone action in the neuroendocrine system: when is the genome involved? In S. Reichlin, R. J. Baldessarini and J. B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 255-268. 13 McEwen, B. S., Stephenson, B. S. and Krey, L. C., Radioimmunoassay of brain tissue and cell nuclear corticosterone, submitted for publication. 14 McEwen, B. S., Weiss, J. M. and Schwartz, L. S., Retention of corticosterone by cell nuclei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471--482. 15 Meyer, J. S., Micco, D. J., Stephenson, B. S., Krey, L. C. and McEwen, B. S., A subcutaneous implantation method for chronic glucocorticoid replacement therapy, PhysioL Behav., (1979) in press. 16 Miller, A. L., Chaptal, C,, McEwen, B. S. and Peck, Jr., E. J., Modulation of high affinity GABA uptake into hippocampal synaptosomes by glucocorticoids, Psychoneuroendocrinology, (1979) in press. 17 Moore, K. E. and Phillipson, O. T., Effects of dexamethasone on phenylethanolamine-N-methyltransferase and adrenaline in the brains and superior cervical ganglion of adult and neonatal rats, J. Neurochem., 25 (1975) 289-294. 18 Moscona, A. A., Control mechanisms in hormonal induction of glutamine synthetase in the embryonic neural retinal. In M. Hamburgh and E. J. W. Barrington (Eds.), Hormones in Development, Appleton-Century-Crofts, New York, 1971, pp. 169-189. 19 Nichol, C. A. and Rosen, F., Adaptive changes in enzymatic activity induced by glucocorticoids. In G. Litwack and D. Kritchevsky (Eds.), Actions of Hormones on Molecular Processes, Wiley, New York, 1964, pp. 234-256. 20 Parvez, H. and Parvez, S., The regulation of monoamine oxidase activity by adrenal cortical steroids, Acta endocr., 73 (1973) 509-517. 21 Sze, P. Y., Neckers, L. and Towle, A. C., Glucocorticoids as a regulatory factor for brain tryptophan hydroxylase, J. Neurochem., 26 (1976) 169-173. 22 Tappaz, M. L., Brownstein, M. J. and Palkovits, M., Distribution of glutamate decarboxylase in discrete brain nuclei, Brain Research, 108 (1976) 371-379.
Brain Research, 166 (1979) 172-175 ((~)Elsevier/North-Holland Biomedical Press
Glucocorticoids and hippocampal enzyme activity
JERROLD S. MEYER*, VICTORIA N. LUINE, RADA I. KHYLCHEVSKAYA** and BRUCE S. McEWEN*** The Rockefeller University, New York, N. Y. 10021 (U.S.A.)
(Accepted December 14th, 1978)
The hippocampus is generally presumed to be a major glucocorticoid target site in the rat brain since it displays a high level of cytosolic binding of corticosterone in vitro 1 and pronounced uptake of this hormone in vivo 14. Autoradiographic experiments have ascertained that much of the hippocampal corticosterone uptake occurs in the nuclei of pyramidal and dentate granule neuronsL Such localization studies, however, have been accompanied by only a few recent experiments investigating the biochemical actions of glucocorticoids in this region of the brain. In one instance, Miller et al. 16 found that adrenalectomy increased the rate of GABA transport into hippocampal synaptosomes while Lee et al. 7 also reported changes in the synthesis of an unidentified hippocampal protein following exposure to corticosterone. These studies did not examine the influence of corticosterone on any hippocampal enzymes, but glucocorticoids are known to exert many of their peripheral metabolic effects via enzyme induction tg. They also control the activities of several enzymes in neural tissues other than the hippocampuslT,ls,20, 21. With these facts in mind, we investigated the possible adrenocortical regulation of a number of key metabolic and neurotransmitter-related enzymes in the rat hippocampus. The results were largely negative, thereby suggesting that other biochemical mechanisms are possibly of greater importance in mediating glucocorticoid action in this target area. Male Sprague-Dawley derived (Charles River, CD strain) rats of 200-300 g were used as subjects. They were group housed under a 14:10 light-dark cycle (lights on at 0500 h) and given lab chow and tap water (or 0.85 ~ saline following adrenalectomy) ad libitum. In most experiments, rats were bilaterally adrenalectomized and then, starting one week later, given a week of replacement therapy or placebo. Replacement was accomplished by either a 100 mg corticosterone pellet implanted subcutaneously 15 or by daily s.c. injections of 0.5 mg corticosterone hemisuccinate. Controls received a * Present address: Department of Psychology, University of Massachusetts, Amherst, Mass. 01035, U.S.A. ** Present address: Institute of General Genetics, U.S,S.R. Academy of Sciences, Moscow 117312, U.S,S.R. *** To whom reprint requests should be sent.
173 cholesterol pellet or saline injections respectively. In one experiment, 3-week adrenalectomized rats were compared to sham-operated controls. At the indicated times, subjects were decapitated and sera collected for corticosterone determination by fluorometry 4 or radioimmunoassay 6. The hippocampus from each animal was dissected over ice and usually homogenized in ice-cold 0.1 M Tris'HC1 buffer, pH 8.0. The buffer was supplemented with 30/~M reduced glutathione and 20 #M pyridoxal phosphate when ~,-aminobutyrate aminotransferase was being assayed. In the case of glutamate decarboxylase, samples were homogenized in 0.1 M phosphate buffer, pH 6.4 supplemented with 1 mM aminoethylisothiouronium bromide (AET). The following enzymes were assayed in crude homogenates by monitoring the appearance or disappearance of nicotinamide adenine dinucleotide (NADH) or NADPH) cofactor in a Farrand filter fluorometer: malate dehydrogenase (E.C. 1.1.1.40, MDH), lactate dehydrogenase (EC 1.1.1.27, LDH), isocitrate dehydrogenase (EC 1.1.1.42, ICDH), succinate dehydrogenase (EC 1.3.99.1, SDH), glucose 6-phosphate dehydrogenase (EC 1.1.1.49, G6PDH), 6-phosphogluconate dehydrogenase (EC 1.1.1.44, 6PGDH), glutamate dehydrogenase (EC 1.4.1.2, GDH), and glycerolphosphate dehydrogenase (EC 1.1.1.8, GPDH) (see Lowry and Passonneau 9 for the general method of enzyme analysis by pyridine nucleotide fluorescence). Other enzymes were assayed fluorometrically with the aid of auxiliary enzymes which utilize pyridine nucleotide cofactors: hexokinase (EC 2.7.1.1, HK), pyruvate kinase (EC 2.7.1.40, PK), glutaminase (EC 3.5.1.2, GLN-ASE), y-aminobutyrate aminotransferase (EC 2.6.1.19, GABA-T), and aspartate aminotransferase (EC 2.6.1.1, ASP-T). The remaining enzymes were measured by radioisotopic methods: glutamate decarboxylase (EC 4.1.1.15, GAD) 22, monoamine oxidase (EC 1.4.3.4, MAO) 11, choline acetyltransferase (EC 2.3.1.6, CAT) 11, and acetylcholinesterase (EC 3.1.1.7, ACE) 5. Substrates and cofactors for all enzymes were present at concentrations which gave maximal in vitro activity. GDH, GAD, GABA-T and CAT were activated prior to assay by treating homogenates with Triton X-100 at a final concentration of 0.1 ~. Homogenate protein concentrations were determined by the method of Lowry et al. 1°. The results are presented in Table I. It is apparent that adrenalectomy and corticosterone replacement affected only the activity of GPDH, an enzyme already known to be regulated by glucocorticoids in other areas of the rat brain 2. Since G P D H appears to be a glial-specific enzyme s, its induction sheds no light on the metabolic function of the neuronal corticosterone uptake system in the hippocampus. However, the presence of this positive effect does provide a standard of comparison for the remaining negative results and establishes the efficacy of our experimental paradigm. Serum corticosterone concentrations at the time of sacrifice ranged from 6-15 #g/100 ml in the injected animals and from 10-25 #g/100 ml in those which received pellet implants. These levels which are in the normal afternoon to evening range for male rats in our lab lz, should have been sufficient to induce any enzymes under physiological control by the adrenal cortex. Of course, many hippocampal enzymes which were not investigated could be modulated by glucocorticoids. However, it seems more likely that the enzyme induction model of estradiol action in the hypothalamus
174 TABLE I Enzyme activities (l~molproduct/g protein~h) in crude hippocampal homogenatesjkom adrenalectomized male rats with or without corticosterone replacement Values are expressed as the mean zk S.E.M. Number of subjects is shown in parentheses. Enzyme
Group ADX
MDH LDH ICDH SDH G6PDH 6PGDH GDH GPDH HK PK GLN-ASE GABA-T ASP-T GAD MAO CAT ACE
153 8329 273 124 98 136 1200 283 2698 2272 695 580 11730 202 45 14 1386
* ADX vs. sham-ADX.
ADX ± CORT ~_ 5 ± 563 ± 14 zk 4 i 4 ± 6 zL- 130 -5:17 ± 134 ± 77 ± 65 ± 25 ± 400 ± 6 :-L 2 ± 1 ± 48
(11) (10) (11) (11) (10) (11) (7) (6) (11) (10) (6) (6) (7) (6) (6) (6) (7)
153 8964 303 122 101 142 1130 481 2823 2528 735 550 12360 212 45 15 1347
-4- 7 (8) ± 649 (8) ± 12 (8) ± 4 (8) ± 8 (8) ± 8 (8) ± 100 (7) ± 49 (6)** ± 172 (8) ± 135 (7) ± 60 (6) ± 45 (6) ± 980 (7) i 6 (6) ± 2 (6) ± 0.5 (6) ± 60 (7)*
** P < 0.01 by t-test.
(see M c E w e n , K r e y a n d Luine 12) does n o t generally apply to adrenal steroids in their target area, the h i p p o c a m p u s . Alternative m e c h a n i s m s c o u l d include the previously cited effects of n e u r o t r a n s m i t t e r u p t a k e a n d p r o t e i n synthesis, the regulation of p r o t e i n p h o s p h o r y l a t i o n , or direct influences o n m e m b r a n e permeability. This research was s u p p o r t e d by N I H G r a n t NS 07080 a n d by a n I n s t i t u t i o n a l G r a n t R F 70095 f r o m the Rockefeller F o u n d a t i o n . J.M. is recipient of U S P H S p o s t d o c t o r a l fellowship NS 05040. We would like to t h a n k B. S. S t e p h e n s o n for expert technical assistance.
1 De Kloet, R., Wallach, G. and McEwen, B. S., Differences in corticosterone and dexamethasone binding in rat brain and pituitary, Endocrinology, 96 (1975) 598-609. 2 De Vellis, J. and Inglish, D., Hormonal control of glycerolphosphate dehydrogenase in the rat brain, J. Neurochem., 15 (1968) 1061-1070. 3 Gerlach, J. L. and McEwen, B. S., Rat brain binds adrenal steroid hormone: radioautography of hippocampus with corticosterone, Science, 175 (1972) 1133-1136. 4 Guillemin, R., Clayton, G. W., Smith, J. D. and Lipscomb, H. S., Measurement of free corticosteroid in rat plasma: physiological validation of a method, Endocrinology, 63 (1958) 349-358. 5 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations, Analyt. Biochem., 64 (1975) 229-238. 6 Krey, L. C., Lu, K.-H., Butler, W. R., Hotchkiss, J., Piva, F. and Knob±l, E., Surgical disconnection of the medial basal hypothalamus and pituitary function in the rhesus monkey. II. GH and cortisol secretion, Endocrinology, 96 (1975) 1088-1093.
1.7.5 7 Lee, K. S., Etgen, A. M. and Lynch, G. S., Corticosterone modification of the synthesis of specific proteins in the central nervous system, Neurosci. Abstr., 3 (1977) 350. 8 Leveille, P. J., DeVellis, J. and Maxwell, D. S., Immunocytochemical localization of glycerol-3phosphate dehydrogenase in rat brain: are oligudendrocytes target cells for glucocorticoids? Neurosci. Abstr., 3 (1977) 333. 9 Lowry, O. H. and Passonneau, J. V., A Flexible System of Enzymatic Analysis, Academic Press, New York, 1972. 10 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. 11 Luine, V. N., Khylchevskaya, R. I. and McEwen, B. S., Effect of gonadal steroids on activities of monoamine oxidase and choline acetylase in rat brain, Brain Research, 86 (1975) 293-306. 12 McEwen, B. S., Krey, L. C. and Luine, V. N., Steroid hormone action in the neuroendocrine system: when is the genome involved? In S. Reichlin, R. J. Baldessarini and J. B. Martin (Eds.), The Hypothalamus, Raven Press, New York, 1978, pp. 255-268. 13 McEwen, B. S., Stephenson, B. S. and Krey, L. C., Radioimmunoassay of brain tissue and cell nuclear corticosterone, submitted for publication. 14 McEwen, B. S., Weiss, J. M. and Schwartz, L. S., Retention of corticosterone by cell nuclei from brain regions of adrenalectomized rats, Brain Research, 17 (1970) 471--482. 15 Meyer, J. S., Micco, D. J., Stephenson, B. S., Krey, L. C. and McEwen, B. S., A subcutaneous implantation method for chronic glucocorticoid replacement therapy, PhysioL Behav., (1979) in press. 16 Miller, A. L., Chaptal, C,, McEwen, B. S. and Peck, Jr., E. J., Modulation of high affinity GABA uptake into hippocampal synaptosomes by glucocorticoids, Psychoneuroendocrinology, (1979) in press. 17 Moore, K. E. and Phillipson, O. T., Effects of dexamethasone on phenylethanolamine-N-methyltransferase and adrenaline in the brains and superior cervical ganglion of adult and neonatal rats, J. Neurochem., 25 (1975) 289-294. 18 Moscona, A. A., Control mechanisms in hormonal induction of glutamine synthetase in the embryonic neural retinal. In M. Hamburgh and E. J. W. Barrington (Eds.), Hormones in Development, Appleton-Century-Crofts, New York, 1971, pp. 169-189. 19 Nichol, C. A. and Rosen, F., Adaptive changes in enzymatic activity induced by glucocorticoids. In G. Litwack and D. Kritchevsky (Eds.), Actions of Hormones on Molecular Processes, Wiley, New York, 1964, pp. 234-256. 20 Parvez, H. and Parvez, S., The regulation of monoamine oxidase activity by adrenal cortical steroids, Acta endocr., 73 (1973) 509-517. 21 Sze, P. Y., Neckers, L. and Towle, A. C., Glucocorticoids as a regulatory factor for brain tryptophan hydroxylase, J. Neurochem., 26 (1976) 169-173. 22 Tappaz, M. L., Brownstein, M. J. and Palkovits, M., Distribution of glutamate decarboxylase in discrete brain nuclei, Brain Research, 108 (1976) 371-379.