ht. J. Devl. Neuroscience, Vol. 2, No. 1, pp. 5540, 1984.
073fst_574X/XJ $3.00+0.00 Pergxnon Press Ltd.
Printed in Great Britain.
ONTOGENY
OF GLUTAMINE D. CHATTERJEE
Department
of Cell Biology,
Indian
Institute (Accepted
SYNTHETASE and P. K.
of Chemical 5 September
IN RAT
BRAIN
SARKAR
Biology,
Jadavpur,
Calcutta
700032, India
1983)
Abstract-The developmental pattern of glutamine synthetase (GS) in rat brain has been studied with respect to that of the cells involved in the synthesis of the enzyme. GS activity is very low until day 13 after birth, rises sharply between days 13 and 15, and slowly thereafter. In contrast, more than half of the total number of protoplasmic astrocytes-the cells involved in synthesis of GS-are formed by day 12, following which the rate of proliferation declines considerably. GS is precociously inducible by cortisol in purified protoplasmic astrocytes, in organ cultures of o-13 day rat brain and in the brains of cortisoladministered 12 day rats. These results and the temporal coincidence of the period of increase of GS with the onset of the function of adrenal cortex suggest that GS activity in the developing rat brain is under steroidal control. Key words: Glutamine
synthetase,
Protoplasmic
astrocytes,
Cortisol.
Glutamine synthetase (GS) is one of the key enzymes of brain because of its involvement in the synthesis of nucleic acids and mucopolysaccharides and in the metabolism of neurotransmitters.28 In the neural retina of the embryonic chick, the enzyme is inducible by adrenocorticosteroids.‘0,‘4,22 Although cortisol is known to induce the synthesis of many enzymes in the rat liver and kidney5 it did not accelerate the development of GS in liver or retina of fetal or neonatal rats7 However, in rat hypothalamus, development of GS activity appears to be sensitive to cortisol.27 Immunohistochemical studies of Norenberg & Martinez16 revealed that in the rat brain, GS has an exclusive glial localization and the enzyme is predominantly in the astrocytes of the gray matter. l5 Since the bulk of the glial cells in the gray matter is represented by protoplasmic astrocytes,12 we monitored the ontogeny of GS and that of the protoplasmic astrocytes to determine if the developmental profile of GS activity in rat brain is temporally coincident with the appearance and proliferation of the protoplasmic astrocytes or is regulated by endogenous adrenal corticosteroids. MATERIALS
AND METHODS
Trypsinization of brain tissue from Wistar albino rats of indicated ages was carried out according to the procedure of Norton & Poduslo17 except that incubation with trypsin was performed for 10 min instead of 60-90 min. This resulted in the selective dissociation of cells of the cortex region containing the gray matter (see Results) representing primarily protoplasmic astrocytes and neurons. Initial fractionation of these two types of cells was carried out by differential centrifugation in ficoll.’ The enriched neuronal and the astrocyte (glial) fractions obtained from the above step, referred to as N and G fractions, were further purified by centrifugation over 40% stock isotonic perco11gradients (density range 0.960-l. 150 gm/ml), prepared according to Pharmacia Fine Chemical Bulletin. Protoplasmic astrocytes were collected from density range 0.980-0.998 gm/ml with a particle purity of 9598% and the neurons from 1.026 to 1.150 gm/ml with a purity of 92-96%. For a comparative study, oligodendrocytes were purified according to the procedure of Fewster et al. * from white matter of rat brain with a particle purity of more than 80%. Neurons were distinguished from the protoplasmic astrocytes by their relatively large cell body and axons, sensitivity to 1% methylene blue stain23 and by the presence of marker enzyme B-galactosidase.25 Protoplasmic astrocytes were characterized by their thick wavy processes around the cell body’ in contrast to the thin filamentous fibres of the fibrous astrocytes, by their sensitivity to 1% cresyl violet*” and also by the presence of marker enzyme, glutamine synthetase.16 Purified preparations of protoplasmic astrocytes and oligodendrocytes were further identified by immunofluorescence staining with antibodies against astrocyte specific glutamine synthetaset6 and oligodendrocyte specific galactocerebroside,21 respectively (Fig. 1). Neither antisera gave a positive test with the purified neuronal preparation. Samples for indirect immunofluorescence assays were prepared as described by Treska et al. 26 55
56
n. Chatterjee and P. K. Sarkar
Cell counts were performed at a magnification of x 400 from fields of at least 2000 cells after staining with cresyl violet which stained the neurons lightly and the astrocytes deeply. Unidentified objects in the fields (ranging from 5 to 8% in all the cases) were disregarded. Intact brains of f&20-day-old rats, freed from blood vessels and meninges, were cultured in 10 ml of medium (99% Tyrode balanced salt solution and 1% penicillin streptomycin mixture) using 25 ml Erlenmeyer flasks after gassing with 95% air-5% carbon dioxide. The wet weight of these tissues ranged from 0.2 to 0.6 gm and in all cases more than 95% of the cells remained viable after culture as determined by Trypan blue exclusion test. ’ ’ GS was assayed as described before”.‘4 and protein was estimated according to Lowry et ul. ‘.’GS specific activity is expressed as pmol of y-glutamyl hydroxamate formed per hour per mg protein.
RESULTS AND DISCUSSION Figure 2a shows the types of cells obtained by 10 min trypsinization of 55-day-old rat brain. In contrast to 60-90 min trypsinization commonly used for tissue disruption,‘.” the 10 mitt treatment selectively dissociates the external gray matter containing primarily the protoplasmic astrocytes (65%) with about 25% neurons and few (5%) broken or unidentified cells. Note that in this preparation, cells of the white matter-the fibrous astrocytes or the oligodendrocytes-are absent or rarely present. Phase contrast microphotographs of the protoplasmic astrocytes and the neuronal perikarya purified by the procedure outlined in Methods are shown in Figs 2b and 2c, respectively. In view of the reports of Norenberg & Martinez ” that GS in rat brain is localized exclusively in the astrocytes and the enzyme is highly enriched in cells of the cortex region,” we examined by direct biochemical assay the relative distribution of GS in gray and white matter of adult rat brain, separated by selective dissection of the tissue. In four independent assays, GS activity of the gray matter was 4.8 2 0.3 (mean + SD.) units/mg protein and that of the white matter was 0.75 t 0.1 (mean +: S.D.) units/mg protein. These results show that over 80% of the GS is localized in the gray matter implying that the protoplasmic astrocytes are the predominant source of this enzyme. The developmental profile of GS in the purified protoplasmic astrocytes as well as in the total cell suspension is shown in Fig. 3. In both cases, the enzyme activity remains very low until day 13 when it increases sharply and after day 20 the activity increases relatively slowly until adult level is
20 Age
40
60
(Days)
Fig. 3. Developmental pattern of GS activity in the total brain cell suspension (o-o) and in purified protoplasmic astrocytes (o-----e). The initial five points represent data for 1, 5, IO. t3 and 15 day rat brain. respectively.
reached. The major increase in the level of GS occurs between days 13 and 15. In contrast to this characteristic pattern of increase in GS activity with age, the developmental profile of the neurons and the protoplasmic astrocytes (Fig. 4) show little or no change in the number of neurons after birth but the number of protoplasmic astrocytes showed marked increase in the first 12 postnatal days following which the rate of proliferation declined considerably. These results are consistent with the reports that while the multiplication of rat brain neurons is almost complete at birth,’ the glial ceils, 80% of which are formed by day 12, continue to proliferate until adulthood.*” Figures 3
I(b)
I(a)
Fig. 1. Identification of protoplasmic astrocytes and oligodendrocytes by immunofluorescence staining. (a) Represents protoplasmic astrocytes stained with anti GS serum (x200); (b) represents oligodendrocytes stained with anti galactocerebroside serum (x200).
210 1
2(b) Fig. 2. Phase contrast photomicrographs of (a) adult rat brain cells derived after 10 min trypsinization of the tissue (x 150), (b) purified protoplasmic astrocytes (x300), (c) purified neuronal perikaryon (x 300).
57
59
Ontogeny of glutamine synthetase in rat brain
1.6 0 Z 0.8 a”
20 Age
40
60
(Days)
Fig. 4. Age-dependent alterations in the number of protoplasmic astrocytes (o-o), neurons and in the ratio (n-n) of these cells in the developing rat brain.
(*-•),
and 4 indicate that there is no correlation between the increase in GS activity and the number of protoplasmic astrocytes, suggesting that some external factor muSt be involved in triggering the natural induction of the enzyme. Since GS has been reported to be inducible by corticosteroids in the chick retina,r0.r4.** chick cerebral hemisphere’s*’ and in culture of rat hypothalamus27 and mouse astrocytes,” we determined the effects of cortisol on the activity of GS in organ cultures of developing rat brain as well as in the purified protoplasmic astrocytes from 6, 9, 13 and 21-day-old rats. The results (Table 1) clearly demonstrate that both intact brain and purified astrocytes become progressively more sensitive to cortisol precociously between days 6 and 13 after birth. Following the normal ontogenic rise of GS activity, e.g. at day 21, exposure to external hormones has no effect on the activity of the enzyme. The greater sensitivity of the intact tissue to the exogenous hormone compared to purified protoplasmic astrocytes is presumably related to loss of specific proteins from cells during trypsinization. At the most sensitive age (day 13), cortisol treatment resulted in an 80% increase in the specific activity of GS in 4 h. Table
1. Inducibility of GS by cortisol protoplasmic astrocytes
in whole rat brain from different ages
and in purified
GS activity prnol GHA/h/mg protein
Tissue/cell
Brain
Protoplasmic astrocytes
Percent increment over control
Age (days)
Without cortisol (control)
With cortisol
6 9 13 21
0.69 2 0.02 0.87 * 0.05 1.10~0.04 2.88 2 0.04
0.80~0.03 1.17-co.o5 2.00?0.06 2.94kO.04
15k 3 35k 6 8OklO 2k 1
6 9 13 21
1.70~0.05 1.80~0.10 1.90*0.10 6.50 k 0.20
1.l?7+0.05 2.23 5 0.10 2.76 t 0.10 6.SO-cO.30
10-c 3 24~ 4 45+ 5 0
Intact brain and purified protoplasmic astrocytes were cultured for 4 h in a medium consisting of 99% Tyrode balanced salt solution and 1% penicillinstreptomycin mixture in presence or absence of cortisol (1 &ml) and GS activity was assayed as described in Methods. Data represents mean 2 S.D. from four experiments.
To determine if the precocious induction of GS as seen in culture above could be achieved in viva, we injected cortisol in the brain ventricle of lZday-old rats (2 tq/g body wt) twice at 24 h intervals. GS activity was assayed 4 h after the second injection. In four different experiments, cortisol elicited an increase in GS activity ranging from 38 to 51% with a mean percent increment of UN2:1-D
D. Chatterjee
60
and P. K. Sarkar
? S.D.). Attempts to confirm the influence of corticosteroids in the regulation of brain GS activity by adrenalectomy of lO-12-day-old rats were unsuccessful since it was impossible to keep the rats alive after operation. However, in adult rats, using four animals for each case, it was found that 6 days after adrenalectomy, GS activity of brain declined from 3.2 rt 0.1 units/mg protein (mean -I-SD.) in sham-operated controls to 2.26 + 0.07 unitslmg protein (mean of;S.D.) in the adrenalectomized animals. We conclude from these experiments that the developmental pattern of rat brain GS is regulated by adrenocorticosteroids. The natural ontogenic rise of the enzyme around days 13-15 is also coin43.7 !I 7.5 (mean
cident
with the onset
Acknowledgement-Thanks one of us (D. Chatterjee)
of the function
of the rat adrenal
are due to the Indian with a Fellowship.
Council
cortex
of Medical
on or about
Research
day 14 afer birth.“,’
for sponsoring
the project
and supporting
REFERENCES 1. Farooq M. & Norton W. T. (1978) A modified procedure for isolation of astrocyte and neuron enriched fraction from rat brain. J. Neurochem. 31,887-894. 2. Fewster M. E., Blackstone S. C. & Ihrig T. J. (lY73) The preparation and characterization of isolated olig~endroglia from bovine white matter. BrailPRes. 63,263-271. 3. Fiske V. M. & Leeman S. E. (1964) Observation on adrenal rhythmicity and associated phenomena in the rat: effect of light on adrenocortical function; maturation of the hypothalamic neurosecretory system in relation to adrenal secretion. Ann. N. Y. Acad. Sci. 117,231-243. 4. Ford D. H. (1973) Selected maturational changes observed in postnatal rat brain. In Progress in Brain Research, Vol. 40 (ed. Ford D. H.), pp. 1-12. Elsevier, Amsterdam. _5 Greengard 0. (1971) Enzyme differentiation in mammalian tissue. Essays Biochem. 7, 159-205. 6. Haltmeyer G. C., Denenberg V. H., Thatcher J. & Zarrow M. X. (1966) Response of adrenal cortex of the neonatal rat after subjection to stress. Nature, Lond. 212, 1371-1373. 7. Herzfeld A. & Raper S. M. (1975) Giutamine synthetase and glutamyltransferase in developing chick and rat tissues. Biol. Neon&a 27, 163-176. 0. S. & Hertz L. (1981) induction by hydrocortisone of glutamine syn8. Juurlink B. H. F., Schousboe A., Jorgensen thetase in mouse primary astrocyte cultures. 1. ~eu~~c~e~. 36,136-142. 9. Kirk D. L. & Moscona A. A. (1963) Synthesis of experimentally induced glutamine synthetase (glutamotransferase activity) in embryonic chick retina in vitro. Devl Biol. 8,341-35’7. in embryonic chick 10. Kovacs S. H. (1977) Early events in the induction of glutamine synthetase activity by hydrocortisone neural retina. In vifro 13,24-30. 11. Kruse P. F. & Patterson M. K. (1973) Tissue Culture: Methods and Applications. Academic Press, New York, 100 pp. 12. Kuffler S. W. & Nichols J. 6. (1977) From Neurons to Brain, p, 257. Sinauer Associates, Stamford. CT. 13. Lowry 0. H., Rosebrough N. J., Farr A. L. & Randall R. J. (lY51) Protein measurement with Folin phenol reagent. .I. biol. Chem. 143,265-275. 14. Moscona A. A. & Piddington R. (1966) Stimulation by hydrocortisone of premature changes in the developmental pattern of glutamine synthetase in embryonic retina. Biochem. biophys. Acta. 121,409-411. M. D. (1979) The distribution of glutamine synthetase in the rat central nervous system. J. Hisfochem. 1s. Norenberg Cytochem. 27,756762. 16. Norenberg M. D. and Martinez H. A. (1979) Fine structure localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161,303-310. 17. Norton W. T. and Poduslo S. E. (1970) Neural soma and whole neuroglia of rat brain: a new isolation technique. Science. Wash. 167,1144-1147. and development of glutamine synthetase in the embryonic cerebral hemisphere. J. 18. Piddington R. (1971) Distribution e;rp. Zool. 177,219-228. 19. Piddington R. (1973) Glutamine synthetase in the avian diencephalon. J. exp. Zool. 184, 167-176. R. (1977) Subcellular distribution of glutamyl transferase activities in embryonic cerebral hemispheres. 20. Piddington Brain Res. 128,505-5 14. 21. Raff M. C.. Mirsky R., Fields K. L., Lisak R. P., Dorfman S. H., Silberberg S., Greoson N. A., Leibowitz S. & Kennedy M. C. (1978) Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture. Nafure, Land. 274,813-816. requirement for the induction of glutamine synthetase in chick em22. Reif Lehrer L. & Amos H. (1968) Hydrocortisone bryo retinas. B~ochem. J. 106,425-430. 23. Rose S. P. R. & Sinha A. K. (1969) Some properties of isolated neuronal cell fractions. J. Neurochrm. 16, 1319-1328. E. & Moscana A. A. (1972) Isolation and characterization of glutamine 24. Sarkar P. K., Fischman D. A.. Goldwasser synthetase from chicken neural retina. J. biol. Chem. 247,7743-7749. of lysosomes in neurons and neuropil and a new neuronal 25. Sinha A. K. & Rose S. P. R. (1972) Compartmentation marker. Brain Res. 39, 181-196. organization in flat epitheloid atid stellate process bearing 26. Treska J. C., Bader M. F. & Auris D. (1982) Microtubular astrocytes in cultures. Neurochem Res. 7,275-286. 27. Vaccaro D. E., Leeman S. E. & Rief Lehrer L. (1979) Glutamine synthetase activity in viva and in primary cell cultures of rat hypothalamus. J. Neurochem. 33,953-957. Vol. 3 (ed. Lajtha A.), p. 3.55. Plenum Press, New York. 28. Vander Berg C. J. (1970) Hand BookofNeurochemistry, A. & Arnold E. B. (1980) Age reIated changes in neuronal and g&al enzyme activities. In Advances in 29. Vernadakis Ceilular Neurobiology, Vol. 1 (eds Fedoroff S. &Hertz L.), p. 261. Academic Press. New York.
073fst_574X/XJ $3.00+0.00 Pergxnon Press Ltd.
Printed in Great Britain.
ONTOGENY
OF GLUTAMINE D. CHATTERJEE
Department
of Cell Biology,
Indian
Institute (Accepted
SYNTHETASE and P. K.
of Chemical 5 September
IN RAT
BRAIN
SARKAR
Biology,
Jadavpur,
Calcutta
700032, India
1983)
Abstract-The developmental pattern of glutamine synthetase (GS) in rat brain has been studied with respect to that of the cells involved in the synthesis of the enzyme. GS activity is very low until day 13 after birth, rises sharply between days 13 and 15, and slowly thereafter. In contrast, more than half of the total number of protoplasmic astrocytes-the cells involved in synthesis of GS-are formed by day 12, following which the rate of proliferation declines considerably. GS is precociously inducible by cortisol in purified protoplasmic astrocytes, in organ cultures of o-13 day rat brain and in the brains of cortisoladministered 12 day rats. These results and the temporal coincidence of the period of increase of GS with the onset of the function of adrenal cortex suggest that GS activity in the developing rat brain is under steroidal control. Key words: Glutamine
synthetase,
Protoplasmic
astrocytes,
Cortisol.
Glutamine synthetase (GS) is one of the key enzymes of brain because of its involvement in the synthesis of nucleic acids and mucopolysaccharides and in the metabolism of neurotransmitters.28 In the neural retina of the embryonic chick, the enzyme is inducible by adrenocorticosteroids.‘0,‘4,22 Although cortisol is known to induce the synthesis of many enzymes in the rat liver and kidney5 it did not accelerate the development of GS in liver or retina of fetal or neonatal rats7 However, in rat hypothalamus, development of GS activity appears to be sensitive to cortisol.27 Immunohistochemical studies of Norenberg & Martinez16 revealed that in the rat brain, GS has an exclusive glial localization and the enzyme is predominantly in the astrocytes of the gray matter. l5 Since the bulk of the glial cells in the gray matter is represented by protoplasmic astrocytes,12 we monitored the ontogeny of GS and that of the protoplasmic astrocytes to determine if the developmental profile of GS activity in rat brain is temporally coincident with the appearance and proliferation of the protoplasmic astrocytes or is regulated by endogenous adrenal corticosteroids. MATERIALS
AND METHODS
Trypsinization of brain tissue from Wistar albino rats of indicated ages was carried out according to the procedure of Norton & Poduslo17 except that incubation with trypsin was performed for 10 min instead of 60-90 min. This resulted in the selective dissociation of cells of the cortex region containing the gray matter (see Results) representing primarily protoplasmic astrocytes and neurons. Initial fractionation of these two types of cells was carried out by differential centrifugation in ficoll.’ The enriched neuronal and the astrocyte (glial) fractions obtained from the above step, referred to as N and G fractions, were further purified by centrifugation over 40% stock isotonic perco11gradients (density range 0.960-l. 150 gm/ml), prepared according to Pharmacia Fine Chemical Bulletin. Protoplasmic astrocytes were collected from density range 0.980-0.998 gm/ml with a particle purity of 9598% and the neurons from 1.026 to 1.150 gm/ml with a purity of 92-96%. For a comparative study, oligodendrocytes were purified according to the procedure of Fewster et al. * from white matter of rat brain with a particle purity of more than 80%. Neurons were distinguished from the protoplasmic astrocytes by their relatively large cell body and axons, sensitivity to 1% methylene blue stain23 and by the presence of marker enzyme B-galactosidase.25 Protoplasmic astrocytes were characterized by their thick wavy processes around the cell body’ in contrast to the thin filamentous fibres of the fibrous astrocytes, by their sensitivity to 1% cresyl violet*” and also by the presence of marker enzyme, glutamine synthetase.16 Purified preparations of protoplasmic astrocytes and oligodendrocytes were further identified by immunofluorescence staining with antibodies against astrocyte specific glutamine synthetaset6 and oligodendrocyte specific galactocerebroside,21 respectively (Fig. 1). Neither antisera gave a positive test with the purified neuronal preparation. Samples for indirect immunofluorescence assays were prepared as described by Treska et al. 26 55
56
n. Chatterjee and P. K. Sarkar
Cell counts were performed at a magnification of x 400 from fields of at least 2000 cells after staining with cresyl violet which stained the neurons lightly and the astrocytes deeply. Unidentified objects in the fields (ranging from 5 to 8% in all the cases) were disregarded. Intact brains of f&20-day-old rats, freed from blood vessels and meninges, were cultured in 10 ml of medium (99% Tyrode balanced salt solution and 1% penicillin streptomycin mixture) using 25 ml Erlenmeyer flasks after gassing with 95% air-5% carbon dioxide. The wet weight of these tissues ranged from 0.2 to 0.6 gm and in all cases more than 95% of the cells remained viable after culture as determined by Trypan blue exclusion test. ’ ’ GS was assayed as described before”.‘4 and protein was estimated according to Lowry et ul. ‘.’GS specific activity is expressed as pmol of y-glutamyl hydroxamate formed per hour per mg protein.
RESULTS AND DISCUSSION Figure 2a shows the types of cells obtained by 10 min trypsinization of 55-day-old rat brain. In contrast to 60-90 min trypsinization commonly used for tissue disruption,‘.” the 10 mitt treatment selectively dissociates the external gray matter containing primarily the protoplasmic astrocytes (65%) with about 25% neurons and few (5%) broken or unidentified cells. Note that in this preparation, cells of the white matter-the fibrous astrocytes or the oligodendrocytes-are absent or rarely present. Phase contrast microphotographs of the protoplasmic astrocytes and the neuronal perikarya purified by the procedure outlined in Methods are shown in Figs 2b and 2c, respectively. In view of the reports of Norenberg & Martinez ” that GS in rat brain is localized exclusively in the astrocytes and the enzyme is highly enriched in cells of the cortex region,” we examined by direct biochemical assay the relative distribution of GS in gray and white matter of adult rat brain, separated by selective dissection of the tissue. In four independent assays, GS activity of the gray matter was 4.8 2 0.3 (mean + SD.) units/mg protein and that of the white matter was 0.75 t 0.1 (mean +: S.D.) units/mg protein. These results show that over 80% of the GS is localized in the gray matter implying that the protoplasmic astrocytes are the predominant source of this enzyme. The developmental profile of GS in the purified protoplasmic astrocytes as well as in the total cell suspension is shown in Fig. 3. In both cases, the enzyme activity remains very low until day 13 when it increases sharply and after day 20 the activity increases relatively slowly until adult level is
20 Age
40
60
(Days)
Fig. 3. Developmental pattern of GS activity in the total brain cell suspension (o-o) and in purified protoplasmic astrocytes (o-----e). The initial five points represent data for 1, 5, IO. t3 and 15 day rat brain. respectively.
reached. The major increase in the level of GS occurs between days 13 and 15. In contrast to this characteristic pattern of increase in GS activity with age, the developmental profile of the neurons and the protoplasmic astrocytes (Fig. 4) show little or no change in the number of neurons after birth but the number of protoplasmic astrocytes showed marked increase in the first 12 postnatal days following which the rate of proliferation declined considerably. These results are consistent with the reports that while the multiplication of rat brain neurons is almost complete at birth,’ the glial ceils, 80% of which are formed by day 12, continue to proliferate until adulthood.*” Figures 3
I(b)
I(a)
Fig. 1. Identification of protoplasmic astrocytes and oligodendrocytes by immunofluorescence staining. (a) Represents protoplasmic astrocytes stained with anti GS serum (x200); (b) represents oligodendrocytes stained with anti galactocerebroside serum (x200).
210 1
2(b) Fig. 2. Phase contrast photomicrographs of (a) adult rat brain cells derived after 10 min trypsinization of the tissue (x 150), (b) purified protoplasmic astrocytes (x300), (c) purified neuronal perikaryon (x 300).
57
59
Ontogeny of glutamine synthetase in rat brain
1.6 0 Z 0.8 a”
20 Age
40
60
(Days)
Fig. 4. Age-dependent alterations in the number of protoplasmic astrocytes (o-o), neurons and in the ratio (n-n) of these cells in the developing rat brain.
(*-•),
and 4 indicate that there is no correlation between the increase in GS activity and the number of protoplasmic astrocytes, suggesting that some external factor muSt be involved in triggering the natural induction of the enzyme. Since GS has been reported to be inducible by corticosteroids in the chick retina,r0.r4.** chick cerebral hemisphere’s*’ and in culture of rat hypothalamus27 and mouse astrocytes,” we determined the effects of cortisol on the activity of GS in organ cultures of developing rat brain as well as in the purified protoplasmic astrocytes from 6, 9, 13 and 21-day-old rats. The results (Table 1) clearly demonstrate that both intact brain and purified astrocytes become progressively more sensitive to cortisol precociously between days 6 and 13 after birth. Following the normal ontogenic rise of GS activity, e.g. at day 21, exposure to external hormones has no effect on the activity of the enzyme. The greater sensitivity of the intact tissue to the exogenous hormone compared to purified protoplasmic astrocytes is presumably related to loss of specific proteins from cells during trypsinization. At the most sensitive age (day 13), cortisol treatment resulted in an 80% increase in the specific activity of GS in 4 h. Table
1. Inducibility of GS by cortisol protoplasmic astrocytes
in whole rat brain from different ages
and in purified
GS activity prnol GHA/h/mg protein
Tissue/cell
Brain
Protoplasmic astrocytes
Percent increment over control
Age (days)
Without cortisol (control)
With cortisol
6 9 13 21
0.69 2 0.02 0.87 * 0.05 1.10~0.04 2.88 2 0.04
0.80~0.03 1.17-co.o5 2.00?0.06 2.94kO.04
15k 3 35k 6 8OklO 2k 1
6 9 13 21
1.70~0.05 1.80~0.10 1.90*0.10 6.50 k 0.20
1.l?7+0.05 2.23 5 0.10 2.76 t 0.10 6.SO-cO.30
10-c 3 24~ 4 45+ 5 0
Intact brain and purified protoplasmic astrocytes were cultured for 4 h in a medium consisting of 99% Tyrode balanced salt solution and 1% penicillinstreptomycin mixture in presence or absence of cortisol (1 &ml) and GS activity was assayed as described in Methods. Data represents mean 2 S.D. from four experiments.
To determine if the precocious induction of GS as seen in culture above could be achieved in viva, we injected cortisol in the brain ventricle of lZday-old rats (2 tq/g body wt) twice at 24 h intervals. GS activity was assayed 4 h after the second injection. In four different experiments, cortisol elicited an increase in GS activity ranging from 38 to 51% with a mean percent increment of UN2:1-D
D. Chatterjee
60
and P. K. Sarkar
? S.D.). Attempts to confirm the influence of corticosteroids in the regulation of brain GS activity by adrenalectomy of lO-12-day-old rats were unsuccessful since it was impossible to keep the rats alive after operation. However, in adult rats, using four animals for each case, it was found that 6 days after adrenalectomy, GS activity of brain declined from 3.2 rt 0.1 units/mg protein (mean -I-SD.) in sham-operated controls to 2.26 + 0.07 unitslmg protein (mean of;S.D.) in the adrenalectomized animals. We conclude from these experiments that the developmental pattern of rat brain GS is regulated by adrenocorticosteroids. The natural ontogenic rise of the enzyme around days 13-15 is also coin43.7 !I 7.5 (mean
cident
with the onset
Acknowledgement-Thanks one of us (D. Chatterjee)
of the function
of the rat adrenal
are due to the Indian with a Fellowship.
Council
cortex
of Medical
on or about
Research
day 14 afer birth.“,’
for sponsoring
the project
and supporting
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