Neurochemical Research (l) 299-312 (1976)
C H A N G E S IN SOME E N Z Y M I C A C T I V I T I E S OF S E P A R A T E D N E U R O N A L AND GLIAL CELL-ENRICHED FRACTIONS FROM RAT BRAINS DURING DEVELOPMENT Y. NAGATA, T. NANBA, AND M. ANDO Department of Physiology, School of Medicine Fujita-Gakuen University, Toyoake, Aichi 470-11, Japan
Accepted March 9, 1976
Bulk-prepared neuronal perikarya and glial fractions h a v e been u s e d to s t u d y d e v e l o p m e n t a l c h a n g e s of s o m e e n z y m i c activities c o n c e r n i n g glycolysis and s y n t h e s i s of neurotransmitters. S o m e w h a t higher p y r u v a t e kinase activity w a s found in neuronal perikarya than in glial cells, and its rapid rise w a s o b s e r v e d during early d e v e l o p m e n t a l stages. Increased K + c o n c e n t r a t i o n or D-glutamate addition to the incubation m e d i u m e n h a n c e d c o n s u m p t i o n of p h o s p h o e n o l p y r u vate. This activation o f the e n z y m e was small j u s t after birth, but it increased in parallel with d e v e l o p m e n t to adult level, w h e r e the activation in glial fractions w a s o v e r twice that in neuronal fractions. Choline acetyltransferase activity w a s found in purified neuronal fractions and increased with age; glutamate decarboxylase w a s also f o u n d in high activity in purified neuronal fractions and increased with d e v e l o p m e n t . H o w e v e r , s o m e e n z y m i c activities were also found in glial fractions, and possibilities of c o n t a m i n a t i o n by s y n a p t o s o m a l , myelin, or other subfractions are discussed.
INTRODUCTION Various methods of bulk separation of neuronal perikarya-enriched and glial cell-enriched fractions from fresh animal brains have been developed in a number of laboratories since Rose (1,2) reported successful achievement of the isolation of metabolically active cell fractions from rat brains (3-6). There still might be some problems and arguments 299 9 1976Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y~ 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permission of the publisher.
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on the assessment of the separation techniques of the neuronal and glial cell fractions in bulk, especially on the yield and purity of the isolated cell samples and the morphological state of the cells. There is inevitable damage of cellular processes and cytoplasmic membranes of the separated brain cell preparations, and some leakage of small molecular constituents inside the cytoplasm. Also, there is some cross contamination of the separated cells by other types of cells or subcellular fractions, such as myelin or synaptosomes. However, for the study of the chemical changes expressed as brain functions, it must be of great importance to work at the cellular level in the brain. Although there might be serious limitations in the separation technique in bulk, it is still useful for research on the biochemical background of the brain activity. We have reported on an improved technique for bulk separation of neuronal cell body-enriched and glial cell-enriched fractions from adult and postnatally developing rat brains that does not use any digestive enzymes or incubation (7). Since the high-molecular protein components of the cells, such as enzymes, might be retained intracellularly even after considerable damage to cellular processes or membranes, we studied some enzyme activities during the course of postnatal development. Changes of pyruvate kinase activity were examined on the isolated neuronal and glial cell fractions during development and in potassium ion-stimulated conditions, since the energy for brain functions is derived" mostly from aerobic oxidation of glucose, and pyruvate kinase is known to regulate the metabolic flow of glycolysis (8). Also activities of two main synthesizing enzymes for neurotransmitter candidates, choline acetyltransferase for acetylcholine and glutamic acid decarboxylase for yaminobutyric acid, have been studied during development in separated brain cell fractions.
EXPERIMENTAL PROCEDURE
Materials Ficoll was obtained from Pharmacia Fine Chemicals, Sweden. [1-14C]Sodium-acetate (specific activity 54.9 mCi/mM), [14C]coenzymeA, and [1-14C]Dg-glutamicacid (specific activity 0.1 mCi/0.27 mg) were purchased from New England Nuclear, U.S.A. Enzyme and substrate preparations for enzymic analysis, such as lactic acid dehydrogenase (LDH), pyruvate kinase (PK), phosphoenolpyruvate (PEP), acetone-dried pigeon liver extract, pyridoxal-5'-phosphate, ADP, and NADP were obtained from Boehringer, Mannheim, Germany,and SigmaBiochemicalCo., U.S.A.
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Experimental Animals White adult rats of Wistar strain of both sexes weighing 200-250 g were used. For the developmental studies, young littermate rats 5, 10, 15, 20, 30, and 40 days old were used.
Preparation o f Separated Brain Cell-Enriched Fractions and Synaptosomes Enriched fractions in neuronal cell body and in glial cells were prepared by a method exactly identical to that described previously (7). The synaptosomal fraction was prepared according to Whittaker and Barker's procedure (9). Chopped whole rat brains were homogenized in 0.32 M sucrose, 10 mM Tris-HC1 buffer, pH 7.4, in a glass-teflon Potter-Elvehjem type tissue homogenizer with a clearance of 0.025 cm. The homogenate was centrifuged for 10 rain at 1,000g. The precipitate was washed twice by suspending in 0.32 M sucrose solution and recentrifuged together; the supernatant fluid was centrifuged for 55 rain at 17,000g to sediment the crude mitochondrial fraction. This fraction was resuspended in 0.32 M sucrose solution, placed over the layer of 0.8 M and 1.2 M sucrose solution, and centrifuged for 120 min at 53,000g. The fraction at the interphase between 0.8 M and 1.2 M sucrose solution was collected, diluted with 0.32 M sucrose solution, and then pelleted by centrifugation for 60 min at 100,000g as synaptosomal fraction. The tissue band at the interphase between 0.32 M and 0.8 M sucrose solution was collected as myelin fraction. In order to obtain the "purified" neuronal and glial cell fractions, the simply separated brain cell fractions were subjected to the regular Whittakers's procedure (9), i.e., the procedure of resuspending the simply separated cell fractions in 0.32 M sucrose solution and layering on 0.8 M and 1.2 M sucrose solution, then centrifuging as described above. Pelleted cell fractions were used as "purified" neuronal mad glial cell fractions.
Determination o f Enzymic Activities Pyruvate Kinase (PK; EC 2.7.1.40). The PK activity was assayed from the amount of utilization of t h e substrate phosphoenolpyruvate (PEP) during incubation at 37~ PEP was measured by enzymic analysis on the deproteinized sample with perchloric acid and neutralized with KOH, by following the decrease of the extinction of light at a wavelength of 366 nm in the presence of ADP, N A D H , L D H , and PK (10). Choline Acetyhransferase (ChAc; EC 2.3.1.6). The activity of ChAc was determined by the method of Fonnum (11). The amount of ['4C]acetylcholine (a4C-ACh) newly synthesized from p4C]coenzyme A or [1-'4C]sodium acetate, with preincubation in the extract of acetone-dried pigeon liver at 37~ for 10 min, was measured by liquid scintillation spectrometer (Beckman, type LS-100 or LS-355) after extraction of labeled ACh into ethylbutyl ketone. Glutamic Acid Decarboxylase (GAD; EC 4.1.1.15). The G A D activity was determined by measuring '4COz liberated from [1-i4C]DL-glutamic acid, by the methods of Roberts and Simonsen (12) and Frontali (13), which was modified for microscale (14). Tissue homogehate was incubated in a small test tube under a nitrogen atmosphere with phosphate buffer, pH 6.5, 100 mM KCI, 5• 10-4 M pyridoxal-5'-phosphate, 0.02 M mercaptoethanol, and [114C]DL-glutamate. The ~4CO2 released in the gas phase of the reaction chamber was absorbed on a small folded disc of filter paper, hanging at the rubber stopper inside the test tube, moistened with concentrated (20%) KOH. To obtain complete evolution of COz
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from the incubation medium and, at the same time, to stop the reaction, a small glass rod with a drop of concentrated H2SO4 on its tip was brought into contact with the incubation mixture. The radioactivity of the alkaline filter paper disc that had absorbed the 14CO2 was measured by Beckman liquid scintillation spectrometer. 2',3'-Cyclic Nueleotide 3'-Phosphohydrolase (CNP). Determination of C N P activity was performed according to the procedure for quantitative paper chromatography by Kurihara and Tsukada (15), after activation treatment of the sample with 0.125% Triton X-100. The amount of protein in the sample was determined by the method of Lowry et al. (16).
RESULTS
Pyruvate Kinase (PK) The amount of PK in the immature cerebral cortex at the tenth day after birth was very low; it increased rapidly in parallel with development. This change of PK activity during postnatal development was quite similar to the data of Takagaki (17). In separated neuronal and glial cell fractions, the change of PK activity showed a similar tendency to increase; i.e., the enzyme activity was low at the stage just after birth, and it increased with development. In the neuronal fraction, PK consistently showed a somewhat higher value than that in the glial cell fraction during the course of cerebral growth (Fig. 1). In the adult brain, PK values in the neuronal and glial fractions were both nearly half that of the cerebral cortex tissue (Fig. 1).
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Fro. 1. Developmental changes of PK activity in cerebral cortex and separated neuronal and glial cell fractions. Values are the means of more than 3 observations; brackets indicate the SE.
ENZYMIC ACTIVITIES IN SEPARATED N E U R O N S A N D GLIA
303
TABLE I STIMULATION OF P K ACTIVITY BY POTASSIUM IONS AND D-GLUTAMATE IN CEREBRAL CORTEX SLICES
PK activity (unit/rag protein)
Change (%)
2.02 -+ 0.15 (3) 2.85 -+ 0.34 (3) 2.48 _+ 0.30 (3)
+41.1 +22.8
Control + KC1 (80 raM) + D-glutamate (5 mM)
In a potassium-rich (80 mM) condition, the PK activity in cerebral cortex slices showed a marked increase: It was more than 40% higher than that of the control. A similar phenomenon was observed with the addition of 5 mM o-glutamate to the incubation medium (Table 1). The increment of PK activity by potassium ions in the neuronal cell body fraction was rather large during the early stage, from the tenth to the twentieth day after birth; then it was gradually reaching the adult level, although actually no stimulation was observed in the 10-day-old brain. On the other hand, the amount of stimulated PK activity by potassium ions was rather high at the tenth day in the glial cell fraction, and it increased continuously with cerebral growth. At the adult stage, the amount of PK stimulation was more than twice as high in the glial cell fraction as in the neuronal fraction (Fig. 2).
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FIG, 2. Developmental changes in stimulation of PK activity by potassium ions in separated neuronal and glial cell fractions. Values are the means of more than 3 observations; brackets indicate the SE.
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Choline Acetyltransferase (ChAc) It has already been reported that ChAc activity in the cerebral cortex was very low at the third day after birth, but it increased very rapidly during postnatal days, reaching the adult level, where the activity was several-fold higher than that of the brain of very early days after birth (]8). The ChAc activity of simply separated neuronal and glial cell fractions showed paradoxical distribution; i.e., a rather higher value of ChAc activity was found in the glial cell fraction than in the neuronal fraction at each developmental stage. The preparations were then subjected to further purification procedures according to the regular Whittaker's method (9) of sucrose density gradient centrifugation on 0.8 M and 1.2 M sucrose solution to remove some contaminated fragments of synaptosomes, myelin, or other subcellular components. The ChAc activity of this purified neuronal perikaraya fraction increased rapidly during very early stages up to the twentieth day after birth, attaining the adult value. In the purified glial cell fraction, ChAc activity increased very slowly in almost the same way, but at a much lower level than that of the neuronal fraction (Fig. 3).
Glutamic Acid Decarboxylase (GAD) Studies on G A B A content and G A D activity in the developing brain tissues that have already been performed have indicated the rapid
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FIG. 3. Developmental changes of ChAc activity in purified neuronal and glial cell fractions from rat brains. Values labeled "neuronal" and "glial" are the means of 2 observations of simply separated brain cell fractions. Values with brackets are the means of 3 experiments; brackets indicate the SE.
ENZYMIC ACTIVITIES IN SEPARATED NEURONS AND GLIA
305
P u r i f i e d Ne u r o n s ii
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+-
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FIo. 4. Developmental changes of GAD activity in purified neuronal and glial cell fractions from rat brains, Values labeled "neuronal" and "glial" are the means of 2 observations of simply separated brain cell fractions. Values with brackets are the means of 3 experiments; brackets indicate the SE.
increase of G A D activity during postnatal development in parallel with the detection and increase of GABA content and GABA-T activity (19,20). G A D was found in highly concentrated states in the synaptosomal fraction [35.3-+ 3.11 SE, /zmol/h per g protein (4)] prepared from adult brain. G A D activity in simply separated neuronal and glial cell fractions during postnatal development seemed very similar; i.e., there was a slight increase in G A D activity in both brain cell fractions. After the subjection of these separated cell preparations to Whittaker's method (9), layering on 0.8 M and 1.2 M sucrose density gradient, and centrifuging to remove myelin and synaptosomal subfractions, purified neuronal and glial cell fractions were examined for G A D activity at each developmental stage. The G A D activity in the purified neuronal fraction was low at the tenth day after birth; it then increased linearly up to the thirtieth day. But in the purified glial fraction, the enzyme activity showed a low value even in the very early days, and little increase with cerebral growth. In the adult, the G A D activity in purified neurons was almost twice that in the glial preparation (Fig. 4).
2',3'-Cyclic Nucleotide 3 '-Phosphohydrolase (CNP) By subjecting the simply separated neuronal and glial cell fractions to further purification treatment, contaminated myelin fragments, as identified by the marker enzyme CNP, were found mainly in the fraction on 0.8 M sucrose solution. In the glial cell fraction, C N P activity was
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found mainly in the myelin fraction, and it was very low in cellular fractions. The purified glial cell fraction contained a little C N P activity, which may mean that this fraction contained some oligodendroglial cells, but the neuronal fraction showed extremely low activity. Although G A D was found to be rich in isolated synaptosomes separated from both simply separated neuronal and glial fractions, the enzyme contained much more of both purified neuronal and glial cell fractions. A larger proportion of synaptosomes could be removed from the glial cell fraction, as examined with G A D activity, than from the neuronal fraction; in other words, more of the synaptosomes could be contaminated in the glial fraction than in the neuronal fraction. The purified neuronal fraction, of course, showed much higher G A D activity than the glial fraction; in the collected myelin fraction separated from both neuronal and glial cell fractions, the G A D activity was very low (Table 2).
DISCUSSION Studies to trace the time course of postnatal enzymic development on separated neuronal and glial cell fractions have been reported by SeUinger and his associates on some lysosomal and mitochondrial enzymes, such as N-acetyl-/3-glucosamidase (E. C. 3.1.1.7), succinate dehydrogenase (E.C. 1.3.99.1), and a-glycerphosphate dehydrogenase (E. C. 1.1.2.1) (21-23). They have followed precisely the yield of brain cell preparations from highly immature (5-10 days) and rather older (1845 days) rat cerebral cortex, and showed that the neuronal perikaraya:glial cell yield ratio decreased with age, which indicates that as the glial proportion matured, the procedure succeeded in isolating a gradually smaller proportion of existing neurons (6). They chose the total R N A content of a fraction as the best expression of the number of cells it contained, because R N A measurements reflect the operational accomplishment of the bulk-isolation procedure (24). There might be arguments for the yield and purity of the brain cells by bulk-separation methods; we measured only the quantitative yield of both neuronal and glial fractions separated from adult brains as the protein basis, and examined the purity of the cell fractions as indicated by the RNA: D N A ratio during development. It was found that the RNA: D N A ratio in early postnatal days could be a little higher than that in the cells separated from adult brains. Accordingly, it is suggested that the separation of the cells from the immature brains could be easier, since they have fewer cellular processes, and the cells could be obtained in
307
ENZYMIC ACTIVITIES IN SEPARATED NEURONS AND GLIA
T A B L E II DEVELOPMENTAL CHANGES OF G A D s AND C N P b ACTIVITIES IN PURIFIED NEURONAL AND GLIAL CELL FRACTIONS FROM SIMPLY SEPARATED BRAIN
CELL FRACTIONS(Data show the mean of more than two experiments) Gli~
Myelin
Synaptosomes
Cells
Days after birth
GAD
10 20 30 adult 10 20 30 adult 10 20 30 adult
3.5 4.1 5.3 6.3 9.9 11.4 13.3 12.8 14.2 17.3 16.6 19.1
Neuronal CNP
9.3 27.3 30.1 8.2 9.4 10.7 2.9 2.2 1.9
GAD 2,3 4.1 6.5 8.2 6.0 5.8 7.9 10.0 18.0 26.3 33.4 36.1
CNP
4.1 8.1 9.5 2.1 3.2 3.9 1.2 0.9 0.7
GAD activity: /zmole/h/g protein. b CNP activity: unit/mg protein.
more intact condition (7). Developmental changes of PK activity in separated neuronal and glial cell fractions indicated that glycolytic activity had been increasing considerably with age in both cell fractions, although rather higher PK activity was shown in the neuronal than in the glial fraction. The PK activity in the separated brain cell fractions prepared from adult animals was about half that of the cerebral cortex tissue, because it was naturally considered that some parts of enzymic protein in the cells could be leaked out during the course of the cell separation procedures. However, the leakage of the PK protein seemed to be smaller in the very early immature developmental stages, since the cells collected from young immature brains appeared to be closer to the intact condition than were those from the adult brains, as already suggested by the R N A : D N A ratio of the separated cells from the developing brains (7). Energy for maintaining brain activity is thought to be supplied almost exclusively by the degradation of glucose through glycolysis and TCA cycle inside the cells. Rolleston and Newsholme (25) reported the intracellular concentrations of glycolytic intermediates measured while undergoing different states of aerobic glycolysis in guinea-pig cerebral cortex slices. They concluded that the reactions catalyzed by hexoki-
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nase, phosphofructokinase, and pyruvate kinase, and possibly that catalyzed by glyceraldehyde 3-phosphate dehydrogenase, are far from equilibrium, and that these four enzymes play regulatory roles for the flow of glycolysis. Previous workers (25,26) have reported that increasing the concentration of potassium ions in the medium decreases the concentration of A T P and creatine phosphate, and increases that of A M P and inorganic phosphate, in cerebral cortex slices. These changes facilitate the activities of the glycolytic enzyme system, and result in an increase of lactate formation in the incubated slices. Takagaki (8) reported that potassium ions were also a factor directly promoting the rate of glycolysis, which acts on the PK step in the brain cortex slices. These reports suggest that PK is considered to be an important enzymic background in the phenomenon of the acceleration of glycolysis by potassium ions--the "potassium effect." However, there are several control points in glycolysis, and the action of high potassium ion concentration and o-glutamate addition on the glycolytic pathway in cerebral cortex may be manifold (27). Our finding that potassium stimulation of PK activity in the separated glial cell fraction was higher than that in the neuronal fraction may suggest that the glial cells contribute considerably to the initial steps of the glucose utilization process, especially in the excitatory state of brain activity. Stimulation of PK activity by potassium ions was found to be somewhat higher in the glial cell fraction than in the neuronal fraction during early developmental stages of the "critical period," when conspicuous changes of morphological and biochemical observations are occurring in the living brain. Spontaneous electrical activities or spreading depression on the cerebral cortex area also begin to be recorded during this important period (28). Transformation of the energy-producing pathway from anaerobic glycolysis to aerobic oxidation has been observed in this stage. This transformation corresponds to the phenomenon of lack of K+-stimulation in glucose utilization and lactate formation by highly immature brain slices before 10 days after birth; the amount of stimulation increased remarkably within 1 month (17). Although ACh is an established neurotransmitter in the neuromuscular junction area, it is also considered to be one of the important putative transmitter candidates even in the brain. The activity of the synthesizing enzyme of ACh, choline acetyltransferase (ChAc), in whole cerebral tissue was very low at the stage just after birth; it increased with remarkable rapidity, reaching the adult level about 1 month later, when the activity was several times higher than that of the immature brain (18). In simply separated brain cell preparations, ChAc activity was found, unexpectedly, to be somewhat higher in the glial cell fraction
ENZYMIC ACTIVITIES IN SEPARATED N E U R O N S AND GLIA
309
than in the neuronal fraction. The cell preparations were subjected to the regular Whittaker's procedure (9) to remove probably contaminated synaptosome or myelin fragments, layering the preparation on 0.8 M and 1.2 M sucrose solution and centrifuging the cells down. ChAc is known to be highly concentrated in synaptosomes, especially in external synaptosome membranes (29). In the purified glial cell fraction thus obtained, a larger amount of contaminated synaptosomes could be removed from the simply separated preparation, and the ChAc activity level would be lower than before. On the other hand, some other contaminants with low ChAc value could be removed from the simply separated neuronal sample to form the purified neuronal fraction, and the enzyme activity was found to be increased, but we have not made a structural check on the contaminants. Consequently, ChAc was f o u n d in high concentration in the purified neuronal fraction, and it increased rather rapidly with cerebral growth within a month. However, some ChAc activity was also found in the purified glial fraction, perhaps because the fraction is still contaminated by synaptosomes or because the glial cells contain some ChAc activity. This problem would be an important one to be studied further. Measurements on G A B A content and G A D activity in developing brains have already been performed (19,20), and the data showing G A B A to be formed mainly in the nerve endings from glutamate through G A D are presented (30-32). This transmitter candidate and its synthesizing enzyme are suggested to be condensed in high concentration in presynaptic preparations (33, 34). Sellstr6m et al. (35) reported that G A D was very highly concentrated in synaptosomes (77.52 txmol/h per g protein) and was at a very low level in neuronal perikarya (11.82 txmol/h per g protein) and glial (7.39 ~mol/h per g protein) preparations, which suggests some contamination of synaptosomes and the true localization of the enzyme in the cells. In our preparation, the amount of G A D activity in synaptosome (35.4 txmol/h per g protein) from adult brain cortex tissue was somehat lower than that of Sellstr6m et al. (35), and much higher enzyme activity was observed in our purified neuronal and glial cell fractions. If parts of nerve endings attached on the surface membrane of neurons could be pinched off at the position of the presynaptic neck, and the synaptic area were on the cellular side, high activity of G A D in the neuronal fraction could be found. On the other hand, Rose showed a similar very low value of G A D in both neuronal and glial (neuropil) preparations (36). Since the G A D activity of simply separated neuronal and glial cell fractions showed similar very slow changes during postnatal development, further purification of the sample by Whittaker's method (9) of
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sucrose density gradient centrifugation was performed to remove contaminated fractions. Thus, the purified neuronal fraction showed a rapid increase of G A D activity within a month, whereas the enzyme activity in the purified glial fraction remained low, and rose very gradually. This finding might suggest that G A D is present both in presynaptic regions and in the neuronal cytoplasm in a much more highly concentrated state. Contaminated myelin fragments, as determined by C N P activity, in both brain cell fractions were small, but the possible presence of G A D activity even in the glial cell fraction could not be completely eliminated. Recently, Mac Donnell and Greengard (37) reported that considerable G A D activity was observed in many nonneural tissues.
ACKNOWLEDGMENTS The authors are grateful to Prof. Y. Tsukada of the Keio University School of Medicine for his helpful comments and discussion. Financial support from the Japanese Ministry of Education, the Japan Medical Association, and Nippon C. H. Boehringer Sohn Co. is gratefully acknowledged. REFERENCES 1. ROSE, S. P. R. (1965) Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells. Nature (London) 206, 621,622. 2. ROSE, S. P. R. (1%7) Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J. 102, 33-43. 3. SATAKE, M., and ABE, S. (1%6) Preparation and characterization of nerve cell perikarya from rat cerebral cortex. J. Biochem. (Tokyo) 59, 72-75. 4. BLOMSTRAND, C., and HAMBERGER, A. (1%9) Protein turnover in cell enriched fractions from rabbit brain. J. Neurochem. 16, 1401-1407. 5. NORTON, W. T., arid PODUSLO, S. E. (1970) Neuronal soma and whole neuroglia of rat brain; A new isolation technique. Science 167, 1144-1146. 6. JOHNSON, D. E., and SELLINGER, O. Z. (1971) Protein synthesis in neurons and glial cells of the developing rat brain; An in vivo study. J. Neurochem. 18, 1445-1460. 7. NAGATA, Y., MIKOSHIBA, K., and TSUKADA, Y. (1974) Neuronal cell body enriched and glial cell enriched fractions from young and adult rat brains; Preparation and morphological and biochemical properties. J. Neurochem. 22,493-503. 8. TAKAGAKI, G. (1968) Control of aerobic glycolysis and pyrnvate kinase activity in cerebral cortex slices. J. Neurochem. 15, 903-916. 9. WHITTAKER, V. P., and BARKER, L. A. (1972) The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, in FRIED, R. (ed.), Methods of Neurochemistry, Vol. 2, Marcel Dekker, Inc., New York, pp. 1-15.
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10. CZOK, R., and ECKERT, L. (1971) Determination of phosphoenolpyruvate with pyruvate kinase, in BERGMEYER, H.-U. (ed.), Methods of Enzymic Analysis, Academic Press, New York, pp. 224--233. 11. FONNUM, F. (1969) Radiochemical microassays for the determination of choline acetyltransferase and acetylcholinesterase activity. Biochem. J. 115, 465-472. 12. ROBERTS, E., and SIMONSEN, D. G. (1963) Some properties of L-glutamic decarboxylase in mouse brain. Biochem. Pharmacol. 12, 113-134. 13. FRONTALI, N. (1964) Brain glutamic acid decarboxylase and synthesis of 3~-aminobutyfic acid in vertebrate and invertebrate species, in RICHTER, D. (ed.), Comparative Neurochemistry, Pergamon Press, Oxford, pp. 185-192. 14. NAGATA, Y., YOKOI, Y., and TSUKADA, Y. (1966) Studies on free amino acid metabolism in excised cervical sympathetic ganglia from the rat. J. Neurochem. 13, 1421-1431. 15. KUmHARA, T., and TSUKADA, Y. (1967) The regional and subcellular distribution of 2',Y-cyclic nucleotide 3'-phosphohydrolase in the central nervous system. J. Neurochem. 14, 1167-1174. 16. LOWRY, O. H., ROSEBROUGH,N. S., FARR, A. L., and RANDALL, R. J. (1951) Protein measurement with Folin phenol reagent. J. Biol. Chem. 193,265-275. 17. TAKAGAKI, G. (1974) Developmental changes in glycolysis in rat cerebral cortex. J. Neurochem. 23,479-487. 18. McCAMAN, R. E., and APRISON, M. H. (1964) The synthesis and catabolic enzyme systems for acetylcholine and serotordne in several discrete areas of the developing rabbit brain, in HIMWICH, W. A., and HIMWICH, H. E. (eds.), Progress in Brain Research, Vol. 9, The Developing Brain, Elsevier Publishing Co., Amsterdam, pp. 220-223. 19. BAXTER, C. F., SCHADE, J. P., and ROBERTS, E. (1960) Maturational changes in cerebral cortex. II. Levels of glutamic acid decarboxylase, gamma-aminobutyric acid and some related amino acids, in ROBERTS,E., BAXTER, C. E., VAN HARREVELD, A., WIERSMA, C. A. D., ADEY, W. R., and KILLAM, K. E. (eds), Inhibition in the Nervous System and Gamma-Aminobutyric Acid, Pergamon Press, New York, pp. 214-218. 20. ROBERTS, E., and KURIYAMA, K. (1968) Biochemical-physiological correlations in studies of 3,-aminobutyric acid system. Brain Res. 8, 1-15. 21. IDOYAGA-VARGAS,V., SANTIAGO,J. C., PETIET, P. D., and SELLIN6ER, O. Z. (1972) The early post-natal development of the neuronal lysosome. J. Neurochem. 19, 25332544. 22. SELLINGER,O. Z., and SANTIAGO,J. C. (1972) Unequal development of two enzymes in neuronal cell body and glial cells of rat cerebral cortex. Neurobiology 2, 133-146. 23. SELLINGER, O. Z., LEGRAND, J., CLOS, J., and OHLSSON, W. G. (1975) Unequal patterns of development of succinate-dehydrogenase and acetylcholinesterase in Purkinje cell bodies and granule cells isolated in bulk from the cerebellar cortex of the immature rat. J. Neurochem. 23, 1137-1144. 24. SELLINGER,O. Z., and AZCUP,RA, J. M. (1974) Bulk separation of neuronal cell bodies and glial cells in the absence of added digestive enzymes, in MARKS, N., and RODNIGHT, R. (eds.), Research Methods in Neurochemistry, Plenum Press, New York, pp. 3-38. 25. ROLLESTONE, F. S., and NEWSHOLME, E. A. (1967) Control of glycolysis in cerebral cortex slices. Biochem. J. 104, 524-533. 26. MCILWAtN, H. (1952) Phosphate of brain during in vitro metabolism; Effect of oxygen,
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27. 28. 29. 30.
31.
32. 33.
34. 35. 36. 37.
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glucose, glutamate, glutamine, and calcium and potassium salts. Biochem. J. 52, 289-295. TAI~ACAK~,G. (1972) Control of aerobic glycolysis in guinea-pig cerebral cortex slices. J. Neurochem. 19, 1737-11751. HIMWICH, W. A. (1962) Biochemical and neurophysiological development of the brain in the neonatal period. Int. Rev. Neurobiol. 4, 117-158. FONNOM, F. (1967) The "compartmentation" of choline acetyltransferase within the synaptosome. Biochem. J. 103,262-270. SALGANICOFF,L., and DE ROBERTIS, E. (1965) Subcellular distribution of the enzymes of the glutamic acid, glutamine and y-aminobutyric acid cycles in rat brain. J. Neurochem. 12, 28%309. FONr~UM, F. (1973) Localization of cholinergic and y-aminobutyric acid containing pathways in brain in BALAZS, R., and CI~MER, J. E. (eds.), Metabolic Compartmentation in the Brain, Halsted, New York, pp. 245-259. FONNUMI F. (1968) The distribution of glutamic decarboxylase and aspartate transami= nase in subcellular fractions of rat and guinea-pig brain. Biochem. J. 106, 401-412. BLOOM, F. E., and IVERSEN, L. L. (1971) Localizing :a3H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography. Nature (London) 229, 628-630. MARTIN, D. L., and SMITh, A. A., III. (1972) Ions and the transport of gammaaminobutyric acid by synaptosomes. J. Neurochem. 19, 841-855. SELLSTROM, A., SJt)RBERG, L.-B., and HAMBERGER, A. (1975) Neuronal and glial system for y-aminobutyric acid metabolism. J. Neurochem. 25, 393-398. ROSE, S. P. R. (1968) Glucose and amino acid metabolism in isolated neuronal and glial fractions. J. Neurochem. 15, 1415-1429. MACDONNELL, P., and GREENGARD, O. (1975) The distribution of glutamate decarboxylase in rat tissues; Isotopic vs. fluorometric assays. J. Neurochem. 24, 615-618.
C H A N G E S IN SOME E N Z Y M I C A C T I V I T I E S OF S E P A R A T E D N E U R O N A L AND GLIAL CELL-ENRICHED FRACTIONS FROM RAT BRAINS DURING DEVELOPMENT Y. NAGATA, T. NANBA, AND M. ANDO Department of Physiology, School of Medicine Fujita-Gakuen University, Toyoake, Aichi 470-11, Japan
Accepted March 9, 1976
Bulk-prepared neuronal perikarya and glial fractions h a v e been u s e d to s t u d y d e v e l o p m e n t a l c h a n g e s of s o m e e n z y m i c activities c o n c e r n i n g glycolysis and s y n t h e s i s of neurotransmitters. S o m e w h a t higher p y r u v a t e kinase activity w a s found in neuronal perikarya than in glial cells, and its rapid rise w a s o b s e r v e d during early d e v e l o p m e n t a l stages. Increased K + c o n c e n t r a t i o n or D-glutamate addition to the incubation m e d i u m e n h a n c e d c o n s u m p t i o n of p h o s p h o e n o l p y r u vate. This activation o f the e n z y m e was small j u s t after birth, but it increased in parallel with d e v e l o p m e n t to adult level, w h e r e the activation in glial fractions w a s o v e r twice that in neuronal fractions. Choline acetyltransferase activity w a s found in purified neuronal fractions and increased with age; glutamate decarboxylase w a s also f o u n d in high activity in purified neuronal fractions and increased with d e v e l o p m e n t . H o w e v e r , s o m e e n z y m i c activities were also found in glial fractions, and possibilities of c o n t a m i n a t i o n by s y n a p t o s o m a l , myelin, or other subfractions are discussed.
INTRODUCTION Various methods of bulk separation of neuronal perikarya-enriched and glial cell-enriched fractions from fresh animal brains have been developed in a number of laboratories since Rose (1,2) reported successful achievement of the isolation of metabolically active cell fractions from rat brains (3-6). There still might be some problems and arguments 299 9 1976Plenum PublishingCorporation, 227 West 17th Street, New York, N.Y~ 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming,recording, or otherwise, without written permission of the publisher.
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on the assessment of the separation techniques of the neuronal and glial cell fractions in bulk, especially on the yield and purity of the isolated cell samples and the morphological state of the cells. There is inevitable damage of cellular processes and cytoplasmic membranes of the separated brain cell preparations, and some leakage of small molecular constituents inside the cytoplasm. Also, there is some cross contamination of the separated cells by other types of cells or subcellular fractions, such as myelin or synaptosomes. However, for the study of the chemical changes expressed as brain functions, it must be of great importance to work at the cellular level in the brain. Although there might be serious limitations in the separation technique in bulk, it is still useful for research on the biochemical background of the brain activity. We have reported on an improved technique for bulk separation of neuronal cell body-enriched and glial cell-enriched fractions from adult and postnatally developing rat brains that does not use any digestive enzymes or incubation (7). Since the high-molecular protein components of the cells, such as enzymes, might be retained intracellularly even after considerable damage to cellular processes or membranes, we studied some enzyme activities during the course of postnatal development. Changes of pyruvate kinase activity were examined on the isolated neuronal and glial cell fractions during development and in potassium ion-stimulated conditions, since the energy for brain functions is derived" mostly from aerobic oxidation of glucose, and pyruvate kinase is known to regulate the metabolic flow of glycolysis (8). Also activities of two main synthesizing enzymes for neurotransmitter candidates, choline acetyltransferase for acetylcholine and glutamic acid decarboxylase for yaminobutyric acid, have been studied during development in separated brain cell fractions.
EXPERIMENTAL PROCEDURE
Materials Ficoll was obtained from Pharmacia Fine Chemicals, Sweden. [1-14C]Sodium-acetate (specific activity 54.9 mCi/mM), [14C]coenzymeA, and [1-14C]Dg-glutamicacid (specific activity 0.1 mCi/0.27 mg) were purchased from New England Nuclear, U.S.A. Enzyme and substrate preparations for enzymic analysis, such as lactic acid dehydrogenase (LDH), pyruvate kinase (PK), phosphoenolpyruvate (PEP), acetone-dried pigeon liver extract, pyridoxal-5'-phosphate, ADP, and NADP were obtained from Boehringer, Mannheim, Germany,and SigmaBiochemicalCo., U.S.A.
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Experimental Animals White adult rats of Wistar strain of both sexes weighing 200-250 g were used. For the developmental studies, young littermate rats 5, 10, 15, 20, 30, and 40 days old were used.
Preparation o f Separated Brain Cell-Enriched Fractions and Synaptosomes Enriched fractions in neuronal cell body and in glial cells were prepared by a method exactly identical to that described previously (7). The synaptosomal fraction was prepared according to Whittaker and Barker's procedure (9). Chopped whole rat brains were homogenized in 0.32 M sucrose, 10 mM Tris-HC1 buffer, pH 7.4, in a glass-teflon Potter-Elvehjem type tissue homogenizer with a clearance of 0.025 cm. The homogenate was centrifuged for 10 rain at 1,000g. The precipitate was washed twice by suspending in 0.32 M sucrose solution and recentrifuged together; the supernatant fluid was centrifuged for 55 rain at 17,000g to sediment the crude mitochondrial fraction. This fraction was resuspended in 0.32 M sucrose solution, placed over the layer of 0.8 M and 1.2 M sucrose solution, and centrifuged for 120 min at 53,000g. The fraction at the interphase between 0.8 M and 1.2 M sucrose solution was collected, diluted with 0.32 M sucrose solution, and then pelleted by centrifugation for 60 min at 100,000g as synaptosomal fraction. The tissue band at the interphase between 0.32 M and 0.8 M sucrose solution was collected as myelin fraction. In order to obtain the "purified" neuronal and glial cell fractions, the simply separated brain cell fractions were subjected to the regular Whittakers's procedure (9), i.e., the procedure of resuspending the simply separated cell fractions in 0.32 M sucrose solution and layering on 0.8 M and 1.2 M sucrose solution, then centrifuging as described above. Pelleted cell fractions were used as "purified" neuronal mad glial cell fractions.
Determination o f Enzymic Activities Pyruvate Kinase (PK; EC 2.7.1.40). The PK activity was assayed from the amount of utilization of t h e substrate phosphoenolpyruvate (PEP) during incubation at 37~ PEP was measured by enzymic analysis on the deproteinized sample with perchloric acid and neutralized with KOH, by following the decrease of the extinction of light at a wavelength of 366 nm in the presence of ADP, N A D H , L D H , and PK (10). Choline Acetyhransferase (ChAc; EC 2.3.1.6). The activity of ChAc was determined by the method of Fonnum (11). The amount of ['4C]acetylcholine (a4C-ACh) newly synthesized from p4C]coenzyme A or [1-'4C]sodium acetate, with preincubation in the extract of acetone-dried pigeon liver at 37~ for 10 min, was measured by liquid scintillation spectrometer (Beckman, type LS-100 or LS-355) after extraction of labeled ACh into ethylbutyl ketone. Glutamic Acid Decarboxylase (GAD; EC 4.1.1.15). The G A D activity was determined by measuring '4COz liberated from [1-i4C]DL-glutamic acid, by the methods of Roberts and Simonsen (12) and Frontali (13), which was modified for microscale (14). Tissue homogehate was incubated in a small test tube under a nitrogen atmosphere with phosphate buffer, pH 6.5, 100 mM KCI, 5• 10-4 M pyridoxal-5'-phosphate, 0.02 M mercaptoethanol, and [114C]DL-glutamate. The ~4CO2 released in the gas phase of the reaction chamber was absorbed on a small folded disc of filter paper, hanging at the rubber stopper inside the test tube, moistened with concentrated (20%) KOH. To obtain complete evolution of COz
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from the incubation medium and, at the same time, to stop the reaction, a small glass rod with a drop of concentrated H2SO4 on its tip was brought into contact with the incubation mixture. The radioactivity of the alkaline filter paper disc that had absorbed the 14CO2 was measured by Beckman liquid scintillation spectrometer. 2',3'-Cyclic Nueleotide 3'-Phosphohydrolase (CNP). Determination of C N P activity was performed according to the procedure for quantitative paper chromatography by Kurihara and Tsukada (15), after activation treatment of the sample with 0.125% Triton X-100. The amount of protein in the sample was determined by the method of Lowry et al. (16).
RESULTS
Pyruvate Kinase (PK) The amount of PK in the immature cerebral cortex at the tenth day after birth was very low; it increased rapidly in parallel with development. This change of PK activity during postnatal development was quite similar to the data of Takagaki (17). In separated neuronal and glial cell fractions, the change of PK activity showed a similar tendency to increase; i.e., the enzyme activity was low at the stage just after birth, and it increased with development. In the neuronal fraction, PK consistently showed a somewhat higher value than that in the glial cell fraction during the course of cerebral growth (Fig. 1). In the adult brain, PK values in the neuronal and glial fractions were both nearly half that of the cerebral cortex tissue (Fig. 1).
~
2.0
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~o
io
DaysafterBirth
~"
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Fro. 1. Developmental changes of PK activity in cerebral cortex and separated neuronal and glial cell fractions. Values are the means of more than 3 observations; brackets indicate the SE.
ENZYMIC ACTIVITIES IN SEPARATED N E U R O N S A N D GLIA
303
TABLE I STIMULATION OF P K ACTIVITY BY POTASSIUM IONS AND D-GLUTAMATE IN CEREBRAL CORTEX SLICES
PK activity (unit/rag protein)
Change (%)
2.02 -+ 0.15 (3) 2.85 -+ 0.34 (3) 2.48 _+ 0.30 (3)
+41.1 +22.8
Control + KC1 (80 raM) + D-glutamate (5 mM)
In a potassium-rich (80 mM) condition, the PK activity in cerebral cortex slices showed a marked increase: It was more than 40% higher than that of the control. A similar phenomenon was observed with the addition of 5 mM o-glutamate to the incubation medium (Table 1). The increment of PK activity by potassium ions in the neuronal cell body fraction was rather large during the early stage, from the tenth to the twentieth day after birth; then it was gradually reaching the adult level, although actually no stimulation was observed in the 10-day-old brain. On the other hand, the amount of stimulated PK activity by potassium ions was rather high at the tenth day in the glial cell fraction, and it increased continuously with cerebral growth. At the adult stage, the amount of PK stimulation was more than twice as high in the glial cell fraction as in the neuronal fraction (Fig. 2).
1.6
~"~~
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~2
~!ii~/.i~}i~i~i:
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S
~0.8
~
~. 0.~,
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I!~11
I
I0
I
/
20 30 Days aftor Birth
/t
I
adult
FIG, 2. Developmental changes in stimulation of PK activity by potassium ions in separated neuronal and glial cell fractions. Values are the means of more than 3 observations; brackets indicate the SE.
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Choline Acetyltransferase (ChAc) It has already been reported that ChAc activity in the cerebral cortex was very low at the third day after birth, but it increased very rapidly during postnatal days, reaching the adult level, where the activity was several-fold higher than that of the brain of very early days after birth (]8). The ChAc activity of simply separated neuronal and glial cell fractions showed paradoxical distribution; i.e., a rather higher value of ChAc activity was found in the glial cell fraction than in the neuronal fraction at each developmental stage. The preparations were then subjected to further purification procedures according to the regular Whittaker's method (9) of sucrose density gradient centrifugation on 0.8 M and 1.2 M sucrose solution to remove some contaminated fragments of synaptosomes, myelin, or other subcellular components. The ChAc activity of this purified neuronal perikaraya fraction increased rapidly during very early stages up to the twentieth day after birth, attaining the adult value. In the purified glial cell fraction, ChAc activity increased very slowly in almost the same way, but at a much lower level than that of the neuronal fraction (Fig. 3).
Glutamic Acid Decarboxylase (GAD) Studies on G A B A content and G A D activity in the developing brain tissues that have already been performed have indicated the rapid
T
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+
Purified Neurons
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E
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~rified
~l,a
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5
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20
30
40
adult
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FIG. 3. Developmental changes of ChAc activity in purified neuronal and glial cell fractions from rat brains. Values labeled "neuronal" and "glial" are the means of 2 observations of simply separated brain cell fractions. Values with brackets are the means of 3 experiments; brackets indicate the SE.
ENZYMIC ACTIVITIES IN SEPARATED NEURONS AND GLIA
305
P u r i f i e d Ne u r o n s ii
30
+-
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i
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FIo. 4. Developmental changes of GAD activity in purified neuronal and glial cell fractions from rat brains, Values labeled "neuronal" and "glial" are the means of 2 observations of simply separated brain cell fractions. Values with brackets are the means of 3 experiments; brackets indicate the SE.
increase of G A D activity during postnatal development in parallel with the detection and increase of GABA content and GABA-T activity (19,20). G A D was found in highly concentrated states in the synaptosomal fraction [35.3-+ 3.11 SE, /zmol/h per g protein (4)] prepared from adult brain. G A D activity in simply separated neuronal and glial cell fractions during postnatal development seemed very similar; i.e., there was a slight increase in G A D activity in both brain cell fractions. After the subjection of these separated cell preparations to Whittaker's method (9), layering on 0.8 M and 1.2 M sucrose density gradient, and centrifuging to remove myelin and synaptosomal subfractions, purified neuronal and glial cell fractions were examined for G A D activity at each developmental stage. The G A D activity in the purified neuronal fraction was low at the tenth day after birth; it then increased linearly up to the thirtieth day. But in the purified glial fraction, the enzyme activity showed a low value even in the very early days, and little increase with cerebral growth. In the adult, the G A D activity in purified neurons was almost twice that in the glial preparation (Fig. 4).
2',3'-Cyclic Nucleotide 3 '-Phosphohydrolase (CNP) By subjecting the simply separated neuronal and glial cell fractions to further purification treatment, contaminated myelin fragments, as identified by the marker enzyme CNP, were found mainly in the fraction on 0.8 M sucrose solution. In the glial cell fraction, C N P activity was
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found mainly in the myelin fraction, and it was very low in cellular fractions. The purified glial cell fraction contained a little C N P activity, which may mean that this fraction contained some oligodendroglial cells, but the neuronal fraction showed extremely low activity. Although G A D was found to be rich in isolated synaptosomes separated from both simply separated neuronal and glial fractions, the enzyme contained much more of both purified neuronal and glial cell fractions. A larger proportion of synaptosomes could be removed from the glial cell fraction, as examined with G A D activity, than from the neuronal fraction; in other words, more of the synaptosomes could be contaminated in the glial fraction than in the neuronal fraction. The purified neuronal fraction, of course, showed much higher G A D activity than the glial fraction; in the collected myelin fraction separated from both neuronal and glial cell fractions, the G A D activity was very low (Table 2).
DISCUSSION Studies to trace the time course of postnatal enzymic development on separated neuronal and glial cell fractions have been reported by SeUinger and his associates on some lysosomal and mitochondrial enzymes, such as N-acetyl-/3-glucosamidase (E. C. 3.1.1.7), succinate dehydrogenase (E.C. 1.3.99.1), and a-glycerphosphate dehydrogenase (E. C. 1.1.2.1) (21-23). They have followed precisely the yield of brain cell preparations from highly immature (5-10 days) and rather older (1845 days) rat cerebral cortex, and showed that the neuronal perikaraya:glial cell yield ratio decreased with age, which indicates that as the glial proportion matured, the procedure succeeded in isolating a gradually smaller proportion of existing neurons (6). They chose the total R N A content of a fraction as the best expression of the number of cells it contained, because R N A measurements reflect the operational accomplishment of the bulk-isolation procedure (24). There might be arguments for the yield and purity of the brain cells by bulk-separation methods; we measured only the quantitative yield of both neuronal and glial fractions separated from adult brains as the protein basis, and examined the purity of the cell fractions as indicated by the RNA: D N A ratio during development. It was found that the RNA: D N A ratio in early postnatal days could be a little higher than that in the cells separated from adult brains. Accordingly, it is suggested that the separation of the cells from the immature brains could be easier, since they have fewer cellular processes, and the cells could be obtained in
307
ENZYMIC ACTIVITIES IN SEPARATED NEURONS AND GLIA
T A B L E II DEVELOPMENTAL CHANGES OF G A D s AND C N P b ACTIVITIES IN PURIFIED NEURONAL AND GLIAL CELL FRACTIONS FROM SIMPLY SEPARATED BRAIN
CELL FRACTIONS(Data show the mean of more than two experiments) Gli~
Myelin
Synaptosomes
Cells
Days after birth
GAD
10 20 30 adult 10 20 30 adult 10 20 30 adult
3.5 4.1 5.3 6.3 9.9 11.4 13.3 12.8 14.2 17.3 16.6 19.1
Neuronal CNP
9.3 27.3 30.1 8.2 9.4 10.7 2.9 2.2 1.9
GAD 2,3 4.1 6.5 8.2 6.0 5.8 7.9 10.0 18.0 26.3 33.4 36.1
CNP
4.1 8.1 9.5 2.1 3.2 3.9 1.2 0.9 0.7
GAD activity: /zmole/h/g protein. b CNP activity: unit/mg protein.
more intact condition (7). Developmental changes of PK activity in separated neuronal and glial cell fractions indicated that glycolytic activity had been increasing considerably with age in both cell fractions, although rather higher PK activity was shown in the neuronal than in the glial fraction. The PK activity in the separated brain cell fractions prepared from adult animals was about half that of the cerebral cortex tissue, because it was naturally considered that some parts of enzymic protein in the cells could be leaked out during the course of the cell separation procedures. However, the leakage of the PK protein seemed to be smaller in the very early immature developmental stages, since the cells collected from young immature brains appeared to be closer to the intact condition than were those from the adult brains, as already suggested by the R N A : D N A ratio of the separated cells from the developing brains (7). Energy for maintaining brain activity is thought to be supplied almost exclusively by the degradation of glucose through glycolysis and TCA cycle inside the cells. Rolleston and Newsholme (25) reported the intracellular concentrations of glycolytic intermediates measured while undergoing different states of aerobic glycolysis in guinea-pig cerebral cortex slices. They concluded that the reactions catalyzed by hexoki-
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nase, phosphofructokinase, and pyruvate kinase, and possibly that catalyzed by glyceraldehyde 3-phosphate dehydrogenase, are far from equilibrium, and that these four enzymes play regulatory roles for the flow of glycolysis. Previous workers (25,26) have reported that increasing the concentration of potassium ions in the medium decreases the concentration of A T P and creatine phosphate, and increases that of A M P and inorganic phosphate, in cerebral cortex slices. These changes facilitate the activities of the glycolytic enzyme system, and result in an increase of lactate formation in the incubated slices. Takagaki (8) reported that potassium ions were also a factor directly promoting the rate of glycolysis, which acts on the PK step in the brain cortex slices. These reports suggest that PK is considered to be an important enzymic background in the phenomenon of the acceleration of glycolysis by potassium ions--the "potassium effect." However, there are several control points in glycolysis, and the action of high potassium ion concentration and o-glutamate addition on the glycolytic pathway in cerebral cortex may be manifold (27). Our finding that potassium stimulation of PK activity in the separated glial cell fraction was higher than that in the neuronal fraction may suggest that the glial cells contribute considerably to the initial steps of the glucose utilization process, especially in the excitatory state of brain activity. Stimulation of PK activity by potassium ions was found to be somewhat higher in the glial cell fraction than in the neuronal fraction during early developmental stages of the "critical period," when conspicuous changes of morphological and biochemical observations are occurring in the living brain. Spontaneous electrical activities or spreading depression on the cerebral cortex area also begin to be recorded during this important period (28). Transformation of the energy-producing pathway from anaerobic glycolysis to aerobic oxidation has been observed in this stage. This transformation corresponds to the phenomenon of lack of K+-stimulation in glucose utilization and lactate formation by highly immature brain slices before 10 days after birth; the amount of stimulation increased remarkably within 1 month (17). Although ACh is an established neurotransmitter in the neuromuscular junction area, it is also considered to be one of the important putative transmitter candidates even in the brain. The activity of the synthesizing enzyme of ACh, choline acetyltransferase (ChAc), in whole cerebral tissue was very low at the stage just after birth; it increased with remarkable rapidity, reaching the adult level about 1 month later, when the activity was several times higher than that of the immature brain (18). In simply separated brain cell preparations, ChAc activity was found, unexpectedly, to be somewhat higher in the glial cell fraction
ENZYMIC ACTIVITIES IN SEPARATED N E U R O N S AND GLIA
309
than in the neuronal fraction. The cell preparations were subjected to the regular Whittaker's procedure (9) to remove probably contaminated synaptosome or myelin fragments, layering the preparation on 0.8 M and 1.2 M sucrose solution and centrifuging the cells down. ChAc is known to be highly concentrated in synaptosomes, especially in external synaptosome membranes (29). In the purified glial cell fraction thus obtained, a larger amount of contaminated synaptosomes could be removed from the simply separated preparation, and the ChAc activity level would be lower than before. On the other hand, some other contaminants with low ChAc value could be removed from the simply separated neuronal sample to form the purified neuronal fraction, and the enzyme activity was found to be increased, but we have not made a structural check on the contaminants. Consequently, ChAc was f o u n d in high concentration in the purified neuronal fraction, and it increased rather rapidly with cerebral growth within a month. However, some ChAc activity was also found in the purified glial fraction, perhaps because the fraction is still contaminated by synaptosomes or because the glial cells contain some ChAc activity. This problem would be an important one to be studied further. Measurements on G A B A content and G A D activity in developing brains have already been performed (19,20), and the data showing G A B A to be formed mainly in the nerve endings from glutamate through G A D are presented (30-32). This transmitter candidate and its synthesizing enzyme are suggested to be condensed in high concentration in presynaptic preparations (33, 34). Sellstr6m et al. (35) reported that G A D was very highly concentrated in synaptosomes (77.52 txmol/h per g protein) and was at a very low level in neuronal perikarya (11.82 txmol/h per g protein) and glial (7.39 ~mol/h per g protein) preparations, which suggests some contamination of synaptosomes and the true localization of the enzyme in the cells. In our preparation, the amount of G A D activity in synaptosome (35.4 txmol/h per g protein) from adult brain cortex tissue was somehat lower than that of Sellstr6m et al. (35), and much higher enzyme activity was observed in our purified neuronal and glial cell fractions. If parts of nerve endings attached on the surface membrane of neurons could be pinched off at the position of the presynaptic neck, and the synaptic area were on the cellular side, high activity of G A D in the neuronal fraction could be found. On the other hand, Rose showed a similar very low value of G A D in both neuronal and glial (neuropil) preparations (36). Since the G A D activity of simply separated neuronal and glial cell fractions showed similar very slow changes during postnatal development, further purification of the sample by Whittaker's method (9) of
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sucrose density gradient centrifugation was performed to remove contaminated fractions. Thus, the purified neuronal fraction showed a rapid increase of G A D activity within a month, whereas the enzyme activity in the purified glial fraction remained low, and rose very gradually. This finding might suggest that G A D is present both in presynaptic regions and in the neuronal cytoplasm in a much more highly concentrated state. Contaminated myelin fragments, as determined by C N P activity, in both brain cell fractions were small, but the possible presence of G A D activity even in the glial cell fraction could not be completely eliminated. Recently, Mac Donnell and Greengard (37) reported that considerable G A D activity was observed in many nonneural tissues.
ACKNOWLEDGMENTS The authors are grateful to Prof. Y. Tsukada of the Keio University School of Medicine for his helpful comments and discussion. Financial support from the Japanese Ministry of Education, the Japan Medical Association, and Nippon C. H. Boehringer Sohn Co. is gratefully acknowledged. REFERENCES 1. ROSE, S. P. R. (1965) Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells. Nature (London) 206, 621,622. 2. ROSE, S. P. R. (1%7) Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells. Biochem. J. 102, 33-43. 3. SATAKE, M., and ABE, S. (1%6) Preparation and characterization of nerve cell perikarya from rat cerebral cortex. J. Biochem. (Tokyo) 59, 72-75. 4. BLOMSTRAND, C., and HAMBERGER, A. (1%9) Protein turnover in cell enriched fractions from rabbit brain. J. Neurochem. 16, 1401-1407. 5. NORTON, W. T., arid PODUSLO, S. E. (1970) Neuronal soma and whole neuroglia of rat brain; A new isolation technique. Science 167, 1144-1146. 6. JOHNSON, D. E., and SELLINGER, O. Z. (1971) Protein synthesis in neurons and glial cells of the developing rat brain; An in vivo study. J. Neurochem. 18, 1445-1460. 7. NAGATA, Y., MIKOSHIBA, K., and TSUKADA, Y. (1974) Neuronal cell body enriched and glial cell enriched fractions from young and adult rat brains; Preparation and morphological and biochemical properties. J. Neurochem. 22,493-503. 8. TAKAGAKI, G. (1968) Control of aerobic glycolysis and pyrnvate kinase activity in cerebral cortex slices. J. Neurochem. 15, 903-916. 9. WHITTAKER, V. P., and BARKER, L. A. (1972) The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, in FRIED, R. (ed.), Methods of Neurochemistry, Vol. 2, Marcel Dekker, Inc., New York, pp. 1-15.
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