Regional and cellular differences in rat brain protein synthesis in vivo and in slices during development

11736-5748/86 $(13.(1(1+11.(1(I Pergamon Journals Ltd. (~) 1986 ISDN

Int. J. Devl. Ne,rosciem'e. Vol. 4. No. 3. pp. 21Y9-215. 1986. Printed in Great Britain.

R E G I O N A L A N D C E L L U L A R DIFFERENCES IN R A T B R A I N PROTEIN SYNTHESIS IN VIVO A N D IN SLICES DURING DEVELOPMENT F. M. SHAHBAZIAN,MYRON JACOBS* and ABEL LAJTHA Center for Neurochemistry, The Nathan S. Kline Institute for Psychiatric Research, Ward's Island, New York, NY 10035 and *Department of Pathobiology and Oral Pathology, College of Dentistry, New York University, New York, NY I(X)I0, U.S.A. (Accepted 3 December 19851

Abstract--We compared the rate of protein synthesis in immature and adult rat brain in vivo to that in brain slices. After the incorporation of a flooding dose of [laC]valine, in vivo and in brain slices, the label in proteins was measured in CNS regions and in neuron- and glia-enriched fractions. In regions in vivo in the adult, incorporation rates in corpus callosum were lower than in other regions, which were similar: in the young, cerebellum showed the highest rates and hypothalamus and cord the lowest. Since hypothalamus and cord were low in the young, there was no change during development in these two areas; in other areas incorporation rates in young were 2-3 times higher than in adult brain proteins. Incorporation rates in slices were lower than in vivo. In the young, cerebellum, olfactory bulb, and cord were close to in vivo, and other areas in slices from young incorporated at 611--90%of in vivo rates. In adult slices incorporation was 5-15% of that in vivo except in olfactory bulb, where it was 30%. in the cellular fractions, incorporation in vivo in young was close in the neuronal and glial fractions; in adults incorporation rates in neurons were higher, as the decrease in development was less in neurons than astrocytes, in slices in young, astrocytes incorporated amino acids at 100% of the in vivo rates, neurons at 611%; in adult slices, incorporation was at only 4-7% of the in vivo rate. The results show that developmental changes in protein metabolism occur in all brain areas and brain cells, with metabolic rates in young 2-3 times that in adult. Incorporation in vitro is less than in vivo: it is close in immature tissue, but is greatly decreased in adult. Although developmental and postmortem differences in rates of synthesis affect most areas and cells, regional and cellular heterogeneity can be found. Key words: Protein synthesis, Development, Slices, Regional differences, Cellular differences.

W e p r e v i o u s l y r e p o r t e d o u r o b s e r v a t i o n that slices from i m m a t u r e b r a i n i n c o r p o r a t e a m i n o acids at a b o u t 80% of the in v i v o rate, w h e r e a s slices from a d u l t b r a i n i n c o r p o r a t e at only 10% of the in v i v o rate. 7 T h e r e a s o n s for this d e v e l o p m e n t a l d i f f e r e n c e - - the relative stability of i n c o r p o r a t i o n in slices from i m m a t u r e b r a i n vs large p o s t m o r t e m c h a n g e s in adult s l i c e s - - a r e not clear at the p r e s e n t time. W e felt that d e t e r m i n a t i o n of v a r i o u s p r o t e i n s f o r m e d in the slice w o u l d p r o v i d e inf o r m a t i o n o n p r o t e i n s that are sensitive a n d resistant to p o s t m o r t e m a l t e r a t i o n s , a n d possibly o n s o m e of the c o n t r o l l i n g factors of c e r e b r a l p r o t e i n m e t a b o l i s m . Such c o n t r o l m e c h a n i s m s have a role n o t o n l y in physiological p r o t e i n t u r n o v e r , b u t also in processes of d e v e l o p m e n t , cell d e a t h , etc. Slices from b r a i n s e e m to be a s u i t a b l e system for s t u d y i n g b r a i n p r o t e i n synthesis. In t e r m s of rates of p r o t e i n synthesis o b t a i n e d by the v a r i o u s t e c h n i q u e s available, i n c o r p o r a t i o n in slices is lower t h a n it is in v i v o b u t higher t h a n in isolated cells a n d cell-free systems. 3 C h a n g e s in p r o t e i n synthesis in b r a i n slices might n o t be the same for all b r a i n proteins. It was r e p o r t e d that, u n d e r stress such as d e c a p i t a t i o n a n d / o r slicing of the b r a i n , synthesis of a few p r o t e i n s is i n d u c e d , while for most p r o t e i n s , it is i n h i b i t e d . 26 T o characterize the h e t e r o g e n e i t y of p r o t e i n s s y n t h e s i z e d in b r a i n tissue, we m e a s u r e d i n c o r p o r a t i o n i n t o n e u r o n s a n d glia, a n d v a r i o u s b r a i n regions.

MATERIALS AND METHODS Animals used

A l b i n o W i s t a r rats g r o w n in o u r l a b o r a t o r y were fed P u r i n a r o d e n t chow No. 5001 ( p r o t e i n , 2 3 % ; fat, 4 . 5 % ; fiber, 6 . 0 % ; ash, 8 . 0 % ; m i n e r a l s , 2 . 5 % ) a d l i b i t u m . A n i m a l s had access to w a t e r at all times. T h e y were kept in dark/light for 12-hr a l t e r n a t i n g periods. Each g r o u p consisted of 209

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both males and females, except where noted. The immature groups consisted of animals 8-10 days old, weighing no more than 25 g. The young adult animals were 4-6 weeks old and weighed 100-150 g. A m i n o acid incorporation in vivo in C N S regions Rats were injected with 3 ml/100 g body wt of 500 mM valine solution containing [t4C]valine5 (uniformly labeled) having a specific activity of 0.038 i~Ci/p, mol. The large dose of valine given in combination with the low metabolism of this amino acid results in several fold increase in brain levels in vivo and in the incubated brain slices to the extent that the tissue specific activity of the free amino acid is that of the administered labeled valine. 5 After 2 hr, the animals were decapitated and the spinal cord and the following brain regions were dissected: olfactory bulbs, frontal cortex, parietal cortex, hippocampus, corpus callosum, striatum, thalamus, hypothalamus, midbrain, pons and medulla, and cerebellum. Previous work has shown that incorporation rates are constant over several hours in vivo 5 and in brain slices. ~' The tissues were washed with saline, chopped with a Mcllwain tissue chopper at a setting of 0.468-mm thickness, precipitated with 10% T C A , and frozen until used. The frozen tissue was thawed and agitated with an Eberbach shaker for 15 rain, centrifuged at 6000 g, and decanted. Then 5 ml of 10% T C A was added 5-10 times and the mixture was shaken for 15 rain, centrifuged, and decanted to remove free valine radioactivity. Following this, about 5 ml of 1 N N a O H was added to each gram of tissue and the mixture was incubated at room t e m p e r a t u r e for 48 hr in a stoppered tube to bring about its disintegration. The mixture was vortexed and sonicated at a setting of 1 A (Branson Sonifier Model S123 with standard tapered i/~- microtip) until uniformity was attained. From the suspension, aliquots were taken for scintillation counting (1 ml tissue suspension in 1 N N a O H , and 10 ml of Dimiscint from National Dianostics) and for protein assay (Lowry protein assay as modified by Peterson).21 A m i n o acid incorporation in slices f r o m C N S regions The same tissues as above were dissected, rinsed with H E P E S medium (see below), and chopped with a Mcllwain tissue chopper at a setting of 0.468-mm thickness. After being weighed on a Mettler analytical balance, the slices were incubated, as described before, ~' in H E P E S (N-2hydroxyethylpiperazine-N-2-ethanesulfonic acid) medium, oxygenated for 2 min 4'~' and 1 mM [14C]valine having an S R A of 1.5 mCi/ixmol for 2 hr in a stoppered Erlenmeyer flask in a water bath shaker (100 cycles/rain) at 37°C. The reaction was then stopped by cooling in an ice bath, the incubation medium was removed, and the tissue was treated as above. Calculations The incorporation of amino acids into protein expressed as percent of protein-bound amino acid replaced per hour was calculated by using the following formulaS: k (protein synthesis rate in percent per hour) = S R A of T C A insoluble fraction (dpm/mg protein at 1 hr)/0.58 (~mol valine per mg protein) x S R A of T C A soluble fraction (dpm/~mol valine). Incorporation in vivo in cell fractions Rats were injected with 3 ml/100 g of a 500 mM valine solution (0.038 txCi/~mol). After 2 hr, the animals were decapitated and the cerebral hemispheres were removed and chopped with the Mcllwain tissue slicer to a thickness of approximately I mm. The four brains in each immature group were pooled and the two brains in each young adult group were pooled for all subsequent treatment. For a morphological description of the cell fractions see Ref. 9, and for a recent m e a s u r e m e n t of protein synthesis in these fractions see Patel et al. 2" Incorporation in I m m a t u r e and thickness setting incubated in 10

slices in cell fractions young adult brains from rats were chopped with the Mcllwain tissue slicer at a of 0.468 mm. The brains of each group as in the above section were pooled and ml of oxygenated H E P E S medium with ! mM valine (4.1) ixCi/~mol) in a

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211

stoppered 50-ml E r l e n m e y e r flask. After 1 hr, the reaction was stopped by cooling and the contents of each flask were centrifuged, decanted, and washed with saline, then recentrifuged and decanted.

Neuron- and astrocyte-rich fractions The brain slices were incubated at 37°C in a water bath shaker in a buffer of 10 mM KH2PO4N a O H ( p H 6) containing 8% glucose, 5% fructose, and 2% Ficoll, to which 0.1% acetylated trypsin (Sigma) had been addedfl The length of the incubation period of immature brain tissue was 40 min; for adult in vivo, 90 min; and for adult in vitro, 60 min. Trypsinization was stopped at the appropriate time for each group by removing the medium, changing to buffer containing 0.1% soybean trypsin inhibitor (Sigma) but no enzyme, and washing three times with fresh inhibitor-free buffer. Most of the cellular elements were freed from one another by forcing the tissue through a nozzle (2.4-mm diameter) and a nylon mesh screen (420-1~m diameter openings) several times by using a slight vacuum. Subsequent treatment involved concentration of the cell suspension, differential centrifugation into several pellets, and step gradient centrifugation of each resuspended pellet to yield layers of cell bodies, processes, disrupted tissue, and subcellular organelles as described by Farooq and Nortonfl A small sample of each fraction containing neuronal and astrocytic cell bodies was examined microscopically using a phase contrast microscope. The remainder of each fraction was precipitated with 10% T C A and washed several times with fresh 10% T C A , and the protein content and radioactivity were determined as before. RESULTS

Protein synthesis in CNS regions Most brain regions in immature animals synthesized protein at rates of about l % / h r , except for the hypothalamus and spinal cord (Table 1), which had rates about half that of other regions. Adult rates of protein synthesis (0.5%/hr) were generally about half those found in immature animals, except for the hypothalamus and spinal cord, in which the rates were similar in immature and adult. Table I. ha vivo rates of protein synthesis in different CNS regions Region

Immature

Adult

Olfactory bulb Frontal cortex Parietal cortex Hippocampus Corpus callosum Striatum Thalamus Hypothalamus Midbrain

1.03-+ 0.06 0.45---0.(14 I. i 2 ± (1.19 0.43__.(I.{14 1.26-+0.04 (1.44± 0.04 1.26-+0.08 (1.39± 0.01 0.93 -+0.18 0.36± 0.04 (I.94±(I.(15 11.37± 0.06 (I.95 _+[1.I I (I.41± 0.06 (I.45 _+0.04 (I.45-+0.08 0.95-+ 0.04 0.45± 0.04

2.3 2.6 2.9 3.2 2.6 2.5 2.3 I.() 2.1

Ponsand medulla Cerebellum Spinal cord

0.94+-0.04 1.28-+ (I.(18 (I.46 +_0.04

1.8 3.(1 1.0

0.51 ± 0 . 0 4 (I.42-+ O.(M (I.45 ± O.(M

I : A ratio

Rates are presented as mean -+S.D. percent protein of total synthesized per hour carried out in 3 independent sets of experiments. Immature (5 days old) and young adult (5 weeks old) rats were given a flooding dose of [14C]valine by i.p. injection. After 2 hr. the animals were killed and dissected, tissues were washed, disintegrated, and analyzed for protein content and radioactivity. When rates of protein synthesis in vitro were c o m p a r e d with rates of intact brain, some regions showed more stability, in that slices from these regions had rates of synthesis similar to those in

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vivo. In the immature rat, the regions of least decrease in synthesis were the olfactory bulbs, cerebellum, and spinal cord (Table 2), whereas the least stable regions with the largest decrease in synthesis postmortem were the corpus callosum, hippocampus, and thalamus. The adult brain regions all show much lower rates of incorporation, in slices as compared to in vivo, with the olfactory bulb slightly more stable than other regions. Table 2. In vitro rates of protein synthesis in CNS regions % In vitro:in vivo Immature Adult

Region

Immature

Adult

I : A ratio

Olfactory bulb Frontal cortex

1.17 ± 0.09 1.01 -+ 0.14

0.130 ± 0.010 0.048 -+ 0.040

9.0 21.0

114 90

29 I1

Parietal cortex Hippocampus Corpus callosum Striatum Thalamus Hypothalamus Midbrain

0.80 ± 0.07 0.46 - 0.(}4 0.40---0.10 0.78 ± 0.14 0.27 ± 0.03 0.42 ± 0.08 0.74±0.04

0.015 0.027 0.061 0.032 0.062 0.044 0.041

53.3 17.0 6.6 24.4 4.4 9.5 18.0

63 37 43 83 28 93 78

3 7 17 9 15 10 9

Pons and medulla

0.72 -+ 0.(16

0.025 ± 0.004

28.8

77

5

Cerebellum Spinal cord

1.65±0.16 (I.50 ± 0. I 0

0.018±0.002 0.026 ± 0.003

91.7 19.2

129 109

4 6

± 0.002 - 0.003 ±0.0(13 ± 0.0()2 ± 0.013 ± 0.007 -+0.006

Rates are presented as mean ± S.D. protein of total synthesized per hour and as percent of that synthesized in vivo carried out in 3 independent sets of experiments. Immature (5 days old) and young adult (5 weeks old) rats were given a flooding dose of [14CIvaline by i.p. injection (in vitro slice method). After 2 hr, the tissues were washed, disintegrated, and analyzed for protein and radioactivity to measure the rates of protein synthesis.

Protein synthesis in cell fractions In vivo in immature brain, neurons and astrocytes had similar rates of protein synthesis, while amino acid incorporation in neurons of adult brain was about three times that found in astrocytes (Table 3). In incubated immature brain slices, the decrease in the rate of protein synthesis was greater in neurons than in astrocytes, whereas in adult slices rates for both neurons and astrocytes were < 10% of in vivo synthesis rates. In vivo neuronal perikarya had higher rates of protein synthesis than brain homogenates, both in immature and in adult intact brain. In the immature brain astrocytes had a higher rate of protein synthesis than the brain homogenate, while in the adult brain rates were about the same (Table 3). Although the rate of protein synthesis of neurons in vitro was lower in both immature and adult rats, the difference was much greater in the adult. For astrocytes, there was almost no decrease in the immature tissue, while in the adult a decrease occurred that was almost as great as for neurons. Table 3. Rates of protein synthesis in purified neuron and astrocyte fractions In vivo Immature(I) Homogenate Neuron fraction IN) Astrocyte fraction (As) N:H As:H

In vitro

% In vitro.'in vivo

Adult (A)

I:A

Immature (I)

Adult (A)

Immature

Adult

1 . 4 9 - 0.19 11.62- 0.04 2.50 ± 11.116 1.55 ± 0.14 2.116± 0.13 0.55 ± 0.03 1.7 2.5 1.4 0.9

2.4 1.6 2.7

1.24 ± (I.31) 1.37 -+ 0.35 2.113 -+ (I.30 1.1 1.6

11.(175± 0.1115 0.1154± (I.080 0.040 + 0.023

83 55 99

12 3.5 7.2

1).7

(1.5

Rates are presented as mean -+ S.D. percent protein of total synthesized per hour carried out in 3 independent sets of experiments. Immature (10 days old) and young adult (4 weeks old) rats were given a flooding dose of [14C]valine either by i.p. injection (in vitro) or by incubation (Or vitro slice method). After 1 or 2 hr, the brain tissue was incubated in a buffer containing trypsin, washed, and fractionated into neurons and astrocytes. The cells were then disintegrated and assayed for protein and radioactivity to measure the rates of protein synthesis.

Regional differences in protein synthesis in development

213

DISCUSSION Regions of adult CNS all exhibited in vivo rates of synthesis of about 0.4%/hr with little variation. It has to be emphasized that this overall figure is the average of heterogenous fractions, those of both high and low turnover rate. In the immature CNS, regions fell into three groups: parietal cortex, frontal cortex, hippocampus, and cerebellum, with rates around 1.2%/hr; olfactory bulb, corpus callosum, striatum, thalamus, midbrain, and pons-medulla, with rates of around 1.0%/hr; hypothalamus and spinal cord, with rates of 0.45%/hr. In the immature brain the hypothalamus and spinal cord exhibited the low rates of protein synthesis found in the adult brain, indicating the likelihood that, at least in the rat, the 5-day-old spinal cord and hypothalamus had already decreased to mature protein synthesis levels. Rates of protein synthesis in other areas in young were two to three times higher than those in adults. This is similar to the results previously found for total brain and also to the data in the literature.17 The rates of protein synthesis in adult in vivo preparations of CNS regions were similar to those reported by Furst et al. ;~ however, they were slightly lower than those reported by Dunlop et al. 8 A comparison of regional heterogeneity of alterations of incorporation rates in slices from brain shows larger differences in the immature as compared to adult brain. In adult slices, rates of protein synthesis in CNS regions were all low (3-17% of in vivo rates) except for the olfactory bulb (29% of in vivo rate). The rates in slices of young brain were more variable; most areas had approximately half of the in vivo rates, but some had 30-50% and others had approximately the same as in vivo rates. Incorporation in the cerebellar slices was at rates that were higher than in vivo values'. Although immature hypothalamus and spinal cord exhibited the same low rates of synthesis in vivo as those of adults, the rates of protein synthesis in in vitro preparations of the tissues from adults was not reduced by 90%, as they were in adult brain areas. These regional differences in protein metabolism might, in part, be explained by different metabolic rates of the two major cell populations, neurons and neuroglia. We examined two representative fractions, one consisting almost entirely of neuronal perikarya, 70% of the other made up of astrocytes. In vivo in adult brain, neurons were found to have a higher synthesis rate than astrocytes or the brain homogenate, whose rates were similar. Using a different method of cell separation, Lisy and Lodin ~8and Hamberger and Sourander ~2 also reported higher synthesis rates for neuronal proteins in mice and rats older than 20 days (Table 4), and demonstrated similar ratios of neurons and astrocytes to homogenate as those in the present investigation. Table 4. Relative rates of protein synthesis in neurons (N), astrocytes (As), and total h o m o g e n a t e (H) Animal

Age (days)

Mode

Duration (min)

N:H

N:As

Reference

Rat Rat Rat Rat Mouse Rat Rat Rat Rat

10 10 II 18 20 21 30 43 90

intrathecal, pulse i.p., flood i.p., flood lntrathecal, pulse i.p., pulse i.p., flood i.p., flood lntrathecal, pulse i.p., flood

10 120 6(1 10 30 60 120 10 60

1.7 1.9 2.4 2.2 2.5 2.0

1.7 1.2 0.5 i .3 2.4 1.5 2.7 0.5 1.6

16 Present results 12 16 18 12 Present results 16 12

Neurons and astrocytes in the immature brain had higher rates of protein synthesis than the homogenate, with neurons again exhibiting a higher rate of metabolism than astrocytes, in agreement with the findings of Johnson and Sellinger, 16 but differing somewhat from the data reported by Hamberger and Sourander 12 (Table 4); the exceptionally high values obtained by the latter investigators for rates of protein synthesis in immature astrocytes suggest the possibility that their astrocytic fraction had a different composition. From the findings it seems that with development the rate of protein synthesis in astrocytes decreases more than in neurons. The consistently lower

214

F . M . Shahbazian et al.

level of p r o t e i n synthesis o f n e u r o n s in v i t r o may be the result of t h ei r h a v i n g g r e a t e r sensitivity to p o s t m o r t e m c h a n g e s than astrocytes. A n u m b e r o f p r o c e d u r e s h a v e b e e n p u b l i s h e d for p r e p a r i n g n e u r o n - and gila-rich fractions 1'2'9"12"14"22"25 using f r a g m e n t a t i o n , i n c u b a t i o n , sieving, and g r a d i e n t c e n t r i f u g a t i o n . T h e r e are v a r i a t i o n s in the m e t h o d o l o g y used by the v ar i o u s l a b o r a t o r i e s , ~"2'9'12"14'22'25 with s o m e steps m o d i f i e d or o m i t t e d , e.g. with i n c u b a t i o n o f slices o m i t t e d , "}'t-~'23'25 or with p r o t e a s e , 9"t4 sucrose, 27 Ficoll, 13 P V C , TM o r glycerol a n d a c e t o n e 24 a d d e d to the i n c u b a t i o n m e d i u m . S o m e c o m p a r i s o n s o f the v a r i o u s m e t h o d s h a v e b e e n m a d e . 14 It is likely that the d i f f e r e n t m e t h o d s yield cell p r e p a r a t i o n s that are not o f identical c o m p o s i tion, but differ in t h e ir m o r p h o l o g i c a l o r m e t a b o l i c characteristics and h a v e d i f f e r e n t types and d e g r e e s o f c o n t a m i n a t i n g fractions. T h e p r o c e d u r e that we used 9 gives a relatively high yield, a low d e g r e e o f c o n t a m i n a t i o n , and g o o d m e t a b o l i c activity. H o w the m e t h o d o f p r e p a r a t i o n a l t e r s the m e a s u r e d a m i n o acid i n c o r p o r a t i o n is not cl ear at the p r e s e n t t i m e , but it is unlikely that the main c h a n g e s o b s e r v e d h e r e in d e v e l o p m e n t and in v i v o / i n v i t r o w o u l d , in o u r n e u r o n a l and glial p r e p a r a t i o n s , be d i f f e r e n t f r o m that of o t h e r p r e p a r a t i o n s . It is o f interest that m a n y C N S p r o t e i n s w e r e s h o w n to be s y n t h e s i z e d in the cell p r e p a r a t i o n s in vitro, w O u r findings show that d e v e l o p m e n t a l c h a n g e s in m e t a b o l i c rates are r e m a r k a b l y similar for m o s t brain p r o t e i n s , and also that p o s t m o r t e m d e c r e a s e s in p r o t e i n synthesis o c c u r in all C N S p r o t e i n s - - b u t the i n c o r p o r a t i o n into a l m o s t all p r o t e i n s is a l t e r e d p o s t m o r t e m in the adult brain to a m u c h g r e a t e r e x t e n t than in the i m m a t u r e organ. REFERENCES I. Althaus H. H., Huttner W. B. and Neuhoff V. (1977) Neurochemical and morphological studies of bulk-isolated rat brain cells. A new approach to the preparation of cerebral neurons. Hoppe-Seyler's Z. Physiol. Chem..$58, 11551159. 2. Clausen J. (19821 Cell isolation. In Ham/book ofNeurochemistrv, Vol. 2 (ed, Lajtha A.), pp. 16.'L-393. Plenum Press, New York. 3. Dunlop D. S. (1978) Measuring protein synthesis and degradation rates in CNS tissue. In Research Methods in Neurochemistry, Vol. 4 (eds Marks N. and Rodnight R.), pp. 91-141. Plenum Press, New York. 4. Dunlop D. S., van Elden W. and Lajtha A. (1974) Measurements of rates of synthesis in rat brain slices. J. Neurochem. 22, 821-830. 5. Dunlop D. S., van Elden W. and Lajtha A. (1975) A method for measuring brain protein synthesis rates in young and adult rats. J. Neurochem. 24, 337-344. 6. Dunlop D. S., van Elden W. and Lajtha A. (19751 Optimum conditions for protein synthesis in incubated slices of rat brain. Brain Res. 99, 303-318. 7. Dunlop D. S., Lajtha A, and Toth J. (1977) Measuring brain protein metabolism in young and adult rats. In Mechanisms, Regulation and Special Function o f Protein Synthesis in the Brain (eds Roberts S., Lajtha A. and Gispen W. H.), pp. 79-96. Elsevier. Amsterdam. 8. Dunlop D. S., van Elden W. and Lajtha A. 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Res. 3, 535-547. 13. Hamberger A., Eriksson O. and Norrby K. (1971) Cell size distribution in brain suspensions and in fractions enriched with neuronal and glial cells. Exp. Cell Res. 67, 38(b-388. 14. Hemminki K., Huttnen M. O. and Jarnefelt J. (1970) Some properties of brain cell suspensions prepared by a mechanical-enzymic method. Brain Res. 23, 23-34. 15. iqbal K. and Tellez-Nagel I. (1972) Isolation of neurons and glial cells from normal and pathological human brain. Brain Res. 45, 296-301. 16. 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. Ig, 1445-14611. 17. Lajtha A. and Dunlop D. S. (19811 Turnover of proteins in the nervous system. Life Sci. 29, 755-767. 18. Lisy V. and Lodin Z. (1973) Incorporation of radioactive leucine into neuronal and glial proteins during postnatal development. Neurobiology 3, 320--326. 19. Nakayama T. (1981) Synthesis of cytoskeletal proteins in bulk-isolated neuronal perikarya. J. Neurochem. 36, 13981405. 20. Patel N. J., Cohen J. and Balazs R. (1984) Protein synthesis in cells isolated from the developing rat cerebellum. Int. J. Devl Neurosci. 3, 287-299.

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