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Biosynthesis of RNA in neuron- and glia-enriched fractions
228
BRAIN RI!SEAR(H
BIOSYNTHESIS OF RNA IN NEURON- AND GLIA-ENRICHED FRACTIONS
P. VOLPE AND A. GIUDITTA
International Laboratory of Genetics and Biophysics, Naples (Italy) (Accepted April 7th, 1967)
INTRODUCTION
One of the main problems of neurobiology concerns the contribution of neuronal and neuroglial cells to the overall cerebral metabolism. In several laboratories, these investigations have been pursued with preparations of single cells obtained by hand dissection and with several ingenious microadaptations of analytical techniques2-5,7-10,18A4. Although these efforts have provided information on the metabolism of these cellular types and have stimulated interest in the hypothesis that their functions are complementary, the low yield of cells obtained has prevented the application of biochemical procedures not suitable for microdeterminations. Recently some of these difficulties have been overcome by the availability of methods which allow the bulk separation of neurons and glia cells 15,16. We have used these techniques for the study of the synthesis of RNA in vivo in 'neuronal' and 'neurogtial' fractions isolated from the cerebral cortex of the rabbit, and have obtained evidence for the occurrence of marked differences in the kinetics of RNA labelling. MATERIALS AND METHODS
Preparation and analysis of'neuronal' and 'neuroglial' fractions The procedure originally described by Rose for the cerebral cortex of the rat 15was applied with minor modifications to the cortex of the rabbit (4 g). Fractions B and C recovered from the gradient (Fig. 1, I) were diluted to 10 ml with a solution containing 10 ~ Ficoll, 0.1 M KC1 and 0.01 M potassium phosphate buffer (pH 7.4) and centrifuged at 100,000 × g for 30 min in a Spinco ultracentrifuge (rotor No. 40). The pellets were resuspended in 5.0 ml of medium A (0.32 M sucrose, 0.l mM CaCI2, 0.05 M Tris, pH 7.5). In separate experiments, we adapted to the rabbit cortex the procedure Abbreviations: SDS, sodium dodecyl sulphate; NDS, 1,5-naphthalene disulphonate; Tris, tris (hydroxymethyl) aminomethane; EDTA, ethylenediaminetetraacetic acid. Brain Research, 6 (1967) 228-240
RNA
229
SYNTHESIS IN NEURONAL FRACTIONS
l A SUCROSE 0 5 M FICOLL 70 %-
B, SUCROSE 1.75 M FIC:OLL 20 o/%
,
L
SUCROSE I 35 M
P
I
]I
Fig. 1. I. Method of Rose. Separation of a cell suspension into four layers by centrifugation in a discontinuous Ficoll-sucrose gradient at 22,500 rev./min for 2 h in rotor SW 25 of the Spinco ultracentrifuge. A, floating layer; B, enriched 'glial' fraction; C, enriched 'neuronal' fraction; D, pellet containing erythrocytes and nuclei. II. Method of Satake and Abe. Preparation of nerve cell bodies by centrifugation in a discontinuous sucrose gradient at 14,5013rev./min for 30 min in rotor SW 25 of the Spinco ultracentrifuge. The narrow band (B') contains heterogeneous material ; at the bottom of the tube the pellet containing nerve cell bodies (P). described by Satake and Abe 16 for the preparation of nerve cell bodies from ratbrain. Approximately 4 g o f cortex cut into small pieces was suspended in 50 ml of acetoneglycerol-water (1 : 1 : 1, v/v) for 30 min at 0-4°C. The tissue, obtained by filtration through cheese-cloth, was gently homogenized in 30 ml o f glycerol-0.25 M sucrose (3 : 1, v/v) using a Potter homogenizer with a teflon pestle. The suspension, passed by means of suction t h r o u g h flannel-cloth, was diluted to 100 ml with R i n g e r - L o c k e solution, and treated according to the Satake and Abe procedure 16. The cell fractions were examined by phase-contrast microscopy and stained with Mayer's haemallum. P h o t o g r a p h s were taken with a Leitz O r t h o m a t microscope. Carbonic anhydrase activity was measured manometrically at 10°C from initial rates lz. Reaction velocities were always proportional to the concentration of enzyme. D N A was determined with a diphenylamine method 6.
Extraction and analysis o f R N A [6-14C]Orotic acid (44.5 m C / m M ) was given subarachnoidally to adult rabbits o f approximately 3-4 kg in a dose o f 100 ¢C, according to the procedure previously
Brahz Research, 6 (1967) 228-240
230
P. V O L P E A N [ ) A, (}IUI)II"fA
described 18. The cell fractions were suspended in 5.0 ml of medium A, and 0.5 ml of a 10~ SDS-5~o NDS solution were added, followed by 5.0 ml of a phenol-water mixture (86 : 14) containing 0.07 M Tris buffer (pH 7,4), 0.14~,, SDS and t mg/ml o-hydroxyquinoline. After extraction for 10 min at 4°C in a shaker, the samples were centrifuged in the cold. The phenol phases and the interphases were re-extracted twice according to the above procedure with equal volumes of medium A at 45°C for 5 min. The aqueous supernatants were mixed, re-extracted once at 4 C with an equal volume of phenol, and brought to a concentration of 0.1 M NaCI and 66 %/i ethanol. After standing overnight at --I 5°C, the precipitated RNA was washed by centrifugation with 80 ~o ethanol, dried and dissolved in 2.0 ml of 10 mM Tris buffer (pH 7.4). Residual DNA was digested by incubation for 15 min at 4"C with 100#g DNAase/ml in presence of 2 mM MgClz. At the end of the incubation, 40 ul of 0. l M EDTA (pH 8.0) were added and the solution extracted with phenol at 4'C for 15 min. RNA was re-precipitated as described above and dissolved in 1.0 ml of 10 mM Naacetate (pH 5.0) containing 0.1 M NaCI and 4 M urea. Sedimentation velocity was analysed by centrifugation at 2°-4°C in 5-20~o sucrose gradients containing 0.1 M NaCI and 4 M urea (15 h at 20,000 rev./min in rotor SW25 ofa Spinco ultracentrifuge). Chemicals Ficoll (mol. weight approx. 400,000) was a product of Pharmacia, Uppsala, Sweden. Glycerol and diphenylamine (Analar) were obtained from the British Drug Houses, Poole, England. DNAase (electrophoretically purified) was received from the Worthington Biochemical Corporation, Freehold, N. J., U.S.A. [6-14C]Orotic acid (44.5 mC/mM) was from the Radiochemical Centre, Amersham, England. Phenol (Carlo Erba, Milan, Italy) was distilled and stored frozen. Sodium dodecylsulphate was from T. Schuchardt, Munich, Germany; 1,5-naphthalene disulphonate from Eastman Organic Chemicals, Rochester 3, N.Y.; sucrose from Merck, Darmstadt, Germany. All reagents were of the purest grade available. RESULTS
' Neuronal' and'neuroglial'fractionsprepared by the method of Rose Morphological and chemical analysis. As described by Rose for the cerebral cortex of the rat 15, four comparable fractions were obtained in the cortex of the rabbit from the final Ficoll-sucrose gradient (Fig. 1, 1). Examination of fractions B and C by phase-contrast microscopy revealed that fraction C contained a significant amount of neurons and of neuron-like cells which were in part damaged (Fig. 2, C), and a variable proportion of naked nuclei and other unidentified material. The cellular content of fraction B was considerably more heterogeneous, with rare neuron-like bodies and it also contained a larger proportion of cell debris. Determination of carbonic anhydrase content in the four fractions showed the following percentage distribution: A, 14.8~; B, 63.3~; C, 11.4~o; D, 10.5~; thus indicating that most Brain Research, 6 (1967) 228-240
RNA SYNTHESIS 1N NEURONAL FRACTIONS
p
231
~
Fig. 2. Phase-contrast micrographs of neuronal fractions prepared by the method of Satake and Abe (P) and Rose (C).
Brain Research, 6 (1967) 228-240
232
P. VOLPE AND A. GIUDITTA
TABLE I CARBONIC ANHYDRASE ACTIVITY OF FRACTIONS
Experiments
B
AND C~ PREPARED BY THE METHOD OF ROSE
/d C02/min per ttg DNA
1 2 3 4 5 6 Average
B
C
10.8 6.7 10.9 6.9 5.8 15.8 9.5
1.3 1.1 0.6 0.0 1.8 3.0 1.3
of the enzyme was present in fraction B. The carbonic anhydrase activity referred to DNA content was determined in several preparations of fractions B and C and found to be consistently higher (5-10 times) in fraction B (Table I). Since it has been shown that this enzyme is localized in glial cells5, this finding indicated that fraction B was enriched in this cellular type and that fraction C was contaminated with glial material to approximately 15~ in comparison with fraction B. The DNA content and the amount of R N A extracted from fractions B and C was found to vary relatively little in each preparation (Table II). Fraction C contained a quantity of DNA and RNA which was more than twice that of fraction B, but the ratio RNA/DNA was approximately similar in both fractions. Kinetics of RNA labelling. The results of gradient centrifugation of the RNA extracted from fractions B and C at different times after injection of [14C]orotic acid are shown in Fig. 3. From the patterns of extinction at 260 m# and of radioactivity, and from the values of specific activity for total RNA calculated therefrom (Table III), the following conclusions were drawn. After 1 h of labelling, the specific activity of fraction C was significantly lower than that of fraction B, particularly for the species of RNA heavier than 28 S. After 3 h, the specific activities of both fractions increased considerably, but the increase was more marked for fraction C, whose specific activity TABLE II DNA
AND
RNA
CONTENT OF CELL FRACTIONS
The optical densities indicate the amount of R N A extracted from each cell fraction.
Method
Cell fractions
Number of experiments
DNA (#g)
RNA ( O.D. 260 mtQ
RNA/DNA
Rose
B C
7 6
168 ± 28.3 476 dz 52.0
5.500 dz 1.040 12.200 ± 1.230
0.032 0.025
B' P
5 3
547 :& 44.0 283 ~ 26.7
7.000 ± 0.720 3.330 ~ 0.100
0.013 0.012
Satake and Abe
Brain Research, 6 (1967) 228-240
R N A SYNTHESIS IN NEURONAL FRACTIONS
233
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Fig. 3. Sedimentation analysis of radioactive RNA extracted from fracitons B and C prepared by the method of Rose at different times after injection of [14C]orotic acid. Continuous line, optical density; dashed line, radioactivity. To facilitate a comparison, the scales of optical density and of radioactivity have been normalized for the two fractions at each labelling time. The patterns obtained at 1 and 14 h refer to RNA samples prepared without the first extraction in the cold.
b e c a m e m o r e than twice that o f fraction B. This prevalence was m o r e evident for the heaviest types o f R N A ( > 28 S). A similar relationship was found at 6 h after labelling. After longer times (14 h), the specific activities o f both fractions showed a further significant increase, which this time was greater for fraction B. As a consequence o f this change, the specific activity o f the latter fraction b e c a m e larger than that o f fraction C and the ratio of specific activities (C/B) approached the value obtained at 1 b. As shown in Table IIl, similar kinetics of labelling were obtained when the incorporated radioactivities of fractions B and C were compared in terms o f D N A content. The essential parallelism between the radioactivity and the optical density profiles at 14 h indicated that m o s t of the labelled R N A was of the ribosomal and soluble types. At earlier times other types of radioactive R N A were present, particularly in the heavy regions o f the gradient ( > 28 S) and in the 18 S region. While the former types represent a mixture of ribosomal precursors and of D N A - l i k e R N A of u n k n o w n function ts, the prevalence of labelling in the latter region should be atTABLE Ill L A B E L L I N G OF T O T A L
RNA
E X T R A C T E D F R O M F R A C T I O N S B A N D C , P R E P A R E D BY T H E M E T H O D OF R O S E ,
AFTER D I F F E R E N T PERIODS OF [ 1 4 C ] O R O T I C A C I D I N C O R P O R A T I O N
The values obtained at 1 and 3 h represent the average of three experiments; those at 6 and 14 h the average of two experiments, with the exception of the value of counts/rain per pg DNA at 6 h which was derived from a single determination. Labelling Counts/min/ RNA time ( O.D. 260 my)
Counts/min/yg DNA
C/B
(I,)
1 3 6 14
Counts/min
Counts/min
B
C
B
C
RNA
DNA
198 835 870 3850
156 1960 1490 2770
9.7 12.6 18.5 76.0
2.8 18.6 38.2 46.5
0.79 2.35 1.75 0.72
0.30 1.50 2.06 0.61
Brain Research, 6 (1967) 228-240
234
P. VOLPE AND A. GIUDITTA
4,000 2,000 w
28s
4,000
o~ 2 . o 0 0 ~ 0
+°°I '+S
3
6
9
12
Hours
Fig. 4. Kinetics of the labelling of the main types of R N A extracted from fractions C and B (Rose) as resolved by sucrose-density centrifugation. R N A types are operationally defined as the regions of the gradient centered around the S values referred to. Values at 1 and 3 h are an average of three experiments; those at 6 and 14h of two experiments. O, C; O, B.
tributed to the rapid synthesis of 18 S rRNA (ref. 18), although the presence of some degradation products cannot be excluded. The data of Fig. 3 were also used to calculate the specific activities of the main types of RNA, resolved by density gradient centrifugation as a function of labelling time (Fig. 4). A different kinetics of incorporation between the two fractions was noted for the heaviest types of RNA ( ~ 28 S) and to a lesser extent for the species sedimenting in the regions of 28 S, 18 S and 4 S. It should be pointed out that the radioactive RNA sedimenting ahead of 28 S after 14 h labelling, represents, for the most part, the heavy tail of 28 S rRNA and cannot therefore be compared to the RNA types with similar sedimentation values observed at shorter times. The specific activity of the RNA present in the 4 S region of the gradient, approached similar values in both cellular fractions at a time (6 h) when the differences for the other types of RNA were still significant. The increase in specific activity observed at 14 h with alt RNA types, particularly for fraction B, will be commented upon in the discussion.
Nerve cell bodies and heterogeneous fraction prepared by the method of Satake and Abe Morphological and chemical analysis. Centrifugation of the cell suspension prepared from rabbit cortex according to the method of Satake and Abe 16 resulted in the separation of a pellet containing nerve cell bodies from a heterogeneous fraction Brain Research, 6 (1967) 228-240
R N A SYNTHESISIN NEURONALFRACTIONS lh
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Fig. 5. Sedimentation analysis of radioactive RNA extracted from fractions B' and P prepared by the method of Satake and Abe at different times of labelling. Other details as in Fig. 3. (Fig. 1, I[). Examination of the pellet by phase-contrast microscopy revealed that it was chiefly composed of fairly well-preserved nerve cells, showing in several cases the beginning o f a neurite (Fig. 2, P). Fraction B' on the other hand was very heterogeneous and showed hardly any intact cellular form. No carbonic anhydrase activity was detected in fraction P, thus indicating that glial contamination was essentially lacking, while band B' contained 20 ~ of the enzyme present in the homogenate. Relatively little variation was found in the content of D N A and in the amount of R N A extracted from fractions P and B' in the different preparations (Table II). The R N A i DNA ratio was essentially the same for both fractions, although fraction P contained less D N A and R N A than fraction B'. Kinetics o f R N A labelling. R N A from fractions P and B' was extracted and analysed by sucrose density gradient centrifugation (Fig. 5) at the same times after injection of [14C]orotic acid as for the cellular fractions prepared by the method of Rose. The specific activities of total R N A calculated from these data are reported in Table IV. At an early time after labelling (1 h), the specific activity of fraction P was only half that of fraction B', which was noticeably richer in types of R N A heavier TABLE IV RNA EXTRACTED FROM FRACTIONS B ' AND P, PREPARED ABE,AFTER DIFFERENT PERIODS OF [14C]OROTIC ACID INCORPORATION
LABELLING OF TOTAL AND
BY THE METHOD OF SATAKE
The values represent the average of two experiments with the exception of those at 6 h which refer to a single analysis. Labelling Counts/min/RNA time ( O.D. 260 mlt ) (h)
1 3 6 14
Counts/min/Hg D NA
P/B'
B'
P
B'
P
Counts~mix RNA
Counts/min DNA
950 2090 1970 2780
474 4950 6040 2330
8.0 30.0 29.5 58.0
5.1 66.0 63.3 24.8
0.50 2.37 3.07 0.84
0.64 2.20 2.14 0.43
Brain Research, 6 (1967) 228-240
236
P. V O L P E A N D A. ~ I I U D I T I A
10001 ~,xx>
g 2ooo1~ / "~ "
28s
,o 28 S
18s
60001 2000~_ /
6000t
4s
/
3 6 9 12 Hours Fig. 6. Kinetics of labelling of the main types of RNA extracted from fractions P and B' (Satake and Abe) resolved by sucrose-density centrifugation. RNA types are operationally defined as the regions of the gradient centered around the S values referred to. The values represent the average of two experiments with the exception of those at 6 h which refer to a single determination, c3, p; O, B'. than 28 S. The opposite situation prevailed after 3 h of labelling, at which time the specific activity of fraction P had increased much more than that of fraction B', the activity thus becoming more than twice as large as the latter's. Only minor changes were present at 6 h as compared with those at 3 h, but at later times (14 h) a marked decrease occurred in the specific activity of fraction P, while that of fraction B' remained essentially unchanged or showed a slight increase. As a consequence of these modifications the P/B' ratio became less than I. Similar results were obtained when the comparison between the two fractions was made in terms of radioactivity incorporated per #g of D N A (Table IV). The specific activities of the main types of RNA resolved by sedimentation analysis were calculated from the data of Fig. 5 and reported in Fig. 6 as a function of time. The largest increase in specific activity occurrrd for the heaviest types of RNA ( > 28 S) of fraction P between 1 and 3-6 h after labelling, while the analogous RNA species of fraction B' showed little or no change. The specific activity of the latter R N A was, however, considerably larger than that of fraction P, 1 h after the injection of [14C]orotic acid. At later times (14 h), the specific activity of the heavy RNA ( > 28 S) of fraction B' remained essentially unchanged but appeared considerably reduced in fraction P. It has already been mentioned that the heavy RNA labelled after 14 h is not to be compared with the rapidly sedimenting type present at earlier times. Similar modifications were evident in the R N A sedimenting in the 28 S and 18 S regions, while little changes occurred in soluble RNA (4 S).
Brain Research, 6 (1967) 228-240
RNA
SYNTHESIS IN NEURONAL FRACTIONS
237
& B'
2
r
6
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10
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.
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.
}1
14
HOURS
Fig. 7. P/B'. Ratio of specific activities of the different types of RNA present in fractions P and B' prepared by the method of Satake and Abe, as a function of time. C/B. The same with fractions C and B prepared by the method of Rose. [3,28 S region ; O, 18 S region; ±, 4 S region.
A more reliable comparison of the variations between fractions P and B' was afforded by plotting the ratio of the specific activities for each type of R N A as a function of time after labelling (Fig. 7). The numerical value of this ratio is independent of many variables encountered in the individual experiments, including the effective dose of precursor incorporated. Comparable increases in the P/B' ratio between 1 and 3-6 h after labelling were obtained for the R N A types sedimenting at 28 S (from 0.98 to 5.55) and at 18 S (from 0.69 to 3.72). As to the R N A present in the 4 S region, although the initial increase in the ratio was of the same magnitude as that of the other types (from 0.26 at 1 h to 2.55 at 3 h), at 6 h the ratio became close to unity, thus indicating the attainment of similar specific activities in both fractions. Comparable results were obtained when the ratio of specific activities was calculated for the types of R N A extracted from fractions C and B prepared by the method of Rose (Fig. 7). Also, in this case, the ratio for the RNA sedimenting in the 4 S region approached unity at 6 h after labelling, when those of the other types of RNA were still significantly higher. DISCUSSION The results presented in this paper indicate that the R N A o f ' n e u r o n a l ' fractions obtained by two different methods of preparation has different kinetics when labelled in vivo from the R N A of 'neuroglial' fractions. The data show that while early on (after I h), 'neuronal' R N A has a lower specific activity than 'glial' RNA, later (3-6 h), Brain Research, 6 (1967) 228-240
238
P. VOLPE A N D A. ( J l U D I T F A
it becomes substantially more labelled. And still later (14 h), the specific activities of both fractions become almost equal. The biological meaning of these observations is for the most part conditioned by the purity and preservation of the cellular fractions from which the RNAs have been extracted. The morphological appearance of the 'neuronal' and 'glial' fractions C and B has been described by Rose 15 for rat cortex. Similar results have been obtained in these experiments carried out with rabbit brain. The distribution of carbonic anhydrase confirmed the expected composition of the 'neuronal' fraction. The appearance of fractions P and B', obtained with a different procedure, has been described by Satake and Abe for the rat 16. Similar fractions have been obtained with rabbit cortex. Fraction P contains for the most part well-preserved nerve cells with a limited degree of contamination by other structures (Fig. 2). The absence of carbonic anhydrase activity in this fraction provides further indication of its purity. Less certainty exists as to the cellular composition of fraction B of Rose and of the heterogeneous fraction (B') of Satake and Abe. Both fractions, and in particular the latter one, contain fragmented cellular material and free nuclei which are not easily attributed to defined cell types. The lack of a suitable chemical marker for nerve cells prevents the assessment of contamination with neuronal components. However, the presence of a large proportion of the total carbonic anhydrase activity in fraction B indicates an enrichment in glial material. Whatever cellular composition may prevail in these fractions, it is striking that similar labelling kinetics have been obtained in 'neuronal' preparations obtained by two completely different methods. The initial lower rate of labelling of 'neuronal' RNA might depend on a slower labelling of the pool of soluble precursors in these cells, which in turn might be due to several possible factors (pool size, permeability, predetermined pathway of entrance of precursors). Since the specific activity of 'glial' RNA increases by a factor of 2-4 between 1 and 6 h, while that of 'neuronal' RNA increases by a factor greater than 10, one should also assume that the wave of radioactivity of the soluble pool which first reaches the 'glial' comlzartment should also disappear rapidly from this compartment while reaching the 'neuronal' space. Determination of the specific activities of the soluble components of each of these fractions cannot be carried out reliably at present, in view of the unsFecified losses which they would suffer during the preparation. A mere difference in the specific activity of the two pools cannot fully explain the observed differences in the specific activities of the two RNAs since in this case all types of RNA should have been affected. On the other hand, these differences were much less evident for 4 S RNA, the specific activity of which was found to be essentially the same in 'neuronal" and 'gliaF fractions at a time (6 h) when the differences in the specific activities of all other types of RNA were maximal or almost maximal (Fig. 7). Another explanation might be that there is a faster rate of incorporation of radioactive precursors into 'glial" RNA. This possibility is supported by recent findings of a higher RNA turnover in glial cells isolated by micromanipulation from the hypoglossal nucleus of the rabbit 2 and is in accord with the results of autoradiographic studies 11. By itself, however, it would not account for the higher specific activity observed in the 'neuronal' fractions at Brain Research, 6 (1967) 228-240
RNA
239
SYNTHESIS IN NEURONAL FRACTIONS
later times after labelling. A migration of formed material from one type of cell to another might also be considered as a possible partial reason for this effect, but it remains only an attractive hypothesis until supported by direct evidence. The most likely explanation, however, may reside in the recognition that newly synthesized cerebral RNA is mainly a mixture of two families of polyribonucleotides, namely the ribosomal precursors and DNA-like RNA is, of which the former types are labelled at a relatively slow rate, while the latter species are more rapidly degraded 1,tv. Since it is known that neuronal RNA is mainly of the cytoplasmic or ribosomal type, while glial RNA is mainly of the nuclear type 9 and therefore likely to synthesize larger amounts of DNA-like RNA, it would be expected that RNA from 'neuronal' fractions would have a lower specific activity at initial times than RNA from 'neuroglial' fractions, while at later times after labelling the rapid degradation of the DNA-like RNA in comparison with the stability of ribosomal RNA would progressively increase the specific activity of 'neuronal' RNA relative to that of'glial' RNA. Another point of interest is the finding that the specific activity of the RNA extracted from the nerve cell fraction prepared by the method of Satake and Abe decreases substantially between 6 and 14 h, while that of the heterogeneous fraction remains essentially constant. This might reflect the presence of longer-lived pools in the latter fraction, but might also be a sign of migration of neuronal RNA in a cellular compartment not recovered with the nerve cell bodies. This compartment might well be represented by axons and nerve endings known to receive materials manufactured in the perikarya. The substantial increase in specific activity of the RNA extracted from the 'neuroglial' fraction B and to a lesser extent from the 'neuronal' fraction C prepared by the method of Rose during the same period of time (from 6 to 14 h) might perhaps reflect the presence of such particulates in these fractions. SUMMARY
The rate of incorporation of 14C-labelled orotic acid injected into the subarachnoidal space of rabbits has been determined in 'neuronal' and 'glial' fractions obtained by two independent methods of preparation. 'Neuronal' RNA becomes labelled at a slower initial rate than 'glial' RNA, but its specific activity increases to levels 2 3 times higher between 3 and 6 h. The ratio of specific activities of'neuronal' v e r s u s 'glial' RNA decreases again at 14 h, approaching values lower than 1. The significance of these findings is discussed in relation to the relative prevalence of ribosomal and nuclear RNA in neurons and in glial cells respectively. ACKNOWLEDGEMENTS
We would like to thank Dr. J. E. Edstr6m for helpful discussion and Mr. Pagliuca for technical assistance.
Brain Research,
6 (1967) 228-240
240
p. VOLPE ANt) A. (;lttDITTA
REFERENCES 1 ATTARDI,G., PARNAS,H., HWANG, M. I. H., AND ATTARDI, B., Giant-size rapidly labeled nuclear ribonucleic acid and cytoplasmic messenger ribonucleic acid in immature duck erythrocytes, J. tool. Biol., 20 (1966) 145-182. 2 DANEHOLT, B., AND BRATTG/~RD,S.-O., A comparison between R N A metabolism of nerve cells and glia in the hypoglossal nucleus of the rabbit, J. Neurochem., 13 (1966) 913-921. 3 EGYHAZI, E., Microchemical fraction of neuronal and glial RNA, Biochim. biophys. Acta, 114 (1966) 516-526. 4 EGYHAZI,E., AND 1-~YDI~N,H., R N A with high specific activity in neurons and glia, Brain Research, 2 (1966) 197-200. 5 GIACOBINI,E., A cytochemical study of the localization of carbonic anhydrase in the nervous system, J. Neurochem., 9 (1962) 169-177. 6 GILES, K. W., AND MYERS, A., An improved diphenylamine method for the estimation of DNA, Nature (Lond.), 206 (1965) 93. 7 HYD~N, H., Quantitative assay of compounds in isolated fresh nerve cells and glial cells from control and stimulated animals, Nature (Lond.), 184 (1959) 433-435. 8 HYD~N, H., Nuclear R N A changes of nerve cells during a learning experiment in rats, Proc. nat. Acad. Sci. (Wash.), 48 (1962) 1366-1373. 9 HYD/:N, I-[., AND EGYHAZI, E., Glial R N A changes during a learning experiment in rats, Proc. nat. A cad. Sci. (Wash.), 49 (1963) 618-623. l0 HYD~N, H., AND PIGON, A., A cytophysiological study of the functional relationship between oligodendroglial cells and nerve cells of Deiters' nucleus, J. Neurochem., 6 (1959) 57-72. 11 KOENt~, H., An autoradiographic study of nucleic acid and protein turnover in the mammalian neuraxis, J. biophys, biochem. Cytol., 4 (1958) 785-792. 12 KREBS, H. A., AND ROUGHTON,F. J. W., Carbonic anhydrase as a tool in studying the mechanism of reaction involving H2COa, COs or HCOa, Biochem. J., 43 (1948) 550-555. 13 LOWRY, O. H., The quantitative histochemistry of the brain histological sampling, d. Histochem. Cytochem., 1 (1953)420-428. 14 ROOTS, B. L., AND JOHNSTON, P. V., Neurons of ox brain nuclei: their isolation and appearance by light and electron microscopy, J. Ultrastruct. Res., 10 (1964) 350-361. 15 ROSE, S. P. R., Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal cells, Nature (Lond.), 206 (1965) 621-622. 16 SATAKE,M., AND ABE, S., Preparation and characterization of nerve cell perikaryon from rat cerebral cortex, J. Biochem. (Tokyo), 59 (1966) 72-75. 17 SCHERRER,K., MARCAUD,L., ZAJDELA,F., LONDON, 1. M., AND GROS, F., Pattern of R N A metabolism in a differentiated cell: a rapidly labeled, unstable 60 S R N A with messenger properties in duck erythrocytes, Proc. nat. Acad. Sci. (Wash.), 56 (1966) 1571-1578. 18 VESCO, C., AND GIUDIT'FA,A., Pattern of R N A synthesis in rabbit brain, Biochim. biophys. Acta, 142 (1967) 385-402.
Brain Research, 6 (1967) 228-240
BRAIN RI!SEAR(H
BIOSYNTHESIS OF RNA IN NEURON- AND GLIA-ENRICHED FRACTIONS
P. VOLPE AND A. GIUDITTA
International Laboratory of Genetics and Biophysics, Naples (Italy) (Accepted April 7th, 1967)
INTRODUCTION
One of the main problems of neurobiology concerns the contribution of neuronal and neuroglial cells to the overall cerebral metabolism. In several laboratories, these investigations have been pursued with preparations of single cells obtained by hand dissection and with several ingenious microadaptations of analytical techniques2-5,7-10,18A4. Although these efforts have provided information on the metabolism of these cellular types and have stimulated interest in the hypothesis that their functions are complementary, the low yield of cells obtained has prevented the application of biochemical procedures not suitable for microdeterminations. Recently some of these difficulties have been overcome by the availability of methods which allow the bulk separation of neurons and glia cells 15,16. We have used these techniques for the study of the synthesis of RNA in vivo in 'neuronal' and 'neurogtial' fractions isolated from the cerebral cortex of the rabbit, and have obtained evidence for the occurrence of marked differences in the kinetics of RNA labelling. MATERIALS AND METHODS
Preparation and analysis of'neuronal' and 'neuroglial' fractions The procedure originally described by Rose for the cerebral cortex of the rat 15was applied with minor modifications to the cortex of the rabbit (4 g). Fractions B and C recovered from the gradient (Fig. 1, I) were diluted to 10 ml with a solution containing 10 ~ Ficoll, 0.1 M KC1 and 0.01 M potassium phosphate buffer (pH 7.4) and centrifuged at 100,000 × g for 30 min in a Spinco ultracentrifuge (rotor No. 40). The pellets were resuspended in 5.0 ml of medium A (0.32 M sucrose, 0.l mM CaCI2, 0.05 M Tris, pH 7.5). In separate experiments, we adapted to the rabbit cortex the procedure Abbreviations: SDS, sodium dodecyl sulphate; NDS, 1,5-naphthalene disulphonate; Tris, tris (hydroxymethyl) aminomethane; EDTA, ethylenediaminetetraacetic acid. Brain Research, 6 (1967) 228-240
RNA
229
SYNTHESIS IN NEURONAL FRACTIONS
l A SUCROSE 0 5 M FICOLL 70 %-
B, SUCROSE 1.75 M FIC:OLL 20 o/%
,
L
SUCROSE I 35 M
P
I
]I
Fig. 1. I. Method of Rose. Separation of a cell suspension into four layers by centrifugation in a discontinuous Ficoll-sucrose gradient at 22,500 rev./min for 2 h in rotor SW 25 of the Spinco ultracentrifuge. A, floating layer; B, enriched 'glial' fraction; C, enriched 'neuronal' fraction; D, pellet containing erythrocytes and nuclei. II. Method of Satake and Abe. Preparation of nerve cell bodies by centrifugation in a discontinuous sucrose gradient at 14,5013rev./min for 30 min in rotor SW 25 of the Spinco ultracentrifuge. The narrow band (B') contains heterogeneous material ; at the bottom of the tube the pellet containing nerve cell bodies (P). described by Satake and Abe 16 for the preparation of nerve cell bodies from ratbrain. Approximately 4 g o f cortex cut into small pieces was suspended in 50 ml of acetoneglycerol-water (1 : 1 : 1, v/v) for 30 min at 0-4°C. The tissue, obtained by filtration through cheese-cloth, was gently homogenized in 30 ml o f glycerol-0.25 M sucrose (3 : 1, v/v) using a Potter homogenizer with a teflon pestle. The suspension, passed by means of suction t h r o u g h flannel-cloth, was diluted to 100 ml with R i n g e r - L o c k e solution, and treated according to the Satake and Abe procedure 16. The cell fractions were examined by phase-contrast microscopy and stained with Mayer's haemallum. P h o t o g r a p h s were taken with a Leitz O r t h o m a t microscope. Carbonic anhydrase activity was measured manometrically at 10°C from initial rates lz. Reaction velocities were always proportional to the concentration of enzyme. D N A was determined with a diphenylamine method 6.
Extraction and analysis o f R N A [6-14C]Orotic acid (44.5 m C / m M ) was given subarachnoidally to adult rabbits o f approximately 3-4 kg in a dose o f 100 ¢C, according to the procedure previously
Brahz Research, 6 (1967) 228-240
230
P. V O L P E A N [ ) A, (}IUI)II"fA
described 18. The cell fractions were suspended in 5.0 ml of medium A, and 0.5 ml of a 10~ SDS-5~o NDS solution were added, followed by 5.0 ml of a phenol-water mixture (86 : 14) containing 0.07 M Tris buffer (pH 7,4), 0.14~,, SDS and t mg/ml o-hydroxyquinoline. After extraction for 10 min at 4°C in a shaker, the samples were centrifuged in the cold. The phenol phases and the interphases were re-extracted twice according to the above procedure with equal volumes of medium A at 45°C for 5 min. The aqueous supernatants were mixed, re-extracted once at 4 C with an equal volume of phenol, and brought to a concentration of 0.1 M NaCI and 66 %/i ethanol. After standing overnight at --I 5°C, the precipitated RNA was washed by centrifugation with 80 ~o ethanol, dried and dissolved in 2.0 ml of 10 mM Tris buffer (pH 7.4). Residual DNA was digested by incubation for 15 min at 4"C with 100#g DNAase/ml in presence of 2 mM MgClz. At the end of the incubation, 40 ul of 0. l M EDTA (pH 8.0) were added and the solution extracted with phenol at 4'C for 15 min. RNA was re-precipitated as described above and dissolved in 1.0 ml of 10 mM Naacetate (pH 5.0) containing 0.1 M NaCI and 4 M urea. Sedimentation velocity was analysed by centrifugation at 2°-4°C in 5-20~o sucrose gradients containing 0.1 M NaCI and 4 M urea (15 h at 20,000 rev./min in rotor SW25 ofa Spinco ultracentrifuge). Chemicals Ficoll (mol. weight approx. 400,000) was a product of Pharmacia, Uppsala, Sweden. Glycerol and diphenylamine (Analar) were obtained from the British Drug Houses, Poole, England. DNAase (electrophoretically purified) was received from the Worthington Biochemical Corporation, Freehold, N. J., U.S.A. [6-14C]Orotic acid (44.5 mC/mM) was from the Radiochemical Centre, Amersham, England. Phenol (Carlo Erba, Milan, Italy) was distilled and stored frozen. Sodium dodecylsulphate was from T. Schuchardt, Munich, Germany; 1,5-naphthalene disulphonate from Eastman Organic Chemicals, Rochester 3, N.Y.; sucrose from Merck, Darmstadt, Germany. All reagents were of the purest grade available. RESULTS
' Neuronal' and'neuroglial'fractionsprepared by the method of Rose Morphological and chemical analysis. As described by Rose for the cerebral cortex of the rat 15, four comparable fractions were obtained in the cortex of the rabbit from the final Ficoll-sucrose gradient (Fig. 1, 1). Examination of fractions B and C by phase-contrast microscopy revealed that fraction C contained a significant amount of neurons and of neuron-like cells which were in part damaged (Fig. 2, C), and a variable proportion of naked nuclei and other unidentified material. The cellular content of fraction B was considerably more heterogeneous, with rare neuron-like bodies and it also contained a larger proportion of cell debris. Determination of carbonic anhydrase content in the four fractions showed the following percentage distribution: A, 14.8~; B, 63.3~; C, 11.4~o; D, 10.5~; thus indicating that most Brain Research, 6 (1967) 228-240
RNA SYNTHESIS 1N NEURONAL FRACTIONS
p
231
~
Fig. 2. Phase-contrast micrographs of neuronal fractions prepared by the method of Satake and Abe (P) and Rose (C).
Brain Research, 6 (1967) 228-240
232
P. VOLPE AND A. GIUDITTA
TABLE I CARBONIC ANHYDRASE ACTIVITY OF FRACTIONS
Experiments
B
AND C~ PREPARED BY THE METHOD OF ROSE
/d C02/min per ttg DNA
1 2 3 4 5 6 Average
B
C
10.8 6.7 10.9 6.9 5.8 15.8 9.5
1.3 1.1 0.6 0.0 1.8 3.0 1.3
of the enzyme was present in fraction B. The carbonic anhydrase activity referred to DNA content was determined in several preparations of fractions B and C and found to be consistently higher (5-10 times) in fraction B (Table I). Since it has been shown that this enzyme is localized in glial cells5, this finding indicated that fraction B was enriched in this cellular type and that fraction C was contaminated with glial material to approximately 15~ in comparison with fraction B. The DNA content and the amount of R N A extracted from fractions B and C was found to vary relatively little in each preparation (Table II). Fraction C contained a quantity of DNA and RNA which was more than twice that of fraction B, but the ratio RNA/DNA was approximately similar in both fractions. Kinetics of RNA labelling. The results of gradient centrifugation of the RNA extracted from fractions B and C at different times after injection of [14C]orotic acid are shown in Fig. 3. From the patterns of extinction at 260 m# and of radioactivity, and from the values of specific activity for total RNA calculated therefrom (Table III), the following conclusions were drawn. After 1 h of labelling, the specific activity of fraction C was significantly lower than that of fraction B, particularly for the species of RNA heavier than 28 S. After 3 h, the specific activities of both fractions increased considerably, but the increase was more marked for fraction C, whose specific activity TABLE II DNA
AND
RNA
CONTENT OF CELL FRACTIONS
The optical densities indicate the amount of R N A extracted from each cell fraction.
Method
Cell fractions
Number of experiments
DNA (#g)
RNA ( O.D. 260 mtQ
RNA/DNA
Rose
B C
7 6
168 ± 28.3 476 dz 52.0
5.500 dz 1.040 12.200 ± 1.230
0.032 0.025
B' P
5 3
547 :& 44.0 283 ~ 26.7
7.000 ± 0.720 3.330 ~ 0.100
0.013 0.012
Satake and Abe
Brain Research, 6 (1967) 228-240
R N A SYNTHESIS IN NEURONAL FRACTIONS
233
3h
I
t3
:=L1.2
"~ ,...., /_.
I00
E ,~ o.~
f.~.
C
~
06 200
)."W ' j
,]
/~ ,"-.. ,'" /_~"/.-, b ', (
oz
c
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lal 1oo
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,
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J
l'~'" / "
/
FRACTION NUMBER
O.S
12
20
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c
Z' lZO~
,! ~'~,
1,0
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, F~ ,,/~,, ' . ~ ..
~00
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0~
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02
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lt.h
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i".
°'6f
1 ....
!/u/
o'r. [~-.---~-~:~ 4
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Fig. 3. Sedimentation analysis of radioactive RNA extracted from fracitons B and C prepared by the method of Rose at different times after injection of [14C]orotic acid. Continuous line, optical density; dashed line, radioactivity. To facilitate a comparison, the scales of optical density and of radioactivity have been normalized for the two fractions at each labelling time. The patterns obtained at 1 and 14 h refer to RNA samples prepared without the first extraction in the cold.
b e c a m e m o r e than twice that o f fraction B. This prevalence was m o r e evident for the heaviest types o f R N A ( > 28 S). A similar relationship was found at 6 h after labelling. After longer times (14 h), the specific activities o f both fractions showed a further significant increase, which this time was greater for fraction B. As a consequence o f this change, the specific activity o f the latter fraction b e c a m e larger than that o f fraction C and the ratio of specific activities (C/B) approached the value obtained at 1 b. As shown in Table IIl, similar kinetics of labelling were obtained when the incorporated radioactivities of fractions B and C were compared in terms o f D N A content. The essential parallelism between the radioactivity and the optical density profiles at 14 h indicated that m o s t of the labelled R N A was of the ribosomal and soluble types. At earlier times other types of radioactive R N A were present, particularly in the heavy regions o f the gradient ( > 28 S) and in the 18 S region. While the former types represent a mixture of ribosomal precursors and of D N A - l i k e R N A of u n k n o w n function ts, the prevalence of labelling in the latter region should be atTABLE Ill L A B E L L I N G OF T O T A L
RNA
E X T R A C T E D F R O M F R A C T I O N S B A N D C , P R E P A R E D BY T H E M E T H O D OF R O S E ,
AFTER D I F F E R E N T PERIODS OF [ 1 4 C ] O R O T I C A C I D I N C O R P O R A T I O N
The values obtained at 1 and 3 h represent the average of three experiments; those at 6 and 14 h the average of two experiments, with the exception of the value of counts/rain per pg DNA at 6 h which was derived from a single determination. Labelling Counts/min/ RNA time ( O.D. 260 my)
Counts/min/yg DNA
C/B
(I,)
1 3 6 14
Counts/min
Counts/min
B
C
B
C
RNA
DNA
198 835 870 3850
156 1960 1490 2770
9.7 12.6 18.5 76.0
2.8 18.6 38.2 46.5
0.79 2.35 1.75 0.72
0.30 1.50 2.06 0.61
Brain Research, 6 (1967) 228-240
234
P. VOLPE AND A. GIUDITTA
4,000 2,000 w
28s
4,000
o~ 2 . o 0 0 ~ 0
+°°I '+S
3
6
9
12
Hours
Fig. 4. Kinetics of the labelling of the main types of R N A extracted from fractions C and B (Rose) as resolved by sucrose-density centrifugation. R N A types are operationally defined as the regions of the gradient centered around the S values referred to. Values at 1 and 3 h are an average of three experiments; those at 6 and 14h of two experiments. O, C; O, B.
tributed to the rapid synthesis of 18 S rRNA (ref. 18), although the presence of some degradation products cannot be excluded. The data of Fig. 3 were also used to calculate the specific activities of the main types of RNA, resolved by density gradient centrifugation as a function of labelling time (Fig. 4). A different kinetics of incorporation between the two fractions was noted for the heaviest types of RNA ( ~ 28 S) and to a lesser extent for the species sedimenting in the regions of 28 S, 18 S and 4 S. It should be pointed out that the radioactive RNA sedimenting ahead of 28 S after 14 h labelling, represents, for the most part, the heavy tail of 28 S rRNA and cannot therefore be compared to the RNA types with similar sedimentation values observed at shorter times. The specific activity of the RNA present in the 4 S region of the gradient, approached similar values in both cellular fractions at a time (6 h) when the differences for the other types of RNA were still significant. The increase in specific activity observed at 14 h with alt RNA types, particularly for fraction B, will be commented upon in the discussion.
Nerve cell bodies and heterogeneous fraction prepared by the method of Satake and Abe Morphological and chemical analysis. Centrifugation of the cell suspension prepared from rabbit cortex according to the method of Satake and Abe 16 resulted in the separation of a pellet containing nerve cell bodies from a heterogeneous fraction Brain Research, 6 (1967) 228-240
R N A SYNTHESISIN NEURONALFRACTIONS lh
3h
6h
f,a':: '°
0 5~
I
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as .
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.
.
.
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.
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200
~
LOOO ~o
01
28
~2
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Lsoo
02
500
~
12
20
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FRACTiON NUMBER
Fig. 5. Sedimentation analysis of radioactive RNA extracted from fractions B' and P prepared by the method of Satake and Abe at different times of labelling. Other details as in Fig. 3. (Fig. 1, I[). Examination of the pellet by phase-contrast microscopy revealed that it was chiefly composed of fairly well-preserved nerve cells, showing in several cases the beginning o f a neurite (Fig. 2, P). Fraction B' on the other hand was very heterogeneous and showed hardly any intact cellular form. No carbonic anhydrase activity was detected in fraction P, thus indicating that glial contamination was essentially lacking, while band B' contained 20 ~ of the enzyme present in the homogenate. Relatively little variation was found in the content of D N A and in the amount of R N A extracted from fractions P and B' in the different preparations (Table II). The R N A i DNA ratio was essentially the same for both fractions, although fraction P contained less D N A and R N A than fraction B'. Kinetics o f R N A labelling. R N A from fractions P and B' was extracted and analysed by sucrose density gradient centrifugation (Fig. 5) at the same times after injection of [14C]orotic acid as for the cellular fractions prepared by the method of Rose. The specific activities of total R N A calculated from these data are reported in Table IV. At an early time after labelling (1 h), the specific activity of fraction P was only half that of fraction B', which was noticeably richer in types of R N A heavier TABLE IV RNA EXTRACTED FROM FRACTIONS B ' AND P, PREPARED ABE,AFTER DIFFERENT PERIODS OF [14C]OROTIC ACID INCORPORATION
LABELLING OF TOTAL AND
BY THE METHOD OF SATAKE
The values represent the average of two experiments with the exception of those at 6 h which refer to a single analysis. Labelling Counts/min/RNA time ( O.D. 260 mlt ) (h)
1 3 6 14
Counts/min/Hg D NA
P/B'
B'
P
B'
P
Counts~mix RNA
Counts/min DNA
950 2090 1970 2780
474 4950 6040 2330
8.0 30.0 29.5 58.0
5.1 66.0 63.3 24.8
0.50 2.37 3.07 0.84
0.64 2.20 2.14 0.43
Brain Research, 6 (1967) 228-240
236
P. V O L P E A N D A. ~ I I U D I T I A
10001 ~,xx>
g 2ooo1~ / "~ "
28s
,o 28 S
18s
60001 2000~_ /
6000t
4s
/
3 6 9 12 Hours Fig. 6. Kinetics of labelling of the main types of RNA extracted from fractions P and B' (Satake and Abe) resolved by sucrose-density centrifugation. RNA types are operationally defined as the regions of the gradient centered around the S values referred to. The values represent the average of two experiments with the exception of those at 6 h which refer to a single determination, c3, p; O, B'. than 28 S. The opposite situation prevailed after 3 h of labelling, at which time the specific activity of fraction P had increased much more than that of fraction B', the activity thus becoming more than twice as large as the latter's. Only minor changes were present at 6 h as compared with those at 3 h, but at later times (14 h) a marked decrease occurred in the specific activity of fraction P, while that of fraction B' remained essentially unchanged or showed a slight increase. As a consequence of these modifications the P/B' ratio became less than I. Similar results were obtained when the comparison between the two fractions was made in terms of radioactivity incorporated per #g of D N A (Table IV). The specific activities of the main types of RNA resolved by sedimentation analysis were calculated from the data of Fig. 5 and reported in Fig. 6 as a function of time. The largest increase in specific activity occurrrd for the heaviest types of RNA ( > 28 S) of fraction P between 1 and 3-6 h after labelling, while the analogous RNA species of fraction B' showed little or no change. The specific activity of the latter R N A was, however, considerably larger than that of fraction P, 1 h after the injection of [14C]orotic acid. At later times (14 h), the specific activity of the heavy RNA ( > 28 S) of fraction B' remained essentially unchanged but appeared considerably reduced in fraction P. It has already been mentioned that the heavy RNA labelled after 14 h is not to be compared with the rapidly sedimenting type present at earlier times. Similar modifications were evident in the R N A sedimenting in the 28 S and 18 S regions, while little changes occurred in soluble RNA (4 S).
Brain Research, 6 (1967) 228-240
RNA
SYNTHESIS IN NEURONAL FRACTIONS
237
& B'
2
r
6
.
10
.
.
.
.
}1
14
HOURS
Fig. 7. P/B'. Ratio of specific activities of the different types of RNA present in fractions P and B' prepared by the method of Satake and Abe, as a function of time. C/B. The same with fractions C and B prepared by the method of Rose. [3,28 S region ; O, 18 S region; ±, 4 S region.
A more reliable comparison of the variations between fractions P and B' was afforded by plotting the ratio of the specific activities for each type of R N A as a function of time after labelling (Fig. 7). The numerical value of this ratio is independent of many variables encountered in the individual experiments, including the effective dose of precursor incorporated. Comparable increases in the P/B' ratio between 1 and 3-6 h after labelling were obtained for the R N A types sedimenting at 28 S (from 0.98 to 5.55) and at 18 S (from 0.69 to 3.72). As to the R N A present in the 4 S region, although the initial increase in the ratio was of the same magnitude as that of the other types (from 0.26 at 1 h to 2.55 at 3 h), at 6 h the ratio became close to unity, thus indicating the attainment of similar specific activities in both fractions. Comparable results were obtained when the ratio of specific activities was calculated for the types of R N A extracted from fractions C and B prepared by the method of Rose (Fig. 7). Also, in this case, the ratio for the RNA sedimenting in the 4 S region approached unity at 6 h after labelling, when those of the other types of RNA were still significantly higher. DISCUSSION The results presented in this paper indicate that the R N A o f ' n e u r o n a l ' fractions obtained by two different methods of preparation has different kinetics when labelled in vivo from the R N A of 'neuroglial' fractions. The data show that while early on (after I h), 'neuronal' R N A has a lower specific activity than 'glial' RNA, later (3-6 h), Brain Research, 6 (1967) 228-240
238
P. VOLPE A N D A. ( J l U D I T F A
it becomes substantially more labelled. And still later (14 h), the specific activities of both fractions become almost equal. The biological meaning of these observations is for the most part conditioned by the purity and preservation of the cellular fractions from which the RNAs have been extracted. The morphological appearance of the 'neuronal' and 'glial' fractions C and B has been described by Rose 15 for rat cortex. Similar results have been obtained in these experiments carried out with rabbit brain. The distribution of carbonic anhydrase confirmed the expected composition of the 'neuronal' fraction. The appearance of fractions P and B', obtained with a different procedure, has been described by Satake and Abe for the rat 16. Similar fractions have been obtained with rabbit cortex. Fraction P contains for the most part well-preserved nerve cells with a limited degree of contamination by other structures (Fig. 2). The absence of carbonic anhydrase activity in this fraction provides further indication of its purity. Less certainty exists as to the cellular composition of fraction B of Rose and of the heterogeneous fraction (B') of Satake and Abe. Both fractions, and in particular the latter one, contain fragmented cellular material and free nuclei which are not easily attributed to defined cell types. The lack of a suitable chemical marker for nerve cells prevents the assessment of contamination with neuronal components. However, the presence of a large proportion of the total carbonic anhydrase activity in fraction B indicates an enrichment in glial material. Whatever cellular composition may prevail in these fractions, it is striking that similar labelling kinetics have been obtained in 'neuronal' preparations obtained by two completely different methods. The initial lower rate of labelling of 'neuronal' RNA might depend on a slower labelling of the pool of soluble precursors in these cells, which in turn might be due to several possible factors (pool size, permeability, predetermined pathway of entrance of precursors). Since the specific activity of 'glial' RNA increases by a factor of 2-4 between 1 and 6 h, while that of 'neuronal' RNA increases by a factor greater than 10, one should also assume that the wave of radioactivity of the soluble pool which first reaches the 'glial' comlzartment should also disappear rapidly from this compartment while reaching the 'neuronal' space. Determination of the specific activities of the soluble components of each of these fractions cannot be carried out reliably at present, in view of the unsFecified losses which they would suffer during the preparation. A mere difference in the specific activity of the two pools cannot fully explain the observed differences in the specific activities of the two RNAs since in this case all types of RNA should have been affected. On the other hand, these differences were much less evident for 4 S RNA, the specific activity of which was found to be essentially the same in 'neuronal" and 'gliaF fractions at a time (6 h) when the differences in the specific activities of all other types of RNA were maximal or almost maximal (Fig. 7). Another explanation might be that there is a faster rate of incorporation of radioactive precursors into 'glial" RNA. This possibility is supported by recent findings of a higher RNA turnover in glial cells isolated by micromanipulation from the hypoglossal nucleus of the rabbit 2 and is in accord with the results of autoradiographic studies 11. By itself, however, it would not account for the higher specific activity observed in the 'neuronal' fractions at Brain Research, 6 (1967) 228-240
RNA
239
SYNTHESIS IN NEURONAL FRACTIONS
later times after labelling. A migration of formed material from one type of cell to another might also be considered as a possible partial reason for this effect, but it remains only an attractive hypothesis until supported by direct evidence. The most likely explanation, however, may reside in the recognition that newly synthesized cerebral RNA is mainly a mixture of two families of polyribonucleotides, namely the ribosomal precursors and DNA-like RNA is, of which the former types are labelled at a relatively slow rate, while the latter species are more rapidly degraded 1,tv. Since it is known that neuronal RNA is mainly of the cytoplasmic or ribosomal type, while glial RNA is mainly of the nuclear type 9 and therefore likely to synthesize larger amounts of DNA-like RNA, it would be expected that RNA from 'neuronal' fractions would have a lower specific activity at initial times than RNA from 'neuroglial' fractions, while at later times after labelling the rapid degradation of the DNA-like RNA in comparison with the stability of ribosomal RNA would progressively increase the specific activity of 'neuronal' RNA relative to that of'glial' RNA. Another point of interest is the finding that the specific activity of the RNA extracted from the nerve cell fraction prepared by the method of Satake and Abe decreases substantially between 6 and 14 h, while that of the heterogeneous fraction remains essentially constant. This might reflect the presence of longer-lived pools in the latter fraction, but might also be a sign of migration of neuronal RNA in a cellular compartment not recovered with the nerve cell bodies. This compartment might well be represented by axons and nerve endings known to receive materials manufactured in the perikarya. The substantial increase in specific activity of the RNA extracted from the 'neuroglial' fraction B and to a lesser extent from the 'neuronal' fraction C prepared by the method of Rose during the same period of time (from 6 to 14 h) might perhaps reflect the presence of such particulates in these fractions. SUMMARY
The rate of incorporation of 14C-labelled orotic acid injected into the subarachnoidal space of rabbits has been determined in 'neuronal' and 'glial' fractions obtained by two independent methods of preparation. 'Neuronal' RNA becomes labelled at a slower initial rate than 'glial' RNA, but its specific activity increases to levels 2 3 times higher between 3 and 6 h. The ratio of specific activities of'neuronal' v e r s u s 'glial' RNA decreases again at 14 h, approaching values lower than 1. The significance of these findings is discussed in relation to the relative prevalence of ribosomal and nuclear RNA in neurons and in glial cells respectively. ACKNOWLEDGEMENTS
We would like to thank Dr. J. E. Edstr6m for helpful discussion and Mr. Pagliuca for technical assistance.
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Brain Research, 6 (1967) 228-240