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The distribution of RNA in the cerebellum of the rabbit
Experimental
Cell Research 28, 501406
501
(1962)
THE DISTRIBUTION
OF RNA IN THE CEREBELLUM OF THE RABBIT J. JARLSTEDT
Institute
of Neurobiology,
Medical
Faculty,
Received February
University
of Gothenburg, Sweden
1, 1962
SEVERAL
investigators have demonstrated the occurrence of two or more populations of Purkinje cells in the cerebellar cortex based upon different chromophilic or ultraviolet-absorbing properties [7, 13, 16, 171. The Purkinje cells are usually seen as a mixture of strongly and lightly absorbing cells. The several hypotheses that have been advanced suggest that these variations reflect cells of various functions or cells during various functional states [l, 2, 71. However, the possibility that these differences in absorbance or basophilia may be artefactual and the consequence of a varying degree of cell shrinkage has certainly not been excluded. Indeed, it is possible that the substance responsible for basophilic staining and absorption properties in ultraviolet light, ribonucleic acid (RNA), is present in similar amounts in all cells. That this may be the case gains some support from Cammermeyer’s recent observations [3] that a certain fraction of Purkinje cells collapses during the preparation of cerebellar tissue for histological investigation as a result of the handling of the tissue. It is evident, therefore, that investigations of RNA changes based upon the information of RNA concentration only, may give erroneous information if shrinkage occurs to varying degrees. In order to exclude the variable of shrinkage, the RNA content per cell must be determined. The present communication reports an investigation of the cerebellar cortical layers with regard to RNA content and concentration, in which ultramicrochemical methods were used. The results obtained are at variance with those in which cytophotometrical methods were employed. They also provide information about RNA in other strata of the cerebeIIar cortex as well as underlying white matter. MATERIAL
AND
METHODS
Rabbits weighing between 1.5-2.2 kg were used. The animals were killed by air emboli and pieces of cerebellum were removed immediately after death and fixed in Carnoy’s solution (i.e. absolute ethanol, chloroform, concentrated acetic acid, 6/3/l by volume) for 90 min. The pieces of tissue were embedded in paraffin and cut Experimental
Cell Research 28
502
J. Jarlstedt
at IO-40 ,LL.RNA was determined in units isolated by micromanipulation and extracted with ribonuclease in an oil chamber, according to EdstrGm [4.6]. The extracts collected were evaporated to dryness on a quartz glass and redissolved in glycerolcontaining buffer, forming lens-shaped drops. The drops were photographed in ultraviolet light at a wavelength of 257 m,& together with a reference system. The amounts of RNA in the ultraviolet-absorbing spots were determined by a photometric procedure [5]. RESULTS
Preliminary investigations were carried out to ascertain whether RNA can be estimated quantitatively in the cerebellar cortex. In view of reports in the literature (see discussion) that cerebellar RNA is particularly unstable after death, an experiment was carried out in which tissue was taken at different time intervals after death. It was found that the tissue could be kept at room temperature for up to 24 hr before fixation without any effect on the RNA values of the Purkinje cells (see Table I). Thus, for the purposes of the procedure used, RNA was sufficiently stable. An investigation was carried out also in order to see whether the fixation process influenced the RNA content of the Purkinje cells. It has been claimed by some authors (see discussion) that Carnoy’s fixative exerts a strong hydrolytic action on RNA and dissolves it already after a few hours’ exposure. Pieces of tissue were fixed for varying times up to 72 hr, and the RNA contents of isolated Purkinje cells were determined (see Table II). The results of this study indicated that the RN,4 content did not decrease as a result of the preparative procedure. In addition, a microchemical control experiment was performed in which pieces of tissue from the cerebellum were analyzed for RNA content. Absorption curves of the extracts were recorded in ultraviolet. They were found to correspond well to the absorption curve recorded for purified yeast RNA. TABLE I. Comparison between the RNA contents of Purkinje cells from pieces of the vermis fixed after varying time periods. Rabbit
weight:
Interval between death and fixation 5 min 2 hr 24 hr Experimental
Cell Research 28
1.7 kg.
Time of fixation:
90 min.
Mean value in wg
n
119+6 lOS+S 109*5
16 11 21
RNA distribution Pieces of tissue taken were analyzed for RNA RNA is highest in the granular layer is lower
503
in rat cerebellum
from different layers of the cerebellar hemispheres content. As shown by Table III, the concentration of Purkinje cells, viz., approximately 2 per cent. The in RNA concentration, viz., half to one-third of the
II. A comparison of RNA values determined in Purkinje cells obtained from pieces of the vermis fixed for varying time periods.
TABLE
Rabbit weight: 1.8 kg. Time of fixation hr
Mean value in wk7
R
15oi7 150*11 145k8 146+6 14456 1381-8
12 12 12 12 12 12
4 14 5 24 72 1 $a
a Tissue pieces were frozen immediately and stored cold for 30 min, placed afterwards in room temperature for 5 min and then fixed in Carnoy’s solution for 90 min. TABLE
III. RNA concentration (per cent, w/v) measured in cerebellar white matter and different layers of the cerebellar cortex. Rabbit weight: 1.8 kg.
Fixation time: 90 min.
Material
Mean value in per cent
n
White matter Granular stratum Molecular stratum Purkinje cells
0.42 + 0.05 0.87 k 0.06 0.28 k 0.03 2.16 +0.14
6 9 11 11
Purkinje cells. It is even lower in the white matter, and lowest in the molecular stratum, where the concentration of RNA was found to be one-tenth of that in the Purkinje cells. It should be kept in mind, however, that the concentration data given in Table III represent quantities of RNA per fixed volume. Considerable shrinkage takes place during the preparative procedure (of the order of 50 per cent of the initial volume) and the values per volume of fresh tissue should consequently be roughly half of those given here. The main value of the concentration data is that they enable comparisons to be made within the material. Experimental
Cell Research 28
J. Jarlstedt
504
The remaining analyses were carried out on Purkinje cells. A comparison was made between cells dissected out from the hemispheres and from the vermis. Table IV shows that there is no difference in RNA content of Purkinje cells from these two regions. TABLE IV. A comparison between RNA values determined in Purkinje cells from pieces of the vermis and the hemispheres. Rabbit weight: 1.8 kg.
Fixation time: 90 min.
Area
Mean value in @pg
n
Hemispheres Vermis
143&G 141*7
24 23
TABLE V. A comparison of RNA values determined in chromophilic and chromophobic Purkinje cells. Cells obtained from pieces of the hemispheres. Rabbit weight: 1.9 kg.
Fixation time: 90 min.
Type of cells
Mean value in p,ug
n
Chromophilic Chromophobic
143110 141+11
12 12
Of interest is the relative RNA content of chromophilic and chromophobic staining Purkinje cells. These two cell types can be distinguished by their peculiar morphology [3], viz., the darkly staining cells are angular and have a shrunken appearance, while the lightly staining cells are rounded and with even outlines. Consequently, it is possible to select representative samples of each cell type in unstained sections. Table V shows that the amount of RNA is the same in the two groups of cells. When all values from the same rabbit are collected to give a distribu-
Fig. l.--Distribution curve. All values collected from a single rabbit. Rabbit weight 1.8 kg.
Experimental
Cell Research 28
RNA distribution
in rat cerebellum
505
tion curve, it is seen (Fig. 1) that this curve has a unimodal distribution. This indicates that the cells belong to a single population with regard to their RNA contents. DISCUSSION
May and Grenell [ 10, 151 noted an extraordinary lability in the RNA of the cerebellum. They found a marked reduction in the content of total nucleic acids, amounting to 37 per cent when the tissue was dissected without being kept on a cold block. Such a relative decrease represents a value exceeding that of the RNA content itself. This indicates that DNA very likely accounts partially for the decrease. Moreover, the same authors state that RNA was reduced by 47 per cent in the cerebellum as a result of storage in the deepfreeze. This marked lability of RNA does not seem likely, as it is well known that the cells in the central nervous system, the cerebellum included, are relatively resistant to autolysis, and that the Purkinje cells retain their basophilic staining properties for a long time after death. The present study lends credence to the correctness of these general observations and contradicts the findings of May and Grenell [lo, 151. In view of the earlier reports on the dissolving effect of Carnoy’s solution on RNA [12, 141, the present findings, showing an unaltered RNA content even after 72 hr of fixation, are worth noting. They are, however, in good agreement with the experience of recent studies on this question, but using other material [5, 61. Consequently, from the preparative point of view, the Purkinje cells show certain advantages, since RNA losses resulting from tissue preparation do not take place. Gaitonde and Richter [8, 91 measured the incorporation of 3%-methionine in different layers of the cerebellum. The correlation between their data and the present results is relatively good. They concluded that the incorporation is particularly prominent in layers exhibiting a high content of cell bodies. However, one discrepancy between their observations and the present findings may be noted. In this study, more RNA was found in the white matter than in the molecular layer, while Gaitonde and Richter found a greater incorporation in the latter layer. The volume of the molecular layer is, to a large extent, composed of dendrites and dendritric ramifications from the Purkinje cells [ll]. Thus it is evident that these dendrites have a relatively low RNA concentration. On the other hand, it is well known that the dendrites are very similar in their proximal parts to perikarya with regard to basophilia and ultraviolet absorption, a Experimental
Cell Research 28
J. Jarlstedt fact which indicates that there may be a proximo-distal gradient in the RNA concentration of the dendrites. As Table V shows, there is no significant difference betwenn darkly and lightly staining Purkinje cells with respect to the RNA content. This is in good agreement with Cammermeyer’s investigation [3], which demonstrated that the chromophilia of some neurons might be due to shrinkage resulting from post-mortem trauma of the cerebellar tissue. Furthermore, the distribution curve for all RNA values obtained from the Purkinje cells of one of the rabbits investigated gives no evidence for two or more cell groups. SUMMARY
A microchemical investigation of the RNA content in different layers of the cerebellar cortical layers as well as that of the underlying white matter has been carried out. The results indicate considerable differences in RNA concentration of the different layers according to the following order of decreasing concentrations: Purkinje cells, granular layer, white matter and molecular layer. The data give no evidence for the existence of two or more populations of Purkinje cells, with regard to RNA content. Neurons from different parts of the cerebellar cortex have identical RNA contents on a per cent/volume basis. I am indebted to Dot. J.-E. Edstrijm for help and suggestions and to Dr. E. Koenig for revising the English text. The work was supported by a grant from the Medical Faculty, University of Gothenburg, Sweden. REFERENCES 1. ATTARDI, G., Expptl. Cell Research Suppl. 4, 25 (1957). 2. BRATTGARD, S.-O. and HYD&N, H., Acta Radiol. 94 (1952). 3. CAMMERMEYER, J., Exptl. Neurol. 2, 379 (1960). 4. EDSTR~M, J.-E., J. Neurochem. 3, 1 (1958).
5. ~ Biochim. et Biophys. Acta 12, 361 (1953). 6. ~ Acta Histochem. Suppl. II, 27 (1959). 7. EINARSSON, L., Am. J. Anat. 53, 141 (1933). 8. GAITONDE, M. K. and RICHTER, D., J. Physiol. 126, 7 D (1954). 9. __ Metabolism of the Nervous System, pp. 449-455, 1957. 10. GRENELL, R. G. and MAY, L., Neurology 8, Suppl. 1 (1958). 11. Handbucb der mikroskopischen Anatomie des Menschen IV/8. Nervensystem. Berlin 1958. 12. HARBERS, E. and NEUMANN, K., Z. Naturforsch. lob, 357 (1955). 13. HYDEN, H., Acta Physiol. &and. 6, Suppl. XVII (1943). 14. LAGERSTEDT, S., Experientia 12, 425 (1956). 15. MAY, L. and GRENELL, R., Proc. Sot. Exptl. Biol. Med. 102, 235 (1958). 16. SAGUCHI, S., Zytologische Studien H 4, 1930. 17. SCH~~MMELFEDER, N., KROGII, R. E. and EBSCHNER, K. J., Histochemie 1, 1 (1958).
Experimental
Cell Research 28
Springer,
Cell Research 28, 501406
501
(1962)
THE DISTRIBUTION
OF RNA IN THE CEREBELLUM OF THE RABBIT J. JARLSTEDT
Institute
of Neurobiology,
Medical
Faculty,
Received February
University
of Gothenburg, Sweden
1, 1962
SEVERAL
investigators have demonstrated the occurrence of two or more populations of Purkinje cells in the cerebellar cortex based upon different chromophilic or ultraviolet-absorbing properties [7, 13, 16, 171. The Purkinje cells are usually seen as a mixture of strongly and lightly absorbing cells. The several hypotheses that have been advanced suggest that these variations reflect cells of various functions or cells during various functional states [l, 2, 71. However, the possibility that these differences in absorbance or basophilia may be artefactual and the consequence of a varying degree of cell shrinkage has certainly not been excluded. Indeed, it is possible that the substance responsible for basophilic staining and absorption properties in ultraviolet light, ribonucleic acid (RNA), is present in similar amounts in all cells. That this may be the case gains some support from Cammermeyer’s recent observations [3] that a certain fraction of Purkinje cells collapses during the preparation of cerebellar tissue for histological investigation as a result of the handling of the tissue. It is evident, therefore, that investigations of RNA changes based upon the information of RNA concentration only, may give erroneous information if shrinkage occurs to varying degrees. In order to exclude the variable of shrinkage, the RNA content per cell must be determined. The present communication reports an investigation of the cerebellar cortical layers with regard to RNA content and concentration, in which ultramicrochemical methods were used. The results obtained are at variance with those in which cytophotometrical methods were employed. They also provide information about RNA in other strata of the cerebeIIar cortex as well as underlying white matter. MATERIAL
AND
METHODS
Rabbits weighing between 1.5-2.2 kg were used. The animals were killed by air emboli and pieces of cerebellum were removed immediately after death and fixed in Carnoy’s solution (i.e. absolute ethanol, chloroform, concentrated acetic acid, 6/3/l by volume) for 90 min. The pieces of tissue were embedded in paraffin and cut Experimental
Cell Research 28
502
J. Jarlstedt
at IO-40 ,LL.RNA was determined in units isolated by micromanipulation and extracted with ribonuclease in an oil chamber, according to EdstrGm [4.6]. The extracts collected were evaporated to dryness on a quartz glass and redissolved in glycerolcontaining buffer, forming lens-shaped drops. The drops were photographed in ultraviolet light at a wavelength of 257 m,& together with a reference system. The amounts of RNA in the ultraviolet-absorbing spots were determined by a photometric procedure [5]. RESULTS
Preliminary investigations were carried out to ascertain whether RNA can be estimated quantitatively in the cerebellar cortex. In view of reports in the literature (see discussion) that cerebellar RNA is particularly unstable after death, an experiment was carried out in which tissue was taken at different time intervals after death. It was found that the tissue could be kept at room temperature for up to 24 hr before fixation without any effect on the RNA values of the Purkinje cells (see Table I). Thus, for the purposes of the procedure used, RNA was sufficiently stable. An investigation was carried out also in order to see whether the fixation process influenced the RNA content of the Purkinje cells. It has been claimed by some authors (see discussion) that Carnoy’s fixative exerts a strong hydrolytic action on RNA and dissolves it already after a few hours’ exposure. Pieces of tissue were fixed for varying times up to 72 hr, and the RNA contents of isolated Purkinje cells were determined (see Table II). The results of this study indicated that the RN,4 content did not decrease as a result of the preparative procedure. In addition, a microchemical control experiment was performed in which pieces of tissue from the cerebellum were analyzed for RNA content. Absorption curves of the extracts were recorded in ultraviolet. They were found to correspond well to the absorption curve recorded for purified yeast RNA. TABLE I. Comparison between the RNA contents of Purkinje cells from pieces of the vermis fixed after varying time periods. Rabbit
weight:
Interval between death and fixation 5 min 2 hr 24 hr Experimental
Cell Research 28
1.7 kg.
Time of fixation:
90 min.
Mean value in wg
n
119+6 lOS+S 109*5
16 11 21
RNA distribution Pieces of tissue taken were analyzed for RNA RNA is highest in the granular layer is lower
503
in rat cerebellum
from different layers of the cerebellar hemispheres content. As shown by Table III, the concentration of Purkinje cells, viz., approximately 2 per cent. The in RNA concentration, viz., half to one-third of the
II. A comparison of RNA values determined in Purkinje cells obtained from pieces of the vermis fixed for varying time periods.
TABLE
Rabbit weight: 1.8 kg. Time of fixation hr
Mean value in wk7
R
15oi7 150*11 145k8 146+6 14456 1381-8
12 12 12 12 12 12
4 14 5 24 72 1 $a
a Tissue pieces were frozen immediately and stored cold for 30 min, placed afterwards in room temperature for 5 min and then fixed in Carnoy’s solution for 90 min. TABLE
III. RNA concentration (per cent, w/v) measured in cerebellar white matter and different layers of the cerebellar cortex. Rabbit weight: 1.8 kg.
Fixation time: 90 min.
Material
Mean value in per cent
n
White matter Granular stratum Molecular stratum Purkinje cells
0.42 + 0.05 0.87 k 0.06 0.28 k 0.03 2.16 +0.14
6 9 11 11
Purkinje cells. It is even lower in the white matter, and lowest in the molecular stratum, where the concentration of RNA was found to be one-tenth of that in the Purkinje cells. It should be kept in mind, however, that the concentration data given in Table III represent quantities of RNA per fixed volume. Considerable shrinkage takes place during the preparative procedure (of the order of 50 per cent of the initial volume) and the values per volume of fresh tissue should consequently be roughly half of those given here. The main value of the concentration data is that they enable comparisons to be made within the material. Experimental
Cell Research 28
J. Jarlstedt
504
The remaining analyses were carried out on Purkinje cells. A comparison was made between cells dissected out from the hemispheres and from the vermis. Table IV shows that there is no difference in RNA content of Purkinje cells from these two regions. TABLE IV. A comparison between RNA values determined in Purkinje cells from pieces of the vermis and the hemispheres. Rabbit weight: 1.8 kg.
Fixation time: 90 min.
Area
Mean value in @pg
n
Hemispheres Vermis
143&G 141*7
24 23
TABLE V. A comparison of RNA values determined in chromophilic and chromophobic Purkinje cells. Cells obtained from pieces of the hemispheres. Rabbit weight: 1.9 kg.
Fixation time: 90 min.
Type of cells
Mean value in p,ug
n
Chromophilic Chromophobic
143110 141+11
12 12
Of interest is the relative RNA content of chromophilic and chromophobic staining Purkinje cells. These two cell types can be distinguished by their peculiar morphology [3], viz., the darkly staining cells are angular and have a shrunken appearance, while the lightly staining cells are rounded and with even outlines. Consequently, it is possible to select representative samples of each cell type in unstained sections. Table V shows that the amount of RNA is the same in the two groups of cells. When all values from the same rabbit are collected to give a distribu-
Fig. l.--Distribution curve. All values collected from a single rabbit. Rabbit weight 1.8 kg.
Experimental
Cell Research 28
RNA distribution
in rat cerebellum
505
tion curve, it is seen (Fig. 1) that this curve has a unimodal distribution. This indicates that the cells belong to a single population with regard to their RNA contents. DISCUSSION
May and Grenell [ 10, 151 noted an extraordinary lability in the RNA of the cerebellum. They found a marked reduction in the content of total nucleic acids, amounting to 37 per cent when the tissue was dissected without being kept on a cold block. Such a relative decrease represents a value exceeding that of the RNA content itself. This indicates that DNA very likely accounts partially for the decrease. Moreover, the same authors state that RNA was reduced by 47 per cent in the cerebellum as a result of storage in the deepfreeze. This marked lability of RNA does not seem likely, as it is well known that the cells in the central nervous system, the cerebellum included, are relatively resistant to autolysis, and that the Purkinje cells retain their basophilic staining properties for a long time after death. The present study lends credence to the correctness of these general observations and contradicts the findings of May and Grenell [lo, 151. In view of the earlier reports on the dissolving effect of Carnoy’s solution on RNA [12, 141, the present findings, showing an unaltered RNA content even after 72 hr of fixation, are worth noting. They are, however, in good agreement with the experience of recent studies on this question, but using other material [5, 61. Consequently, from the preparative point of view, the Purkinje cells show certain advantages, since RNA losses resulting from tissue preparation do not take place. Gaitonde and Richter [8, 91 measured the incorporation of 3%-methionine in different layers of the cerebellum. The correlation between their data and the present results is relatively good. They concluded that the incorporation is particularly prominent in layers exhibiting a high content of cell bodies. However, one discrepancy between their observations and the present findings may be noted. In this study, more RNA was found in the white matter than in the molecular layer, while Gaitonde and Richter found a greater incorporation in the latter layer. The volume of the molecular layer is, to a large extent, composed of dendrites and dendritric ramifications from the Purkinje cells [ll]. Thus it is evident that these dendrites have a relatively low RNA concentration. On the other hand, it is well known that the dendrites are very similar in their proximal parts to perikarya with regard to basophilia and ultraviolet absorption, a Experimental
Cell Research 28
J. Jarlstedt fact which indicates that there may be a proximo-distal gradient in the RNA concentration of the dendrites. As Table V shows, there is no significant difference betwenn darkly and lightly staining Purkinje cells with respect to the RNA content. This is in good agreement with Cammermeyer’s investigation [3], which demonstrated that the chromophilia of some neurons might be due to shrinkage resulting from post-mortem trauma of the cerebellar tissue. Furthermore, the distribution curve for all RNA values obtained from the Purkinje cells of one of the rabbits investigated gives no evidence for two or more cell groups. SUMMARY
A microchemical investigation of the RNA content in different layers of the cerebellar cortical layers as well as that of the underlying white matter has been carried out. The results indicate considerable differences in RNA concentration of the different layers according to the following order of decreasing concentrations: Purkinje cells, granular layer, white matter and molecular layer. The data give no evidence for the existence of two or more populations of Purkinje cells, with regard to RNA content. Neurons from different parts of the cerebellar cortex have identical RNA contents on a per cent/volume basis. I am indebted to Dot. J.-E. Edstrijm for help and suggestions and to Dr. E. Koenig for revising the English text. The work was supported by a grant from the Medical Faculty, University of Gothenburg, Sweden. REFERENCES 1. ATTARDI, G., Expptl. Cell Research Suppl. 4, 25 (1957). 2. BRATTGARD, S.-O. and HYD&N, H., Acta Radiol. 94 (1952). 3. CAMMERMEYER, J., Exptl. Neurol. 2, 379 (1960). 4. EDSTR~M, J.-E., J. Neurochem. 3, 1 (1958).
5. ~ Biochim. et Biophys. Acta 12, 361 (1953). 6. ~ Acta Histochem. Suppl. II, 27 (1959). 7. EINARSSON, L., Am. J. Anat. 53, 141 (1933). 8. GAITONDE, M. K. and RICHTER, D., J. Physiol. 126, 7 D (1954). 9. __ Metabolism of the Nervous System, pp. 449-455, 1957. 10. GRENELL, R. G. and MAY, L., Neurology 8, Suppl. 1 (1958). 11. Handbucb der mikroskopischen Anatomie des Menschen IV/8. Nervensystem. Berlin 1958. 12. HARBERS, E. and NEUMANN, K., Z. Naturforsch. lob, 357 (1955). 13. HYDEN, H., Acta Physiol. &and. 6, Suppl. XVII (1943). 14. LAGERSTEDT, S., Experientia 12, 425 (1956). 15. MAY, L. and GRENELL, R., Proc. Sot. Exptl. Biol. Med. 102, 235 (1958). 16. SAGUCHI, S., Zytologische Studien H 4, 1930. 17. SCH~~MMELFEDER, N., KROGII, R. E. and EBSCHNER, K. J., Histochemie 1, 1 (1958).
Experimental
Cell Research 28
Springer,