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DNA content of mouse cerebellar layers
226
Brain Research, 50 (1973) 226-229 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
DNA content of
nmum
c ~ r
layers
H O W A R D S. MAKER, G E R A R D M. L E H R E R AND CIPORA WEISS
Division of' Neurochemistry, Department of Neurology, The Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029 (U.S.A.) (Accepted October 31st, 1972)
During late maturation and aging significant changes occur in the enzyme content and metabolism of mouse cerebellum10. Since several of these changes are in opposite directions in the different layers, they may be obscured ff whole cerebellum is studied. In order to disthaguish changes related to celt proliferation or migration from those occurring in established cells, we measured cell density by assaying DNA in the molecular, granular and white matter layers of mouse cerebeUum in prepubertal, adult and aged mices. With the aid of a stereomieroscope, samples of molecular and granular layers and white matter weighing from 0.5 to 2.0 /~g were dissected from frozen-dried sections of cerebella of C57B1/6J mice aged 1, 8 and 30 monthsXl. The Purkinje cell layer which contains polyploid cells was discarded. At 1 month the cerebellum appears mature. Eight months is one-third of the median life span and 30 months is well beyond the median life span of this strain. The samples were weighed on a quartz fiber balance and lipid was extracted on teflon-covered glass carriers as previously described 11. A Folch solvent extraction (chloroform-methanol-water, 25:12.6:1.6) rather than a less polar solvent was used in order to extract the ~nglioside which reacts in the DNA assay 7. The small amount of ganglioside not extracted by this solvent20 did not appear significantly to affect the results. The contribution of residual deoxysugars (fucose, sialic acid) in glycoprotein is unknown but probably small. The concentrations of DNA per rag of dry weight and lipid-free dry weight in the molecular, granular and white matter layers of mice aged 1, 8 and 30 months are shown in Table I. There is a significant decrease in cell density in the molecular and white layers between 1 and 8 months whether expressed on a dry weight or lipid-free dry weight basis. This is particularly marked in the white matter where the cell density in the adult drops to 50 % of that at 1 month. Between 8 and 30 months only small changes occur in the cerebeUar eortical layers but there is a considerable increase in the cellularity of the white matter, although the DNA concentration never reaches the level present prior to the completion o f myelination. Methods for the assay of DNA are based on the determination of one of 3 components: phosphorus, ultraviolet light absorbing bases or deoxyribose. Most methods have serious disadvantages related to incomplete extraction, degradation or
227
SHORT C O M M U N I C A H O N S
TABLE I DNA IN MOUSECEREBELLARLAYERS DNA as #g/rag dry weight and #g/rag lipid-free dry weight in molecular and granular layers and white matter of mouse cerebellum. Figures in parentheses are standard errors of the mean among 6-10 animals. The 1-month-old animals were selected from 3 litters. The starred values for the lipid corrected data indicate significant differences (P < 0.05) from the preceding age. #g D N A / m g dry weight
Molecular Granular White
#g D N A / m g L F D weight
1 me.
8 mos.
30 mos.
1 me.
8 mos.
30 mos.
10.9 (0.3) 85.5 (3.0) 6.76 (0.62)
7.54 (O.78) 84.0 (3.3) 3.02 (0.22)
8.44 (0.62) 88.5 (4.2) 3.98 (0.39)
17.9 (0.5) 127.5 (4.5) 23.0 (2.1)
12.7" (1.3) 120.0" (4.8) 11.7* (0.9)
14.3 (1.0) 129.0" (6.1) 16.8* (1.7)
p o o r separation from interfering substancesg, 14. Brain tissue in particular shows significant variation of D N A content depending on the assay employed le. On the microchemical scale these difficulties are compounded. The method of Kissane and Robins is probably most reliable 15 since it does not require prior extraction of D N A or separation from RNA. In the present adaptation 7, the extraction of lipid which contains sialic acid (the greatest potential source of error) is probably more thorough than in the original method. Since the D N A content per diploid cell of many mammals~(including man and mouse) is similar is, the D N A concentration is an index of the cell packing density. In general, the cell density decreases as brains become more complex 4. However, the cell density of adult mouse cerebellar white matter does not differ significantly from that of rat 1,t5, rabbit tl or human 4 cerebral white matter. The interspecies decline in cell density of cortical regions with brain development is probably related to neuropil expansion. In both mouse and rat brain, cell proliferation and migration are still occurring at the time of birth is. The D N A content of whole rodent brain reaches a plateau at 15 days of age 1~'. The cerebellum, which undergoes its major growth after birth in these animals, appears morphologically mature at 21 days of age is. Since both whole brain and cerebellum increase in volume after this time, one might expect a decrease in cell density concurrent with increase in the volume of neuropil in late maturation 19. Increase in volume and decrease in cell density in the frontal cortex of the albino rat between 28 and 150 days of age have been described2,19. The present study indicates a similar change in mouse cerebellum occurring after its apparent morphological maturation. Despite some exceptionst, 21, most investigators have found a decline in total D N A and cell number in rodent cortex late in maturation. We have found a 30 % decrease in total D N A concentration in the neuropil-rich molecular layer of cerebellum between 1 and 8 months of age. This layer is devoid of myelinated fiberslL
228
SHORT COMMUNICATIONS
The decline in D N A is unchanged when the data are expressed on a lipid-free dry weight basis. In contrast, the highly cellular granular layer maintains an almost constant cell concentration over this period. The separation of neuronal perikarya from one another m a y be even more marked than is implied by total D N A levels if glial proliferation continues at this stage. The D N A concentration of the adult cerebellar white matter is one-half of that at 1 m o n t h of age. The lipid concentration changes little over this interval 11 and the decrease in cell concentration over this period can be related to the increase in the volume of myelin 6. The 30-40 % increase in cell density in the white matter which we found between 8 and 30 months is most likely due to continued glial proliferation 2, rather than a relative loss of lipid or contraction o f the white matter volume. The fact that the D N A content o f the cortical layers of aged mice remains constant does not, in itself, indicate that there is no change in the cell population since a loss o f neurons may be accompanied by an equivalent glial increase or by contraction of the neuropil volume. In the rat cerebral cortex the neuronal packing density does not change t h r o u g h two years of age 2 but in the aged h u m a n a decline in neuron n u m b e r occurs b o t h in the cerebrumZ and cerebellumL The relation o f these changes to the process o f aging in the h u m a n is complex, since age-associated pathological processes such as vascular disease may play a role in the neuronal loss. The study was supported by the National Institute o f Neurological Diseases and Stroke G r a n t No. NS 06224 and by the Benjamin Miller Memorial G r a n t No. 293 for Research on Multiple Sclerosis, National Multiple Sclerosis Society.
1 BAss, N. H., NETSKY, M. G., AND YOUNG, E., Effect of neonatal malnutrition on developing cerebrum. I. Microchemical and histologic study of cellular differentiation in the rat, Arch. Neurol. Chic., 23 (1970) 289-302. 2 BRIZZEE,K. R., VOGT, J., AND KHARETCHO,X., Postnatal changes in glial neuron index witha comparison of methods of cell enumeration in the white rat. In D. P. PURPURAANDJ. P. SCnAD~ (Eds.), Growth and Maturation of the Brain, Progr. Brain Res., Vol. 4, Elsevier, Amsterdam, 1964, pp.136-146. 3 BRODY,H., Organization of the cerebral cortex. III. A study of aging in the human cerebral cortex, J. comp. Neurol., 102 (1955) 511-516. 4 EAYRS,J. T., AND GOOOHF,AD, B., Postnatal development of the cerebral cortex of the rat, J. "Anat. (Lond.), 93 (1959) 388-402. 5 ELLIS,R. S., Norms for some structural change in the human cerebellum from birth to old age, J. comp. Neurol., 32 (1920) 1-33. 6 HADDARA,M. A., ANDNOOREDDIN,M. A., A quantitative study on the postnatal development of the cerebellar vermis of mouse, J. comp. Neurol., 128 (1966) 245-253. 7 H~ss, H. H., AND THALHEIMER, C., Microassay of biochemical structural components in nervous tissues. I. Extraction and partition of lipids and assay of nucleic acids, Y. Neurochem., 12 (1965) 193-204. 8 K.IS,SANE,J. M., ANDROBINS,E,, The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. bioL Chem., 234 (1958) 184-188. 9 LOVTRUP,S., Chemical determination of DNA in animal tissues, Acta Biochim. Pol., IX (I962) 411-424. 10 MAKER,H. S., LEHRER,G. M., SILIDES,D. J., AND WEISS,C., Energy metabolism of cerebellar cortex and white matter during late maturation, Neurology (Minneap.), 21 (I971) 444.
SHORT COMMUNICATIONS
£L~
11 MAKER,H. S., LEHRER, G. M., AND WEISS,C., Regional chloroform-methanol-water solutes in mouse cerebellum, Brain Research, 39 (1972) 260-263. 12 MANDEL,P., REIN. H., HARTH-EDEL, S., AND MAP,DELL, R., Distribution and metabolism of ribonucleic acid in the vertebrate CNS. In D. RICHTER (Ed.), Comparative Neurochemistry, Pergamon Press, New York, 1964, pp. 149-163. 13 MIALE,L., AND SIDMAN,R. L., An autoradiographic analysis of histogenesis in the mouse cerebellum, Exp. Neurol., 4 (1961) 277-296. 14 MUNRO,H. N., AND FLECK,A., The determination of nucleic acids. In D. GLICK(Ed.), Methods of Biochemical Analysis, Vol. 14, Interscience Publ., New York, 1966, pp. 113-176. 15 RAPPAPORT,D. A., FRITZ, R. R., AND MYERS,J. L., Nucleic acids. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 1, Plenum Press¢ New York, 1969, pp. 101-119. 16 SANTEN,R. J., AND AGRANOFF,B. W., Studies on the estimation of deoxyribonucleic and ribonucleic acid in rat brain, Biochim. biophys. Acta (Amst.), 72 (1963) 251-262. 17 SMrrH, K. R., JR., The cerebellar cortex of the rabbit. An electron microscopic study, J. cutup. Neurol., 121 (1963) 459484. 18 SOBER,H. A. (Ed.), Handbook of Biochemistry, 2nd ed., Chemical Rubber Co., Cleveland, Ohio, 1970, pp. 112-113. 19 SUGITA,N., Comparative studies on the growth of the cerebral cortex, V. Part I. On the area of the cortex and number of cells in a unit volume, J. cutup. Neurol., 29 (1918) 61-68. 20 SUZUKI, K., The pattern of mammalian brain gangliosides. I. Evaluation of extraction procedures, J. Neurochem., 12 (1965) 629-638. 21 UZMAN,L. L., AND RUMLEY,M.~k., Changes in the composition of the developing mouse brain during early myelination, J. Neui'ochem., 3 (1958) 170-184.
Brain Research, 50 (1973) 226-229 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
DNA content of
nmum
c ~ r
layers
H O W A R D S. MAKER, G E R A R D M. L E H R E R AND CIPORA WEISS
Division of' Neurochemistry, Department of Neurology, The Mount Sinai School of Medicine of the City University of New York, New York, N.Y. 10029 (U.S.A.) (Accepted October 31st, 1972)
During late maturation and aging significant changes occur in the enzyme content and metabolism of mouse cerebellum10. Since several of these changes are in opposite directions in the different layers, they may be obscured ff whole cerebellum is studied. In order to disthaguish changes related to celt proliferation or migration from those occurring in established cells, we measured cell density by assaying DNA in the molecular, granular and white matter layers of mouse cerebeUum in prepubertal, adult and aged mices. With the aid of a stereomieroscope, samples of molecular and granular layers and white matter weighing from 0.5 to 2.0 /~g were dissected from frozen-dried sections of cerebella of C57B1/6J mice aged 1, 8 and 30 monthsXl. The Purkinje cell layer which contains polyploid cells was discarded. At 1 month the cerebellum appears mature. Eight months is one-third of the median life span and 30 months is well beyond the median life span of this strain. The samples were weighed on a quartz fiber balance and lipid was extracted on teflon-covered glass carriers as previously described 11. A Folch solvent extraction (chloroform-methanol-water, 25:12.6:1.6) rather than a less polar solvent was used in order to extract the ~nglioside which reacts in the DNA assay 7. The small amount of ganglioside not extracted by this solvent20 did not appear significantly to affect the results. The contribution of residual deoxysugars (fucose, sialic acid) in glycoprotein is unknown but probably small. The concentrations of DNA per rag of dry weight and lipid-free dry weight in the molecular, granular and white matter layers of mice aged 1, 8 and 30 months are shown in Table I. There is a significant decrease in cell density in the molecular and white layers between 1 and 8 months whether expressed on a dry weight or lipid-free dry weight basis. This is particularly marked in the white matter where the cell density in the adult drops to 50 % of that at 1 month. Between 8 and 30 months only small changes occur in the cerebeUar eortical layers but there is a considerable increase in the cellularity of the white matter, although the DNA concentration never reaches the level present prior to the completion o f myelination. Methods for the assay of DNA are based on the determination of one of 3 components: phosphorus, ultraviolet light absorbing bases or deoxyribose. Most methods have serious disadvantages related to incomplete extraction, degradation or
227
SHORT C O M M U N I C A H O N S
TABLE I DNA IN MOUSECEREBELLARLAYERS DNA as #g/rag dry weight and #g/rag lipid-free dry weight in molecular and granular layers and white matter of mouse cerebellum. Figures in parentheses are standard errors of the mean among 6-10 animals. The 1-month-old animals were selected from 3 litters. The starred values for the lipid corrected data indicate significant differences (P < 0.05) from the preceding age. #g D N A / m g dry weight
Molecular Granular White
#g D N A / m g L F D weight
1 me.
8 mos.
30 mos.
1 me.
8 mos.
30 mos.
10.9 (0.3) 85.5 (3.0) 6.76 (0.62)
7.54 (O.78) 84.0 (3.3) 3.02 (0.22)
8.44 (0.62) 88.5 (4.2) 3.98 (0.39)
17.9 (0.5) 127.5 (4.5) 23.0 (2.1)
12.7" (1.3) 120.0" (4.8) 11.7* (0.9)
14.3 (1.0) 129.0" (6.1) 16.8* (1.7)
p o o r separation from interfering substancesg, 14. Brain tissue in particular shows significant variation of D N A content depending on the assay employed le. On the microchemical scale these difficulties are compounded. The method of Kissane and Robins is probably most reliable 15 since it does not require prior extraction of D N A or separation from RNA. In the present adaptation 7, the extraction of lipid which contains sialic acid (the greatest potential source of error) is probably more thorough than in the original method. Since the D N A content per diploid cell of many mammals~(including man and mouse) is similar is, the D N A concentration is an index of the cell packing density. In general, the cell density decreases as brains become more complex 4. However, the cell density of adult mouse cerebellar white matter does not differ significantly from that of rat 1,t5, rabbit tl or human 4 cerebral white matter. The interspecies decline in cell density of cortical regions with brain development is probably related to neuropil expansion. In both mouse and rat brain, cell proliferation and migration are still occurring at the time of birth is. The D N A content of whole rodent brain reaches a plateau at 15 days of age 1~'. The cerebellum, which undergoes its major growth after birth in these animals, appears morphologically mature at 21 days of age is. Since both whole brain and cerebellum increase in volume after this time, one might expect a decrease in cell density concurrent with increase in the volume of neuropil in late maturation 19. Increase in volume and decrease in cell density in the frontal cortex of the albino rat between 28 and 150 days of age have been described2,19. The present study indicates a similar change in mouse cerebellum occurring after its apparent morphological maturation. Despite some exceptionst, 21, most investigators have found a decline in total D N A and cell number in rodent cortex late in maturation. We have found a 30 % decrease in total D N A concentration in the neuropil-rich molecular layer of cerebellum between 1 and 8 months of age. This layer is devoid of myelinated fiberslL
228
SHORT COMMUNICATIONS
The decline in D N A is unchanged when the data are expressed on a lipid-free dry weight basis. In contrast, the highly cellular granular layer maintains an almost constant cell concentration over this period. The separation of neuronal perikarya from one another m a y be even more marked than is implied by total D N A levels if glial proliferation continues at this stage. The D N A concentration of the adult cerebellar white matter is one-half of that at 1 m o n t h of age. The lipid concentration changes little over this interval 11 and the decrease in cell concentration over this period can be related to the increase in the volume of myelin 6. The 30-40 % increase in cell density in the white matter which we found between 8 and 30 months is most likely due to continued glial proliferation 2, rather than a relative loss of lipid or contraction o f the white matter volume. The fact that the D N A content o f the cortical layers of aged mice remains constant does not, in itself, indicate that there is no change in the cell population since a loss o f neurons may be accompanied by an equivalent glial increase or by contraction of the neuropil volume. In the rat cerebral cortex the neuronal packing density does not change t h r o u g h two years of age 2 but in the aged h u m a n a decline in neuron n u m b e r occurs b o t h in the cerebrumZ and cerebellumL The relation o f these changes to the process o f aging in the h u m a n is complex, since age-associated pathological processes such as vascular disease may play a role in the neuronal loss. The study was supported by the National Institute o f Neurological Diseases and Stroke G r a n t No. NS 06224 and by the Benjamin Miller Memorial G r a n t No. 293 for Research on Multiple Sclerosis, National Multiple Sclerosis Society.
1 BAss, N. H., NETSKY, M. G., AND YOUNG, E., Effect of neonatal malnutrition on developing cerebrum. I. Microchemical and histologic study of cellular differentiation in the rat, Arch. Neurol. Chic., 23 (1970) 289-302. 2 BRIZZEE,K. R., VOGT, J., AND KHARETCHO,X., Postnatal changes in glial neuron index witha comparison of methods of cell enumeration in the white rat. In D. P. PURPURAANDJ. P. SCnAD~ (Eds.), Growth and Maturation of the Brain, Progr. Brain Res., Vol. 4, Elsevier, Amsterdam, 1964, pp.136-146. 3 BRODY,H., Organization of the cerebral cortex. III. A study of aging in the human cerebral cortex, J. comp. Neurol., 102 (1955) 511-516. 4 EAYRS,J. T., AND GOOOHF,AD, B., Postnatal development of the cerebral cortex of the rat, J. "Anat. (Lond.), 93 (1959) 388-402. 5 ELLIS,R. S., Norms for some structural change in the human cerebellum from birth to old age, J. comp. Neurol., 32 (1920) 1-33. 6 HADDARA,M. A., ANDNOOREDDIN,M. A., A quantitative study on the postnatal development of the cerebellar vermis of mouse, J. comp. Neurol., 128 (1966) 245-253. 7 H~ss, H. H., AND THALHEIMER, C., Microassay of biochemical structural components in nervous tissues. I. Extraction and partition of lipids and assay of nucleic acids, Y. Neurochem., 12 (1965) 193-204. 8 K.IS,SANE,J. M., ANDROBINS,E,, The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. bioL Chem., 234 (1958) 184-188. 9 LOVTRUP,S., Chemical determination of DNA in animal tissues, Acta Biochim. Pol., IX (I962) 411-424. 10 MAKER,H. S., LEHRER,G. M., SILIDES,D. J., AND WEISS,C., Energy metabolism of cerebellar cortex and white matter during late maturation, Neurology (Minneap.), 21 (I971) 444.
SHORT COMMUNICATIONS
£L~
11 MAKER,H. S., LEHRER, G. M., AND WEISS,C., Regional chloroform-methanol-water solutes in mouse cerebellum, Brain Research, 39 (1972) 260-263. 12 MANDEL,P., REIN. H., HARTH-EDEL, S., AND MAP,DELL, R., Distribution and metabolism of ribonucleic acid in the vertebrate CNS. In D. RICHTER (Ed.), Comparative Neurochemistry, Pergamon Press, New York, 1964, pp. 149-163. 13 MIALE,L., AND SIDMAN,R. L., An autoradiographic analysis of histogenesis in the mouse cerebellum, Exp. Neurol., 4 (1961) 277-296. 14 MUNRO,H. N., AND FLECK,A., The determination of nucleic acids. In D. GLICK(Ed.), Methods of Biochemical Analysis, Vol. 14, Interscience Publ., New York, 1966, pp. 113-176. 15 RAPPAPORT,D. A., FRITZ, R. R., AND MYERS,J. L., Nucleic acids. In A. LAJTHA(Ed.), Handbook of Neurochemistry, Vol. 1, Plenum Press¢ New York, 1969, pp. 101-119. 16 SANTEN,R. J., AND AGRANOFF,B. W., Studies on the estimation of deoxyribonucleic and ribonucleic acid in rat brain, Biochim. biophys. Acta (Amst.), 72 (1963) 251-262. 17 SMrrH, K. R., JR., The cerebellar cortex of the rabbit. An electron microscopic study, J. cutup. Neurol., 121 (1963) 459484. 18 SOBER,H. A. (Ed.), Handbook of Biochemistry, 2nd ed., Chemical Rubber Co., Cleveland, Ohio, 1970, pp. 112-113. 19 SUGITA,N., Comparative studies on the growth of the cerebral cortex, V. Part I. On the area of the cortex and number of cells in a unit volume, J. cutup. Neurol., 29 (1918) 61-68. 20 SUZUKI, K., The pattern of mammalian brain gangliosides. I. Evaluation of extraction procedures, J. Neurochem., 12 (1965) 629-638. 21 UZMAN,L. L., AND RUMLEY,M.~k., Changes in the composition of the developing mouse brain during early myelination, J. Neui'ochem., 3 (1958) 170-184.