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DNA content in nerve-cell nucleus. A biochemical and cytophotometric study of the rat cerebrum
NeuroscienceVol. 16, No. 1, pp. 45-41, 1985 Printed in Great Britain
03064522/85$3.00+ 0.00 Pergamon Press Ltd IBRO
DNA CONTENT IN NERVE-CELL NUCLEUS. BIOCHEMICAL AND CYTOPHOTOMETRIC STUDY OF THE RAT CEREBRUM
A
V. MARES,* J. CRKOVSKA,~ T. L. MARSAK~ and S. .!?T~PEK~ *Institute of Physiology, Czechoslovak Academy of Sciences, Videtiski 1083, 142 20 Prague 4, Czechoslovakia; tThe First Department of Medical and Forensic Chemistry, Charles University, Prague, Czechoslovakia; and SInstitute of Developmental Biology, Academy of Sciences, Moscow, U.S.S.R. Abstract-The cerebral nuclei of 30-day-old rats were separated by a two-step gradient centrifugation into the fractions of large and small nuclei. The DNA content per nucleus was determined biochemically and cytophotometrically in these fractions as well as in the non-separated cerebellar and liver-cell nuclei used as reference cells. The DNA content of large and/or mainly neuronal, and the small and/or glial cell-enriched nuclei were 6.74 k 0.51 and 6.21 -f. 0.30 pg, repsectively, and in the cerebellum 6.10 + 0.28 pg per nucleus. The DNA-content values determined cytophotometrically in Feulgen-stained samples of large and small bulk isolated cortical nuclei ranged within the diploid limits, indicated by a part of a liver-cell popufation measured in parallel. No evidence for the large scale existence of an “extra-DNA” reported earlier (Bregnard, Knueset and Kuenzle (1975) ~~~oc~e~~e 43,s~61; Bregnard, Kuende and Ruth (1977) .Expl Cell Res. 107,151-157; Kuenzle, Bregnard, Hiibschner and Ruth (1978)Ex$ Celf Res. 113, 151-l 601in cortical neurons has thus been obtained. INTRODUCTION
as all nerve cells of the cerebral cortex of rats have been reported to possess an “extra-DNA” With respect to the potential amounting to 3.X. ‘n2~‘* biological significance of such an exceptional enlargement of the neuronal genome, as well the pitfall8 involved in the determination of DNA content in nerve-cell nuclei,‘*5*‘5we considered it necessary to reinvestigate these findings by cytophotomet~c and bi~hemical methods. portance
Surprisingly enough, the content of DNA in a nervecell nucleus has not yet been definitely established. Cytophotometric data accumulated during the 1960s postulated that large neurons of various species, e.g. cerebellar Purkinje ceils, pyramidal neurons of the ~pp~mpus, spinal cord motone~ons, etc., contain a tetraploid (4C) amount of DNA (for review Ref. 11). Subsequent r~nv~tigations3,s,8,1~1* using improved cytophotometric techniques, autoradiography and biochemical estimation of DNA in bulk-isolated cells have, however, failed to confirm the earlier observations. In the case of the cerebellar Purkinje cells, for instance, none or only a very small number of nuclei is at present reported to exceed the diploid (2C) DNA level.3~4~8J3-‘5 Recently, the problem of DNA in nerve cells has, however, been ressurected and has increased in im-
*The data obtained was presented to the Cxechoslovak Physiological Society, Prague, 1%%1.~
EXPERIMFZIAL PROCEDURES Animals Wistar rats were fed on a standard diet DOS-2b, kept under natural day:night-light cycle and killed by cervical dislocation at age 30 days. Bulk isolation qf nuclei and biochemical estimation of DNA The forebrain hemispheres and cerebellum of 10 rats were dissected and homogenized free of meninges in 2.0moi/l sucrose in 0.001 mol/l potassium phosphate buffer (containing 0.001 mol/l MgC!,) in every experiment listed in Table I. The cerebral nuclet were separated in two fractions by a
Table 1. DNA content in nerve-cell nuclei isolated from the rat brain Method* Experiment Experiment Experiment Experiment Experiment Mean f S.E.
I (D) 2 3 4 5
(D) (D) (G-M) (C-L)
Cerebral cortex (pg/nucIeus) Small nuclei Large nuclei
Cerebellum (pg/nucleus)
5.06 6.06 7.90 8.10 6.60
5.07 6.22 6.74 6.71 6.30
6.07 5.29 6.12 6.90
6.74 f 0.5 I
6.21 f 0.30
6.10 f 0.28
*Method of DNA determination.
D, DNA determined according to rn~ifi~ method of Dische;’ G-M, DNA determined according to Gifes and Myers;9 C-L, DNA determined according to Croft and Lubran6 45
46
V. Mares
two-step centrifugation procedure in a sucrose gradient.” A fraction of large nuclei, including the nuclei of neurons and large astrocytes, and of smaller nuclei were obtained. For estimation of DNA three methods (A-C) were used. (A) One milhhtre of nuclear suspension (0.32 mol/l sucrose) was treated with 1 ml of trichloroacetic acid (100 g/l) at 0’ C for IOmin. The precipitate was dissolved in 0.3 ml NaOH (0.3 mol/l) and then 2 ml of trichloroacetic acid were added (50 g/l) for IO min (0 C). The precipitate was supplemented with 2 ml trichloroacetic acid (50 g/l) and hydrolysed at 90 C for 20 min in a closed glass ampule. The DNA content of the supernatant was determined by diphenylamine according to Dische’ in glass-closed ampules. (B) isolated nuclei were resuspended in Ringer’s solution and I ml of the suspension was hydrolysed at 70°C for 15 min in 5 ml of perchloric acid (100 g/l) in tightly closed ampules. The DNA in the supernatant was determined by diphenylamine using the method of Giles and Myers.!’ (C) The nuclei were treated as in (B) and DNA in the supernatant was determined by diphenylamine according to Croft and Lubran.’ Calf thymus DNA (Sigma) served as a standard for both methods. Feulgen staining and cyrophotometry Small pieces of the liver of 4 rats were squashed and, together with smears of the bulk-isolated cells, fixed in 96% ethanol or Carnoy solution. The cells were hydrolysed in 5N HCI at 37°C for IOmin and then stained with Schiffs reagent3,16 at room temperature for 60 min. The slides were subsequently washed twice in fresh bisulphite solution, rinsed in water and dehydrated. The DNA content was measured with a Vickers MS6 microdensitometer; a detailed description was given earlier.’ RESULTS Biochemical
data (Table
1)
The mean content of DNA per nucleus in the fractions of small and large cortical nuclei as well as the cerebellum, as calculated from five independent isolations of cells and DNA determinations by three and methods, is 6.21 k 0.30, 6.74 f 0.5 1 6.10 k 0.28 pg respectively (Table 1). Higher DNA values were, however, obtained when nuclei unintentionally passed through centrifugation in isotonic sucrose (45 min) before separation on the sucrose gradient. As an influence of DNA released from the nuclei undergoing cell lysis was suspected in this case, we have had a brief look at the number of nuclei stored in isotonic sucrose in an ice-water bath. It was found that the nuclear counts started declining immediately after isolation (by 30% within 30 min) and the DNA/nucleus values exceeded diploid amounts of DNA in spite of washing samples immediately before counting nuclei and determining the DNA. When disruption of nuclei was further enwith a homogenization hanced (by a mild Teflon-glass homogenizer, 200-p M clearance, seven strokes by hand followed by regular washing), the DNA/nucleus values reached l&l 9 pg. Cytophotometric
data (Fig.
I)
The values of Feulgen-DNA amounts measured in the large and small cortical nuclei isolated by centrifugation are shown in Figs l(A,B). The peaks of their frequency distributions correspond well to the peak
et al.
A
B 15 IO 5 LUL 220
260
300 yr
15 10 5 LJllL 220
260
A
300
L_ 500
2c
%O
A
560
UZ
1000
a
SC
Fig. 1. The frequency distribution of DNA-content values determined cytophotometrically. Smears of the (A) large (n = 101) and (B) small nuclei (n = 101) of the telencephalon of 30-day-old rats isolated by centrifugation (sampled from a paralled biochemical study). (C) Liver cells (n = 100). Abscissa: DNA in arbitrary units. 2C, 4C and 8C indicate peaks of the distribution of diploid, tetraploid and octoploid population of liver cells. Ordinate: number of measured nuclei.
of the diploid part of the liver-cell population (Fig. IC). In the liver two additional peaks, corresponding to the cells with tetraploid and octoploid amount of DNA, are apparent in the histogram. DISCUSSION
The data on the DNA content in cortical neurons measured biochemically do not indicate significant differences in the mean values of the small and large, i.e. glial and neuronal nuclei-enriched fractions, as well as of the cerebellum, and they fit well to the diploid amount of DNA in rat cells (for review, see Ref. 6). Similarly the cytophotometric data obtained indicate that the nerve-cell nuclei of the cortex of young-adult rats examined in the present study possess diploid amounts of DNA. The presence of an “extra-DNA” amounting to 3.5C observed by others,‘,2.‘0 could therefore not be confirmed. The explanation of this discrepancy is rather difficult. A critical step in the biochemical determination of the nuclear DNA content is isolation and counting of cell nuclei, as shown by the fact that about one-third of the nuclei undergoes disruption within a half an hour after their isolation. This interval is not much longer than the time necessary for determination of the number of isolated nuclei before starting with the DNA
determination.
A proper
control
of the release
DNA content in nerve-cell nuclei
of DNA from nuclei is difficult because of a different duration of the isolation procedure for neurons and reference cells, a possible difference in the resistivity of various types of nuclei, as well as possible variations in adhesivity of the released DNA to neuronal and non-neuronal nuclei resistant to washing. An uncontrolled influence of some of these factors is thus to be suspected for the small variations of DNA/number values in our individual experiments and also for the difference between the present and earlier reports on a large amount of “extraDNA”‘.‘.“’ The experimental procedure that we used for the isolation of nerve-cell nuclei differed, however, in some respect from that used by authors reporting “extra-DNA”.” In the latter study nervecell nuclei were isolated only after previous separation of nerve-. and glial-cell bodies. This intermediate step may lead to enrichment of the neuronal fraction by some minority cell type such as, e.g. Betz pyramidal neurons of the motor cortex or pyramidal neurons of the hippocampus which had been pre-
47
viously shown cytophotometrically to contain an overdiploid amount of DNA. “J*J~ Nevertheless a recent cytophotometric studyi revealed that the majority of pyramidal neurons of the hippocampus in youngadult rats is also diploid. Recent cytophotometric studies3.8.‘3on the DNA content of Purkinje cells have also revealed that a large volume of neuronal nuclei can introduce serious errors in the cytophotometric or cytofluorometric measurements and may have led to an overestimation of the true DNA content of cortical neurons in earlier reports on “extra-DNA”.‘.2 However, the aim of this paper is not to disclose the existence of a small number of cells in the cerebral cortex with a hyperdiploid or tetraploid amount of DNA, as in e.g. the Purkinje cell population,3.‘5 which might have escaped biochemical detection in bulkisolated fractions or changes in DNA content which would be exceeding the limits of the whole nucleus cytophotometry. In fact our data do not support the existence of a mass amount of “extra-DNA” as reported earlier.‘.2.‘0
REFERENCES 1. 2
Breanard A., Knuesel A. and Kuenzle C. C. (1975) Are all the neuronal nuclei oolvn1oid? Hisrochemie 43. 5961. Bregnard A., Kuenzle C. C. and Ruth F. (1977) Cytophotometric and autoradiographic’ evidence for post-natal DNA synthesis in neurons of the rat cerebral cortex. Expl Cell Res. 107, 151-157.
3. Brodsky V. Ja., Marshak T. L., Mare: V., Lodin Z., Fiilop Z. and Lebedev E. A. (1979) Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. A cytophotometric study. Hisrochemistry 59, 233-248. 4. Cohen J., Mare8 V. and Lodin Z. (1973) DNA content of purified preparations of mouse Purkinje neurons isolated by a velocity sedimentation technique. J. Neurochem. 20, 651-657. 5. Crkovska J., Stlpek S., Marshak T. L. and Mares V. (1981) Does an “extra DNA” exist in control neruons? Physiologiu bohemoslov. (abstr.) 30, 166. 6. Croft D. N. and Lubran M. (1965) The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washing. Eiochem. J. 95, 612-621. 7. Dische Z. (1930) Faber einige neue charakteristische Farbereationen der Thymonukleinsaure und eine Mikromethode zur Bestimmung derselben in tierischen Organen mit Hilfe dieser Reaktionen. Mikrochemie 8, 432. 8. Fujita S., Fukuda M., Kitamura T. and Yoshida S. (1972) Two-wavelength scanning method in Feulgen cytophotometry. Acta Histochem. Cytochem. 5, 146152. 9. Giles K. W. and Myers A. (1965) An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206, 93. 10. Kuenzle C. C., Bregnard A., Hiibschner U. and Ruth F. (1978) Extra DNA in forebrain neurons. Expl Cell Res. 113, 151-160. 11. Lapham L. W., Lentz R. D., Woodward D. J., Heifer B. J. and Herman C. I. (1971) Postnatal development of tetraploid DNA content in the Purkinje neurons of the rat: an aspect of cellular differentiation. In Cellular Aspecrs of Neural Growth and D~firenriufion, Vol. 14 (ed. Please D.), pp. 61-61. University of California Press, Los Angeles. 12. Lentz R. D. and Lapham L. W. (1969) A quantitative cytochemical study of the content of neurons of rat cerebellar cortex. J. Neurochem. 16, 379-384. 13. MareS V. and van der Ploeg M. (1980) Cytophotometric re-investigations of DNA content in Purkinje cells of the rat cerebellum, Histochemistry 69, 161-166. 14. MareS V., Lodin Z. and Sacha J. (1973) A cytochemical and autoradiographic study of nuclear DNA in mouse Purkinje cells. Brain Res. 53, 273-289. 15. Marshak T. L., Mares V. and Brodsky V. Ja. (1978) The number of Purkinje cells with increased amount of DNA in the rat cerebellum. Cirologiu, U.S.S.R. 20, 651-656. 16. Marshak T. L., MareS V., Stipek S. and Crkovska J. (1983) On the problem of “extra-DNA” in the rat brain neurons. A cytochemical study. (In Russian.) Citologia, U.S.S.R. 25, 539545. 17. Martinek J., Kyjonka F., Crkovska J. and Stipek S. (1978) Fractionation of rat telencephalic cell nuclei isolated at different stages of postnatal development. Folia morph. 26, 321-325. 18. McIlwain D. L. and Capps-Covey P. (1976) The nuclear DNA content of large ventral spinal neurons. J. Neurochem. 27, 109-I 12. 19. Novakova V., Sandritter W. and Schlueter G. (1970) DNA content of neurons in rat central nervous system. Expl Cell Res. 60, 454456. (Accepted 27 March 1985)
03064522/85$3.00+ 0.00 Pergamon Press Ltd IBRO
DNA CONTENT IN NERVE-CELL NUCLEUS. BIOCHEMICAL AND CYTOPHOTOMETRIC STUDY OF THE RAT CEREBRUM
A
V. MARES,* J. CRKOVSKA,~ T. L. MARSAK~ and S. .!?T~PEK~ *Institute of Physiology, Czechoslovak Academy of Sciences, Videtiski 1083, 142 20 Prague 4, Czechoslovakia; tThe First Department of Medical and Forensic Chemistry, Charles University, Prague, Czechoslovakia; and SInstitute of Developmental Biology, Academy of Sciences, Moscow, U.S.S.R. Abstract-The cerebral nuclei of 30-day-old rats were separated by a two-step gradient centrifugation into the fractions of large and small nuclei. The DNA content per nucleus was determined biochemically and cytophotometrically in these fractions as well as in the non-separated cerebellar and liver-cell nuclei used as reference cells. The DNA content of large and/or mainly neuronal, and the small and/or glial cell-enriched nuclei were 6.74 k 0.51 and 6.21 -f. 0.30 pg, repsectively, and in the cerebellum 6.10 + 0.28 pg per nucleus. The DNA-content values determined cytophotometrically in Feulgen-stained samples of large and small bulk isolated cortical nuclei ranged within the diploid limits, indicated by a part of a liver-cell popufation measured in parallel. No evidence for the large scale existence of an “extra-DNA” reported earlier (Bregnard, Knueset and Kuenzle (1975) ~~~oc~e~~e 43,s~61; Bregnard, Kuende and Ruth (1977) .Expl Cell Res. 107,151-157; Kuenzle, Bregnard, Hiibschner and Ruth (1978)Ex$ Celf Res. 113, 151-l 601in cortical neurons has thus been obtained. INTRODUCTION
as all nerve cells of the cerebral cortex of rats have been reported to possess an “extra-DNA” With respect to the potential amounting to 3.X. ‘n2~‘* biological significance of such an exceptional enlargement of the neuronal genome, as well the pitfall8 involved in the determination of DNA content in nerve-cell nuclei,‘*5*‘5we considered it necessary to reinvestigate these findings by cytophotomet~c and bi~hemical methods. portance
Surprisingly enough, the content of DNA in a nervecell nucleus has not yet been definitely established. Cytophotometric data accumulated during the 1960s postulated that large neurons of various species, e.g. cerebellar Purkinje ceils, pyramidal neurons of the ~pp~mpus, spinal cord motone~ons, etc., contain a tetraploid (4C) amount of DNA (for review Ref. 11). Subsequent r~nv~tigations3,s,8,1~1* using improved cytophotometric techniques, autoradiography and biochemical estimation of DNA in bulk-isolated cells have, however, failed to confirm the earlier observations. In the case of the cerebellar Purkinje cells, for instance, none or only a very small number of nuclei is at present reported to exceed the diploid (2C) DNA level.3~4~8J3-‘5 Recently, the problem of DNA in nerve cells has, however, been ressurected and has increased in im-
*The data obtained was presented to the Cxechoslovak Physiological Society, Prague, 1%%1.~
EXPERIMFZIAL PROCEDURES Animals Wistar rats were fed on a standard diet DOS-2b, kept under natural day:night-light cycle and killed by cervical dislocation at age 30 days. Bulk isolation qf nuclei and biochemical estimation of DNA The forebrain hemispheres and cerebellum of 10 rats were dissected and homogenized free of meninges in 2.0moi/l sucrose in 0.001 mol/l potassium phosphate buffer (containing 0.001 mol/l MgC!,) in every experiment listed in Table I. The cerebral nuclet were separated in two fractions by a
Table 1. DNA content in nerve-cell nuclei isolated from the rat brain Method* Experiment Experiment Experiment Experiment Experiment Mean f S.E.
I (D) 2 3 4 5
(D) (D) (G-M) (C-L)
Cerebral cortex (pg/nucIeus) Small nuclei Large nuclei
Cerebellum (pg/nucleus)
5.06 6.06 7.90 8.10 6.60
5.07 6.22 6.74 6.71 6.30
6.07 5.29 6.12 6.90
6.74 f 0.5 I
6.21 f 0.30
6.10 f 0.28
*Method of DNA determination.
D, DNA determined according to rn~ifi~ method of Dische;’ G-M, DNA determined according to Gifes and Myers;9 C-L, DNA determined according to Croft and Lubran6 45
46
V. Mares
two-step centrifugation procedure in a sucrose gradient.” A fraction of large nuclei, including the nuclei of neurons and large astrocytes, and of smaller nuclei were obtained. For estimation of DNA three methods (A-C) were used. (A) One milhhtre of nuclear suspension (0.32 mol/l sucrose) was treated with 1 ml of trichloroacetic acid (100 g/l) at 0’ C for IOmin. The precipitate was dissolved in 0.3 ml NaOH (0.3 mol/l) and then 2 ml of trichloroacetic acid were added (50 g/l) for IO min (0 C). The precipitate was supplemented with 2 ml trichloroacetic acid (50 g/l) and hydrolysed at 90 C for 20 min in a closed glass ampule. The DNA content of the supernatant was determined by diphenylamine according to Dische’ in glass-closed ampules. (B) isolated nuclei were resuspended in Ringer’s solution and I ml of the suspension was hydrolysed at 70°C for 15 min in 5 ml of perchloric acid (100 g/l) in tightly closed ampules. The DNA in the supernatant was determined by diphenylamine using the method of Giles and Myers.!’ (C) The nuclei were treated as in (B) and DNA in the supernatant was determined by diphenylamine according to Croft and Lubran.’ Calf thymus DNA (Sigma) served as a standard for both methods. Feulgen staining and cyrophotometry Small pieces of the liver of 4 rats were squashed and, together with smears of the bulk-isolated cells, fixed in 96% ethanol or Carnoy solution. The cells were hydrolysed in 5N HCI at 37°C for IOmin and then stained with Schiffs reagent3,16 at room temperature for 60 min. The slides were subsequently washed twice in fresh bisulphite solution, rinsed in water and dehydrated. The DNA content was measured with a Vickers MS6 microdensitometer; a detailed description was given earlier.’ RESULTS Biochemical
data (Table
1)
The mean content of DNA per nucleus in the fractions of small and large cortical nuclei as well as the cerebellum, as calculated from five independent isolations of cells and DNA determinations by three and methods, is 6.21 k 0.30, 6.74 f 0.5 1 6.10 k 0.28 pg respectively (Table 1). Higher DNA values were, however, obtained when nuclei unintentionally passed through centrifugation in isotonic sucrose (45 min) before separation on the sucrose gradient. As an influence of DNA released from the nuclei undergoing cell lysis was suspected in this case, we have had a brief look at the number of nuclei stored in isotonic sucrose in an ice-water bath. It was found that the nuclear counts started declining immediately after isolation (by 30% within 30 min) and the DNA/nucleus values exceeded diploid amounts of DNA in spite of washing samples immediately before counting nuclei and determining the DNA. When disruption of nuclei was further enwith a homogenization hanced (by a mild Teflon-glass homogenizer, 200-p M clearance, seven strokes by hand followed by regular washing), the DNA/nucleus values reached l&l 9 pg. Cytophotometric
data (Fig.
I)
The values of Feulgen-DNA amounts measured in the large and small cortical nuclei isolated by centrifugation are shown in Figs l(A,B). The peaks of their frequency distributions correspond well to the peak
et al.
A
B 15 IO 5 LUL 220
260
300 yr
15 10 5 LJllL 220
260
A
300
L_ 500
2c
%O
A
560
UZ
1000
a
SC
Fig. 1. The frequency distribution of DNA-content values determined cytophotometrically. Smears of the (A) large (n = 101) and (B) small nuclei (n = 101) of the telencephalon of 30-day-old rats isolated by centrifugation (sampled from a paralled biochemical study). (C) Liver cells (n = 100). Abscissa: DNA in arbitrary units. 2C, 4C and 8C indicate peaks of the distribution of diploid, tetraploid and octoploid population of liver cells. Ordinate: number of measured nuclei.
of the diploid part of the liver-cell population (Fig. IC). In the liver two additional peaks, corresponding to the cells with tetraploid and octoploid amount of DNA, are apparent in the histogram. DISCUSSION
The data on the DNA content in cortical neurons measured biochemically do not indicate significant differences in the mean values of the small and large, i.e. glial and neuronal nuclei-enriched fractions, as well as of the cerebellum, and they fit well to the diploid amount of DNA in rat cells (for review, see Ref. 6). Similarly the cytophotometric data obtained indicate that the nerve-cell nuclei of the cortex of young-adult rats examined in the present study possess diploid amounts of DNA. The presence of an “extra-DNA” amounting to 3.5C observed by others,‘,2.‘0 could therefore not be confirmed. The explanation of this discrepancy is rather difficult. A critical step in the biochemical determination of the nuclear DNA content is isolation and counting of cell nuclei, as shown by the fact that about one-third of the nuclei undergoes disruption within a half an hour after their isolation. This interval is not much longer than the time necessary for determination of the number of isolated nuclei before starting with the DNA
determination.
A proper
control
of the release
DNA content in nerve-cell nuclei
of DNA from nuclei is difficult because of a different duration of the isolation procedure for neurons and reference cells, a possible difference in the resistivity of various types of nuclei, as well as possible variations in adhesivity of the released DNA to neuronal and non-neuronal nuclei resistant to washing. An uncontrolled influence of some of these factors is thus to be suspected for the small variations of DNA/number values in our individual experiments and also for the difference between the present and earlier reports on a large amount of “extraDNA”‘.‘.“’ The experimental procedure that we used for the isolation of nerve-cell nuclei differed, however, in some respect from that used by authors reporting “extra-DNA”.” In the latter study nervecell nuclei were isolated only after previous separation of nerve-. and glial-cell bodies. This intermediate step may lead to enrichment of the neuronal fraction by some minority cell type such as, e.g. Betz pyramidal neurons of the motor cortex or pyramidal neurons of the hippocampus which had been pre-
47
viously shown cytophotometrically to contain an overdiploid amount of DNA. “J*J~ Nevertheless a recent cytophotometric studyi revealed that the majority of pyramidal neurons of the hippocampus in youngadult rats is also diploid. Recent cytophotometric studies3.8.‘3on the DNA content of Purkinje cells have also revealed that a large volume of neuronal nuclei can introduce serious errors in the cytophotometric or cytofluorometric measurements and may have led to an overestimation of the true DNA content of cortical neurons in earlier reports on “extra-DNA”.‘.2 However, the aim of this paper is not to disclose the existence of a small number of cells in the cerebral cortex with a hyperdiploid or tetraploid amount of DNA, as in e.g. the Purkinje cell population,3.‘5 which might have escaped biochemical detection in bulkisolated fractions or changes in DNA content which would be exceeding the limits of the whole nucleus cytophotometry. In fact our data do not support the existence of a mass amount of “extra-DNA” as reported earlier.‘.2.‘0
REFERENCES 1. 2
Breanard A., Knuesel A. and Kuenzle C. C. (1975) Are all the neuronal nuclei oolvn1oid? Hisrochemie 43. 5961. Bregnard A., Kuenzle C. C. and Ruth F. (1977) Cytophotometric and autoradiographic’ evidence for post-natal DNA synthesis in neurons of the rat cerebral cortex. Expl Cell Res. 107, 151-157.
3. Brodsky V. Ja., Marshak T. L., Mare: V., Lodin Z., Fiilop Z. and Lebedev E. A. (1979) Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. A cytophotometric study. Hisrochemistry 59, 233-248. 4. Cohen J., Mare8 V. and Lodin Z. (1973) DNA content of purified preparations of mouse Purkinje neurons isolated by a velocity sedimentation technique. J. Neurochem. 20, 651-657. 5. Crkovska J., Stlpek S., Marshak T. L. and Mares V. (1981) Does an “extra DNA” exist in control neruons? Physiologiu bohemoslov. (abstr.) 30, 166. 6. Croft D. N. and Lubran M. (1965) The estimation of deoxyribonucleic acid in the presence of sialic acid: application to analysis of human gastric washing. Eiochem. J. 95, 612-621. 7. Dische Z. (1930) Faber einige neue charakteristische Farbereationen der Thymonukleinsaure und eine Mikromethode zur Bestimmung derselben in tierischen Organen mit Hilfe dieser Reaktionen. Mikrochemie 8, 432. 8. Fujita S., Fukuda M., Kitamura T. and Yoshida S. (1972) Two-wavelength scanning method in Feulgen cytophotometry. Acta Histochem. Cytochem. 5, 146152. 9. Giles K. W. and Myers A. (1965) An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206, 93. 10. Kuenzle C. C., Bregnard A., Hiibschner U. and Ruth F. (1978) Extra DNA in forebrain neurons. Expl Cell Res. 113, 151-160. 11. Lapham L. W., Lentz R. D., Woodward D. J., Heifer B. J. and Herman C. I. (1971) Postnatal development of tetraploid DNA content in the Purkinje neurons of the rat: an aspect of cellular differentiation. In Cellular Aspecrs of Neural Growth and D~firenriufion, Vol. 14 (ed. Please D.), pp. 61-61. University of California Press, Los Angeles. 12. Lentz R. D. and Lapham L. W. (1969) A quantitative cytochemical study of the content of neurons of rat cerebellar cortex. J. Neurochem. 16, 379-384. 13. MareS V. and van der Ploeg M. (1980) Cytophotometric re-investigations of DNA content in Purkinje cells of the rat cerebellum, Histochemistry 69, 161-166. 14. MareS V., Lodin Z. and Sacha J. (1973) A cytochemical and autoradiographic study of nuclear DNA in mouse Purkinje cells. Brain Res. 53, 273-289. 15. Marshak T. L., Mares V. and Brodsky V. Ja. (1978) The number of Purkinje cells with increased amount of DNA in the rat cerebellum. Cirologiu, U.S.S.R. 20, 651-656. 16. Marshak T. L., MareS V., Stipek S. and Crkovska J. (1983) On the problem of “extra-DNA” in the rat brain neurons. A cytochemical study. (In Russian.) Citologia, U.S.S.R. 25, 539545. 17. Martinek J., Kyjonka F., Crkovska J. and Stipek S. (1978) Fractionation of rat telencephalic cell nuclei isolated at different stages of postnatal development. Folia morph. 26, 321-325. 18. McIlwain D. L. and Capps-Covey P. (1976) The nuclear DNA content of large ventral spinal neurons. J. Neurochem. 27, 109-I 12. 19. Novakova V., Sandritter W. and Schlueter G. (1970) DNA content of neurons in rat central nervous system. Expl Cell Res. 60, 454456. (Accepted 27 March 1985)