Differences in Brain Determined by
Cell Flow
Nuclear DNA Cytometry
as
Takao HOSHINO,Kenneth T. WHEELER, Kazuhiro NOMURA Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, California
Summary Nuclei
selected
isolated
by
DNA
in
DNA
their
the in
from
DNA
of
observation Key
peak
words:
be
gray DNA is not nuclear
in
of
matter, as
e.g.
gray
matter,
than
the
diameters
at
this cell
canine
of
appear
Betz
possible
DNA,
flow
two
or
to
examination
in
may
contain
biological
nuclei
amount to
nuclei
neuronal a
their regard
of
contrast some
of
distinct
with
a "hyperdiploid"
that
were
amount
regard
amount)
12-16 ƒÊm
cells,
The
with
man)
the
that
either
suggest
and and
diploid
carried
results
and
FCM.
would
which
These
pyramidal
(rat,
Microscopic
those
with
6-8 ƒÊm.
by
brain
more
that
nuclei
measured
isolation,
It
Acriflavin-Feulgen.
of
understood
tissue
Acriflavin-Feulgen,
cerebral 10%
by
brain with
measured.
the
revealed
diameters
normal
was
exist
stained
primarily with
cerebral
amount
cells
of
stained
(approximately
population
consisted
other the
to each
areas
grinding, nucleus
diploid content
ability
sorted
various tissue
individual of
absolute
of
of
each
populations
to
from
means
in
nuclei
"hyperdiploid"
significance
of
this
time. nuclear
The genetic information of a cell is carried in its nuclear deoxyribonucleic acid (DNA) which exerts its control over all cellular functions via the manufacture of messenger ribonucleic acid (RNA). The amount of DNA in a nucleus of a nondividing cell is usually constant throughout a cell's lifespan. In most dividing cells the DNA content immediately after division represents the diploid chromosomal complement (2C); this increases during the DNA synthetic phase to double that amount (4C), and then returns to the diploid state again after mitosis. However, some non-dividing cells have been found to contain a 4C or tetraploid amount of DNA in both normal and abnormal tissues. For exam ple, it has been demonstrated by cytophotomet ric analysis that liver contains many tetraploid cells.26) It has also been discovered by cytopho tometry that some cells in the central nervous system have a 4C DNA comple ment.10.14,15,16.17,22,24)
cytometry
Using the cytophotometric method, one can determine the DNA content of each cell type in a particular tissue ,18,19)but it is time consuming and the results are somewhat unsatisfactory in terms of resolution and statistical validity. Usually the coefficient of variation in each peak is so great that slight differences in amount of DNA cannot be quantitated. Recent develop ments in biophysics have resulted in a more efficient and accurate method of analyzing the DNA content in each cell, namely by flow cyto meter (FCM) analysis .27) The FCM technique measures the amount of DNA per cell by quan titating the intensity of the fluorescence emitted by a DNA bound dye as the cells flow rapidly past a high intensity laser beam. From this data a histogram can be constructed showing the frequency with which these DNA contents ap pear in the cell population. The histogram (number of cells vs. intensity of fluorescence) has a single narrow peak when the entire population consists of diploid cells. Multiple peaks can be found at positions relative to the intensity of their fluorescence when cells with tetraploid
(4C), octaploid (8C), etc., DNA contents exist in the population. If there are cells synthesizing DNA for duplication, their relative fluorescence values fall between these peaks. This paper de scribes preliminary results of the FCM analysis of nuclei isolated from representative areas of the cerebral hemisphere and the cerebellum in rat, dog and man. Method and Material Fresh pieces of brain tissue from rats and dogs were taken immediately after the animals were sacrificed. Human brain tissue was ob tained from lobectomized material during brain tumor surgery. Selected portions from these specimens were processed as follows: A 50-100 mg sample of tissue was homoge nized in a Dounce tissue grinder (pestle b) in 4 ml of ice-cold saline G and then filtered through a layer of 15 ym NITEX (Tetko Inc., Elmsford, NY). The homogenate was centri fuged at 4°C for 5 minutes at 100 rpm (221g) in a refrigerated IEC-PR6 centrifuge, the super natant was removed, and the pellet was re suspended in 2 ml of ice-cold saline G. This solution was then layered with a pipette on top of 30 ml of 40% sucrose containing 1.5 mM CaCl2 in a 50 ml conical centrifuge tube and, finally, recentrifuged for 15 minutes at 4,000 RPM (3536g). The supernatant was re moved with a pipette, the nuclei were re suspended in 5 ml of saline G and were then fixed with 5 ml of 20% formalin. These samples could then be stored at 4°C for several weeks before they were stained. To stain the isolated nuclei, the DNA was hydrolyzed for 20 minutes in 4N HCl at room temperature and then ex posed to acriflavin (0.02g acriflavin, 0.5g K2S205 in 10 ml of 0.5 N HCl and 90 ml of distilled water) in the dark at room temperature for 20 minutes .13)The stained specimens were analyzed on the Lawrence Livermore Lab oratory FCM. Results Nuclei from cerebral gray matter Figure 1A shows a representative FCM profile obtained from nuclei located in the ante rior half of the rat cerebrum. There is a double peak in the 2C position where diploid cells are
Fig. 1 FCM analysis of nuclei isolated from rat: A) cerebrum, B) cerebellum. The relative fluores cence intensity values of 1.0 and 2.0 correspond to the position of nuclei with a 2C and 4C DNA content, respectively.
expected, as well as a few nuclei in the 4C DNA position. The double peak in the 2C DNA position has been observed in every profile ob tained from more than 20 animals examined to date. Although the ratio of the height of the peaks varies from animal to animal, the position of the two 2C peaks relative to one another is always the same (the second peak occurs at a position equivalent to 11.5% more DNA than the first peak). A similar double peak at the 2C DNA position was present in profiles obtained from canine cerebral gray matter, canine cau date nucleus and human cerebral gray matter (Fig. 2A, B and Fig. 3A). Therefore, it appears that two distinct populations of diploid cells exist in the rat cerebrum, and in both canine and human cerebral gray matter, either with regard to absolute DNA content or with regard to ability to stain with Acriflavin-Feulgen. The absence of such a double 2C peak in the FCM profile of nuclei isolated from the cerebellum of rat (Fig. 2C) and dog (Fig. 2D), or from the cerebral white matter of dog (Fig. 2C) and human (Fig. 3B) makes it unlikely that this is due to an artifact of the isolation technique. There are only a few individual cerebral nuclei with DNA contents between 2C and 4C (Fig. 1A). This indicates that only a few cells in the cerebrum are undergoing DNA synthesis at any
Fig. 2 FCM analysis of nuclei isolated from canine: A) cerebral gray matter, B) caudate nucleus, C) cerebral white matter and D) cerebel lum. The relative intensity values of 1.0 and 2.0 have the same meaning as in Fig. 1.
Fig. 3 FCM analysis of nuclei isolated from human: A) cerebral gray matter and B) cerebral white matter. The relative intensity values of 1.0 and 2.0 have the same meaning as in Fig. 1.
particular time and virtually all of the cells appear arrested in a diploid state. Microscopic examination of the sorted nuclei from the 4C position revealed that most of these nuclei ex isted as doublets.
ulation, represented by the Purkinje cells, which might contain a true 4C DNA complement. Since over 85% of cells in the cerebellar hemi sphere are internal granular layer neurons, a majority of the nuclei which comprise the 2C DNA peak should originate from these cells.
Nuclei from cerebellum and cerebral white matter
Analysis of population in double peak
The DNA distributions of nuclei isolated from the rat cerebellum (Fig. 1B), canine cere bellum (Fig. 2D), the white matter of canine cerebrum (Fig. 2C) and the white matter of human cerebral hemisphere (Fig. 3B) exhibit only one peak in the 2C position and thus are distinctly different from profiles exhibited by gray matter nuclei. In addition, there is a rea sonably prominent population with an apparent 4C DNA composition and essentially no nuclei located between those two peaks. Micro scopic examination of the sorted rat cerebellar nuclei with a 4C DNA composition revealed approximately 35% singlets and 65% doublets. However, microspectrophotometric analysis of the singlet nuclei from the sorted 4C population showed that all of these nuclei had a 2C DNA composition; therefore, the singlets must have originated from the separation of doublets after they had passed through the laser beam. As long as this doublet problem remains it will not be possible to observe the 0.6-0.8% of the pop
The nuclei located in either peak of the double 2C peak in human and rat cerebrum were sorted with a Becton-Dickenson sorter and examined by fluorescence microscopy. As is seen in Fig. 4A and 5A, nuclei sorted from the left peak revealed very uniform round or oval-shaped nuclei with diameters of 6-8 µm. In contrast, nuclei sorted from the right peak (Fig. 4B, Fig. 5B) consisted primarily of nuclei with diameters of 12-16 pm as measured from photographs. Discussion The FCM and cell sorter system we have used routinely gives a coefficient of variation (CV), of 3.5% or less,9)permitting the two populations in cerebral gray matters to be distinguished and sorted separately with only minor difficulty.281 However, one major obstacle had to be over come to make the FCM technique a useful analytic tool. Suspensions of either single cells or reasonably intact isolated nuclei are required
Fig. 4 Sorted rat nuclei: A) from the left side of the double peak. Most have diameters of 6-8 µm. B) from the right side of the double peak. Most have diameters of 12-16 ,aim. (x 350)
Fig. 5 Sorted human nuclei: A) from the left side of the double peak; most have diameters of 6-8 µm. B) sorted nuclei from the right side of the double peak; most have diameters of 12-16 µm. ( x 350)
for FCM analysis. The usual mincing procedure with or without trypsinization was not very successful in dissociating tissue with many en tangled fibrils, such as long axons, dendrites and astrocytic processes. In an effort to overcome this difficulty we modified a nuclei isolation procedure reported by Kato, et al.,121and pre pared suspensions of nuclei from various parts of brain tissue in rat, dog and man. Recovery of intact nuclei with the tissue grinding method reported is more than 70%,12) and no selective destruction correlated with any specific DNA content was observed.") Microscopic obser vation indicates that the nuclei in the homo genate immediately after tissue grinding are singlets. Nevertheless, the formation of doublets
or triplets at the end of the whole procedure is clearly shown in most of the profiles analyzed. We made several efforts to separate these mul tiplet nuclei prior to FCM analysis by sonicating them after fixation or by homogenation and centrifugation without CA" or Mg' + in the presence of detergent and EDTA, but none of these attempts was successful. Although solving this problem is critical if this method is to be used for cell kinetic studies, it does not introduce any serious error into our current study.
From the results obtained with FCM analysis, it is possible to conclude that some neuronal cells in the cerebrum carry slightly more than the normal diploid amount of DNA (hyperdiploid, approximately 10% more chromatin than nor
mal diploid content) in their nuclei. Most of these hyperdiploid nuclei were 12-16 µm in diameter, which is consistent with nuclei from pyramidal or Betz neurons in the cerebral gray matter.5' 12)We may need more proof to identify the nuclei of larger diameters as those of py ramidal and/or Betz neurons, since some as trocytes contain nuclei of similar size. However, since the latter cells constitute a very minor proportion of the population in normal brain, it is reasonable to consider most of the larger nuclei as being of neuronal origin. 1-12) This increased amount of DNA could be ascribed to a nucleolar satellite of small size which contains DNA and is common in the nuclei of nerve cells .21)However, this nucleolar satellite is conspicuous in the females of many species while in the male it is smaller or absent. Its existence is not a sufficient explanation for our findings since the cerebral tissue of males also has a similar hyperdiploid population. Al ternatively, the apparently distinct populations of diploid cells in gray matter of cerebrum may simply reflect differing affinities for the Acriflavin-Feulgen stain.' 2,3,4,6,20) The de crease in Feulgen stainability of spermatozoa during maturation in the epididymis is well documented and has been correlated with the degree of chromatin condensation.'°8' Using the dye-binding technique, investigators from different laboratories obtained values for DNA content of mature spermatozoa in ejaculates that were 20-50% lower than expected, while the estimated values obtained from spermatids agreed well with their known DNA content.' 8) On the other hand, some structural alterations induced in DNA intensify the DNA-feulgen reaction, causing increased dye binding to an identical amount of DNA.29) If differing dye binding affinities are responsible for our results, it would be of interest to verify that DNA in some neuronal nuclei is structurally or func tionally different from that in the nuclei of other cell types in brain. If the results are due to variability in dye-binding, this may imply that chromatin is less condensed in the cells we call "hyperdiploid ." Although the technique does not at present allow us to distinguish between these possibilities of differences in structure versus amount of DNA, it is evident by FCM analysis that a reproducible difference does exist.
We already know that all nerve cells in the brain do not possess identical characteristics. For example, the internal granular layer cells, which are also neurons, have nuclei which are smaller than those of pyramidal or Betz neu rons, but nevertheless contain a 2C DNA com plement. Similarly, Purkinje cells in the cerebel lar hemisphere in a wide variety of mammals, some pyramidal neurons in the hippocampus' o) and 17% of glial cells in the Purkinje layer of the cerebellum23) have been reported to contain a tetraploid amount of DNA. Initial reports of such a 4C nuclear DNA complement in human Purkinje cells,16), however, have recently been refuted. 22) Scholtz25) has recently found by means of microspectrophotometry that in the rabbit ap proximately 3% of the occipital lobe neurons carry a tetraploid amount of nuclear DNA. His preliminary results indicate that no neurons posessing this tetraploid complement of DNA could be found in animals which had been rendered experimentally blind for a period of time prior to sacrifice. Although it is not clear whether these tetraploid neurons die or become diploid as a result of the induced blindness, it is an interesting observation since it suggests that an increased amount of nuclear DNA may have a close relationship with function for some cell types. Further investigations will be required to elucidate a similar functional distinction be tween the diploid and hyperdiploid population of cells observed in the cerebral gray matter in our studies. Since the pyramidal or Betz neurons are unable to proliferate but are very active in either nerve conduction or transmitter synthesis, our current observations may help to charac terize the biological behavior of these unique neurons. Acknowledgements This work was supported in part by United State Public Health Service Grants CA-13525 and CA-19992. We thank J. W. Gray who provided Flow-Cytometry at Lawrence Liver more Laboratory, University of California, also Barbara Riddle and Leslie Williams for editorial assistance. References 1) Agrell, I., Bergquist, H. A.: Cytochemicalevi dence for varied DNA complexesin the nuclei
of
undifferentiated
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604-606,1962 2)
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cation
3)
Bergquist,
and
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M.
of
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acid
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and
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16)
multipli
Comp.
Feulgen-deoxyribonucleic
chid
stud
cell
differentiation.
189-198,
meristematic
4)
H.
DNA-Complexes
Cell
K.
cells
Res.
A.,
61:
in
of
the
or
191-198,
Kjellstrand,
T.
T.:
during
Feulgen
43:
123-130,
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17)
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P.
depolymerisation
hydrolysis.
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A
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Blinkov,
S.
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M.,
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tables.
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19)
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Brachet, of
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Garcia,
A.
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10)
to
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pp.
Gray,
J.
F.
in
the
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et
al.:
by 21: L.
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of
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Kato,
T.,
Kurosawa,
from
the
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Paper
assortment Biol.
on
32:
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variability vances F.
pp.
and
and ed.)
L.
occurrence
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in
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L.
DNA
L.,
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Crissman,
New
H. despite
number,
in
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Biology" York,
Johnstone,
the
two
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25)
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their J.
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in in
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cat
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(E.
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22)
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11)
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fluorometry. C.
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125-151, W.,
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DNA content of giant fibrous astrocytes, with implications concerning the nature of these cells , J. Neuropath. Exptl. Neurol. 23: 419-430, 1964 Lapham, L. W.: The tetraploid DNA content of normal human Purkinje cells and its develop ment during the perinatal period. A quantitative cytochemical study. Excerpta Medica Int. Con gress Series 100: 445-449, 1965 Lapham, L. W.: Tetraploid DNA content of Purkinje neurons of human cerebellar cortex, Science, 159: 310-312, 1968 Leuchtenberger, C.: Quantative determination of DNA in cells by Feulgen microspectropho tometry, in "General Cytochemical Methods" Vol. 1 (J. F. Danielle, ed.) New York, Academic Press Inc. pp. 219-278, 1958 Leuchtenberger, C., Leuchtenberger, R., Davis, A. M.: A microspectrophotometric study of the deoxyribose nucleic acid (DNA) content in cells of normal and malignant human tissues. Am. J. Path. 30: 65-85, 1954 Mittermayer, C., Madreiter, H., Lederer, B., et al.: Differential acid hydrolysis of euchromatin and heterochromatin (biochemical, histochemi cal and morphological studies). Beitr. Path. 143: 157-171,1971 Moore, K. L., Barr, M. L.: Morphology of the nerve cell nucleus in mammals, with special reference to the sex chromatin. J. Comp. Neurol. 98: 213-231, 1953 Mann, D. M. A., Yates, P. D.: Polyploidy in the human nervous system. I. The DNA content of neurones and glia of the cerebellum. J. Neurol. Sci. 18: 183-196, 1953 Mann, D. M. A., Yates, P. D.: Polyploidy in the human nervous system. II. Studies of the glial cell populations of the Purkinje cell layer of the human cerebellum. J. Neurol. Sci. 18: 197-205, 1973 Sandritter, W., Novakova, V., Pilny, J., Kiefer, G.: Cytophotometrische Messungen des Nukleinsaure and Proteingehaltes von Gang lienzellen der Ratte wahrend der post natalen Entwicklung and in Alter, Zeitschrift fur Zell forschung 80: 145-152, 1967 Scholtz, C.: Presented at the 52nd Annual meet ing of American Association of Neuropatho logist, June 11-13, 1976. San Francisco, Calif . 1976 Schwartz, F. J.: The development in the human liver of multiple deoxyribose nucleic acid (DNA) classes and their relationship to the age of the individual, Chromosoma 8: 53-72, 1956 Van Dilla, M. A., Trujillo, T. T., Mullaney, P. E., et al.: Cell microfluorometry: A method for rapid fluorescence measurement. Science 163:
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Cell Flow
Nuclear DNA Cytometry
as
Takao HOSHINO,Kenneth T. WHEELER, Kazuhiro NOMURA Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, California
Summary Nuclei
selected
isolated
by
DNA
in
DNA
their
the in
from
DNA
of
observation Key
peak
words:
be
gray DNA is not nuclear
in
of
matter, as
e.g.
gray
matter,
than
the
diameters
at
this cell
canine
of
appear
Betz
possible
DNA,
flow
two
or
to
examination
in
may
contain
biological
nuclei
amount to
nuclei
neuronal a
their regard
of
contrast some
of
distinct
with
a "hyperdiploid"
that
were
amount
regard
amount)
12-16 ƒÊm
cells,
The
with
man)
the
that
either
suggest
and and
diploid
carried
results
and
FCM.
would
which
These
pyramidal
(rat,
Microscopic
those
with
6-8 ƒÊm.
by
brain
more
that
nuclei
measured
isolation,
It
Acriflavin-Feulgen.
of
understood
tissue
Acriflavin-Feulgen,
cerebral 10%
by
brain with
measured.
the
revealed
diameters
normal
was
exist
stained
primarily with
cerebral
amount
cells
of
stained
(approximately
population
consisted
other the
to each
areas
grinding, nucleus
diploid content
ability
sorted
various tissue
individual of
absolute
of
of
each
populations
to
from
means
in
nuclei
"hyperdiploid"
significance
of
this
time. nuclear
The genetic information of a cell is carried in its nuclear deoxyribonucleic acid (DNA) which exerts its control over all cellular functions via the manufacture of messenger ribonucleic acid (RNA). The amount of DNA in a nucleus of a nondividing cell is usually constant throughout a cell's lifespan. In most dividing cells the DNA content immediately after division represents the diploid chromosomal complement (2C); this increases during the DNA synthetic phase to double that amount (4C), and then returns to the diploid state again after mitosis. However, some non-dividing cells have been found to contain a 4C or tetraploid amount of DNA in both normal and abnormal tissues. For exam ple, it has been demonstrated by cytophotomet ric analysis that liver contains many tetraploid cells.26) It has also been discovered by cytopho tometry that some cells in the central nervous system have a 4C DNA comple ment.10.14,15,16.17,22,24)
cytometry
Using the cytophotometric method, one can determine the DNA content of each cell type in a particular tissue ,18,19)but it is time consuming and the results are somewhat unsatisfactory in terms of resolution and statistical validity. Usually the coefficient of variation in each peak is so great that slight differences in amount of DNA cannot be quantitated. Recent develop ments in biophysics have resulted in a more efficient and accurate method of analyzing the DNA content in each cell, namely by flow cyto meter (FCM) analysis .27) The FCM technique measures the amount of DNA per cell by quan titating the intensity of the fluorescence emitted by a DNA bound dye as the cells flow rapidly past a high intensity laser beam. From this data a histogram can be constructed showing the frequency with which these DNA contents ap pear in the cell population. The histogram (number of cells vs. intensity of fluorescence) has a single narrow peak when the entire population consists of diploid cells. Multiple peaks can be found at positions relative to the intensity of their fluorescence when cells with tetraploid
(4C), octaploid (8C), etc., DNA contents exist in the population. If there are cells synthesizing DNA for duplication, their relative fluorescence values fall between these peaks. This paper de scribes preliminary results of the FCM analysis of nuclei isolated from representative areas of the cerebral hemisphere and the cerebellum in rat, dog and man. Method and Material Fresh pieces of brain tissue from rats and dogs were taken immediately after the animals were sacrificed. Human brain tissue was ob tained from lobectomized material during brain tumor surgery. Selected portions from these specimens were processed as follows: A 50-100 mg sample of tissue was homoge nized in a Dounce tissue grinder (pestle b) in 4 ml of ice-cold saline G and then filtered through a layer of 15 ym NITEX (Tetko Inc., Elmsford, NY). The homogenate was centri fuged at 4°C for 5 minutes at 100 rpm (221g) in a refrigerated IEC-PR6 centrifuge, the super natant was removed, and the pellet was re suspended in 2 ml of ice-cold saline G. This solution was then layered with a pipette on top of 30 ml of 40% sucrose containing 1.5 mM CaCl2 in a 50 ml conical centrifuge tube and, finally, recentrifuged for 15 minutes at 4,000 RPM (3536g). The supernatant was re moved with a pipette, the nuclei were re suspended in 5 ml of saline G and were then fixed with 5 ml of 20% formalin. These samples could then be stored at 4°C for several weeks before they were stained. To stain the isolated nuclei, the DNA was hydrolyzed for 20 minutes in 4N HCl at room temperature and then ex posed to acriflavin (0.02g acriflavin, 0.5g K2S205 in 10 ml of 0.5 N HCl and 90 ml of distilled water) in the dark at room temperature for 20 minutes .13)The stained specimens were analyzed on the Lawrence Livermore Lab oratory FCM. Results Nuclei from cerebral gray matter Figure 1A shows a representative FCM profile obtained from nuclei located in the ante rior half of the rat cerebrum. There is a double peak in the 2C position where diploid cells are
Fig. 1 FCM analysis of nuclei isolated from rat: A) cerebrum, B) cerebellum. The relative fluores cence intensity values of 1.0 and 2.0 correspond to the position of nuclei with a 2C and 4C DNA content, respectively.
expected, as well as a few nuclei in the 4C DNA position. The double peak in the 2C DNA position has been observed in every profile ob tained from more than 20 animals examined to date. Although the ratio of the height of the peaks varies from animal to animal, the position of the two 2C peaks relative to one another is always the same (the second peak occurs at a position equivalent to 11.5% more DNA than the first peak). A similar double peak at the 2C DNA position was present in profiles obtained from canine cerebral gray matter, canine cau date nucleus and human cerebral gray matter (Fig. 2A, B and Fig. 3A). Therefore, it appears that two distinct populations of diploid cells exist in the rat cerebrum, and in both canine and human cerebral gray matter, either with regard to absolute DNA content or with regard to ability to stain with Acriflavin-Feulgen. The absence of such a double 2C peak in the FCM profile of nuclei isolated from the cerebellum of rat (Fig. 2C) and dog (Fig. 2D), or from the cerebral white matter of dog (Fig. 2C) and human (Fig. 3B) makes it unlikely that this is due to an artifact of the isolation technique. There are only a few individual cerebral nuclei with DNA contents between 2C and 4C (Fig. 1A). This indicates that only a few cells in the cerebrum are undergoing DNA synthesis at any
Fig. 2 FCM analysis of nuclei isolated from canine: A) cerebral gray matter, B) caudate nucleus, C) cerebral white matter and D) cerebel lum. The relative intensity values of 1.0 and 2.0 have the same meaning as in Fig. 1.
Fig. 3 FCM analysis of nuclei isolated from human: A) cerebral gray matter and B) cerebral white matter. The relative intensity values of 1.0 and 2.0 have the same meaning as in Fig. 1.
particular time and virtually all of the cells appear arrested in a diploid state. Microscopic examination of the sorted nuclei from the 4C position revealed that most of these nuclei ex isted as doublets.
ulation, represented by the Purkinje cells, which might contain a true 4C DNA complement. Since over 85% of cells in the cerebellar hemi sphere are internal granular layer neurons, a majority of the nuclei which comprise the 2C DNA peak should originate from these cells.
Nuclei from cerebellum and cerebral white matter
Analysis of population in double peak
The DNA distributions of nuclei isolated from the rat cerebellum (Fig. 1B), canine cere bellum (Fig. 2D), the white matter of canine cerebrum (Fig. 2C) and the white matter of human cerebral hemisphere (Fig. 3B) exhibit only one peak in the 2C position and thus are distinctly different from profiles exhibited by gray matter nuclei. In addition, there is a rea sonably prominent population with an apparent 4C DNA composition and essentially no nuclei located between those two peaks. Micro scopic examination of the sorted rat cerebellar nuclei with a 4C DNA composition revealed approximately 35% singlets and 65% doublets. However, microspectrophotometric analysis of the singlet nuclei from the sorted 4C population showed that all of these nuclei had a 2C DNA composition; therefore, the singlets must have originated from the separation of doublets after they had passed through the laser beam. As long as this doublet problem remains it will not be possible to observe the 0.6-0.8% of the pop
The nuclei located in either peak of the double 2C peak in human and rat cerebrum were sorted with a Becton-Dickenson sorter and examined by fluorescence microscopy. As is seen in Fig. 4A and 5A, nuclei sorted from the left peak revealed very uniform round or oval-shaped nuclei with diameters of 6-8 µm. In contrast, nuclei sorted from the right peak (Fig. 4B, Fig. 5B) consisted primarily of nuclei with diameters of 12-16 pm as measured from photographs. Discussion The FCM and cell sorter system we have used routinely gives a coefficient of variation (CV), of 3.5% or less,9)permitting the two populations in cerebral gray matters to be distinguished and sorted separately with only minor difficulty.281 However, one major obstacle had to be over come to make the FCM technique a useful analytic tool. Suspensions of either single cells or reasonably intact isolated nuclei are required
Fig. 4 Sorted rat nuclei: A) from the left side of the double peak. Most have diameters of 6-8 µm. B) from the right side of the double peak. Most have diameters of 12-16 ,aim. (x 350)
Fig. 5 Sorted human nuclei: A) from the left side of the double peak; most have diameters of 6-8 µm. B) sorted nuclei from the right side of the double peak; most have diameters of 12-16 µm. ( x 350)
for FCM analysis. The usual mincing procedure with or without trypsinization was not very successful in dissociating tissue with many en tangled fibrils, such as long axons, dendrites and astrocytic processes. In an effort to overcome this difficulty we modified a nuclei isolation procedure reported by Kato, et al.,121and pre pared suspensions of nuclei from various parts of brain tissue in rat, dog and man. Recovery of intact nuclei with the tissue grinding method reported is more than 70%,12) and no selective destruction correlated with any specific DNA content was observed.") Microscopic obser vation indicates that the nuclei in the homo genate immediately after tissue grinding are singlets. Nevertheless, the formation of doublets
or triplets at the end of the whole procedure is clearly shown in most of the profiles analyzed. We made several efforts to separate these mul tiplet nuclei prior to FCM analysis by sonicating them after fixation or by homogenation and centrifugation without CA" or Mg' + in the presence of detergent and EDTA, but none of these attempts was successful. Although solving this problem is critical if this method is to be used for cell kinetic studies, it does not introduce any serious error into our current study.
From the results obtained with FCM analysis, it is possible to conclude that some neuronal cells in the cerebrum carry slightly more than the normal diploid amount of DNA (hyperdiploid, approximately 10% more chromatin than nor
mal diploid content) in their nuclei. Most of these hyperdiploid nuclei were 12-16 µm in diameter, which is consistent with nuclei from pyramidal or Betz neurons in the cerebral gray matter.5' 12)We may need more proof to identify the nuclei of larger diameters as those of py ramidal and/or Betz neurons, since some as trocytes contain nuclei of similar size. However, since the latter cells constitute a very minor proportion of the population in normal brain, it is reasonable to consider most of the larger nuclei as being of neuronal origin. 1-12) This increased amount of DNA could be ascribed to a nucleolar satellite of small size which contains DNA and is common in the nuclei of nerve cells .21)However, this nucleolar satellite is conspicuous in the females of many species while in the male it is smaller or absent. Its existence is not a sufficient explanation for our findings since the cerebral tissue of males also has a similar hyperdiploid population. Al ternatively, the apparently distinct populations of diploid cells in gray matter of cerebrum may simply reflect differing affinities for the Acriflavin-Feulgen stain.' 2,3,4,6,20) The de crease in Feulgen stainability of spermatozoa during maturation in the epididymis is well documented and has been correlated with the degree of chromatin condensation.'°8' Using the dye-binding technique, investigators from different laboratories obtained values for DNA content of mature spermatozoa in ejaculates that were 20-50% lower than expected, while the estimated values obtained from spermatids agreed well with their known DNA content.' 8) On the other hand, some structural alterations induced in DNA intensify the DNA-feulgen reaction, causing increased dye binding to an identical amount of DNA.29) If differing dye binding affinities are responsible for our results, it would be of interest to verify that DNA in some neuronal nuclei is structurally or func tionally different from that in the nuclei of other cell types in brain. If the results are due to variability in dye-binding, this may imply that chromatin is less condensed in the cells we call "hyperdiploid ." Although the technique does not at present allow us to distinguish between these possibilities of differences in structure versus amount of DNA, it is evident by FCM analysis that a reproducible difference does exist.
We already know that all nerve cells in the brain do not possess identical characteristics. For example, the internal granular layer cells, which are also neurons, have nuclei which are smaller than those of pyramidal or Betz neu rons, but nevertheless contain a 2C DNA com plement. Similarly, Purkinje cells in the cerebel lar hemisphere in a wide variety of mammals, some pyramidal neurons in the hippocampus' o) and 17% of glial cells in the Purkinje layer of the cerebellum23) have been reported to contain a tetraploid amount of DNA. Initial reports of such a 4C nuclear DNA complement in human Purkinje cells,16), however, have recently been refuted. 22) Scholtz25) has recently found by means of microspectrophotometry that in the rabbit ap proximately 3% of the occipital lobe neurons carry a tetraploid amount of nuclear DNA. His preliminary results indicate that no neurons posessing this tetraploid complement of DNA could be found in animals which had been rendered experimentally blind for a period of time prior to sacrifice. Although it is not clear whether these tetraploid neurons die or become diploid as a result of the induced blindness, it is an interesting observation since it suggests that an increased amount of nuclear DNA may have a close relationship with function for some cell types. Further investigations will be required to elucidate a similar functional distinction be tween the diploid and hyperdiploid population of cells observed in the cerebral gray matter in our studies. Since the pyramidal or Betz neurons are unable to proliferate but are very active in either nerve conduction or transmitter synthesis, our current observations may help to charac terize the biological behavior of these unique neurons. Acknowledgements This work was supported in part by United State Public Health Service Grants CA-13525 and CA-19992. We thank J. W. Gray who provided Flow-Cytometry at Lawrence Liver more Laboratory, University of California, also Barbara Riddle and Leslie Williams for editorial assistance. References 1) Agrell, I., Bergquist, H. A.: Cytochemicalevi dence for varied DNA complexesin the nuclei
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