Acta histochem. 69,206-216 (1981) Istituto di Istologia, Embl'iologia e Antropologia dell'Universita Centro di Studio per I'Istochimiea del C.N.R. presso l'Universita di Pavia (Italy) Cytochemical study of chromatin changes in Purkinje cell population as markers of rat cerebellar histogenesis By GRAZIELLA BERNOCCRI and ELDA SCRERINI With 3 figures (Received December 3, 1980) Summary In the population of rat cerebellum Purkinje cells, }<'pulgen·DN A contents higher than 20 aee present starting from the 9th to the 12th day of postnatal life. This stage of histogenesis corre- sponds to the time when other histological and cytochemical parameters suggest that important functional changes are taking place in the cerebellum. Hyperdiploid values (H2c) gradually increase during the subsequent histogenesis, involving also the physico-chemical state of chl'omatin. The key steps of histogenesis suggested by our investigations might be a starting point to reexamine the problem of tritiated thimidine incorporation, in order to demonstrate the possible synthesis of extra-DNA. Introduction Feulgen-DNA contents exceeding the diploid value in cerebral cortex neurons is presently ascribed to an extrasynthesis of DNA, demonstrated by labeling with tritiated thimidine [(3HTdr); (BREGNARD et al. 1977, 1979, KUENZLE et al. 1978)J. On the contrary, the problem of similar high contents of Feulgen-DNA in the Purkinje neurons of the cerebellar cortex is more complex. In these neurons in spite of a few attempts (MARES et al. 19n, MANUELIDIS and MANUELIDIS 1974) no 3HTdr incorporation has yet been demonstrated. This suggests that, if any extrasynthesis takes place at all, it requires more complicated mechanisms. On the other hand, through the use of all available techniques of photometric correction (BERNOCCRI et al. 1979, 1980, BRODSKY et al. 1980), the existence of various levels of Feulgen-DNA contents (from 2c to 4c; c is DNA content of haploid chromosome set) in the population of Purkinje cells of adult rat cerebellum has been demonstrated. This finding agrees with the report by several authors on the presence of different functional states within this cell population (LANGE 1966, EBEL 1975, BERNOCCRI et al. 1975, 1976, 1979, BERNOCCRI and MANFREDI ROMANINI 1977, BERNOCCRI and SCRERINI 1980, SCRERINI et al. 1979, 1981, PFISTER and GORNE 1978). Acta histochem. 69,206-216 (1981) Istituto di Istologia, Embl'iologia e Antropologia dell'Universita Centro di Studio per I'Istochimiea del C.N.R. presso l'Universita di Pavia (Italy) Cytochemical study of chromatin changes in Purkinje cell population as markers of rat cerebellar histogenesis By GRAZIELLA BERNOCCRI and ELDA SCRERINI With 3 figures (Received December 3, 1980) Summary In the population of rat cerebellum Purkinje cells, }<'pulgen·DN A contents higher than 20 aee present starting from the 9th to the 12th day of postnatal life. This stage of histogenesis corre- sponds to the time when other histological and cytochemical parameters suggest that important functional changes are taking place in the cerebellum. Hyperdiploid values (H2c) gradually increase during the subsequent histogenesis, involving also the physico-chemical state of chl'omatin. The key steps of histogenesis suggested by our investigations might be a starting point to reexamine the problem of tritiated thimidine incorporation, in order to demonstrate the possible synthesis of extra-DNA. Introduction Feulgen-DNA contents exceeding the diploid value in cerebral cortex neurons is presently ascribed to an extrasynthesis of DNA, demonstrated by labeling with tritiated thimidine [(3HTdr); (BREGNARD et al. 1977, 1979, KUENZLE et al. 1978)J. On the contrary, the problem of similar high contents of Feulgen-DNA in the Purkinje neurons of the cerebellar cortex is more complex. In these neurons in spite of a few attempts (MARES et al. 19n, MANUELIDIS and MANUELIDIS 1974) no 3HTdr incorporation has yet been demonstrated. This suggests that, if any extrasynthesis takes place at all, it requires more complicated mechanisms. On the other hand, through the use of all available techniques of photometric correction (BERNOCCRI et al. 1979, 1980, BRODSKY et al. 1980), the existence of various levels of Feulgen-DNA contents (from 2c to 4c; c is DNA content of haploid chromosome set) in the population of Purkinje cells of adult rat cerebellum has been demonstrated. This finding agrees with the report by several authors on the presence of different functional states within this cell population (LANGE 1966, EBEL 1975, BERNOCCRI et al. 1975, 1976, 1979, BERNOCCRI and MANFREDI ROMANINI 1977, BERNOCCRI and SCRERINI 1980, SCRERINI et al. 1979, 1981, PFISTER and GORNE 1978).
Transcript
Acta histochem. 69,206-216 (1981)
Istituto di Istologia, Embl'iologia e Antropologia dell'Universita
Centro di Studio per I'Istochimiea del C.N.R. presso l'Universita di Pavia (Italy)
Cytochemical study of chromatin changes in Purkinje cell population as markers of rat cerebellar histogenesis
By GRAZIELLA BERNOCCRI and ELDA SCRERINI
With 3 figures
(Received December 3, 1980)
Summary
In the population of rat cerebellum Purkinje cells, }<'pulgen·DN A contents higher than 20
aee present starting from the 9th to the 12th day of postnatal life. This stage of histogenesis corre
sponds to the time when other histological and cytochemical parameters suggest that important
functional changes are taking place in the cerebellum.
Hyperdiploid values (H2c) gradually increase during the subsequent histogenesis, involving
also the physico-chemical state of chl'omatin.
The key steps of histogenesis suggested by our investigations might be a starting point to
reexamine the problem of tritiated thimidine incorporation, in order to demonstrate the possible
synthesis of extra-DNA.
Introduction
Feulgen-DNA contents exceeding the diploid value in cerebral cortex neurons is presently ascribed to an extrasynthesis of DNA, demonstrated by labeling with tritiated thimidine [(3HTdr); (BREGNARD et al. 1977, 1979, KUENZLE et al. 1978)J. On the contrary, the problem of similar high contents of Feulgen-DNA in the Purkinje neurons of the cerebellar cortex is more complex. In these neurons in spite of a few attempts (MARES et al. 19n, MANUELIDIS and MANUELIDIS 1974) no 3HTdr incorporation has yet been demonstrated. This suggests that, if any extrasynthesis takes place at all, it requires more complicated mechanisms.
On the other hand, through the use of all available techniques of photometric correction (BERNOCCRI et al. 1979, 1980, BRODSKY et al. 1980), the existence of
various levels of Feulgen-DNA contents (from 2c to 4c; c is DNA content of haploid
chromosome set) in the population of Purkinje cells of adult rat cerebellum has been demonstrated.
This finding agrees with the report by several authors on the presence of different
functional states within this cell population (LANGE 1966, EBEL 1975, BERNOCCRI et al. 1975, 1976, 1979, BERNOCCRI and MANFREDI ROMANINI 1977, BERNOCCRI and SCRERINI 1980, SCRERINI et al. 1979, 1981, PFISTER and GORNE 1978).
Acta histochem. 69,206-216 (1981)
Istituto di Istologia, Embl'iologia e Antropologia dell'Universita
Centro di Studio per I'Istochimiea del C.N.R. presso l'Universita di Pavia (Italy)
Cytochemical study of chromatin changes in Purkinje cell population as markers of rat cerebellar histogenesis
By GRAZIELLA BERNOCCRI and ELDA SCRERINI
With 3 figures
(Received December 3, 1980)
Summary
In the population of rat cerebellum Purkinje cells, }<'pulgen·DN A contents higher than 20
aee present starting from the 9th to the 12th day of postnatal life. This stage of histogenesis corre
sponds to the time when other histological and cytochemical parameters suggest that important
functional changes are taking place in the cerebellum.
Hyperdiploid values (H2c) gradually increase during the subsequent histogenesis, involving
also the physico-chemical state of chl'omatin.
The key steps of histogenesis suggested by our investigations might be a starting point to
reexamine the problem of tritiated thimidine incorporation, in order to demonstrate the possible
synthesis of extra-DNA.
Introduction
Feulgen-DNA contents exceeding the diploid value in cerebral cortex neurons is presently ascribed to an extrasynthesis of DNA, demonstrated by labeling with tritiated thimidine [(3HTdr); (BREGNARD et al. 1977, 1979, KUENZLE et al. 1978)J. On the contrary, the problem of similar high contents of Feulgen-DNA in the Purkinje neurons of the cerebellar cortex is more complex. In these neurons in spite of a few attempts (MARES et al. 19n, MANUELIDIS and MANUELIDIS 1974) no 3HTdr incorporation has yet been demonstrated. This suggests that, if any extrasynthesis takes place at all, it requires more complicated mechanisms.
On the other hand, through the use of all available techniques of photometric correction (BERNOCCRI et al. 1979, 1980, BRODSKY et al. 1980), the existence of
various levels of Feulgen-DNA contents (from 2c to 4c; c is DNA content of haploid
chromosome set) in the population of Purkinje cells of adult rat cerebellum has been demonstrated.
This finding agrees with the report by several authors on the presence of different
functional states within this cell population (LANGE 1966, EBEL 1975, BERNOCCRI et al. 1975, 1976, 1979, BERNOCCRI and MANFREDI ROMANINI 1977, BERNOCCRI and SCRERINI 1980, SCRERINI et al. 1979, 1981, PFISTER and GORNE 1978).
Cytochemical study of chromatin changes 207
Based on such "heterogeneity" it is reasonable to interpret Feulgen-DNA values as the expression of a different degree of chromatin compaction (and therefore chromatin activity) (BERNoccHI et al. 1980) even if the present absence of evidence in favour or against the hypothesis that chromatin activity implies also an extrasynthesis of DNA.
It is possible that the coexistence in adults of variously functioning chromatins makes 3HTdr labeling difficult, since we do not yet know the time and the rate
according to which the various neurons reach functional maturity or settle down to a probably temporary chromatin inactivity.
In effect, all the autoradiographic studies carried out so far have failed to take
into account the critical stages of Purkinje cell maturation, connected with nuclear
heterogeneity. In the interpretation of the biology of the neuron this aspect can not be disregarded.
Our goals are therefore (1) to detect these critical stages and, most important (2) to establish whether chromatin modifications simultaneously affect the cell population as a whole or occur gradually during histogenesis.
Materials and methods
We studied the offspring of 12 primiparous, 3·month-old Wistar rats at 4, 9, 12, 16, 22, and
30 days of life. Only litters of comparable size were examined and all were adjusted to 8 pups
at the time of birth. At each stage 4 newborn male rats from 2 mothers (2 each) were studied. At each stage the body weight of newborns was fairly similar.
After fixation by immersion in 4 % formaldehyde in water pH = 7 for 3 days and embedding in paraffin, the cerebella were cross-sectioned in histochemical sequences of 10 slides (4 to 6
sections per slide) at a thickness of 8 tun (4,9, 12 days) and 12 pm (16, 22, 30 days). On 2 slides of each series (1st and 6th), each bearing vermis and hemisphere sections at dif
ferent levels, the Feulgen reaction was carried out according to the method described by BERNOOCHI et al. (1976a), with 5 N HCI at 23°C for 1 h (which time falls into the optimal range of DNA availability according to BERNOCCHI et al. 1980) and Schiff reagent (DE TOMASI in PEARSE, 1975)
for 45 min. We decided to work on sedions because they allow a correct probing of the whole cell popula
tion. Indeed, it is worth pointing out that no profound difference was noticed between measure
ments on sections (either fixed by immersion or by perfusion) and on isolated cells or nuclei (BERNOCCHI et al. 1976, 1979, 1980, SCHERINI 1978).
Thc Feulgen-DNA content and its distribution area were evaluated in Purkinje cells and in
the small granular cells of the inner layer as control of the 2c value.
At each stage we examined 100 Purkinje cells and 80 granular cells from 4 rats except at
4 days when we measured 40 Purkinje cells.
Evaluations of Feulgen-DNA (in arbitrary units) and the simultaneous measurements of
the area (in arbitrary unitH) occupied by the Feulgen-DNA (integrated area) were performed
with a Vickers M86 integmting scanning micro densitometer under the following conditions:
A = 550 ± 5 nm; threshold 0.041); dry condenser; objective X 100; ocular X 10; scanning spot
0.4 11m (spot 2); mask variable "ccQI'ding to the size of the nucleus and including a minimum
amount of eytoplasm in the measurement field (MANN et al. 1978).
1) "Ve chose sueh a, sensitivity threshold since, in the electronic configuration of Vickers
M86, density values lower than O.D. = 0.04 must be essentially considered as background noise
and non specific coloration.
Cytochemical study of chromatin changes 207
Based on such "heterogeneity" it is reasonable to interpret Feulgen-DNA values as the expression of a different degree of chromatin compaction (and therefore chromatin activity) (BERNoccHI et al. 1980) even if the present absence of evidence in favour or against the hypothesis that chromatin activity implies also an extrasynthesis of DNA.
It is possible that the coexistence in adults of variously functioning chromatins makes 3HTdr labeling difficult, since we do not yet know the time and the rate
according to which the various neurons reach functional maturity or settle down to a probably temporary chromatin inactivity.
In effect, all the autoradiographic studies carried out so far have failed to take
into account the critical stages of Purkinje cell maturation, connected with nuclear
heterogeneity. In the interpretation of the biology of the neuron this aspect can not be disregarded.
Our goals are therefore (1) to detect these critical stages and, most important (2) to establish whether chromatin modifications simultaneously affect the cell population as a whole or occur gradually during histogenesis.
Materials and methods
We studied the offspring of 12 primiparous, 3·month-old Wistar rats at 4, 9, 12, 16, 22, and
30 days of life. Only litters of comparable size were examined and all were adjusted to 8 pups
at the time of birth. At each stage 4 newborn male rats from 2 mothers (2 each) were studied. At each stage the body weight of newborns was fairly similar.
After fixation by immersion in 4 % formaldehyde in water pH = 7 for 3 days and embedding in paraffin, the cerebella were cross-sectioned in histochemical sequences of 10 slides (4 to 6
sections per slide) at a thickness of 8 tun (4,9, 12 days) and 12 pm (16, 22, 30 days). On 2 slides of each series (1st and 6th), each bearing vermis and hemisphere sections at dif
ferent levels, the Feulgen reaction was carried out according to the method described by BERNOOCHI et al. (1976a), with 5 N HCI at 23°C for 1 h (which time falls into the optimal range of DNA availability according to BERNOCCHI et al. 1980) and Schiff reagent (DE TOMASI in PEARSE, 1975)
for 45 min. We decided to work on sedions because they allow a correct probing of the whole cell popula
tion. Indeed, it is worth pointing out that no profound difference was noticed between measure
ments on sections (either fixed by immersion or by perfusion) and on isolated cells or nuclei (BERNOCCHI et al. 1976, 1979, 1980, SCHERINI 1978).
Thc Feulgen-DNA content and its distribution area were evaluated in Purkinje cells and in
the small granular cells of the inner layer as control of the 2c value.
At each stage we examined 100 Purkinje cells and 80 granular cells from 4 rats except at
4 days when we measured 40 Purkinje cells.
Evaluations of Feulgen-DNA (in arbitrary units) and the simultaneous measurements of
the area (in arbitrary unitH) occupied by the Feulgen-DNA (integrated area) were performed
with a Vickers M86 integmting scanning micro densitometer under the following conditions:
A = 550 ± 5 nm; threshold 0.041); dry condenser; objective X 100; ocular X 10; scanning spot
0.4 11m (spot 2); mask variable "ccQI'ding to the size of the nucleus and including a minimum
amount of eytoplasm in the measurement field (MANN et al. 1978).
1) "Ve chose sueh a, sensitivity threshold since, in the electronic configuration of Vickers
M86, density values lower than O.D. = 0.04 must be essentially considered as background noise
and non specific coloration.
208 G. BEHXOCCHI and E. SCHE HIXI
The microdensitometr ie recording was canied out only on whole nuclei , carefully chosen for
seet ion microdensitometry according to LENTZ and LA P HAM (1969). The background sett ing was obta ined with t he spot position close to t ho nucleus on a white
cytoplasmic area in ordor to avoid non-specific light absorption as suggested by MIKSCHE et al.
(1979) for Vickers microdensitometcr. The correction of the error conneded with stray-light (GOLDSTEIX 1970, 1971, BED I and
GOLDSTEIN 1976) was obtained by correction for glare (4 %). The area of distribution of F eulgen-positive m aterial was estimated for the number of points
with absorbance higher than the instrumental t hreshold chosen by the operator (integrated area). The Feulgen-D~A/integrated arca ratio therefore represents the status of chromatin conden
sation. Nuelear areas r,um 2] of the same nudei used fo r measuring Feulgen-DNA were obtained after
diame ter l'eeording with ;t Loi tz filar micrometer by the following formul a :
A N = d l X d 2 X n/4 X 1\:.2
where d l and d 2 are the perpendicular diameters and K is a constant dependent on instrument setting.
The comparison bet ween different stages was carried out for each measurement (Feulgen-DNA , integrated area, nuclear area , Feulgen-DNA/ integrated area ratio ) using the GOSSET'S
("STUDENT'S") t test.
Results
Table 1 shows the mean values (± s.e.) of the Feulgen-DNA content of Purkinje cells, of the distribution area of Feulgen-DNA (integrated area) of the nuclear area and of the ratio of the two parameters at different stages.
The 2c value of granular cells was constant at the time of transition from one stage to the subsequent one.
Based on these findings, in the comparison between the Feulgen-DNA of Purkinje cells and that of granular cells, we regarded t he Feulgen-DNA content of granular cells at t he same stage as 2c.
The increase of t he average value beyond 2c starts at 9 days and is significant at 12 days when adult values have already been reached.
Correspondingly the Feulgen-DNA distribution range (Fig. 1) not only shifts towards high values beginning on day 12 but is typically an expression of the coexistence of widely different values together with pictures which, from the 12th day onwards, are comparable with those previously described in adults animals.
The :Feulgen-DN A integrated area ratio (Table 1) is regarded by us as the expression of the degree of chromatin compaction (and therefore of its activity). This ratio undergoes deep changes around the 16th day, which appears to suggest t hat at that time chromatin decondensation is higher.
In Figs. 2 and 3 an analytical examination of the Feulgen-DNAfintegrated area ratio should take into account the A, B, C and D quadrants, limited by (a) a line parallel to the axis of t he ordinates which marks t he limits of Feulgen-DNA contents of Purkinje cells at t he first stage considered and, thus, by reference to the 2c Feulgen-DNA content of granules, the limit of the diploid value, and (b) the line parallel to the axis of abscissae, marking the limit of Fe ulgen-DNA distribution values of Purkinje cells at 4 days.
208 G. BEHXOCCHI and E. SCHE HIXI
The microdensitometr ie recording was canied out only on whole nuclei , carefully chosen for
seet ion microdensitometry according to LENTZ and LA P HAM (1969). The background sett ing was obta ined with t he spot position close to t ho nucleus on a white
cytoplasmic area in ordor to avoid non-specific light absorption as suggested by MIKSCHE et al.
(1979) for Vickers microdensitometcr. The correction of the error conneded with stray-light (GOLDSTEIX 1970, 1971, BED I and
GOLDSTEIN 1976) was obtained by correction for glare (4 %). The area of distribution of F eulgen-positive m aterial was estimated for the number of points
with absorbance higher than the instrumental t hreshold chosen by the operator (integrated area). The Feulgen-D~A/integrated arca ratio therefore represents the status of chromatin conden
sation. Nuelear areas r,um 2] of the same nudei used fo r measuring Feulgen-DNA were obtained after
diame ter l'eeording with ;t Loi tz filar micrometer by the following formul a :
A N = d l X d 2 X n/4 X 1\:.2
where d l and d 2 are the perpendicular diameters and K is a constant dependent on instrument setting.
The comparison bet ween different stages was carried out for each measurement (Feulgen-DNA , integrated area, nuclear area , Feulgen-DNA/ integrated area ratio ) using the GOSSET'S
("STUDENT'S") t test.
Results
Table 1 shows the mean values (± s.e.) of the Feulgen-DNA content of Purkinje cells, of the distribution area of Feulgen-DNA (integrated area) of the nuclear area and of the ratio of the two parameters at different stages.
The 2c value of granular cells was constant at the time of transition from one stage to the subsequent one.
Based on these findings, in the comparison between the Feulgen-DNA of Purkinje cells and that of granular cells, we regarded t he Feulgen-DNA content of granular cells at t he same stage as 2c.
The increase of t he average value beyond 2c starts at 9 days and is significant at 12 days when adult values have already been reached.
Correspondingly the Feulgen-DNA distribution range (Fig. 1) not only shifts towards high values beginning on day 12 but is typically an expression of the coexistence of widely different values together with pictures which, from the 12th day onwards, are comparable with those previously described in adults animals.
The :Feulgen-DN A integrated area ratio (Table 1) is regarded by us as the expression of the degree of chromatin compaction (and therefore of its activity). This ratio undergoes deep changes around the 16th day, which appears to suggest t hat at that time chromatin decondensation is higher.
In Figs. 2 and 3 an analytical examination of the Feulgen-DNAfintegrated area ratio should take into account the A, B, C and D quadrants, limited by (a) a line parallel to the axis of t he ordinates which marks t he limits of Feulgen-DNA contents of Purkinje cells at t he first stage considered and, thus, by reference to the 2c Feulgen-DNA content of granules, the limit of the diploid value, and (b) the line parallel to the axis of abscissae, marking the limit of Fe ulgen-DNA distribution values of Purkinje cells at 4 days.
'Y. 70
50
30
10
% 70
10
% 70
50
30
10
Cytochemical study of chromatin changes
12
n , , ,
, , , ,
20
, days
28
9 days
28
12 days
:J1D:~~ I L..J I
, , , , , II , •
12 20 28
Feulgen DNA (a.u.l
'Y. 70
50
30
10
0/. 70
50
30
10
% 70
50
30
10
r - , , , , ,
" days
lfUl ,/I'~ 12 20 28
, , , "
, , , , , ,
22 days
30 days
;'~ , , : ' , , , ,
1/ \ ,
12 20 28
209
Fig_ 1. Feulgen-DNA of PW'kinje cells (-) and internal granular cells (---) distributions at different stages of postnatal cerebellar histogenesis. Ordinate : % frequency.
T able 1. Mean values (± s.e.) of the parameters studied. The differences between 2 subsequent stages are reported (**: P < 0.01; ***: P < 0.001). The F eulgen-DNA content of Purkin je cells increases significantly at 12 days in comparison to the F eulgen-DNA of granular cells at ull stages and of Purkinje cells a t 4- 9 days, thereafter remaining at about the same levels. At 4 days 110 measurement of F eulgen-DNA on internal granular cells was carried out because they were often overlapped and not clearly identifiable
Stages Feulgen-DNA (a.u.) INTEGRATED NUCLEAR F eulgen-DNA/
Granular cells Purkinje cells AREA (a.u.) AREA [.um2] INTEGRATED Purkinje cells Purkinje cells AREA
Fig_ 1. Feulgen-DNA of PW'kinje cells (-) and internal granular cells (---) distributions at different stages of postnatal cerebellar histogenesis. Ordinate : % frequency.
T able 1. Mean values (± s.e.) of the parameters studied. The differences between 2 subsequent stages are reported (**: P < 0.01; ***: P < 0.001). The F eulgen-DNA content of Purkin je cells increases significantly at 12 days in comparison to the F eulgen-DNA of granular cells at ull stages and of Purkinje cells a t 4- 9 days, thereafter remaining at about the same levels. At 4 days 110 measurement of F eulgen-DNA on internal granular cells was carried out because they were often overlapped and not clearly identifiable
Stages Feulgen-DNA (a.u.) INTEGRATED NUCLEAR F eulgen-DNA/
Granular cells Purkinje cells AREA (a.u.) AREA [.um2] INTEGRATED Purkinje cells Purkinje cells AREA
iHT~~-r.~-r'~_'~~i~I~~~.~-r.-r~.~C __ _ II 13 IS 17 19 21 2] 2S 27
Feulgen DNA (a.u.)
Fig. 2.
Fig. 2 to 3. Feulgen -DNA and integrated area (a.u.) in Purk in je nell population at different histogenetic st ages. The line parallel to the axis of the ordinat e m arks the limits of Feulgen -DNA contents of Purkillje coils a t 4th day of postnatal life and, thus, by reference to the 20 Feulgen-DNA content of gl"anuh1r cells, the limit of 20 values. The line parallel to t he axis of abscissa marks the limits of integrated areas (Felligen- DN A distribution areas) of P urkinje cells a t 4th
iHT~~-r.~-r'~_'~~i~I~~~.~-r.-r~.~C __ _ II 13 IS 17 19 21 2] 2S 27
Feulgen DNA (a.u.)
Fig. 2.
Fig. 2 to 3. Feulgen -DNA and integrated area (a.u.) in Purk in je nell population at different histogenetic st ages. The line parallel to the axis of the ordinat e m arks the limits of Feulgen -DNA contents of Purkillje coils a t 4th day of postnatal life and, thus, by reference to the 20 Feulgen-DNA content of gl"anuh1r cells, the limit of 20 values. The line parallel to t he axis of abscissa marks the limits of integrated areas (Felligen- DN A distribution areas) of P urkinje cells a t 4th
,... :i aj .......
Cytochemical study of chromatin changes
20 L 0 0
B .0 0 o 0
: .. ., . 0
.00
.... 0
• 0 0
• • D : 5+---------------------o~o-o~~------~ • • -:
day. The A quadrant corresponds to the range of 2c Feulgen-DNA values under the initial COll
densation state; quadrant B will comprise 20 highly decondensed neurons; quadrant C the nuclei
with a Feulgen-DNA content exceeding 20 and highly condensed chromatin, while quadrant D
will be occupied by nuclei with Feulgen-DNA values above 2c and decondensed chromatin.
14*
212 G. BERNOOOHI and E. SOHERINI
Therefore: the A quadrant corresponds to the range of 2c Feulgen-DNA values
under the initial condensation state; quadrant B will comprise 2c highly decondensed neurons; quadrant C the nuclei with a Feulgen-DNA content exceeding 2c and highly condensed chromatin, while quadrant D will be occupied by nuclei with Feulgen-DNA values above 2c and decondensed chromatin.
Therefore the cells which fall into quadrants Band D exhibit a higher decondensation (which can be interpreted as a higher degree of activity), while those in A and C are more condensed (less active).
It is interesting to note the gradual filling of D and C after the 9th day, in coincidence with the appearance of H2c values, and to point out the following characteristic:
l. on the 12th day there is a large group of H2c nuclei whose Feulgen-DNA distribution area is unchanged while Feulgen-DNA content is increased, chromatin being therefore more condensed; there are also 2c or slightly above 2c nuclei with a larger area, where chromatin is less condensed;
2. on the 16th day Feulgen-DNA content distribution is already similar to that of the adult but most nuclei (both 2c and H2c) are highly decondensed;
3. on the 22nd day the situation is the same as on 30th day, i.e. the same as in adult rat (BERNOCCHI et al. 1976b).
The size of the nuclei was found to increase significantly between 4 to 9 and 12 to 16 days, maintaining the same value at 22 and 30 days (Table 1). On the contrary, between 9 and 12 days nuclear dimensions do not change, their low variability suggesting, unlike in the other stages, that nuclear areas are homogeneous.
Discussion
By applying the most useful microdensitometric technique for Vickers according to recent df1ta of MIKSCRE et al. (1979) we are able to confirm that in the rat the Purkinje cell population
exhibits an average increase in the Feulgen·DN A content and to show that this increase begins
at the early stages of postnatal life. A significant increase appears to take place between the 9th and the 12th day, in agreement with the early data of SANDRITTER et al. (1967), but in partial disagreement with those of LENTZ and LAPRAM (1970). These last authors reported "tetraploidy" even at 7 days of postnatal life. As a matter of fact, in our investigations we too observed an increase in the average value of Feulgen-DN A content between the 4th and the 9th day: this
increase, however, was not significant and was probably due to the presence of a few H2c cells
in respect to the 2c cells. In this connection, we would once again like to stress the importance
of examining many vcrmis and hemisphere sections, when the Purkinje population is involved
(SCHERlNI ct al. 1979) because the very high variability of some histochemical parameters within
the cerebellum. This could account for the discrepances between the results of LENTZ and LAPHAM
(1970) and ours.
The new results of this work stem on the one hand from the analysis of the distribution of
Feulgen-DNA contents only and, on the other, from the analysis of Feulgen-DNA in relation
with the integrated area, i.e. the area of distribution of Feulgen positive material (Figs. 1, 2; Table 1).
This analysis shows that at certain stages Feulgen-DNA changes could be related only to changes in chromatin condensation and/or structure and others possibly also to extra synthesis of DNA.
212 G. BERNOOOHI and E. SOHERINI
Therefore: the A quadrant corresponds to the range of 2c Feulgen-DNA values
under the initial condensation state; quadrant B will comprise 2c highly decondensed neurons; quadrant C the nuclei with a Feulgen-DNA content exceeding 2c and highly condensed chromatin, while quadrant D will be occupied by nuclei with Feulgen-DNA values above 2c and decondensed chromatin.
Therefore the cells which fall into quadrants Band D exhibit a higher decondensation (which can be interpreted as a higher degree of activity), while those in A and C are more condensed (less active).
It is interesting to note the gradual filling of D and C after the 9th day, in coincidence with the appearance of H2c values, and to point out the following characteristic:
l. on the 12th day there is a large group of H2c nuclei whose Feulgen-DNA distribution area is unchanged while Feulgen-DNA content is increased, chromatin being therefore more condensed; there are also 2c or slightly above 2c nuclei with a larger area, where chromatin is less condensed;
2. on the 16th day Feulgen-DNA content distribution is already similar to that of the adult but most nuclei (both 2c and H2c) are highly decondensed;
3. on the 22nd day the situation is the same as on 30th day, i.e. the same as in adult rat (BERNOCCHI et al. 1976b).
The size of the nuclei was found to increase significantly between 4 to 9 and 12 to 16 days, maintaining the same value at 22 and 30 days (Table 1). On the contrary, between 9 and 12 days nuclear dimensions do not change, their low variability suggesting, unlike in the other stages, that nuclear areas are homogeneous.
Discussion
By applying the most useful microdensitometric technique for Vickers according to recent df1ta of MIKSCRE et al. (1979) we are able to confirm that in the rat the Purkinje cell population
exhibits an average increase in the Feulgen·DN A content and to show that this increase begins
at the early stages of postnatal life. A significant increase appears to take place between the 9th and the 12th day, in agreement with the early data of SANDRITTER et al. (1967), but in partial disagreement with those of LENTZ and LAPRAM (1970). These last authors reported "tetraploidy" even at 7 days of postnatal life. As a matter of fact, in our investigations we too observed an increase in the average value of Feulgen-DN A content between the 4th and the 9th day: this
increase, however, was not significant and was probably due to the presence of a few H2c cells
in respect to the 2c cells. In this connection, we would once again like to stress the importance
of examining many vcrmis and hemisphere sections, when the Purkinje population is involved
(SCHERlNI ct al. 1979) because the very high variability of some histochemical parameters within
the cerebellum. This could account for the discrepances between the results of LENTZ and LAPHAM
(1970) and ours.
The new results of this work stem on the one hand from the analysis of the distribution of
Feulgen-DNA contents only and, on the other, from the analysis of Feulgen-DNA in relation
with the integrated area, i.e. the area of distribution of Feulgen positive material (Figs. 1, 2; Table 1).
This analysis shows that at certain stages Feulgen-DNA changes could be related only to changes in chromatin condensation and/or structure and others possibly also to extra synthesis of DNA.
Cytochemical study of chromatin changes 213
More exactly, the stages studied mark specific steps of postnatal histogenesis:
1. Between the 4th and 9th day, when the first H2 c values appear, a general decondensation
of chromatin attended by an increase of tho nuclear area is observed;
2. Between the 9th and 12th day, when two new populations can be found, namely a less
abundant population with 2c or slightl y ovor 2c Feulgen-DNA contents and decondensed chromatin (ext ra-'DNA synthet izing?), and a more abundant population with H2c Feulgen-DNA and
condensed chromatin (extra-DNA synthetized 1); nuclear areas appear to be unchanged a s com
pared to t he previous period. This finding is quite interesting since it might suggest a possible extrasynthesis of DNA.
Based on our data the negative results obtained in the rat by autoradiography by MANuELIDIs
and MANUELIDIS (1974) can be criticized since these authors injected 3HTdr up to the 9th day only, which explains why they failed to find any labeling in Purkinje cells.
3. The period between the 12th to 11lth and 22nd day including a time (16th day) when the
F eulgen-DNA content distribut ion is similar to tha t of t he 12th day (few 2c cells, many H2 c
cells, rare values close to 4c); however, chrom'1tin as a whole becomes decondensed and the nuclear
area increases. It is interesting to emp hasize that between the 12th and the 16th day 30 % of Purkinje cells
exhibit cytoplasmic eosinophilia which might be rela t ed to protein accumulation (BERNoccHI
and SCHERINI 1980). This would expla in the higher act ivity of chroma tin, demonstrated by its
decondensation. On the contrary, the f'Lct that at 22 days nuclei are most ly condensed might suggest an inactiva
tion of chromatin in many nuclei. This might be linked to the attainment of the percentage of
eosinophilic cells (70 to 80 %) typical of adult animals which occurs a t this time. More general remflrk s which can be drawn from our data are the following. In the first place,
already by the second week of life the nuclear popula tion appears to be heterogeneous, as we
have already demonstrated also by two wave lengths microdensitometry in the adult rat cerebel
lum (BERNoccHI 1975, BERXOCCHI et al. 1976b, 1979, 1980, BERNoccHI and MANFREDI ROMANINI 1977, SCHERINI 1978) and also in agreement with BRODSKY et al. (1974, 1979, 1980) who, however,
found 2c and H2 c pel'centages slightly different from ours. In the second place our data on the
slow increase of the F eulgen-DNA content in the cell population suggest tha t such synthesis
might also be slow (i.c- affecting the populat ion little by little), and thus difficult to detect by 3HTdr incorporation.
Difficulties m ay be rela ted, in addition to the choice of critical st ages, to the fact that H2c cells a t the early stages of histogenesis are not only few but also scattered t hroughout the cerebellum. This can be overcome by screening the whole Purkinje cell populat ion (SCHERINI et al. 1979).
Another limitation of autoradiographic techniques is that many H2c cells might exhibit extrasYllthesis of a very low fraction of DNA. In this connection, CAMEROX et al. (1978) have developed a highly sensitive method which allowed them to demonstrat e the synthesis of a low fraction of D NA in adult mouse Purkinje cells, thus supporting the con cept of DNA instability
in these neurons.
In condllsion, on the ba~is of the~e remarks and of t he modes of appea mnce of early H2c values, the period we intend to examine (in the future) by 3HTdr incorporation is the one between day 4 and day 12 focusing our attention in particular on days 9 to 12.
Finally , with regard to t he process of maturation of the Purkinje neuron, the first signs of changes a t nuclear level which do not a ffect the entire cell population and r andomly appear in some cells only are in line with t ho results which indicate that the differentiation of these
n eurons do not occur simultaneously in the population as a whole (ALTMAx 1972). In particular nuclear changes between 9th and 12th day, observed here on the basis of Feulgen-DN A content, are supported by numerous histological and physiological data indicati ng that impor tant neuronal and extraneuronal modifications t ake p lace within the second week of li fe. In this period synapto-
Cytochemical study of chromatin changes 213
More exactly, the stages studied mark specific steps of postnatal histogenesis:
1. Between the 4th and 9th day, when the first H2 c values appear, a general decondensation
of chromatin attended by an increase of tho nuclear area is observed;
2. Between the 9th and 12th day, when two new populations can be found, namely a less
abundant population with 2c or slightl y ovor 2c Feulgen-DNA contents and decondensed chromatin (ext ra-'DNA synthet izing?), and a more abundant population with H2c Feulgen-DNA and
condensed chromatin (extra-DNA synthetized 1); nuclear areas appear to be unchanged a s com
pared to t he previous period. This finding is quite interesting since it might suggest a possible extrasynthesis of DNA.
Based on our data the negative results obtained in the rat by autoradiography by MANuELIDIs
and MANUELIDIS (1974) can be criticized since these authors injected 3HTdr up to the 9th day only, which explains why they failed to find any labeling in Purkinje cells.
3. The period between the 12th to 11lth and 22nd day including a time (16th day) when the
F eulgen-DNA content distribut ion is similar to tha t of t he 12th day (few 2c cells, many H2 c
cells, rare values close to 4c); however, chrom'1tin as a whole becomes decondensed and the nuclear
area increases. It is interesting to emp hasize that between the 12th and the 16th day 30 % of Purkinje cells
exhibit cytoplasmic eosinophilia which might be rela t ed to protein accumulation (BERNoccHI
and SCHERINI 1980). This would expla in the higher act ivity of chroma tin, demonstrated by its
decondensation. On the contrary, the f'Lct that at 22 days nuclei are most ly condensed might suggest an inactiva
tion of chromatin in many nuclei. This might be linked to the attainment of the percentage of
eosinophilic cells (70 to 80 %) typical of adult animals which occurs a t this time. More general remflrk s which can be drawn from our data are the following. In the first place,
already by the second week of life the nuclear popula tion appears to be heterogeneous, as we
have already demonstrated also by two wave lengths microdensitometry in the adult rat cerebel
lum (BERNoccHI 1975, BERXOCCHI et al. 1976b, 1979, 1980, BERNoccHI and MANFREDI ROMANINI 1977, SCHERINI 1978) and also in agreement with BRODSKY et al. (1974, 1979, 1980) who, however,
found 2c and H2 c pel'centages slightly different from ours. In the second place our data on the
slow increase of the F eulgen-DNA content in the cell population suggest tha t such synthesis
might also be slow (i.c- affecting the populat ion little by little), and thus difficult to detect by 3HTdr incorporation.
Difficulties m ay be rela ted, in addition to the choice of critical st ages, to the fact that H2c cells a t the early stages of histogenesis are not only few but also scattered t hroughout the cerebellum. This can be overcome by screening the whole Purkinje cell populat ion (SCHERINI et al. 1979).
Another limitation of autoradiographic techniques is that many H2c cells might exhibit extrasYllthesis of a very low fraction of DNA. In this connection, CAMEROX et al. (1978) have developed a highly sensitive method which allowed them to demonstrat e the synthesis of a low fraction of D NA in adult mouse Purkinje cells, thus supporting the con cept of DNA instability
in these neurons.
In condllsion, on the ba~is of the~e remarks and of t he modes of appea mnce of early H2c values, the period we intend to examine (in the future) by 3HTdr incorporation is the one between day 4 and day 12 focusing our attention in particular on days 9 to 12.
Finally , with regard to t he process of maturation of the Purkinje neuron, the first signs of changes a t nuclear level which do not a ffect the entire cell population and r andomly appear in some cells only are in line with t ho results which indicate that the differentiation of these
n eurons do not occur simultaneously in the population as a whole (ALTMAx 1972). In particular nuclear changes between 9th and 12th day, observed here on the basis of Feulgen-DN A content, are supported by numerous histological and physiological data indicati ng that impor tant neuronal and extraneuronal modifications t ake p lace within the second week of li fe. In this period synapto-
214 G. BERNOCCHI and E. SCHERINI
genesis is quite active (ALT~IAN 1972), the climbing fiber response is the same as in the adult
(PURO and WOODWARD 1977). the dendl"ite tree becomes more evident (ALTMAN 1972). fluorescent
nervouS terminals containing biogenic amines gathet' around Purkinje cells (BERNOCCHI et at
1978) and the endoplasmic reticulum as well as the r ibosomes become organized into Nissl bodies
(LODIN et al. 1968, ALTMAN 1972). Subsequently, between the 12th and the 16th day, another
indicator of the h eterogen eity of the Purkinje cell population, namely cytoplasmic eosinophilia.
starts to be observed (BERNoccHI and SCHERINI 1980). At this point a Purkinje matUl'at ion pattern can be outlined: the establishment of syn aptic
contacts together with the presence of various enzymatic activities (WOODWARD et al. 1969,
SCHERINI and BERNOCCHI 1980) leads or is related to a change in chromatin activity; this process
is then followed by protein accumulation, responsible for the cytoplasmic eosinophilia. As pre·
viously smmised (BERNOCCHI and SCHERDII 1980) this last could be not only a "significant arti·
fact", but the true expression of a s tage of different synthetic activity.
Acknowledgements
We thank Prof. M. G. MANFREDI ROMaNINI for criticism of the manuscript. We wish to
thank Mr, S, VENERONI and Mt's , T, BOSINI VENERONI for technical assistance. The Wi star
rats were supplied by Laboratories Zambon, Milano (Italy).
Literature
ALTMAN, J., Postnatal development of the cerebellar cortex: in the rat, II: Phases in the matura·
tion of Purkinje cells and the molecular layer. J. Compo Nemol. 145.399-464 (1972).
BEDI, K. S., and GOLDSTEIN, D. J .• Apparent anomalies in nuclear Feulgen DNA contents.
Role of systematic microdensitometric errors. J. Cell BioI. 71, 68 - 88 (1976).
BERNOCCHI, G., Contcnuto in DNA e a rea nucleare dei n euroni durante l'istogenesi cerebellare
del ratto. 1st. Lomb. Sci. Lett. 109, 143-161 (1975). CRABI, M., and MAZZINI. G., Biogenic amincs during postnatal deveJoplTlent of rat cerebellum. Cell. Mol. BioI. 23, 317 - 325 (1978).
DE STEFANO, G. F., PORCELLI, F., REDI, C. A., and MANFREDI ROMANINI, M. G., Feulgen
reaction hydrolysis kinetics in the interphase and metaphase. The Nucleus 19, 141-149 (1976a).
DE VIVO, M. L., e DE STEFANO, G. F .• Individuazione istochimica di due aspetti morfo· funzionali delle cellule di Purkinje nel ccrvelletto di ratto. 1st. Lomb. Sci. Lett. no, 17 - 34 (1976b).
and MANFREDI ROMANINI, M. G., Chromatin cytochemistry as a tool for the functional inter
pretation of Purkinje's cells in rat cCI'ebellum. Riv. Istoch. Norm. Pat. 21, 131-142 (1977).
REDI, C. A., and SCHERINI. E .• Feulgen DNA content of the Purkinje neuron: "Diploid"
OJ· "Tetraploid"? Bas. Appl. H istoehem. 23, 65-70 (1979).
REDI, C. A., SCHERINI, E., BOTTIROLI, G., and FREITAS. 1., On the chromatin availability
to hydrolysis of the Feulgen reaetion in Purkinje cell population : Microdensitometric and Microfluorometric data. Bas. Appl. Histochem. 24, 121-134 (19HO).
et SCHERINI, E., Experience sur les neurones "eosinophiles". Influence de la malnutrition
prote ique maternelle sur leur apparition dans l'histogenese cerebelleuse du rat. Acta Neuro· pathol. (Berl.) 50, 159-161 (1980).
BREGNARD, A., KUENZLE, C. C., and RucH, F., Cytophotometric and autoradiographic evidence for postnatal DNA synthesis in n eurons of the rat cerebral cortex. Exp. Cell Res. 107, 151-157 (1977).
214 G. BERNOCCHI and E. SCHERINI
genesis is quite active (ALT~IAN 1972), the climbing fiber response is the same as in the adult
(PURO and WOODWARD 1977). the dendl"ite tree becomes more evident (ALTMAN 1972). fluorescent
nervouS terminals containing biogenic amines gathet' around Purkinje cells (BERNOCCHI et at
1978) and the endoplasmic reticulum as well as the r ibosomes become organized into Nissl bodies
(LODIN et al. 1968, ALTMAN 1972). Subsequently, between the 12th and the 16th day, another
indicator of the h eterogen eity of the Purkinje cell population, namely cytoplasmic eosinophilia.
starts to be observed (BERNoccHI and SCHERINI 1980). At this point a Purkinje matUl'at ion pattern can be outlined: the establishment of syn aptic
contacts together with the presence of various enzymatic activities (WOODWARD et al. 1969,
SCHERINI and BERNOCCHI 1980) leads or is related to a change in chromatin activity; this process
is then followed by protein accumulation, responsible for the cytoplasmic eosinophilia. As pre·
viously smmised (BERNOCCHI and SCHERDII 1980) this last could be not only a "significant arti·
fact", but the true expression of a s tage of different synthetic activity.
Acknowledgements
We thank Prof. M. G. MANFREDI ROMaNINI for criticism of the manuscript. We wish to
thank Mr, S, VENERONI and Mt's , T, BOSINI VENERONI for technical assistance. The Wi star
rats were supplied by Laboratories Zambon, Milano (Italy).
Literature
ALTMAN, J., Postnatal development of the cerebellar cortex: in the rat, II: Phases in the matura·
tion of Purkinje cells and the molecular layer. J. Compo Nemol. 145.399-464 (1972).
BEDI, K. S., and GOLDSTEIN, D. J .• Apparent anomalies in nuclear Feulgen DNA contents.
Role of systematic microdensitometric errors. J. Cell BioI. 71, 68 - 88 (1976).
BERNOCCHI, G., Contcnuto in DNA e a rea nucleare dei n euroni durante l'istogenesi cerebellare
del ratto. 1st. Lomb. Sci. Lett. 109, 143-161 (1975). CRABI, M., and MAZZINI. G., Biogenic amincs during postnatal deveJoplTlent of rat cerebellum. Cell. Mol. BioI. 23, 317 - 325 (1978).
DE STEFANO, G. F., PORCELLI, F., REDI, C. A., and MANFREDI ROMANINI, M. G., Feulgen
reaction hydrolysis kinetics in the interphase and metaphase. The Nucleus 19, 141-149 (1976a).
DE VIVO, M. L., e DE STEFANO, G. F .• Individuazione istochimica di due aspetti morfo· funzionali delle cellule di Purkinje nel ccrvelletto di ratto. 1st. Lomb. Sci. Lett. no, 17 - 34 (1976b).
and MANFREDI ROMANINI, M. G., Chromatin cytochemistry as a tool for the functional inter
pretation of Purkinje's cells in rat cCI'ebellum. Riv. Istoch. Norm. Pat. 21, 131-142 (1977).
REDI, C. A., and SCHERINI. E .• Feulgen DNA content of the Purkinje neuron: "Diploid"
OJ· "Tetraploid"? Bas. Appl. H istoehem. 23, 65-70 (1979).
REDI, C. A., SCHERINI, E., BOTTIROLI, G., and FREITAS. 1., On the chromatin availability
to hydrolysis of the Feulgen reaetion in Purkinje cell population : Microdensitometric and Microfluorometric data. Bas. Appl. Histochem. 24, 121-134 (19HO).
et SCHERINI, E., Experience sur les neurones "eosinophiles". Influence de la malnutrition
prote ique maternelle sur leur apparition dans l'histogenese cerebelleuse du rat. Acta Neuro· pathol. (Berl.) 50, 159-161 (1980).
BREGNARD, A., KUENZLE, C. C., and RucH, F., Cytophotometric and autoradiographic evidence for postnatal DNA synthesis in n eurons of the rat cerebral cortex. Exp. Cell Res. 107, 151-157 (1977).
Cytochemical study of chromatin changes 215
RUCH, F., LUTZ, H., and KUENZLE, C. C., Histones and DNA increase synchronously in
neurons during early postnatal development in the rat forebrain cortex. Histochemistry. 61, 271-279 (1979).
BRODSKY, V. JA., AGROSKIN, L. C., LEBEDEV, E. A., MARSHAK, T. L., PAPAJAN, G. V., SEGAL, O. L., SOKOLOVA, G. A., and JARYGIN, K. N., Stability and variations of the amount of DNA
in a population of cerebellum cells. J. Gen. BioI. (USSR) 35, 917 -925 (1974). KUDRYAVTSEV, B. N., and MARSHAK, T. L., Determination of DNA surplus in the nuclei
of some cerebellar Purkinje cells with different cytochemical methods. Bas. AppI. Histoch.
24, 401-408 (1980). MARSHAK, T. L., MARES, V., LODIN, Z., FULOP, Z., and LEBEDEV, E. V., Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. Histochemistry. 59, 233-248 (1979).
CAMERON, 1. L., POOL, M. R. H., and HOAGE, T. T., Low level incorporation of tritiated thymidine
into the nuclear DNA of Purkinje neurons of adult mice. Cell Tissue Kinet. 12, 445 - 451 (1979).
EBEL, E. J., Dark neurons: a significant artifact. The influence of maturational state of neurons
on the occurrence of the phenomenon. Acta Neuropath. (Berl.) 33, 271-273 (1975).
KUENZLE, C. C., BREGNARD, A., HUB SCHERER, U., and RUCH, F., Extra DNA in forebrain cortical
neurons. Exp. Cell Res. 1I3, 151-160 (1978).
LANGE, W., Uber einen fiirberisch darstelbaren Dimorphismus del' Purkinjezellen bei del' Maus.
Acta Histochem. (Jena). 23, 31-39 (1966).
LENTZ, R. D., and LAPHAM, L. W., Postnatal development of tetraploid DNA content in rat Purkinje cells: A quantitative cytochemical study. J. NeuropathoI. Exp. Neurol. 29,43-56 (1970).
LODIN, Z., FALTIN, .J., and MARES, V., Distribution of nucleic acid in the course of the ontogenetic
development of the nerve cells. Folia Morphol. 16, 171-183 (1968).
MANN, D. M. A., YATES, P.O., and BARTON, C. M., The DNA content of Purkinje cells in mammals. J. Compo Neurol. 180, 345-348 (1978).
MANuELIDIs, L., and MANUELIDIS, E. E., On the DNA content of cerebellar Purkinje cells in vivo and in vitro. Exp. Neurol. 43,192-206 (1974).
MARES, V., LODIN, Z., and SACHA, J., A cytochemical and autoradiographic study on nuclear DNA in mouse Purkinje cells. Brain Res. 53, 273-289 (1973).
MIKSCHE, J. P., DHILLON, S. S., BERLYN, G. P., and LANDAUER, K . • T., Nonspecific light loss and intrinsic DNA variation problems associated with Feulgen DNA cytophotometry. J. Histochem. Cytochem. 27, 1377 -1379 (1979).
PEARSE, A. G. E., Histochemistry theoretical and applied. Vol. 1. Edinburgh, London, New York, Churchill Ltd. 1975.
VON PFISTER, C., und GORNER, R., Zur Spezifitat des fluorescenzhistochemischen Nachweises del' y-Aminobuttersaure (GABA). Acta Histochem. 63, 80-88 (1978).
PURO, D. G., and WOODWARD, D. J., Maturation of evoked climbing fiber imput to rat cerebellar
SANDRITTER, \V., NOVAKOVA, V., PILNY, J., and KIEFER, G., Cytophotometrische Messungen des Nuklein-Saure und Proteingehaltes von Ganglienzellen del' Ratte wahrend del' postnatalen Entwicklung und im Alter. Z. Zellforsch. 80, 145-152 (1967).
SCHERINI, E., Effetto della fissazione per perfusione sui contenuto in DNA (materiale Feulgen
positivo) delle ccllule di Purkinje nel cervelletto <Ii ratto. Riv. Istoch. Norm. Pat. 22, 55-60 (1978).
and BERNOCCHI, G., A degradative step of GABA metabolism during postnatal cerebellar ontogenesis. Histochemical study of Succinic Semialdehyde Dehydrogenase in the Purkinje cell population. VIth Int. Histochem. Cytochem. Congr., Brighton, August 17 -22 (1980).
Cytochemical study of chromatin changes 215
RUCH, F., LUTZ, H., and KUENZLE, C. C., Histones and DNA increase synchronously in
neurons during early postnatal development in the rat forebrain cortex. Histochemistry. 61, 271-279 (1979).
BRODSKY, V. JA., AGROSKIN, L. C., LEBEDEV, E. A., MARSHAK, T. L., PAPAJAN, G. V., SEGAL, O. L., SOKOLOVA, G. A., and JARYGIN, K. N., Stability and variations of the amount of DNA
in a population of cerebellum cells. J. Gen. BioI. (USSR) 35, 917 -925 (1974). KUDRYAVTSEV, B. N., and MARSHAK, T. L., Determination of DNA surplus in the nuclei
of some cerebellar Purkinje cells with different cytochemical methods. Bas. AppI. Histoch.
24, 401-408 (1980). MARSHAK, T. L., MARES, V., LODIN, Z., FULOP, Z., and LEBEDEV, E. V., Constancy and variability in the content of DNA in cerebellar Purkinje cell nuclei. Histochemistry. 59, 233-248 (1979).
CAMERON, 1. L., POOL, M. R. H., and HOAGE, T. T., Low level incorporation of tritiated thymidine
into the nuclear DNA of Purkinje neurons of adult mice. Cell Tissue Kinet. 12, 445 - 451 (1979).
EBEL, E. J., Dark neurons: a significant artifact. The influence of maturational state of neurons
on the occurrence of the phenomenon. Acta Neuropath. (Berl.) 33, 271-273 (1975).
KUENZLE, C. C., BREGNARD, A., HUB SCHERER, U., and RUCH, F., Extra DNA in forebrain cortical
neurons. Exp. Cell Res. 1I3, 151-160 (1978).
LANGE, W., Uber einen fiirberisch darstelbaren Dimorphismus del' Purkinjezellen bei del' Maus.
Acta Histochem. (Jena). 23, 31-39 (1966).
LENTZ, R. D., and LAPHAM, L. W., Postnatal development of tetraploid DNA content in rat Purkinje cells: A quantitative cytochemical study. J. NeuropathoI. Exp. Neurol. 29,43-56 (1970).
LODIN, Z., FALTIN, .J., and MARES, V., Distribution of nucleic acid in the course of the ontogenetic
development of the nerve cells. Folia Morphol. 16, 171-183 (1968).
MANN, D. M. A., YATES, P.O., and BARTON, C. M., The DNA content of Purkinje cells in mammals. J. Compo Neurol. 180, 345-348 (1978).
MANuELIDIs, L., and MANUELIDIS, E. E., On the DNA content of cerebellar Purkinje cells in vivo and in vitro. Exp. Neurol. 43,192-206 (1974).
MARES, V., LODIN, Z., and SACHA, J., A cytochemical and autoradiographic study on nuclear DNA in mouse Purkinje cells. Brain Res. 53, 273-289 (1973).
MIKSCHE, J. P., DHILLON, S. S., BERLYN, G. P., and LANDAUER, K . • T., Nonspecific light loss and intrinsic DNA variation problems associated with Feulgen DNA cytophotometry. J. Histochem. Cytochem. 27, 1377 -1379 (1979).
PEARSE, A. G. E., Histochemistry theoretical and applied. Vol. 1. Edinburgh, London, New York, Churchill Ltd. 1975.
VON PFISTER, C., und GORNER, R., Zur Spezifitat des fluorescenzhistochemischen Nachweises del' y-Aminobuttersaure (GABA). Acta Histochem. 63, 80-88 (1978).
PURO, D. G., and WOODWARD, D. J., Maturation of evoked climbing fiber imput to rat cerebellar
SANDRITTER, \V., NOVAKOVA, V., PILNY, J., and KIEFER, G., Cytophotometrische Messungen des Nuklein-Saure und Proteingehaltes von Ganglienzellen del' Ratte wahrend del' postnatalen Entwicklung und im Alter. Z. Zellforsch. 80, 145-152 (1967).
SCHERINI, E., Effetto della fissazione per perfusione sui contenuto in DNA (materiale Feulgen
positivo) delle ccllule di Purkinje nel cervelletto <Ii ratto. Riv. Istoch. Norm. Pat. 22, 55-60 (1978).
and BERNOCCHI, G., A degradative step of GABA metabolism during postnatal cerebellar ontogenesis. Histochemical study of Succinic Semialdehyde Dehydrogenase in the Purkinje cell population. VIth Int. Histochem. Cytochem. Congr., Brighton, August 17 -22 (1980).
216 G. BERNOCClU ,md E. SCHERINI, Cytochemical study of chrom.atin changes
SCHERINI, E., BOLCHI, F., BIGGIOGERA, M., "and BERNOCCHI, G., Further evidence for different morphofunctional aspects in the Pnrkinje cell population of adult rat cerebellum. An ultra· structural study. J. Submicr. Cytol., 13, 17 -29 (1981). FOR)IENTI, F., eBERNocCHI, G., L'eosinofilia citopiasma tica dei neuroni di Purkinje : Analisi distribuzionale n elle diffel'onti aroe cerebellari. Arch. It. Anat. Embriol. 84, 389 - 397 (1979).
SUMNER, A. T., EVANS, J . , and BUCKLAXD, R. A., Mechanism involved in the banding of chromo· somes with quinacrine and Giem~a. I: the effects of fixa tion in methanol·acetic acid. Exp. Cell Res. 81,214-222 (1973).
WOODWARD, D. J., HOFFER, B . J., and LAPHAM, L. W., Postnata l development of electrical and enzyme histochemical activity in Purkinje cells. Exp. Neurol. 23, 120-139 (1969).
Address: G. BERNOCCHI, Istituto di Istologia, Embriologia e AntI'opologia Piazza Botta , 10, I· 27100 Pavia.
216 G. BERNOCClU ,md E. SCHERINI, Cytochemical study of chrom.atin changes
SCHERINI, E., BOLCHI, F., BIGGIOGERA, M., "and BERNOCCHI, G., Further evidence for different morphofunctional aspects in the Pnrkinje cell population of adult rat cerebellum. An ultra· structural study. J. Submicr. Cytol., 13, 17 -29 (1981). FOR)IENTI, F., eBERNocCHI, G., L'eosinofilia citopiasma tica dei neuroni di Purkinje : Analisi distribuzionale n elle diffel'onti aroe cerebellari. Arch. It. Anat. Embriol. 84, 389 - 397 (1979).
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Address: G. BERNOCCHI, Istituto di Istologia, Embriologia e AntI'opologia Piazza Botta , 10, I· 27100 Pavia.
