POSTNATAL CHANGES IN THE CONTENT OF BASIC AND ACIDIC PROTEINS IN RAT BRAIN ASTRiOGLfA AND ~L~G~DENDRUGL~A VmnA. taboratory
KLENIKCWA,
I_ Z,
PEVZNER
of Functional Neurochemistry, Pavlov Institute of Physiology of the Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R.
and OLGA
h&XAREK
~epartrne~~ of Neurology. Medical Academy. P~z&,
P&and
Abstract--Carnoy-fixed parat% sections of the brain white matter of S-45-day-old Wistar rats were stained with Fast Green FCT-; pi-l 2.6. for acidic fanionicf proteins or with hep~~u~A~c~an Blue for basic fcationicf proteins. Roth the hj~~hem~~l cokxxr reactions for the proteins were found to be
specifjc and to obey the ~ou~~r-Lamb~rt_B~r‘s law, C~tos~~ophoto~trj~ determinations have shown that ‘within the period between the 5th and 8th day after birth, the content both of acidic and of basic proteins per ceh in the astrocytes and o~igodendroglj~ytes of rat corpus callosum is markedly increased. In the course of subsequent postnatal development, the protein content in the neurophal cell decreased. this decrease being steeper for the basic proteins than for the acidic ones.
Based on these c~tosspectrophotometric results, as well as on previous autoradiographic data, a ~g~esti~n is put forward c~u~rniu~ two successive su#~pulati~us of ghal cells in the rat brain in the course of early postnatal development.
IN THE last few years much attention has been paid for reviews: Bm, 1969; S~KXJTER,1970; MwRE, to the metabolism of cerebral proteins in the course 1973; PALWD~N er al., 1976). Ta avoid d~~cuItjes in of brain development (for literature, see P~CAREVA~ the d~~~t~ati~n between large ghai c&s and small 1972; HIMWCH. l973; PALLADIN, BELfK & PC&YAK- nesirona the corpus calfosum was chosen as a conOVA, 1974). The majority of these studies have &aft venient brain area where neurons are absent. with the proteins extracted from the whole cerebra1 tissue homogenate. However, two main kinds of brain cetls, neurons and glia. are always present, and these EXPERYMENTAL PRQCEDURES two cell populations are fikely to differ from each Five-, g-, I?-, 25- and 4Sday-old Wistar rats of both other with respect tu a number of b~~herni~~ sexes were rapid@ d~pj~~~ without anaesthesia. The properties, ioc~ud~~g the de~~~~~rnen~a~ dynamics of brain was quiekjy taken out, fixed for f h in a chilled Carnoy solution fcthanol-chloroform-acetic acid. 6:3 : 1q by protein metabolism. The data ia the literature which volume). dehydrated and embedded in paraffin. deal with developmental changes in the macromoleFive pm thick frontal para!% sections were stained for cular metabolism of @al cells are, as a rule. derived acidic or basic proteins. To reveal acidic proteins, the se+ from the analysis of whole neurogiial ~pu~atjons
witb~ut separate consideration of the metabofism of jndividua~ types af the neur~~i~a. astrocytes and ohgodendrogbocytes. As far as we know. there are no data in the literature on the subject of a comparison of postnatal changes in the macromolecular metabo* tism of astro~~a and o~j~~~drog~ia from the same
animal. To study this question, the qu~tjtat~ve
cyto-
cbemieal approach seems to be tke most prorn~sjn~ because it makes it possible visually to identify the particular eel) type under invest~~atjan. Therefore, the aim of the present work was to eompare the developmental pattern of protein content per celt in astrocytes and o~~godendrog~j~~~. Acidic fanionic) and basic @ationic) proteins were anatysed
separately by means of quantjtatjve cytochemistry since these proteins have been shown to play different regulatory roles in the nervous tissue metabolism (see
tions were incubated for trOmin at room temperature in @.I% Fast Green FCF in 1% acetic acid, pW 2.6 (T&ITCH, 1966). To reveal basic proteins, the sections were treated for 3 h at 37°C with the saline containing 0.1 M Tris-Htl buffer, pN 10.0, 4000 i.u. sodium heparinate, and 0.2 M ethylenediamine tetra-acetic acid, then the sections were incubated for 6Qmin at room temperature in l”?; Akian Bfue, plf f&f &A,AtintE& IfRfERE,1973) Optical densities of the stained &al c&s in the sections were determined at &Burn with the aid of double-beam
recording microspectrophototneter MUF-5
(BAKMAREV,
Popav, S~vo~rsov % S~ZIRWOV. 1964) with a diameter of the ~a~~hrorna~jc ~hot~~etr~~ spot of 1 pm. The wave~~gth for the cytos~ctrophotometry was chosen at some distance from the ~orr~sp~nd~n~ spectral maxima (Fig. If, otherwise the optical density values were too high for accurate measurement. Optical density correlates only with a concentration of the hghtabsorbing substance. To find its total content per eel) the DAVYDOVA,
XARIJBINA,
VERA A. KLENIKOVA. L. 2. PEVZNER and OLGA MULAREK
1188
rather weak, at pH 6.5 negative. If histones had been extracted by treatment of the sections with HCI (0.2 N HCI for 60 min at room temperature; or 0.1 N HCI for 3 h at 30°C; or 1 N HCI for I@-20min at 60 C) the subsequent staining of the sections with heparineAlcian Blue resulted in no colour reaction at all. The extraction of histones from the sections with HCl using any of three schemes described above did not change the optical density of the cells stained for acidic proteins with Fast Green FCF, pH 2.6. On the other hand, on esterification of protein carboxylic groups by treatment of the sections with methanolconcentrated HCI for 5 h at 56‘C. the sections remained uncoloured when stained with Fast Green.
loo
60
60 % 40
20 i
loo-
80-
Applicability
of the histochemical
co/our
reactions
to
cytospectrophotometry 40-
20I
, I soo340580
I
I 620
I 660
I 700
nm
FIG.
1.
Spectral curves of the histochemical dyes in the
stained sections of rat brain. The ordinate shows optical density as a percentage of the maximal absorption taken as 100%. The absicissa shows the wavelength (in nm).
optical density of each cell that was measured was multiplied by the corresponding cell volume. The latter was calculated according to the formula of the rotational ellip soid: V = n/6 Dd’ where D and d corresponded to large and small diameters of the cell. The diameters were
measured under the microscope with the aid of the eyepiece micrometer MOV-I-15”.
Each age group of rats consisted of 6 animals. 27-30 cells of each type being measured from each rat. All the calculations as well as the statistical treatment of the data obtained according to Student’s r test were performed using a digital computer. Some of the sections were stained using a Nissl method with Toluidine Blue or thionine or with haematoxylineosine to distinguish the compared ceil types, astrocytes and oligodendrogliocytes. They were distinguished morphologically according to their size. the form and size of their nuclei. the presence and the number of nucleoli. chromatin aggregations, density of staining. etc. (MORI & LEBLOND.
1970).
Quantitative (cytospectrophotometric) determinations with the aid of any colour reactions are possible only in the case of these reactions obeying first. Bouger-Lambert’s law. i.e. a proportionality between the intensity of the cell staining and the path length of the absorbed light, and, second, Beer’s law. i.e. a proportionality between the concentration of the substance within the cell and the optical density of the dye. The former law was checked when cells were measured in sections of different thickness. As seen from Fig. 2, a linearity could be in fact demonstrated in this case. The latter law was examined in two different ways: in sections and in model systems. In sections, an indirect proof according to Hardy’s principle (see SANDRI~R, KIEFER& RICK, 1966) was obtained: if Beer’s law is obeyed the form of spectral curves in various points of the cell with different concentrations of the dye should be the same when calculated for the same relatiive absorption maximum. Sufficiently good coincidence of these spectral curves was observed, when we recorded spectra in small 1.0
0.9 Q6
F t
0
Acidic proteins J
/f
0.6 . as.
Material
Alcian Blue was purchased from Schuchardt, Mtinchen, Bundesrepublik Deutschland; Fast Green FCF from E. Merck AG. Darmstadt, Bundesrepublik Deutschland; heparin from Reanal, Budapest, Hungary.
RESULTS Control
of specificity
of the histochemical
reactions
The most intensive staining of basic proteins with heparin-Alcian Blue was observed at pH 10.0. At pH 9.0 the colour reaction was only moderate, at pH 8.0
FIG. 2. Dependence of the optical density of stained proteins in rat brain histological sections on the section thickness.
1189
Postnatal changes in neuroglia protein content
rti
”
’ ’ ”
’ ”
’ ’ ” lo
5
IS
’ ’ ”
’ ’ 1 20
mg/m\
Postnatal
Fm. 3. Dependence of the optical density of the stained polyvinylformal films soaked with proteins on the protein concentration.
areas in cells with different optical densities. In model
systems. films made of 0.5oi, polyvinylformal were soaked with different concentrations (from 5 to 25 m&ml) of crystalline serum albumin or lyophilized brain histones. Spectral curves of these proteinsoaked films stained with the histochemical colour reactions applied were rather close (Alcian Blue) or completely identical (Fast Green) to the curves registered from the stained cells of brain sections. Cytospectrophotometry of the films confirmed the linearity between the concentration of the proteins and the optical density of their complex with the corresponding histochemical dyes (Fig 3). * Comparison cgtes
ofbasic
and acidic protein content in astro-
and oligodendrogliocytes
of adult rats
The 45-day-old rats were taken as being adult. Our previous morphological and cytochemical studies confirmed that their brains were quite mature (see WENDER,KOZIK, MULAREK& O~ARZEWSKA,1974). As seen from Table 1, the total content of basic and acidic proteins per ccl1 was higher in astrocytes than in oligodendrogliocytes. This difference is due mainly to a much higher volume of each astrocyte compared with that of the oligodendrogliocyte. Meanwhile, the optical density known to correlate linearly to the protein concentration value, i.e. protein content per cell volume unit in this particular case, was somewhat lower in astrocytes than in oligodendrogliocytes
(Table 1).
TABLE
1.
PROTEIN C~N~NT
Type of neuroglial cells and kind of proteins Basic proteins Oligodendrogliocytes Astrocytes Acidic proteins Oligodendrogliocytes Astrocytes
The optical density of the dye bound to acidic proteins was twice as high as that of the dye bound to basic proteins (Table 1). Since the extinction coefllcients of the two dyes applied did not Seem to differ markedly (see Fig. 3) it is not unreasonable to suggest tentatively that both the astrocytes and the oligodendrogliocytes are richer in acidic proteins than in basic ones. in the content
of basic
proteins
In 5-day-old rats, the content of basic proteins per cell both in astrocytes and in oligodendrogliocytes of the corpus callosum was higher than in adult, 45-dayold, animals (Fig. 4). By the 8th day of postnatal development, this difference became still more pronounced. Later on, the content of basic proteins decreased so that it was even lower in IFday-old than in 5-day-old rats (particularly in oligodendrogliocytes). By 25 days after the birth, no statistically significant differences were observed as compared with 45-day-old rats (Fig. 4). Postnatal
changes
in the content
of acidic
proteins
As seen from Fig 5, the content of acidic proteins per cell was close to the adult level in 5-day-old rats but increased by the 8th day after the birth. This increase was quantitatively less pronounced than that of basic protein content (see Fig. 4). In the course of subsequent postnatal development, the acidic protein content decreased only slightly. Its content. unlike the basic protein content, was markedly higher than the adult level as late as in 25-day-old rats. The accumulation of acidic proteins in 8- and lFday-old animals was quantitatively greater in astrocytes than in oligodendrogliocytes (Fig 5). Analysis
ofglial
cell populations
with
respect
to protein
con tent
Histograms of the distribution of glial cells according to their basic (Fig 6) and acidic (Fig. 7) protein content have demonstrated that in S-day-old rats, the population both of astrocytes and of oligodendrogliocytes in the corpus callosum is sufficiently homo-
THENEun00~l~L OF ADULT WETAR
IN
changes
CELLS RAPS
OF WE
cowus
CALL~~UM
Optical density (x 100)
Cell volume (in pm3)
Protein content per cell (in arbitrary units)
40.9 * 1.2 23.8 + 0.6
4.9 f 0.2 13.2 + 0.5
197.4 f 11.2 295.4 * 10.8
65.4 f 1.4 42.0 f 0.7
5.3 + 0.2 13.9 + 0.4
338.6 + 17.6 563.0 f 16.0
As adult animals. 45-day-old rats were taken. Each mean is obtained from 160 to 180 cells from 6 animals.
VERAA. KLENIKOVA.L. Z PEVZNERand OLGA MULAREK
1190
Basic proteirn, dnys
FIG. 4. Postnatal changes in the basic protein content per cell in the glial cells of rat corpus callosum. The ordinate shows per cent deviation from the adult rat level (45-day-old animals) taken as 1003;. The abscissa shows the age of the rats.
geneous. As early as in May-old rats, however, a marked heterogeneity appeared: not only the mean value of protein content increased but the dispersion of individual cells with respect to their protein content became much higher both in oligodendroglia and particularly in astroglia. By the 17th day after the birth, the bulk of oligodendroghocytes and particuiarly of astrocytes rich in basic proteins disappeared, so the astroglial and oligodendroglial cell populations were again rather homogeneous as far as the basic protein content is concerned (Fig. 6). Distribution of the glial cells with respect to their acidic protein still remained heterogeneous in 17- and 25-day-old rats, the homogeneity of the cell populations being restored only by the 45th day after the birth in astroglia and to a lesser degree in oligodendroglia (Fig. 7). DISCUSSION Advantages and disadvantages of proteins in glial cells
of histochemical
studies
This problem was previously discussed in detail by be added that no other method but quantitative cytochemistry enables us at present to carry out a separate biochemical PEVZNER (1972). Here it should
analysis of several subpopulations of ghal cells in the same brain area. At the same time, it is to be taken into account that histochemical staining has some shortcomings. One of them is a lower resolution possible with histochemical dyes compared with histological ones. The most reliable identification of glial cell types, particularly in the developing brain, can be achieved by a combination of neuromorphological, impregnational and electron microscopic procedures. Otherwise the differentiation of glial cells in the preparations stained, for instance, only with histochemical dyes should be considered with caution. However, MORI& LEBLOND(1970) claim that just the very morphological parameters which we used in our work (form and size of the cells, density of staining, number of nucleoli. etc.) make possible a reliable identification of oligodendroglial cells, and even their subdivision into several classes of cells. Nevertheless, we cannot help thinking that the similarity observed in the whole pattern of postnatal protein content changes between astrocytes and oligodendrogliocytes should be considered as preliminary. This could. on the one hand, reflect an actual similarity in the dynamics of postnatal maturation of macromolecular metabolism
-Kx) t
FE. 5. Postnatal changes in the acidic protein content per cell in the glial cells of rat corpus callosum. All designations as those in Fig. 4.
Postnatal changes in neurogIia protein content
1191
4
0
%
16
t6
12 8 4
I2 b
0
3
t6 12 6 4 0
I6 12 6 4 0
3
3
4
4
0
0
I2
IP 8 4 0
6 4 0 I
’
’
’
t
1
t
’
02 histogram of astroplial and o~xo~ndro~a~ cells according to tbtir basic protein content per cell. The ordinate shows the penxntage of the cell number with corresponding protein content as compared to the total number of the cells measured taken as lO@oi, The abscissa shows the protein content per cell (in arbitrary units).
0
2
6
4
FIG. 6. ~jst~buti~
I2 B
B
Pi
4
4
0
0
12
I2
6 4
8 4
0
0
8
6
4
4
0
a
4 0
4 0
20 16 12
20
6
B
+i
I6 I2
4
4
0
0 AC&l& proieiis 1 0
1
1
2
i
1
4
2
i
6
0-
Fig. 7. Distribution histogram of astrogtial and otigodendroglial cells according to their acidic protein content per cell A11designations as those in Fig. 6.
1192
VERAA. KLENIKOVA. L. 2. PEVZNER and OLGAMULAREK
within these two types of glial cells. On the other hand, however, some uncertainty cannot be excluded, at least partly, in distinguishing astrocytes from oligodendrogliocytes in histochemical preparations. This point still needs further investigation. The second disadvantage of the methods used is that the histochemical dyes reveal only individual chemical residues within the whole protein molecules. This difficulty, however, is met in biochemical studies on proteins, too. All the known methods of biochemical determinations of protein, such as u.v.-absorption, the ninhydrin method, the Folin reaction, etc. also determine some particular amino acids or reactive groups of proteins. It cannot be excluded that the proportion of these residues does not change in the course of development in a way different from the bulk of the brain protein molecules. Finally, it should be borne in mind that, although some arguments in favour of the specificity of the histochemical dyes used are presented above (Figs 2 and 3). we still ignore the particular proteins which are revealed with the histochemical colour reactions. Each of these histochemical reactions results from the binding of the corresponding dye to a group of proteins rather than to an individual protein. Within each group, the proportion of the individual proteins which are likely to differ in their extinction coefficients can vary in the course of postnatal development. Therefore, this point should also be considered with some caution. Differences
in
postnatal
between astroglia
changes
of protein
content
and oligodendrogiia
The postnatal dynamics of changes in the basic protein are almost identical in astrocytes and in oligodendrogliocytes of rat corpus callosum (Fig 4). The postnatal changes in the content of acidic protein per cell in these two types of neuroglial cells (Fig. 5) are also rather similar. In fact, the differences which are revealed between astrocytes and oligodendrogliocytes with respect to the protein content are of a quantitative rather than of a qualitative character. Indeed, the degree of an initial postnatal accumulation of basic proteins in Sday-old rats as compared with adult animals is much higher in oligodendrogliocytes than in astrocytes (Fig. 4). As far as the increase in the acidic protein content is concerned, its degree, on the contrary, is somewhat higher in astroglia than in oligodendroglia (Fig. 5). DijJerences in postnatal und basic glial
changes of the content
of acidic
proteins
As seen from a comparison of Figs 4 and 5, the whole pattern of changes in postna ’ proteins is characterized by a similar feature: an mcrcase by the 5th day after the birth followed by a decrease by the 45th day of postnatal development. This adult level of glial protein content per cell is, however, achieved much earlier with respect to basic proteins rather
than to acidic ones. In this connection, it is worth recalling that for the last decade a great attention has been paid to brain-specific proteins. All of these proteins are acidic (BOGOCH, 1969; MOORE. 1973: PALLADINer al., 1976), and at least two of them have been shown immunochemically to be localized preferably in neuroglial cells (TABUCHI & KIIWH, 1975: HAGLID. HAMBERGER.HANSON. HYD~N, ~ERSSON& RBNNB;~CK,1976; BIGNAMI& DAHL, 1977). In the rat. unlike the chick, rabbit and guinea-pig, these glial acidic proteins, S-100 and glial fibrillary protein. appear in the brain as late as the 4th-5th day after the birth (BIGNAMI & DAHL, 1975; HYD~N & R~JNNBXCK,1975). It is possible that the postnatal accumulation of acidic proteins which according IO our data is particularly pronounced in astroglia (Fig. 5) can be accounted for, at least partly, by biosynthesis of glial fibrillary acidic protein. This protein. as follows from the work of BIGNAMI& DAHL (1975 : 19771 is peculiar to astrocytes. Heterogeneity
of glial cell population
in the developiny
rat corpus callosum The observation which, in our opinion, merits a detailed discussion is that a period of intensive accumulation of acidic and basic proteins in the neuroglial cells of the corpus callosum is followed by a different period. The latter is characterized by a return of the content of these proteins to the level which is equal to or even somewhat lower than the level peculiar to 5-day-old rats. Two main reasons are likely to account for this decrease in protein content: an intensive degradation of the proteins within the same cell population and,/or an appearance of a new subpopulation of the neuroglial cells with a lower protein content per cell. The autoradiographic approach can be of value in answering this question. Previously we used intraperitoneal C3H]thymidine injection to rats of similar ages (WENDERet al., 1974). It has been shown that two peaks of DNA synthesis seem to occur in the glial cells of the corpus callosum in the course of postnatal development. The first peak corresponded to the 5th. the second to the 12th day after the birth. This appears to indicate two main waves of glial cell proliferation in the corpus callosum. After the proliferation of the cells, it takes some days for the whole transcription-translation process to be completed. Therefore, it can be expected that the definitive protein pattern peculiar to these glial subpopulations should occur somewhat later. In fact, this protein pattern Seems to be formed by the beginning of the 2nd postnatal week for the first subpopulation of neuroglial cells and by the end of the 1st postnatal month for the second one (Figs 4 and 5). The appearance of the second subpopulation of glial cells in the developing corpus callosum might explain the heterogeneity of the glial cell population which is clearly seen in histograms beginning from the 8th day of postnatal development (Figs 6 and 7).
Postnatal changes in neuroglia protein content A similar pattern of glial cell proliferation was demonstrated autoradiographically by BIESOLD. BRUCKNER& MARES(1976) for the gliogenesis in the rat lateral geniculate nucleus. Perhaps such a bimodal pattern is a general feature of postnatal development of mammalian neuroglia which results from a particu-
1193
lar function of neuroglial cells, namely their participation in postnatal myelinogenesis.
Acknowledgements-We are very grateful to Mrs TAMARA P. MOR~S~VAfor her skilful technical assistance.
REFERENCES F. M., DAWDOVA M. I.. ZARUBINAI. L., Popov A. I., SKVORTSOV G. E. & SMIRNOV V. A. (1964) Microspectrophotometer for ultraviolet and visible spectra (MUF-5). Cytologiya (Leningrad) 6, 114-120 (in Russian). BIE~LD D.. BR~~CKNER G. & MARES V. (1976) An autoradiographic study of gliogenesis in the rat lateral geniculate nucleus (LGN). Brain Res. 104. 295-302. BIONAMIA. & DAHL D. (1975) Astroglial protein in the developing spinal cord of the chick embryo. Dec. Biol. 44. 204 209. BIONAMIA. &-DAHL D. (1977) Specificity of the glial fibrillary acidic protein for astroglia. J. Histochem. Cytochem. 25. 466468. B(JGOCHS. (1969) Proteins. In Hattdbook of Neurochemistry (ed. LA!THA A.). Vol. 1. pp. 75-92. Plenum Press. New York. D~ITCH A. D. (1966) Cytophotometry of proteins. In Introduction 10 Quantitative Cyrochemistry (ed. WIED G. L.), pp. 451-468. Academic Press. New York. HACLIUK. G.. HAMBERGER A.. HANSSONH.-A.. HYD~N H.. PERSSONL. & R~~NNBACK L. (1976) Cellular and subcellular distribution of the S-100 protein in rabbit and rat central nervous system. J. Neurosci. Res. 2, 175-191. HIMWICHW. (Ed.) (1973) Biochemisrry of the Developing Brain. Marcel Dekker, New York. HSII~N H. & R~~NNBAC~ L. (1975) S-100 on isolated neurons and glial cells from rat, rabbit and guinea-pig during early postnatal development. Neurobiology 5, 291-302. LABELLEJ. L. & BRIEREN. (1971) Staining of nuclear basic proteins without deoxyribonucleic acid hydrolysis using heparin and alcian blue. Actu hisrochem. 41, 338-348. MOOREB. W. (1973) Brain-specific proteins. In Proreins of Ihe Nervous System (eds SCHNUDERD. J. et a/.). pp. I-12. Raven Press. New York. MORI S. & LEBLONDC. P. (1970) Electron microscopic identification of three classes of oligodendrocytes and a preliminary study of their proliferative activity in the corpus callosum of young rats. J. camp. Neurol. 139, l-30. PALLADINA. V.. BELIKYA. V. & PCILYAKOVA N. M. (1976) Proteins of the Brain and Their Metabolism. Plenum Press. New York. PFVZNERL. Z. (1972) Macromolecular changes within neuron-neuroglia unit during behavioral events. In Macromolecules und Behavior. 2nd edn (ed. GAITO J.). pp. 335-358. Appleton-Century-Crofts, New York. PIGAREVAZ. D. (1972) Biochemistry of the Deoeloping Brain. Meditsina, Moscow (in Russian). SANURITTER W., KIEFERG. & RICK W. (1966) Gallocyanin chrome alum. In Introduction to Quantitative Cytochemisrr!, (ed. WIED G. L.), pp. 295-324. Academic Press, New York. SHOOTERE. M. (1970) Some aspects of gene expression in the nervous system. In 7he Neurosciences. Second Stud!, Progrum (ed. SCHMITTF. 0.). pp. 812-827. The Rockefeller University Press, New York. TAIX~~HIK. & KIRSCHW. M. (1975) lmmunocytochemical localization of S-100 protein in neurons and glia of hamster cerebellum. &air, Res. 92, 175-180. WENDEHM., KOZIK M.. MULAREK0. & O~ARZEWSKAE. (1974) Incorporation of C3H]thymidine into neuroglial cells in the course of myelinogenesis. Folia histochem. cytochem. 12, 115-124. BAKHAREV
(Accepted
26 January
1979)
KLENIKCWA,
I_ Z,
PEVZNER
of Functional Neurochemistry, Pavlov Institute of Physiology of the Academy of Sciences of the U.S.S.R., Leningrad, U.S.S.R.
and OLGA
h&XAREK
~epartrne~~ of Neurology. Medical Academy. P~z&,
P&and
Abstract--Carnoy-fixed parat% sections of the brain white matter of S-45-day-old Wistar rats were stained with Fast Green FCT-; pi-l 2.6. for acidic fanionicf proteins or with hep~~u~A~c~an Blue for basic fcationicf proteins. Roth the hj~~hem~~l cokxxr reactions for the proteins were found to be
specifjc and to obey the ~ou~~r-Lamb~rt_B~r‘s law, C~tos~~ophoto~trj~ determinations have shown that ‘within the period between the 5th and 8th day after birth, the content both of acidic and of basic proteins per ceh in the astrocytes and o~igodendroglj~ytes of rat corpus callosum is markedly increased. In the course of subsequent postnatal development, the protein content in the neurophal cell decreased. this decrease being steeper for the basic proteins than for the acidic ones.
Based on these c~tosspectrophotometric results, as well as on previous autoradiographic data, a ~g~esti~n is put forward c~u~rniu~ two successive su#~pulati~us of ghal cells in the rat brain in the course of early postnatal development.
IN THE last few years much attention has been paid for reviews: Bm, 1969; S~KXJTER,1970; MwRE, to the metabolism of cerebral proteins in the course 1973; PALWD~N er al., 1976). Ta avoid d~~cuItjes in of brain development (for literature, see P~CAREVA~ the d~~~t~ati~n between large ghai c&s and small 1972; HIMWCH. l973; PALLADIN, BELfK & PC&YAK- nesirona the corpus calfosum was chosen as a conOVA, 1974). The majority of these studies have &aft venient brain area where neurons are absent. with the proteins extracted from the whole cerebra1 tissue homogenate. However, two main kinds of brain cetls, neurons and glia. are always present, and these EXPERYMENTAL PRQCEDURES two cell populations are fikely to differ from each Five-, g-, I?-, 25- and 4Sday-old Wistar rats of both other with respect tu a number of b~~herni~~ sexes were rapid@ d~pj~~~ without anaesthesia. The properties, ioc~ud~~g the de~~~~~rnen~a~ dynamics of brain was quiekjy taken out, fixed for f h in a chilled Carnoy solution fcthanol-chloroform-acetic acid. 6:3 : 1q by protein metabolism. The data ia the literature which volume). dehydrated and embedded in paraffin. deal with developmental changes in the macromoleFive pm thick frontal para!% sections were stained for cular metabolism of @al cells are, as a rule. derived acidic or basic proteins. To reveal acidic proteins, the se+ from the analysis of whole neurogiial ~pu~atjons
witb~ut separate consideration of the metabofism of jndividua~ types af the neur~~i~a. astrocytes and ohgodendrogbocytes. As far as we know. there are no data in the literature on the subject of a comparison of postnatal changes in the macromolecular metabo* tism of astro~~a and o~j~~~drog~ia from the same
animal. To study this question, the qu~tjtat~ve
cyto-
cbemieal approach seems to be tke most prorn~sjn~ because it makes it possible visually to identify the particular eel) type under invest~~atjan. Therefore, the aim of the present work was to eompare the developmental pattern of protein content per celt in astrocytes and o~~godendrog~j~~~. Acidic fanionic) and basic @ationic) proteins were anatysed
separately by means of quantjtatjve cytochemistry since these proteins have been shown to play different regulatory roles in the nervous tissue metabolism (see
tions were incubated for trOmin at room temperature in @.I% Fast Green FCF in 1% acetic acid, pW 2.6 (T&ITCH, 1966). To reveal basic proteins, the sections were treated for 3 h at 37°C with the saline containing 0.1 M Tris-Htl buffer, pN 10.0, 4000 i.u. sodium heparinate, and 0.2 M ethylenediamine tetra-acetic acid, then the sections were incubated for 6Qmin at room temperature in l”?; Akian Bfue, plf f&f &A,AtintE& IfRfERE,1973) Optical densities of the stained &al c&s in the sections were determined at &Burn with the aid of double-beam
recording microspectrophototneter MUF-5
(BAKMAREV,
Popav, S~vo~rsov % S~ZIRWOV. 1964) with a diameter of the ~a~~hrorna~jc ~hot~~etr~~ spot of 1 pm. The wave~~gth for the cytos~ctrophotometry was chosen at some distance from the ~orr~sp~nd~n~ spectral maxima (Fig. If, otherwise the optical density values were too high for accurate measurement. Optical density correlates only with a concentration of the hghtabsorbing substance. To find its total content per eel) the DAVYDOVA,
XARIJBINA,
VERA A. KLENIKOVA. L. 2. PEVZNER and OLGA MULAREK
1188
rather weak, at pH 6.5 negative. If histones had been extracted by treatment of the sections with HCI (0.2 N HCI for 60 min at room temperature; or 0.1 N HCI for 3 h at 30°C; or 1 N HCI for I@-20min at 60 C) the subsequent staining of the sections with heparineAlcian Blue resulted in no colour reaction at all. The extraction of histones from the sections with HCl using any of three schemes described above did not change the optical density of the cells stained for acidic proteins with Fast Green FCF, pH 2.6. On the other hand, on esterification of protein carboxylic groups by treatment of the sections with methanolconcentrated HCI for 5 h at 56‘C. the sections remained uncoloured when stained with Fast Green.
loo
60
60 % 40
20 i
loo-
80-
Applicability
of the histochemical
co/our
reactions
to
cytospectrophotometry 40-
20I
, I soo340580
I
I 620
I 660
I 700
nm
FIG.
1.
Spectral curves of the histochemical dyes in the
stained sections of rat brain. The ordinate shows optical density as a percentage of the maximal absorption taken as 100%. The absicissa shows the wavelength (in nm).
optical density of each cell that was measured was multiplied by the corresponding cell volume. The latter was calculated according to the formula of the rotational ellip soid: V = n/6 Dd’ where D and d corresponded to large and small diameters of the cell. The diameters were
measured under the microscope with the aid of the eyepiece micrometer MOV-I-15”.
Each age group of rats consisted of 6 animals. 27-30 cells of each type being measured from each rat. All the calculations as well as the statistical treatment of the data obtained according to Student’s r test were performed using a digital computer. Some of the sections were stained using a Nissl method with Toluidine Blue or thionine or with haematoxylineosine to distinguish the compared ceil types, astrocytes and oligodendrogliocytes. They were distinguished morphologically according to their size. the form and size of their nuclei. the presence and the number of nucleoli. chromatin aggregations, density of staining. etc. (MORI & LEBLOND.
1970).
Quantitative (cytospectrophotometric) determinations with the aid of any colour reactions are possible only in the case of these reactions obeying first. Bouger-Lambert’s law. i.e. a proportionality between the intensity of the cell staining and the path length of the absorbed light, and, second, Beer’s law. i.e. a proportionality between the concentration of the substance within the cell and the optical density of the dye. The former law was checked when cells were measured in sections of different thickness. As seen from Fig. 2, a linearity could be in fact demonstrated in this case. The latter law was examined in two different ways: in sections and in model systems. In sections, an indirect proof according to Hardy’s principle (see SANDRI~R, KIEFER& RICK, 1966) was obtained: if Beer’s law is obeyed the form of spectral curves in various points of the cell with different concentrations of the dye should be the same when calculated for the same relatiive absorption maximum. Sufficiently good coincidence of these spectral curves was observed, when we recorded spectra in small 1.0
0.9 Q6
F t
0
Acidic proteins J
/f
0.6 . as.
Material
Alcian Blue was purchased from Schuchardt, Mtinchen, Bundesrepublik Deutschland; Fast Green FCF from E. Merck AG. Darmstadt, Bundesrepublik Deutschland; heparin from Reanal, Budapest, Hungary.
RESULTS Control
of specificity
of the histochemical
reactions
The most intensive staining of basic proteins with heparin-Alcian Blue was observed at pH 10.0. At pH 9.0 the colour reaction was only moderate, at pH 8.0
FIG. 2. Dependence of the optical density of stained proteins in rat brain histological sections on the section thickness.
1189
Postnatal changes in neuroglia protein content
rti
”
’ ’ ”
’ ”
’ ’ ” lo
5
IS
’ ’ ”
’ ’ 1 20
mg/m\
Postnatal
Fm. 3. Dependence of the optical density of the stained polyvinylformal films soaked with proteins on the protein concentration.
areas in cells with different optical densities. In model
systems. films made of 0.5oi, polyvinylformal were soaked with different concentrations (from 5 to 25 m&ml) of crystalline serum albumin or lyophilized brain histones. Spectral curves of these proteinsoaked films stained with the histochemical colour reactions applied were rather close (Alcian Blue) or completely identical (Fast Green) to the curves registered from the stained cells of brain sections. Cytospectrophotometry of the films confirmed the linearity between the concentration of the proteins and the optical density of their complex with the corresponding histochemical dyes (Fig 3). * Comparison cgtes
ofbasic
and acidic protein content in astro-
and oligodendrogliocytes
of adult rats
The 45-day-old rats were taken as being adult. Our previous morphological and cytochemical studies confirmed that their brains were quite mature (see WENDER,KOZIK, MULAREK& O~ARZEWSKA,1974). As seen from Table 1, the total content of basic and acidic proteins per ccl1 was higher in astrocytes than in oligodendrogliocytes. This difference is due mainly to a much higher volume of each astrocyte compared with that of the oligodendrogliocyte. Meanwhile, the optical density known to correlate linearly to the protein concentration value, i.e. protein content per cell volume unit in this particular case, was somewhat lower in astrocytes than in oligodendrogliocytes
(Table 1).
TABLE
1.
PROTEIN C~N~NT
Type of neuroglial cells and kind of proteins Basic proteins Oligodendrogliocytes Astrocytes Acidic proteins Oligodendrogliocytes Astrocytes
The optical density of the dye bound to acidic proteins was twice as high as that of the dye bound to basic proteins (Table 1). Since the extinction coefllcients of the two dyes applied did not Seem to differ markedly (see Fig. 3) it is not unreasonable to suggest tentatively that both the astrocytes and the oligodendrogliocytes are richer in acidic proteins than in basic ones. in the content
of basic
proteins
In 5-day-old rats, the content of basic proteins per cell both in astrocytes and in oligodendrogliocytes of the corpus callosum was higher than in adult, 45-dayold, animals (Fig. 4). By the 8th day of postnatal development, this difference became still more pronounced. Later on, the content of basic proteins decreased so that it was even lower in IFday-old than in 5-day-old rats (particularly in oligodendrogliocytes). By 25 days after the birth, no statistically significant differences were observed as compared with 45-day-old rats (Fig. 4). Postnatal
changes
in the content
of acidic
proteins
As seen from Fig 5, the content of acidic proteins per cell was close to the adult level in 5-day-old rats but increased by the 8th day after the birth. This increase was quantitatively less pronounced than that of basic protein content (see Fig. 4). In the course of subsequent postnatal development, the acidic protein content decreased only slightly. Its content. unlike the basic protein content, was markedly higher than the adult level as late as in 25-day-old rats. The accumulation of acidic proteins in 8- and lFday-old animals was quantitatively greater in astrocytes than in oligodendrogliocytes (Fig 5). Analysis
ofglial
cell populations
with
respect
to protein
con tent
Histograms of the distribution of glial cells according to their basic (Fig 6) and acidic (Fig. 7) protein content have demonstrated that in S-day-old rats, the population both of astrocytes and of oligodendrogliocytes in the corpus callosum is sufficiently homo-
THENEun00~l~L OF ADULT WETAR
IN
changes
CELLS RAPS
OF WE
cowus
CALL~~UM
Optical density (x 100)
Cell volume (in pm3)
Protein content per cell (in arbitrary units)
40.9 * 1.2 23.8 + 0.6
4.9 f 0.2 13.2 + 0.5
197.4 f 11.2 295.4 * 10.8
65.4 f 1.4 42.0 f 0.7
5.3 + 0.2 13.9 + 0.4
338.6 + 17.6 563.0 f 16.0
As adult animals. 45-day-old rats were taken. Each mean is obtained from 160 to 180 cells from 6 animals.
VERAA. KLENIKOVA.L. Z PEVZNERand OLGA MULAREK
1190
Basic proteirn, dnys
FIG. 4. Postnatal changes in the basic protein content per cell in the glial cells of rat corpus callosum. The ordinate shows per cent deviation from the adult rat level (45-day-old animals) taken as 1003;. The abscissa shows the age of the rats.
geneous. As early as in May-old rats, however, a marked heterogeneity appeared: not only the mean value of protein content increased but the dispersion of individual cells with respect to their protein content became much higher both in oligodendroglia and particularly in astroglia. By the 17th day after the birth, the bulk of oligodendroghocytes and particuiarly of astrocytes rich in basic proteins disappeared, so the astroglial and oligodendroglial cell populations were again rather homogeneous as far as the basic protein content is concerned (Fig. 6). Distribution of the glial cells with respect to their acidic protein still remained heterogeneous in 17- and 25-day-old rats, the homogeneity of the cell populations being restored only by the 45th day after the birth in astroglia and to a lesser degree in oligodendroglia (Fig. 7). DISCUSSION Advantages and disadvantages of proteins in glial cells
of histochemical
studies
This problem was previously discussed in detail by be added that no other method but quantitative cytochemistry enables us at present to carry out a separate biochemical PEVZNER (1972). Here it should
analysis of several subpopulations of ghal cells in the same brain area. At the same time, it is to be taken into account that histochemical staining has some shortcomings. One of them is a lower resolution possible with histochemical dyes compared with histological ones. The most reliable identification of glial cell types, particularly in the developing brain, can be achieved by a combination of neuromorphological, impregnational and electron microscopic procedures. Otherwise the differentiation of glial cells in the preparations stained, for instance, only with histochemical dyes should be considered with caution. However, MORI& LEBLOND(1970) claim that just the very morphological parameters which we used in our work (form and size of the cells, density of staining, number of nucleoli. etc.) make possible a reliable identification of oligodendroglial cells, and even their subdivision into several classes of cells. Nevertheless, we cannot help thinking that the similarity observed in the whole pattern of postnatal protein content changes between astrocytes and oligodendrogliocytes should be considered as preliminary. This could. on the one hand, reflect an actual similarity in the dynamics of postnatal maturation of macromolecular metabolism
-Kx) t
FE. 5. Postnatal changes in the acidic protein content per cell in the glial cells of rat corpus callosum. All designations as those in Fig. 4.
Postnatal changes in neurogIia protein content
1191
4
0
%
16
t6
12 8 4
I2 b
0
3
t6 12 6 4 0
I6 12 6 4 0
3
3
4
4
0
0
I2
IP 8 4 0
6 4 0 I
’
’
’
t
1
t
’
02 histogram of astroplial and o~xo~ndro~a~ cells according to tbtir basic protein content per cell. The ordinate shows the penxntage of the cell number with corresponding protein content as compared to the total number of the cells measured taken as lO@oi, The abscissa shows the protein content per cell (in arbitrary units).
0
2
6
4
FIG. 6. ~jst~buti~
I2 B
B
Pi
4
4
0
0
12
I2
6 4
8 4
0
0
8
6
4
4
0
a
4 0
4 0
20 16 12
20
6
B
+i
I6 I2
4
4
0
0 AC&l& proieiis 1 0
1
1
2
i
1
4
2
i
6
0-
Fig. 7. Distribution histogram of astrogtial and otigodendroglial cells according to their acidic protein content per cell A11designations as those in Fig. 6.
1192
VERAA. KLENIKOVA. L. 2. PEVZNER and OLGAMULAREK
within these two types of glial cells. On the other hand, however, some uncertainty cannot be excluded, at least partly, in distinguishing astrocytes from oligodendrogliocytes in histochemical preparations. This point still needs further investigation. The second disadvantage of the methods used is that the histochemical dyes reveal only individual chemical residues within the whole protein molecules. This difficulty, however, is met in biochemical studies on proteins, too. All the known methods of biochemical determinations of protein, such as u.v.-absorption, the ninhydrin method, the Folin reaction, etc. also determine some particular amino acids or reactive groups of proteins. It cannot be excluded that the proportion of these residues does not change in the course of development in a way different from the bulk of the brain protein molecules. Finally, it should be borne in mind that, although some arguments in favour of the specificity of the histochemical dyes used are presented above (Figs 2 and 3). we still ignore the particular proteins which are revealed with the histochemical colour reactions. Each of these histochemical reactions results from the binding of the corresponding dye to a group of proteins rather than to an individual protein. Within each group, the proportion of the individual proteins which are likely to differ in their extinction coefficients can vary in the course of postnatal development. Therefore, this point should also be considered with some caution. Differences
in
postnatal
between astroglia
changes
of protein
content
and oligodendrogiia
The postnatal dynamics of changes in the basic protein are almost identical in astrocytes and in oligodendrogliocytes of rat corpus callosum (Fig 4). The postnatal changes in the content of acidic protein per cell in these two types of neuroglial cells (Fig. 5) are also rather similar. In fact, the differences which are revealed between astrocytes and oligodendrogliocytes with respect to the protein content are of a quantitative rather than of a qualitative character. Indeed, the degree of an initial postnatal accumulation of basic proteins in Sday-old rats as compared with adult animals is much higher in oligodendrogliocytes than in astrocytes (Fig. 4). As far as the increase in the acidic protein content is concerned, its degree, on the contrary, is somewhat higher in astroglia than in oligodendroglia (Fig. 5). DijJerences in postnatal und basic glial
changes of the content
of acidic
proteins
As seen from a comparison of Figs 4 and 5, the whole pattern of changes in postna ’ proteins is characterized by a similar feature: an mcrcase by the 5th day after the birth followed by a decrease by the 45th day of postnatal development. This adult level of glial protein content per cell is, however, achieved much earlier with respect to basic proteins rather
than to acidic ones. In this connection, it is worth recalling that for the last decade a great attention has been paid to brain-specific proteins. All of these proteins are acidic (BOGOCH, 1969; MOORE. 1973: PALLADINer al., 1976), and at least two of them have been shown immunochemically to be localized preferably in neuroglial cells (TABUCHI & KIIWH, 1975: HAGLID. HAMBERGER.HANSON. HYD~N, ~ERSSON& RBNNB;~CK,1976; BIGNAMI& DAHL, 1977). In the rat. unlike the chick, rabbit and guinea-pig, these glial acidic proteins, S-100 and glial fibrillary protein. appear in the brain as late as the 4th-5th day after the birth (BIGNAMI & DAHL, 1975; HYD~N & R~JNNBXCK,1975). It is possible that the postnatal accumulation of acidic proteins which according IO our data is particularly pronounced in astroglia (Fig. 5) can be accounted for, at least partly, by biosynthesis of glial fibrillary acidic protein. This protein. as follows from the work of BIGNAMI& DAHL (1975 : 19771 is peculiar to astrocytes. Heterogeneity
of glial cell population
in the developiny
rat corpus callosum The observation which, in our opinion, merits a detailed discussion is that a period of intensive accumulation of acidic and basic proteins in the neuroglial cells of the corpus callosum is followed by a different period. The latter is characterized by a return of the content of these proteins to the level which is equal to or even somewhat lower than the level peculiar to 5-day-old rats. Two main reasons are likely to account for this decrease in protein content: an intensive degradation of the proteins within the same cell population and,/or an appearance of a new subpopulation of the neuroglial cells with a lower protein content per cell. The autoradiographic approach can be of value in answering this question. Previously we used intraperitoneal C3H]thymidine injection to rats of similar ages (WENDERet al., 1974). It has been shown that two peaks of DNA synthesis seem to occur in the glial cells of the corpus callosum in the course of postnatal development. The first peak corresponded to the 5th. the second to the 12th day after the birth. This appears to indicate two main waves of glial cell proliferation in the corpus callosum. After the proliferation of the cells, it takes some days for the whole transcription-translation process to be completed. Therefore, it can be expected that the definitive protein pattern peculiar to these glial subpopulations should occur somewhat later. In fact, this protein pattern Seems to be formed by the beginning of the 2nd postnatal week for the first subpopulation of neuroglial cells and by the end of the 1st postnatal month for the second one (Figs 4 and 5). The appearance of the second subpopulation of glial cells in the developing corpus callosum might explain the heterogeneity of the glial cell population which is clearly seen in histograms beginning from the 8th day of postnatal development (Figs 6 and 7).
Postnatal changes in neuroglia protein content A similar pattern of glial cell proliferation was demonstrated autoradiographically by BIESOLD. BRUCKNER& MARES(1976) for the gliogenesis in the rat lateral geniculate nucleus. Perhaps such a bimodal pattern is a general feature of postnatal development of mammalian neuroglia which results from a particu-
1193
lar function of neuroglial cells, namely their participation in postnatal myelinogenesis.
Acknowledgements-We are very grateful to Mrs TAMARA P. MOR~S~VAfor her skilful technical assistance.
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(Accepted
26 January
1979)
