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S-100 in the central nervous system of rat, rabbit and guinea pig during postnatal development
Brain Research, 123 (1977) 331-345
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
331
S-100 IN THE CENTRAL NERVOUS SYSTEM OF RAT, RABBIT AND GUINEA PIG DURING POSTNATAL DEVELOPMENT
K. G.
HAGLID, H.-A. HANSSON and L. RONNB~CK
Institute of Neurobiology, Universityof Giiteborg, Giiteborg (Sweden)
(Accepted July 6th, 1976)
SUMMARY The accumulation o f the brain-specific S-100 protein has been studied during postnatal development of rat, rabbit and guinea pig quantitatively, using immunoelectrophoresis, and qualitatively, by immunoelectron microscopy. Newborn guinea pigs show high levels of S-100. The distribution was similar to that of adult animals with an enrichment of S-100 to the postsynaptic membranes and to the astrocytic filaments. The neuronal plasma membranes as well as the neuronal nuclear membranes, astrocytic and oligodendroglial plasma membranes, also showed a specific activity for S-100. The amount of S-100 increased linearly from birth until the 3rd and 4th postnatal week of rabbit and rat, respectively. During the 2nd and 3rd week rabbit and rat nervous systems showed an accumulation of S-100, especially in the postsynaptic membranes and in the astrocytic filaments. In this study we present evidence that the S-100 protein quantitatively and ultrastructurally appears according to a pattern which parallels the maturation of brain, showing adult characteristics already at birth in early developing brains (guinea pig) and a change towards adult pattern after birth in late developing brains (rat and rabbit). In the latter two species change towards an adult S-100 distribution pattern proceeds during the postnatal period concomitant with the enzymatic and electrophysiologicai maturation of the brain.
INTRODUCTION A number of brain proteins have been isolated during the past 10 years4, ~o,as, 40,4a,52. The acidic S-100 protein 3s has been most extensively studied. It is present both in glial cellsl-a,7,13,27,a4,47 and in neuronslT,ls,22,2a,27-ao,4a,51 of the central
332 nervous system. We have earlier localized S-100 to the postsynaptic membranes by immunoelectron microscopy17, and to the surface of isolated neurons by immunofluorescence zs,3°, by immunoelectron microscopy23 and by Sepharose 4B spherules, to which anti-S-100 antibodies were coupled29. We have also studied the appearance of S-100 on neuronal cell membranes during postnatal development30. In this paper we correlate changes in the levels of the S-100 protein and its appearance in brain ultrastructures during postnatal maturation of early (guinea pig) and late developing (rat and rabbit) central nervous systems. MATERIALS AND METHODS
Animals Rats, rabbits and guinea pigs during early postnatal period were used in this study (newborn, 1-6 weeks old and adult animals).
Preparation of the S-IO0 protein S-100 was prepared according to Moore as, except that all buffers during purification contained 2.5 mM EDTA and 0.1 mM 2-mercaptoethanol. The purity of the protein was controlled by disc electrophoresis (15 ~ polyacrylamide)11,42, by electrophoresis in a continuous buffer system (7.5 ~ polyacrylamide)5, and by electrophoresis in SDS (15~ polyacrylamide)4L No other proteins were discovered in the electrophoretic pattern.
Production of antiserum against the beef S-IO0 protein Antibodies to beef-brain S-100 were prepared, using as antigen the protein conjugated to methylated bovine serum albumin 4~. To 1 ml of 0.15 M NaC1, containing 1 mg of S-100, was added 0.1 ml of 1 ~ (w/v) methylated bovine serum albumin. S-100 methylated bovine serum albumin flocculent suspension (!. 1 ml) was emulsified with an equal volume of complete Freund's adjuvant and injected into the toepads and hind subcutis of albino rabbits, once weekly for 4 weeks. Ten weeks after the last injection, a single intravenous injection of the S-100-methylated bovine serum albumin in 1.1 ml of 0.15 M NaCI was given as a booster. Ten days after the intravenous injection the animals were bled through the marginal ear vein, and the blood was collected for antiserum. The specificity of the antiserum was tested by complement fixation 31,41 against different organ extracts such as liver, kidney, spleen, lung and blood. A 10,000-fold higher level of antigen expressed on protein bases was found in brain. Immunoelectrophoresis in agarose (see below) revealed a single precipitation line against brain homogenate and beef S-100 protein in the most anodal part of the gel not present in extracts from other organs, even if the protein amount of the organ extracts were at the load limit of the agarose gel electrophoresis.
Preparation of antisera for immunoelectron microscopy The antiserum against S-100 was labelled with peroxidase by mixing antiserum with glutaraldehyde and horseradish peroxidase (type VI, Sigma Chemical Co., St. Louis, Mo. U.S.A.) in optimal proportions 17,1s. This mixture was shaken for 90 min at
333 room temperature. The anti-beef S-100 rabbit serum conjugated with peroxidase was separated from free peroxidase by elution from a G-75 Sephadex column (Pharmacia Fine Chemicals Co., Uppsala, Sweden). The fractions containing the peroxidaselabeled proteins were identified by their absorption at 280 nm. The conjugate was absorbed with liver powder 2~ (anti-S-100-peroxidase-liver abs.). Half of this liverabsorbed anti-S-100 antiserum was absorbed with beef S-100 protein by incubation in a shaking waterbath for 1 h at 37 °C, and for 12 h at + 4 °C. The S-100-absorbed conjugate was thereafter centrifuged for 30 min at 10,000 × g. The pellet containing the S-100-anti-S-100 precipitate was discarded. The procedure was repeated several times until the antiserum was free from antibodies against S-100 as confirmed by complement fixation at the nanogram levelal,aL The S- 100-absorbed conjugate (anti-S- 100peroxidase-liver-S-100 abs.) was used as a control during immunoelectron microscopy. Conjugated sera were stored at q- 4 °C and were stable for two weeks after preparation.
Specificity tests of the antigen-antibody reaction The specificity of the antiserum was tested in the following ways. (1) A single precipitation band was obtained when the antiserum was tested by double diffusion in 1 ~ agar against purified S-100. (2) Microcomplement fixation31,41 at the nanogram level with the antiserum prepared detected S-100 only in brain and gave a positive result for pure S-100. No activity was found in other tissues, including liver, kidney, spleen, lung and blood, unless 10,000 times higher extract concentrations (mg protein/ ml) were used.
Preparation of tissue materials for immunoeleetrophoresis The brains of the animals were divided into three equal parts, frontal, middle and dorsal. Surface (mostly grey matter) and deeper (mostly white matter) layers of the cortex were dissected out on ice from all animals, except for newborn and 1-week-old rats and rabbits where a 1 mm thick layer from frontal, middle and dorsal cortex was removed. Cerebellum and brain stem under the IVth ventricle were also used in all animals. The tissue samples were weighed and quickly frozen in tight fitting boxes at --80 °C until used. The samples were homogenized in 20:1 (v/w), (10:1 v/w for rat and rabbit 1 and 7 days old) barbital buffer (pH 8.6, ionic strength 0.02) containing 2.5 mM EDTA and 0.1 mM 2-mercaptoethanol, and centrifuged for 60 min at 105,000 × g. The supernatants which contained soluble proteins were used for quantitative immunoelectrophoresis described below. After 2-mercaptoethanol had been removed by lyophilization, protein was determined according to Lowry et al. z5 both in supernatants and homogenates.
Quantitative immunoelectrophoresis The quantitation of S-100 was carried out according to the procedure of Laurel132 modified for this (soluble, low molecular weight S-100) protein by Stavrou et al. 5°. The immunoelectrophoresis was performed in 1 ~ Litex agarose (Glostrup, Denmark) containing 1 ~ polyethylene glycol (M.W. 6000), in barbital buffer (pH
334 8.6, ionic strength 0.02), containing 2.5 m M EDTA, 0.1 m M 2-mercaptoethanol and 0 . 4 ~ anti-beef S-100 rabbit serum for 6 h at a current of 1 mA/cm at 15 °C.
lmmunoelectron microscopic procedure The animals were anaesthetized with ether or Mebumal and fixed by perfusion via the ascending aorta during l0 min. Tissue pieces from the frontal, parietal and visual lobes, from the optic nerve and from the cerebellum were dissected, fixed by immersion for a further 1-2 h and then rinsed in cold 0.15 M cacodylate buffer, pH 7.2, containing 1 mM EDTA. The fixation solution contained 4 ~ freshly prepared formaldehyde and 1 m M EDTA in 0.15 M cacodylate buffer (pH 7.2). In some experiments 0.1 ~ picric acid was added to the fixative. The fixed and rinsed tissue pieces were sectioned in a Vibratome (Oxford), at a nominal thickness of 20 #m. The sections were incubated for 45 min in anti-S-100-peroxidase-liver abs. conjugate or in anti-S100-peroxidase-liver-S-100 abs. conjugate in serial dilutions with 0.15 M cacodylate buffer, pH 7.2, containing 1 m M EDTA, until the difference between the test and the control antiserum was optimal. The sections were rinsed in buffer and incubated in cacodylate buffer containing 0.05 ~o 3,3-diaminobenzidine and 0.01 ~ hydrogen peroxide 16. After rinsing, the sections were postfixed in 2 ~ osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol and embedded in Epon. Thin sections were prepared on a LKB Ultrotome III and studied in a Siemens Elmiscope 1 A. Sections with S-100-absorbed antiserum (dilution of antiserum and absorbed antiserum 1:350) were used as controls. RESULTS To rule out the possibility that the observed changes in the levels of S-100 during development were simply the result of alterations in water content in the brain, levels of S-100 have been expressed both as/zg S-100 per g wet weight (Fig. 1) and as #g of S-100 protein per mg of total protein (Fig. 2). Our values of S-100 in the three adult animal types are higher than values previously reported in the literature 24,3a. This might be due to differences in extraction procedures. We avoided Ca 2+ effects on S-100 protein during the homogenization of the material, since it has been reported that Ca 2+ increases the level of tissue-bound S-100 ~9. We also used osmotic shock to release S-100 protein from the homogenized material. Preliminary results indicate that if osmotic shock was compared to isotonic sucrose containing 0.1 ~ Triton as extraction solution, a 2-fold larger amount of S- 100 per g wet weight tissue was liberated by osmotic shock as compared with the sucroseTriton extraction methodlL That osmotic shock treatment is a more effective extraction procedure than high or low ionic strength solutions 4s indicates that the S-100 might be trapped inside cellular compartments. In order to determine whether S-100 from rat and guinea pig brain had different relative mobilities, supernatants from frontal cortex and cerebellum were run on 7.5 polyacrylamide gel electrophoresis together with pure S-100 as standards, according to Calissano et al). After destaining of the gels, densitometry 21 was made on the front
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Age days Fig. ]. Diagrams showing the accumulation of S-|00 protein (yg S-]00/g wet weight) during postnatal development of (A) rat, (B) rabbit and (C) guinea pig. F, frontal brain, superficial part; M, middle brain, superficial part; D, dorsal brain, superficial part; C, cerebellum; BS, brain stem under the IVth ventricle. More or less parallel curves were obtained regarding S-]00 accumulation in deeper parts of frontal, middle and dorsal brain. The differences between values from superfficial and deeper parts of brain were not statistically significant, probably due to difficulties of obtaining only grey and w h i t e m a t t e r a t dissection. S . D . s a r e i n d i c a t e d .
bands which react immunologically with S-100 antiserum. The relative mobilities of the front band from guinea pig frontal cortex and cerebellum was 1.096, i.e. somewhat higher than that of rat brain frontal cortex and cerebellum (1.085) while pure S-100 from bovine whole brain had a relative mobility of 1.083 as determined in our electrophoresis system. The amount of protein in the front band expressed per mg protein on
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Fig. 2. Similar diagram to that in Fig. 1. In this diagram S-100 accumulation has been expressed as fig S-100/mg soluble protein. A: rat; B: rabbit and C: guinea pig.
the gel is shown in Table I. The results are comparable to, although lower than those obtained using quantitative immunoelectrophoresis, indicating a high content of S-100 in guinea pig brain already at birth. The possibility that guinea pig S-100 has a higher mobility on agarose (1%) gel electrophoresis and therefore higher values are obtained from guinea pig brain when determined immunoelectrophoretically cannot be excluded. It is also a possibility that immunological species differences in the absence of Ca 2+ as in our immunoelectrophoretic system are larger than the small immunological species difference previously reported for the S-100 protein measured in a sys-
337 TABLE I Protein content (l~g) in front band expressed per mg total protein on the gel (7.5 % polyacrylamide gel electrophoresis as described in the text} i~g protein in front band ( S-IO0) per rng total protein Cerebellum
Frontal cortex
Rat Newborn 7 days 14 days Adult
0 11.5 23.5 54.5
0 5.0 12.3 36.4
Guinea pig Newborn 7 days 14 days Adult
66.0 78.2 90.9 156.6
34.0 55.2 70.5 89.5
tem which contains Ca 2+. Due to this the S-100 protein is expressed in beef-S-100 protein equivalents. In guinea pig brain, rather high levels o f S-100 were already f o u n d at birth as determined by immunoelectrophoresis 5°. The a m o u n t o f S-100 increased 2-3-fold f r o m birth until adult age (Fig. 1). Using immunoelectron microscopy with anti-S-100
Fig. 3. Synapses from frontal cortex of guinea pig, 2 days old. A heavy anti-S-100 peroxidase activity is seen in the postsynaptic membrane (arrow). × 39,000.
338 antiserum conjugated with peroxidase, neuronal plasma membranes from newborn guinea pigs showed a moderate electron opacity. The anti-S-100 peroxidase precipitates were localized all through the thickness of the membrane as far as could be judged at high magnifications. The intensity of the staining varied along the nerve cell cytoplasmic membranes. In the cerebral cortex, the postsynaptic membranes showed the strongest antiS-100 peroxidase activity of all structures of newborn guinea pigs (Fig. 3). The presynaptic membrane had a moderate electron opacity. The membranes of most synaptic vesicles were not stained above the background level. Astrocytes and oligodendrocytes were defined according to Peters et al. 44. The immunoelectron microscopic investigation in newborn guinea pig showed a clear-cut localization of S-100 protein in the plasma membrane. Astrocytic processes showed a distinct presence of S-100 protein along the filaments. Oligodendrocytes appeared to have a definite S-100 content in their plasma membranes. Vascular endothelial ceils, basal membranes and pericytes showed no presence of S-100 protein. The luminal surface of the endothelial cells showed a non-specific peroxidase activity. In the newborn rat brain, however, no S-100 could be detected by quantitative immunoelectrophoresis ~0. After treatment of tissue sections for electron microscopy, neither nerve cells nor glial cells from 1-day-old rats showed any specific anti-S-100 peroxidase activity. Synapses of newborn rats were rarely detected without previous contrasting with lead citrate and uranyl acetate because they lacked anti-S-100 peroxidase activity. The results, using the immunoelectron microscopic technique, are in good correlation with results obtained by quantitative immunoelectrophoresis. The amount of S-100 increased steadily from the first week until the end of the fourth week of postnatal life (Fig. 1). The highest levels and the most rapid rise of S-100 was found in the cerebellum and in the brain stem, while the rise in S-100 was less pronounced in frontal, middle and dorsal brain. A weak but specific staining, mostly in the plasma membrane and in the mitochondria was observed in 7-day-old rat neurons. By the 14th day the plasma membrane had low to moderate activity while specific staining continued to be found in mitochondrial membranes, nuclear membrane, and nucleoplasm. The intensity of the peroxidase activity increased after 14 days and was comparable to that of adult rats la at the age of 3-4 weeks. At that age the cell membranes of neurons showed specific activity of variable intensity while the nuclear membrane, nucleoplasm and mitochondria showed a specific staining. By the 7th day of postnatal life of the rat, a weak but specific activity was seen both in the pre- and postsynaptic membranes (Fig. 4). A more adult appearance of the peroxidase staining was seen in 14-day-old rats where the postsynaptic membrane showed a strong activity (Fig. 5). The presynaptic membrane had a moderate electron opacity. A specific staining of the astrocytic perivascular endfeet in the form of numerous minute granular precipitates among the filaments was seen in 1-week-old rats.
Fig. 4. A: frontal cortex from 3-day-old rat treated for detection of the S-100 protein. No significant specific staining is visible, x 49,000. B: synapse from rat frontal cortex (9 days postnatally). Both pre-(arrow) and postsynaptic (arrow head) electron opacity is seen indicating the presence of S-100. × 67,000.
340
Fig. 5. a: synapse from 14-day-old rat frontal cortex, showing an increased electron opacity in the postsynaptic membrane (arrow) compared to the presynaptic membrane. This indicates an accumulation of S-100 postsynaptically. × 67,000. b: adult rat frontal cortex incubated in anti-S-100 peroxidase as described in the text. An accumulation of S-100 is seen in the postsynaptic membranes, x 53,000.
341
Fig. 6. Astrocyte from the optic nerve of a 17-day-old rabbit with activity of S-100 in several of its filaments. The nucleus of the astrocyte to the left. x 89,000. Fig. 7. Optic nerve from adult rabbit showing an astrocyte with a specific anti-S-100 peroxidase reaction in its filaments, x 63,000.
342 The plasma membranes, some mitochondria and occasionally a few of the endoplasmic reticular membranes of both astrocytes and oligodendrocytes from 1-week-old rats showed a specific peroxidase activity. From the second week until at the end of the fourth week of postnatal life of the rat a more intense electron opacity was seen in the locations described above. S-100 could already be found in newborn rabbits with the highest amounts in cerebellum and brain stem as determined by immunoelectrophoresis (Fig. 1). The rise was linear until the end of the 3rd week of postnatal life. The immunoelectron microscopic staining pattern of sections from rabbit brain was similar to that of rat during postnatal development. On the 7th day of postnatal life, the neuronal plasma membranes showed scattered deposits of anti-S-100 peroxidase, and the synapses had a weak but specific activity both pre- and postsynaptically. A more intense electron opacity of anti-S-100 peroxidase was visible on some parts of the neuronal plasma membranes from rabbits of 2 weeks age. A marked peroxidase activity was seen in the postsynaptic membrane at this age of the animal. In astrocytes and oligodendrocytes a staining of 7-day-old rabbits was seen in the plasma membrane, scattered profiles of the endoplasmic reticulum membranes and in some mitochondria. The astrocytic perivascular endfeet and other processes in the neuropil showed a wide range of activity among their filaments (Fig. 6). Sections from adult rabbits showed a similar distribution of S-100, but the anti-S-100 peroxidase staining was more intense, especially in the astrocytic endfect (Fig. 7). DISCUSSION Discrete stages in the maturation of mammalian brain during ontogeny have been described. These include the attainment of adult number of neurons, a period of accelerated growth of the brain during which extensive morphological maturation occurs, and a final period of consolidation of varying duration that leads to the mature adult central nervous system. There occurs an accumulation of the S-100 protein during postnatal development in the brains of rat 24,47,51, of mouse 8, of the avian optic tectum 6, and of the chick spinal cord 9. S-100 has also been studied during development of the embryonic chick brain 15, as well as during human foetal developmental These quantitative data are in agreement with the appearance of the S-100 protein on isolated neuronal plasma membranes and in glial cells during early postnatal development of rat, rabbit, and guinea pig, as studied by the immunofluorescence technique z0. Wide variation in the stages of maturation among animals of various species at the same conceptual age has complicated the study of the differential maturation of the nervous system 37. In more highly developed species it is difficult to delineate precisely the 'critical period of brain growth '26. In some species, e.g. guinea pig, this is a prenatal phenomenon. In others, e.g. rat and rabbit, it occurs after birth. Rat and rabbit are poorly developed at birth both physically and behaviourally while guinea pig is already well-developed at birth. Brain from newborn guinea pigs showed a distribution and an amount of S-100 which were similar to that of adult
343 animals. Furthermore, the adult type of synaptic localisation, i.e. high levels in the postsynaptic membrane, could be demonstrated in newborn guinea pig brain (Fig. 3). In contrast, in rabbit and rat brain S-100 accumulates rapidly during the first 3-4 weeks of postnatal life. The most rapid accumulation in rabbit and rat occurs in cerebellum and brain stem rather than in cerebrum. The rapid accumulation of S-100 protein in brain stem and cerebrum of the rat occurs at the end of the period marked by proliferation of neuroblasts, and during glial proliferation. The rapid increase in levels of S-100 extends through the period of glial proliferation, and continues after most cell division has terminated. Spontaneous electrical activity and accelerated morphological development of the rat brain does not occur until 7-12 days after birth 10. During this postnatal period, the first appearance of adult type of ultrastructural distribution of S-100 protein could be demonstrated, with increased levels of S-100 protein in neuronal membrane, especially in the postsynaptic part. The change in content of the S-100 protein in developing rat brain also parallels the appearance of enzymatic activities which are thought to be unique to the nervous system, and begins at a time when there appears to be a shift from rapid growth to functional maturation s. It has previously clearly been shown that during development of, for example, rat brain, the synaptosomes are a privileged site of interaction for S-100 protein, because the synapse contains specific S-100 protein receptors 12. However, it has been shown that this probably tissue-bound part of the S-100 protein might be antigenically inactivated during immunoelectron microscopic procedures by a too-heavy fixation ae. ACKNOWLEDGEMENTS This work was supported by the Swedish Cancer Society Grants 262-B73-04X73; 96 and No. 262-B-74-05X-74; 76; by the Swedish Medical Research Council (B75-12X-2548-07), by the Medical Faculty of G6teborg, Wilhelm and Martina Lundgren's Foundation and Ollie and Elof Ericsson's Foundation. The authors are indebted to Eng. Ulla Svedin and Eng. Gunnar Sundberg for excellent technical assistance.
REFERENCES 1 Benda, P., Protrine S-100 et cellules gliales du rat, Rev. neuroL, 118 (1968) 364-367. 2 Benda, P., Protrine S-100 et tumeurs crrrbrales humaines, Rev. neurol., 118 (1968) 368-372. 3 Benda, P., Lightbody, J., Sato, G., Levine, L. and Sweet, W., Differentiated rat gial cell strain in tissue culture, Science, 161 (1968) 370-371. 4 Bennett, G. S. and Edelman, G. M., Isolation of an acidic protein from rat brain, J. biol. Chem., 243 (1968) 6234-6241. 5 Calissano, P., Moore, B. W. and Friesen, A., Effect of calcium ion on S-100, a protein of the nervous system, Biochemistry, 8 (1969) 4318-4326. 6 Cicero, T. J., Cowan, W. M. and Moore, B. W., Changes in the concentrations of the two brainspecific proteins, S-100 and 14-3-2, during the development of the avian optic tectum, Brain Research, 24 (1970) 1-10. 7 Cicero, T. J., Cowan, W. M., Moore, B. W. and Suntzeff, F., The cellular localization of the two brain specific proteins, S-100 and 14-3-2, Brain Research, 18 (1970) 25-34.
344 8 Cicero, T. J., Ferrendelli, J. A., Suntzeff, V. and Moore, B. W., Regional changes in CNS levels of the S-100 and 14-3-2 proteins during development and aging of the mouse, J. Neurochem., 19 (1972) 2119-2125. 9 Cicero, T. J. and Province, R. R., The levels of the brain-specific proteins, S-100 and 14-3-2, in the developing chick spinal cord, Brain Research, 44 (1972) 294-298. 10 Crain, S. M., Development of electrical activity in cerebral cortex of albino rat, Proc. Soc. exp. Biol. (N. Y.), 81 (1952) 49-51. 11 Davis, B. J., Disc electrophoresis. 11. Method and application to human serum proteins, Ann. N.Y. Acad. Sci., 121 (1964) 404427. 12 Donato, R., Michetti, F. and Miani, N., Soluble and membrane-bound S-100 protein in cerebral cortex synaptosomes. Properties of the S-100 receptor, Brain Research, 98 (1975) 561-573. 13 Edstr6m, A., Haglid, K. G., Kanje, M., R6nnb~ick, L. and Walum, E., Morphological alterations and increase of S-100 protein in cultured human glioma cells deprived of serum, Exp. Cell Res., 83 (1973) 426-429. 14 Eng, L. F. and Kosek, G. C., Light and electron microscopic localization of the glial fibrillary acidic protein and S-100 protein by immunoenzymatic techniques, Trans. Amer. Soc. Neurochem., 5 (1974) 160. 15 Friedman, H. P. and Wenger, B. S., Accumulation of an organ-specific protein during development of the embryonic chick brain, J. Ernbryol. exp. Morph., 23 (1970) 289-297. 16 Graham, R. C., Jr. and Karnovsky, M. J., The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultastructural cytochemistry by a new technique, J. Histochem. Cytochem., 14 (1966) 291-302. 17 Haglid, K. G., Hamberger, A., Hansson, H.-A., Hyd6n, H., Persson, L. and R~nnb~ick, L., S 100 protein in synapses of the central nervous system, Nature (Lond.), 251 (1974) 532-534. 18 Haglid, K. G., Hamberger, A., Hansson, H.-A., Hyd6n, H., Persson, L. and R6nnb~ick, L., Cellular and sul~cellular distribution of the S 100 protein in rat and rabbit central nervous system, J. Neurosci. Res., (1976) in press. 19 Haglid, K. G. and R~Snnb~ick, L., unpublished results. 20 Haglid, K. G., R6nnback, L. and Stavrou, D., Purification of soluble glycoproteins from human nervous tissue and liver, J. Neurochem., 24 (1975) 1053-1057. 21 Haglid, K. G. and Stavrou, D., Water-soluble and pentanol-extractable proteins in human brain normal tissue and human brain tumours, with special reference to S 100 protein, J. Neurochem., 20 (1973) 1523-1532. 22 Haglid, K. G., Stavrou, D., R6nnback, L., Carlsson, C.-A. and Weidenbach, W., The S-100 protein in water-soluble and pentanol extractable form in normal human brain and tumours of the human nervous system. A quantitative study, J. neurol. Sci., 20 (1973) 103-111. 23 Hansson, H.-A., Hyd6n, H. and Rtinnb~ick, L., Localization of S 100 protein in isolated nerve cells by immunoelectron microscopy, Brain Research, 93 (1975) 349-352. 24 Herschman, H. R., Levine, L. and DeVellis, J., Appearance of a brain-specific antigen (S-100 protein) in the developing rat brain, J. Neurochem., 18 (1971) 629 633. 25 Hijmans, W., Schuit, H. R. E. and Klein, F., An immunofluorescence procedure for the detection of intracellular immunoglobulins, Clin. exp. lrnmunol., 4 (1969) 457-472. 26 Himwich, W. A., In C. C. Pfeiffer and J. R. Smythies (Eds.), International Review of Neurobiology, Vol. 4, Academic Press, New York, 1962, p. 151. 27 Hyd6n, H. and McEwen, B. S., A glial protein specific for the nervous system, Proc. nat. Acad. Sci. (Wash.), 55 (1966) 354-358. 28 Hyd6n, H. and R6nnback, L., Membrane-bound S 100 protein on nerve cells and its distribution, Brain Reseach, 100 (1975) 615 628. 29 Hyd6n, H. and R/SnnbSck, L., The brain specific S 10~) protein on neuronal cell membranes, (1976) submitted for publication. 30 Hyd6n, H. and ROnnb~ick, L., S 100 on isolated neurons and glial cells from rat, rabbit and guinea pig during early postnatal development, Neurobiology, 5 (1975) 291-302. 31 Kabat, E. and Mayer, M., In E. Kabat and M. Mayer (Eds.), Experimental lmmunochemistry, 2nd ed., Thomas, Springfield, I11., 1961, pp. 136-158. 32 Laurell, C.-B., Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies, Analyt. Biochem., 15 (1966) 45-52. 33 Levine, L. and Moore, B. W., Structural relatedness of a vertebrate brain acidic protein as measured immunochemically, Neurosci. res. Progr. Bull., 3 (1965) 18-22.
345 34 Lightbody, J., Pfeiffer, S. E., Kornblith, P. L. and Herschman, H., Biochemically differentiated clonal human glial cells in tissue culture, J. Neurobiol., 1 (1970) 411417. 35 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 36 Matus, A. and Mughal, S., Immunohistochemical localisation of S-100 protein in brain. In K. G. Haglid, A. Hamberger, H.-A. Hansson, H. Hyd6n, L. Persson and L. R/Snnback (Eds.), Reply, Nature (Lond.), 258 (1975) 746-749. 37 Mcllwain, H., Biochemistry and the Central Nervous System, 2nd ed. Little, Brown, Boston, Mass., 1959, p. 186. 38 Moore, B. W., A soluble protein characteristic of the nervous system, Biochem. biophys. Res. Commun., 19 (1965) 739-744. 39 Moore, B. W., Brain specific proteins. In D. J. Schneider (Ed.), Proteins of the Nervous System, Raven Press, New York, 1973, pp. 1-12. 40 Moore, B. W. and McGregor, D., Chromatographic and electrophoretic fractionation of soluble proteins of brain and liver, J. biol. Chem., 240 (1965) 1647-1653. 41 Moore, B. W. and Perez, V. J., Complement fixation for antigens on a picogram level, J. Immunol., 96 (1966) 1000-1005. 42 Ornstein, L., Disc electrophoresis. I. Background and theory, Ann. N.Y. Acad. Sci., 121 (1964) 321-349. 43 Packman, P. M., Blomstrand, C. and Hamberger, A., Disc electrophoretic separation of proteins in neuronal, glial and subcellular fractions from cerebral cortex, J. Neurochem., 18 (1971) 1-9. 44 Peters, A., Palay, S. L. and Webster, H. deF., The Fine Structure of the Nervous System, The Cells and Their Processes, New York Hoeber Medical Division, Harper and Row, New York, 1970. 45 Plescia, O. J., Braun, W. and Palczuk, N. C., Production of antibodies to denatured deoxyribonucleic acid (DNA), Proc. nat. Acad. Sci. (Wash.), 52 (1964) 279-285. 46 Ramirez, G., Haglid, K. G., Karlsson, B. and R6nnb~ick, L., Purification and characterization of a soluble glycoprotein from rat brain (GM-50-C), FEBS Lett., 38 (1974) 143-146. 47 Rapport, M. M., Laev, H., Mahadik, S. and Graf, L., Immunohistochemical appearance of the S-100 protein in developing rat brain, Trans. Amer. Soc. Neurochem., 5 (1974) 58. 48 Rusca, G., Calissano, P. and Alema, S., Identification of a membrane-bound fraction of the SI00 protein, Brain Research, 49 (1972) 223-227. 49 Shapiro, A. L., Vifiuela, E. and Maizel, Jr., J. V., Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels, Biochem. biophys. Res. Commun., 28 (1967) 815-820. 50 Stavrou, D., Ltibbe, I. und Haglid, K. G., Immunelektrophoretische Quantifizierung des hirnspezifischen S 100 Proteins, Acta neuropath. (BerL), 29 (1974) 275-280. 51 Sviridov, S. M., Korochkin, L. I., Ivanov, V. N., Maletskaya, E. I. and Bakhtina, T. K., Immunohistochemical studies of S-100 protein during postnatal ontogenesis of the brain of two strains of rats, J. Neurochem., 19 (1972) 713-718. 52 Warecka, K. and Bauer, H., Studies on 'brain-specific' proteins in aqueous extracts of brain tissue, J. Neurochem., 14 (1967) 783-787. 53 Zuckerman, J. E., Herschman, H. R. and Levine, L., Appearance of a brain specific antigen (the S-100 protein) during human foetal development, J. Neurochem., 17 (1970) 247-251.
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
331
S-100 IN THE CENTRAL NERVOUS SYSTEM OF RAT, RABBIT AND GUINEA PIG DURING POSTNATAL DEVELOPMENT
K. G.
HAGLID, H.-A. HANSSON and L. RONNB~CK
Institute of Neurobiology, Universityof Giiteborg, Giiteborg (Sweden)
(Accepted July 6th, 1976)
SUMMARY The accumulation o f the brain-specific S-100 protein has been studied during postnatal development of rat, rabbit and guinea pig quantitatively, using immunoelectrophoresis, and qualitatively, by immunoelectron microscopy. Newborn guinea pigs show high levels of S-100. The distribution was similar to that of adult animals with an enrichment of S-100 to the postsynaptic membranes and to the astrocytic filaments. The neuronal plasma membranes as well as the neuronal nuclear membranes, astrocytic and oligodendroglial plasma membranes, also showed a specific activity for S-100. The amount of S-100 increased linearly from birth until the 3rd and 4th postnatal week of rabbit and rat, respectively. During the 2nd and 3rd week rabbit and rat nervous systems showed an accumulation of S-100, especially in the postsynaptic membranes and in the astrocytic filaments. In this study we present evidence that the S-100 protein quantitatively and ultrastructurally appears according to a pattern which parallels the maturation of brain, showing adult characteristics already at birth in early developing brains (guinea pig) and a change towards adult pattern after birth in late developing brains (rat and rabbit). In the latter two species change towards an adult S-100 distribution pattern proceeds during the postnatal period concomitant with the enzymatic and electrophysiologicai maturation of the brain.
INTRODUCTION A number of brain proteins have been isolated during the past 10 years4, ~o,as, 40,4a,52. The acidic S-100 protein 3s has been most extensively studied. It is present both in glial cellsl-a,7,13,27,a4,47 and in neuronslT,ls,22,2a,27-ao,4a,51 of the central
332 nervous system. We have earlier localized S-100 to the postsynaptic membranes by immunoelectron microscopy17, and to the surface of isolated neurons by immunofluorescence zs,3°, by immunoelectron microscopy23 and by Sepharose 4B spherules, to which anti-S-100 antibodies were coupled29. We have also studied the appearance of S-100 on neuronal cell membranes during postnatal development30. In this paper we correlate changes in the levels of the S-100 protein and its appearance in brain ultrastructures during postnatal maturation of early (guinea pig) and late developing (rat and rabbit) central nervous systems. MATERIALS AND METHODS
Animals Rats, rabbits and guinea pigs during early postnatal period were used in this study (newborn, 1-6 weeks old and adult animals).
Preparation of the S-IO0 protein S-100 was prepared according to Moore as, except that all buffers during purification contained 2.5 mM EDTA and 0.1 mM 2-mercaptoethanol. The purity of the protein was controlled by disc electrophoresis (15 ~ polyacrylamide)11,42, by electrophoresis in a continuous buffer system (7.5 ~ polyacrylamide)5, and by electrophoresis in SDS (15~ polyacrylamide)4L No other proteins were discovered in the electrophoretic pattern.
Production of antiserum against the beef S-IO0 protein Antibodies to beef-brain S-100 were prepared, using as antigen the protein conjugated to methylated bovine serum albumin 4~. To 1 ml of 0.15 M NaC1, containing 1 mg of S-100, was added 0.1 ml of 1 ~ (w/v) methylated bovine serum albumin. S-100 methylated bovine serum albumin flocculent suspension (!. 1 ml) was emulsified with an equal volume of complete Freund's adjuvant and injected into the toepads and hind subcutis of albino rabbits, once weekly for 4 weeks. Ten weeks after the last injection, a single intravenous injection of the S-100-methylated bovine serum albumin in 1.1 ml of 0.15 M NaCI was given as a booster. Ten days after the intravenous injection the animals were bled through the marginal ear vein, and the blood was collected for antiserum. The specificity of the antiserum was tested by complement fixation 31,41 against different organ extracts such as liver, kidney, spleen, lung and blood. A 10,000-fold higher level of antigen expressed on protein bases was found in brain. Immunoelectrophoresis in agarose (see below) revealed a single precipitation line against brain homogenate and beef S-100 protein in the most anodal part of the gel not present in extracts from other organs, even if the protein amount of the organ extracts were at the load limit of the agarose gel electrophoresis.
Preparation of antisera for immunoelectron microscopy The antiserum against S-100 was labelled with peroxidase by mixing antiserum with glutaraldehyde and horseradish peroxidase (type VI, Sigma Chemical Co., St. Louis, Mo. U.S.A.) in optimal proportions 17,1s. This mixture was shaken for 90 min at
333 room temperature. The anti-beef S-100 rabbit serum conjugated with peroxidase was separated from free peroxidase by elution from a G-75 Sephadex column (Pharmacia Fine Chemicals Co., Uppsala, Sweden). The fractions containing the peroxidaselabeled proteins were identified by their absorption at 280 nm. The conjugate was absorbed with liver powder 2~ (anti-S-100-peroxidase-liver abs.). Half of this liverabsorbed anti-S-100 antiserum was absorbed with beef S-100 protein by incubation in a shaking waterbath for 1 h at 37 °C, and for 12 h at + 4 °C. The S-100-absorbed conjugate was thereafter centrifuged for 30 min at 10,000 × g. The pellet containing the S-100-anti-S-100 precipitate was discarded. The procedure was repeated several times until the antiserum was free from antibodies against S-100 as confirmed by complement fixation at the nanogram levelal,aL The S- 100-absorbed conjugate (anti-S- 100peroxidase-liver-S-100 abs.) was used as a control during immunoelectron microscopy. Conjugated sera were stored at q- 4 °C and were stable for two weeks after preparation.
Specificity tests of the antigen-antibody reaction The specificity of the antiserum was tested in the following ways. (1) A single precipitation band was obtained when the antiserum was tested by double diffusion in 1 ~ agar against purified S-100. (2) Microcomplement fixation31,41 at the nanogram level with the antiserum prepared detected S-100 only in brain and gave a positive result for pure S-100. No activity was found in other tissues, including liver, kidney, spleen, lung and blood, unless 10,000 times higher extract concentrations (mg protein/ ml) were used.
Preparation of tissue materials for immunoeleetrophoresis The brains of the animals were divided into three equal parts, frontal, middle and dorsal. Surface (mostly grey matter) and deeper (mostly white matter) layers of the cortex were dissected out on ice from all animals, except for newborn and 1-week-old rats and rabbits where a 1 mm thick layer from frontal, middle and dorsal cortex was removed. Cerebellum and brain stem under the IVth ventricle were also used in all animals. The tissue samples were weighed and quickly frozen in tight fitting boxes at --80 °C until used. The samples were homogenized in 20:1 (v/w), (10:1 v/w for rat and rabbit 1 and 7 days old) barbital buffer (pH 8.6, ionic strength 0.02) containing 2.5 mM EDTA and 0.1 mM 2-mercaptoethanol, and centrifuged for 60 min at 105,000 × g. The supernatants which contained soluble proteins were used for quantitative immunoelectrophoresis described below. After 2-mercaptoethanol had been removed by lyophilization, protein was determined according to Lowry et al. z5 both in supernatants and homogenates.
Quantitative immunoelectrophoresis The quantitation of S-100 was carried out according to the procedure of Laurel132 modified for this (soluble, low molecular weight S-100) protein by Stavrou et al. 5°. The immunoelectrophoresis was performed in 1 ~ Litex agarose (Glostrup, Denmark) containing 1 ~ polyethylene glycol (M.W. 6000), in barbital buffer (pH
334 8.6, ionic strength 0.02), containing 2.5 m M EDTA, 0.1 m M 2-mercaptoethanol and 0 . 4 ~ anti-beef S-100 rabbit serum for 6 h at a current of 1 mA/cm at 15 °C.
lmmunoelectron microscopic procedure The animals were anaesthetized with ether or Mebumal and fixed by perfusion via the ascending aorta during l0 min. Tissue pieces from the frontal, parietal and visual lobes, from the optic nerve and from the cerebellum were dissected, fixed by immersion for a further 1-2 h and then rinsed in cold 0.15 M cacodylate buffer, pH 7.2, containing 1 mM EDTA. The fixation solution contained 4 ~ freshly prepared formaldehyde and 1 m M EDTA in 0.15 M cacodylate buffer (pH 7.2). In some experiments 0.1 ~ picric acid was added to the fixative. The fixed and rinsed tissue pieces were sectioned in a Vibratome (Oxford), at a nominal thickness of 20 #m. The sections were incubated for 45 min in anti-S-100-peroxidase-liver abs. conjugate or in anti-S100-peroxidase-liver-S-100 abs. conjugate in serial dilutions with 0.15 M cacodylate buffer, pH 7.2, containing 1 m M EDTA, until the difference between the test and the control antiserum was optimal. The sections were rinsed in buffer and incubated in cacodylate buffer containing 0.05 ~o 3,3-diaminobenzidine and 0.01 ~ hydrogen peroxide 16. After rinsing, the sections were postfixed in 2 ~ osmium tetroxide in cacodylate buffer, dehydrated in a graded series of ethanol and embedded in Epon. Thin sections were prepared on a LKB Ultrotome III and studied in a Siemens Elmiscope 1 A. Sections with S-100-absorbed antiserum (dilution of antiserum and absorbed antiserum 1:350) were used as controls. RESULTS To rule out the possibility that the observed changes in the levels of S-100 during development were simply the result of alterations in water content in the brain, levels of S-100 have been expressed both as/zg S-100 per g wet weight (Fig. 1) and as #g of S-100 protein per mg of total protein (Fig. 2). Our values of S-100 in the three adult animal types are higher than values previously reported in the literature 24,3a. This might be due to differences in extraction procedures. We avoided Ca 2+ effects on S-100 protein during the homogenization of the material, since it has been reported that Ca 2+ increases the level of tissue-bound S-100 ~9. We also used osmotic shock to release S-100 protein from the homogenized material. Preliminary results indicate that if osmotic shock was compared to isotonic sucrose containing 0.1 ~ Triton as extraction solution, a 2-fold larger amount of S- 100 per g wet weight tissue was liberated by osmotic shock as compared with the sucroseTriton extraction methodlL That osmotic shock treatment is a more effective extraction procedure than high or low ionic strength solutions 4s indicates that the S-100 might be trapped inside cellular compartments. In order to determine whether S-100 from rat and guinea pig brain had different relative mobilities, supernatants from frontal cortex and cerebellum were run on 7.5 polyacrylamide gel electrophoresis together with pure S-100 as standards, according to Calissano et al). After destaining of the gels, densitometry 21 was made on the front
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Age days Fig. ]. Diagrams showing the accumulation of S-|00 protein (yg S-]00/g wet weight) during postnatal development of (A) rat, (B) rabbit and (C) guinea pig. F, frontal brain, superficial part; M, middle brain, superficial part; D, dorsal brain, superficial part; C, cerebellum; BS, brain stem under the IVth ventricle. More or less parallel curves were obtained regarding S-]00 accumulation in deeper parts of frontal, middle and dorsal brain. The differences between values from superfficial and deeper parts of brain were not statistically significant, probably due to difficulties of obtaining only grey and w h i t e m a t t e r a t dissection. S . D . s a r e i n d i c a t e d .
bands which react immunologically with S-100 antiserum. The relative mobilities of the front band from guinea pig frontal cortex and cerebellum was 1.096, i.e. somewhat higher than that of rat brain frontal cortex and cerebellum (1.085) while pure S-100 from bovine whole brain had a relative mobility of 1.083 as determined in our electrophoresis system. The amount of protein in the front band expressed per mg protein on
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the gel is shown in Table I. The results are comparable to, although lower than those obtained using quantitative immunoelectrophoresis, indicating a high content of S-100 in guinea pig brain already at birth. The possibility that guinea pig S-100 has a higher mobility on agarose (1%) gel electrophoresis and therefore higher values are obtained from guinea pig brain when determined immunoelectrophoretically cannot be excluded. It is also a possibility that immunological species differences in the absence of Ca 2+ as in our immunoelectrophoretic system are larger than the small immunological species difference previously reported for the S-100 protein measured in a sys-
337 TABLE I Protein content (l~g) in front band expressed per mg total protein on the gel (7.5 % polyacrylamide gel electrophoresis as described in the text} i~g protein in front band ( S-IO0) per rng total protein Cerebellum
Frontal cortex
Rat Newborn 7 days 14 days Adult
0 11.5 23.5 54.5
0 5.0 12.3 36.4
Guinea pig Newborn 7 days 14 days Adult
66.0 78.2 90.9 156.6
34.0 55.2 70.5 89.5
tem which contains Ca 2+. Due to this the S-100 protein is expressed in beef-S-100 protein equivalents. In guinea pig brain, rather high levels o f S-100 were already f o u n d at birth as determined by immunoelectrophoresis 5°. The a m o u n t o f S-100 increased 2-3-fold f r o m birth until adult age (Fig. 1). Using immunoelectron microscopy with anti-S-100
Fig. 3. Synapses from frontal cortex of guinea pig, 2 days old. A heavy anti-S-100 peroxidase activity is seen in the postsynaptic membrane (arrow). × 39,000.
338 antiserum conjugated with peroxidase, neuronal plasma membranes from newborn guinea pigs showed a moderate electron opacity. The anti-S-100 peroxidase precipitates were localized all through the thickness of the membrane as far as could be judged at high magnifications. The intensity of the staining varied along the nerve cell cytoplasmic membranes. In the cerebral cortex, the postsynaptic membranes showed the strongest antiS-100 peroxidase activity of all structures of newborn guinea pigs (Fig. 3). The presynaptic membrane had a moderate electron opacity. The membranes of most synaptic vesicles were not stained above the background level. Astrocytes and oligodendrocytes were defined according to Peters et al. 44. The immunoelectron microscopic investigation in newborn guinea pig showed a clear-cut localization of S-100 protein in the plasma membrane. Astrocytic processes showed a distinct presence of S-100 protein along the filaments. Oligodendrocytes appeared to have a definite S-100 content in their plasma membranes. Vascular endothelial ceils, basal membranes and pericytes showed no presence of S-100 protein. The luminal surface of the endothelial cells showed a non-specific peroxidase activity. In the newborn rat brain, however, no S-100 could be detected by quantitative immunoelectrophoresis ~0. After treatment of tissue sections for electron microscopy, neither nerve cells nor glial cells from 1-day-old rats showed any specific anti-S-100 peroxidase activity. Synapses of newborn rats were rarely detected without previous contrasting with lead citrate and uranyl acetate because they lacked anti-S-100 peroxidase activity. The results, using the immunoelectron microscopic technique, are in good correlation with results obtained by quantitative immunoelectrophoresis. The amount of S-100 increased steadily from the first week until the end of the fourth week of postnatal life (Fig. 1). The highest levels and the most rapid rise of S-100 was found in the cerebellum and in the brain stem, while the rise in S-100 was less pronounced in frontal, middle and dorsal brain. A weak but specific staining, mostly in the plasma membrane and in the mitochondria was observed in 7-day-old rat neurons. By the 14th day the plasma membrane had low to moderate activity while specific staining continued to be found in mitochondrial membranes, nuclear membrane, and nucleoplasm. The intensity of the peroxidase activity increased after 14 days and was comparable to that of adult rats la at the age of 3-4 weeks. At that age the cell membranes of neurons showed specific activity of variable intensity while the nuclear membrane, nucleoplasm and mitochondria showed a specific staining. By the 7th day of postnatal life of the rat, a weak but specific activity was seen both in the pre- and postsynaptic membranes (Fig. 4). A more adult appearance of the peroxidase staining was seen in 14-day-old rats where the postsynaptic membrane showed a strong activity (Fig. 5). The presynaptic membrane had a moderate electron opacity. A specific staining of the astrocytic perivascular endfeet in the form of numerous minute granular precipitates among the filaments was seen in 1-week-old rats.
Fig. 4. A: frontal cortex from 3-day-old rat treated for detection of the S-100 protein. No significant specific staining is visible, x 49,000. B: synapse from rat frontal cortex (9 days postnatally). Both pre-(arrow) and postsynaptic (arrow head) electron opacity is seen indicating the presence of S-100. × 67,000.
340
Fig. 5. a: synapse from 14-day-old rat frontal cortex, showing an increased electron opacity in the postsynaptic membrane (arrow) compared to the presynaptic membrane. This indicates an accumulation of S-100 postsynaptically. × 67,000. b: adult rat frontal cortex incubated in anti-S-100 peroxidase as described in the text. An accumulation of S-100 is seen in the postsynaptic membranes, x 53,000.
341
Fig. 6. Astrocyte from the optic nerve of a 17-day-old rabbit with activity of S-100 in several of its filaments. The nucleus of the astrocyte to the left. x 89,000. Fig. 7. Optic nerve from adult rabbit showing an astrocyte with a specific anti-S-100 peroxidase reaction in its filaments, x 63,000.
342 The plasma membranes, some mitochondria and occasionally a few of the endoplasmic reticular membranes of both astrocytes and oligodendrocytes from 1-week-old rats showed a specific peroxidase activity. From the second week until at the end of the fourth week of postnatal life of the rat a more intense electron opacity was seen in the locations described above. S-100 could already be found in newborn rabbits with the highest amounts in cerebellum and brain stem as determined by immunoelectrophoresis (Fig. 1). The rise was linear until the end of the 3rd week of postnatal life. The immunoelectron microscopic staining pattern of sections from rabbit brain was similar to that of rat during postnatal development. On the 7th day of postnatal life, the neuronal plasma membranes showed scattered deposits of anti-S-100 peroxidase, and the synapses had a weak but specific activity both pre- and postsynaptically. A more intense electron opacity of anti-S-100 peroxidase was visible on some parts of the neuronal plasma membranes from rabbits of 2 weeks age. A marked peroxidase activity was seen in the postsynaptic membrane at this age of the animal. In astrocytes and oligodendrocytes a staining of 7-day-old rabbits was seen in the plasma membrane, scattered profiles of the endoplasmic reticulum membranes and in some mitochondria. The astrocytic perivascular endfeet and other processes in the neuropil showed a wide range of activity among their filaments (Fig. 6). Sections from adult rabbits showed a similar distribution of S-100, but the anti-S-100 peroxidase staining was more intense, especially in the astrocytic endfect (Fig. 7). DISCUSSION Discrete stages in the maturation of mammalian brain during ontogeny have been described. These include the attainment of adult number of neurons, a period of accelerated growth of the brain during which extensive morphological maturation occurs, and a final period of consolidation of varying duration that leads to the mature adult central nervous system. There occurs an accumulation of the S-100 protein during postnatal development in the brains of rat 24,47,51, of mouse 8, of the avian optic tectum 6, and of the chick spinal cord 9. S-100 has also been studied during development of the embryonic chick brain 15, as well as during human foetal developmental These quantitative data are in agreement with the appearance of the S-100 protein on isolated neuronal plasma membranes and in glial cells during early postnatal development of rat, rabbit, and guinea pig, as studied by the immunofluorescence technique z0. Wide variation in the stages of maturation among animals of various species at the same conceptual age has complicated the study of the differential maturation of the nervous system 37. In more highly developed species it is difficult to delineate precisely the 'critical period of brain growth '26. In some species, e.g. guinea pig, this is a prenatal phenomenon. In others, e.g. rat and rabbit, it occurs after birth. Rat and rabbit are poorly developed at birth both physically and behaviourally while guinea pig is already well-developed at birth. Brain from newborn guinea pigs showed a distribution and an amount of S-100 which were similar to that of adult
343 animals. Furthermore, the adult type of synaptic localisation, i.e. high levels in the postsynaptic membrane, could be demonstrated in newborn guinea pig brain (Fig. 3). In contrast, in rabbit and rat brain S-100 accumulates rapidly during the first 3-4 weeks of postnatal life. The most rapid accumulation in rabbit and rat occurs in cerebellum and brain stem rather than in cerebrum. The rapid accumulation of S-100 protein in brain stem and cerebrum of the rat occurs at the end of the period marked by proliferation of neuroblasts, and during glial proliferation. The rapid increase in levels of S-100 extends through the period of glial proliferation, and continues after most cell division has terminated. Spontaneous electrical activity and accelerated morphological development of the rat brain does not occur until 7-12 days after birth 10. During this postnatal period, the first appearance of adult type of ultrastructural distribution of S-100 protein could be demonstrated, with increased levels of S-100 protein in neuronal membrane, especially in the postsynaptic part. The change in content of the S-100 protein in developing rat brain also parallels the appearance of enzymatic activities which are thought to be unique to the nervous system, and begins at a time when there appears to be a shift from rapid growth to functional maturation s. It has previously clearly been shown that during development of, for example, rat brain, the synaptosomes are a privileged site of interaction for S-100 protein, because the synapse contains specific S-100 protein receptors 12. However, it has been shown that this probably tissue-bound part of the S-100 protein might be antigenically inactivated during immunoelectron microscopic procedures by a too-heavy fixation ae. ACKNOWLEDGEMENTS This work was supported by the Swedish Cancer Society Grants 262-B73-04X73; 96 and No. 262-B-74-05X-74; 76; by the Swedish Medical Research Council (B75-12X-2548-07), by the Medical Faculty of G6teborg, Wilhelm and Martina Lundgren's Foundation and Ollie and Elof Ericsson's Foundation. The authors are indebted to Eng. Ulla Svedin and Eng. Gunnar Sundberg for excellent technical assistance.
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