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Demonstration of acidic polyanions in certain glial cells during postnatal rat brain development
356
Braiu Research, 73 (1974) 356-361 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Demonstration of acidic polyanions in certain glial cells during postnatal rat brain, deYelopment
I. A. L A M P E R T AND P. D. LEWIS
Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 (Great Britain) (Accepted March 7th, 1974)
In the developing rat cerebrum, cell formation continues on a massive scale in the early postnatal period, as many as half of the total cells being acquired in the first 3 weeks after birth 3. The source of many of these is the periventricular matrix layer, which, as a persistent and mitotically active subependymal layer, continues to produce cells throughout life 1,s. The natural history of the subependymal cell, and the mechanism of its transformation into differentiated neuroglia, is poorly understood, although the existence of this transformation in the postnatal rat brain has been shown by autoradiography 9,1~. It is known that a variety of embryonic cells produce acid mucopolysaccharides in tissue culture 6. In recent years the Alcian blue-'critical electrolyte concentration' (C.E.C.) method15,16 has proved to be a sensitive and reliable technique for the demonstration of such substances in sections of tissues in which they are known to occur. The present study was initiated in an attempt to discover, by means of this histochemical method, more information about the maturation of the undifferentiated cells of the subependymal layer. Albino rats of mixed strains aged 1, 6 and 12 days, and 3, 6, 7 and 12 weeks were killed under chloroform anaesthesia by aortic perfusion-fixation with formolacetic acid (1%o glacial acetic acid in 1 0 ~ neutral formalin) 5. After 24 h postfixation in neutral formalin, brains were bisected through the mesencephalon. Paraffin sections of cerebrum at levels AA~ and BB1~9, and of midcerebellum and upper pons, were cut and brought to distilled water. Sections were then stained for 20 h at 18 °C in solutions of magnesium chloride of molarity 0.05, 0.20, 0.45, 0.50, 0.55, 0.60 and 0.65 M with Alcian blue (Raymond Lamb, London), 0.05 ~ (w/v) in 0.025 M sodium acetate buffer at pH 5.61~. Stained sections were washed in corresponding dye-free salt-buffer solutions and distilled water, dehydrated in alcohol, cleared in xylene, and mounted in synthetic resin. Duplicate sections were treated with ovine testicular hyaluronidase (Type III, Sigma Chemical Co.), made up in solutions of 600 units/ml in Sorensen's buffer (pH 6.8), for 3 h at 37 °C, after which they were stained as above. Control sections were treated with buffer alone before staining. Haematoxylin- and eosinstained sections were also prepared.
+ +
+ +
+
7 weeks
12 weeks
+ (occasional cell)
21 days
6 weeks
+ +
± +
12 days
1 day 6 days
+
++
+ +
+ (occasional cell)
+ +
± +
0
++ (corpus callosum; internal capsule) ++ (temporal white matter)
++ (corpus callosum) 0
-4+ + (corpus callosum)
0
0
0
0
0
± + (occasional neurone)
Other cells
++
++
+ + + +
External granular layer
Glia in fibre tracts
Subependymal layer
Ependyma and choroid plexus
Cerebellum
Cerebrum
O = absent; ± = very weak, barely visible; + = moderate staining; + + -- intense staining.
STAINING OF RAT BRAIN BY ALCIAN BLUE IN 0.45 M MAGNESIUM CHLORIDE
TABLE I
++ (scattered cells in foliar white matter and fibre tracts) ++ (scattered cells in central white matter and inferior cerebeUar peduncle) 0
++
0
-I- 4-
0 ±
0
0
0
++ (scattered glia throughout puns) 0
4+ (Purkinje and granule ceils)
± ++ (inferior cerebellar peduncle; deep cerebellar white matter) 0
± +
++
Other cells
Glia in fibre tracts
Ependyma and choroM plexus
L~ L~ '-...I
0
0
m
358
SHORT COMMUNICATIONS
Fig. 1. a: corpus callosum. Cytoplasm of glial cells, mainly in rows, is intensely stained, b: posterior horn o f lateral ventricle. Ependymal and glial cytoplasm is stained. Rats aged 6 weeks. Alcian blue in 0.45 M MgCI2. ,', 400.
SHORT COMMUNICATIONS
359
Sections stained in 0.05 and 0.20 M magnesium chloride showed extensive colouration of glial and neuronal cell bodies and of neuropil. With concentrations of 0.45 M and above, at which only highly sulphated polyanions are stained 15, staining of neuropil was barely perceptible and no nuclei were stained. In contrast, the cytoplasm of certain cells, both in cerebrum and cerebellum, was intensely stained; from their situation and morphology in these and parallel haematoxylin--eosin sections, nearly all of the Alcian blue-stained cells were identified as neuroglial. Their topographic and age distribution is summarised in Table I. It can be seen that the subependymal layer showed an increase in staining intensity of its cells up to 12 days and that (except for the anomalous 21 day results) staining intensity was maintained up to 7 weeks, after which it declined. The pattern of staining in ependyma and choroid plexus at varying ages was similar. Intensely stained glial cells in fibre tracts, sometimes in rows and resembling interfascicular oligodendroglia, were numerous in animals aged 6 and 12 days, but less numerous at 6 and 7 weeks, when they were more caudally distributed (Fig. 1). They were absent in 12-week-old rats. Ceils of the cerebellar external granular layer were intensely stained up to the time of disappearance of the layer at 21 days. Neurones in the cerebellar cortex were moderately stained in 6-day-old rats; at the same age some neurones in the basal ganglia and in the pyramidal cell layer of the hippocampus were similarly stained. Cytoplasmic staining, as described above, was strong in 0.50 and 0.55 M magnesium chloride, but was very weak or absent at 0.60 M. In contrast, mast cells in the leptomeninges, meningeal connective tissue, blood vessel walls and choroid plexus basement membranes showed intense staining at concentrations of magnesium chloride up to 0.65 M. Hyaluronidase treatment removed stainable material from glial cells, blood vessel walls and meninges, but left mast cell granules intact. Control treatment with buffer alone had no effect on staining. Staining with Alcian blue at magnesium chloride concentrations of 0.45 M and above demonstrates the presence of a sulphated polyanion, and the abolition of staining by prior hyaluronidase treatment suggests strongly that this is an acidic glycosaminoglycan. This evidence, together with a C.E.C. 15 of 0.50 M, favours the identification of the predominantly stained substance in our experiments as chondroitin sulphate C. It should be said that results with hyaluronidase must be regarded as supportive, rather than conclusive proof of identification, for destruction of specific substrates other than chondroitin sulphate C, or nonspecific destruction by possible contaminant enzymes, could result in structural alterations and loss of other materials possibly present. Acid mucopolysaccharides, mainly chondroitin sulphate and including chondroitin sulphate C, have been found in several chemical studies of the brainT,H, 17, but conclusive identification of the cytoplasmic Alcian blue-stained material demonstrated here can come only from its biochemical analysis. We interpret our findings as showing the presence of acid glycosaminoglycan (including chondroitin sulphate C) in the cells of the germinal layers of the postnatal rat brain. To our knowledge, this is the first specific localisation of this group of substances by a histochemical technique in this situation. It is well known that
360
SHORT COMMUNICATIONS
developing immature tissues contain glycosaminogtycan, and acid mucopolysaccharides (staining at a high C.E.C.) have been shown in cultures of 'spongioblasts' from disaggregated fetal mouse brain 12. It could be argued that staining of subependymal and external granular layers reflects the immaturity of their component cells, and that persistent staining of scattered subependymal cells in older animals is related to the persistent - - if declining - - mitotic activity and presumed gliogenesis at this site. Intense staining of ependyma and choroid plexus epithelium may be linked to the highly specialised nature of these glial cell layers. However, in the present state of our knowledge, it is hard to envisage what function this histochemical feature might be related to. A high concentration of chondroitin sulphate has been noted previously in choroid plexus 14, and the observations reported here suggest that this may be in the epithelial cell cytoplasm as well as in basement membranes. The most striking finding in the present study is, however, the staining of parenchymal neuroglial cells, many of which are clearly interfascicular oligodendroglia, in different fibre tracts at different ages. This staining appears to be correlated, in temporal terms, with myelin formation, for it is insignificant at birth and becomes prominent only at the end of the first postnatal week, in the period immediately preceding the phase of rapid myelination4, is - - which is accompanied by glycosaminoglycan synthesisL Ling and Leblond 1° have shown an increase in numbers of 'dark' oligodendrocytes in the rat corpus callosum between 3 weeks and 5 months, and a reciprocal fall in numbers of 'light' and 'medium' (immature) oligodendroglia. It is possible that these immature cells, which are presumably differentiating into mature oligodendroglia, are the cells demonstrated in the corpus callosum and elsewhere in the present study. It may be premature to equate Alcian blue staining of cells in fibre tracts with the deposition of myelin, but this possibility certainly merits further consideration.
This work was supported by a research grant from the Medical Research Council (P.D.L.).
1 ALLEN, E., The cessation of mitosis in the central nervous system of the albino rat, J. comp. Neurol., 22 (1912) 547-568. 2 BALAZS,R., BROOKSBANK,B. W. L., PATEL,A. J., JOrtNSON,A. L., AND WILSON, D. m., Incorporation of [35S]sulphate into brain constituents during development and the effects of thyroid hormone on myelination, Brain Research, 30 (1971) 273-293. 3 BAL~.ZS,R., Kov~,cs, S., COCKS,W. A., JOHNSON,A. L., ANDEAVRS,J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: postnatal cell formation, Brain Research, 25 (1971) 555-570. 4 BASS,N. H., NETSKV,M. G., ANDYOUNG,E., Microchemical studies of postnatal development in rat cerebrum. 2. Formation of myelin, Neurology (Minneap.), 19 (1969) 405-414. 5 CAVANAGH,J. B., AND LEWIS,P. D., Perfusion-fixation, colchicine, and mitotic activity in the adult rat brain, J. Anat. (Lond.), 104 (1969) 341-350. 6 DINGLE,T. J., ANDWEBB,M., Mucopolysaccharide metabolism in tissue culture. In E. N. W~LLMER(Ed.), Cells and Tissues in Culture. Methods, Biology and Physiology, Vol. 1, Academic Press, New York, 1965, pp. 353-396. 7 GOLt)BeRG,J. M., ANt)CUNNINGHAM,W. L., Incorporation of [asS]sulphate into the glycosaminoglycans of rat brain, Bioehem. J., 120 (1970) 15P.
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361
8 HOPEWELL,J. W., A quantitative study of the mitotic activity in the suhependymal plate of adult rats, Cell Tiss. Kinet., 4 (1971) 273-278. 9 LEWIS, P. D., The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia, Brain, 91 0968) 721-738. 10 LING, E.-A., AND LEBLOND, C. P., Investigation of gliai cells in semithin sections. II. Variation with age in the numbers of the various glial cell types in rat cortex and corpus caUosum, J. comp. Neurol., 149 (1973) 73-82. 11 MARGOLIS,R. U., Acid mucopolysaccharides and proteins of bovine whole brain, white matter and myelin, Biochim. biophys. Acta (Amst.), 141 (1967) 91-102. 12 Moss, C. A., Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures, Hi~tochem. J., 5 (1973) 547-556. 13 PATERSON,J. A., PRIVAT,A., LING, E. A., AND LEBLOND,C. P., Investigation of glial cells in semithin sections. III. Transformation of subependymal cells into glial cells, as shown by radioautograpby after aH-thymidine injection into the lateral ventricle of the brain of young rats, J. comp. NeuroL, 149 (1973) 83-102. 14 SINGH, M., AND BACHHAWAT,B. K., The distribution and variation with age of different uronic acid-containing mucopolysaccharides in brain, J. Neurochem., 12 (1965) 519-525. 15 SCOTT, J. E., Histochemistry of Alcian blue. III. The molecular biological basis of staining by Alcian blue 8 GX and analogous phthalocyanins, Histochemie, 32 (1972) 191-212. 16 SCOTT,J. E., AND DORLING, J., Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions, Histochemie, 5 (1965) 221-233. 17 Vos, J., KURIYAMA,K., AND ROBERTS,E., Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain, Brain Research, 12 (1969) 172-179. 18 WELLS, M. A., AND DITTMER, J. C., A comprehensive study of the postnatal changes in the concentration of the lipids of developing rat brain, Biochemistry, 6 (1967) 3169-3175. 19 ZEMAN, W., AND INNES, J. R. M., Craigie's Neuroanatomy of the Rat, Academic Press, New York, 1963, pp. 216-217
Braiu Research, 73 (1974) 356-361 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Demonstration of acidic polyanions in certain glial cells during postnatal rat brain, deYelopment
I. A. L A M P E R T AND P. D. LEWIS
Department of Histopathology, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 (Great Britain) (Accepted March 7th, 1974)
In the developing rat cerebrum, cell formation continues on a massive scale in the early postnatal period, as many as half of the total cells being acquired in the first 3 weeks after birth 3. The source of many of these is the periventricular matrix layer, which, as a persistent and mitotically active subependymal layer, continues to produce cells throughout life 1,s. The natural history of the subependymal cell, and the mechanism of its transformation into differentiated neuroglia, is poorly understood, although the existence of this transformation in the postnatal rat brain has been shown by autoradiography 9,1~. It is known that a variety of embryonic cells produce acid mucopolysaccharides in tissue culture 6. In recent years the Alcian blue-'critical electrolyte concentration' (C.E.C.) method15,16 has proved to be a sensitive and reliable technique for the demonstration of such substances in sections of tissues in which they are known to occur. The present study was initiated in an attempt to discover, by means of this histochemical method, more information about the maturation of the undifferentiated cells of the subependymal layer. Albino rats of mixed strains aged 1, 6 and 12 days, and 3, 6, 7 and 12 weeks were killed under chloroform anaesthesia by aortic perfusion-fixation with formolacetic acid (1%o glacial acetic acid in 1 0 ~ neutral formalin) 5. After 24 h postfixation in neutral formalin, brains were bisected through the mesencephalon. Paraffin sections of cerebrum at levels AA~ and BB1~9, and of midcerebellum and upper pons, were cut and brought to distilled water. Sections were then stained for 20 h at 18 °C in solutions of magnesium chloride of molarity 0.05, 0.20, 0.45, 0.50, 0.55, 0.60 and 0.65 M with Alcian blue (Raymond Lamb, London), 0.05 ~ (w/v) in 0.025 M sodium acetate buffer at pH 5.61~. Stained sections were washed in corresponding dye-free salt-buffer solutions and distilled water, dehydrated in alcohol, cleared in xylene, and mounted in synthetic resin. Duplicate sections were treated with ovine testicular hyaluronidase (Type III, Sigma Chemical Co.), made up in solutions of 600 units/ml in Sorensen's buffer (pH 6.8), for 3 h at 37 °C, after which they were stained as above. Control sections were treated with buffer alone before staining. Haematoxylin- and eosinstained sections were also prepared.
+ +
+ +
+
7 weeks
12 weeks
+ (occasional cell)
21 days
6 weeks
+ +
± +
12 days
1 day 6 days
+
++
+ +
+ (occasional cell)
+ +
± +
0
++ (corpus callosum; internal capsule) ++ (temporal white matter)
++ (corpus callosum) 0
-4+ + (corpus callosum)
0
0
0
0
0
± + (occasional neurone)
Other cells
++
++
+ + + +
External granular layer
Glia in fibre tracts
Subependymal layer
Ependyma and choroid plexus
Cerebellum
Cerebrum
O = absent; ± = very weak, barely visible; + = moderate staining; + + -- intense staining.
STAINING OF RAT BRAIN BY ALCIAN BLUE IN 0.45 M MAGNESIUM CHLORIDE
TABLE I
++ (scattered cells in foliar white matter and fibre tracts) ++ (scattered cells in central white matter and inferior cerebeUar peduncle) 0
++
0
-I- 4-
0 ±
0
0
0
++ (scattered glia throughout puns) 0
4+ (Purkinje and granule ceils)
± ++ (inferior cerebellar peduncle; deep cerebellar white matter) 0
± +
++
Other cells
Glia in fibre tracts
Ependyma and choroM plexus
L~ L~ '-...I
0
0
m
358
SHORT COMMUNICATIONS
Fig. 1. a: corpus callosum. Cytoplasm of glial cells, mainly in rows, is intensely stained, b: posterior horn o f lateral ventricle. Ependymal and glial cytoplasm is stained. Rats aged 6 weeks. Alcian blue in 0.45 M MgCI2. ,', 400.
SHORT COMMUNICATIONS
359
Sections stained in 0.05 and 0.20 M magnesium chloride showed extensive colouration of glial and neuronal cell bodies and of neuropil. With concentrations of 0.45 M and above, at which only highly sulphated polyanions are stained 15, staining of neuropil was barely perceptible and no nuclei were stained. In contrast, the cytoplasm of certain cells, both in cerebrum and cerebellum, was intensely stained; from their situation and morphology in these and parallel haematoxylin--eosin sections, nearly all of the Alcian blue-stained cells were identified as neuroglial. Their topographic and age distribution is summarised in Table I. It can be seen that the subependymal layer showed an increase in staining intensity of its cells up to 12 days and that (except for the anomalous 21 day results) staining intensity was maintained up to 7 weeks, after which it declined. The pattern of staining in ependyma and choroid plexus at varying ages was similar. Intensely stained glial cells in fibre tracts, sometimes in rows and resembling interfascicular oligodendroglia, were numerous in animals aged 6 and 12 days, but less numerous at 6 and 7 weeks, when they were more caudally distributed (Fig. 1). They were absent in 12-week-old rats. Ceils of the cerebellar external granular layer were intensely stained up to the time of disappearance of the layer at 21 days. Neurones in the cerebellar cortex were moderately stained in 6-day-old rats; at the same age some neurones in the basal ganglia and in the pyramidal cell layer of the hippocampus were similarly stained. Cytoplasmic staining, as described above, was strong in 0.50 and 0.55 M magnesium chloride, but was very weak or absent at 0.60 M. In contrast, mast cells in the leptomeninges, meningeal connective tissue, blood vessel walls and choroid plexus basement membranes showed intense staining at concentrations of magnesium chloride up to 0.65 M. Hyaluronidase treatment removed stainable material from glial cells, blood vessel walls and meninges, but left mast cell granules intact. Control treatment with buffer alone had no effect on staining. Staining with Alcian blue at magnesium chloride concentrations of 0.45 M and above demonstrates the presence of a sulphated polyanion, and the abolition of staining by prior hyaluronidase treatment suggests strongly that this is an acidic glycosaminoglycan. This evidence, together with a C.E.C. 15 of 0.50 M, favours the identification of the predominantly stained substance in our experiments as chondroitin sulphate C. It should be said that results with hyaluronidase must be regarded as supportive, rather than conclusive proof of identification, for destruction of specific substrates other than chondroitin sulphate C, or nonspecific destruction by possible contaminant enzymes, could result in structural alterations and loss of other materials possibly present. Acid mucopolysaccharides, mainly chondroitin sulphate and including chondroitin sulphate C, have been found in several chemical studies of the brainT,H, 17, but conclusive identification of the cytoplasmic Alcian blue-stained material demonstrated here can come only from its biochemical analysis. We interpret our findings as showing the presence of acid glycosaminoglycan (including chondroitin sulphate C) in the cells of the germinal layers of the postnatal rat brain. To our knowledge, this is the first specific localisation of this group of substances by a histochemical technique in this situation. It is well known that
360
SHORT COMMUNICATIONS
developing immature tissues contain glycosaminogtycan, and acid mucopolysaccharides (staining at a high C.E.C.) have been shown in cultures of 'spongioblasts' from disaggregated fetal mouse brain 12. It could be argued that staining of subependymal and external granular layers reflects the immaturity of their component cells, and that persistent staining of scattered subependymal cells in older animals is related to the persistent - - if declining - - mitotic activity and presumed gliogenesis at this site. Intense staining of ependyma and choroid plexus epithelium may be linked to the highly specialised nature of these glial cell layers. However, in the present state of our knowledge, it is hard to envisage what function this histochemical feature might be related to. A high concentration of chondroitin sulphate has been noted previously in choroid plexus 14, and the observations reported here suggest that this may be in the epithelial cell cytoplasm as well as in basement membranes. The most striking finding in the present study is, however, the staining of parenchymal neuroglial cells, many of which are clearly interfascicular oligodendroglia, in different fibre tracts at different ages. This staining appears to be correlated, in temporal terms, with myelin formation, for it is insignificant at birth and becomes prominent only at the end of the first postnatal week, in the period immediately preceding the phase of rapid myelination4, is - - which is accompanied by glycosaminoglycan synthesisL Ling and Leblond 1° have shown an increase in numbers of 'dark' oligodendrocytes in the rat corpus callosum between 3 weeks and 5 months, and a reciprocal fall in numbers of 'light' and 'medium' (immature) oligodendroglia. It is possible that these immature cells, which are presumably differentiating into mature oligodendroglia, are the cells demonstrated in the corpus callosum and elsewhere in the present study. It may be premature to equate Alcian blue staining of cells in fibre tracts with the deposition of myelin, but this possibility certainly merits further consideration.
This work was supported by a research grant from the Medical Research Council (P.D.L.).
1 ALLEN, E., The cessation of mitosis in the central nervous system of the albino rat, J. comp. Neurol., 22 (1912) 547-568. 2 BALAZS,R., BROOKSBANK,B. W. L., PATEL,A. J., JOrtNSON,A. L., AND WILSON, D. m., Incorporation of [35S]sulphate into brain constituents during development and the effects of thyroid hormone on myelination, Brain Research, 30 (1971) 273-293. 3 BAL~.ZS,R., Kov~,cs, S., COCKS,W. A., JOHNSON,A. L., ANDEAVRS,J. T., Effect of thyroid hormone on the biochemical maturation of rat brain: postnatal cell formation, Brain Research, 25 (1971) 555-570. 4 BASS,N. H., NETSKV,M. G., ANDYOUNG,E., Microchemical studies of postnatal development in rat cerebrum. 2. Formation of myelin, Neurology (Minneap.), 19 (1969) 405-414. 5 CAVANAGH,J. B., AND LEWIS,P. D., Perfusion-fixation, colchicine, and mitotic activity in the adult rat brain, J. Anat. (Lond.), 104 (1969) 341-350. 6 DINGLE,T. J., ANDWEBB,M., Mucopolysaccharide metabolism in tissue culture. In E. N. W~LLMER(Ed.), Cells and Tissues in Culture. Methods, Biology and Physiology, Vol. 1, Academic Press, New York, 1965, pp. 353-396. 7 GOLt)BeRG,J. M., ANt)CUNNINGHAM,W. L., Incorporation of [asS]sulphate into the glycosaminoglycans of rat brain, Bioehem. J., 120 (1970) 15P.
SHORT COMMUNICATIONS
361
8 HOPEWELL,J. W., A quantitative study of the mitotic activity in the suhependymal plate of adult rats, Cell Tiss. Kinet., 4 (1971) 273-278. 9 LEWIS, P. D., The fate of the subependymal cell in the adult rat brain, with a note on the origin of microglia, Brain, 91 0968) 721-738. 10 LING, E.-A., AND LEBLOND, C. P., Investigation of gliai cells in semithin sections. II. Variation with age in the numbers of the various glial cell types in rat cortex and corpus caUosum, J. comp. Neurol., 149 (1973) 73-82. 11 MARGOLIS,R. U., Acid mucopolysaccharides and proteins of bovine whole brain, white matter and myelin, Biochim. biophys. Acta (Amst.), 141 (1967) 91-102. 12 Moss, C. A., Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures, Hi~tochem. J., 5 (1973) 547-556. 13 PATERSON,J. A., PRIVAT,A., LING, E. A., AND LEBLOND,C. P., Investigation of glial cells in semithin sections. III. Transformation of subependymal cells into glial cells, as shown by radioautograpby after aH-thymidine injection into the lateral ventricle of the brain of young rats, J. comp. NeuroL, 149 (1973) 83-102. 14 SINGH, M., AND BACHHAWAT,B. K., The distribution and variation with age of different uronic acid-containing mucopolysaccharides in brain, J. Neurochem., 12 (1965) 519-525. 15 SCOTT, J. E., Histochemistry of Alcian blue. III. The molecular biological basis of staining by Alcian blue 8 GX and analogous phthalocyanins, Histochemie, 32 (1972) 191-212. 16 SCOTT,J. E., AND DORLING, J., Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions, Histochemie, 5 (1965) 221-233. 17 Vos, J., KURIYAMA,K., AND ROBERTS,E., Distribution of acid mucopolysaccharides in subcellular fractions of mouse brain, Brain Research, 12 (1969) 172-179. 18 WELLS, M. A., AND DITTMER, J. C., A comprehensive study of the postnatal changes in the concentration of the lipids of developing rat brain, Biochemistry, 6 (1967) 3169-3175. 19 ZEMAN, W., AND INNES, J. R. M., Craigie's Neuroanatomy of the Rat, Academic Press, New York, 1963, pp. 216-217