0736-5748/83 $3.00+0.00 Pergamon Press Ltd. 1983 ISDN
Int. J. Devl. Neuroscience, Vol. 1, No. 6, pp. 411-425, 1983. Printed in Great Britain.
I M M U N O H I S T O C H E M I C A L A N D BIOCHEMICAL A P P R O A C H E S TO THE D E V E L O P M E N T OF N E U R O G L I A IN THE CNS, WITH SPECIAL R E F E R E N C E TO C E R E B E L L U M M. S.
GHANDOUR,*
O. K. LANGLEY*and J. CLOSI
*Centre de Neurochimie du CNRS, 5 rue Blaise Pascal, 67084 Strasbourg C6dex and tLaboratoire de Physiologie Compar6e, Universit6 des Sciences et Techniques du Languedoc, Place E. Bataillon 34060 Montpellier C6dex, France (Accepted 10 August 1983)
Abstraet--Immunocytochemical methods have in recent years played a more important role in investigations of the development and function of glial cells in the nervous system because of their potential to distinguish between different cell populations. This short review attempts to highlight the value of this approach and summarizes the major cell-type markers currently available. These include, for the astrocyte, GFA protein, S-100 protein, vimentin, aa-enolase and a-2 glycoprotein. For the oligodendrocyte, myelin basic protein, the Wolfgram proteins, 2',3'-cyclic nucleotide 3'-phosphohydrolase, myelin associated glycoprotein, proteolipid protein, galactocerebroside, carbonic anhydrase and glycerol 3phosphate dehydrogenase and other glial cell markers recognized by monoclonal antibodies are discussed. The application of these techniques to the study of the developing brain (and in particular the rodent cerebellum) are reviewed. It has proved possible to follow the development of distinct populations of astrocytes and oligodendrocytes from a very precocious age to the adult situation, thus providing new insight on the relationship between glial cells and neurons during normal and abnormal histogenesis. Key words: Astrocyte markers, Oligodendrocyte markers, Glial cells development, Cerebellum.
The glial (or supporting) cells in the central nervous system (CNS) were first described as a population distinct from neurons by Virchow. 141 The origin of glial cells from neuroepithelia was firstly and firmly established by the classical study of R a m o n y Cajal. 113 There are two distinct populations of glial cells, oligodendrocytes and astrocytes, 31'1°°'1°1 whose histological and ultrastructural characteristics are well known in both adult 6'12'13'56'83'90'9s'107'113'127'140 and immature nervous systems. 5,14,30,75,88,89,107,113,123,124,128,135,140 Because it contains relatively few neuronal cell types located in three distinct layers, the cerebellum represents an attractive region of the nervous system for biochemical and morphological analysis. Detailed data concerning glial cell organization in adult cerebellum are available in the literature.6,34,99,113,127 In this organ, the protoplasmic astroglia is represented by Bergmann glial cells (or Golgi epithelial cells) and by velamentous astrocytes in the granular layer (Figs 1 and 2), while fibrous astrocytes are present essentially in the white matter (Figs 3 and 4). A large n u m b e r of oligodendrocytes are present in the white matter (Fig. 5) with fewer in the granular and Purkinje cell layers. Oligodendrocytes are rarely observed in the deep part of the molecular layer. 45'48'99'134 Whereas the development of neuronal cell types in the cerebellum is relatively well known, 2~ morphological studies on cerebellar glia development, based on gold sublimate, Golgi or silver impregnations and electron microscopy are comparatively scarce and have provided an incomplete picture. 8'17'18'26'30'74'75'107'113 Recognition of astrocytes and oligodendrocytes during the early stages of cerebellar d e v e l o p m e n t by classical morphological methods remains difficult and equivocal. F u r t h e r m o r e , the ability of the cerebellar external granular layer (germinative layer) to produce both neuronal and glial cells is still in dispute. 29"41'50'84"107"128"134 In recent years, particular attention has been paid to the importance of glial elements in the histogenesis of nervous tissue, particularly of the cerebellar cortex, and to considerations of the role of glia in both physiological and pathological conditions during ontogenesis. 13,34,58,64,98,101,112,121 During the same period, the identification and characterization of some specific glial cell components and the availability of cell type-specific antibodies conjugated to visual labels have allowed an easier distinction of the different neuroglial cell types, particularly in the cerebellum. This p a p e r concentrates on most recent data concerning some glial cell markers during cerebellar development, particularly in the rat and mouse where it is essentially postnatal. Some problems encountered with immunohistological techniques are described elsewhere. 44'45'1°2'139 411
412
M.S. Ghandour eta1. G L I A L C E L L M A R K E R S AND N E U R O G L 1 A
Astrocyte markers Glialfibrillary acidic protein (GFA). G F A was isolated firstly from gliosed human brain.35 Other sources for G F A isolation were also used.22-24 G F A is considered the best currently available astrocyte marker o n CNS, 9"36'37"117particularly in cerebellum, and is associated with the glial filaments in astrocyte cytoplasm. S 1O0protein. The soluble acidic protein S 10087 is localized in glial cells.21'36'37'43'49"76's2 A putative minor localization in neurons has not been clearly demonstrated. 32"33,53,54'59'13s Recently, a systematic study at light and electron microscopic levels on S100 protein localization in the rat cerebellum showed that this protein is not only exclusively located in glial cells,21,49,50 but can be used as an exclusive marker for astrocytes. 49"5° aa-Enolase. One of the enolase isoenzymes, called aa-enolase or non-neuronal enolase (NNE) has been found to be specific to glial cells, 51'71"78"119'12° while the brain specific enolase or -y~/enolase or 14-3-2 protein is located in neurons. 47"51'7°'1°3'119A2° The two forms of enolase are immunologically distinct.11"51"63'77"145Recently, a detailed study of aa-enolase localization at light and electron microscopic levels in rat cerebellum showed that this isoenzyme is present only in astroglia.51,71 Other astrocyte markers. A number of other antigens are also present only in astrocytes of CNS, for example, glutamine synthetase. 92'93 In the cerebellum the protoplasmic astrocytes and Bergmann glia, which are located in the gray matter, contain more glutamine synthetase than the fibrous astrocytes in the white matter. 92 The ~-2 glycoprotein, isolated by Warecka 142 from human brain has also been found to be a specific astroglial marker in cerebellum. 52 Monoclonal antibodies, designated anti-M1 and anti-C1 were also detected in astrocytes of cerebellum. 6s'126 In contrast, butyrylcholinesterase can be considered as a general marker for glial cells. 43"62"137 Vimentin, another intermediate filament 1°'25'~36 protein appears to be a marker for immature astrocyte.10"25 Immunohistochemical localization for S100 protein, oLa-enolase and G F A is represented by Figs 6, 7 and 9, respectively. The markers of adult cerebellar astroglia are indexed in Fig. 10. Astrocyte development In rat cerebellum the first studies of astrocyte development by immunohistochemistry with antibodies, particularly with those directed against GFA, were limited to Bergmann glia development. 9 Astrocyte formation has also been studied in both monkey and human brain with this antibody. 15.73. More recently, the development of all types of astrocytes in rat cerebellum has been described in detail. 49'5° Data are now available for GFA, S100 protein, ~ - e n o l a s e , anti-Ml and anti-C1 development patterns in this organ. To our knowledge, no developmental study has so far been published for glutamine synthetase and a-2 glycoprotein. At birth a number of immature astrocytes in the medullary layer of the rat cerebellum and a large number of Bergmann fibers are stained for S100 protein; s° in contrast the presence of G F A protein is limited to a few cells and to some Bergmann fibres. 5° This suggests that S100 protein is present earlier in immature astrocytes than GFA. The number of astrocytes (S100-containing cells), particularly those located in the white matter, increased significantly from days 4 to 7, when the Bergmann glial cells align themselves in the neighbourhood of Purkinje cells. From days 3 to 4 after birth the perikarya of Bergmann glial cells and astrocytes in the future granular layer and white matter are well stained for GFA. By day 7 a large number of astrocytes (GFA-containing cells) appear in the internal granular layer and in the white matter (Figs 14 and 15). During the second week after birth the developmental patterns as shown with anti-Sl00 parallel those with anti-GFA antibodies. By day 7 astrocytes in the white matter are stained with the monoclonal antibodies antiM1 and anti-C1.68,126 Bergmann glia and the other protoplasmic astrocytes in the granular layer are transiently stained for MI from day 10 after birth to the end of week 4, while only the Bergmann glia is still stained for C1 after day 10. 68"126
Figs 1-5. All glial cell types are shown in these midsagittal sections of the cerebellar vermis of 21-day-old rats, impregnated according to the Golgi- Cox method. Abbreviations: ML, molect~lar layer; GL, granular layer; WM, white mater; P, Purkinje cells; PCL, Purkinje cell layer; ol, oligodendrocyte; f.ast fibrous astrocyte; p.ast, protoplasmic astrocyte; BG, Bergmann glia. Fig. 1. Several Bergmann astrocytes, near Purkinje cell bodies in the molecular layer and one fibrous astrocyte in the internal granular layer. Fig. 2. One protoplasmic astrocyte in the internal granular layer. Figs 3 and 4. Two fibrous astrocytes !n the internal granular layer and in the white matter. Fig 5. One oligodendrocyte in the white matter.
413
Figs G9. Captions
on p. 417.
414
i
y
Figs 11-16. Captions on p. 418.
415
Neuroglia in adult and developing cerebellum
417
ML
PCL
GL
WM
oliqodendrocyte
astrocyte GFA protein S 100-protein
e
~ a¢.enolase glutamine synthetase *
Int. J. Devl. Neuroscience, Vol. 1, No. 6, pp. 411-425, 1983. Printed in Great Britain.
I M M U N O H I S T O C H E M I C A L A N D BIOCHEMICAL A P P R O A C H E S TO THE D E V E L O P M E N T OF N E U R O G L I A IN THE CNS, WITH SPECIAL R E F E R E N C E TO C E R E B E L L U M M. S.
GHANDOUR,*
O. K. LANGLEY*and J. CLOSI
*Centre de Neurochimie du CNRS, 5 rue Blaise Pascal, 67084 Strasbourg C6dex and tLaboratoire de Physiologie Compar6e, Universit6 des Sciences et Techniques du Languedoc, Place E. Bataillon 34060 Montpellier C6dex, France (Accepted 10 August 1983)
Abstraet--Immunocytochemical methods have in recent years played a more important role in investigations of the development and function of glial cells in the nervous system because of their potential to distinguish between different cell populations. This short review attempts to highlight the value of this approach and summarizes the major cell-type markers currently available. These include, for the astrocyte, GFA protein, S-100 protein, vimentin, aa-enolase and a-2 glycoprotein. For the oligodendrocyte, myelin basic protein, the Wolfgram proteins, 2',3'-cyclic nucleotide 3'-phosphohydrolase, myelin associated glycoprotein, proteolipid protein, galactocerebroside, carbonic anhydrase and glycerol 3phosphate dehydrogenase and other glial cell markers recognized by monoclonal antibodies are discussed. The application of these techniques to the study of the developing brain (and in particular the rodent cerebellum) are reviewed. It has proved possible to follow the development of distinct populations of astrocytes and oligodendrocytes from a very precocious age to the adult situation, thus providing new insight on the relationship between glial cells and neurons during normal and abnormal histogenesis. Key words: Astrocyte markers, Oligodendrocyte markers, Glial cells development, Cerebellum.
The glial (or supporting) cells in the central nervous system (CNS) were first described as a population distinct from neurons by Virchow. 141 The origin of glial cells from neuroepithelia was firstly and firmly established by the classical study of R a m o n y Cajal. 113 There are two distinct populations of glial cells, oligodendrocytes and astrocytes, 31'1°°'1°1 whose histological and ultrastructural characteristics are well known in both adult 6'12'13'56'83'90'9s'107'113'127'140 and immature nervous systems. 5,14,30,75,88,89,107,113,123,124,128,135,140 Because it contains relatively few neuronal cell types located in three distinct layers, the cerebellum represents an attractive region of the nervous system for biochemical and morphological analysis. Detailed data concerning glial cell organization in adult cerebellum are available in the literature.6,34,99,113,127 In this organ, the protoplasmic astroglia is represented by Bergmann glial cells (or Golgi epithelial cells) and by velamentous astrocytes in the granular layer (Figs 1 and 2), while fibrous astrocytes are present essentially in the white matter (Figs 3 and 4). A large n u m b e r of oligodendrocytes are present in the white matter (Fig. 5) with fewer in the granular and Purkinje cell layers. Oligodendrocytes are rarely observed in the deep part of the molecular layer. 45'48'99'134 Whereas the development of neuronal cell types in the cerebellum is relatively well known, 2~ morphological studies on cerebellar glia development, based on gold sublimate, Golgi or silver impregnations and electron microscopy are comparatively scarce and have provided an incomplete picture. 8'17'18'26'30'74'75'107'113 Recognition of astrocytes and oligodendrocytes during the early stages of cerebellar d e v e l o p m e n t by classical morphological methods remains difficult and equivocal. F u r t h e r m o r e , the ability of the cerebellar external granular layer (germinative layer) to produce both neuronal and glial cells is still in dispute. 29"41'50'84"107"128"134 In recent years, particular attention has been paid to the importance of glial elements in the histogenesis of nervous tissue, particularly of the cerebellar cortex, and to considerations of the role of glia in both physiological and pathological conditions during ontogenesis. 13,34,58,64,98,101,112,121 During the same period, the identification and characterization of some specific glial cell components and the availability of cell type-specific antibodies conjugated to visual labels have allowed an easier distinction of the different neuroglial cell types, particularly in the cerebellum. This p a p e r concentrates on most recent data concerning some glial cell markers during cerebellar development, particularly in the rat and mouse where it is essentially postnatal. Some problems encountered with immunohistological techniques are described elsewhere. 44'45'1°2'139 411
412
M.S. Ghandour eta1. G L I A L C E L L M A R K E R S AND N E U R O G L 1 A
Astrocyte markers Glialfibrillary acidic protein (GFA). G F A was isolated firstly from gliosed human brain.35 Other sources for G F A isolation were also used.22-24 G F A is considered the best currently available astrocyte marker o n CNS, 9"36'37"117particularly in cerebellum, and is associated with the glial filaments in astrocyte cytoplasm. S 1O0protein. The soluble acidic protein S 10087 is localized in glial cells.21'36'37'43'49"76's2 A putative minor localization in neurons has not been clearly demonstrated. 32"33,53,54'59'13s Recently, a systematic study at light and electron microscopic levels on S100 protein localization in the rat cerebellum showed that this protein is not only exclusively located in glial cells,21,49,50 but can be used as an exclusive marker for astrocytes. 49"5° aa-Enolase. One of the enolase isoenzymes, called aa-enolase or non-neuronal enolase (NNE) has been found to be specific to glial cells, 51'71"78"119'12° while the brain specific enolase or -y~/enolase or 14-3-2 protein is located in neurons. 47"51'7°'1°3'119A2° The two forms of enolase are immunologically distinct.11"51"63'77"145Recently, a detailed study of aa-enolase localization at light and electron microscopic levels in rat cerebellum showed that this isoenzyme is present only in astroglia.51,71 Other astrocyte markers. A number of other antigens are also present only in astrocytes of CNS, for example, glutamine synthetase. 92'93 In the cerebellum the protoplasmic astrocytes and Bergmann glia, which are located in the gray matter, contain more glutamine synthetase than the fibrous astrocytes in the white matter. 92 The ~-2 glycoprotein, isolated by Warecka 142 from human brain has also been found to be a specific astroglial marker in cerebellum. 52 Monoclonal antibodies, designated anti-M1 and anti-C1 were also detected in astrocytes of cerebellum. 6s'126 In contrast, butyrylcholinesterase can be considered as a general marker for glial cells. 43"62"137 Vimentin, another intermediate filament 1°'25'~36 protein appears to be a marker for immature astrocyte.10"25 Immunohistochemical localization for S100 protein, oLa-enolase and G F A is represented by Figs 6, 7 and 9, respectively. The markers of adult cerebellar astroglia are indexed in Fig. 10. Astrocyte development In rat cerebellum the first studies of astrocyte development by immunohistochemistry with antibodies, particularly with those directed against GFA, were limited to Bergmann glia development. 9 Astrocyte formation has also been studied in both monkey and human brain with this antibody. 15.73. More recently, the development of all types of astrocytes in rat cerebellum has been described in detail. 49'5° Data are now available for GFA, S100 protein, ~ - e n o l a s e , anti-Ml and anti-C1 development patterns in this organ. To our knowledge, no developmental study has so far been published for glutamine synthetase and a-2 glycoprotein. At birth a number of immature astrocytes in the medullary layer of the rat cerebellum and a large number of Bergmann fibers are stained for S100 protein; s° in contrast the presence of G F A protein is limited to a few cells and to some Bergmann fibres. 5° This suggests that S100 protein is present earlier in immature astrocytes than GFA. The number of astrocytes (S100-containing cells), particularly those located in the white matter, increased significantly from days 4 to 7, when the Bergmann glial cells align themselves in the neighbourhood of Purkinje cells. From days 3 to 4 after birth the perikarya of Bergmann glial cells and astrocytes in the future granular layer and white matter are well stained for GFA. By day 7 a large number of astrocytes (GFA-containing cells) appear in the internal granular layer and in the white matter (Figs 14 and 15). During the second week after birth the developmental patterns as shown with anti-Sl00 parallel those with anti-GFA antibodies. By day 7 astrocytes in the white matter are stained with the monoclonal antibodies antiM1 and anti-C1.68,126 Bergmann glia and the other protoplasmic astrocytes in the granular layer are transiently stained for MI from day 10 after birth to the end of week 4, while only the Bergmann glia is still stained for C1 after day 10. 68"126
Figs 1-5. All glial cell types are shown in these midsagittal sections of the cerebellar vermis of 21-day-old rats, impregnated according to the Golgi- Cox method. Abbreviations: ML, molect~lar layer; GL, granular layer; WM, white mater; P, Purkinje cells; PCL, Purkinje cell layer; ol, oligodendrocyte; f.ast fibrous astrocyte; p.ast, protoplasmic astrocyte; BG, Bergmann glia. Fig. 1. Several Bergmann astrocytes, near Purkinje cell bodies in the molecular layer and one fibrous astrocyte in the internal granular layer. Fig. 2. One protoplasmic astrocyte in the internal granular layer. Figs 3 and 4. Two fibrous astrocytes !n the internal granular layer and in the white matter. Fig 5. One oligodendrocyte in the white matter.
413
Figs G9. Captions
on p. 417.
414
i
y
Figs 11-16. Captions on p. 418.
415
Neuroglia in adult and developing cerebellum
417
ML
PCL
GL
WM
oliqodendrocyte
astrocyte GFA protein S 100-protein
e
~ a¢.enolase glutamine synthetase *
