Immunohistochemical demonstration of S-100 protein and GFA protein in interstitial cells of rat pineal gland

Brain Research, 140 (1978) 1-13 ~) Elsevier/North-Holland Biomedical Press

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Research Reports

I M M U N O H I S T O C H E M I C A L D E M O N S T R A T I O N OF S-100 PROTEIN AND GFA PROTEIN IN INTERSTITIAL CELLS OF RAT PINEAL G L A N D

M. MOLLER, A. INGILD and E. BOCK Anatomy Department B, University of Copenhagen, DK-2100 Copenhagen 0 and the Protein Labor~;tory, University of Copenhagen, Sigurdsgade 34, DK-2200 Copenhagen N (Denmark)

(Accepted April 27th, 1977)

SUMMARY The presence of the glial marker proteins, the S-100 and the glial fibrillary acidic (GFA) protein, in the pineal gland was investigated in the rat. Using both the indirect peroxidase-labelled immunoglobulin technique and the unlabelled antibody enzyme (PAP) method, we observed few scattered S-100 and GFA positive cells in the pineal. The number, location and morphology of these cells suggest they are the pineal interstitial cells. This indicates that the interstitial cells are of neuroectodermal origin, possibly macroglial cells themselves. INTRODUCTION The mammalian pineal gland contains two types of cells. The dominant cell is the pinealocyte, believed to synthesize and release several hormones that antagonize the anterior pituitary lobe 9. The other type is the interstitial cell. In the rat 2,26, interstitial cells are few and usually situated near the pericapillary spaces. Milofsky 1~ considered these cells astrocytes based on electron microscopic observations of an abundance of microfilaments both in the perikaryon and in cell processes. However, the ultrastructure of these cells differs in many ways from the classical astrocyte in the brain~, ')~. Therefore the classification of the interstitial cell requires further clarification. During the past decade several proteins, claimed to be unique for nervous tissue, were isolated from the brain. The S-100 protein was isolated by Moore 1~ and found present in glial cells la. The glial fibrillary acidic protein (GFA) was isolated by Eng et al. 1~, and located in fibrous astrocytes 5. The localization of these proteins to specific cells was due to the development of highly sensitive immunohistochemical methods.

in the present study we use both the indirect peroxidase-labelled immunoglobulin technique and the unlabelled antibody enzyme method (PAP) 22, to locate the S-100 and GFA proteins in rat pineal glands. We demonstrate that both S-100 and G F A proteins are present in the interstitial cells. This indicates that these cells are macroglial or neuroectodermal in origin. MATERIAL AND METHODS Preparation of antisera

Human brain GFA was kindly supplied by Dr. L. F. Eng, Palo Alto, Calif. U.S.A., and antiserum was raised in rabbits. Antiserum raised against GFA isolated from human spinal cord was a gift from Drs. Dahl and Bignami, Boston, Mass., U.S.A. Purified ox brain S-100 and antiserum against ox brain S-100 were kindly supplied by Dr. K. G. Haglid, Gothenburg, Sweden, and antiserum against ox brain S-100 was raised in rabbits. An antiserum against ox brain micro-tubuli, raised in rabbits, was a gift from Dr. O. Behnke, Copenhagen, Denmark. Antisera against whole rat brain and whole humain brain were prepared as described by Bock et al 6. Normal swine serum, swine anti-rabbit lgG, peroxidase-labelled swine antirabbit IgG, and soluble complexes of horseradish peroxidase-rabbit anti-horseradish peroxidase (PAP) were obtained from Dako-immunoglobulins A/S, Copenhagen. The antibody titre of swine anti-rabbit IgG was 400 #g/ml, i.e. one ml of antibody precipitated 400 #g of rabbit IgG as determined by Sewell titration 2°. PAP has been prepared according to SternbergerZL The peroxidase content was 0.75 g/1. The antibody content was 1.98 g/l as estimated from photometric measurements. At pH 7.2 the r~tcm = 1.38. For peroxidase extinction coefficient of rabbit lgG one g/l was taken as ~27s F l'm ~ 0.720 and ~4o3 ~tc,, == 2.237 (one g/l, pH 7.2). the values used were ~278 Control o f antisera Immunoprecipitation. The specificity of the anti S-100 and anti GFA antisera

was tested by means of crossed immunoelectrophoresis with intermediate gel 3. Forebrain was homogenized in detergent containing mediumL The homogenate was centrifuged 6 × 106g × min and the supernatant was used for the immunoelectrophoresis as follows: supernatant containing 50-100 #g protein was submitted to ordinary agarose gel electrophoresis (first dimension electrophoresis) applying l0 V/cm for 40 min at 14-16 °C. After rotating the electric field 90 °, the electrophoresis was continued into an intermediate gel strip, containing the antiserum under investigation (antiS- 100 or anti-GFA). Above this gel strip another antibody-containing gel, the reference gel, was moulded. During the second dimension electrophoresis (1.5 V/cm, 18-20 h at 14-16 °C), the antigens migrate through the intermediate gel and thereafter through the reference gel. Depending upon the titre and the specificity of the antiserum in the intermediate gel, the antigens will be (a) completely, (b) partially or (c) not at all retarded by immunoprecipitation in the intermediate gel. This is reflected in the reference precipitate pattern by (a) absence of a precipitate, (b) fusion of reference and intermediate gel precipitates or (c) no change in the reference precipitate pattern as determined by comparing the test plate with an obligatory control plate with preimmune serum in the intermediate gel.

Preparation of purified antigens and absorption of antisera Human GFA was purified by hydroxylapatite chromatography as described by Dahl and Bignami a°. Rat S-100 was prepared by anionchromatography. Eight rat forebrains were homogenized in Tris • barbital buffer, 0.02 M, 5 m M EDTA, pH 8.6. The homogenate was centrifuged 6 × 10~g x rain. (NH4)2SO4 was added to the supernatant, 30 g/100 ml. Twenty-four hours later the solution was centrifuged 105 g x rain. The resulting supernatant was dialysed against a sodium acetate buffer 0.05 M, 1 m M EDTA, pH 5.6. Column chromatography was performed using 3 g DEAE Sephadex (Pharmacia, Sweden), suspended and equilibrated against the acetate buffer (approx. bed vol. 100 ml). The dialysed supernatant was applied on the column and proteins were stepwise eluted by acetate buffer, containing 1 m M EDTA, with increasing ionic strength (1-= 0.05, 0.1, 0.2, 0.4, 0.7). The S-100 protein was thereafter eluted from the column by 2 M NaC1 in acetate buffer, 0.05 M, 1 m M EDTA, pH 5.6. The purified antigens were mixed with the corresponding antiserum in varying amounts and the mixtures were allowed to react for 20-25 h at room temperature. After centrifugation (6 x lOSg × min) the supernatants were removed and used as control antisera.

Preparation of tissues for immunohistochemistry Forty-five albino Wistar rats of either sex were used. The rats were killed by decapitation or by ether inhalation. The brains were quickly removed from the skull. Two to three mm thick tissue blocks of the cerebral cortex, cerebellum and the pineal gland were dissected out. Cryostat sections. Some tissue blocks were immediately frozen in a COz-expansion cooler (Pearse-Slee) and 6-#m-thick cryostat sections were cut at --20 °C and placed on gelatinized glass slides. The sections were then fixed in acetone or l°~] methanol-free formaldehyde (prepared from paraformaldehyde) in phosphate buffered physiological saline (PBS) for 10 rain or I h at 4 °C (pH 7.4). Other freshly dissected tissue blocks were immediately fixed in acetone or 1 '~'o methanol-free formaldehyde in PBS (pH 7.4), for 10 rain or I h at 4 °C. Some of these blocks were immediately frozen in a CO~-expansion cooler and 6-/~m cryostat sections were cut and placed on gelatinized glass slider. Paraffin sections. The rest of the blocks were fixed in 1 ~ formaldehyde (pH 7.4) for 1 h, quickly dehydrated in increasing concentrations of ethanol and, via xylol, embedded in paraffin in vacuo. Six-/~m-thick sections were cut, placed on gelatinized glass slides and deparaffinized in xylol.

lmmunohistochemical staining In cryostat and paraffin sections endogenous peroxidase activity was blocked in some sections by incubation with 0.3 ~ hydrogen peroxide in methanol for 30 min 23. The sections were then incubated with swine serum, diluted 1/5 in PBS, for 10 min to decrease non-specific protein binding. These steps were followed by the specific immunohistochemical staining.

The indirect peroxidase-labelled immunoglobulin technique. The sections were sequentially incubated in 30-min steps in the following reagents: (1) specific rabbit antisera (anti-S-100, anti-GFA, and anti-microtubuli), (2) peroxidase-labelled swine anti-rabbit IgG (diluted 1/20). The unlabelled antibody enzyme (PAP) technique. The sections were sequentially incubated in 30-min steps in the following reagents: (1) specific rabbit antisera (antiS-100, anti-GFA, and anti-microtubuli), (2) swine anti-rabbit lgG (diluted 1/10), and (3) PAP (diluted 1/20). All the dilutions in the above-mentioned techniques were performed in PBS. The immunohistochemical stainings were performed with the specific rabbit antisera (anti-S-100, anti-GFA and anti-microtubuli) in the following PBS-dilutions: 1/5, 1/10, 1/20, 1/100, 1/500 and 1/1000. Each step was followed by washing in PBS for 10 min. By a 5-15 min reaction with a mixture containing 0.05 % 3,Y-diaminobenzidine tetrahydrochloride and 0.005/,~ hydrogen peroxide in 0.05 M Tris • HCI buffer (pH 7.6), the peroxidase bound to the tissues formed the characteristic brown insoluble reaction product. After a 15-min wash in distilled water, the sections were treated with 2 % OsO4 in water for 30 min, washed in tap water and mounted in glycerol gelatin. Some sections were lightly counter-stained with iron hematoxylin before mounting.

Control of specificity of the immuno-histochemical staining Immunohistochemical staining of both paraffin and cryostat sections was undertaken, where one of the following reagents was omitted : (1) specific rabbit antiserum (S-100 or GFA), (2) swine anti-rabbit IgG and (3) PAP. In some controls (4) the specific antiserum was replaced by rabbit serum; controls were also performed where (5) GFA and S-100 antisera, absorbed by GFA and S-100 proteins, were used; the staining of neurons seen with (6) the specific antiserum against microtubuli also served as a control for the methods used. RESULTS Immunohistochemical staining with the PAP as well as the indirect technique was effective on both the cryostat and paraffin sections. The best results were obtained on the cryostat sections, which were postfixed with acetone for 10 min and incubated with the specific rabbit antisera in the dilution 1/1000. If specific antisera in dilutions 1/5 were used, an unspecific staining of the nucleus and the nerve tracts was present. In our light microscopical experiments the PAP technique did not stain the cells more than the indirect technique. The paraffin sections gave a morphology very superior to the cryostat sections, but the sensitivity of the method decreased.

Control of antisera The specificity of the S-100 antiserum and the G F A antiserum was controlled by crossed immunoelectrophoresis with intermediate gel3. Both antisera reacted with only one antigen which was identified using purified S-100 and GFA. Figs. 1 and

Figs. 1 and 2. Extract of whole rat brain subjected to crossed immunoelectrophoresis with intermediate gel. The applied amount of total protein was 60 pg. The reference antiserum in the upper gel was antiwhole rat brain (anti-RB), code 0874, 20/~l/sq.cm. The intermediate gel in Fig. 1 (O) contained preimmune serum, 3.5/tl/sq.cm, and in Fig. 2 anti S-100, code 2075, 3.5 pl/sq.cm. Figs. 3 and 4. Extract of human brain cortical matter subjected to crossed immunoelectrophoresis with intermediate gel. The applied amount of total protein was 200/~g. The reference antiserum in the upper gel was anti-whole human brain (anti-HB) code 0874, 25/d/sq.cm. The intermediate gel (O) in Fig. 3 contained preimmune serum, 4,6 t~l/sq.cm and in Fig. 4 anti-GFA, code 0473, 4,6 ttl/sq.cm.

2 show a crossed immunoelectrophoresis with intermediate gel of S-100 antiserum using rat brain extract as reference antigen and anti-whole rat brain as reference antiserum. Fig. 1 demonstrates the reference pattern with preimmune serum in the intermediate gel. Fig. 2 shows the test plate with S-100 antiserum in the intermediate gel. One single immunoprecipitate, corresponding to the S-100 protein, disappeared from the reference gel and became apparent in the intermediate gel, demonstrating the specificity of the antiserum. Figs. 3 and 4 show a crossed immunoelectrophoresis with intermediate gel of GFA antiserum using human brain extract as reference antigen and anti-whole human brain as reference antiserum. Fig. 3 shows the reference pattern and Fig. 4 shows the test plate with anti-GFA in the intermediate gel. One immunoprecipitate corresponding to GFA disappeared from the reference gel and became apparent in the intermediate gel. In Figs. 5 and 6 the identity between GFA antiserum produced by us (antiGFA 1) and GFA antiserum produced by Drs. Dahl and Bignami (anti-GFA 2) is demonstrated. Both anti°GFA I and anti-GFA 2 gave a characteristic GFA immunoprecipitate with a sharp anodal part and a double contoured cathodal part. This was especially pronounced for GFA 2. Double contour of immunoprecipitates is often encountered using crossed immunoelectrophoresis (e.g. serum alpha 2 macroglobulin). It is a characteristic of the specific antigen-antibody precipitate and must not be interpreted as an indication of contaminating antibodies with other specificities. In Figs. 7 and 8 the identity between S-100 antiserum produced by us (anti-S100 1) and the S-100 antiserum produced by Dr. Haglid (anti-S-100 2) is demonstrated. Control sections Sections stained with the immunob, istochemical procedures, where one of the reagents was omitted showed no peroxidase reaction product in the cells, When the antisera were used, where the antibodies were absorbed by the specific antigens, the sections showed only slight gllal cell staining. When cortex cerebri was treated with anti-microtubuli serum, neuronal staining was present in all the cortical layers (Fig. 12). From the cell bodies radially-oriented, apical dendrites emerged. The sections from the pineal gland showed only light staining of all cells with anti-microtubuli. Demonstration of the S-IO0 protehl Cerebral cortex and cerebellum. Many cells located throughout the whole cerebral cortex were stained with S-100 antiserum (Fig. 9). These cells had a typical glial morphology with brown peroxidase stained perikarya from which long-branched processes extended (Fig. 9, inset). Often the S-100 positive cells were arranged as neuronal satellite cells. In the corpus callosum itself many single cells, possibly oligodendroglial cells, were stained. A peroxidase-stained limiting membrane was evident around the capillaries.

Figs. 5 and 6. Extract of human brain cortical matter subjected to crossed immunoelectrophoresis with intermediate gel. The applied amount of total protein was 80/~g. The reference antiserum, antiGFA-1, in the upper gel was anti-GFA, code 0473, 4/tl/sq.cm. The intermediate gel (O) in Fig. 3 contained preimmune serum, 10 t~l/sq.cm, and in Fig. 4 anti-GFA 2 (prepared by Dahl and Bignami), 10/~l/sq. cm. Fig. 7 and 8. Extract of human brain cortical matter subjected to crossed immunoelectrophoresis with intermediate gel. The applied amount of total protein was 100/~g. The reference antiserum anti S-100 I, in the upper gel was anti S-100, code 2075, 3.5/tl/sq.cm. The interinediate gel (O) in Fig. 7 contained preimmune serum, 7/tl/sq.cm and in Fig. 8 anti-S-100 2, (prepared by Dr. Haglid), 7/~l/sq.cm.

in the cerebellum m a n y peroxidase-stained cells were a p p a r e n t in the g r a n u l a r layer and few in the m o l e c u l a r layer. The p e r i k a r y a o f the Purkinje cells were surrounded by stained satellite cells. In a d d i t i o n the radially-oriented Bergmann glial fibers in the m o l e c u l a r layer were lightly stained. Many, p r o b a b l y oligodendroglial cells, were located between the nerve tracts. Pineal gland. Cells that stained with S-100 antiserum were few c o m p a r e d with the total n u m b e r o f cells in the pineal. A c o n c e n t r a t i o n o f stained cells occurred in the region o f the pineal gland close to the pineal stalk but stained cells were scattered t h r o u g h o u t the gland. These cells had brown peroxidase staining o f the cell bodies (Fig. 10), and from the cell b o d y 2-6 stained cellular processes emerged. These pro-

Fig. 9, Cortex cerebri with peroxidase staining for the S-100 protein (antiserum dilution I/5). Many dark stained astrocytes are seen throughout the hemisphere. Note the unspecific staining of corpus callosum (CC). 95. Inset: two astrocytes from Fig. 5. , 265. Fig. 10. The pineal gland with peroxidase staining for the S-100 protein. Several cells tarrows) with slender processes are stained. P, perivascular space. Antiserum dilution 1/20. 95,

cesses often a p p r o a c h e d the perivascular space, where they frequently m a d e a barrier between the perivascular space and the pineal p a r e n c h y m a .

Demonstration of the GFA protein Cerebral cortex, l m m u n o h i s t o c h e m i c a l staining o f sections from cortex cerebri (Fig. 13) with a n t i - G F A serum resulted in staining o f m a n y cell bodies, from which long t o r t u o u s processes emerged. These cells were spread t h r o u g h o u t the cortex cere-

Fig. 11. lmmunohistochemical reaction for GFA protein on the cerebellum. Note the Bergmann glial fibers in the molecular layer (M). G, granular layer. Antiserum dilution 1/1000. ~ 100. Fig. 12. Control section from cortex cerebri stained with the anti-microtubuli antiserum (dilution 1/20). Note the dark stained neurons (arrows). × 95.

Fig. 13. l m m u n o h i s t o c h e m i c a l reaction for the G FA protein on a cerebral cortical area. T h e bended arrows point to positive stained astrocytes with long slender cellular processes. A n t i s e r u m dilution l J 1000. x 425. Fig. 14. T h e pineal gland with peroxidase staining for the G F A protein. Several triangular stained cells (straight arrows) are seen. C u r v e d arrow indicates area from which the pineal stalk has been t o r n away. A n t i s e r u m dilution 1/20. 90. Fig. 15. High power magnification o f one of the cells in Fig. 9. T h e arrow shows ,t very dark stained triangular cell with a process propagating to a capillary (C). A n t i s e r u m dilution I,;Y3 600.

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bri and were often located as satellite cells around the neurons. No difference in staining intensities between the G F A positive cells was observed. Many bipolar cells with their long axis parallel with the nerve tracts were scattered throughout the corpus callosum. In the cerebellum the radial Bergmann glial fibers were stained together with few cells in the molecular layer (Fig. 11). Many big multipolar cells were seen in the granular layer, and smaller, often bipolar, cells were observed in the central nerve tract area. Pineal gland. Staining of pineal sections with anti-GFA serum demonstrated peroxidase-stained cells identical in localization and morphology to the cells seen after staining with S-100 antiserum (Figs. 14 and 15). DISCUSSION

The interstitial cells in the mammalian pineal gland were described 50 years ago. These cells were clearly visualized by metal impregnation techniques used for astroglial cells and were therefore classified as astrocytes. The first ultrastructural studies on the mammalian pineal gland of rat 15, rabbit 25, cow and sheep 1, and cat and monkey 24 described the interstitial cells as astrocytes of the fibrillar types. This description was based on the abundance of intracellular filaments in cell processes. More extensive ultrastructural studies in the rat z,26 questioned whether or not this cell was equivalent to the fibrillar astrocyte in the central nervous system. Both Wolfe 2~ and Arstila" observed that the ultrastructure of the interstitial cell was unlike the fibrillar astrocyte in the brain, especially because of the high content of heterogenous dark bodies. It was also suggested that the interstitial cell was not of neuroectodermal origin. However, in recent ultrastructural studies of the pineal gland in the pocket gopher 21 and the ground squirrel iv the interstitial cells are described as classical fibrillary astrocytes. In these papers the authors claim that some of the interstitial cells might be oligodendroglial ceils. Autoradiographic studies 19 have shown that the interstitial cells in the rat pineal gland are able to concentrate 7-aminobutyric acid. The interstitial cells share that ability with brain glial cells. By modern immunohistochemical methods it is possible to localize specific proteins in cells. It was our purpose to add immunological evidence to the morphological discussion by examining whether or not two glial marker proteins, the S-100 and the G F A protein, are present in the interstitial cells. We have used both the indirect immunoperoxidase technique and the PAP method 22. This last methods should have an enhanced specificity and sensitivity compared to methods, where enzymes are covalently linked to antibodies 8. The G F A protein is localized in both fibrillar and protoplasmatic astrocytes 14. Ultrastructural studies ~2 suggest this protein is associated with the astrocytic filaments. The S-100 protein is also localized in glial cells but this protein is present both in astrocytes and oligodendrocytes 14. In addition some studies have suggested that a fraction of the S-100 is located in neuronal membranO 8.

12 In our study we find staining of many multipolar cells in the cerebral cortex and especially in the granular layer of cerebellum, both with the G F A and S-100 antisera. In addition both sera stain bipolar cells in the corpus callosum and the central tract area of cerebellum. We can confirm earlier studies 14, that a fibrillar pattern is observed in cells stained with the GFA antiserum, in contrary to S-100-stained cells, but in our study the S-100 antisera in addition to perikarya also stained the cell processes. However, it is difficult only on the morphology of the peroxidase-stained cells to distinguish between astroglial and oligodendroglial cells. In the pineal gland we observed few scattered cells stained with GFA and S-100 antisera. One possibility is, that these cells are the interstitial cells. Based on three lines of evidence: (1) paucity of stained cells compared to the total number of glandular cells, (2) the ovoid to triangular shape of the cell bodies and (3) processes of stained cells penetrate the parenchyma to the perivascular space, we believe they are the classical interstitial cells. However, a definitive proof has to be made by immunoelectronmicroscopy. The immunohistochemical procedure itself is delicate. The tissue proteins (S-100, GFA) we have worked with are especially difficult because they do not tolerate long fixation times. Unspecific staining of the edges and any protuding parts of the section present problems; however, preincubation with swine serum reduced these. Unspecific staining of nerve tracts and nuclei was always a problem when the antisera used for immunohistochemistry were too concentrated. This unspecific staining always disappeared when highly diluted antisera were used. Our study demonstrates that two glial marker proteins, GFA and S-100, are present in certain cells in the rat pineal gland. Several factors indicate that G F A and S-100 positive cells are the classical interstitial cells of the pineal gland. The hypothesis that these cells might be macroglial cells or of macroglial origin is thus supported. ACKNOWLEDGEMENTS The skillful technical assistance by Mrs. Lone Andersen, Anni Andersen and Jette Nording is gratefully acknowledged. The advice, suggestions and help from the staffat the Histochemical Laboratory, Anatomy Department A, University of Copenhagen and financial support of the Carlsberg Foundation, the Danish Cancer Society and the P. Carl Petersen's Foundation is gratefully acknowledged.

REFERENCES 1 Anderson, E., The anatomy of bovine and ovine pineals, J. Ultrastruct. Res., Suppl. 8 (1965). 2 Arstila, A. U., Electron microscopic studies on the structure and histochemistry of the pineal gland of the rat, Neuroendocrinology, 2, Suppl. (1967). 3 Axelsen, N. H., Intermediate gel in crossed and in fused rocket immunoelectrophoresis, Scand. J. ImmunoL, 2, Suppl. 1 (1973) 71-77. 4 Bargman, W., Die Epiphysis Cerebri. In W. v. M611endorff(Ed.), Handbuch der Mikroskopischen Anatomie des Menschen, Bd. I/1/4, Springer Verlag, Berlin, 1943, pp. 309-502.

13 5 Bignami, A., Eng, L. F., Dahl, D. and Uyeda, C. T., Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence, Bruin Research, 43 (1972) 429435. 6 Boek, E., Mellerup, E. T. and Rafaelsen, O. J., Antigen-antibody crossed electrophoresis of water soluble rat brain proteins, J. Neurochem., 18 (1971)2435-2440. 7 Bock, E., Jorgensen, O. S. and Morris, S.J., Antigen-antibody crossed electrophoresis of rat brain synaptosomes and synaptic vesicles: correlation to water-soluble antigens from rat brain, J. Neurochent., 22 (1974) 1013-1017. 8 Burns, J., Background staining and sensitivity of the unlabelled antibody-enzyme (PAP) method. Comparison with the peroxidase-labelled antibody sandwich method using formalin fixed paraffin embedded material, Histochemistry, 43 (1975) 291-294. 9 Cardinali, D. P., Melatonin and the endocrine role of the pineal organ. In V. H. T. James and L. Martini (Eds.), Current Topics h7 Experimental Endocrinology, Vol. 2, Academic Press, New York, 1974, pp. 107-128. t0 Dahl, D. and Bignami, A., Glial fibrillary acidic protein from normal and gliosed human brain. Demonstration of multiple related polypeptides, Biochim. biophys. Acta (Amst.), 386 (1975) 41 51. 11 Eng, L. F., Vanderhaeghen, J. J., Bignami, A. and Gerstl, B., An acidic protein isolated from fibrous astrocytes, Brain Researeh, 28 (1971) 351 354. 12 Eng, L. F., Kosek, J. C. and Miles, L. E. M., lmmunohistologic and immunoradiometric study with brain specific proteins, Trans. Amer. Soe. Neurochem., 6 (1975) 75. 13 Hyden, H. and McEwen, B. S., A glial protein specific for the nervous s:ystem, Proc. nat. Aead. Sei. (Wash.), 55 (1966) 354 358. 14 Ludwin, S. K., Kosek, J. C. and Eng, L. F., The topographical distribution of S-100 and G F A proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidaselabelled antibodies, J. comp. Neurol., 165 (1976) 197 208. 15 Milofsk~,, A., The fine structure of the pineal in the rat, with special reference to parenchyma, Anat. Rec., 127 (1957)435 436. 16 Moore, B.W., A soluble protein characteristic of the nervous system, Bioehem. biophys. Res. CommuH., 19 (1965) 739 744. 17 Povlishock, J. T., Kriebel, R. M. and Seibel, H. R., A light and electron microscopic study of the pineal gland of the ground squirrel, Citellus trideeemlineatus, Amer. J. Anat., 143 (1975) 465-484. 18 R6nnb/.ick, L., Studies on S-100, a brain-specific protein, during maturation and in the adult nervous system of rat, rabbit and guinea pig, Thesis, Gothenburg, 1975. 19 Schon, F. and Kelly, J. S., The characterisation of [aH]GABA uptake into the satellite glial cells of rat sensory ganglia, Brain Research, 66 (1974) 289 300. 20 Sewell, M. M. H., A semi-quantitative technique using the LKB immunodiffusion apparatus, Sci. Tools, 14 (1976) 11. 21 Sheridan, M. N. and Reiter, R. J., The fine structure of the pineal gla~ld in the pocket gopher, Geo,o's bursariu.~, Amer. J. Anat., 136 (1973) 363 382. 22 Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J. and Meyer, H. G., The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen antibody complex (horseradish peroxidase antihorseradish peroxidase) and its use in identification of spirochete:~, J. Histochem. Cytochem., 18 (1970) 315-333. 23 Streefkerk, J. G., 1nhibition of erythroc:,te pseudoperoxidase activity by treatment with hydrogen peroxide following methanol, J. Histochem. Cvtochem., 20 (1972) 829 831. 24 Wartenberg, H., The mammalian pineal organ: electron microscopic studies on the fine structure of pinealocytes, glial cells and on the perivascular compartment, Z. Zell[orsch., 86 (1968) 74 97. 25 Wartenberg, H. and Gusek, W., Licht- und Elektronenmikroskopische Beobachtungen fiber die Struktur der Epiphysis Cerebri des Kaninchens, Progr. Brain Res., 10 (1965) 296-315. 26 Wolfe. D. E., The epiphysial cell: an electron-microscopic study of the intercellular relationship and intercellular morphology in the pineal body of the albino rat, Progr. Brain Res., 10 (1965) 332 376.