Cerebellar cell surface antigens of mouse brain

Proc. Nat. Acad. Sci. USA

Vol. 72, No. 10, pp. 4110-4114, October 1975 Immunology

Cerebellar cell surface antigens of mouse brain (reaggregated cell culture/cerebellar mutants/differentiation/cytotoxicity/immunofluorescence)

NICHOLAS W. SEEDS Department of Biophysics & Genetics and Department of Psychiatry, University of Colorado Medical Center, Denver, Colo. 80220

Communicated by Theodore T. Puck, July 11, 1975

ABSTRACT Reaggregated cells from 6- to 8-day-old mouse cerebella have been used to raise antibodies in rabbits. The interaction of these antibodies with cerebellar cell surface components was assessed by cytotoxicity of 5lCr-labeled cerebellar cell cultures and indirect immunofluorescence. A quantitative comparison of the relative amount of antigen on cells from other mouse tissues, brain regions, cerebella of various aged mice and mutant mice, and other animal species, as well as several clonal cell lines of nervous system origin, was made. A fixed subthreshold concentration of antiserum was adsorbed with increasing numbers of dissociated cells or amounts of particulate tissue prior to incubation with complement and 5tCr-labeled cerebellar target cells. Mouse thymus, spleen, liver, and heart tissue possess negligible adsorbing capacity, whereas kidney and sperm gave some adsorption. Of the brain regions examined, only cerebellum removed all immunofluorescence and cytotoxic activity, whereas other regions removed less than 90%, suggesting the possibility of cerebellar specific antigens on certain cell types. Only mouse and rat cerebellum gave measurable adsorptions, and this capacity decreased with increasing age. Although cerebellar mutants (staggerer, weaver, and nervous) possessed similar adsorptive capacity, glioma and neuroblastoma clonal cell lines differed measurably in their adsorption; only the mouse neuroblastoma clones displayed significant adsorption of the antiserum. Reaggregated brain cell cultures undergo extensive biochemical differentiation and synaptogenesis resembling in vivo brain maturation in both rate and extent of development (1-4). Furthermore, a more defined system of reaggregates from specific brain regions, notably the cerebellum, displays biochemical differentiation that reflects normal cerebellar maturation (5). It has been suggested that development observed in these reaggregated cell cultures may be related to the reestablishment of specific cell interactions that are favored by this culture technique (6). Although the initial aggregate is a random mixture of cells, cell migration leads to distinct cellular arrays characteristic of the tissue of origin (7, 8). Cells from various brain regions will segregate from one another, and cells from the same region can reorganize into histotypic structures (9, 10). Furthermore, cells from certain neurological mutants characterized by abnormal cell migration show the same abnormality in vitro (11). One may propose that these migratory and sorting out activities of brain cells, as well as synaptogenesis, are related to the presence and distribution of specific cell surface molecules that distinguish cell classes, and possibly different developmental stages of a particular cell class. If so, the sensitivity and specificity of the immune system may allow us to distinguish various cell types and stages of differentiation within the nervous system. Such antisera could be used as a tool in cell isolation, identification, and manipulation of brain cell cultures. Abbreviations: anti-Cbl, rabbit antiserum against mouse cerebellum; Ab, antibody; C', complement; NRbS, normal rabbit serum.

An immunological approach is not unique. Recently, Akeson and Herschman (12) demonstrated a differentiation antigen on mouse neuroblastoma that was also found in mouse brain. Martin (13) also described an antigen shared by neuroblastoma and brain. Using the mouse glioma G-26 (14), Schachner (15) has prepared an antiserum that reacts with brain and appears to be glial specific. However, our approach has been different, and is similar to that of Goldschneider and Moscona (16) for chick retina. Antisera were prepared to dissociated neonatal cerebellar cells of normal and mutant mice (17). The cell surface specificity of these antisera has been assayed directly by cell cytotoxicity and immunofluorescence on these same dissociated brain cells. The first of these antisera, one to normal cerebellum, is characterized in this report. MATERIALS AND METHODS Mice. All the mice used in these studies were from our breeding colonies and are derived from stocks of the Jackson Laboratory, Bar Harbor, Maine. The parental strains used here are C57B1/6J, weaver [B6CBA(Wv/+)], staggerer [C57B1(++Sg/dse+)], and nervous [C3HeB/J(Nr/+)]. Cell Cultures. The aggregate cultures were prepared from 6- to 8-day-old mouse cerebella as described for fetal brain (4). However, the second trypsinization was omitted, and large pieces of tissue were dispersed by repeated pipeting in basal Eagle's medium containing 10% fetal calf serum. The dissociated cerebellar cells were allowed to reaggregate for 4 hr at 370 prior to use. For some immunofluorescent observations these same cerebellar cells were cultured on collagen-coated coverslips for 4-6 days. Mouse glioma G-26 cells were the generous gift of Dr. John Minna (NIH) and the clonal cell lines, neuroblastoma N18 and N103 (18, 19), as well as the rat glioma C6 (20), were maintained in logarithmic growth in 150-mm Falcon dishes with 25 ml of Dulbecco's modification of Eagle's medium containing 10% fetal calf serum. Antisera. For the initial injection approximately 4 X 107 cerebellar cells from equal numbers of C57B1/6J and the B6/CBA(Wv/+) mice were mixed with an equal volume of Freund's adjuvant and administered by combined intraperitoneal and footpad injection of rabbits. All subsequent injections consisted of 2 to 4 X 107 cerebellar cells without adjuvant and were performed at 2-to 4-week intervals. The rabbits were not bled until 8 days after the third injection and then at 8-10 days after all subsequent boosts. Cytotoxicity Test. Dissociated cerebellar cells described above were incubated with 100 ,uCi of Na251CrO4 during the fourth hour of reaggregation. The cells were washed four times and dispersed in basal Eagle's medium to 5 X 106 cells per ml. A 50-,ul aliquot of cells was incubated with an equal volume of diluted antiserum at 40 for 90 min. Then 4110

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Table 1. Tissue specificity of anti-Cbl Adsorbing tissue A. Mouse Heart

Spleen Liver Kidney Brain B. Mouse Sperm Neural retina Brain

% Residual cytotoxicity 100 100 100 88 48 81 43 42

A 1:16 dilution of anti-Cbl was adsorbed at 4° for 90 min with a washed low-speed particulate fraction from various mouse tissues containing identical amounts of protein. The adsorbed serum was assessed for residual cytotoxic activity on 51Cr-labeled cerebellar cells.

the cells were incubated at 370 for 60 min with 50 ,l of guinea pig complement (diluted 1:3) selected for low toxicity. Saline (150 Mil) was added and the cells were pelleted by centrifugation. A 200-,l aliquot of the supernatant was placed in a vial containing 1 ml of H20, 10 ml of Triton/toluene/Permafluor (1:2:0.135); radioactivity was determined in a Searle Mark III liquid scintillation spectrometer. The antibody (Ab)-dependent release of 51Cr, determined by subtracting the 51Cr released when only complement (C') was present, and compared to the 3X freeze-thaw controls that represent maximal release, gives the % kill defined as: cpm released with Ab and C' -cpm released with only C' 100 X cpm released 3 X freeze-thaw - cpm released with C' Adsorbed antiserum activity is usually expressed as % residual cytotoxicity, which is defined as: 100 net cpm released with adsorbed diluted serum net cpm released with unadsorbed diluted serum For the cerebellar cells, high concentrations of antiserum released as much 51Cr as the 3X freeze-thaw treatment, or >85% of the total 51Cr associated with the cells. Adsorption. Antisera were adsorbed with either particulate portions of tissue homogenate or pure cell preparations. Tissue homogenates (1:10) were prepared in Saline 1 (0.138 M NaCl, 5.4 mM KCI, 1.1 mM Na2HPO4, 1.1 mM KH2PO4, 12 mM glucose, and 0.9 mM CaC12) using five strokes of a loose pestle and one stroke with a tight pestle in a Dounce Homogenizer. The homogenate was centrifuged for 10 min at 400 X g and the supernatant discarded. The pellet, containing mostly membrane and some whole cells, was washed several (4-6) times with Saline 1 until the supernatants were clear, followed by a final wash in basal Eagle's medium with 5% fetal calf serum. The protein content of this particulate fraction was determined by the procedure of Lowry et al. (21). Antisera were adsorbed for 90 min at 4° with increasing amounts of the particulate fraction, then centrifuged; the antiserum was aspirated. Adsorptions with neonatal cells or cultured cells were performed on increasing numbers of packed cells in a manner analogous to that for particulate tissue; however, the data are plotted as a ratio of the number of adsorbing cells (A) per 50 Al of antiserum to the number

40~~~~~

N Rb S

1/2

1/32 1/ OAns 1/s12 Antisera Concentration

FIG. 1. Cytotoxicity of antisera against cerebellum. A typical titration curve of rabbit antiserum against mouse cerebellum for cytotoxic activity against 2.5 X 105 cerebellar cells from 6- to 8day-old mice. Cytotoxicity of anti-Cbl exhaustively adsorbed with mouse thymus cells and normal rabbit serum (NRbS) is also shown.

of 51Cr-labeled cerebellar target cells (T) (usually 2.5 X 105/50-Ml assay mixture). Indirect Immunofluorescence. Partially reaggregated cells or surface cultures on collagen-coated coverslips were washed three times with Isotris buffer [0.01 M Tris-HCl (pH 7.4), 0.14 M NaCl, 0.15 mM CaCl2, 0.5 mM MgSO4] incubated with 50 Ml of diluted antiserum for 30 min at room temp., washed three times with Isotris buffer, and incubated with 100 Ml of 1:8 diluted fluorescein-isothiocynate conjugated goat antibody to rabbit gamma globulin (Meloy Labs, Springfield, Va.) for 30 min. Control samples with normal rabbit serum, cerebellum adsorbed rabbit antiserum, or without rabbit serum were always used. The cells and coverslips were well washed, then mounted in glycerol-Gelvatol (Monsanto Chemical Co.) and observed with a Leitz Ortholux epi-fluorescence microscope equipped with the appropriate accessories. Attempts to fix the cells with glutaraldehyde or formaldehyde prior to incubation with the antibody gave high background fluorescence. RESULTS The cytotoxic activity of serum from rabbits injected with cerebellar cells (anti-Cbl) as compared to normal rabbit serum is clearly shown in Fig. 1. The unadsorbed serum has a titer endpoint of >1:1200 for 2.5 X 105 cerebellar target cells and is quite tissue specific prior to any adsorption. Extensive adsorption with thymus has a slight effect on the titer and reduces the maximal cerebellar cell kill by only 15%; thus the rabbit antibodies are directed primarily at antigens other than those shared by both brain and thymus, such as Thy 1.2 and H-2b (22, 23). The tissue specificity of the anti-Cbl was tested by adsorbing a fixed subthreshold concentration of antiserum (1:16) with increasing amounts of a particulate fraction containing cell and membrane protein. Table 1 shows the relative adsorptive capacity of several adult tissues. At a protein concentration where brain adsorbs approximately 50% of the cytotoxic activity, spleen, heart, and liver fail to adsorb any activity and kidney complexes 12% of the activity. Even at much higher concentrations, spleen, heart, and liver tissue fail to reduce cytotoxicity. In other studies sperm removed 19% of the cytotoxicity, whereas neural retina adsorption was similar to that of adult brain.

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Proc. Nat. Acad. Sci. USA 72 (1975) Table 2. Adsorption with dissociated mouse cells Cells

Liver (6 days) Thymus Medulla Cortex Cerebellum

% Residual cytotoxicity

94 93 48 13 0

A 1:16 dilution of anti-Cbl was adsorbed at 4° for 90 min with 30 x 105 dissociated cells from 6-day-old mouse tissue, then assessed for residual cytotoxicity on 2.25 x 105 5iCr-labeled cerebellar cells. Antisera Concentration

FIG. 2. Residual cerebellar specific cytotoxicity. Anti-Cbl extensively adsorbed against neonatal mouse liver and cerebral cortex was examined for residual cytotoxicity. The titration curve of serum subjected to additional adsorptions with cerebral cortex (Liv. + Cor. + Cor.) is also shown. serum

The regional specificity of the anti-Cbl was examined at a fixed antiserum concentration (1:16) using increasing numbers of dissociated cells from 6-day-old mice rather than particulate fractions of tissue membranes. Table 2 shows that at an adsorbing cell to target cell ratio (A/T) of about 13, where cerebellum gives complete adsorption, liver and thymus cells adsorb very little activity whereas cells from the medulla and cortex have quite different adsorptive capacities. Increased numbers of adsorbing cells give very little reduction in these values. Multiple adsorption of anti-Cbl with massive amounts of liver and cerebral cortex tissue leads to a residual cytotoxicity of 10-15% (Fig. 2). Similar results are found when a fixed concentration of antiserum is used and the amount of adsorbing tissue is increased. Since there is an apparent plateau, the residual cytotoxicity may represent cerebellar specific or unique antigens. It was of interest to examine whether the relative amount of these cell surface antigens underwent a change during brain development; therefore, the adsorptive capacity of membrane protein from the adult cerebellum was compared to the 6-day-old cerebellum. Fig. 3 shows that the adult tissue has about 1/4 the adsorptive capacity of the neonatal tissue. This difference may represent the loss or masking of an*Adult *6 Day

100

~80T 60 a:

40

20-

0-

0.03

0.1

0.3

1.0

3.0

Protein (mg)

FIG. 3. Relative adsorptive capacity of neonatal and adult cerebellum. A 1:16 dilution of anti-Cbl was adsorbed with increasing amounts of a washed particulate fraction from 6-day-old and adult mouse cerebellum and assessed for residual cytotoxicity.

tigenic sites during development or simply the dilution of the antigen by late forming cell types that lack the antigen, possibly glial cells or granule neurons. In addition, a small residual cytotoxic activity is consistently found with anti-Cbl adsorbed by adult cerebellum that. may represent antigen exposed only on the young developing cells. However, at these high concentrations of adsorbing material and low cytotoxicity the assay is not sensitive enough to make a definite statement about a differentiation antigen. The possibility that the anti-Cbl possessed components specific for certain cell types within the cerebellum was examined by using cerebellar mutants that show a paucity of certain cell types. Two of these mutants, staggerer and weaver, show a greatly reduced granule cell population and another mutant, nervous, is characterized by Purkinje cell death. A difference in the adsorptive capacity of cerebellar tissue from these mutants would be expected if the relative amounts of the antigens varied greatly among these cell types. Quantitative adsorption of anti-Cbl with mutant tissue is compared to their normal litter mates in Fig. 4; however, there is no marked difference between the adsorptive capacity of normal and mutant cerebellar tissue. Since several tumor cell lines from the rodent nervous system have been used to generate antisera that also reacts with brain (12, 13, 15), it was of interest to see whether these Clonal cell lines possessed any of the brain specific cell surface antigens. Quantitative adsorptions of anti-Cbl were carried out with increasing cell numbers (Fig. 5). At an adsorbing cell to target cell ratio where cerebellum has removed all the cytotoxic activity, mouse neuroblastoma clones remove approximately 50% of the activity. In contrast, the glial cell lines remove less than 10% of the cytotoxic activity at cell ratios as high as 40:1. This result is especially surprising since the G26 cells are of the same C57B1/6 origin as the reaggregated cells and retain histocompatibility antigens characteristic of this strain. In addition, primary cultures of mouse embryo fibroblasts showed adsorptions similar to the glioma cells, while cultures of mouse teratoma, human neuroblastoma, and glioma failed to adsorb any cytotoxic activity at these cell concentrations (results not shown). The partial reaction with mouse sperm (Table 1) suggested that the brain antigen(s) may be similar to that specified by the T-locus of mouse (24); however, colonies of undifferentiated stem cells from the primitive teratocarcinoma (25) fail to adsorb any cytotoxic activity. Lack of adsorption by the human neuroblastoma is not surprising since the rabbit anti-mouse Cbl is relatively species specific, showing partial crossreactivity with rat cerebellum, but not with hamster, human, or chicken at high levels of membrane protein. Thus, these results with clonal cell lines suggest that the anti-Cbl may be specific for nerve cells.

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100-A Weaver Cbl * Nervous Cbl V Normal Chi so4

-0

60

40

0

O.0S

1.0

0.2

Protein

30

(mg)

FIG. 4. Relative adsorptive capacity of cerebellar tissue from neurological mutants.. A 1:16 dilution of anti-Cbl was adsorbed with increasing amounts of a washed particulate fraction from cerebella of young (14-19 days old) mutant mice or their normal littermates and assessed for residual cytotoxicity.

Indirect imnmunofluorescence studies with fluoresceinconjugated goat anti-rabbit gamma globulin and rabbit antiCbl extensively adsorbed against liver and cerebral cortex indicate that about 15% of the cells show a bright ring membrane fluorescence (Fig. 6A) and another 15-20% show a weak membrane fluorescence. These latter cells contain the antigens, but presumably their density is not high enough for bound antibody to fix complement and produce substantial cell kill. To get a visual idea of the 15-30% cerebellar subpopulation that possesses these antigens, cerebellar cells from 6-day-old mice have been grown in surface culture on collagen-coated coverslips. Phase contrast microscopy shows these cultures to contain a lower layer of epithelial or fibroblastic cells covered with astrocytes and neuronal-like cells. Liver adsorbed serum reacts strongly with many of the cells and outlines the cell bodies and fiber tracts (Fig. 6B); however, it gives very little reaction with the fibroblast-like cells on the bottom. Furthermore, neuroblastoma clones N18 and

Adsorption Cells Target Cells FIG. 5. Adsorptive capacity of neurological tumor cell lines. A 1:16 dilution of anti-Cbl adsorbed with increasing numbers of cultured tumor cell lines (mouse glioma G26, rat glioma C6, and mouse neuroblastoma N18 and N103) was assessed for cytotoxic activity on 2.5 X 105 cerebellar target cells.

N103 show membrane fluorescence with liver adsorbed serum but not serum adsorbed with cerebral cortex. AntiCbl exhaustively adsorbed against liver and cortex has a much reduced fluorescence with the cultures and is confined to patches on a limited number of cells, many of which have mono- and bipolar processes characteristic of neurons (Fig. 6C). The limited fluorescence is readily demonstrated in Fig. 6C, where the entire field contains cell bodies or processes and only two cells are fluorescent. DISCUSSION neonatal mouse cerebellum have from cells Reaggregated been used to generate an antiserum that has a high degree of specificity for cells of the nervous system. The studies presented here have shown that (i) 80% of the cerebellar cells

FIG. 6. Immunofluorescence of mouse cerebellar cells with anti-Cbl serum. Indirect immunofluorescence was performed as described in Materials and Methods. Ring fluorescence of partially reaggregated cells using anti-Cbl adsorbed with mouse liver and cerebral cortex (A). Membrane fluorescence of 5-day-old cerebellar cell culture on a collagen-coated coverslip with liver adsorbed anti-Cbl (B). Membrane fluorescence of similar culture with liver and cortex adsorbed anti-Cbl (C). Bars on figures equal 25 ,m.

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have cell surface antigen(s) that are not found on other tissues such as thymus, spleen, heart, and liver; (ii) 10-30% of these cerebellar cells have antigen(s) not found in other brain regions; (ill) the relative amount of the antigen(s) decreases during cerebellar development; and (iv) the antigen(s) appears to be concentrated on neurons. The apparent concentration of the Cbl-antigen on neurons is suggested by the high adsorption capacity of neuron-rich tissues (cerebellum, cerebrum, and neural retina) and limited adsorption by non-brain tissues and reduced adsorption by brain regions containing relatively fewer neurons (i.e., medulla). The differential adsorption displayed by neuroblastoma and glioma cell cultures (Fig. 5) also supports this concept; however; one must be cautious since these are tumor-derived cells and they have had ample time to change their cell surface properties during growth in vitro. Although morphology of cultured cells can be deceiving, the immunofluorescence studies also suggest that the antisera is reacting primarily with neuron-like cells. However, the adsorption displayed by two non-neural tissues, sperm and kidney, is somewhat disconcerting. Sperm cells are known to possess cell surface antigens common to the early embryo (24), and such antigens may also be retained on brain cells. Adsorption ability of the mesodermally derived kidney is not readily explained on embryological grounds; however, antisera prepared against mouse neuroblastoma cells also react with kidney tissue (13). The relative decline in the adsorptive capacity of cerebellum with increasing maturation (Fig. 3) may be related to several processes. The antigen(s) may be involved in cerebellar morphogenesis, and as cell migration is completed and maturation proceeds the need for this cell surface moiety diminishes and it becomes masked or lost. Cell surface differences between adult and neonatal cerebellum are suggested by the decreased reaggregateability of cells from adult cerebellum and their inhibitory effect on reaggregation of neonatal cells (refs. 6-9; Seeds, unpublished). Furthermore, the mouse cerebellum increases 4- to 8-fold in mass from postnatal day 8 to adulthood, which represents both cell proliferation and cell growth. Granule cells represent the major increase in cell number, with a small contribution from stellate neurons and glia. Dendritic growth of Purkinje cells and formation of parallel fibers by granule cells are also responsible for the increased mass of the cerebellum. The absence or reduction of the antigen(s) on these late-forming cells, as well as a differential distribution of the antigen on the cell surface of all cerebellar cells, could produce this difference between adult and neonatal cerebellum. Although the adsorption by cerebellar mutants failed to show a differential distribution of the antigen(s) on granule and Purkinje cells (Fig. 4), 10-30% of the cells from neonatal cerebellum possess antigen(s) not found on other brain cells. The identity of this subpopulation of cells is not yet known; however, more sensitive techniques of gradient fractionation (26) and fluorescent cell separation (27) should allow identification of the specific cell types involved. These studies have supported the initial proposal that the properties of cell sorting and migration, as well as the behavior of differentiated and developing cells as demonstrated in

Proc. Nat. Acad. Sci. USA 72 (1975)

reaggregating cultures, may be related to the presence and distribution of specific molecules on the cell surface. Whether the antigen(s) studied here is related to these processes has yet to be demonstrated; however, our preliminary experiments show that the anti-Cbl prevents stable aggregate formation of cerebellar cells. Further characterization of antiCbl and other antisera against brain should offer a better understanding of the cell surface's role in cell interaction during brain development. I thank Dr. John J. Cohen for numerous stimulating discussions and suggestions and Drs. J. Lehman, K. Prasad, and J. Perkins for the mouse teratoma, human neuroblastoma, and glioma cells, respectively. The dedicated technical assistance of Mrs. Barbara Fonda is greatly appreciated, as are the space and facilities of the Eleanor Roosevelt Institute for Cancer Research. This research was supported in part by Grants NS-09818 and CA-15549 and a Career Development Award K04-GM-40170 from the USPHS. 1. Seeds, N. W. (1971) Proc. Nat. Acad. Sci. USA 68,1858-1861. 2. Seeds, N. W. & Gilman, A. G. (1971) Science 174,292. 3. Seeds, N. W. & Vatter, A. E. (1971) Proc. Nat. Acad. Sci. USA 68,3219-3222. 4. Seeds, N. W. (1975) J. Biol. Chem. 250,5455-5458. 5. Seeds, N. W. (1974) Trans. Am. Soc. Neurochem. 5, 55. 6. Moscona, A. A. (1973) in Cell Biology in Medicine, ed. Biltar, E. (Wiley-Interscience, New York), pp. 571-591. 7. Seeds, N. W. (1973) in Tissue Culture of the Nervous System, ed. Sato, G. (Plenum Press, New York), pp. 35-54. 8. Moscona, A. A. (1961) in Growth in Living Systems, ed. Farrow, M. (Basic Books, New York,) pp. 197-220. 9. Garber, B. B. & Moscona, A. A. (1972) Dev. Biol. 27,217-234. 10. DeLong, G. R. (1970) Dev. Biol. 22,563-575. 11. DeLong, G. R. & Sidman, R. L. (1970) Dev. Biol. 22,584-600. 12. Akeson, R. & Herschman, H. R. (1974) Proc. Nat. Acad. Sci. USA 71, 187-191. 13. Martin, S. E. (1974) Nature 249,71-73. 14. Zimmerman, H. M. (1955) Am. J. Pathol. 31, 1-29. 15. Schachner, M. (1974) Proc. Nat. Acad. Sci. USA 71, 17951799. 16. Goldschneider, I. & Moscona, A. A. (1972) J. Cell Biol. 53, 435-449. 17. Seeds, N. W. (1974) J. Cell Biol. 63, 307a. 18. Seeds, N. W., Gilman, A. G., Amano, T. & Nirenberg, M. W. (1970) Proc. Nat. Acad. Sci. USA 66,160-168. 19. Amano, T., Richelson, E. & Nirenberg, M. (1972) Proc. Nat. Acad. Sci. USA 69,258-263. 20. Benda, P., Lightbody, J., Sato, G., Levine, L. & Sweet, W. (1968) Science 161, 311-315. 21. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J.

(1951) J. Biol. Chem. 193,265-275. 22. Schachner, M. & Hammerling, U. (1974) Brain Res. 73, 362371. 23. Acton, R. T., Morris, R. J. & Williams, A. F. (1974) Eur. J. Immunol. 4, 598-602. 24. Artzt, A., Bennett, D. & Jacob, F. (1974) Proc. Nat. Acad. Sci. USA 71, 811-814. 25. Lehman, J. M., Speers, W. C., Swartzendruber, D. E. & Pierce, G. B. (1974) J. Cell Physiol. 84, 13-28. 26. Sellinger, 0. A., Legrand, J., Clos, J. & Ohlsson, W. G. (1974) J. Neurochem. 23,1137-1144. 27. Banner, W. A., Hulett, H. R., Sweet, R. G. & Herzenberg, L. A. (1972) Rev. Sci. Instrum. 43,404-409.