DEVELOPMENTAL

BIOLOGY

Immunological

72, 308-319 (1979)

Studies on the Purkinje Cells from Rat and Mouse Cerebella

I. Evidence for Antibodies Characteristic of the Purkinje Cells JACQUES

MALLET,

RICHARD

CHRISTEN,

AND JEAN-PIERRE

CHANGEUX

Neurobiologie Moltkulaire et Laboratoire Associe’ Centre National de la Recherche Scientifique, Interactions Molkdaires et Cellulaires, Institut Pasteur, 75015 Paris, France Received December 29, 1978; accepted in revised form March 28, 1979 Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with lz51 and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ‘2”I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ‘251-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ‘251-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively. INTRODUCTION

During the development of the nervous system, various cell types are positioned and connected in an orderly manner, and it is commonly believed that characteristic molecules of the cell surface play a role in determining such an organization. An analysis of the differences in membrane composition between various cell types or organs may lead to the identification of such molecules and in considering how to define these differences, an immunological appreach appears to be a method of choice. For instance, in such attempts, antisera have been raised against tumors and cloned cell lines derived from nervous tissue (Akeson and Hers&man, 1974; Coakham, 1974; Fields et al., 1975; Lee et al., 1977; Martin, 1974; Schachner, 1973,1974; Schachner and

Carnow, 1975). These populations of cells provide a rather homogeneous antigenic material but suffer the drawback that they may differ in several respects from their normal counterparts. Nevertheless, using antisera raised in mice against a rat tumor cell line, Brockes et al. (1977) have defined a surface antigen which is present on Schwann cells but not on fibroblasts. Other investigators have used as antigens fragments of brain tissue (Raiteri et al., 1972; Schachner et al., 1975, 1976; Seeds, 1975; Toh and Cauchi, 1974; Zimmermann and Schachner, 1976) or subcellular fractions of the same origin (Bock et al., 1974; Herschman et al., 1972; Jorgensen and Bock, 1974; Mickey et al., 1971). Under these conditions, the antigenic material used derives from a variety of cell types.

308 OOlZ-1606/79/100308-12$02.00/O Copyright 0 1979 by Academic Press, All rights of reproduction in any form

Inc. reserved

MALLET,

CHRISTEN,

AND CHANGEUX

Recently, taking advantage of the availability of bulk isolation of the three major cell types from brain, Poduslo et al. (1977) have obtained cell surface antisera specific to rat neurons and to lamb oligodendroglia. In the present study, large cells isolated from cerebella of 12- to E-day rats (i.e., mainly Purkinje neurons) were used as antigens. Using an in vitro binding assay and taking advantage of the mouse mutant, Purkinje cell degeneration (pcd),’ in which Purkinje cells are missing in the adult cerebellum, we define a class of antibodies which are directed against antigens characteristic of the Purkinje cell. MATERIALS

AND

METHODS

Animals The pcd mutation was kept on a moderately inbred background congenic for C 57 BL/G-J (N = 4). Mice originating from the Jackson laboratory were raised at the Pasteur Institute. The pcd/pcd homozygotes were produced by intercrossing +/pcd heterozygotes. Soon after the identification of homozygotes, the progenies were culled to increase the survival of the homozygotes by reducing competition with the normal littermates. Animals from the C57 BL/6 strain were used as controls. Antisera and Immunoglobulins For each injection 5-10 x lo5 Purkinje cells were purified from 20 cerebella of 12to 16-day rats by the method of Sellinger et aE. (1974) with minor modifications as already described (Mallet et al., 1976). Before the injection, the cells were washed three times with 10 ml of 0.01 M phosphate buffer, pH 7.4, 0.15 M NaCl (PBS) to remove the bovine serum albumin (BSA) used during the purification. The following two immunization procedures were used with Bouscat young adult rabbits. ’ Abbreviations used: pcd, Purkinje cell degeneration; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PMSF, phenyhnethylsulfonylfluoride.

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Hyperimmunization. The Purkinje cells were resuspended in 1 ml of PBS and emulsified with 1 ml of complete Freund’s adjuvant with a loose Teflon-glass homogenizer. Multiple injections were performed intradermally, as superficially as possible, at Days 1-5-9-13. A boosting was given 2 weeks later and then at intervals of at least 3 weeks by injecting the cells intravenously and intradermally without Freund’s adjuvant. Bleeds were taken 6-8 days following the immunization, starting after the fifth injection. Two rabbits were immunized according to this scheme and the different bleedings were not pooled. Intravenous immunization. One rabbit was immunized exclusively by intravenous injections without Freund’s adjuvant on Days 1-8-14-21-36 and then at various intervals of at least 3 weeks. Bleeds were also taken 6-8 days following the immunization, starting after the fifth injection. The sera from the different bleedings were not pooled. The rabbit died 6 months after the first injection with its back legs paralyzed. Purification

of Immunoglobulins

IgG and IgA were purified as follows: To 20 ml of gently stirred antiserum, 13.5 ml of saturated (NH&SO4 was added dropwise, and the mixture was left for about 20 hr at room temperature. A heavy precipitate formed. The mixture was then centrifuged at 4000g for 20 min and the supernatant discarded. The precipitate was washed twice with approximately 15 ml of a 40% saturated (NH& SO4 solution. The antibody-containing precipitate was mixed with a small amount of water and transferred to a 5-ml dialysis bag. The dialysis was performed at 4°C for 2 x 12 hr against distilled water, 1 X 24 hr against 0.050 M Na acetate, 0.021 M acetic acid, pH 5.0 (acetate buffer), 2 x 12 hr against distilled water, and 1 x 24 hr against acetate buffer, pH 5.0. During dialysis a precipitate of lipoproteins formed and was removed by centrifugation. The supernatant was applied on a column con-

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taining 5 ml of DEAE-Sephadex A 50 equilibrated with acetate buffer and eluted with approximately 10 ml of acetate buffer. Finally, the immunoglobulins were dialyzed against PBS containing 15 mM NaN3. Control sera were obtained from rabbits before immunization and were purified in the same way. Preparation

of lz51-Labeled IgG

The immunoglobulins were iodinated by the chloramine-T method (Greenwood et al., 1963) up to a specific activity of 4-5 $Zi/pg, corresponding to the incorporation of about 0.3 I atom per IgG molecule. Proteins were determined by the method of Lowry et al. (1951). Purification of lz51-Labeled Anti Purkinje Antibodies An homogenate of 10 mg of 12- to 17-day rat cerebellar membranes in 200 ~1PBS, 1% BSA was incubated at 4”C, for 30 min with 200 ~1 of nonimmune IgG (15 mg/ml) and 2 hr with 500 pl of the original 1251gG(0.6 mg/ml and about 2 mCi). The homogenate was then centrifuged for 20 min at 100,000g and washed once with 10 ml of PBS, 1% BSA. The membranes were then homogenized for a few minutes at 4°C in 2.5 ml of 0.2 M HCl glycine, pH 2.9, containing 1% BSA, and centrifuged for 10 min at 100,OOOg. The supernatant, which contained about 2% of the initial radioactivity, was mixed with 500 ~1 of 1 M KZHPOI, 1% BSA to raise the pH and was finally dialyzed against several changes of PBS supplemented with 0.02% NaN3. Before use the 1251-labeled IgG were centrifuged at 100,OOOgfor 10 min in a Beckman air-driven ultracentrifuge. Immunodiffusion

Tests

The antisera were analyzed by Ouchterlony’s double diffusion test (Ouchterlony et al., 1968) with 1% agarose in PBS buffer containing 1% of the neutral detergent Berol, on microscope slides at room temperature for 48 hr. The agarose was dried to a

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line film and stained with Coomassie brilliant blue. Immunoperoxidase Assay The indirect immunoperoxidase method (Avrameas, 1972) was carried out at various dilutions of serum on cell smears. Purified cells were resuspended in a small volume of 7.5% polyvinyl-pyrrolidone, 1% BSA. A fine droplet of the suspension was deposited on a microscope slide (washed in acetone, alcohol, and distilled water) and spread on a small surface with a coverslip. The slides were dried at 37°C for 30 min. The cells were then fixed for 15 min at room temperature in 4% paraformaldehyde in 0.2 A4 cacodylate buffer, pH 7.4, and washed three times with PBS. The cells can be stored for several days before use in this buffer in the presence of 0.02% NaNa (w/v). Slides were incubated for 40 min at room temperature with the sera and washed four times for 2 hr with PBS. They were then incubated with sheep anti-rabbit antibodies coupled with peroxidase (Institut Pasteur Production, dilution l/20) for 40 min and washed four times during 2 hr with PBS. The peroxidase was revealed by the method of Graham and Karnovsky (1966) by incubating the slides for 5 min in a 0.1 M TrisHCl buffer, pH 7.6, containing 0.1% (w/v) of 3,3’-diaminobenzidine tetrahydrochloride and 0.01% H202. Cells were then dehydrated in graded ethanol series, cleared in xylene, and mounted permanently in Permount (SO-P-16, Fisher Scientific Company). For control studies, slides were treated as described above except that the sera were from nonimmunized rabbits. Preparation

of Membranes

Ten Sprague-Dawley rats of 12-15 days were killed without anesthesia and their cerebella immediately dissected and added to 10 ml of an ice-cold solution of 0.32 M sucrose in 3 mJ4 MgCl2 and 10 mM TrisHCl, pH 7.3. All subsequent operations were performed at 4°C. The tissue was homogenized in a motor-driven glass-Tef-

MALLET,

CHRISTEN,

AND CHANGEUX

lon Potter homogenizer with 25 up-anddown stokes. The homogenate was then centrifuged at SOOg for 10 min. The pellet was washed twice with the aforementioned sucrose solution. The supernatants were pooled, diluted three times with bidistilled water, and centrifuged twice for 45 min at 100,OOOg. Membranes from rat liver, heart, and brain, or from normal or pcd mutant mouse cerebella were prepared as described above, and the volumes of the various solutions were adjusted to the same wet weight of starting tissue. Absorption

of Antisera

IgG was absorbed with crude membrane preparations prepared as described above from 12- to 17-day rats. The sera were either mixed with membranes resuspended in a PBS, 1% BSA solution or directly homogenized with membrane pellets when dilution of the sera was not necessary. The absorptions were carried out at 4°C for 2 hr with shaking and the sera recovered by centrifugation at 100,OOOg for 30 min.

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scintillation fluid. The radioactivity retained on the filter in the absence of membrane fragments was always estimated in parallel experiments in which all the components of the experimental mixture were present except the membrane fragments. This background radioactivity was found to be proportional to the total amount of free ‘251-labeled IgG and did not vary significantly within a given batch of filters. It ranged from 0.02 to 0.05% of the total radioactivity of the sample. This quantity was always substracted from the number of counts retained on the filter in the presence of membrane fragments. RESULTS

Ouchterlony

Reaction

The presence of antibodies directed against cerebellar membranes was tested by the Ouchterlony’s double diffusion method after dissolution of the membranes

Binding of “‘I-Labeled IgG from the AntiPurkinje Cell Serum to Cerebellar Membranes 1251-Labeled IgG and the membranes were diluted in PBS containing 1% BSA, 0.02% NaNa, 10e4 M phenylmethylsulfonylfluoride (PMSF), and 0.15% zymofren (SPECIA, Paris). Typically increasing amounts of membranes (100 ~1) were mixed with 30 ~1 of nonimmune serum (15 mg/ml) and agitated at 4°C for 30 min. Then 75 ~1 of ‘251-labeled IgG (in general, 7.5 pg/ml) was added and shaking continued for 24 hr. Aliquots (2 X 90 ~1) of the mixture were rapidly filtered on a Millipore filter (HAWP 02500) previously equilibrated with PBS, 10% BSA solution overnight and mounted in a suction apparatus. The filters were then washed four times with 5 ml of PBS. Radioactivity bound to particles trapped on the filter was counted in a liquid scintillation counter using toluene-POPOP as a

FIG. 1. Ouchterlony double diffusion in 1% agarose gel and PBS buffer containing 1% Berol. The antiPurkinje cell serum was placed in the central well and allowed to react with serial dilutions of 14-day cerebellar membranes solubilized with Berol. (A membrane pellet containing about 5 mg protein was treated overnight at 4°C with 300 ~1 of PBS containing 0.02% NaNa, 0.15% Zymofren, 10m4 PMSF, and 1% Berol. The insolubilized particles were removed by centrifugation at 100,OOOg for 5 min with a Beckman air-driven microfuge.) The dilutions clockwise from well A are, respectively, 1, %, L/4, times the concentration in this well.

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t

20

40

60 I

B

80

100 120 pg MEMBRANES

140

160

180

200

TIME (HOURS)

FIG. 2. (A) Specificity of the binding of ‘251-labeled anti-Purkinje cell IgG to cerebellar membranes. The binding was performed as described under Materials and Methods, except that nonimmune serum was not added (0). Effect of preincubating the membranes with increasing amounts of anti-Purkinje IgG (A) and with nonimmune IgG (m). (B) Effect of time on the binding of ‘ZsI-labeled anti-Purkinje cell IgG to cerebellar membranes. The binding was performed as described under Materials and Methods with 30 pg of cerebellar membranes. in 1%Berol. Several precipitation lines were observed (Fig. 1) with the sera raised by hyperimmunization against purified rat Purkinje ceI.Is. Only a faint line was noticed with the serum raised by intravenous injection. Ah the following studies were carried out with sera from hyperimmunized rabbits.

Binding of ‘251-Labeled IgG from AntiPurkinje Cell Serum to Cerebellar Membranes Quantitative information on the strength and specificity of the anti-Purkinje cell sera was obtained by a direct binding assay. Increasing amounts of cerebellar membranes were added to a fixed amount (gen-

MALLET,

CHRISTEN,

Rat and Mouse Cerebellar Purkinje

AND CHANGEUX

erally 7.5 pg) of ‘251-labeled IgG. The 1251labeled IgG bound to the membranes was estimated by Millipore ultrafiltration (see Methods). Figure 2A shows the binding curve obtained with the unabsorbed antiPurkinje cell serum. The binding is time dependent (Fig. 2B), and at equilibrium about 3% of the ‘251-labeledIgG bind to an excess of cerebellar membranes. To test the specificity of the binding toward the antiPurkinje cell antibodies, inhibition experiments were carried out with nonlabeled immune and preimmune sera. A partial (about 30%) inhibition was obtained when the membranes were preincubated with the nonimmune serum, while preincubation with the immune serum reduced the binding up to 85% (Fig. 2A). Clearly the immune serum contains a population of antibodies which are not present in the nonimmune serum and have been raised subsequently to the immunization with the Purkinje cells. In the following binding assays,membranes were preincubated with a lOOO-fold excess of nonimmune serum in order to detect only the immunoglobulins produced by the immune response.

Organ Specificity Serum

of the Anti-Purkinje

Cell

The ‘251-labeled IgG purified from the anti-Purkinje cell serum was absorbed with high amounts of various membrane preparations. After each absorption an aliquot of the supernatant was used to perform a binding assay with cerebellar membranes. The results are shown in Fig. 3. A first absorption with a mixture of liver and heart membranes removed 71% of the ‘251-labeledanti-Purkinje antibodies. A second absorption with the same membranes removed an additional 9.5%.2A first absorption with cerebrum membranes caused a 13% decrease of the remaining 1251-labeled antibodies. A second absorption with the *A third absorption was not performed in this experiment, but other binding assays not reported here indicated that the second absorption was indeed complete.

20

40

60

Cells, I

80

100

313

1x I

pg MEMBRANES

anti-Purkinje cell FIG. 3. Binding of “‘I-labeled IgG on rat cerebellar membranes after successive absorptions. The absorptions were carried out with various membrane suspensions as described under Materials and Methods, starting with 500 ~1 of ‘?-labeled anti-Purkinje cell IgG (0.5 mg/ml and about 1 mC). Each absorption causes a serum dilution of 1.2- to 1.5fold. About %o of the initial amount of ‘251-labeled IgG was kept after each absorption to perform binding assays as described under Materials and Methods. Unabsorbed serum (V), serum absorbed once (m) and twice (0) with 4 mg of liver and heart membranes, then once (0) and twice (0) with 4 mg of cerebrum membranes, and finally once (A) and twice (A) with 2 mg of cerebellum membranes. For all binding assays the same amount of ‘251-labeled IgG was placed in each tube (about 10” cpm).

same membranes had no effect. Finally, about 6.5% of the total ‘251-labeledantibodies was absorbed on cerebellar membranes. A second absorption with cerebellar membranes did not cause a further decrease of the remaining antibodies. Therefore 6.5% of the antibodies which recognize cerebellar membranes are directed specifically against cerebellar antigens.

Purification of the Antibodies against Purkinje Cells

Directed

In order to increase the ratio of specific to nonspecific binding of ‘251-labeled IgG, antibodies directed against Purkinje cells were purified by absorption on cerebellar membranes and released at pH 2.9 (see

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FIG. 4. Binding of enriched ‘251-labeled anti-Purkinje cell antibodies to rat cerebellar membranes after various absorptions. Anti-Purkinje cell antibodies were enriched as described under Materials and Methods by selective absorption and subsequent elution from cerebellar membranes. Absorptions and binding assays were as described in the legend to Fig. 3 and under Materials and Methods, taking into account that the enriched ‘*‘Ilabeled anti-Purkinje cell IgG represented about 2% of the original ‘251-labeled IgG. Unabsorbed serum (0). About 5 ag of the enriched ‘251-labeled anti-Purkinje cell antibodies was absorbed with 8 mg of liver and heart membranes (A), then with 4 mg of cerebrum membranes (u), and finally with 2 mg of cerebellum membranes

Methods). The released ‘251-labeled IgG course of the absorptions, we can estimate that liver plus heart, cerebrum, and cerewas then thoroughly absorbed with liver and heart, cerebrum, and cerebellar mem- bellum membranes absorbed out succesbranes. The results of these successive ab- sively 75.8, 17.4, and 6.8% of the purified ‘251-labeled IgG.3 These figures compare sorptions are reported in Fig. 4. After puritlcation about 48% of the total a These figures are obtained as follows: If one deof ?-labeled anti-Purkinje ‘251-labeled IgG bound to an excess of cer- fines as n the percentage against cereebellar membranes. After successive ab- antibodies which is directed specifically brum and cerebellum antigens versus the total unabsorptions with a mixture of liver and heart sorbed “‘I-labeled IgG population, the value of z is membranes and with cerebrum mem- given by the equation x/(52 + x) = 18.3/100 and is branes, only approximately 18.3 and 5.9% equal to 11.6. As only 48% of the ‘251-labeled IgG are directed against Purkinje cells, the perof the 1251-labeledIgG bound to an excess antibodies of cerebellar membranes. Taking into ac- centage of antibodies removed by the fast absorption is then 75.8. Similarly we have computed that absorpcount that 52% of the ‘251-labeled IgG do tions with cerebrum and cerebellum membranes renot bind to cerebellar membranes, and will moved successively about 17.4 and 6.8% of the antialways remain in the supernatant in the Purkinje antibodies.

MALLET,

CHRISTEN,

AND CHANGEUX

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315

well with those obtained from absorptions carried out with the total population of IgG. Moreover, at least in the case of the absorption by cerebrum membranes, the ratio of specific to nonspecific binding appear; significantly increased.

Binding Assays with Membranes from the Cerebellum of Normal and Purkinje Cc 11 Degeneration ($cd) Mutant Mice The specificity of the anti-Purkinje c#!ll serum toward the Purkinje cell was assessed by making use of the cerebellua from pcd/pcd mouse. The pcd/pcd indivib uals lose 99% of the Purkinje cells postnstally (Mullen et al., 1976); in addition, it has been shown (Mullen, 1977) that the pcd locus exerts its primary effect within the Purkinje cell. First, a comparative binding assay was performed with rat and mou,;e cerebellar membranes (Fig. 5). At low mer nbrane concentrations the two curves look similar. However, the binding curve dolie with mouse cerebellum reaches a plate:tu at a lower membrane concentration th:tn that done with rat cerebellum. Binding curves done with cerebella membranes from normal and pcd/pcd rn,i-

r

1

2c

JIM MEMBRANES

FIG. 5. Comparative binding of ‘251-labeled antiPurkinje cell IgG to rat and mouse cerebellar membranes. Binding assays were performed as described under Materials and Methods to the same concentration of rat and mouse membranes.

/ .

. /o/o-o

ormal and PCD

g,a-ra-8-y 10

3 -.l

10

20

30

40

pg MEMBRANES

FIG. 6. Comparative binding of enriched ‘251-labeled anti-Purkinje cell antibodies to normal and pcd/ pcd mouse cerebellar membranes. Binding assays were carried out as described under Materials and Methods and in the legend to Fig. 4, using the same sera as those used to perform experiments represented in that figure. (A) Unabsorbed antibodies. In this case each point represents the mean value obtained from two separate experiments. (B) Antibodies were absorbed on heart and liver membranes. (C) Antibodies were further absorbed with cerebrum membranes (m and q ), and finally, in order to determine the nonspecific binding, antibodies were absorbed with cerebellum membranes (0 and 0).

tant mice with unabsorbed and absorbed enriched lz51-labeled anti-Purkinje antibodies are compared in Fig. 6. Before absorption (Fig. 6A) a small difference between the two curves was already apparent. After absorption on liver and heart membranes the difference became more significant. About 17% of the antibodies did not bind to pcd/pcd cerebellar membranes (Fig. 6B).

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FIG. 7. Indirect immunoperoxidase reaction of a mixture of purified small cells, mostly granule (g), and large cells, mostly Purkinje (P) cells, with (A) IgG (1 mg/ml) directed against purified Purkinje cells: When the IgG was 10 times more dilute the staining of the small cells was very faint, while the large cells were still heavily stained. The inset represents the reaction with nonimmune IgG (1 mg/ml). (B) IgG (1 mg/ml) after absorption with heart, liver, and cerebrum membranes. About 6 mg of IgG was successively absorbed with 130 mg of a mixture of heart and liver membranes and 100 mg of cerebrum membranes as described under Materials and Methods. Sera were diluted in PBS containing 1% BSA to neutralize the antibodies directed against BSA. (The bar represents 20 pm.) Finally, after absorption on cerebrum this fraction increased up to 75% (Fig. 6C). The binding to pcd/pcd cerebellar membranes was nevertheless still significant compared to the nonspecific binding, which was identical with membranes prepared from normal and mutant cerebella. The differences observed between pcd and normal mice cerebella unambiguously show that the anti-Purkinje cell serum contains antibodies directed against antigens which are specific to the Purkinje cell.

Indirect

Peroxidase

Staining

The specificity of the anti-Purkinje cell serum was further tested by the indirect peroxidase technique on smears of purified “large” cells and granule cells. Before absorption both the granule and the “large” cells were labeled (Fig. 7A). After full absorption with membranes from heart, liver,

and cerebrum, only the “large” positively (Fig. 7B).

cells reacted

DISCUSSION

The purpose of this study was to demonstrate the existence of antigens characteristic of a given category of neurons, the Purkinje cells of the cerebellum. To perform this task antisera have been raised against a population of “large cells” purified from a suspension resulting from the mechanical dissociation of cerebella from 12to 15-day-old rats. Unlike other methods, the one used in this work does not include a trypsinization step and is therefore expected to preserve surface antigens to a large extent. However, the fractionation method involves a centrifugation step and cells are separated mostly according to their size and shape. The suspension should therefore contain mostly the largest cells

MALLET,

CHRISTEN,

AND CHANGEUX

present in the cerebellum, the Purkinje cells, although Golgi cells and neurons from the cerebellar deep nuclei might also be present in significant amounts. Moreover, the homogenization procedure is expected to split axons and dendrites from the cells bodies, and the antibodies have therefore been raised against cell somas. For simplicity the sera raised under these conditions have been referred to as anti-“Purkinje cell.” The serum with the highest titer of antibodies was obtained by the method of hyperimmunization; by Ouchterlony diffusion test it gives several precipitation lines against cerebellar membranes solubilized by nondenaturing detergents. This particular serum has been used throughout this study. The detection of antibodies directed against cerebellum specific antigens was done by a direct binding method using 1251labeled IgG and a crude preparation of membrane fragments prepared from fresh cerebellum. Thus, only membrane-bound antigens were assayed under these conditions. Absorption of the total population of ‘251-labeled immunoglobulins by liver, heart, and cerebrum membranes shows that in the immune serum the majority of the antibodies are directed against determinants which are not specific to the cerebellum. In other words, most of the membrane-bound antigens from cerebellar membranes are common to other categories of cells. Only about 6% of the population of IgG which recognizes cerebellar membranes is left in solution after absorption on cerebrum. These IgGs are considered as “specific” to antigens present in the cerebellum but are absent (or present in much lower amounts) in the cerebrum. Since the cell suspension injected into the rabbit contained mostly Purkinje cells, are these antigens present only on this class of cell? To answer this question we have taken advantage of the pcd neuropathological

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mutant from the mouse. In the homozygous pcd/pcd animals almost 99% of the Purkinje cells degenerate postnatally in the cerebellum, but the Golgi cells and the neurons from the deep cerebellar nuclei persist. First, it was confirmed that a significant cross-reactivity exists between rat and mouse cerebellum. Second, a difference was noticed between the binding curves done with membranes prepared from normal and mutant cerebellum which was amplified after purification of the IgG on cerebellar membranes; after cerebrum absorption the binding of ‘251-labeled IgG to pcd/pcd cerebella was 25% of that found with normal animals. Without ambiguity, antibodies directed specifically against Purkinje cell antigens are present in the serum. This conclusion is supported by the observation that the immunoperoxidase reaction carried out with the fully absorbed serum stains only the “large cells” in a suspension of dissociated cerebellar cells. However, a significant fraction of the cerebellum-specific antibodies (about 25%) still binds to pcd/pcd cerebellar membranes and is therefore assumed to correspond to antigens present on other categories of cells in the cerebellum. The indirect peroxidase staining shows that they are not present on the granule cells, and evidence is given in the following paper that some reaction takes place at the level of the deep cerebellar nuclei (Woodhams et al., 1979). Furthermore, the corresponding antibodies can be absorbed with brain stem membranes. A population of antibodies fully specific to Purkinje cell antigens can therefore be obtained. The antigens corresponding to the cerebellum-specific antibodies have not yet been identified; in particular, is the PAM) protein (Mallet et al., 1975, 1976) recently found as a characteristic component of the Purkinje cells by electrophoresis in SDSpolyacrylamide gels part of the antigens that we have serologically defined? The Purkinje cell-specific antibodies can be of use as cell markers in cultures of

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explants or dissociated cells. Work is in progress to establish the subcellular localization of the cerebellum-specific antigens and, particularly, to see if they are surface antigens. Specific cell surface antigens are likely to play a role in the various cell interactions leading to the establishment of the neuronal networks in the cerebellum, and the availability of the corresponding antisera would provide a tool to analyze these interactions. We thank Dr. S. Avrameas for his advice and suggestions at the early stage of the work and Drs. J. C. Antoine, C. Bon, B. Gilbert, and P. Pradelles for useful discussions and comments. We are also grateful to Dr. J. L. Guenet for his help in the breeding of the mutant mice. This research was supported by grants from the Institut National de la Sante et de la Recherche Medicale, the Centre National de la Recherche Scientifique (ATP Petits Mammiferes), the Delegation G&i&ale a la Recherche Scientifique et Technique, the College de France, and the Commissariat a l’Energie Atomique. REFERENCES AKESON, R., and HERSCHMAN, H. R. (1974). Neural antigens of morphologically differentiated neuroblastoma cells. Nature 249,620-623. AVRAMEAS, S. (1972). Enzyme markers: Their linkage with proteins and use in immuno-histochemistry.

Histochem. J. 4, 321-330. BOCK, E., JORGENSEN, Antigen antibody brain synaptosomes tion to water-soluble

0. S., and MORRIS, S. J. (1974). crossed electrophoresis of rat and synaptic vesicles: Correlaantigens from rat brain. J.

Neurochem. 22,1013-1017. BROCKES, J. P., FIELDS, K. L., and RAFF, M. C. (1977). A surface antigenic marker for rat Schwann cells.

Nature 266.364-366. COAKHAM, H. (1974). Surface antigen(s) common to human astrocytoma celIs. Nature 250, 328-330. FIELDS, K. L., GOSLING, C., MEGSON, M., and STERN, P. L. (1975). New cell surface antigens defined by tumors of the nervous systems. Proc. Nat. Acad.

Sci. USA 72,1296-1300. GRAHAM, R. C., and KARNOVSKY, M. J. (1966). The early stages of absorption of injected horse-radish peroxidase into the proximal tubules of mouse kidney. Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302. GREENWOOD, F. C., HUNTER, W. M., and GLOVER, J. S. (1963). The preparation of 13’1-labelled human growth hormone of high specific radioactivity. Riothem. J. 89, 114-123.

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HERSCHMAN, A. (1972). organelIes.

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