Proc. Nati. Acad. Sci. USA Vol. 87, pp. 528-532, January 1990 Neurobiology

Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes (basket cells/cerebellum/synapse)

GABOR TIGYI, CARLOS MATUTE*, AND RICARDO MILEDIt Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, Irvine, CA 92717

Contributed by Ricardo Miledi, September 29, 1989

ABSTRACT A method was developed to produce monoclonal antibodies to brain cell antigens by using Xenopus oocytes as immunological vectors. The method consists in injecting Xenopus oocytes with rat brain mRNA to express foreign proteins and using the oocytes for immunization. Immunizations were preceded by immunotolerization of mice to antigens of native oocyte membranes. With this approach we generated a set of monoclonal antibodies that are specific markers for the cerebellar "pinceau"-a unique complex synapse formed between basket cell terminals and the initial segment of the Purkinje cell axon. Our findings reveal an immunoreactivity highly localized at the pinceau and its late expression beginning at postnatal day 19 during cerebellar development.

5 days after injection (14, 15). Batches of 50-100 injected oocytes were used to prepare, by sucrose density gradient centrifugation (16), a membrane fraction to immunize mice. Newborn BALB/c mice were rendered immunotolerant (17) to native oocyte membrane antigens, or 4- to 6-week-old animals were further "tolerized" with the aid of cyclophosphamide (ref. 18; Sigma; 100 mg/kg of body weight) and membrane antigens from native Xenopus oocytes (500 ,ug of protein per tolerization). Mice that were rendered immunoparalytic with the above procedures were immunized intraperitoneally with membranes (1 mg of protein per immunization) prepared from rat cerebral cortex mRNA-injected oocytes, injected with a booster 2 weeks later, and used for fusion 4 days following the last booster. Spleen cells from these animals were fused with SP2/0-Ag/14 myeloma cells by standard procedures (19, 20), and the resulting hybridomas were raised as described (21). Of 16 fusions performed with slightly varied immunization protocols, more than 7000 hybridoma wells were tested on rat brain parasagittal frozen sections. Sections were incubated with hybridoma-conditioned supernatants or diluted ascites fluid (1:5000) and processed for immunoperoxidase staining by using the biotin-avidin system (Vectastain ABC kit, mouse IgM specific; Vector Laboratories) with the 3,3'diaminobenzidine HCI (DAB) chromogen. In some cases to improve the detectability of the immunoreactivity, the DAB reaction product was intensified with silver (22, 23). The intensification procedure increases the detectability of bound peroxidase traces by about 2 orders of magnitude. An initial screening was made to select colonies that produced antibodies recognizing rat brain antigens. Subsequently, immunoperoxidase staining was performed on various rat and Xenopus tissues, and those hybridomas producing antibodies that reacted only with rat brain tissue were characterized further. Positive colonies were cloned by limiting dilution, and cells were then frozen or injected into mice for ascites production. Isolation of cerebellar subfractions for biochemical characterization of the antigen will be described in detail elsewhere. Briefly, cerebella from adult Sprague-Dawley rats were homogenized, and the slow-speed supernatant fraction was ultracentrifuged at 105 x g to remove the cytosolic fraction. The pellet was resuspended in 0.1 M Tris buffer (pH 7.4) containing 1 M NaCl and ultracentrifuged as before to solubilize the nonintegral membrane protein fraction. The remaining pellet was solubilized with 10 mM 3-[(3-

Cerebellar basket cells provide the major axo-somatic and axo-axonic synaptic input to Purkinje cells (1). The descending basket cell axons form the en passant synapses that make the pericellular basket around the soma of the Purkinje cell and then proceed to the initial segment of the Purkinje cell axon, where they form a paintbrush-like axo-axonic synapse-the "pinceau," as Ramon y Cajal termed it (2). No other example of this voluminous and complex synapse is known in the mammalian nervous system. The exact function of the pinceau is not yet known, although it has been proposed that it mediates chemical and electrical inhibition of the Purkinje cell (3-6); and loss of basket cells has been shown to cause severe locomotor deficits (7). Production of monoclonal antibodies (mAbs) to the diverse molecular constituents of the nervous system is contributing greatly to our knowledge of the molecular basis of brain function (8-12). We now report an approach used to produce a set of mAbs recognizing an epitope present almost exclusively in the cerebellar pinceau terminals and not detected in any of the other four types of synaptic contacts made by the cerebellar basket cell axons. These mAbs were obtained by using Xenopus oocytes as immunological expression vectors for mRNA isolated from rat cerebral cortex and some of the underlying tissue.

MATERIALS AND METHODS Detailed description of the methods and procedures will be presented elsewhere. Briefly, oocytes from Xenopus laevis were microinjected with 50 ng of poly(A)+ mRNA extracted from the cerebral cortex of adult Sprague-Dawley rats by established procedures (13). To ascertain the translation of the foreign mRNA, electrophysiological responses elicited by the major neurotransmitters and activation of voltagegated membrane channels were recorded in the oocytes about

cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) detergent and ultracentrifuged to obtain the mem-

Abbreviations: mAb, monoclonal antibody; DAB, 3,3'-diaminobenzidine hydrochloride. *Present address: Department of Neurosciences, Universidad del Pais Vasco, 48940 Lejona, Spain. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Neurobiology: Tigyi et al. brane protein fraction. All three fractions were tested for mAb 2G6 binding by the ELISA technique. Most of the immunoreactivity was detected in the nonintegral membrane protein fraction; therefore, this fraction was used for gel permeation chromatography on a Superose 6 column in a fast protein liquid chromatography (FPLC) apparatus (Pharmacia) in 0.1 M Tris HCl/0.5 M NaCl, pH 7.4. Aliquots from the chromatography fractions were applied to coat ELISA plates (Beckman) and assayed for mAb 2G6 ascites (1:2000) binding by using peroxidase-labeled rabbit anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark). The immunoreactivity was quantitated with a Titertek microplate photometer at 505-nm wavelength and was normalized for the protein content of each fraction as estimated by their optical absorbance at 280 nm. The column was calibrated by using thyroglobulin, ferritin, and bovine serum albumin (Pharmacia) as molecular weight standards.

RESULTS Of all colonies tested, 440 hybridomas were found to produce antibodies reactive with rat brain antigens. Some of these antibodies displayed interesting patterns of immunoreactivity in the brain and will be described elsewhere. Here we concentrate on 4 hybridomas that produced antibodies that labeled structures at the base of the Purkinje cells, on the border of the granular cell layer. Although obtained from different fusions, all 4 hybridomas produced IgM-type antibodies and gave identical immunohistological labeling. The staining pattern for 1 of these antibodies is illustrated in Fig. 1. High-power magnification (x640) of the immunostained sections showed the mAb 2G6 immunoreactivity located mainly at the base of every Purkinje cell, surrounding the initial 15-20 gm of their axons (Fig. 1C). Thus, mAb 2G6 was found to label structures that resemble the pinceau terminals (1-3), while no immunoreactivity was detected in the cerebral cortex or in other parts of the rat central nervous system. No immunohistochemical binding of this mAb was seen in liver, kidney, lung, or skeletal muscle tissues of the rat. Moreover, mAb 2G6 did not stain sections prepared from native noninjected oocytes or from the brain of the Xenopus. However, injected oocytes showed a weak but definite immunostaining. mAb 2G6 stained the pinceau terminals in the mouse, rabbit, and human cerebella, indicating that both the pinceau and the antigen are well conserved among these species. Immunochemical experiments aimed at identifying the antigen by conventional methodology (immunoblotting) failed to provide clear data because of the lability of the epitope to biochemical extraction and separation techniques. However, when cerebellar homogenates were fractionated by ultracentrifugation and assayed for binding of mAb 2G6 in ELISA, the immunoreactivity was enriched in the cerebellar membrane fraction. The immunoreactivity could be solubilized from the membrane fraction by using 1 M sodium chloride, suggesting that the antigen belongs to the nonintegral membrane protein fraction. Since immunoblots from native or sodium dodecyl sulfate/polyacrylamide gels did not reveal clearly the identity of the antigen, we applied a different technique to estimate the native molecular weight of the immunoreactive substance. Proteins in the nonintegral membrane protein fraction were separated by high-resolution gel filtration on a Superose 6 column. Subsequently, ELISA plates were coated with the eluted fractions and assayed for binding of mAb 2G6. The peak of the immunoreactivity was found in a fraction that corresponded to Mr 1,000,000 (Fig. 2). This relatively large molecular weight suggests that, in its native form, the antigen defined by mAb 2G6 is probably part of a large molecular complex. The developmental appearance of the 2G6 immunoreactivity was assessed from embryonic day 10 to postnatal day

Proc. Natl. Acad. Sci. USA 87 (1990)

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30 in Sprague-Dawley rats (Fig. 3). The 2G6 immunoreactivity first appeared around postnatal day 19 in the archicerebellum within the nodulus and was detected only after silver intensification of the DAB reaction product, while the conventional methodology failed to detect this early production of the antigen. After postnatal day 20 both the intensity and the number of immunopositive pinceau terminals increased, and after postnatal day 23 the 2G6 immunoreactivity could be detected throughout the cerebellum. The pattern found in the adult rat brain was attained by postnatal day 30.

DISCUSSION The origin of the progenitor lymphoblasts of these unique and highly specific antibody-producing hybridomas remains an intriguing question. The presence of immunoreactivity in mRNA-injected oocytes and the lack of cross-reactivity with native noninjected Xenopus oocytes and other frog tissues suggests that these mAbs originated from an immune response directed against antigens that are not present in the native oocyte but are expressed in the oocytes injected with cerebral mRNA. But then, why is the staining localized in the cerebellum? Several explanations could account for this phenomenon: low sensitivity of the immunohistological technique, uptake of the antigen by the pinceau, amplified translation by the oocytes, etc. It is interesting that others have also reported mAbs to unique "cerebellar" antigens after using spinal cord tissue or cerebral cortex synaptosomal plasma membranes for immunization (12, 17). Nonetheless, the answer to the origin of the 2G6 antigen will not be clear until the nature of the antigen is fully established. It should be interesting also to see what mAb staining patterns are obtained when oocytes injected with cerebellar mRNA are used for immunization. Alternatively, it could be thought that mAb 2G6 is a product of an autoreactive lymphoblast activated as a result of the repeated immunization and tolerization. This might be suggested by the fact that mAb 2G6 is an IgM with K light chains, a structure that is often found in autoantibodies (24-26). However, natural autoantibodies are known for their wide polyspecificity, recognizing various antigens in different tissues, which contrasts with the specificity of mAb 2G6. Furthermore, no antibodies with this unique staining pattern were detected when mice were "sham-immunized" with complete Freund's adjuvant or when hybridomas were generated after immunization with native oocyte membranes and other antigens following the use of similar drastic immunotolerization. In addition, other researchers (8-12, 16-18) used a large variety of neural antigens and immunization procedures and found interesting cerebellum-specific antibodies, but none were like the one described here. Thus, our findings reveal a previously undescribed cellspecific antigen defined by a set of monoclonal antibodies. It is interesting that this epitope not only is cell-specific but also (within the basket cells) is found mainly in the most terminal axon branches that surround the initial segment of the Purkinje cell axon. The comparative lack of immunoreactivity in the pericellular baskets as well as in the other synaptic contacts made between basket cell axons and Purkinje cell dendrites suggests the accumulation of a highly specific antigenic structure in the pinceau. In the rat, the formation of the basket cells peaks on postnatal day 6 or 7, and is completed by day 13 (7, 27). The downward branches of the basket cell axons develop their pericellular terminals between days 8 and 15 (28), and the characteristic cytoarchitecture and synaptic connectivity of the cerebellum is thought to be fully developed by postnatal day 25 (29). In contrast, our findings suggest that in the rat the molecular maturation of the pinceau terminal is not complete before the end of week 4. Interestingly, there are some

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Proc. Natl. Acad. Sci. USA 87 (1990)

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FIG. 1. (A) Binding of mAb 2G6 to a parasaggital section of the adult cerebellum viewed with an image analyzer. The dot-like immunoreactivity is present throughout the cerebellar folia, and it is localized to the border of the molecular (MO) and granule cell (GR) layers. The meninges (M), containing some nonspecifi1~~~cally bound peroxidase activity, mark the contours of the cerebellar cortex. WM, white matter. (Bar = 1 mm.) (B) A folium immunostained with mAb 2G6. The antigen is found beneath every Purkinje cell (arrowheads) at the typical site of the paint-brush terminals (arrows). The sections were counterstained with Mayer's hematoxylin. Abbreviations are as in A. (Bar = 125 ,um.) (C) Higher power (X640) view of the pinceauterminal region. At this magnification the Purkinje cell (P) axon can be followed as it crosses the im-

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Other abbreviations are as in A. (Hematoxilin counterstain; bar = 20 ,um.) (D) A drawing of C showing the various cellular elements. Abbreviations are as in A and C. The asterisks between the arrowheads label the immunopositive pinceau complex.

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complex cerebellar motor-behavioral skills (e.g., jumping, rearing without support, and head-down descent from a rope; see refs. 30 and 31) that develop with a time course similar to the development of the 2G6 antigen. Much remains to be done to define the ultrastructural localization as well as the nature and functional role of this antigen and of the pinceau complex itself. Perhaps the mAbs that we produced will help to elucidate some of these questions and contribute also to our knowledge about the molecular genetics of a highly specific synaptic marker. Finally, it seems that Xenopus oocytes, which are very efficient in expressing foreign proteins, may be profitably used to generate other unique antibodies. Our results with whole cerebral cortex mRNA show that the oocytes can be used as immunological vectors.

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We are very grateful to Ms. Cindy Asselin for her excellent assistance with the immunohistochemistry, and we thank Drs. C. Sotelo, J. Fallon, J. Marshall, and H. Killackey for their valuable comments regarding this manuscript. This work was supported by a grant from the Klingenstein Foundation and by Grant NS23284 from the National Institutes of Health. 1. Palay, L. S. & Chan-Palay, V. (1974) Cerebellar Cortex: Cytology and Organization (Springer, New York). 2. Ramon y Cajal, S. (1911) Histologie du Systeme Nerveux de L'homme et des Vertebres; Reprinted 1955 by Consejo Superior de Investigaciones Cientificas, Madrid. 3. Eccles, J. C., Ito, M. & Szentagothai, J. (1%7) The Cerebellum as a Neuronal Machine (Springer, New York). 4. Sotelo, C. & Llinas, R. (1972) J. Cell Biol. 36, 271-289. 5. Korn, H. & Axelrad, H. (1980) Proc. Natl. Acad. Sci. USA 77, 6244-6247.

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Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.

A method was developed to produce monoclonal antibodies to brain cell antigens by using Xenopus oocytes as immunological vectors. The method consists ...
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