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Monoclonal Antibodies to Specific Astroglial and Neuronal Antigens Reveal the Cytoarchitecture of the Bergmann Glia Fibers in the Cerebellum’

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Monoclonal Antibodies to Specific Astroglial and Neuronal Antigens Reveal the Cytoarchitecture of the Bergmann Glia Fibers in the Cerebellum’ 0270.6474/84/0401-0265$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 4, No. 1, pp. 265-273 Printed in U.S.A. January 1984 MONOCLONAL ANTIBODIES TO SPECIFIC ASTROGLIAL AND NEURONAL ANTIGENS REVEAL THE CYTOARCHITECTURE OF THE BERGMANN GLIA FIBERS IN THE CEREBELLUM’ ANGEL L. DE BLAS Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York 11794 Received May 11, 1983; Revised August 22, 1983; Accepted August 22, 1983 Abstract The cytoarchitecture of the cerebellar Bergmann fibers in the adult rat was investigated. Two monoclonal antibodies, one specific for the Bergmann fibers and astrocyte processes and the other specific for the cell bodies and dendrites of the Purkinje cells as well as an antiserum to the glial fibrillary acidic protein, were used in immunocytochemical peroxidase-antiperoxidase assays. The Bergmann fibers are revealed as columns organized in long vertical palisades parallel to the longitudinal plane of the folium. The palisades are not continuous; instead they are formed by sets of two to six aligned Bergmann fibers. Each of these sets of Bergmann fibers is separated from its longitudinally aligned neighbors by gaps. Each Bergmann fiber is formed by a bundle of two to four Bergmann glia processes which frequently show a helical organization. These results help to reconcile the different views on the organization of the .Bergmann fibers derived from the’studies done with the light microscope versus those done with the electron microscope. The Bergmann glia may play a fundamental role in directing the geometrical organi- zation of the cerebellar constituents. Rambn y Cajal (1911, vol. II, pp. l-106) described the the molecular layer perpendicular to the pial surface. histology of the cerebellum and the geometrical arrange- Rambn y Cajal (1911, vol. II, p. 70) noticed that the ment, orientation, and development of its cellular con- Bergmann fibers are organized in parallel palisades. This stituents. This and other studies (i.e., Braitenberg and model was supported later by the immunofluorescence Atwood, 1958; Eccles et al., 1967; Palay and Chan-Palay, studies of Bignami and Dahl (1974a, Fig. 6) using an 1974) indicate that the dendritic arborization of each antiserum to the glial fibrillary acidic protein (GFAP). Purkinje cell has a planar configuration and is oriented Altman’s (1975) results also supported Ram6n y Cajal’s in the transverse plane of the folium. This orientation is interpretation. However, the palisade organization of the also shared by the axons and dendrites of the stellate Bergmann fibers was not reported in studies on cerebel- and basket neurons. In contrast, the parallel fibers which lar preparations examined with the electron microscope are formed by the axons of the granule neurons are (i.e., Rakic, 1971; Swarz and Del Cerro, 1977). Further- oriented in the longitudinal plane of the folium. Thus more, Palay and Chan-Palay (1974, p. 299) stated: “as- the parallel fibers are perpendicular to the dendritic trees cending processes of neighboring cells (Bergmann glia) of the Purkinje cells. are all interdigitated to form a dense forest rather than Much less attention has been focused on the geomet- the alternating or intercalated palisades described by rical organization of the Bergmann glia. These cerebellar Ram6n y Cajal.” cells have their cell bodies in the Purkinje cell layer and In this communication I present a study on the cy- their fibers (Bergmann fibers) oriented radially through toarchitecture of the Bergmann fibers based on differ- ential interference contrast light microscopy immuno- cytochemistry. I have used a monoclonal antibody spe- 1 I wish to thank Holly M. Cherwinski for her excellent technical cific for Bergmann fibers and astrocyte processes, an help. I also thank Dr. Harvey Karten for the use of some of his antiserum to GFAP, and a monoclonal antibody specific laboratory facilities and for his comments and discussions. I am grateful to Stephen Mernoff and Linda Cerracchio for their help in the prepa- for the Purkinje cell bodies and dendrites. These results ration of the manuscript. This research was supported by Grant NS show that (i) the Bergmann fibers are organized in 17708 from the National Institute of Neurological and Communicative palisades, and (ii) these palisades are parallel to the Disorders and Stroke and by a Basil O’Connor starter research grant longitudinal plane of the folium. A model for the cytoar- from the National Foundation-March of Dimes. chitectural organization of the Bergmann fibers is pre- 265 266 De Blas Vol. 4, No. 1, Jan. 1984 sented. I have also made an attempt to reconcile the antiserum to GFAP that is also specific for Bergmann apparent discrepancies among the results obtained with glia and astrocytes (Dahl and Bignami, 1973), and (iii) a the light and electron microscopes. I believe that the monoclonal antibody (B-6A2) which in the cerebellum discrepancies result from using the name Bergmann fiber specifically recognizes Purkinje cell bodies and dendrites. for describing two different structures. Each of the Berg- Figure 1 shows the reactivity of these antibodies with mann fibers described by the light microscopists is the cerebellum. The monoclonal antibody B-9G7 (Fig. 1, formed by a bundle of thin Bergmann processes. These B and D) and the antiserum to GFAP (Fig. 1E) show a Bergmann processes were also called Bergmann fibers similar pattern of reactivity; both recognize Bergmann by the electron microscopists. fibers and astrocyte processes. Nevertheless, B-9G7 binds to a cytoskeleton molecule other than GFAP which is Materials and Methods specifically found in these glial cells (A. L. de Blas, R. 0. The monoclonal antibodies B-9(37 and B-6A2 were Kuljis, and H. M. Cherwinski; submitted for publication). obtained after immunizing BALB/c mice with synapto- It should also be noticed that most of the stained ele- somal plasma membranes from the rat cerebral cortex ments in the molecular layer are Bergmann fibers. There (A. L. de Blas, R. 0. Kuljis, and H. M. Cherwinski, are also a few astrocyte processes that have their cell submitted for publication). They were produced accord- bodies in the granular cell layer (see also Bignami et al., ing to the method described by De Blas et al. (1981) 1980). In contrast, the monoclonal antibody B-6A2 in the using the myeloma line P3X63AgB. For immunocyto- cerebellum binds specifically to the cell bodies and main chemistry, Sprague-Dawley rats were perfused with per- dendrites of the Purkinje cells (Fig. 1, A and C). No iodate/lysine/4% paraformaldehyde fixative (McLean other cerebellar structures bind this antibody. and Nakane, 1974). Fixed and frozen brains were sliced Bergmann glia fibers are organized in parallel palisades. (25 pm thick) with a microtome. The slices were incu- Figure 2 shows horizontal sections (parallel to the pial bated for 12 to 36 hr at 4°C with B-9G7 or B-6A2 hybri- surface) of the molecular layer of the cerebellum stained doma culture medium to which Triton X-100 was added with the antibody B-9G7. In Figure 20 the antiserum to to a final concentration of 0.3% (v/v). The rabbit anti- GFAP was used instead. The stained elements are seg- serum to GFAP was used at a dilution of 1:5,000 with ments of the Bergmann fibers. 0.3% Triton X-100 in 0.1 M phosphate buffer, pH 7.4. These results indicate that the Bergmann fibers are The tissue slices were then sequentially incubated with organized in parallel palisades of columns. The columns rabbit anti-mouse IgG serum diluted 1:lOO (or goat anti- are generally organized in sets (of two to six aligned rabbit IgG for slices treated with rabbit antiserum to columns). These sets are aligned and separated from GFAP) and mouse (or rabbit for anti-GFAP) peroxidase- each other by a gap of 9 pm (average value). The columns antiperoxidase complex (PAP) diluted l:lOO, each for 45 are seen as dots in the sections that are perfectly perpen- min at 22°C. The dilutions were done with 0.3% Triton dicular to the Bergmann fibers (Fig. 2, A, C, and D). The X-100 in 0.1 M phosphate buffer, pH 7.4, and the washes distance between two parallel palisades has an average with the same buffer without Triton X-100. The perox- value of 9.5 pm. When the sections are not perfectly idase reaction was developed with 0.05% (w/v) diami- perpendicular to the Bergmann fibers it can be seen that nobenzidine tetrahydrochloride, 0.01% (v/v) HzOz, each column (or Bergmann fibers to the light microsco- 0.03% (w/v) cobalt chloride, and 0.03% (w/v) nickel pists) is composed of several closely packed Bergmann ammonium sulfate in 0.1 M phosphate buffer, pH 7.4, at processes (Fig. 2, B, E, and F). Figure 2F shows that the 22”. The nickel and cobalt ions were used for intensifi- Bergmann fibers and processes are frequently twisted cation of the enzymatic reaction (Adams, 1981). The and curled forming helical structures (see also Figs. 1, D tissue sections were mounted on glass slides and incu- and E and 3B). bated with 0.05% 0~0~ for 15 sec. The conditioned Figure 3A shows the distribution of the neuronal cell culture medium of the parental myeloma P3X63AgB and bodies in the molecular layer with respect to the Berg- a nonimmune rabbit antiserum were used for controls. mann fiber palisades. Figure 3B shows some important The tissue sections processed with the control antibodies features of the organization of these fibers (see also Fig. showed no immunocytochemical staining. When indi- 1, D and E): (i) The Bergmann fibers are straight col- cated, the mounted sections were counterstained with umns that extend from near the Purkinje cell body layer acidic cresyl violet (5 gm/liter) for 2 min at 22°C.
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