A monoclonal antibody identifies a novel epitope surrounding a subpopulation of the mammalian central neurons

NeuroscienceVol. 29, No. 3, PP. 645457, Printedin Great Britain

0306-4522/89 $3.00 + 0.00 Pergamon Press plc 0 1989 IBRO

1989

A MONOCLONAL ANTIBODY IDENTIFIES A NOVEL EPITOPE SURROUNDING A SUBPOPULATION OF THE MAMMALIAN CENTRAL NEURONS E.

WATANABE,*

S. C.

FUJITA,t$

F. MURAKAMI, * M.

HAYASH@

*Department of Biophysical Engineering, Faculty of Engineering

and M.

MATSUMURA~

Science, Osaka University, Toyonaka,

Osaka 560, Japan TDepartment of Pharmacology, Gunma University School of Medicine, Maebashi 371, Japan SDepartment of Physiology, Primate Research Institute, Kyoto University, Inuyama 484, Japan TDepartment of Neurophysiology, Primate Research Institute, Kyoto University, Inuyama 484, Japan Abstract-A monoclonal antibody was obtained by immunizing mice with an extract of monkey brain. The monoclonal antibody 473 stained a small subpopulation of neurons in various regions of monkey and rat central nervous system. The perimeters of neuronal somata and the proximal parts of dendrites bound the antibody. Electron microscopic analysis showed that the immunoreactivity was associated with the outer surface of the cell. The immunoreactivity in the rat cerebral cortex appeared gradually during the second four weeks after birth. The antibody stained fetal cartilages but otherwise was specific to the nervous system. Experiments on the stability of the immunoreactivity to enzymatic and chemical treatments of the sections suggest that the antigen molecule is of proteoglycan nature.

Torpedo synaptosomes, Tor 23, binds to the perimeter of a restricted set of neurons in the rat central nervous system. I3 Two more MAbs of this type were recently described.7~‘6~28 No evidence is yet available concerning the function of these intriguing antigens. Is the apparent similarity of these MAbs fortuitous, or perhaps a manifestation of a hypothetical family of epitopes that is involved in neuronal interactions? In our extensive library of MAbs generated against immunogens from chicken and mammalian nervous systems, several were found which, in their staining of central neurons, appeared to form a common group with the MAbs mentioned above. Studies are being made on these MAbs in the hope that clarification of neuroanatomical and molecular relationships among their antigens and with those of reported MAbs will contribute to answering the question posed above. Here we report on an immunohistochemical analysis of the first of our antibodies, MAb 473, in the central nervous system of the monkey and rat. This antibody stains the perimeter of only a subpopulation of mammalian central neurons. Some experiments pertaining to the chemical nature of the antigen are also described.

Since Sperry hypothesized chemoaffinity as the basis of specificity of neuronal connectivity,*’ the possible molecular distinctness of individual or groups of neurons has long attracted serious interest. Although neurotransmitters with related enzymes and neuropcptides are well known to occur in specific subsets

of neurons,” it was with the advent of the monoclonal antibody (MAb) technique’* that it became possible to explore the extent of the chemical diversity of neurons and to study the nature of the molecules responsible for it. Thus, in their landmark contribution, Zipser and McKay showed that a homogenate of the nervous tissue could be used as an immunogen to obtain a panel of antibodies that bound to subsets of neurons.*’ This was followed by a number of studies reporting MAbs selective or specific to subregions of the nervous system.27 We also reported, previously, MAbs that delineate divisions in the chick embryonic spinal cord,3 rabbit olfactory system,“.i9 and rabbit forebrain.‘* Among the numerous MAbs reported, a class of antibodies is notable in that it recognizes antigens on the surface of a small subset of central neurons. MAb Cat-301, originally generated with immunogen from cat spinal cord,” stains a subpopulation of neurons in mammalian cerebral cortex.5.6 MAbs VCl.1 and VC5.1, produced against a homogenate of cat visual cortex, define a subpopulation of GABAergic neurons in the cortex.*.*’ An MAb made against

fTo whom correspondence should be addressed. Abbreuiations: EDTA, ethylenediaminetetra-acetate; MAb, monoclonal antibody; TBS, Tris-buffered saline.

EXPERIMENTAL PROCEDURES Antibodies The MAb 473A12 studied in this work arose from a hybridoma experiment using mice immunized with an extract of monkey cerebrum. Cerebral hemispheres were taken from an adult Japanese monkey (Macaca ficara &caru) killed by blood letting from the artery under deep pentobarbital (Nembutal) anesthesia (30 mg/kg, intraperitoneal). The tissue (43 g) was extracted with boiling 2 M acetic acid (200ml). The extract was concentrated and conjugated to

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keyhole limpet hemocyanin (Sigma) by glutaraldehyde. The product was dialysed against saline, emulsified in Freund’s complete adjuvant, and injected intraperitoneally into BALB/c mice. Each mouse received material corresponding to 1 g of original tissue. The mice were boosted twice during the subsequent 3 months. The hybridoma experiments were conducted according to Oi and Herzenberg.‘2 Myeloma cells of PAI strain (obtained from Dr S. Ohta) were used. Culture supernatants of colony-bearing wells were tested using sections of fixed monkey neocortex and rat spinal cord by immunofluorescence method as described below. Out of 1077 tested, immunostaining was detected in 66. MAb 473Al2 was unique in staining a subset of central neurons. The hybridoma line was cloned by limiting dilution, and determined to be an IgA by an Ouchterlony double diffusion typing kit (Miles). MAb 473Al2 did not bind to hemocyanin as tested in a dot binding assay, although some others from the same fusion did so. In the following, this antibody will be designated as MAb 473 for brevity. Hybridoma culture supernatant was concentrated about 30 times by ammonium sulfate precipitation, followed by dialysis against Tris-buffered saline (TBS; 10 mM Tris-HCI buffer, pH 7.4, containing 0.13 M NaCl, 5 mM KCI, 5 mM NaN,, and 1 mM EDTA). Aliquots were stored at - 80°C. For most of the experiments the concentrated antibody solution was used at six times dilution in TBS with 0.3% Triton X-100 added, although supernatants from aged cultures contained sufficient concentration of the antibody to give virtually saturating levels of staining intensity. The MAb 8C5 used in dual staining shown Fig. 2 was described elsewhere.4 The antibody is an IgG2a. and specific to nuclei of eukaryotic cells.

Immunofluorescence histochemistry For histochemical studies, the experimental animals were fixed by transcardial perfusion under deep pentobarbital (Nembutal) anesthesia. Three monkeys were bled, perfused first with 0.9% NaCl or 0.1 M phosphate buffer, pH 7.4, then with the chilled fixative solution either of Zamboni’s (2% paraformaldehyde, 0.2% picric acid in 0.1 M phosphate buffer, two animals) or 4% paraformaldehyde in 0. I M phosphate buffer, pH 7.4. Staining with MAb 473 was the same regardless of the two fixatives. The dissected brain and cervical spinal cord were immersed in the same fixative overnight in the refrigerator; they were then washed and cryoprotected in several changes of phosphate-buffered saline (12 mM phosphate buffer, pH 7.2, with 0.9% NaCl) containing 20 or 30% sucrose. For albino rats (Wistar). mice (BAiB/c), and a domestic cat, the fixative was 3.5% formaldehyde in 0.1 M phosphate buffer, pH 7.4. Rat fetuses and chick embryos were fixed by immersion overnight in chilled phosphate-buffered saline containing 3.5% formaldehyde. Appropriate blocks of required tissues were frozen embedded in OCT Compound (Miles). Sections of 13pm thickness were cut in a Tissue Tek II cryostat microtome (Miles) at around - 2O”C, and picked up onto egg-albumin subbed coverglasses. Such sections were stored until use with silica gel desiccant in the refrigerator. All immunostaining steps were performed at room temperature. The sections on the coverglass were covered with the MAb solution for approx. 2h, then rinsed in TBS for IS min. They were then incubated for 30 min with fluorescein-conjugated second antibody against mouse IgG (H + L) (Cappel) diluted 70 times in TBS. For the simulta-

neous dual staining reproduced in Fig. 2, the second antibody was a mixture of rhodamine-conjugated anti-mouse IgA and fluorescein-conjugated anti-mouse Fc (Cappel). For sections of rat tissues, the second antibody not cross reactive to rat immunoglobulin (Cappel) was used at 50 times dilution. After a 30-min rinse in TBS, the coverglasses were mounted onto slides with a drop of 90% glycerol in

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trl

TBS, and viewed and photographed with a Nikon Fluophot or Optiphot microscope under appropriate epifluorescencc optics. Spent medium from myeloma culture was used in place of MAb solutions for negative controls of immunostatning. Sections of these controls were virtually free of Ruorescein or rhodamine fluorescence. Brain sections from monkeys and l-year-old rats showed scattered punctate orange fluorescence of lipofuscin-like granules. Although negative controls are not shown in most of the figures, Fig. 7C and D serves to illustrate the specific immunofluorescence and a low level of tissue autofluorescence. Some of the sections studied by immunofluorescence were subsequently stained with Thionin. The photomicrographic prints from the Nissl-stained material were compared by superimposition with those of immunofluorescence micrographs of corresponding fields. Profiles of major blood vessels were helpful in registering the two images to identify immunopositive cells on the Nissl-stained sections. To test the nature of the antigen molecule, the sections were pretreated in one of the following ways prior to immunostaining. (i) Dry sections were incubated in a mixture of chloroform and methanol (2: 1) for 1h at room temperature. (ii) Sections were covered with 50 mM acetate buffer, pH 5.0, containing 50 mM sodium metaperiodate and 0.1 M NaCl for 1 h ai 4°C in the dark.24 (iii) Sections were incubated with chondroitinase ABC (0.08-2 U/ml. protease-free. Seikagaku Kogyo) in 0.1 M TrissHCl buffer. pH 8.0, containing 50 mM sodium acetate, 0.9% NaCl, and 0.1% bovine serum albumin, for 2 h at 37 C. Under these conditions immunoreactivities toward MAb 95H2” (binding to a 60,000 mol. wt neuronal protein) and MAb 82ElO’ (specific to neurofilament) were not affected. (iv) Sections were incubated in 0.5 N NaOH for 14 h at room temperature. For these experiments cryostat sections were picked up onto coverglasses subbed with 2% neoprene solution in toluene. Under such conditions glycosaminoglycan chains are expected to be eliminated from the core polypeptides of proteoglycans.’

Electron microscop? Adult rats weighing 200-225 g were deeply anesthetized with pentobarbital, and perfused transcardialy with 0.1 M phosphate-buffered saline, pH 7.4, followed by a fixative containing 4% paraformaldehyde, 0.5% glutaraldehyde and 0.002% CaCl,. After leaving the brain in situ for 1 h, the sensorimotor cortex was dissected out and cut with a Microslicer (Dosaka EM, Kyoto) at 50pm. Then the sections were incubated with MAb 473 overnight, and processed by the avidinbiotin-peroxidase method (Vector) using diaminobenzidine as the chromogen. Following immunocytochemical procedures, the sections were further fixed in osmium tetroxide. dehydrated and flat embedded in Epon (TAAB). Sections were observed with a light microscope and ultrathin sections were cut, stained with uranyl acetate, and examined with an electron microscope (Jeol IOO-CX).

Immunoblotting Monkey cortical tissue was homogenized and boiled in 10 times tissue weight of the sample buffer containing 2.3% sodium dodecyl sulfate.*’ Procedures for slab gel electrophoresis, blotting onto nitrocellulose sheet, and immunodetection were as described previously.’ RESULTS

Immunoreactive

cells in the monkey

cerebral cortex

MAb 473 was originally discovered by its characteristic staining of sections from the monkey cerebral

cortex, where the immunoreactivity outlined a subpopulation of neurons (Fig. 1). The immunostaining

Monoclonal antibody to a subset of central neurons

Fig. 1.Cellsof monkey cerebral cortex in sections stained with MAb473 by immunofluore~n~ (A, C, E). Sections were su~~~ntly stained by N&l, and ~rms~nding fields are reproduced on the right (B, D, F). (A, B) Motor cortex in the vicinity of layer V. (C, D) Somatosensory cortex in the vicinity of layers III and IV. (E, I?) Primary visual cortex in the vicinity of layer IV. Asterisks mark carresponding images of the blood vessels. To aid comparison, arrowheads in the right panels point to some of the immunopositive cells; others can be located by trigonometry. Arrowheads in A point to the strongly stained cells mentioned in the text. Arrows in B, D, and F indicate examples of neurons negative toward MAb 473. Sections were cut perpendicnlar to the pial surface. Supe&ial toward top of the panels. Scale bar represents 200~rn.

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had a granular appearance that surrounded a cell profile with a neuronal morphology. The size of the stained cells ranged from about 10 to more than 50 pm. In an overwhelming number of instances the cytoplasm and nucleus of the stained cells were free of detectable immunoreactivity. Proximal portions of dendrites were stained, but no obvious axonic staining was observed, nor was any staining seen in the white matter. Figure 1 shows representative immunostaining of sections from the motor (A), somatosensory (C), and visual (E) areas on the left column, with the same microscopic fields after Nissl staining shown on the right (B, D, F). Comparison of the fluorescence and Nissl results reveals that the immunopositive cells comprise but a small subset of neurons. In a count made for a representative field in area 3b, 18% of the Nissl-stained neurons were positive for MAb 473. Some pyramidal cells including Betz cells were positive (Fig. IA and C), while the majority of and more prominently stained cells were of a non-pyramidal type. The immunopositive cell bodies were, as a rule, stained continuously along their entire perimeter, except where emerging thick dendrites had been cut. The intensity of stain was relatively constant along

the cell perimeter for a particular cell. This indicatss that variation among the stained cells in the intensity of stain is not a technical artefact, for example due to locally altered permeability toward antibody reagent, but rather reflects actual variation in the amount of the antigen expressed by the cell It also suggests that the level of antigen expression is not dictated locally by the apposed neighbors of the cell in question, but is controlled by the stained cell. There appear to be at least two subclasses among the immunopositive neurons: the majority with medium to weak stain, and a smaller class with strong stain. Three such strongly stained cells can be seen in Fig. 1A (arrowheads). None of the immunopositive neurons with pyramid-like triangular morphology was strongly stained, and most of the intensely stained cells had round or fusiform perikarya and lacked an obvious apical dendrite. In the visual cortex (Fig. IE) staining of pyramidal cells was not readily apparent, and the cells with pyramid-like triangular Nissl profile were generally devoid of the immunostain. A number of morphological subtypes of nonpyramidal cells are known.“.‘~ Assi~ment of the MAb 473-positive cells to those subtypes is not possible at present, as the distal portions of the

Fig. 2. Distribution of MAb 473-positive cells among cortical layers in the monkey. (A) Somatosensory cortex, (C) visual cortex. The sections were simultaneously stained for cell nuclei using MAb 8CX4 and the same microscopic fields are reproduced in panels B and D. See Experimental Procedures for technical details. Scale bar represents 200 brn.

Mon~Ional

antibody to a subset of centrai neurons

649

cells. Antibodies Cat-3019 and VC5.12 label the dendrites are not stained by this antibody. The light microscopic appearance of MAb 473-positive cells is Lugaro cells in the cat cerebellum. In the cervical spinal cord, positive cells were seen thus similar to that of cells positive to Cat-301,5*6*9 in the ventral and middle layers of the gray matter VCI .l and VCS. 1: and MAb 6A2.i6 (Fig. 3E). Nissl-stained sections indicated the exis~is~ri~~~ion of monoclo~i antibody 473-positive celis tence of some large immunopositive cells in the in the monkey central nervous system ventral horn. Three other cortical areas of the monkey were Immunoreactive cells in the rat central nervous system examined. The prefrontal, primary olfactory and The histological characteristics of MAb 473 reauditory areas all contained immunopositive cells of vealed above in the monkey central nervous system similar appearance. The density of the positive cells, however, was higher in the somatosensory and visual are intriguing, but do not readily give clues to the functions subserved by the antigen molecules. It will cortices than in other areas studied. be necessary to extend further studies to animal Distribution along the thickness of the cortex was species which are more accessible to experimentation examined in the somatosensory (Fig. 2A and B) and than the monkey. MAb 473 was found to cross react primary visual areas (Fig. 2C and D). Sections in the brains of the cat, rat, and mouse, but not of shown in Fig. 2 had been simultaneously stained with the chicken. Appearance of the stained neurons in the MAb 473 and MAb 8(X4 specific to cell nuclei. The bound MAbs were independently visualized by three mammals was similar to that in the monkey. Distribution of MAb 473-positive neurons was rhodamine- or fluorescein-conjugated class specific studied in the rat (Fig. 4). In the neocortex the second antibodies. The distribution of cell nuclei positive cells were a small subset of neurons, and not helped to define the cortical laminae. restricted to particular layers (Fig. 4A), although they The two areas had a similar distribution of positive were relatively more abundant in certain layers decells. No immunoreactivity was seen in the molecular pending upon the cortical areas. Variation among the layer (layer I) nor in the white matter. The immunoareas seemed more marked than in the monkey. The positive ceils were rather rare in layer II, but broadly cingulate cortex was relatively rich in immunodistributed over layer III through the upper stratum positive cells, while the allocortex ventral to the of layer VI (Fig. 2). This is in contrast to the case with rhinal sulcus (entorhinal and amygdala areas) did not Cat-301, for which the immunopositive cells densely contain positive cells. Nissl staining indicated that the occur in layer VI, and to a lesser extent in layers BIB positive cells were generally non-pyramidal (Fig. 4A and IV, of the monkey area 17.‘.* vs B). In the hippocampus and dentate gyrus, clearly The somatosensory and visual areas were also studied in tangential sections stained with MAb 473. positive cells were not seen. Subiculum, however, contained many positive cells (Fig. 4C). No obvious patterns were seen in disposition of the In the thalamus positive cells were mainly clustered immunoreactive ceils or direction of stained portions in the reticular thalamic nucleus. Cells here were of the dendrites. Outside the neocortex, the hippocampal formation, relatively weakly stained, and a diffuse appearance of cerebellum, and spinal cord were examined (Fig. 3). immunoreactivity in the neuropil was a characteristic of this region (Fig. 4D). A number of positive cells No clear staining was observed with the cells of the were also noted in the red nucleus (Fig. 4E). dentate gyrus. In the hippocampus, a small number In the cerebellar cortex, the immunoreactivity was of horizontally oriented cells in the outer rim of the stratum oriens were positive. In the subiculum, more associated only with rare ceils in the granule cell stained cells were seen broadly dist~buted (Fig. 3A). layer, as it was in the monkey (Fig. 4F). These cells, These cells were non-pyramidal (Fig. 3A vs 8). however, occupied a deeper position compared with In the cerebellar nuclei many large strongly stained the monkey counterpart, often lying in the bottom cells were seen (Fig. 3C). Here Nissl staining showed half of the thickness of the layer. that most of the large cells were labeled. In addition On the other hand, many positive cells were found to staining of cell bodies, an extensive meshwork of in the cerebellar and vestibular nuclei. Figure 4G and process-like structure, presumably dendrites, was H illustrates that most but not all large neurons of the strongly immunopositive. lateral vestibular nucleus are positive with MAb 473, The only ceils stained in the cerebellar cortex were whereas the majority of the smaller neurons are not. rare cells in the granular layer (Fig. 3D). The cell Besides the nuclei mentioned, solitary immunobodies were usually situated directly below the Furpositive cells were seen in various regions of the kinje cell layer, or in some cases somewhat deeper in diencephalon and the rest of the brain stem. Immunothe granule cell layer. A horizontal process was often reactivity in the rat spinal cord was not readily seen to extend from the cell body, and less frequently apparent; weakly positive large anterior horn cells an additional process was seen to course toward the were seen in some of the sections. No neuronal white matter. These cells are probably the Lugaro element was immunopositive in the retina, where only celi~.~) It is not possible at present to know whether a weak staining was seen on the inner limiting MAb 473 stains all or only a subset of the Lugaro membrane. In the olfactory bulb, some fibrous stain

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Fig. 3. MAb 473-positive cells in other regions of monkey central nervous system. (A. B) Subiculum. The same field after Nissl staining is shown in B. Asterisks indicate a blood vessel. Arrowheads in B poinl to the immunopositive cells. (C) A deep cerebellar nucleus. Note an extensive meshwork of dendrite-like immunopositive structures. (D) Cerebellar cortex. m, molecular layer; p. Purkinje cell iayer: g. granule cell layer; w, white matter. Smail dots, particularly in or near the Purkinje cell layer. are due to lipofuscin-like material with orange autofluorescence. (E) Ventral horn of the cervical spinal cord. Lateral to right. Scale bar represents lOO/tm for D, and 200pm for A-C and E.

was seen external to the olfactory regions of the bulb.

glomeruli

in certain

I)evelopmental appearance of the immunoreactiuity in the rat brain Developmental and ultrastructural studies are indispensable to an understanding of the physiological

function

of the antigen

molecules

originally

defined

by an MAb. Development of the immunorea~tivity toward MAb 473 was studied in the sensttrimotor cortex and subiculum of the rat. Fetuses at the 17th and 18th days of gestation, and animals of 0. 14. 28. 35. 42 and 55 days, 14 weeks, and 12 months of age were examined. No immunoreactivity was detected

Monoclonal antibody to a subset of central neurons

Fig. 4. MAb 473-positive cells in the rat central nervous system. (A, B) Cerebral cortex, somatosensory area. Asterisks indicate corresponding blood vessels. Arrowheads in B (Nissl staining) point to some of the immunopositive ceils. (C) Subiculum. (D) Thalamic reticular nucleus. (E) Red nucleus. (F) Cerebellar cortex. m, molecular layer; p, Purkinje cell layer; g, granule cell layer; w, white matter. Dots in Eurkinje cell layer are due to autofluorescence of lipofuscin-like material. (G, H) Lateral vestibular nucleus. Note that most of the large neurons are positive. An arrow in H (Nissl staining) points to a large neuron negative to MAb 473. All micrographs were from coronal sections. Dorsal toward top in C-E, G, and H. Scale bar represents lOO/lm for F, and 2OOfim for A-E, G and H.

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Fig. 5. Development of MAb 473 immunor~ctivity in the rat cerebral cortex. (A) 14 days after birth; (B) 35 days; (C) 55 days; (D) 14 weeks. Scale bar represents 200pm. Arrowheads in B point toward emerging immunopositive neurons.

up to two weeks (Fig. 5A). In the 4- and S-week materials a weak staining of neuronal somata was observed in the neocortex, and their number was smaller than in the adult (Fig. 5I3, arrowheads). The intensity of staining and the number of stained cells increased through the sixth week, to attain levels comparable to those of adult at around 8 weeks (Fig. 5C and D). One-year-old animals were similar to younger adults in these respects. A similar time course was seen in the subiculum. The antigen to MAb 473 is thus expressed by the central neurons at later stages of development of the brain, perhaps concurrent with functional maturation. It is interesting that the antigen for Cat-301 has also been found to be expressed relatively late during development of the cat spinal cord, gradually increasing during the first 3 weeks after birth.’

An immunoelectron microscopic study of MAb 473 was made on the cerebral cortex of adult rats. In agreement with the fight microscopic observation, the immuno~roxidase reaction product was found on a small number of cehs with neuronal ultrastructural profile. The reaction products encircled the cell body (Fig. 6A), and were found along the outer surface of the cell membrane (Fig. 68, arrowheads). Reaction products were not found in the synaptic terminals (Fig. 6B, asterisk) and the synaptic regions, although definite localization of the antigen must await further studies. The antigens for Cat-301 and VCI. 1 have similarly been localized in the outer surface of neurons excluding the synaptic clefts.n,2”

Monoclonal antibody to a subset of central neurons Immunoreactivity outside the central nervous system

Various tissues from the rat were studied immunohistochemically for presence of immunoreactivity toward MAb 473. No immunoreactivity was detected in the dorsal root ganglion or diaphragm including its neuromuscular junctions as identified by fluoresceinconjugated a-bungarotoxin. Nor was any staining observed in the thigh muscles, adrenal gland, kidney, pituitary, liver, intestine, or skin from the back. Strong fluorescence, however, was found in the

653

cartilaginous tissues of the fetal rat (Fig. 7A). All cartilaginous tissues of the fetal rat encountered were stained. Overall intensity of the stain, however, varied depending upon the regions of the tissue, and from cell to cell in a particular region. The immunofluorescence outlined the chondocytes (Fig. 7A), but the precise subcellular localization remains to be established. Cartilaginous tissues of adult rats were largely negative. Nasal, bronchial, and articular cartilages as well as that of the ear were devoid of staining except for occasional

Fig. 6. Electron micrographs of neurons in the rat cerebral cortex immunostained with MAb 473. (A) An electron micrograph of a neuron in the cerebral cortex. Electron dense reaction products rim the cell soma. Scale bar represents 2 pm. (B) A high-power micrograph showing electron dense reaction products near the outer surface of a neuron (arrowheads). Note that synaptic terminals are free of reaction products (asterisks). Scale bar represents 1 pm.

Fig. 7. Staining of cartilages by MAb 473 (A, B), and susceptibility of both neuronal and cartilaginous immunoreactivity to chondroitinase ABC (C-F). (A) A cartilaginous tissue in the head of 1&day rat fetus. (B) Cross section of vertebra of 7-day chick embryo. SC,spinal cord: n, notochord. (C) Immuno~sit~~e ceils of monkey cerebral cortex after control treatment with buffer alone. (D) A neighboring section after treatment with chondroitinase ABC as detailed in Experimental Procedures. (E) Chicken vertebra from IS-day embryo, after control buffer treatment. (F) A neighboring section after the enzyme treatment. Treatment with buffer alone did not affect the immunoreactivity to MAb 473. Scale bar represents 100 pm for A. C. and D. and 200pm fur B, E, and F.

clusters of weakly positive cells in the articular cartilages. In spite of the observation that the chicken brain lacks the immunopositive neurons, strong staining of

cartilage cells was also observed in the chick embryos.

Figure 7B shows the vertebral cartilage of a 7-day chick embryo. Note that strongly positive cells occur intermixed with negative or weakly positive cells. The

Monoclonal antibody to a subset of central staining was strong in certain regions of bone primordia in more developed embryos as well (see Fig. 7E). There seem to be certain spatial patterns to the regions rich in positive cells, but the matter remains to be investigated. Nature of the antigen molecule

Several preliminary attempts were made to generally assess the chemical nature of the antigen molecule or the antigenic determinant (epitope) to MAb 473. Histological sections were treated with ~hlorofo~-methanol mixture, or with sodium metaperiodate under the conditions detailed in Experimental Procedures. The MAb 473 immunoreactivity of both neurons and cartilages persisted after both treatments. These results argue against a glycolipid nature of the antigen, and against a direct participation of carbohydrates bearing a-diols in the epitope. Furthermore, repeated attempts failed to detect immunoreactive bands in the electrophoretic blot of monkey brain homogenate. Since the antigen in the central nervous system was found to occur extracellularly (Fig. 6), and it proved quite abundant in cartilaginous tissues (Fig. 7A and B), a possibility of proteoglycan was then considered. The neuronal as well as cartilaginous immunoreactivity was indeed obliterated by prior treatment of the sections with protease-free chondroitinase ABC under the conditions described in Experimental Procedures (Fig. 7D and F). Staining was not affected by treatment with the buffer alone (Fig. 7C and E). The immunoreactivity was aIso lost by treatment of the sections with 0.5 N NaOH under the conditions (see Experimental Procedures) in which immunoreactivity toward MAbs 95H23 and 82E103 were not affected. The results above, taken as a whole, indicate that the molecule responsible for staining with MAb 473 is a proteoglycan or some closely related chemical species. DISCUSSION The immunohistochemical studies described above show that the antigen molecule for MAb 473 occurs associated with the cell surface of a small subpopulation of central neurons in the monkey and rat. The immunopositive cells do not fall in a particular morpholo~~al class, and their occurrence and abundance depend on regions of the central nervous system. Outside the nervous system the only tissue found to express significant immunoreactivity was cartilaginous tissue of the rat fetus and chick embryo. Experiments testing the effect of chemical and enzymatic treatment of histological sections indicate that the antigen is a proteoglycan or a related substance, The light microscopic appearance of the stained cells and ultrastructural localization of the antigen for MAb 473 are quite similar to those reported for

neurons

655

There is certain similarity in antibody Cat-301. 6~8,9~1s distribution of the neurons positive to the two antibodies: Cat-301 stains the Lugaro cells in the cat cerebellum,9~15 and MAb 473 appears to stain the equivalent cells in the cerebellum of the monkey, and possibly in the rat. However, in the monkey visual of cortex, there occurs a heavy accumulation Cat-301-positive cells in layer VI with a less marked concentration in layers BIB and IV,5,6 while MAb 473-positive cells are not concentrated in a particular layer (Fig. 2C). Cat-301-positive neurons of the rat cerebral cortex are restricted to the hippocampal formation6 while MAb 473-positive neurons occur in the rat neocortex but barely in the hippocampal formation. In the rat thalamus, Cat-301-positive neurons occur in the intralaminar nuclei,6 but MAb 473-positive neurons are mostly found in the reticular nucleus. It is likely that the two antigens are related but distinct, and belong to a common family. In this respect, it is interesting that recently the antigen for Cat-301 was suggested to be a proteoglycan, although no reasoning was given. 26 A direct comparison by dual staining would be rewarding. Antibodies VCl.1 and VC5.1 also outline a subpopulation of neurons in the cat central nervous system.2 These two antibodies stain the same population of cells in the cerebral cortex, although they stain different neuronal systems in the retina or cerebellum. Lateral geniculate nucleus is not stained by either antibody; they are thus distinct from Cat-301.2 They are also likely to be different from MAb 473, which does not stain neuronal elements in the retina. Different sets of multiple proteins with molecular weight ranging between 95,000 and 170,000 are considered to be the antigen moiecules bound by VCl.1 and VC5.1.’ Recentiy, VCI .I-positive cells in the cat visual cortex were shown to constitute a subset of GABAergic neurons.” Recent reports described MAb 6A2 which similarly stained a small subpopulation of neurons in the human neocortex, hippocampal formation, and spinal cord. ‘J’ Another antibody, 4F4, was described as staining the Golgi apparatus of most neurons in the mouse brain, but, in a distinct neuronal subpopulation, the cell membrane as well.28 It would be interesting to compare these and other MAbs discussed above directly, in the same experimental animal. Since all such antibodies bind only to a small subset of neurons, it is tempting to speculate that there exists in the central neural tissue an epitope system of enormous molecular diversity, such that each neuronal subset expresses certain combinations of the epitopes. If this is the case, one would expect to find many more antibodies that mark yet different neuronal subsets. We have indeed obtained recently several MAbs of the type discussed above. Compared with MAb 473, some of these appear to define a different though partly overlapping subset of

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neurons. These MAbs will be described in subsequent reports. At least two approaches will be important in the clarification of the physiological functions of the antigen molecules. One is to establish their chemical identity. When the chemical structure is known, it will be possible to introduce the purified or synthetic antigen, or polyclonal antibodies raised against it, into physiological preparations, developing animals, or neuronal cultures. The other approach is to study possible alterations in expression of the immunoreactivity in the brains with perturbed physiology

caused by surgical or pharmacological interventions. As MAb 473 cross reacts in the mouse. it should hc interesting to also study the brains of the many neurological mutants available. thank Drs K. Mori and K. Obata for interest and helpful discussions and Dr Y. Hotta for encouragement. Thanks are also due to Mrs Y. Roppongi for expert photography, and to Mrs Y. Aoki for preparation of the manuscript. This work was supported by the Special Coordination Funds of the Science and Technology Agency of the Japanese Government, and by the Primate Research Institute of Kyoto University. Acknowledgements-We

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