NeuroscienceVol. 41, No. 1, pp. 277-285, 1991 Printed in Great Britain

0306-4522/91$3.00+ 0.00 Pergamon Press plc 0 1991IBRO

A MONOCLONAL ANTIBODY RAISED AGAINST THE MAUTHNER CELL ALSO RECOGNIZES SOME RETICULAR NEURONS A. TRILLER,*~ M.-A. NICOLA,t E. COUDRIER,$ D. LOUVARD$ and H. KoRNt tNeurobiologie

Cellulaire (INSERM U261) and SBiologie des Membranes (UA CNRS 040270) Institut Pasteur, 25 rue du Dr-Roux, 75015 Paris, France

monoclonal antibody was raised against dissected Mauthner cells of goldfish, Carassius (mAb 222C2) recognized in this neuron a determinant that was localized on the soma of the Mauthner cell in front of the axon hillock and on the dorsomedial portion of the initial third of its ventral dendrite. When observed with electron microscopy, the staining was associated with polyribosomes and with the reticulum, close to the Golgi cisternae. The antibody also labelled other large neurons (1Wpm) of the nuclei reticularis superior, medialis and inferior. In these cells, patchy immunolabelled elements could be detected, dispersed within cytoplasm. They did not exhibit the characteristic topological distribution observed in the Mauthner cell. On the basis of their size and location, this group of neurons may send axons to the spinal cord. No staining was observed in other areas of the brainstem, or in other structures such as the cerebellum or the optic tectum. The expression of this antigenic molecule in Mauthner and reticular cells suggests that these two sets of neurons are functionally and/or ontogenetically related. Although the molecular and functional ‘characteristics of the antigenic molecule have not been determined, this antibody should be a useful marker for further developmental studies. Abstract-A

aurutu.s. The immunoglobulin

The Mauthner cell (M-cell) exists as a single pair of neurons in the bramstem of most fishes, including teleosts. These neurons can be identified by size, shape and localization33.42 and also by electrophysioFunctionally, the M-cell is relogical properties. 8~Lo sponsible for a stereotyped behaviour, the initial phase of the startle reflex.’ The singularity of this cell raises two types of question. First, are there any molecules specific to this neuron and/or to others within the brainstem? In fact, Bullock’ predicted that other identifiable neurons would be discovered in the teleost brain. They would be comparable to the population of well-characterized cells described in the lamprey brainstem35,36 which project to the spinal cord. Second, what is the phylogenetic relationship of the M-cell with adjacent neurons, i.e. reticular and/or vestibular cells, which also send axons to the spinal cord? Inputs and spinal projections alone may not be sufficient to differentiate these vestibular and reticulospinal neurons from the M-cell. Despite the fact that they differ morphologically and functionally from the M-cell, some of these neurons, not yet identified, may also be able to generate behaviours

*To whom correspondence should be addressed. Abbreviutions: ABC, avidin-biotin-peroxidase complex; Ig, immunoglobulin; mAb, monoclonal antibody; Mcell, Mauthner cell; MLF, medial longitudinal fasciculus; PB, phosphate buffer; PBS, phosphate-buffered saline; SAM-TRITC, sheep anti-mouse tetramethylrhodamine isothiocyanate.

that are very similar to the escape reflex initiated by the M-celL6,** In an attempt to address these questions, we have raised a monoclonal antibody against the M-cell. Zipser and McKay first suggested,4’ and numerous later studies3~9,1*~13~18~!9,*5,~ confirmed, that subsets of cells may express antigens specific for morphological or functional subclasses of neurons. In our case, we expected to find an antibody that would recognize either the M-cell specifically, or groups of neurons (that include the M-cell) with common functional and/or morphological characteristics. EXPERIMENTAL PROCEDURES Preparation of antigens and immunizarion

After anaesthesia with tricaine (MS222 Sandoz), the brains from goldfish (Carassius uurutus, 5dcm in length) were removed and immersed in formaldehyde (4%) in 120 mM phosphate-buffered saline (PBS) for 6 h at 4°C. They were incubated overnight in 40% sucrose in PBS at @C. Microtome frozen sections (80pm) were stained with Cresvl Thionine (1%) for visualization of the M-cell. This neuron and its immediate surrounding tissue, which included impinging boutons and adjacent astrocytes,29 were dissected by hand under a microscope (Fig. 1). The selected tissue was frozen in liquid nitrogen and stored at -70°C. One mouse BALB/C was immunized with 200 cells suspended in 5 ~1 of PBS. A “single shot” intrasplenic injection, as described by Spitz et aZ.,38 was preferred. Briefly, the mouse was anaesthetized with ether and the spleen was exposed through a small skin incision (l-l .5 cm in length). A needle was inserted deeply in the spleen and the antigen was inoculated as the needle was pulled out. The spleen was then replaced in the peritoneal cavity, which was sutured in two planes. 217

A.

278

TRILLER

Fig. 1. Schematic drawing of a frontal section of the brainstem passing through the vestibular nerve at the level from which the M-cell was dissected. The main nuclei present on this plane of the brain are represented on the lefthand side. Tissues were removed at about 100pm around the M-cell (right side) and the collected (hatched) area was kept for immunization. dt, descending trigeminal tract; mm, nucleus reticularis medialis; vestc, vestibular complex; vest.n, vestibular nerve.

Antibody

For both light and ultrastructural observations, the “Vectastain” (Vector Lab., Inc., Burlingame, CA) avidinbiotin-peroxidase complex (ABC) procedure2” was chosen to visualize the monoclonal antibody (mAb) bound to the tissue. More precisely, after overnight incubation at 4°C with the primary antibody, the slices were kept for 2 h in biotinylated horse anti-mouse IgG (1:200), washed, and treated for 2 h with the avidin-biotin-peroxidase reagent (Vectastain, ABC). The presence of the complex was revealed after the addition of 0.03% diaminobenzidine tetrachloride in 0.05 M Tris, pH 7.4, and 0.006% H,O,. The reaction time (5-15min at room temperature) was controlled under the light microscope and stopped when a light brown colouration of the background appeared. For light microscopy, the sections were dehydrated in graded ethanol, cleared in xylene and mounted with Eukitt (Kindler, Freiburg, RFA) before examination with a microscope equipped with Nomarski interferential optics. For ultrastructural observations, the sections were posthxed with 2% osmium tetroxide in PB, dehydrated and flat-embedded in Araldite. Ultrathin sections were counterstained with a Reynolds lead solutio# and examined with a Philips CM 10 electron microscope. Control staining experiments were performed with culture supernatant from non-secreting cells or from cultures producing IgM of undefined specificity. As expected, this procedure resulted in no labelling.

production

RESULTS

Three days later the spleen was removed under deep anaesthesia and the splenocytes were fused with Sp2/0 myeloma cells. Hybrydoma cells were plated on 24-well tissue culture plates and grown in an appropriate medium. The production and the idiotype of the antibody in the culture supernatants were determined with an ELISA test. Screening

et al.

and histological

characterization

of antibody

The possibility that a secreting hybridoma recognized the M-cell was examined by indirect immunofluorescence labelling experiments. After anaesthesia with tricame, fishes (1&12cm) were perfused intracardially with 4% paraformaldehyde in 0.12 M phosphate buffer (PB); the brains were removed, postfixed in the same fixative for 4 h and immersed overnight in 40% sucrose PB. In order to block the free aldehyde groups, frozen sections (60nm) were dipped in 0.25% ammonium chloride dissolved in PBS. The sections were subsequently incubated overnight at room temperature with the culture supernatant which, at this stage, may contain several antibodies. They were ultimately reacted for 2 h with tetramethylrhodamine-coupled sheep anti-mouse IgG (1: 200; SAM-TRITC, Cappel Laboratories). Between each step, the sections were extensively washed in PBS. Finally they were mounted with Moviol (Hoechst, Frankfurt, RFA) and viewed with a Zeiss microscope equipped with epifluorescence optics and Zeiss filters selective for rhodamine. The selected wells were sub-cloned by the limiting-dilution method at an average density of 0.5 cells per well (Falcon 96 well trays) and re-examined with the same fluorescence procedure. Immunoperoxidase

labeling

The cytological distribution of the antigen was also examined with light and electron microscopy after staining using immunoperoxidase techniques. For light microscopy, fishes were perfused, postfixed and sectioned as for fluorescence. For electron microscopy, a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in PB (for 20 min), followed by 4% paraformaldehyde alone (20 min) in the same buffer was preferred for the perfusion. In this case the brains were sectioned (50pm) with a Vibratome and the slices were kept in ice-cold PBS.

From the 600 hybrid colonies obtained from a single fusion experiment, the 300 that secreted immunoglobulins were screened by indirect immunofluorescence. Eleven monoclonal antibodies stained cells in the goldfish brainstem. The ELISA technique showed that these antibodies all belonged to the IgM class. The supernatant of eight clones stained glial cells and three of them recognized neurons. Among the last group, one clone named 222C2 produced an immunoglobulin that recognized the M-cell more specifically. In this paper, we describe the staining properties of this monoclonal antibody. Similar labelling was consistently observed in 11 fishes stained for indirect immunofluorescence, nine for enzymatic reaction and for light microscopy, and three other animals at the ultrastructural level. Label&g

of the Mauthner

cell

The antibody 222C2 marked discontinuous patches within the M-cell, as shown in Fig. 2. Their size varied between 0.5 and 3 pm, and they predominated at the somatic level between the axon hillock and the nucleus of the cell (Fig. 2A, C). In some sections (Fig. 2D), the patches could be observed at the border of the cell, which was visualized with Nomarski interferential optics. The reaction product was also always present in the initial third of the ventral dendrite (Fig. 2B, E). It was located on the border of this process, at its dorsal and more medial portion, on the convex side of the cell. Occasionally, a weak immunoreactivity, present as smaller (< 0.3 pm) dots, was also observed within the lateral dendrite (not illustrated here). This was also located under the plasma membrane. The characteristic

Monoclonal antibody against teieost’s M-ceil

Fig. 2. Discontinuous labelling of the M-cell obtained with mAb 222C2. (A), (B) Immunofluorescence (arrow) within the soma and the large ventral dendrite. The staining is concentrated around the nucleus, at the emergence of the axon hillock, under the axon cap (which is delineated by dotted lines in A), and in the proximal third of the ventral dendrite (delineated by dashed lines in B). (C)-(E) Intr~ll~ar looalixation of the immuno~cti~ty demonstrated with a peroxidase reaction and observed with Nomarski optics. (C) Clusters (arrows) of enzymatic reaction product accumulated in the soma between the nucleus and the axon hillock. The boundaries of the axon cap are indicated by dashed lines. (D) Labelhng (arrows) adjacent to the intracellular side of the limit (arrowheads) of the cell, at the junction between the soma and the ventral dendrite. (E) Reaction product (arrow) within the ventral dendrite. (F) Schematic drawing of the topological distribution of staining (hatched areas). Circled letters refer to the corresponding pictures of this figure. AC, axon cap; AH, axon hillock; Ax, axon; n, nucleus; S, soma; VD, ventral dendrite. Calibration bars: (AHE) = 40 pm.

279

280

A. TRILLERet ul.

Fig. 3. Ultrastructural evidence that the antigenic determinant is localized intracellularly in the M-cell soma. (A), (B) Accumulation of electron-dense material (arrow) at about 3 pm from the surface of the cell which is in contact with several axonal terminals (T). The labelling is associated with ribosomes and reticulum (crossed arrow), and is in the vicinity of Golgi cistemae (triangle). (C), (D) Other examples showing that the immunoprecipitate (arrow) and the Golgi apparatus (triangle) are associated. (A), (C) and (B), (D) Low and high power magnification, respectively. Calibration bars: (At-fD) = 1 pm.

pattern of labelling distribution is summarized in Fig. 2F. The precise intracellular localization of the antigenie determinant was assessed with electron microscopy (Fig. 3). The reaction product was associated with clusters of polyribosomes and endo-

plasmic reticulum (Fig. 3A, B). In some sections (Fig. 3C, D), the immunoprecipitate was close to one side of the Golgi apparatus. It must be stressed here that the presence of electron-dense material at a given location does not prove that the antigen is indeed associated with a given structure. It remains possible

Monoclonal antibody against teleost’s M-cell

Fig. 4. Labelhng of reticular neurons with mAb 222C2 and absence of staining in the vestibular and cerebellar cells. (A), (B) Immunofluorescence showing the discontinuous and randomly distributed patches of fluorescence (arrows) within the soma (s) of reticular neurons and extending slightly in the dendrite (de). Low and high power magnification, respectively. (C)Another example of a reticular cell with a dotted labelling (arrows) following immunoperoxidase treatment. (D) Electron micrograph showing the association of the immunoprecipitate (arrow) with the Golgi body (crossed arrow). (E), (F) Lack of labelling in vestibular (V) neurons and in Purkinje (P) cells, the latter being located between the granular (Gr) and molecular (MO) layer of the cerebellum. (C), (E) and (F) were observed with Nomarski optics. Calibration bars: (A), (C), (E) and (F) = 15 pm; (B) = 7.5 pm; (D) = 0.5 pm.

281

282

A. TRILLER

that the reaction product, the oxidized diaminobenzidine, may have been translocated from the locus where the peroxidase molecules were actually bound. Labelling of other brainstem neurons

Other cells located within the reticular formation were also stained by antibody 222C2. They were of variable size ranging from 10 to 4O/*m in diameter. The large ones (2&40 pm) were either multipolar or had two larger dendrites with orientations roughly parallel to those of the M-cell. The smaller neurons (lG20pm) were multipolar. In all of them, the punctate staining (Fig. 4AC) was scattered within the cytoplasm of the soma and in the initial portion of the dendrites. The reticular neurons may have been sectioned obliquely, and thus an organized pattern could have been missed. We observed no organized pattern of antigenic sites in transversal and sagittal sections; in fact the labelling was rather scattered. This suggests that reticular cells do not exhibit the distinct localization seen in the M-cell. The size of the labelled dots ranged from 0.2 to 0.5 pm. In addition, a faint brownish staining could be observed within the cytoplasm of these cells. This cytosolic background may result from an artifactual diffusion of the reaction product. Electron microscopy confirmed that the immunolabelled cells were always neurons and showed that the immunoprecipitate was associated with clusters of ribosomes and reticulum cisternae, often beside the Golgi apparatus (Fig. 4D). The antibody did not recognize all cells of the reticular formation. The conditions for the determination of the proportion of immunostained neurons were not optimal for several reasons. First, due to their high molecular weight, IgMs have a low penetration in tissue. This leads to an underestimation of the number of neurons containing the antigenic determinant. Second, the unlabelled neuronal elements were identified by a faint brownish staining of the cytoplasm and on the basis of their size (larger than 10pm) and shape. Their number was therefore greatly underestimated. Recognizing these limitations we estimate that the antigen recognized 35 + 11% (n = 4) of reticular neurons located within 300 pm of the ventral dendrite on sections where the dendrite was present. One of them is shown in Fig. 5A and B, in the region corresponding to the anterior pole of the nucleus reticular medialis.30B37Whatever the exact proportion of stained neurons, this indicates that some soma with identical shape and size did not contain antigenic determinant. Figure 5C and D shows this in a section 350 pm caudal to the preceding one, where only two of a group of four reticular neurons are labelled. Although their proportion has not been quantified in other regions, the cells with the antigenic determinant were scattered within the ventrolateral column of the reticular nuclei (Fig. 5) extending rostrocaudally from the anterior peduncle of the cerebellum to the posterior portion of the facial lobe (the latter also being named tuber impar

et al

facialis). This column ‘o,37includes: rostrocaudally (i) the nucleus reticularis superior which extends from the level of the secondary gustatory nucleus to the M-cell, (ii) the nucleus reticularis medialis which runs from the level of the M-cell to the anterior portion of the vagal lobe and (iii) the nucleus reticularis inferior from the caudal limit of the nucleus reticularis medialis to the caudal portion of the facial lobe. Finally, no dotted staining was ever detected at the limit of the optical resolution, with both fluorescent and peroxidase reactions, in other structures. Such was the case for example in the vestibular nuclei (Fig. 4E), in the cerebellum (Fig 4F), in the vagal lobe, in the optic tectum or in any of the other cerebral regions of the goldfish brain. In the rat CNS no labelling could be obtained in the spinal cord, in the brainstem and/or in the cerebellum, but this may be related to species specificity DISCUSSlON

We report here that a monoclonal antibody raised against the M-cell of teleosts also binds to some reticular neurons. Thus these two apparently distinct classes of cells, which have not previously been related on the basis of morphological criteria, may share a common origin and/or possess similar physiological properties. IdentiJication of antibody 222C2

Our procedures for preparing the antigen and screening were governed by (i) the low amount of available material since there are only two M-cells per fish and (ii) our intention to use the mAb for immunocytochemistry. In this study, as in others,‘8,‘9.4t the antigen was fixed with aldehyde and screening with indirect immunofluorescence was also done with fixed tissue. It would have been extremely difficult to prepare a sufficient amount of fresh homogenized M-cell membrane for both immunization and screening. As confirmed here,“.38 a single shot intrasplenic injection using a low amount of antigens was sufficient to elicit an immune response. Indeed, in this case the injected material was directly localized in the target organ which secretes the immunoglobulin. However, one disadvantage was that this procedure produced IgMs, which are not as good immunoreactants as IgGs. The high molecular weight of IgMs diminishes their penetration in tissues, and IgMs may also show non-specific binding. Due to the chemical modification of the antigen by the aldehyde, we were unable to characterize biochemically the involved determinant(s) which was either (i) denatured during western blot attempts, or (ii) in its native form, during immunoprecipitation experiments. Although great care was taken, the M-cell fraction we injected to prepare antibodies almost certainly included afferent boutons, glia and the so-called axon cap, which are adjacent in this region. This is why other clones were also raised. They are not studied

Monoclonal antibody against teleost’s M-cell

283

I Fig. 5. Location of labelled neurons in the brainstem. (A)-(D) Examples from the same brainstem of immunoreactive reticular neurons stained with peroxidase reaction and observed with Nomarski optics. (A), (B) Labelled cell (arrow) in the vicinity of the M-cell ventral dendrite (VD). The midline is indicated by a farge arrow. (C), (D) Group of neurons marked 1, 2, 3 and 4 observed seven sections (350pm) posterior to (A), (B). Cells 1 and 2 present the characteristic dotted labelling, and cells 3 and 4 are not stained. (E) Schematic drawing of a transverse section at the ievel of the M-cell soma, passing through the vestibular complex (vest.c), the descending trigeminal tract (dt) and the nucleus reticularis medialis (nrm). (F) Parasagittal scheme, showing that the observed stained cells are present within a column dotted area of reticular neurons extending from the anterior peduncle of the cerebellum to the rostra1 portion of the facial lobe. Note that this column includes the nuclei reticularis superior (nrs), medialis (mm) and inferior (nri). Calibration bars: (A) and (C) = 50 pm; (B) and (D) = 20 pm. here in detail, but we found that they stained glia or

ubiquitous

constituents

of nerve cells.

Int~~celiular moralization of the antigen Numerous monoclonal

antibodies specific to given

cell types have been obtained against either surface2,‘fi7.‘9*‘7or intracellular4~‘7~2s determinants of neurons. In most cases of intracellular staining, the labelling was evenly distributed within the cytoplasm. In one case a discontinuous punctate staining, similar

284

A. TRILLER EI al.

to that reported in this study, was described at the light microscopic level in the dorsal root ganglion of the rat.23 The distribution of this intracellular reaction resembled that of the Golgi apparatus. In this report, the immunoreactant was a monoclonal antibody, which had initially been raised against a clone of the mouse embryonal carcinoma cell line PCl3. In our work, the frequent association of the staining with the Golgi apparatus suggests that the molecule to which mAb 222C2 was bound might be glycosylated and, possibly, exported to the plasma membrane. However, the antigen was never detected at the cellular surface. This might result either if the molecule is not expressed or if its conformation and/or relationship with the plasma membrane prevents antibody binding. The subcellular distribution pattern of the immunostaining was different in the M-cell and in the reticular neurons. However, the discontinuous labelling in the soma and proximal dendrites of reticular cells is also compatible with a localization of the antibody in the vicinity of the Golgi apparatus of these cells. Signt$cance

of the labelling

It has been suggested ‘J~ that the M-cell is a displaced neuron of the Deiters (or lateral vestibular) nucleus. This is probably not true since the M-cell sends its axon in the opposite medial longitudinal fasciculus (MLF) as do some reticular neurons (Metcalfe, personal communication). This projection contrasts with that of vestibular neurons which project down the spinal cord through the ipsilateral MLF. This hodologic criterion suggests that the M-cell may belong to the group of reticular neurons21’31.36 (see also Ref. 39). Our observation of a phenotypic expression of a common antigen in Mcells and in reticular neurons reinforces this notion. Recently, M-cell “segmental homolgues”26,27 have been described in the brainstem of the teleost

zebrafish. Two other large reticulospinal neurons were related to the M-cell on the basis of ontogenetic and hodologic arguments. A pair of cells ventromedial and caudal to the M-cell has been describedZ4 in the goldfish medulla. This neuron is comparable to the M-cell since its axon also decussates to the MLF and it has two main dendrites, lateral and ventral bifurcated ones. However, since we observed many more labelled cells the localization of mAb 222C2 was not associated with segmental homologues of the M-cell. However, since neurons were not double labelled to identify the segmental homologues, specific patterns of organization may have been missed. The longitudinal distribution of the stained reticular neurons favours the classical notion that the basic organization of the brainstem consists of a number of longitudinally arranged zones, which correspond to functional columns. 3o Since only a fraction of neurons in one column was stained, additional criteria may be necessary to refine this concept. One may be related to the function of the M-cell and reticular neurons, in triggering an escape or startle reflex.6s’2However, the reticulospinal neurons which are responsible for this vital behaviour when the M-cell is destroyed6 have not yet been clearly identified. Even so, the regions32 of the hindbrain where reticulospinal neurons were found are the three reticular nuclei where immunolabelled cells were observed. Although we have not demonstrated that the stained reticular cells project to the spinal cord, their distribution and size are compatible with this notion.j2 In summary, the mAb recognizes a molecule specific to the M-cell and a subgroup of reticular neurons. Although the function and nature of this molecule remain unclear, its restricted expression suggests that mAb 222C2 will be a useful marker for further

developmental

studies.

Acknowledgements-We thank R. Miles for his helpful criticisms and help with the English editing.

REFERENCES

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25 September

1990)

A monoclonal antibody raised against the Mauthner cell also recognizes some reticular neurons.

A monoclonal antibody was raised against dissected Mauthner cells of goldfish, Carassius auratus. The immunoglobulin (mAb 222C2) recognized in this ne...
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