A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish

Brain Research 901 (2001) 117–127 www.elsevier.com / locate / bres

Research report

A novel monoclonal antibody recognizes a previously unknown subdivision of the habenulo-interpeduncular system in zebrafish Kaori Tomizawa, Hiromi Katayama, Hiroshi Nakayasu* Department of Biology, Faculty of Science, Okayama University, Okayama 700 -8530, Japan Accepted 13 February 2001

Abstract The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We prepared a monoclonal antibody (6G11) which very specifically recognizes only a part of this system. 6G11 is a monoclonal antibody prepared from a neuronal membrane protein in adult zebrafish brain. In western blot analysis of the adult zebrafish brain, the antibody recognized a 95 kDa protein, and the class of the antibody was determined to be IgM. The 6G11 antigen was not detected in zebrafish muscle, intestine, testis or ovary. A group of neurons stained by the 6G11 antibody was located in the caudomedial part of the zebrafish habenula. The 6G11-immunopositive neurons extended their axons into the fasciculus retroflexus (FR). One group of immunopositive neurons projected toward the interpeduncular nucleus (IPN), especially to the intermediate and the central subnucleus (type 1 neuron). The other group projected to the ventral midline at the level of the raphe nucleus; these axons passed ipsilaterally beside the IPN and converged in the ventral midline under the raphe nucleus (type 2 neuron). Both type 1 and type 2 fibers are relatively minor components of the FR. Little has previously been known about this topological pattern in any species. The 6G11 monoclonal antibody could be a useful tool for expanding knowledge of the habenulo-interpeduncular system.  2001 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Staining, tracing, and imaging techniques Keywords: Fasciculus retroflexus; Habenula; Interpeduncular nucleus; Fish; Immunohistochemistry

1. Introduction The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of practically all vertebrates, from lamprey to human. The habenula in zebrafish is located dorsally in the most caudal diencephalon, and the interpeduncular nucleus (IPN) is located in the ventrocaudal mesencephalon. The fasciculus retroflexus (FR) is the main paired fasciculus connecting the habenula and the IPN. Previously, these fascicles have been studied in detail using Golgi staining or neuronal tracers [15,16,33,37,38]. The FR originates from the habenula and projects mainly to the IPN, although it has other minor projections to several regions of the mesen-

*Corresponding author. Tel.: 181-86-251-8630; fax: 181-86-2517876. E-mail address: [email protected] (H. Nakayasu).

cephalon, such as the raphe nucleus and the central gray [6,37,38]. It has been well demonstrated that the IPN and the habenula of mammals can be divided into several subnuclei. This has been demonstrated by electron microscopy [1,11,16] and by the distribution patterns of neurotransmitters, particularly substance P, choline acetyltransferase, calcium-binding proteins, and other neurotransmitter specific enzymes [2,5,12,17]. It has also been reported that the fish IPN can be divided into several areas, which contain many kinds of neurotransmitters and functional proteins [3,20,24,26,30–32,34]. Although it has been known that there is a tight connection between the habenula and the IPN, the topological relationship between their subnuclei is still controversial. It has been difficult to analyze by conventional methods, since the distribution of neurotransmitters and enzymes overlaps at some subnuclear levels, and it is difficult to limit the placement of tracers to a single

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02313-7

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subnucleus or group of neurons. Yet a detailed topological study would be indispensable for identifying groups of fibers or neurons. In the present paper, we report a topological study of the habenulo-interpeduncular system of zebrafish using a new method. We focus on an antigen which exists in a group of fibers in the FR, and which is recognized by a monoclonal antibody, 6G11. This antibody reveals the existence of a group of neurons that project from the caudomedial part of the habenula to the midline regions of the IPN and to the ventral midline under the raphe nucleus.

2. Materials and methods

2.1. Preparation of the neuronal membrane fraction from zebrafish brain

serum (Sigma) according to the Gibco manual. They were selected in a HAT medium (OPTI medium containing 4% fetal calf serum, 5 mM hypoxanthine, 0.02 mM aminopterin and 0.8 mM thymidine) for more than 10 days to remove surviving myeloma cells from the hybridoma cells, and cloned by a limited dilution method in 96-well plates (Falcon). Usually, conditioned medium in which the myeloma cells had been grown for a week was mixed with the same volume of fresh OPTI medium, because otherwise the hybridomas grew very slowly. The detection of a positive hybridoma clone was performed by a micro-scale immunoblot system using the zebrafish synaptic membrane fraction (1340-mm blot). On an average, we could isolate 10–20 positive clones from one immunized mouse. These hybridomas were frozen in liquid nitrogen or allowed to grow for the preparation of ascitic fluid.

2.3. Preparation of ascites Neuronal membranes from zebrafish brain were prepared according to the method of Nakayasu et al. [22] for the preparation of a bovine brain neuronal membrane fraction. The zebrafish were maintained at 28.58C under a common light cycle (14 h light and 10 h dark) [35]. Adult zebrafishes (about 5 or 6 months after fertilization) were anesthetized in cold water. The skulls were removed from the heads in an ice box, and the isolated brains were homogenized in a microtube with a plastic stick at 08C in 10 mM Tris–HCl (pH 7.4), containing 0.25 M sucrose, 1 mM ethylenediamine tetraacetic acid (EDTA), and the protease inhibitors leupeptin and antipain (1 mg / ml). The homogenate was centrifuged at 28003g for 2 min at 4 o C to remove the nuclear fraction and any undestroyed cells. The supernatant was collected, and the precipitate was re-homogenized in the same buffer and re-centrifuged. The supernatants containing the synaptic membranes centrifuged at 10 0003g for 10 min at 48C. The precipitate (crude synaptic membrane fraction) was washed twice with the same buffer, and was used for immunization of animals for antibody production and for immunoblots.

2.2. Production of hybridomas The neuronal membrane fraction from 100 fishes was suspended in 5 ml of PBS (phosphate-buffered saline; 10 mM sodium phosphate buffer, pH 7.4, containing 0.14 M NaCl) and emulsified with the same volume of Freund’s complete adjuvant [14]. The water-in-oil emulsion was injected into 20 male BALB / c mice (more than 5 weeks old) at day 1. The same amount of the antigen was emulsified with an incomplete adjuvant and injected into the same mice at day 15, day 29, and day 44. In some cases the final immunization was omitted. Three days after the final injection, spleen cells from the mice were prepared and fused with P3U1 myeloma cells using polyethyleneglycol. The hybridoma cells were grown in an OPTI medium (Gibco) supplemented with 2–4% fetal calf

About 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane) was injected into male BALB / c mice at least 5 weeks before the injection of the hybridoma cells. Positive hybridoma cells (about 10 7 cells) were injected intraperitoneally. After 1–2 weeks, the ascites were collected and centrifuged at 10 0003g for 10 min to remove the pristane and cellular structures. The ascites were stored at 2808C until use, without any more purification of the antibody. For the working solution, the ascites were mixed with an equal volume of glycerol and stored at 2308C.

2.4. Immunoblotting SDS–polyacrylamide gel electrophoresis (SDS–PAGE; 60340 mm gels) and western blotting were carried out by the methods of Nakayasu et al. [23]. Brain, muscle, intestine and ovary were prepared from one zebrafish and testis from two fish in order to run on ten gels. Following electrophoresis, a nitrocellulose filter of the same size as the gel was overlaid on the gel, and the protein bands were transferred onto the filter at 0.5 A for 30 min. The membrane filter was then blocked by shaking in a solution of 10 mM Tris–HCl (pH 7.4), containing 0.1% Tween 20 (polyoxyethylene sorbitan monolaurate) and 0.14 M NaCl (TBS-Tween) in a 10-cm dish. The monoclonal antibody (ascites), designated 6G11, was diluted with the same buffer at 1:1000, and incubated with the filter at 48C overnight. The filter was washed four times with the same buffer, then incubated with alkaline phosphatase-conjugated secondary antibody (1:10 000 dilution in TBSTween buffer). Two secondary antibodies were used, an alkaline phosphatase-conjugated goat anti-mouse IgM, and an alkaline phosphatase-conjugated goat anti-mouse IgG. After washing with the same buffer, the antigenic band was visualized using BCIP and NBT as alkaline phosphatase substrates [25]. For screening the monoclonal antibody, a small blot (1340 mm) was incubated in a 24-well

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plate with 100 ml of the culture medium from the hybridoma cells.

2.5. Protein staining by colloidal gold Colloidal gold particles (25 nm) were prepared by boiling 0.01% HAuCl 4 containing 0.04% sodium citrate? 2H 2 O for 15 min. After cooling, polyethyleneglycol (0.03%) and Tween 20 (0.05%) were added to stabilize the gold particles [22]. The pH of the solution was then adjusted to 3.0. The colloidal gold suspension was kept at room temperature. The Western blot was blocked, washed twice with distilled water, and incubated with 10 ml of the colloidal gold solution overnight.

2.6. Immunohistochemistry The procedure for sections has been previously described [28,29]. Adult fishes were anesthetized in cold

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water. The brain was carefully dissected out of the skull and fixed with 4% freshly depolymerized paraformaldehyde containing 4% sucrose at 48C overnight and washed well in 0.1 M phosphate buffer containing 4% sucrose. The fixed brain was embedded in O.C.T compound (Tissue Tek), frozen on a cryostat, and cut into sections about 18 mm in thickness. The sections were put onto a glass slide coated with 1.5% gelatin and 0.15% CrK(SO 4 ) 2 ?2H 2 O, and air-dried at 558C. The sections were rinsed with PBS, treated with 0.3% H 2 O 2 dissolved in absolute methanol for 15 min in order to inhibit endogenous peroxidase activity, and washed well with PBS. The sections were treated with PBS / Tween / DMSO solution (0.1 M sodium phosphate, pH 7.3, 0.2% Tween 20 and 1% DMSO (dimethylsulfoxide) containing 0.1% Triton X-100 for 5 min and washed well with the same buffer without Triton X-100. Then non-specific protein binding sites were blocked with PBS / Tween / DMSO, containing 2% Tween 20, for 30 min.

Fig. 1. DiI tracing of the habenulo-interpeduncular system of zebrafish. (Top left) Illustration of a sagittal section after the injection of DiI into the habenula. (A) A sagittal section containing the IPN shows the DiI-labeled FR and IPN. (B) A coronal section in the IPN after the DiI tracing. (C) Illustration of the subnuclear organization of the IPN (dorsal, central, intermediate, and lateral nuclei) in a coronal section. Bars5200 mm.

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The procedure for immunostaining was adapted from the methods of the Microwave Cookbook for Microscopists [19]. The sections were soaked with the ascites diluted 1:10 in PBS / Tween / DMSO solution containing 0.1% Triton X-100, and then microwaved for 14 min, at 12– 20% power, which provides a temperature up to 408C (EMS-820, Electron Microscopy Sciences). After being washed with the same buffer three times, the sections were then microwaved with peroxidase-conjugated goat antimouse IgM antibody (1:80) in PBS / Tween / DMSO solution, and washed well with PBS / Tween / DMSO and with 0.1 M sodium phosphate buffer (pH 7.3) for 10 min. The slices were pre-soaked with diaminobenzidine (DAB)heavy metal staining solution: (0.04% DAB, 0.1 M Tris– HCl (pH 7.4), 0.5% DMSO and 0.45% Ni(NH 4 ) 2 (SO 4 ) 2 for 5 min. To this solution, H 2 O 2 was added to a final concentration of 0.003%. The enzymatic reaction was stopped by washing with 0.1 M sodium phosphate buffer (pH 7.3), and then with distilled water, and the slices were dehydrated in series of alcohols, cleared in xylene, and mounted with Canada balsam. For immunofluorescent microscopy, the sections were incubated with FITC-conjugated goat anti-mouse IgM antibody (1:100) in PBS / Tween / DMSO solution for 2 h at room temperature after incubation with the 6G11 monoclonal antibody. After being washed with the same buffer, the sections were mounted with MOVIOL (Calbiochem), then viewed with a Zeiss Axiophot using an appropriate filter set. Photographs were taken by a CCD camera (Photometrics KAF-1400) using V for Windows (Photometrics), arranged in Photoshop (Adobe), and printed by a Pictrography 3000 (Fujifilm).

2.7. DiI tracing Zebrafish were anesthetized in cold water and perfused transcardially with PBS. The brain were carefully dissected out of the skull and fixed with 4% freshly depolymerized paraformaldehyde containing 4% sucrose and 0.1% EDTA at 48C overnight. A glass micropipette, containing a 5% solution of DiI (1,19-dioctadecyl-3,3,39,39-tetramethylindocarbocyanine perchlorate, LAMBDA) in N,N-dimethylformamide (DMF) and driven by a micromanipulator (Narishige, Japan), was used to deliver a minute amount of tracer into the both habenular nuclei. The brains were then left in 0.1 M phosphate buffer containing 0.1% EDTA for 3–4 weeks at 288C.

3. Results

3.1. The cytoarchitecture of the habenulointerpeduncular system of zebrafish In zebrafish, the habenula is located in the epithalamus and consists of dorsal and ventral parts. The habenulae of

Fig. 2. 6G11 antibody staining on Western blots of membrane fractions of adult zebrafish brain and the other tissues. (A) The 6G11 antibody only recognizes a 95 kDa band in the lane containing whole brain proteins when the incubation was carried out with anti-mouse IgM secondary antibody (lane 3). There is no detectable band when incubation was performed without the primary monoclonal antibody together with mouse IgM and IgG secondary antibody (lane 4), or with the primary monoclonal antibody with mouse IgG secondary antibody (lane 5). Lane 1, molecular weight markers. The blots are stained with colloidal gold for detection of whole proteins (lane 2). (B) Western blots of membrane fractions of adult zebrafish muscle, intestine, testis and ovary. The 6G11 specific band is not detected in these tissues. M, molecular weight markers; G, whole proteins stained with colloidal gold.

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zebrafish are symmetrically shaped. After DiI injection into the habenula, the FRs was heavily stained. The label extended caudoventrally from the habenula towards the IPN (Fig. 1A). The axons stained by DiI terminated on the IPN in zebrafish brain. The stained IPN could be divided into four subnuclei: two midline subnuclei (dorsal, central), and two bilateral subnuclei: (intermediate, lateral) as shown in Fig. 1B and C.

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between the medial habenula and the IPN. The other regions were immunonegative. In order to confirm that the labeled fibers are FR, we double-stained the fibers with DiI injected into the habenula and fluorescence immunostaining with 6G11 (Fig. 4A–C). From this it was determined that the 6G11 antibody recognizes only a subset of the fibers in the FR (Fig. 4B).

3.4. The projection pattern of 6 G11 -positive fibers 3.2. Characterization of the 6 G11 monoclonal antibody Western blots were made with tissue from brain, muscle, intestine, testis and ovary, and hybridized with the 6G11 antibody (Fig. 2A). When visualized with an anti-mouse IgM secondary antibody, 6G11 recognized a band of 95 kDa (lane 3). In some cases a 30 or 21 kDa band was also seen, which we suspect to be a degradative product. A control run in which the primary antibody was omitted (lane 4) showed no detectable band. When alkaline phosphatase-conjugated anti-mouse IgG was used as the secondary antibody, there was also no detectable band on the blot (lane 5). This indicates that 6G11 is an IgM class of antibody, recognizing a protein of about 95 kDa. The existence of the 6G11 antigen was further analyzed with other organs (muscle, intestine, testis, and ovary) in Western blot (Fig. 2B). Tested organs except for brain were invisible to bands recognized by the 6G11 antibody. Consequently, it was suggested that the 6G11 antigen specially expressed in the nervous system of zebrafish.

3.3. The 6 G11 monoclonal antibody identifies a fiber group of the FR Fig. 3A shows a mid-sagittal section of adult zebrafish brain. Our 6G11 monoclonal antibody labels a fascicle running rostrodorsal-to-caudoventral in the mesencephalon

The 6G11-positive fibers arose from the posteromedial part of the medial habenula, shown in coronal and horizontal sections in Fig. 5A and B. The origin of the fibers occurred symmetrically across in a small cluster in the medial habenula. The cell bodies of origin were small and round in shape, and were gathered tightly in a small cluster (Fig. 5C). The 6G11-positive fibers originating from the cluster in the habenula formed a small bundle as they converged caudal to the habenula. Proceeding posteriorly, the 6G11-positive fibers passed ventral to the commissura posterior and medial to the oculomotor nerve roots and then ran orthogonally through the ansulate commissure, which was located immediately rostral to the IPN (data not shown). The descending 6G11-positive fascicle then separated into several groups of bundles in the IPN (Fig. 6), which could be categorized into two types based on their projections.

3.4.1. Type 1 projection The first group of axons ran along the dorsoventral axis in the intermediate and the central subnucleus of the IPN and terminated in the ventral surface (Fig. 6A, and see Fig. 8). A sagittal section of the IPN shows the fibers approached the IPN dorsally and curved ventrally (Fig. 6B, arrowhead) and caudally (Fig. 6B, arrow). These axons were thick and did not branch.

Fig. 3. The staining pattern of the 6G11 antibody in zebrafish brain. The 6G11 antibody very specifically stains the FR between the habenula (Hab) and the IPN, but does not stain other regions of the brain. Ce, cerebellum; Mo, medulla oblongata; Ra, rapfe nucleus; Tel, telencephalon; Bar5200 mm.

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Fig. 4. Double staining (DiI tracing and 6G11 immunostaining) in a sagittal section. (A) A sagittal section containing the FR was stained by the 6G11 antibody. (B) The images in A and C, superimposed. The 6G11-positive fibers in A (green) are a subset of the FR (C, red). (C) DiI labeling of the FR after an injection into the habenula (same section as in A). Bar550 mm.

Fig. 5. Localization of 6G11-positive neurons in the habenula. (Top left) Schematic representation of the coronal section regions shown in A and B. Hab, FR and IPN are indicated by gray. (A) A horizontal section containing the habenula. 6G11 labeling is found in the posterior habenula. (B) A coronal section containing the habenula. 6G11 labeling is located in the medial part of habenula. (C) Localization of 6G11-positive neurons. The 6G11-positive neurons form a tightly packed cluster in the habenula. Chab, commissura habenularum; TeO, tectum opticum. Bars5100 mm in A and B; 25 mm in C.

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Fig. 6. Termination of 6G11-positive fibers in the IPN. (A) A coronal section in the IPN. The 6G11-positive fibers run along the dorsoventral axis at the midline of the IPN and also on the side of IPN (white arrows). (B) A sagittal section in the IPN. Some 6G11-positive fibers arrive at the IPN and then curve ventrally (arrowhead) and caudally (arrow). (C) A coronal section in the rostral-most part of raphe nucleus (caudal-most end of the IPN). The fluorescent 6G11-positive fibers terminate thickly in ventrorostral-most part of raphe nucleus. (D) A horizontal section at the bottom of IPN. The 6G11-positive fibers pass around the IPN. Ra, raphe nucleus. Bars5200 mm in A and C; 50 mm in B and D.

3.4.2. Type 2 projection Other fibers, indicated by the arrow in Fig. 6A, extended caudally and terminated on the ventrocaudal-most end of the IPN under the raphe nucleus (Fig. 6C). As shown in Fig. 6D, the left and right 6G11-positive fibers finally converged at the midline at the ventral-most part of the IPN and the raphe nucleus. The termination of the fibers was too dense to see individual axons. A high proportion of 6G11-positive fibers belongs to type 2. These 6G11positive fibers did not project to other regions. To observe the course of the type 2 axons in detail, a section was cut parallel to the direction of the fibers (Fig. 7). The projection ipsilaterally passed in side of the IPN and innervated the ventrocaudal-most end of IPN. The portion of the axons near the habenula was not visible in these sections. Immunostaining with the 6G11 antibody demonstrated

the presence of two types of immunopositive fibers in the FR. While both arise from the medial-caudal part of the habenula, the type 1 projection innervates the midline part of the IPN, including the intermediate and the central subnucleus, and the type 2 axons innervate the ventrocaudal-most end of the IPN. These two projections represent only a small part of the FR, however, and did not reach the dorsal subnuclei of the IPN, which receive a projection from the habenula (Fig. 1A).

4. Discussion In this paper, we have analyzed the structure and topology of the zebrafish habenulo-IPN system. We have described a new brain-specific antibody, 6G11, and have shown that its staining pattern defines a previously un-

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and goldfish, the 6G11-fibers were discernible (data not shown). The staining pattern is similar to that of zebrafish. While, the 6G11-fibers were not discernible in mouse brain (data not shown).

4.1. Subdivision of the zebrafish habenula and interpeduncular nucleus

Fig. 7. Running pattern of 6G11-positive fibers. The sections were cut as indicated in the illustration (top). The 6G11-positive fibers originate from the habenula (Hab), then pass ipsilaterally around the IPN to innervate the ventrocaudal region of the IPN. Bar5100 mm.

identified subdivision of the FR. We have also used tract tracing to clarify the subdivisions of the habenula and the IPN. The habenulo-interpeduncular system is an evolutionarily conserved structure found in the brain of almost all vertebrates. We performed the 6G11-immunostaining of other vertebrate group. In Medaka fish (Oryzias latipes)

It has been reported that the habenula in some species of non-mammal is asymmetrical as shown by tracing, morphological and neurochemical approaches [7–10,37,38]. In fish, right nucleus of larval lamprey is markedly larger [37] and that of trout is also somewhat larger [38]. We could not detect such distinct asymmetry in the zebrafish habenula. Wullimann et al. [36] did not describe the asymmetry of the habenula, likewise. The 6G11-positive staining pattern was also symmetrical. Andres et al. [1], using mainly Nissl-stained sections, detected the presence of five distinct subnuclei in the medial habenular complex of rat, and ten subnuclei in the lateral habenular complex, but little has been known about the subnuclear organization of the habenular complex in fish. As far as the zebrafish habenula is concerned, there is only one report describing that the habenula is divided into two nuclei in dorsoventral axis: the medial and the lateral [36]. Since the report is thought that the two nuclei are divided by the cytoarchitecture using Bodian and Nissl staining, it is unclear whether the two nuclei are completely identical to the two of mammal habenula. According to the their definition of nuclei, we have shown that 6G11positive cells were located only in the caudal part of the medial nucleus. Moreover, in goldfish, the distribution of substance P-like immunoreactivity showed the highest levels in the peripheral part of the ventral area [31]. In trout, only ventral portion contained a significant number of somatostatinergic fibers [3]. These reports suggest the existence of subnuclear organization of fish habenula. The subdivision of the IPN is also well known in mammals based on ultrastructure and neurochemical anatomy [1,2,5,11,12,16,17]. In general, mammalian IPN are subdivided into three midline subnuclei (rostral, central, and apical) and three bilateral subnuclei (intermediate, lateral and dorsal lateral). Again, little has been known about the subnuclear organization of the fish IPN, although ˜ and Anadon ´ [38] found by use of tract tracing that Yanez the rainbow trout IPN consists of dorsal and ventral areas, separated by glia and a band of fibers. Using Nissl staining, it was shown the feature that large cells gathered in midline, however the existence of distinct subnuclei has not been discernible. We found that the zebrafish interpeduncular nucleus consists of four subnuclei (dorsal, central, intermediate and lateral) by the labeling pattern of DiI, which diffused from the habenula to the IPN (Fig. 1B and C). Thus, it seems that the subnuclear organization of zebrafish IPN is similar to that of mammals and the

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organization system is constructed from the date of early vertebrate.

4.2. The 6 G11 antibody subdivides the FR in zebrafish The FR is composed of several heterologous types of fibers having different origins and terminals. In general, these fibers pass through the FR and reach almost all of the subnuclei of the IPN, the raphe nucleus, the ventral tegmental nuclei, and the central gray [6,37,38]. Although injection of DiI in the habenula led to the labeling of all subnuclei of the IPN (Fig. 1B), 6G11-positive fibers extended only to the central and intermediate subnuclei (Fig. 6A), and not to the dorsal or lateral subnuclei. Double-labeling with DiI tracing and immunofluorescence showed the 6G11-positive fibers to be a unit of the FR arising from the union of several smaller bundles (Fig. 4A–C). Thus, our results indicate that the novel 6G11 antigen, present on neuronal membranes, identifies a previously unknown subdivision of the FR of zebrafish.

4.3. The projection pattern of 6 G11 -positive fibers Unilateral lesion and tracing studies of the habenula have revealed that the FR innervates its target regions either ipsilaterally or bilaterally [4,6,13,16,37]. It has been reported that the projection originating from the ventral part of the rat medial habenula forms both ipsilateral and ˜ ´ [37] bilateral connections [4,16]. Yanez and Anadon observed that the FR of larval lampreys bifurcates to form two fascicles, which extend caudally in the mesencephalon and rostral medulla oblongata. We found that some caudal

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6G11-positive fibers projected ipsilaterally around the IPN and a smaller projection entered the IPN. Our findings are therefore in good agreement with those from larval lamprey. However, it is unclear whether the lamprey projection contained or is completely identical to the caudal 6G11-positive projection, because the location of the neurons in lamprey, which extend to the caudal mesencephalon is unknown. In the IPN, many FR axons do not travel a straight path and traverse from side to side [15,18]. It has also been reported that a minority of FR fibers terminate in the lateral regions of the caudal half of the IPN, where they branch [15]. In contrast, the 6G11-positive fibers ran along dorsoventral axis, and thus were perpendicular to the traversing core axons in the intermediate and the central subnuclei of the IPN. Moreover, we were unable to observe branching and varicosities on these axons. Since the FR does not have scattered neurons along its caudal course, it is probable that the fibers contact long, ventrally directed dendrites of the IPN and the region under the raphe nucleus. We propose that the major FR fibers identified by the 6G11 antibody are a new subdivision of FR.

4.4. The topological organization of the habenulointerpeduncular system in view of 6 G11 staining pattern The 6G11-positive neurons arose from the caudomedial part of the habenula and terminated in two regions: the midline part of the IPN (the intermediate and the central subnucleus) and the ventral midline surface under the raphe nucleus (Figs. 6 and 8). It is well known that the

Fig. 8. Illustration of the two immunoreactive habenulo-IPN pathways in zebrafish.

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major projection to the IPN originates from the medial habenular nucleus [4–6,16,21,27]. The terminals from the ventromedial part of the medial habenula were preferentially observed in the intermediate and the central subnuclei of the IPN [4,16,27]. One projection of the 6G11positive neurons (from caudomedial part of the habenula to midline part of the IPN) was in good agreement with these results. It has also been reported that the FR originating from the habenula projects beyond the IPN in some species [6,18,37,38]. In mammal, projections arose from the lateral habenular nucleus especially extend to the periaqueductal gray, substantia nigra, and ventral tegmental area. The 6G11-positive fibers through the FR did not project to the regions except the IPN and under the raphe nucleus. The results indicated that the projection of the 6G11-positive cells located in the medial part of the habenula was in good agreement with these reports. In other fish species, the FR of trout extended to beyond the IPN, the fiber terminated in all raphe nucleus and the central gray [38]. The FR of larval lampreys converges at the midline beyond the level of the trigeminal motor nucleus [37]. The 6G11-positive two fascicles seemed to gather in the region under the raphe nucleus, and did not extend to the medulla. Thus, we have shown that the habenulo-IPN system is arranged in a topological manner by the use of a novel monoclonal antibody, 6G11. This antibody recognizes a 95 kDa antigen that is expressed specifically in the brain, and which delineates a previously unknown subdivision of the FR.

[6]

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Acknowledgements [17]

The excellent technical support provided by Dr A. Niida and Dr Y. Okada is gratefully acknowledged. We also thank all members in this lab for their helpful advice and assistance.

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