Structure and chemical organization of the accessory olfactory bulb in the goat.код для вставкиСкачать
THE ANATOMICAL RECORD 290:301–310 (2007) Structure and Chemical Organization of the Accessory Olfactory Bulb in the Goat KAZUTAKA MOGI,1,2 KATSUYASU SAKURAI,1,3 TORU ICHIMARU,1,2 SATOSHI OHKURA,1 YUJI MORI,2 AND HIROAKI OKAMURA1* 1 Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba, Japan 2 Laboratory of Veterinary Ethology, The University of Tokyo, Tokyo, Japan 3 Laboratory of Animal Physiology, Tohoku University, Sendai, Japan ABSTRACT The structure and chemical composition of the accessory olfactory bulb (AOB) were examined in male and female goats. Sections were subjected to either Nissl staining, Klüver-Barrera staining, lectin histochemistry, or immunohistochemistry for nitric oxide synthase (NOS), neuropeptide Y (NPY), tyrosine hydroxylase (TH), dopamine b-hydroxylase (DBH), and glutamic acid decarboxylase (GAD). The goat AOB was divided into four layers: the vomeronasal nerve layer (VNL), glomerular layer (GL), mitral/tufted (M/T) cell layer (MTL), and granule cell layer (GRL). Quantitative and morphometric analyses indicated that a single AOB contained 5,000–8,000 putative M/T cells with no sex differences, whereas the AOB was slightly larger in males. Of the 21 lectins examined, 7 speciﬁcally bound to the VNL and GL, and 1 bound not only to the VNL, but also to the MTL and GRL. In either of these cases, no heterogeneity of lectin staining was observed in the rostrocaudal direction. NOS-, TH-, DBH-, and GAD-immunoreactivity (ir) were observed in the MTL and GRL, whereas NPY-ir was present only in the GRL. In the GL, periglomerular cells with GAD-ir were found in abundance, and a subset of periglomerular cells containing TH-ir was also found. Doublelabeling immunohistochemistry revealed that virtually all periglomerular cells containing TH-ir were colocalized with GAD-ir. Anat Rec, 290:301– 310, 2007. Ó 2007 Wiley-Liss, Inc. Key words: accessory olfactory bulb; goat; immunohistochemistry; lectin histochemistry Pheromones are chemical signals mainly involved in social communication between conspeciﬁcs (Brennan, 2001; Halpern and Martinez-Marcos, 2003). It is generally thought that pheromone signals are received by the vomeronasal organ (VNO) and then initially processed at the accessory olfactory bulb (AOB). Although most mammalian species possess an AOB, its location, shape, size, and structure differ substantially between species (Meisami and Bhatnagar, 1998). The AOB is composed of various types of neurons such as the mitral/tufted (M/T) cells, the periglomerular and granule cells, and a small number of short axon cells. The chemical composition of the AOB has been examined using several histochemical methods. Lectin histochemistry was used to demonstrate marked interspecies variation in lectin binding patterns in the rat (Ichikawa Ó 2007 WILEY-LISS, INC. et al., 1992, 1994; Takami et al., 1992b; Sugai et al., 2000), mouse (Salazar et al., 2001), hamster (Taniguchi et al., 1993), opossum (Shapiro et al., 1995), pig (Salazar et al., 2000), and sheep (Salazar et al., 2000, 2003). Furthermore, it has been found that lectin binding is segregated into several subdivisions of rostrocaudal extent in *Correspondence to: Hiroaki Okamura, Laboratory of Neurobiology, National Institute of Agrobiological Sciences, Tsukuba 81-29-305-8602, Japan. Fax: 81-29-838-8610. E-mail: email@example.com Received 21 July 2006; Accepted 28 November 2006 DOI 10.1002/ar.20505 Published online 15 February 2007 in Wiley InterScience (www. interscience.wiley.com). 302 MOGI ET AL. rodents (Takami et al., 1992b; Taniguchi et al., 1993; Ichikawa et al., 1994; Sugai et al., 2000; Salazar et al., 2001) and opossum (Shapiro et al., 1995), but not in herbivores (Salazar et al., 2000, 2003). Using immunohistochemistry, various neurotransmitters have been found distributed in the AOB. For example, a large population of the periglomerular cells contains glutamic acid decarboxylase (GAD), an enzyme that converts glutamic acid to gamma-aminobutyric acid (GABA), in the rat (Mugnaini et al., 1984; Takami et al., 1992a; Quaglino et al., 1999) and dog (Nakajima et al., 1998). The granule cells contain GAD or nitric oxide synthase (NOS) in the rat (Quaglino et al., 1999), mouse (Kishimoto et al., 1993), hamster (Davis, 1991), and dog (Nakajima et al., 1998). In the short axon cells, NOS or neuropeptide Y (NPY) has been detected in the rat (Matsutani et al., 1988), guinea pig (Matsutani et al., 1989), and hamster (Nakajima et al., 1996). In goats, both releaser (Sasada et al., 1983) and primer (Chemineau, 1987) pheromone actions have been described for male–female interactions. For example, in the ‘‘male effect,’’ pheromones produced by males induce out-of-season ovulation in anestrous females (Chemineau, 1987). Further, a putative pheromone receptor gene has been identiﬁed in the VNO (Wakabayashi et al., 2002); however, only a small number of studies have described the morphology of the AOB, showing the laminar structure and distribution of G-proteins in adult (Takigami et al., 2000) and developing (Takigami et al., 2004a) goats. Therefore, we conducted morphometric analyses and histochemical examinations to clarify further the structure and chemical composition of the goat AOB. Comparative analysis of AOB morphology could contribute to our understandings of the sophistication of pheromone communication in this species. MATERIALS AND METHODS Animals We used eight adult Shiba goats (Capra hircus) of each sex that were maintained in the National Institute of Livestock and Grassland Science (Tsukuba, Japan). Male goats were orchidectomized at 6 months of age or older. Female goats were ovariectomized as adults. Animals were not used for experimentation for at least 4 months following castration. Goats were kept in individual pens and fed daily with maintenance amounts of dry hay and formula feed. Water was available ad libitum. At the time of sacriﬁce, the goats were over 2 years of age, and the body weights of males and females ranged from 25 to 42 kg and 25 to 28 kg, respectively. All experimental procedures were approved by the Committee on the Care and Use of Experimental Animals of the National Institute of Agrobiological Sciences, Japan. Tissue Preparations Goats were killed with an overdose of sodium pentobarbital (25 mg/kg body weight). The head was perfused bilaterally through the carotid arteries with 4 L of 10 mM phosphate-buffered saline (PBS) containing 3,000 U heparin/L and 0.7% sodium nitrite, followed by 5 L of ﬁxative consisting of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The olfactory bulb was removed from each goat brain and was immersed in the same ﬁxative without glutaraldehyde overnight at 48C, then in 20% sucrose in 0.1 M phosphate buffer at 48C until it sank. The olfactory bulbs of ﬁve males and ﬁve females were cut sagittally at 50 mm on a freezing microtome. Serial sections were collected in cryoprotectant solution (Watson et al., 1986) and kept at 208C. The olfactory bulbs of the three remaining goats of each sex were cut sagittally at 10 mm on a cryostat. Every eighth section was collected and mounted on gelatin-coated slides. After drying, slides were kept at 208C. Nissl and Klüver-Barrera Staining Every sixth section of all free-ﬂoating sections was washed extensively with PBS containing 0.5% Triton X100 (PBST) and processed for Nissl staining with 0.5% cresyl violet. After observation of the Nissl-stained section, additional sections around the medial and lateral edges of the AOB were similarly processed for Nissl staining to deﬁne the boundaries of the AOB. Sections were mounted on gelatin-coated slides, dehydrated, cleared, and coverslipped. The remaining free-ﬂoating sections were used for lectin histochemistry or immunohistochemistry. All cryostat sections were processed for KlüverBarrera staining. After rinsing with PBST, sections were stained with 1% luxsol fast blue and 1% cresyl violet. Lectin Histochemistry Twenty-one biotinylated lectins (lectin Kit I, II, and III; Vector Laboratory, Burlingame, CA) were used. The sugar moieties speciﬁcally bound with these lectins are listed elsewhere (Ichikawa et al., 1992; Taniguchi et al., 1993). After washing with PBST, free-ﬂoating sections were treated with 3% H2O2 in methanol for 15 min and subsequently incubated with one of the biotinylated lectins (10–30 mg/ml in PBST) for 1 hr. and the avidinbiotin complex (20 ml each/ml PBST; Elite Kit; Vector Laboratory) at room temperature (RT) with gentle shaking. Each step was followed by three 15-min washes with PBST. After the last wash, sections were immersed in 50 mM Tris-HCl (pH 7.6), then reacted with chromogen solution consisting of 3,30 -diaminobenzidine (DAB, 0.8 mg/ml), nickel sulfate (0.4 mg/ml), cobalt chloride (1.5 mg/ml), and 0.01% H2O2 in 50 mM Tris-HCl (pH 7.6) for 8 min. In some sections, lectins were visualized using the chromogen solution without nickel and cobalt ions, and the sections were brieﬂy counterstained with 0.5% cresyl violet. The optimum concentration of each lectin was determined in a preliminary experiment. The speciﬁcity of binding was examined following the method previously mentioned (Shapiro et al., 1995). In brief, biotinylated lectins were incubated with either saline, 10 mg/ml Nacetyl-galactosamine, or 10 mg/ml N-acetyl-glucosamine for 1 hr at RT, then used for lectin histochemistry as described above. Immunohistochemistry Free-ﬂoating sections were rinsed with PBST and treated with 3% H2O2 in methanol for 15 min. After extensive rinsing with PBST, they were incubated with 10% normal goat serum in PBST containing 1% bovine ORGANIZATION OF GOAT ACCESSORY OLFACTORY BULB serum albumin and 0.02% sodium azide (PBST-BSA) for 1 hr. Sections were then incubated with an antibody to either tyrosine hydroxylase (TH; 1:1,000; Protos Biotech, New York, NY), dopamine b-hydroxylase (DBH; 1:800; Sigma, St. Louis, MO), GAD (1:30,000; Biogenesis, Kingston, NH), brain type NOS (1:2,000; Transduction Laboratory, Lexington, KY), or NPY (1:1,000; Biogenesis) in PBST-BSA for 72 hr at 48C. Following three 15-min washes with PBST, sections were incubated with biotinylated goat anti-rabbit IgG (5 ml/ml PBST-BSA containing 1% normal goat serum) for 3 hr and avidin-biotin complex solution (10 ml each/ml PBST) for 1 hr. Each step was followed by three 15-min washes with PBST. After the last wash, sections were immersed in 50 mM Tris-HCl (pH 7.4), then reacted with chromogen solution consisting of DAB (0.4 mg/ml) and 0.0025% H2O2 in 50 mM Tris-HCl for 8 min. The reaction was stopped by immersing sections in 50 mM Tris-HCl. All reactions were performed at RT unless otherwise stated. Some sections were subjected to double immunoﬂuorescence staining. Free-ﬂoating sections were rinsed with PBST and incubated with PBST-BSA containing 10% normal goat serum, 10% normal donkey serum, and 10% fetal bovine serum for 1 hr. Sections were then incubated with a solution containing a monoclonal antibody to TH (1:1,000; Protos Biotech) and the polyclonal antibody to GAD (1:5,000) in PBST-BSA for 48 hr at 48C. After three 15-min washes with PBST, sections were incubated with a solution containing indocarbocyanine (CY3)-labeled goat anti-mouse IgG (4 ml/ml) and ﬂuorescein isothiocyanate (FITC)-labeled donkey anti-rabbit IgG (40 ml/ml) for 3 hr at RT. Sections were washed three times with water, mounted on gelatin-coated slides, and coverslipped with water-soluble mounting medium (Vector Laboratory). Slides were observed under a confocal microscope (FV300; Olympus, Tokyo, Japan). Omission of the primary or secondary antibodies during the staining process served as controls to indicate the speciﬁcity of the staining reaction. None of the control sections showed speciﬁc staining. Data Analysis The dimensions of the goat AOB were analyzed using Nissl-stained free-ﬂoating sections of ﬁve males and ﬁve females. All sections were observed under a microscope, and those containing the AOB were photographed. Maximum lengths in anteroposterior and dorsoventral directions were obtained by measurement on the photograph. The maximum length in the mediolateral direction was given as the distance between two sections containing the medial and lateral edges of the AOB. The sizes of representative cells in the AOB were analyzed using cryostat sections from two males and two females. Two sections containing the median aspect of the AOB were selected from each goat. The sizes of cells containing TH-, NOS-, and NPY-immunoreactivity (ir) were analyzed in eight immuno-stained free-ﬂoating sections that were randomly selected from four goats. Sections were photographed, and the major and minor axes of the cells were measured. The number of M/T cells throughout the AOB was analyzed in cryostat sections from three males and three females. Large round or oval cells that contained intensely stained nucleoli (a typical example is shown in 303 Fig. 1E) were considered M/T cells. Two observers independently counted the number of cells in the AOB under a microscope, and the average value was assigned to each section. In each goat, the total number of M/T cells in a single AOB was obtained by multiplying the summed values of each section by the frequency of seriation (in this case, eight) as described previously (PérezLaso et al., 1997). All values are expressed as mean 6 SEM. Sex differences of measurements were analyzed statistically using Student’s t-tests, and P < 0.05 was considered to be signiﬁcant. RESULTS General Morphology The AOB was located on the caudal and dorsomedial aspects of the olfactory bulb and is semioval in the goat (Fig. 1A). The size of the AOB was summarized in Table 1. Relatively large variation was observed among individuals in each measure, even within the same sex. A slight but signiﬁcant difference was observed between sexes in the maximum length in the mediolateral direction, but no other comparisons were signiﬁcant. The goat AOB was divided into four layers: the vomeronasal nerve layer (VNL), glomerular layer (GL), M/T cell layer (MTL), and granule cell layer (GRL; Fig. 1B). The VNL was composed of unmyelinated vomeronasal nerve-ﬁber bundles and small ﬂat cells (minor axis, 1.71 6 0.03 mm; major axis, 10.37 6 0.12 mm; n ¼ 240). The GL contained several forms of periglomerular cell (minor axis, 4.97 6 0.07 mm; major axis, 6.07 6 0.08 mm; n ¼ 240), which seemed to surround the glomerular proper. The boundary of each glomerulus, however, was less clear (Fig. 1C) compared to the distinct glomeruli in the main olfactory bulb (MOB). In the MTL, the cell density was relatively sparse. Round or oval cells (minor axis, 10.83 6 0.7 mm; major axis, 19.74 6 0.28 mm; n ¼ 120) containing intensely stained nucleoli in this layer were thought to be M/T cells, which are output neurons in the AOB (Fig. 1D). The putative M/T cells did not align to form a distinct layer, but rather were scattered mainly at the ventral portion of the MTL. The GRL contained numerous round cells (minor axis, 4.82 6 0.06 mm; major axis, 6.09 6 0.07 mm; n ¼ 240). These cells were likely granule cells or glial cells. The cell density of the GRL in the AOB was lower than that in the MOB (Fig. 1A). Because some M/T cells were located intermingled with these small cells, the boundary between the MTL and GRL was somewhat unclear. The lateral olfactory tract (lot) passed under the GRL (Fig. 1B). The number of putative M/T cells in the unilateral AOB was estimated as 7,350 6 755 (n ¼ 3) and 5,403 6 529 (n ¼ 3) in the male and female, respectively. Although the female AOB appeared to contain fewer M/T cells, the difference between the sexes was not statistically signiﬁcant. Lectin Histochemistry Using 21 lectins, we observed several staining patterns in the goat AOB (Table 2). No differences in lectin staining patterns were evident between the sexes. Seven lectins, i.e., Soybean agglutinin (SBA), Dolichos biﬂorus agglutinin (DBA), Erythrina crystagali lectin (ECL), 304 MOGI ET AL. Fig. 1. Photomicrographs of sections showing the goat accessory olfactory bulb (AOB). A: Free-ﬂoating section stained by cresyl violet. The laminar structure is less clear in the AOB than in the main olfactory bulb (MOB). The olfactory ventricle (ov) can be seen at the center of the olfactory bulb. B: Klüver-Barrera-stained cryostat section. The lateral olfactory tract (lot), consisting of myelinated bundles of ﬁbers, passes under the granule cell layer (GRL). C: High-power view of the glomerular layer (GL) in B. Several small cells loosely surround cellsparse regions (asterisks). D: Mitral/tufted (M/T) cell layer (MTL) and GRL in B. Arrows in the MTL show putative M/T cells, and several types of small cells can be seen in the GRL. The boundary between the MTL and GRL, however, is less clear. E: High-power view of the putative M/T cells in the MTL. Scale bars ¼ 2 mm (A); 200 mm (B); 40 mm (C and D); and 20 mm (E). Lycopersicon esculentum lectin (LEL), Ricinus communis agglutinin I (RCA), succinylated wheat germ agglutinin (s-WGA), and Vicia villosa agglutinin (VVA), bound to the entire area of the VNL and GL, with no heteroge- neity of staining in the rostrocaudal direction (Fig. 2). Among these, only DBA bound exclusively to the AOB and not to the MOB (Fig. 2A). In some cases such as ECL, patch-like staining was observed, with the diame- 305 ORGANIZATION OF GOAT ACCESSORY OLFACTORY BULB TABLE 1. Maximum length of AOB in three directions Direction Male (n ¼ 5) Female (n ¼ 5) a Anteroposterior Range (mm) Mean 6 SEM Range (mm) Mean 6 SEM 3.2 3.9 2.8 3.6 Layer DBA VVA SBA LEL s-WGA ECL RCA 120 BSL-II 1.4 2.0 1.2 1.6 – 2.2 6 0.2 – 1.7 6 0.1a Dorsoventral 1.1 1.1 0.7 1.1 – 1.1 6 0.0 – 1.5 6 0.2 P < 0.05, male vs female (t test). TABLE 2. Lectin binding patterns in the AOB Lectin – 4.5 6 0.2 – 4.2 6 0.3 Mediolateral VNL GL MTL/GRL þþ þþ þþ þ þþ þ þ þþ þþ þþ þþ þ þþ þþ þ þþ þþ, strong staining; þ, moderate staining; , no or background staining. ter of each patch in the range of 30 to 80 mm (Fig. 2C). The binding of VVA in the AOB was associated with the vomeronasal nerve or its accompanying structure, and thus nerve bundles in the VNL and ﬁne ﬁbers in the GL were clearly delineated (Fig. 2E). In addition, this lectin produced several small dots with diameters of approximately 1 to 2 mm on the surface of small cells in the GL (Fig. 2F). Each cell usually contained one or two dots. VVA binding was also observed in the nerve and glomerular layers in the MOB (not shown) in the same manner as the SBA binding (Fig. 2B). In contrast to the above seven lectins, Bandeiraea simplicifolia lectin II (BSL-II) bound not only to the VNL, but also to the MTL and GRL in the goat AOB (Fig. 2G). Only the glomerular proper in the GL was nearly completely devoid of staining by this lectin (Fig. 2H). Reaction products of BSL-II seemed to consist of several thick ﬁbers, and some of them extended from the GRL to the lot. Once again, rostrocaudal segregation of the staining pattern was not observed in either layer. Two lectins, i.e., Sophora japonica agglutinin and Ulex europaeus agglutinin, produced no staining even at high concentrations (50–100 mg/ml). The remaining 11 lectins, including wheat germ agglutinin (WGA), bound nonspeciﬁcally to all regions of the olfactory bulb. Although some of these lectins seemed to stain the VNL and/or GL slightly darker than the other areas, deﬁnitive identiﬁcation was not possible. sent primary dendrites running parallel to the layer. Their morphology suggested that these were short axon cells. In the GRL, several thick ﬁbers with varicosity showed intense NPY-ir (Fig. 3B). A small number of cells with spindle-shaped somata (minor axis, 10.81 6 0.09 mm; major axis, 20.71 6 0.93 mm; n ¼ 20) showed NPY-ir and sent one or two long primary dendrites running parallel to the layer. The other layers of the AOB were completely devoid of staining. GAD-ir was detected in the GL, MTL, and GRL (Fig. 3C), with the GL in particular having a relatively large number of small cells that showed GAD-ir. Thin ﬁbers were projected in various directions (Fig. 3E), and some of these ﬁbers appeared to arborize within the glomerulus, indicating that GAD-positive cells in the GL were periglomerular cells. Because dense plexuses formed by numerous GAD-positive ﬁbers were homogeneously distributed in the MTL and GRL, the boundary between two layers and the presence of cells containing GAD-ir were difﬁcult to identify. A small portion of cells in the GL showed TH-ir (Fig. 3D). These cells were round or oval in shape (minor axis, 5.87 6 0.13 mm; major axis, 8.39 6 0.15 mm; n ¼ 94). Some TH-positive cells bore dendrites with glomerular arborization (Fig. 3F), indicating that they were periglomerular cells. TH-positive cells were observed throughout the AOB, ranging from 30 to 70 cells in one section in the median part of the AOB. Fibers with TH-ir were sparsely distributed in the MTL and GRL, but no TH-positive cells were observed. Fibers with varicosity in the MTL and GRL showed DBH-ir (not shown). They were homogenously distributed in both layers and did not extend to other layers. Cells with DBH-ir were not present in any layer. Double-labeling immunohistochemistry showed that somata of TH-positive periglomerular cells and the glomerular arborization originating from TH-positive cells contained GAD-ir (Fig. 3G–I). The concomitant presence of GAD-ir was apparent in all TH-positive periglomerular cells. The majority of GAD-positive cells, however, did not possess TH-ir. In the MOB, TH-positive cells were abundant around the glomerulus, and GAD-ir was also observed in some populations of these cells (not shown). Immunohistochemistry NOS-ir was detected in the MTL and GRL (Fig. 3A). The MTL showed a faint NOS-positive background, whereas the GRL contained a dense network of strongly stained ﬁbers. In the GRL, numerous NOS-positive cells with small round somata, corresponding to granule cells, formed a cluster. In addition, larger, more intensely stained cells (minor axis, 11.17 6 0.38 mm; major axis, 21.42 6 0.94 mm; n ¼ 55) were scattered in the GRL and DISCUSSION General Morphology The goat AOB, composed of four layers, was semioval in shape and located in the dorsomedial aspect of the olfactory bulb (Fig. 1). The VNL was relatively thick, presumably reﬂecting a rich supply of sensory neuron ﬁbers from the VNO (Meisami and Bhatnagar, 1998). Periglomerular cells were scattered in the GL and were not Fig. 2. Photomicrographs of sections stained by Dolichos biﬂorus agglutinin (DBA; A), Soybean agglutinin (SBA; B), Erythrina crystagali lectin (ECL; C), succinylated wheat germ agglutinin (s-WGA; D), Vicia villosa agglutinin (VVA; E and F), and Bandeiraea simplicifolia lectin II (BSL-II; G and H). Sections in E through H were counterstained with cresyl violet, and relatively dark staining in the AOB indicates a positive reaction product by the respective lectin. VVA also weakly stained the lot in the MOB (E). F: High-power view of the GL in E. Two types of VVA reaction products can be seen: a round accumulation of thick ﬁbers (asterisk) and a dot-like structure on small cells (arrows) around the former. H: High-power view of G. The BSL-II staining appears to consist of a plexus of thick ﬁbers that do not extend into the glomerular proper (asterisk). Scale bars ¼ 1 mm (A and B); 500 mm (C, D, E, and G); 20 mm (F); and 100 mm (H). ORGANIZATION OF GOAT ACCESSORY OLFACTORY BULB 307 Fig. 3. Photomicrographs of sections showing the distribution of immunoreactivity (ir) in the goat AOB. A: Nitric oxide synthase (NOS)ir. B: Neuropeptide Y (NPY)-ir. C and E: Glutamic acid decarboxylase (GAD)-ir. D and F: Tyrosine hydroxylase (TH)-ir. E and F are highpower views of the GL in C and D, respectively. G and H: Confocal micrographs of a section double-stained for GAD-ir (G) and TH-ir (H) in the GL. I: Colocalization of two immunoreactive substances is visible as a yellow product in a merged image of G and H. Arrows indicate immunoreactive cells and asterisks denote immunoreactive dendrites with the glomerular arborization in E through I. Scale bars ¼ 500 mm (A–D); 50 mm (E–I). regularly arranged around each glomerulus as in other mammals (Meisami and Bhatnagar, 1998). Although the general morphology of the goat AOB is similar to that of the sheep (Tillet et al., 1987; Jansen et al., 1998; Salazar et al., 2000, 2003), the distribution of M/T cells seems to differ between these two herbivores. In the sheep, M/T cells align to form an obvious M/T cell layer (Salazar et al., 2000), whereas in the goat, they were scattered in the ventral portion of the layer and some were surrounded by small granular or glial cells. Such cell arrangement is rather analogous to that of the ferret AOB in which one layer contains intermingled granule and M/T cells and no lamination into individual MTL and GRL is apparent (Kelliher et al., 2001), or the dog AOB in which the MTL and GRL seem to be indistinguishable (Nakajima et al., 1998). In the goat AOB, the lot was located under the GRL. This location differs from that in the rat AOB, in which the lot passes between the MTL and GRL. We analyzed several morphometric characteristics of the AOB in gonadectomized female and male goats. Gonadectomy in adult goats should have little effect on AOB 308 MOGI ET AL. morphology because the organizational action of steroids on the vomeronasal system is achieved only during brain development (Guillamon and Segovia, 1997). The dimensions of the goat AOB (Table 1) were equivalent to or slightly larger than those of the rat AOB (Takami and Graziadei, 1991). The numbers of putative M/T cells in the AOB were approximately 5,000 and 7,000 in the female and male, respectively. These numbers are also equivalent to reported values in intact female (Valencia et al., 1986) and male (Pérez-Laso et al., 1997) rats. The size of the AOB (Segovia et al., 1984) and the number of M/T cells (Valencia et al., 1986; Pérez-Laso et al., 1997) have been shown to be larger in male than in female rats. Whereas we did observe a similar tendency in the goat AOB, only the maximum length in the mediolareral direction differed signiﬁcantly between the sexes. Therefore, unlike in the rat, the goat AOB does not appear to be clearly differentiated between the sexes. Lectin Binding Patterns Of the 21 lectins examined, eight speciﬁcally bound to the AOB in the goat (Table 2). The binding of some lectins was similar to those previously reported. For example, LEL stained the goat AOB similar to other mammals such as the mouse (Salazar et al., 2001), rat (Ichikawa et al., 1992), hamster (Taniguchi et al., 1993), sheep, and pig (Salazar et al., 2000). However, several lectins produced characteristic binding patterns in the goat AOB. First, although the labeling of DBA occurs in both the AOB and the MOB of the sheep (Salazar et al., 2000), it was restricted exclusively to the AOB of the goat, suggesting that carbohydrate moieties in the olfactory bulb differ even between very closely related species. Second, in contrast to the above, VVA bound to both the AOB and the MOB of the goat, whereas it has been shown that carbohydrate residues identiﬁed by VVA are unique to the vomeronasal axons and not to the olfactory axons in a number of other animals, including the rat (Ichikawa et al., 1992; Takami et al., 1992b) and opossum (Shapiro et al., 1995). Furthermore, VVA also bound to cells surrounding the glomerulus in the AOB (Fig. 2F). This result conﬁrms the previous ﬁnding that VVA produces small dots in the GL of the rat AOB (Takami et al., 1992b) and further shows that the small dots represent speciﬁc structures on the cell, possibly the periglomerular cell. Third, BSL-II bound not only to the VNL, but also to the MTL and GRL in the goat AOB (Fig. 2G). To the best of our knowledge, the binding of this lectin in the AOB has been shown previously only in the VNL and GL (Halpern and Martinez-Marcos, 2003); this is the ﬁrst report demonstrating the speciﬁc afﬁnity of the MTL/GRL to lectin. It has been demonstrated that lectin binding is segregated into several subdivisions of rostrocaudal extent in various species (Takami et al., 1992b; Taniguchi et al., 1993; Ichikawa et al., 1994; Shapiro et al., 1995; Salazar et al., 2001), which is thought to reﬂect functional segregation of the AOB (Sugai et al., 2000). However, the segregation of staining was not observed in any of the eight lectins that speciﬁcally bound to the goat AOB. Takigami et al. (2000, 2004b) found in the goat, and subsequently in four other mammals, that the AOB is uniform in terms of the rostrocaudal distribution of G-protein. Our lectin histochemistry data strongly support their suggestion that the segregated structure of AOB is not a common feature in mammalian species. Immunohistochemistry Our results demonstrated laminar distributions of NOS-, NPY-, GAD-, TH-, and DBH-ir in the goat AOB. The distributions were similar to those found in other mammals, but some distributions unique to the goat were also observed. Although the boundary between the MTL and the GRL in the goat AOB was obscure in the Nissl-stained section as noted above, NOS immunohistochemistry clearly discriminated between the two layers, with the MTL weakly stained and the GRL heavily stained (Fig. 3A). The high density of NOS-ir in the GRL might consist of NOS-positive granule cells and their numerous dendrites, as reported in the mouse (Kishimoto et al., 1993) and dog (Nakajima et al., 1998). The segregation of the two layers was further evident in the distribution of NPY-ir. NPYpositive ﬁbers were observed only in the GRL, and not in the MTL (Fig. 3B). This distribution of NPY-ir in the goat is comparable to that in the rat (Matsutani et al., 1988) and guinea pig (Matsutani et al., 1989), both of which have AOBs with ﬁve distinct layers. It has been demonstrated in various mammals such as the rat (Mugnaini et al., 1984; Takami et al., 1992a) and dog (Nakajima et al., 1998) that GAD is contained in a subset of the periglomerular cells and in a large population of granule cells. Although the dense plexuses in the GRL prevented us from identifying each GAD-positive somata (Fig. 3C), it is plausible that granule cells containing GAD occur in the goat AOB as well. Periglomerular cells with GAD-ir, however, were frequently observed, indicating that GABAnergic modulation takes place at the glomerulus of the goat AOB, as in other mammals. We observed as many as 70 periglomerular cells with TH-ir per section in the goat AOB. In rodents, the occurrence of TH-positive periglomerular cells seems to be very low and has been described as ‘‘rare’’ in the mouse (Rosser et al., 1986), ‘‘rare’’ (Mugnaini et al., 1984) or ‘‘limited to a single cell’’ in the rat (Baker, 1986), and ‘‘extremely rare’’ in the hamster (Davis and Macrides, 1983). Therefore, the goat AOB seems to contain relatively abundant periglomerular cells with TH-ir compared to the AOB in rodents. Because DBH-ir was absent in the GL, these cells might be dopaminergic. Double-labeling immunohistochemistry revealed that virtually all TH-ir colocalized with GAD-ir not only in the somata of the periglomerular cell, but also in the glomerular arborization (Fig. 3I). Dopamine and GABA might therefore simultaneously modulate functions in some glomeruli. The coexistence of these two neurotransmitters has been demonstrated in the rat MOB (Kosaka et al., 1985), and this was also conﬁrmed in the goat MOB; however, the presence of GABAnergic/dopaminergic periglomerular cells in the mammalian AOB has not yet been described. The role of these cells in signal transduction at the glomerulus is far from clear, but a similar type of modulation of the glomerulus may occur in both the MOB and the AOB of the goat. Functional Implications Our results demonstrated that the structure and chemical composition of the AOB are well maintained in ORGANIZATION OF GOAT ACCESSORY OLFACTORY BULB the goat. Further, a putative pheromone receptor gene has been identiﬁed in the VNO of the goat (Wakabayashi et al., 2002). This evidence suggests that the AOB plays a critical role as the primary processing center of pheromone signal transduction in the goat. It should be noted, however, that the ‘‘male effect,’’ the typical pheromone action in the goat (Chemineau, 1987) and sheep (Martin et al., 1986), does not appear to depend on the vomeronasal system (Cohen-Tannoudji et al., 1989). Knowing the type of pheromone signals or possibly odor signals conveyed to the AOB and the parts of the central brain that receive the information from the AOB may be of great importance in elucidating the function of the goat AOB. In conclusion, we demonstrated that the goat AOB was divided into four layers: VNL, GL, MTL, and GRL; that a single AOB contained 5,000–8,000 putative M/T cells with no sex differences; that some lectins showed binding patterns unique to the goat AOB, and in these cases, no heterogeneity of lectin binding was observed in the rostrocaudal direction; that NOS-, TH-, DBH-, and GAD-ir occurred in the MTL and GRL, whereas NPY-ir was present only in the GRL; and that a subset of periglomerular cells contained TH-ir and virtually all of them were colocalized with GAD-ir. These results contribute to our understandings of the sophistication of pheromone communication in this species. ACKNOWLEDGMENTS The authors thank Ms. Y. Sakairi for technical assistance and the staff of the National Institute of Livestock and Grassland Science for providing care of the animals. 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