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Structure and chemical organization of the accessory olfactory bulb in the goat.

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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: hokamu@affrc.go.jp
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.
This work was supported in part by Grants-in-Aid for
Scientific Research (15GS0306) from the Japan Society
for the Promotion of Science and the Animal Behavior
Program from the Ministry of Agriculture, Forestry and
Fisheries, Japan.
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