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General organization of the perinatal and adult accessory olfactory bulb in mice.

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THE ANATOMICAL RECORD PART A 288A:1009–1025 (2006)
General Organization of the Perinatal
and Adult Accessory Olfactory
Bulb in Mice
IGNACIO SALAZAR,* PABLO SANCHEZ-QUINTEIRO,
JOSE MANUEL CIFUENTES, AND PATRICIA FERNANDEZ DE TROCONIZ
Department of Anatomy and Animal Production, Unit of Anatomy and Embryology,
Faculty of Veterinary, University of Santiago de Compostela, Lugo, Spain
ABSTRACT
The vomeronasal system is currently a topical issue since the dual functional specificity, vomeronasal system-pheromones, has recently been questioned. Irrespective of the tools used to put such specificity in doubt, the
diversity of the anatomy of the system itself in the animal kingdom is probably of more importance than has previously been considered. It has to be
pointed out that a true vomeronasal system is integrated by the vomeronasal
organ, the accessory olfactory bulb, and the so-called vomeronasal amygdala.
Therefore, it seems reasonable to establish the corresponding differences
between a well-developed vomeronasal system and other areas of the nasal
cavity in which putative olfactory receptors, perhaps present in other kinds
of mammals, may be able to detect pheromones and to process them. In consequence, a solid pattern for one such system in one particular species needs
to be chosen. Here we report on an analysis of the general morphological
characteristics of the accessory olfactory bulb in mice, a species commonly
used in the study of the vomeronasal system, during growth and in adults.
Our results indicate that the critical period for the formation of this structure
comprises the stages between the first and the fifth day after birth, when the
stratification of the bulb, the peculiarities of each type of cell, and the final
building of glomeruli are completed. In addition, our data suggest that the
conventional plexiform layers of the main olfactory bulb are not present in the
accessory bulb. Anat Rec Part A, 288A:1009–1025, 2006. Ó 2006 Wiley-Liss, Inc.
Key words: vomeronasal system; accessory olfactory bulb;
growth; anatomy; mouse
Odors are detected in the majority of mammals by
means of two systems, the main olfactory system (MOS)
and the vomeronasal system (VNS) (Halpern, 1987;
Brennan, 2001; Halpern and Martı́nez-Marcos, 2003),
which, despite some differences, basically share a common pattern of organization (Mori, 1987). Each consists
of a sensory epithelium, two olfactory bulbs, and different telencephalic structures. This similarity results in
the olfactory information being processed in a like manner in both systems, although there are also some specific morphological differences, and at molecular level
different genes are involved in the transduction mechanism (Buck and Axel, 1991; Dulac and Axel, 1995; Mombaerts, 1999).
The main olfactory (MOB) and the accessory olfactory bulbs (AOB), which are the first relay stations in
Ó 2006 WILEY-LISS, INC.
each system, play a key role in the physiological mechanism of olfaction. Within the bulbs, glomeruli organize the corresponding input and output information
Grant sponsor: Ministry of Education and Science, Spain;
Grant number: BFU2004-01004; Grant sponsor: Council of
Innovation, Industry and Commerce, Xunta de Galicia; Grant
number: PGIDIT05PXIC26103PN.
*Correspondence to: Ignacio Salazar, Department of Anatomy
and Animal Production, Unit of Anatomy and Embryology, Faculty of Veterinary, USC, 27002 Lugo, Spain. Fax: 34-982285939.
E-mail: anigsabe@lugo.usc.es
Received 15 April 2006; Accepted 10 June 2006
DOI 10.1002/ar.a.20366
Published online 4 August 2006 in Wiley InterScience
(www.interscience.wiley.com).
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SALAZAR ET AL.
and are considered the true functional unit of the bulbs
(Graziadei and Monti-Graziadei, 1986; Valverde et al.,
1992; Hildebrand and Shepherd, 1997; Bozza et al.,
2002).
The MOB is a structure that has been very well studied at different morphological and physiological levels
(Shepherd and Greer, 1998; López Mascaraque and de
Castro, 2002), but this is not quite the case for the AOB.
Albeit during the last 2 decades, progress in the knowledge of the VNS in general and of the AOB in particular
has been considerable (Hayashi et al., 1993; Taniguchi
and Kaba, 2001; Araneda and Firestein, 2006), there are
still some specific aspects of the AOB that require further exploration. Among the issues in need of precision
is the exact morphology of the AOB, especially when it
is obtained from mice. No authors with the exception of
Hinds (1968a, 1968b) have dealt with the AOB of the
mouse as a whole, although some researchers have studied partial aspects of its morphology in this species
(Wilson and Raisman, 1981; von Campenhausen et al.,
1997; del Punta et al., 2002).
Other experimental animals, such as rats, guinea pigs,
and rabbits, have in fact been used to define the general
characteristics of the AOB (Crosby and Humphrey, 1939;
Allison, 1953; Mori, 1987; Takami and Graziadei, 1991).
The whole AOB has also been studied during development in the opossum (Shapiro et al., 1995), a marsupial
mammal in which an extensive reliance on chemosensory cues is presumed. However, when choosing a morphological pattern for the AOB, the selected species
could be the mouse because its genome has been completed (http://www.ensembl.org), and the possibility of
working with transgenic animals represents a wonderful
challenge.
In an attempt to fill, at least in part, the possible gaps
in our knowledge of the AOB in mice, the aim of the
present work is to focus attention on the study of the
general morphological characteristics of AOB as a whole.
The transition from the AOB during growth to the adult
AOB has been considered of special interest, as it provides a detailed description of the more relevant events
that take place during the critical period that covers the
last gestational days and the first days after birth. Once
the characteristic layers of the AOB have appeared and
strengthened, few changes, if any, have been observed in
relation to the AOB of the adult.
MATERIALS AND METHODS
Animals
Female BALB/c mice were bred, housed singly, and
mated in the Animal House of the Veterinary Faculty
(University of Santiago de Compostela; registry no.
15003AE). Embryonic age was calculated from the time
at which the vaginal plug was evident (embryonic day
0). On embryonic days 15–19, pregnant females were
placed under deep anesthesia and their fetuses removed.
Newborn mice were also examined on postnatal days 0–
12, as were a number of adult males and females. In
what follows, ‘‘En’’ and ‘‘Pn’’ respectively denote embryonic and postnatal ages of n days. For each developmental age, at least three specimens were examined.
All experiments were performed in accordance with
the regulations and laws of the European Union (86/609/
EEC) and Spain (RD 223/1998) for the care and handling of animals in research. All efforts were made to
minimize animal suffering and limit the number of animals used.
Processing of Olfactory Bulbs
Paraffin. The fetuses were fixed directly over 24 hr
in 4% paraformaldehyde or Bouin’s fluid, while newborn,
juvenile, and adult mice were washed and perfused with
the same fixatives before their heads were removed and
the whole brain dissected out and immersed in the fixative. After microdissection (when necessary), the tissue
of interest was embedded in paraffin wax. In the case of
smaller specimens aged E15–E19, the half rostral part
of the brain was cut into transverse or sagittal sections
10 mm thick.
Cryosectioning. After 4% paraformaldehyde perfusion, brains were postfixed for a further 2 hr and then
cryoprotected by immersion overnight in 30% sucrose in
O.1 M phosphate buffer saline (PBS) at 48C. A freezing
microtome was used to cut 40 mm sagittal sections
through the rostral half of the brain. The sections were
collected in PB 0.1 M, pH 7.2, and stored at 48C until
their free-floating processing.
Lectin Histochemistry Protocol
Two lectins, Ulex europaeus agglutinin I (UEA-I) and
Lycopersicum esculentum agglutinin (LEA), were
obtained as biotin conjugates from Sigma (St. Louis,
MO) and detected using a Vectastain avidin-biotin peroxidase complex (ABC) biotin detection kit (Vector, Burlingame, CA). L-fucose and N-acetylglucosamine are the
sugar specificity to UEA-I and LEA, respectively. Both
lectins are considered very good markers for the study
of the AOB in mice; UEA-I labels exclusively the vomeronasal nerve layer and glomerular layer of the AOB,
while LEA labels the same layers in both bulbs, but with
the special feature that in the AOB only the anterior
part is labeled (Salazar et al., 2001; Salazar and Sánchez Quinteiro, 2003).
Paraffin sections were dewaxed and transferred to
phosphate buffer (PB) and then incubated for 30 min at
room temperature with 2% bovine serum albumin in 0.1
M Tris buffer (pH 7.2); incubated for 24 hr at 48C with
lectin at various dilutions in 0.1 M Tris buffer containing 2% bovine serum albumin; washed for 2 3 10 min in
PB; incubated for 90 min at room temperature with Vectastain ABC reagent (1:250 in PB); washed for 10 min in
PB; and washed for 5 min with Tris-HCl (pH 7.6).
Visualization of peroxidase activity by incubation in a
solution containing 0.05% 3,3-diaminobenzidine (DAB)
and 0.003% H2O2 in 0.2 M Tris-HCl buffer (pH 7.6) was
monitored under a microscope; the reaction was
quenched with 0.2 M Tris-HCl buffer. All lectinless control sections were unlabeled and no specific binding was
observed in controls with presaturated lectins.
Immunohistochemistry Protocol
The following specific markers were used: olfactory
marker protein (OMP; generously provided by Dr. F.
Margolis, University of Maryland), growth-associated
protein 43 (GAP-43; Sigma), microtubule-associated pro-
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PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
TABLE 1. Specific information related to the antibodies used
Antibodies
Source
Catalogue
number
Type*/Mode**
Clone
Anti-OMP
Anti-GAP-43
Anti-MAP-2
Dr. F. Margolis
SIGMA
SIGMA
–
G9264
M4403
Policlonal raised in goat
Monoclonal
Monoclonal
Anti-GFAP
DAKO
Z0334
Policlonal raised in rabbit
Biotinylated Anti-Mouse
IgG (HþL)
Biotinylated Anti-Rabbit
IgG (HþL)
VECTOR
BA-2000
Raised in horse
VECTOR
BA-1000
Raised in goat
7B10
HM-2
Binding/References
VNra/Margolis (1972)
VNra/Skene (1989)
Dendrites
npk/Matus (1988)
Astrocytes/Bignami
et al. (1972)
*Antibody.
**Species in which the secondary antibody is raised. VNra, Vomeronasal receptor axons; Npk, Neuroperikarya.
teins 2 (MAP-2; Sigma), and glial fibrillary acidic protein
(GFAP; Dako; Table 1).
Dilutions and Incubations
The final working dilutions used in the paraffinembedded sections were OMP, 1:500; GAP-43, 1:8,000;
MAP-2, 1:300; GFAP, 1:250. Free-floating sections were
incubated at higher dilutions: OMP, 1:10,000; GAP-43,
1:10,000; MAP-2, 1:500; GFAP, 1:1,000.
The paraffin sections were transferred after dewaxing
to PB. Then sections were incubated for 30 min at room
temperature with 5% horse normal serum containing 2%
bovine serum albumin in PB. Cryostat sections were
incubated for 30 min at room temperature with 0.2%
Triton X-100 containing 2% bovine serum albumin in
PB. From that point, the immunohistochemistry was
similar for slide-mounted and free-floating sections.
In both cases, sections were sequentially incubated in
primary antibody for 24 hr at 48C, biotinylated secondary antibody for 1 hr, ABC (Vectastain reagent) for 2 hr,
and visualized either with DAB or Vector VIP (SK-4600;
Burlingame, CA) following standard procedures for the
visualization of horseradish peroxidase complex (HRP).
For double immnunostaining, a sequential twice-repeated enzyme-labeled method was employed. Between
both immunolabelings, the sections were subjected to
treatment with 0.01 M glycine solution (pH 2.2) for
5 min. In selecting the most suitable dye for the HRPH2O2 in order to visualize the immunoreaction, products
DAB, Vector SG (SK-4700) and VIP were combined. No
nonspecific staining was observed in control sections
incubated with either normal horse serum or without
primary antibodies.
In most of the cases, sections embedded in paraffin
were alternatively stained by Nissl and labeled by lectins and other specific markers, and also some sections
were lightly counterstained with cresyl violet.
Image Acquisition and Processing
Digital microscopy images were captured by using a
Karl Zeiss Axiocam MRc5 digital camera. All images
were processed using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA). When necessary, images were
adjusted for contrast and brightness to equilibrate light
levels, cropped, resized, and rotated for purpose of presentation.
RESULTS
In order to facilitate the following description of the
stratified structure of the AOB, reference will be made
to the conventional arrangement of strata as seen in the
MOB, despite the variations between both bulbs. The
classical nomenclature usually employed for the MOB is
nerve layer (NL), which is substituted here by vomeronasal nerve layer (VNNL), glomerular layer (GlL), external plexiform layer (EPL), mitral/tufted cell layer (M/
TcL), internal plexiform layer (IPL), and granular layer
(GrL).
Development and Growth
Fetus, newborn and juvenile: Nissl sections.
Given the objectives of this work, we may begin our
comments at stage E15. Figures 1–3 show a sequence of
events that characterizes the progressive organization of
the AOB. With regard to the topography, from the beginning the accessory bulb occupies the same position as in
adults, while its size and form show only slight differences. The size is proportionally bigger in young samples
than in older (proportionally to the size of the MOB),
the external contour changes from convexity to concavity, and the internal appearance is determined by the
number and distribution of its cells. Measurements and
quantification have been studied separately (data not
shown).
E15–P0 (Fig. 1). The most relevant morphological
detail in these stages is the presence of a prominent
mass of cells, the future mitral/tufted (M/T) cells,
bounded by two zones, which we will call the intermediate superficial zone (IsZ) and the intermediate deep zone
(IdZ), where very few cells are present. Externally, the
developing VNNL is beginning to appear, while internally a densely stained group of proliferating cells,
smaller than those of the main mass, occupies an extensive zone adjacent to the ventricular cavity.
Progressively, the VNNL shows greater definition, and
granular cells replace the group of cells situated in the
deep area of the bulb. Meanwhile, the distribution of
the mass of cells previously mentioned begins to show
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SALAZAR ET AL.
Fig. 1. Nissl-stained sagittal sections of the rostral telencephalon
of fetuses (left anterior, right posterior-orientated; A–H) give precise information about the characteristic form and topography of the AOB
(frames; left column). Higher-power photomicrographs (right column)
of previous sections showing the beginnings of stratification in the
developing AOB. Future M/T cells (asterisks) lead the process of layer
formation, and close to them are the two intermediates zones, IsZ and
IdZ (curve arrows, in F and H). I and J: General view of the comparative layers of the AOB (I) and the MOB (J) at birth. Scale bars ¼
100 mm (B and D); 250 mm (all others).
Fig. 2. Nissl-stained sagittal sections of the AOB (left anterior, right
posterior-orientated) from birth to postnatal day 5. A–C: The thickness
of intermediate zones is indicated by broken lines. D: Limits of IsZ are
established by basal periglomerular cells (arrowheads) and M/T cells
(asterisk). E and F: The vomeronasal nerve and granular layers are
easily identified; the topography of the basal periglomerular cells are
indicated by double arrows; the IdZ evolves into the LOT. G and
H: Higher magnifications of E and F allow the verification of the size
and typical morphology of the periglomerular cells (arrowheads) and
M/T cells (asterisks). E and G are frozen sections; the rest are paraffin
sections. Scale bars ¼ 100 mm (G and H); 250 mm (all others).
1014
SALAZAR ET AL.
Fig. 3. Different images of Nissl-stained sagittal sections of the
AOB (left anterior, right posterior-orientated), once the main changes
have occurred. A and B: Basal periglomerular cells (double arrows)
are considered an interesting reference to establish limits. C: Same
image as B, in which the layers of the AOB are indicated. D: Higher-
power photomicrograph showing details of different cells present in
the AOB, except those of the GrL. E: At 12 postnatal day, the structure of the AOB is close to the adult bulb. A is frozen section; the rest
are paraffin sections. Scale bars ¼ 100 mm (D); 250 mm (all others).
the appearance of the M/T cells. Nevertheless, few
changes take place in the two intermediate zones in this
phase of development.
numerous, albeit scattered and arranged so as to form a
slightly concave curve.
Moreover, intermediate zones are also affected by the
general changes to the bulb. Both zones are especially
evident in overstained cryostat sections because of the
different degree of stain of the whole bulb. In the IsZ,
the presence of a group of small and intensely stained
cells can be observed, the periglomerular basal cells.
Superficially to them is the future GlL, characterized by
the presence of a reduced number of small and medium
cells that never outline glomeruli. Deeply to the periglomerular basal cells would be the EPL, although it is
very difficult, if not impossible, to establish clear limits
between the periglomerular basal cells and M/T cells,
because in this narrow and irregular space there are
usually sparse and scattered, small, medium, and large
P1–P5 (Fig. 2). These stages are considered the
transitional period in the morphological organization of
the laminar architecture of the AOB, as they establish
the transition from immature to mature AOB. The vomeronasal nerve and granular layers are differentiated.
The VNNL mainly consists of small, elongated, and randomly oriented cells showing considerable thickness,
especially in the middle of the bulb. The cells of the GrL
are small, round, and densely stained and packed
and are distributed in rows. The M/T cells, whose perikarya are quite evident, are lightly stained and still
PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
1015
Fig. 4. VNNL and GlL stained for different markers in sagittal sections of the AOB (left anterior, right posterior-orientated) from birth to
postnatal day 5. A: Lower-power photomicrograph of the AOB stained
with GAP-43; other structures adjacent to the AOB are also immunoreactive. Note the glomeruli of the MOB, open arrow. B: The anterior
part of the AOB is the only tissue of this bulb stained by LEA lectin.
C: Higher magnification of B, in which some vomeronasal axons end
in the glomeruli (arrowheads). D: Double-labeled UEA-I and MAP-2
of the AOB, with indication of the M/T cell layer. E: Extension and
arrangement of the VNNL and GlL stained by OMP, with the shade of
some glomeruli more intensely labeled (arrows). F: Glomeruli (arrows)
are filled with dense axons in the MOB (same preparation as E).
G–I: GAP-43 immnunoreactivity showing the general distribution of the
vomeronasal axons ending in the anterior (H) and posterior (I) parts of
the AOB. J–L: GAP-43 immunoreactivity in sagittal sections of the AOB
(J and K) and MOB (L); data useful to compare the different images
of glomeruli in both bulbs (arrows). Scale bars ¼ 50 mm (C, F, and L);
100 mm (D, E, H, I, and K); 250 mm (A, B, G, and J).
cells. The M/TcL is better defined now than in previous
stages.
Similarly, the lateral olfactory tract (LOT) fully occupies the IdZ, where there are only a few cells of small
and medium size, with the GrL in the following deep
stratum. No other space between the M/T cells and GrL
is observed.
periglomerular cells are sparse and do not outline glomeruli, albeit a considerable increase in the number of
periglomerular basal cells establish a sharp limit between the GlL and the rest of the bulb (Fig. 3A, B,
and E). In the meanwhile, progressive reduction of the
M/TcL is evident, with its cells sparse and not forming
true rows. The LOT remains invariable. The existence
of plexiform layers is quite doubtful, although the same
narrow, irregular, and heterogeneous space as before is
also possible to identify in some sections at these
stages.
P6–P12 (Fig. 3). Changes previously described are
corroborated or confirmed at P6 and later stages, and
the general organization of the AOB is close to that of
the adult (Fig. 8). Frozen sections of the whole AOB
(Fig. 3A) clearly show the IsZ, which will be occupied
by most of the glomeruli; other glomeruli are intermingled in the VNNL due to the glomeruli not being
organized into a defined row. It is also evident that
Lectins and inmmunohistochemistry sections.
With regard to the transitional period considered previously, lectins, OMP, and GAP-43 showed a similar pattern of reactivity in the VNNL and GlL. Then sections
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SALAZAR ET AL.
Fig. 5. Lower (left column) and higher (right column) magnifications
of sagittal sections of the AOB (left anterior, right posterior-orientated),
from birth to postnatal day 4, showing MAP-2 immunoreactivity. A, C,
and E: Note the dendritic process plexus in the IsZ (between double
arrows). B and D: Dendritic processes do not form glomerular structures (arrows) at first stages. F: At P4, dendritic glomeruli (arrows) are
more evident. Soma of M/T cells are also shown (arrowheads; B, D, and
F). Scale bars ¼ 50 mm (B and D); 100 mm (F); 250 mm (A, C, and E).
with these markers were selected indistinctly. Dendrites
were identified using MAP-2, and the population of
astrocytes was observed by GFAP.
P2 (Fig. 4D). Both the separation of the VNNL and
GlL from the rest and a discrete population of glomeruli
are intensively labeled by UEA-I, an excellent marker
specifically for the study of the AOB.
Vomeronasal axons.
PO (Fig. 4A). At birth, GAP-43 immunoreactivity
clearly establishes the separation between the future
VNNL and GlL and the rest of the AOB.
P1 (Fig. 4B and C). LEA, which is characterized by
labeling the VNNL and GlL only in the rostral part of
the AOB as well as the same layers of the MOB, labeled
some glomeruli-like structures, not very well isolated.
The VNNL cannot easily be separated from the GlL.
P3 (Fig. 4E and F). Although OMP labels the
VNNL and GlL of the AOB clearly, it is interesting to
point out the different immnunoreactivity between the
corresponding areas stained in the AOB and MOB. This
difference is observed, on the one hand, because of the
extension of the vomeronasal receptor neurons axons
(AOB; Fig. 4E) and the olfactory receptor neurons axons
(MOB; Fig. 4F), and on the other hand, the different
appearance of both glomeruli (Fig. 4E and F).
PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
Fig. 6. Sagittal sections of the AOB (left anterior, right posterior-orientated) at the critical stage of P5 (A–D). A and B: MAP-2 immunoreactivity showing soma of the M/T cells (arrowheads), their dendrites
(open arrows), and dendritic glomeruli (arrows). C: Double-labeled
LEA/MAP-2 demonstrated the areas affected by the vomeronasal
1017
axons and the dendritic projection. D: Double immnunostaining GAP43/MAP-2 photomicrograph showing the definitive participation of the
vomeronasal axons and dendrites in the formation of the glomeruli.
E and F: Higher magnifications of A and B. Scale bars ¼ 50 mm (B);
150 mm (E and F); 250 mm (A, C, and D).
1018
SALAZAR ET AL.
Fig. 7. GFAP immunoreactivity at lower (left column) and higher
(right column) magnifications of sagittal sections of the AOB (left anterior, right posterior-orientated) from birth to postnatal day 5. A and B:
Immature astrocytes (arrows) mainly located in the thickness of the
IsZ. C–F: Topography of astrocytes as before and processes of some
beginning to form glomeruli (arrowheads). G and H: Astrocytes are
densely stained and show the typical appearance of glomeruli in the
AOB (arrowheads). Scale bars ¼ 100 mm (B, D, F, and H); 250 mm (A,
C, E, and G).
PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
1019
Fig. 8. Transverse (A) and sagittal (B) sections of the most rostral
pole of the telencephalon in adult mice (the AOB is framed), with the
corresponding magnifications of the AOB (C and D). Nissl-stained.
Schematic representation of the arrival of the vomeronasal nerves at the
AOB (E, left side) and of the distribution of vomeronasal axons within it,
shown in a partial sagittal section of the AOB, limited by periglomerular
basal cells (arrows; E, right side). FC, frontal cortex; VNNs, vomeronasal
nerves. Scale bars ¼ 250 mm (C and D); 1000 mm (A and B).
P4 (Fig. 4G–I)/P5 (Fig. 4J–L). At these two stages
of the transitional period, GAP-43 stains not only the
VNNL and GlL but also the corresponding axons
enclosed in the LOT (Fig. 4G and J); the wide unstained
space is the location for the M/TcL and theoretically for
the plexiform layers. Albeit some glomeruli are distinguishable in the deep glomerular zone (Fig. 4H, I, and
K), there is not a sharp limit between the VNNL and
the GlL, as is quite evident in the MOB (Fig. 4L). These
immunohistochemistry data corroborate that the glomeruli are neither organized into a defined row nor perfectly
isolated from the VNNL, as Nissl material showed.
are stained in the AOB at these stages of development.
Just below the VNNL, that is to say in the IsZ, dendritic
processes form a plexus that grows gradually according
to age (Fig. 5A, C, and E). The growth of this plexus
means a bigger extension over the VNNL and simultaneously the organization of individual units, with the typical club-shaped appearance of glomeruli (Fig. 5B, D, F).
Dendrites.
P0/P4 (Fig. 5). The two predominant expressions of
MAP-2, neuron perykarya and dendrites (Matus, 1988),
P5 (Fig. 6). Neuron perikarya, dendrites, and their
processes are clearly immunoreactive for MAP-2 (Fig. 6A
and B). At this age, the contribution of the dendritic processes to the formation of glomerular structures is quite
evident (Fig. 6B). Because LEA lectin labels only the anterior part of the VNNL and GlL in the AOB, doublestained LEA/MAP-2 gives precise information about the
projection of the vomeronasal receptor neuron axons (anterior half) and the extension of the dendritic processes
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SALAZAR ET AL.
Fig. 9. Nissl-stained sagittal sections of the AOB (A) and the MOB (B) showing the different number
and distribution of periglomerular cells around glomeruli (arrows) in adult mice. Scale bars ¼ 250 mm.
(posterior part; Fig. 6C). Double-immunostained MAP-2/
GAP-43 shows the whole AOB stained and allows the
conjunction of axons and dendritic processes to be seen
(Fig. 6D). Higher magnification of the two previous figures are also useful to delimitate the corresponding
layers of this bulb (Fig. 6E and F).
Astrocytes.
P0 (Fig. 7A and B). Sections of the AOB from newborn mice express GFAP in a few astrocytes located in
the granular layer, some of them with short processes,
in the deep part of the IsZ, which are occasionally intermingled in the mass of cells that characterizes the typical image of the AOB at this stage.
P1/P2 (Fig. 7C and D). Not only does the number
of astrocytes increase but their size and processes are
more and more evident, while their distribution remains
the same as before; the most superficial branches of
astrocytes organize small and globular structures.
P3/P4 (Fig. 7E and F). Progressively, GFAP-positive astrocytes show neat cell bodies and branches,
whose highest immunoreactivity corresponds to the base
of the GlL, where the populations of periglomerular cells
are highest.
P5 (Fig. 7G and H). Densely labeled astrocytes and
their processes are identified in the GlL and also in
other layers of the accessory bulb; projections of these
astrocytes close to the glomeruli are typical at these
stages. No significant changes are seen in older samples,
apart from a logical increase in the number of these cells.
Adults. In adults, the AOB is a structure that is
semilentil in shape, located in the superior, central, and
most posterior part of the MOB, and consequently covered by the frontal pole of the brain. It measures about
650 3 700 3 1,100 microns in height, width, and length,
respectively, values that have been calculated in postfixed material. Transverse and sagittal sections clearly
show its topography and other general anatomical
details (Fig. 8A and B). Vomeronasal axons run between
the two main bulbs, reach the AOB laterally, in the central part of it, and then branch out (Fig. 8C).
Although the base of the GlL is well established by
the presence of a considerable number of periglomerular
cells, and consequently the separation between this
layer and the next is generally quite evident, the glomeruli of the AOB do not follow the typical pattern displayed in the MOB due to the scarcity and the disorganized distribution of the periglomerular cells and even, in
some cases, their absence around the glomeruli (Fig. 9).
As a result of the heterogeneity of the periglomerular
cells, the boundary of the glomeruli is neither clearly
nor uniformly defined in this bulb. Nevertheless, when
using different markers, it is possible to identify the contribution of the terminals of the vomeronasal nerves, of
the dendrites of the M/T cells, and of periglomerular
cells that participate in the organization of true glomeruli (Fig. 10).
Mitral/tufted cells, which in all cases show a large
soma, are sparse and dispersed, and consequently M/T
cells are not organized into rows (Fig. 11A and B); for
the same reason, it is also complicated to define a limit
between the M/TcL and the EPL/IPL; in fact, it seems
that some M/T cells invade these strata. It would probably be better to refer to undefined spaces or bands
rather than true plexiform layers. Similarly to the P6–
P12 stages, the presumptive EPL is present with the
same characteristics as before, and in thin paraffin sections this layer is easier to identify in some preparations
(Fig. 8). Dendrites from M/T cells that cross the EPL,
and which are typical in the MOB, are only occasionally
found in the AOB, in sections stained by the Nissl
method (Fig. 11C and D). Finally, the LOT, as a relatively wide band of white matter, goes through the AOB
and is located between the M/TcL and GrL.
DISCUSSION
Techniques and Methodology
It is noteworthy that the expression of different
markers, lectins and antibodies, shows variations depending on the processing of the olfactory bulb, that is
to say, paraffin or frozen sections, as well as in relation
to the fixatives employed, in our case paraformaldehyde
and Bouin. Moreover, small variations introduced in
the protocols employed could also modify the results
obtained.
PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
Fig. 10. Sagittal sections of the AOB in adult mice. A: Anterior part
of the AOB labeled with LEA showing glomeruli (arrows) more densely
stained than the adjacent tissue and their disordered distribution.
B: MAP-2-labeled dendrites; their superficial process form glomeruli
(arrows). C: GFAP-labeled astrocytes somata (arrows) and processes,
more densely stained in the proximal (solid arrowheads) than in the
distal (open arrowheads) parts. Scale bars ¼ 100 mm (C); 250 mm (A
and B).
The differences between paraffin and cryostat sections
are very well known in morphology (Baker, 1966), and
these differences are also applicable to the case of the
AOB of mice. However, because of the reduced size of
the structure in this animal, it is not possible to obtain
enough sections to be stained alternatively with different techniques. In this situation, we have opted to use
only frozen material for GFAP, frozen brains and material embedded in paraffin for Nissl, and samples embedded in paraffin for the rest. We have also verified that
using Bouin as fixative to lectins, GAP-43, and OMP
1021
gives better results, while paraformaldehyde was selected for Nissl and MAP-2.
With regard to the use of lectins, it is interesting to
remember that in some cases lectins with the same putative sugar affinity can result in different labeling patterns (Shapiro et al., 1995). In the same study, the
authors mentioned that ‘‘care must be taken in the
interpretation of results from different lectins, different
species, and different systems,’’ a statement with which
we agree. Specifically, in the study of the AOB in
rodents, numerous technical details can modify the
degree of labeling, as was largely discussed previously
(Salazar and Sánchez Quinteiro, 1998).
In relation to the antibodies employed in the present
work, we have tested three different chromogens (DAB,
VIP, and SG) and no significant differences were
detected in the corresponding sections. Moreover, we
have verified that the typical expression for GFAP
(Bignami et al., 1972) and MAP-2 (Matus, 1988) was
regularly shown in the AOB at all ages studied.
Nevertheless, according to the protocol that we followed, our results show that the immunoreactivity for
the other two antibodies differs a little from what was
expected. GAP-43 stains systematically the same tissue
in all postnatal samples, an interesting issue because
this antibody is especially recommended for the identification of growing axons (Skene, 1989), albeit literature
informs that other brain tissue is also reactive in particular circumstances (Verhaagen et al., 1989; Mosevitsky,
2005).
Albeit OMP was first considered a ‘‘gold standard
marker’’ exclusively for mature olfactory receptor neurons (Bailey et al., 1999), it has also been applied to
vomeronasal receptor neurons in the opossum (Shnayder
et al., 1993) and in adult mice and rats (Jia and Halpern, 1996). Our results corroborate that OMP labels
the two most superficial layers of both bulbs after birth,
but with considerable difference in the degree of intensity between AOB and MOB, even at high concentrations (Fig. 4E and F). As similar differences in the
immunoreactivity were observed in adults (results not
shown) and neurogenesis of AOB precedes that of the
MOB (Hinds, 1968a; Brunjes and Frazier, 1986), it suggests that the low density in the VNNL and GlL of the
AOB is not related to age but to the peculiarities in the
organization of the mentioned layers.
Form and Topography of AOB
During growth, the form of the AOB changes from an
external convexity to a concavity and this variation
forces cells to be closer, and consequently it is quite difficult for cells to align themselves in rows. This is particularly evident in the M/T cells but also in other populations of the AOB, which organize strata or layers, as in
the case of GlL (see transversal sections of the bulb).
Another interesting issue concerns the topography of
the AOB and the characteristic arrival of the vomeronasal
axons to it, which could be related to the morphological
and functional division of this bulb. Using different methodologies and strategies, it has been demonstrated that
there is a functional/topographical relationship between
the sensory epithelium of the VNO and the AOB (Imamura
et al., 1985; Jia and Halpern, 1996; Sugai et al., 2000;
Dulac and Torello, 2003). The vomeronasal receptors
1022
SALAZAR ET AL.
Fig. 11. Sagittal sections of the AOB (left column) and MOB (right column) in adult mice. A and
B: Comparison between form, size, and distribution of the M/T cells of the AOB (brackets) and the mitral
cells (Mcs) of the MOB (brackets). C: Dendrites from the M/T cells in the AOB (arrows) are sparse.
D: Dendrites of the mitral cells are numerous and uniformly orientated (arrowheads). Scale bar ¼ 250 mm.
situated in the apical epithelium of the VNO send information to the anterior AOB, while those receptors located in
the basal part send their axons to the posterior AOB, that
is to say, between both structures, VNO/AOB, there is a
zone-to-zone projection. Recently, an elegant experiment
has been published about the same issue (Knöll et al.,
2003). This topographical or zonal organization was previously established between the sensory epithelium of the
main olfactory system and the MOB, although in this case
four main zones were defined: I, II, III, and IV (Vassar
et al., 1993; Ressler et al., 1994; Yoshihara et al., 1997;
Mori et al., 2000).
As a consequence of this kind of arrangement, it was
thought that there could be a corresponding expression
for the olfactory information during development. Some
authors demonstrated that the formation of the olfactory
bulb (olfactory glomeruli) occurred following a distinct
rostrocaudal gradient in the MOB (Bailey et al., 1999).
Others authors suggest that the same rostrocaudal gradient is applicable to the AOB in the rat (Schwarting
et al., 1992) and even in the mouse (von Campenhausen
et al., 1997).
Our present and previous (Salazar et al., 2001; Salazar and Sánchez Quinteiro, 2003) studies have confirmed the division of the AOB into rostral and caudal
parts, but this fact does not necessarily imply its formation (glomeruli) following the same gradient during development. It is true that LEA labels only the anterior
AOB but other lectins, the BSI-B4 for example, stain
exclusively the posterior division at the same stages.
Moreover, dendritic processes revealed by MAP-2 extend
superficially both in the rostral and caudal part, while
the information obtained by Nissl material is uniform
and does not reveal anything conclusive.
Microscopy and Stratification of AOB
Hinds’s publications (1968a, 1968b) were fundamental
to a better understanding of some aspects of the development of the MOB and AOB in the mouse. Moreover, his
publications benefited from the consideration of the
bulbs in this species as a whole. In our case, we agree
with Hinds’s results with regard to the formation of the
AOB from a mass of cells, which appear early in development and later evolve into M/T cells and also the
stratified organization of its final structure. However,
there are discrepancies between Hinds’s work and our
study, mainly due to the difference in our objectives, and
the use of different methodologies and strategies.
By means of autoradiography, at the time an innovative technique, Hinds (1968a) focused his study on the
definition of the time of origin of neuroblasts and neurons, and glioblasts and neuroglia in both bulbs. Hinds
did not pay special attention to either the morphological
characteristics of the cells or their distribution into
organized layers.
As regards the presence or not of mitral and/or tufted
cells in the AOB of mice, there is clear evidence of a
mass of cells in the middle of the AOB, although it is
not easy to fit these cells into one group or the other.
Cajal (1902) thought that their morphology suggested
that these cells were typical tufted cells, while Hinds
PERINATAL AND ADULT ACCESSORY OLFACTORY BULB
(1968a) was of the opinion that the same cells, which
had a time of origin more similar to the mitral cells of
the MOB than to the tufted cells, were in fact mitral
cells. By means of a simple comparison between this
group of cells in the AOB and the mitral cells of the
MOB in the same samples, we consider this group of
cells to be closer to being tufted (size, arrangement, and
distribution) than mitral (topography) cells. In dealing
with this controversy, most authors have opted for the
term ‘‘mitral/tufted cells.’’ Further morphological information will address the true nature of these cells.
In any case, it may be stated that there is general
agreement that the so-called M/C cells send multiple
dendrites to different glomeruli (Cajal, 1902; Allison,
1953; Takami and Graziadei, 1991). We have corroborated this in mice (results not shown).
Likewise, Cajal (1902) considered the glomeruli of the
AOB to be characterized by ‘‘the lack of definition, indistinctness, and small size of the glomerular territories,
which, rather than independent plexus, form a continuous plexiform band, outlined in festoons or unevenness
on the deep side.’’ He added that this organization of the
glomeruli was quite similar to the same structures in
some reptiles and amphibians.
It is true that the most relevant detail of the glomeruli
morphology in the AOB of the mouse is its lack of definition, although not exclusively due to their size, but to
the paucity of the periglomerular cells, most of which
are located on the basal part of the GlL. Our data suggest that some glomeruli of the AOB have the same or
similar size as those of the MOB (data not shown).
Finally, the last comment of the present discussion is
devoted to the stratification of the AOB, a controversial
issue mainly when attempts are made to generalize for
all mammals. The diversity in the mammalian AOB is
so huge (Meisami and Bhatnagar, 1998) that it is inadequate to extrapolate data from one species to another.
For example, concerning the plexiform layers, unpublished results of Meisami and Bhatnagar (1998) indicate
that in mammals with large and well-developed AOBs,
both plexiform layers do exist. On the contrary, Cajal
(1902) concluded that the mentioned layers were absent.
Classical comparative studies of the olfactory system
(Crosby and Humphrey, 1939; Allison, 1953) included
the EPL in its description of the AOB but not the IPL.
Another interesting example, which affects the stratification of the AOB, is the topography of the LOT, situated through or under the accessory bulb.
It is noteworthy that, the obvious morphological diversity of the AOB among mammals, corroborated histochemically and immnunohistochemically (Halpern et al., 1998),
is unresolved and could have functional implications.
In mice, a meticulous observation of the paraffin and
cryostat series of the AOB reveals sections in which
there is a narrow irregular space between the periglomerular basal cells and the M/T cells, the EPL, and
other sections in which it is completely absent. Due to
the variability of this space, the existence of the EPL is,
in our opinion, a subjective issue. In this sense, to
enclose both presumptive plexiform layers in the M/TcL
would be an alternative to be considered.
The most evident limits in the general stratification of
the AOB in mice are represented by the important population of periglomerular basal cells and by the presence
of the LOT.
1023
Fig. 12. Schematic representation of the development of layers formation in the AOB in mice. A: Before birth, E16. B: After birth, P3.
C: Adult. fVNNL, future vomeronasal layer; fGrL, future granular layer.
Scale bars ¼ 250 mm.
Conclusions
In spite of its size, the only hindrance to the accurate
study of the AOB in mice, we are convinced that it is
possible, and necessary, to build a consistent morphological model of the AOB in this species.
Furthermore, given the general characteristics previously considered, we are led to the conclusion that the
AOB in mice is an undefined structure in terms of cell
characteristics and distribution, more markedly so if it
1024
SALAZAR ET AL.
is compared with the MOB. It is noteworthy, however,
that the general stratification of the AOB in mice may
be more clearly understood following the steps in its
transition from nonnatum to juvenile and adult mice
(Fig. 12). Likewise, at stage P5, the definitive building of
glomeruli is concluded.
ACKNOWLEDGMENTS
The authors thank M. Otero, L. Bellón, and J. Castiñeira for their excellent technical assistance, and J. Curtin for the revision of the English text. Dr. N. Vandenberghe provided helpful comments on this manuscript
and contributed to valuable discussions. The comments
of two anonymous reviewers greatly improved the clarity
of the manuscript.
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