THE JOURNAL OF COMPARATIVE NEUROLOGY 438:468 – 489 (2001)
Ultrastructural Localization of GProteins and the Channel Protein TRP2
to Microvilli of Rat Vomeronasal
Receptor Cells
BERT PH. M. MENCO,1* VIRGINIA McM. CARR,1 PATRICK I. EZEH,1
EMILY R. LIMAN,2 AND MAYA P. YANKOVA1
1
Department of Neurobiology and Physiology, Northwestern University, Evanston,
Illinois 60208-3520
2
Department of Biological Sciences/Neurobiology, University of Southern California,
Los Angeles, California 90089-2520
ABSTRACT
Microvilli of vomeronasal organ (VNO) sensory epithelium receptor cells project into the
VNO lumen. This lumen is continuous with the outside environment. Therefore, the microvilli
are believed to be the subcellular sites of VNO receptor cells that interact with incoming
VNO-targeted odors, including pheromones. Candidate molecules, which are implicated in VNO
signaling cascades, are shown to be present in VNO receptor cells. However, ultrastructural
evidence that such molecules are localized within the microvilli is sparse. The present study
provides firm evidence that immunoreactivity for several candidate VNO signaling molecules,
notably the G-protein subunits Gi␣2 and Go␣, and the transient receptor potential channel 2
(TRP2), is localized prominently and selectively in VNO receptor cell microvilli. Although Gi␣2
and Go␣ are localized separately in the microvilli of two cell types that are otherwise indistinguishable in their apical and microvillar morphology, the microvilli of both cell types are
TRP2(⫹). VNO topographical distinctions were also apparent. Centrally within the VNO sensory
epithelium, the numbers of receptor cells with Gi␣2(⫹) and Go␣(⫹) microvilli were equal. However, near the sensory/non-sensory border, cells with Gi␣2(⫹) microvilli predominated. Scattered
ciliated cells in this transition zone resembled neither VNO nor main olfactory organ (MO)
receptor cells and may represent the same ciliated cells as those found in the non-sensory part of
the VNO. Thus, this study shows that, analogous to the cilia of MO receptor cells, microvilli of
VNO receptor cells are enriched selectively in proteins involved putatively in signal transduction.
This provides important support for the role of these molecules in VNO signaling. J. Comp.
Neurol. 438:468 – 489, 2001. © 2001 Wiley-Liss, Inc.
Indexing terms: immunolocalization; freeze substitution; olfaction; signal transduction; Gi␣2; Go␣;
transient receptor potential channel
In several classes of vertebrates and insects, there are
two olfactory pathways that play different but overlapping
roles. One of these detects predominantly gender and conspecific exuded odors, whereas the other detects more
general odors (Hildebrand and Shepherd, 1997; Johnston
2000). In vertebrates there is some overlap between the
perceptive modalities of main (MO) and vomeronasal olfactory organs (VNO; Johnston, 2000). However, it is assumed that in most vertebrates, the MO has evolved to
detect general odors whereas the VNO is specialized for
endocrine responses to odors and prey recognition, the
latter especially in some reptiles (Halpern, 1987; Keverne,
1999; Dulac, 2000; Johnston, 2000).
© 2001 WILEY-LISS, INC.
The signaling pathways of MO and VNO are parallel,
but individual components differ (Berghard et al., 1996;
Wu et al., 1996; Tirindelli et al., 1998). Although a subset
Grant sponsor: NIH-NIDCD; Grant number: DC02491 (BPhMM); Grant
number: DC02774 (VMcMC); Grant number: DC04564 (ERL); Grant sponsor: NSF; Grant number: IBN-0094709 (BPhMM).
*Correspondence to: Dr. Bert Ph. M. Menco, Department of Neurobiology
and Physiology, O.T. Hogan Hall, 2153 North Campus Drive, Northwestern University, Evanston, IL 60208-3520.
E-mail: [email protected]
Received 28 November 2000; Revised 19 April 2001; Accepted 4 July
2001
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
469
TABLE 1. Antibodies Tested for Their Fine Structural Presence in VNO Tissues1
Antibodies
Gi␣12
Gi␣1-32
Gi␣1,22
Go␣2
TRP22
Supplier
Catalog no.
Peptide
Santa Cruz
I-20, sc-391
Biotechnology3
Santa Cruz
C-10, sc-262
Biotechnology
Calbiochem4
371723-S
Santa Cruz
K-20, sc-387
Biotechnology
Fusion protein as described by Liman et al. (1999)
Polyclonal or
monoclonal
93-112
Polyclonal
345-354
Polyclonal
245-254
105-124
Polyclonal
Polyclonal
Peptide sequence
Highly divergent internal region,
IDFGDSARADDARLQLFVLAG
C-terminal, KNNLKECGLY
C-terminal, KNNLKDCGLF
Divergent internal region,
KMVCDVVSRMEDTEPFSAEL
Polyclonal
1
Antibodies that made a case for VNO signaling at the level of receptor cell microvilli are listed. Blocking controls, using the antigenic peptides, were performed for the antibodies
listed here.
2
Antibodies affinity purified.
3
Santa Cruz, CA.
4
La Jolla, CA.
TABLE 2. Antibodies Tested for Their Fine Structural Presence in VNO Tissues1
Antibodies
Gi␣22
Go␣4
Gs␣4
Gs␣2
Golf␣2
G␣q/114
G␣q/112
Type II adenylyl cyclase2
Type III adenylyl cyclase2
OCNC1 (subunit 1, olfactory cyclic nucleotide gated channel)2
IP3 (inositol 1,4,5 trisphosphate) receptor4
Olfactory marker protein4
Supplier
Chemicon3
NEN5
NEN6
Calbiochem7
Jones and Reed (1989)6,8
NEN
Santa Cruz Biotechnology9
Santa Cruz Biotechnology
Bakalyar and Reed (1990)6,8
Matsuzaki et al. (1999)6,10
See Kalinoski et al. (1994)11
Margolis (1982)12
Catalog no.
MAB3077
NEI-804
NEI-805
372732-S
DJ6.3AP
NEI-809
C-19, sc-392
C-20, sc-587
HAB-1
Polyclonal or
monoclonal
Monoclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
Polyclonal
1
Antibodies that gave ambiguous or negative results in the VNO are listed. These include antibodies to MO signaling proteins (see footnote 6). Blocking controls, using the antigenic
peptides, were only performed for the antibodies in Table 1.
Antibodies affinity purified.
3
Temecula, CA. This antibody labeled dendritic endings and microvilli of VNO receptor cells in a fine structural study that made use of pre-embedding immunocytochemistry
(Matsuoka et al., 2001).
4
Not purified antisera.
5
Boston, MA.
6
Same as used in the MO. Labeled predominantly olfactory receptor cell cilia (Menco et al., 1994; Menco, 1997a; Matsuzaki et al., 1999).
7
La Jolla, CA.
8
Gift of Dr. R.R. Reed, Johns Hopkins University, Baltimore, MD.
9
Santa Cruz, CA.
10
Gift of Dr. G.V. Ronnett, Johns Hopkins University, Baltimore, MD.
11
Gift of Dr. D.L. Kalinoski, University of Miami, FL.
12
Gift of Dr. F.L. Margolis, University of Maryland, Baltimore, MD. This antibody worked well in an other fine structural study, but that study made use of pre-embedding
immunocytochemistry (Johnson et al., 1993). For MO, see Menco (1994).
2
of MO receptor cells may make use of a different cascade
(Meyer et al., 2000), the signaling proteins that are activated sequentially by the interaction between odors and
odorant receptors are the same for the majority of receptor
cells. These proteins include the specific GTP-binding (or
G)-protein Golf, type III adenylyl cyclase (AC), and olfactory cyclic nucleotide gated channels (OCNCs; Buck,
2000). All of these molecules are particularly abundant in
specialized cilia (Menco, 1997a; Menco and Morrison,
2002).
VNO receptor cells bear, for still unclear reasons, microvilli instead of cilia (for rat: Vaccarezza et al., 1981).
Also differing from the situation in the MO are results
from immunocytochemical studies on G-proteins that
showed that rodents have two major populations of VNO
receptor cells that each makes use of a different signaling
cascade. One cell type expresses ␣-subunits of the heterotrimeric G-protein Gi2, the other ␣-subunits of Go (Jia and
Halpern, 1996). Both of these are fairly closely related
(Fig. 1 on p. 297 in Watson and Arkinstall, 1994), are
pertussis-toxin sensitive, and may play a role in a phospholipase C (PLC)-dependent phosphoinositide hydrolysis
(Watson and Arkinstall, 1994), also in the VNO (Dulac,
2000; Holy et al., 2000). Immunocytochemistry showed the
Gi␣2(⫹) and Go␣(⫹) cells to be evenly distributed along the
length of the VNO neuroepithelium. However, somata of
Gi␣2-expressing cells are localized more superficially in
the VNO neuroepithelium than somata of Go␣-expressing
cells (Jia and Halpern, 1996), a finding confirmed by in
situ hybridization (Berghard and Buck, 1996). Light microscopic (LM) studies with different antibodies further
demonstrated that immunoreactivity (IR) for both Gi␣2
and Go␣ occurs at the luminal edge of the epithelium,
which is consistent with antigen localization in receptor
cell apices and microvilli (Berghard and Buck, 1996).
There is a correlation between the expression of each of
the VNO G-proteins and two heptahelical odorant superfamilies, V1R for Gi␣2 and V2R for Go␣ (Herrada and
Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). (A third family, V3R [Pantages and Dulac,
2000], appears to be a group within the V1R superfamily
[Mombaerts, 2001].) Furthermore, in situ hybridization
demonstrated that a member of the transient receptor
potential (TRP) channel family, TRP2, is amply present in
both Gi␣2(⫹) and Go␣(⫹) cells. Immunocytochemistry in
isolated single rat VNO receptor cells demonstrated that
TRP2 and Gi␣2 are highly restricted in what are presumed
to be receptor cell microvillar structures. Therefore, it was
proposed that TRP2 is a likely candidate for current generation in VNO receptor cells (Liman et al., 1999). This
470
B.Ph.M. MENCO ET AL.
Fig. 1. Immunoblots for affinity-purified antibodies to Go␣ (A),
Gi␣1,2 (B), Gi␣1-3 (C), and Gi␣1 (D) in pellet (P) and supernatant (S)
membrane preparations of rat VNO epithelia. Antibodies to Go␣, Gi␣1,
and Gi␣1-3 were used at dilutions of 1:200 and antibodies to Gi␣1,2 at a
dilution of 1:1,000. Bands of appropriate molecular weight (kDa) are
marked with an arrow. All antibodies, apart from those to Gi␣1,
labeled proteins of the appropriate weights. For S, the listed aliquots
correspond to 13 ␮g (4 ␮l) to 52 ␮g (16 ␮l). For P, they correspond to
2.5 ␮g (2 ␮l) to 27.5 ␮g (22 ␮l).
channel may be active in the VNO across a wide variety of
species, including some reptiles (Murphy et al., 2001).
V1R(⫹)/Gi␣2(⫹) and V2R(⫹)/Go␣(⫹) VNO receptor cells
project to a distinct region of the accessory olfactory bulb
(AOB), the VNO’s first relay station in the brain. V1R(⫹)/
Gi␣2(⫹) cells project anteriorly and V2R(⫹)/Go␣(⫹) cells
project posteriorly (Berghard and Buck, 1996; Jia and
Halpern, 1996; Herrada and Dulac, 1997; Matsunami and
Buck, 1997; Ryba and Tirindelli, 1997; Sugai et al., 1997;
Halpern et al., 1998; Wekesa and Anholt, 1999). The
V1R(⫹)/Gi␣2(⫹) signaling cascade may be involved in the
recognition of gender-specific volatile pheromones in rodents. The V2R(⫹)/Go␣(⫹) cascade may subserve recognition of conspecific nonvolatile proteinaceous compounds
that need not be gender or strain specific (Cavaggioni et
al., 1999; Inamura et al., 1999; Keverne, 1999; Krieger et
al., 1999; Dulac, 2000).
Fine structural (immuno)cytochemical characterizations of VNO receptor cells have shown that apices and
microvilli of rat VNO receptor cells contain Gi␣2 and Go␣
(Matsuoka et al., 2001) and, in a subset of these cells,
putative receptor VN6 (Takigami et al., 1999). mRNA for
another VNO receptor, V2R-8, has been localized in microvillous receptor cells in the gold fish (Anderson et al.,
1999). Optimally preserving tissue antigenicity, by using
freeze-substitution electron microscopy (EM), combined
with postembedding immunogold immunocytochemistry
(Gilkey, 1993; Griffiths, 1993; Menco, 1995), this study
demonstrates that Gi␣2, Go␣, and TRP2 are expressed
much more abundantly in the VNO sensory microvilli
than in other parts of the VNO receptor cell apices. Moreover, at the level of these microvilli, each of these proteins
has its own peculiar distribution across the epithelial surface (preliminary reports: Menco, 1997b; Menco and
Yankova, 1999).
MATERIALS AND METHODS
Animals
Forty adult male Sprague Dawley rats (Harlan, Indianapolis, IN), 2–3 months old, were used in this study. All
Fig. 2. Frontal sections through VNO showing sensory and nonsensory epithelia (s and ns in A and D). Sensory surfaces demonstrate
IR for purified rabbit polyclonal antibodies to Gi␣1-3 (A,D; 1/1,000),
Gi␣1,2 (B,E; 1/100), and Go␣ (C,F; 1/1,000) (arrows). For controls, PBS
was used instead of primary antibodies (G,H). I: Schematic map that
links the LM survey presented here with the fine structural topographical part of the study (Figs. 6A,B, 8-10; the manner in which the
regions are numbered is explained in the Materials and Methods).
Immunoabsorption with excess antigenic peptide suppressed the labeling in all cases (Figs. 7A,B). The boxed areas in A-C and G are
presented at higher magnifications in D-F and H. VNO sensory sur-
face IR was more intense for Gi␣1-3 (A,D) than for Gi␣1,2 (B,E) and for
Go␣ (C,F). Despite this, we elected to use the Gi␣1,2 antibodies rather
than those to Gi␣1-3 for most of the study to demonstrate Gi␣2(⫹) cells
because of problems of cross-reactivity with Go␣ (see beginning of
Results section). The arrowheads point to the non-sensory epithelium,
where anti-Gi␣1,2 gave some reaction, most likely background, as it
could not be blocked with excess antigenic peptide. Areas labeled 1, 2,
19, and 20 (I) consist mainly of transitional epithelium (Figs. 6B and
8). Upper case indicators in A and I: D, dorsal; L, lateral; V, ventral;
M, medial. Scale bars ⫽ 100 ␮m.
472
B.Ph.M. MENCO ET AL.
Fig. 3. Electron micrograph of the neurosensory epithelial surface
of a conventionally fixed rat VNO embedded in Epon. The surface
contains apices of receptor cell dendrites (asterisks) and of nearby
supporting cells (triangles). The latter tend to be somewhat more
electron opaque than the former. The dendritic apices often show
procedures described below were performed in accordance
with Federal and NIH animal use guidelines and
institution-approved protocols.
Antibodies and peptides
All antibodies were rabbit polyclonal IgGs, unless mentioned specifically. Antibodies used to localize Gi␣2, Go␣,
and TRP2 in the VNO receptor microvilli are listed in
Table 1. Antibodies to the same proteins that did not work
well, antibodies to other proteins believed to be present in
VNO receptor cells (Berghard and Buck, 1996; Wekesa
and Anholt, 1997), and antibodies to MO signaling proteins used as controls (Menco, 1997b) are listed in Table 2.
The antigenic peptides used to generate the Gi␣1,2 and
Gi␣1-3 antibodies differed from the corresponding region of
Go␣ in four and three amino acids, respectively (Watson
clusters of centrioles (curved arrows). Thin microvilli sprouting from
these apices are located close to the epithelial surface (straight arrows). The supporting cells have thicker and straighter microvilli
(serpentine-shaped arrows), the course of which is roughly perpendicular to that of the receptor cell microvilli. Scale bar ⫽ 1 ␮m.
Fig. 4. Antibodies to Gi␣1,2 (A; 1/10) and Go␣ (B; 1/10) bound to
microvilli (straight arrows) of VNO receptor cells. The cell’s apices
(large asterisks), including their membranes (marked by the straight
lines parallel to the apices with large asterisks), as well as apices and
microvilli of nearby receptor cells (small asterisks), and apices (triangles) and microvilli (serpentine-shaped arrows) of supporting cells
display virtually no labeling. There is a gradient of reduced microvillar labeling in the direction of neighboring cells away from apices of
cells with labeled microvilli, suggesting microvillar dilution at some
distance from the cells from which these microvilli sprout (see also
Figs. 8 and 9 and Tables 3 and 4). The curved arrows mark VNO
receptor cell centrioles. Apart from Figure 6D, all tissues in this and
subsequent figures were perfusion fixed with paraformaldehyde, rapidly frozen, freeze substituted, and low temperature embedded in
Lowicryl K11M. The gold particles were 10 nm across. Scale bars ⫽
1 ␮m.
Figure 4
Fig. 5. VNO section as in Figure 4, but immunoreacted with TRP2
(1/10). Although the apical membranes of the receptor cell dendrites
(indicated by a short straight line parallel to the apex of one of the
cells marked with a large asterisk; its centrioles are marked by a
curved arrow) may have bound some of the antibodies, most IR was
found on the receptor cell microvilli (straight arrows). In contrast to
the situation shown in Figure 4, where microvilli of only some of the
receptor were labeled, anti-TRP2 appears to have labeled the microvilli of all the receptor cells (large asterisks; see also Fig. 11).
Supporting cell apical (triangle) structures, including microvilli
(serpentine-shaped arrow), were TRP2(-). The gold particles were 15
nm across. Scale bar ⫽ 1 ␮m.
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
and Arkinstall, 1994). Antibody and peptide dilutions are
listed in the appropriate figure captions.
Immunoblots: tissue preparation
Twenty rats were anesthetized deeply with 85 mg/kg
sodium pentobarbital (Anthony Products, Arcadia, CA),
intraperitoneally (ip). Dissected VNO tissues were frozen
rapidly in liquid nitrogen and stored at ⫺80°C. For the
membrane preparation (Mishra, 1986), thawed tissues
were homogenized in 50 mM Tris (pH 7.4), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM p-tosyl-Larginine methyl ester (TAME), 1 mM dithiothreitol (DTT),
320 mM sucrose, 1 mM ethylenediamine tetra-acetic acid
(EDTA), and 10 mM sodium metavanadate, by using a
Tissumizer威 homogenizer (Tekmar, Cincinnati, OH). The
sample was then centrifuged at 550 ⫻ g for 5 minutes at
4°C with a J2-21 centrifuge (Beckman, Palo Alto, CA). The
pellet was discarded. The supernatant was centrifuged at
23,000 ⫻ g for 15 minutes at 4°C. The 23,000 ⫻ g pellet
was resuspended. The pellet and supernatant were aliquoted (100 ␮l). The protein content of each was determined by using the Bradford (1976) method for the supernatant (3.3 ␮g/␮l) and pellet (1.2 ␮g/␮l). The aliquoted
samples were then stored at ⫺80°C for immunoblotting
assays.
Immunoblots
After 10% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), samples were transferred to
polyvinylidene fluoride (PVDF) membranes (Bio-Rad,
Hercules, CA; Harlow and Lane, 1988). Transfers were
blocked with 5% nonfat dry milk (1 hour). IR was assessed
with secondary peroxidase-conjugated goat-anti-rabbit
(GAR) IgG (1:5,000; Jackson ImmunoResearch Labs.,
West Grove, PA), chemiluminescence reagent (NEN, Boston, MA), and Kaleidoscope prestained standards (BioRad) for molecular weights.
LM: tissue preparation
Deeply anesthetized adult rats were perfused transcardially with freshly prepared phosphate-buffered saline
(PBS), pH 7.0, followed by Bouin’s fixative. The heads,
trimmed of lower jaws, teeth, soft palates, skin, and muscle, were postfixed in fresh fixative overnight at 4°C,
rinsed several times in 50% alcohol, decalcified in RDO威
(Apex Engineering, Plainfield, IL), rinsed in running water, dehydrated through ethanol, cleared in Histosol威 (National Diagnostics, Atlanta, GA), and embedded in Paraplast威 (Sherwood Medical, St. Louis, MO). Sections
(coronal, 10 ␮m) through the VNO and AOB were
mounted on silanated slides (slides coated with 3⬘aminopropyl triethoxy silane, Sigma, St. Louis).
LM: immunocytochemistry
Deparaffinized (xylene) sections were rehydrated in a
graded series of ethanol, preincubated as directed at room
temperature, and incubated with the affinity-purified antibodies at appropriate dilutions in PBS for 2 hours at
37°C. IR was visualized by using the rabbit Vectastain
ABC Elite威 kit (Vector, Burlingame, CA) with diaminobenzidine as the chromogenic agent.
EM: conventional methods
Deeply anesthetized rats were perfused with Karnovsky’s (1965) aldehyde fixative in 0.1 M sodium cacody-
475
late, pH 7.4. Dissected VNOs were further fixed (2 hours),
rinsed, postfixed in 1% OsO4, block stained with 3% uranyl acetate (UAc) in 50% ethanol, dehydrated, infiltrated
with Epon (Polysciences, Warrington, PA) through propylene oxide, and embedded in Epon. Poststained (3% UAc
in distilled H2O) and counterstained (lead citrate; Reynolds, 1963) ultrathin sections (Leica/Reichert Ultracut S,
Vienna, Austria) were examined in a JEM-100-CX II
transmission EM at 120 kV (JEOL, Tokyo, Japan).
EM: rapid freezing and freeze substitution
Some antibodies work better in fixed tissues, others in
unfixed tissues (Menco, 1995). For the preparation of unfixed tissue, animals were asphyxiated with CO2 (1,000
pounds per square inch, 30 – 45 seconds, until there was no
heartbeat). VNOs were frozen rapidly on a liquid nitrogencooled copper block (Gentleman Jim Quick-Freezing System, Energy Beam Sciences, Agawam, MA; Phillips and
Boyne, 1984). Other animals were perfusion fixed with 4%
paraformaldehyde ⫹ 0.15 mM CaCl2 in 100 mM Sorenson’s phosphate buffer, pH 7.2. VNOs were further fixed
overnight at 4°C by using the same fixative but in 100 mM
bicarbonate (pH 9.4), washed, cryoprotected in sucrose/
glycerol mixtures, and frozen rapidly in liquid propane by
using the Gentleman Jim. Unfixed tissues were freeze
substituted (Leica/Reichert CS Auto) through acetone and
fixed tissues through methanol (both at ⫺80°C). The hydrophilic methacrylate resin Lowicryl K11M (Chemische
Werke Lowi G.m.b.H., Waldkraiburg, Germany), which
polymerizes with ultraviolet light at low temperatures,
was used for infiltration (⫺80°C to ⫺60°C) and embedding
(⫺60°C to ⫹25°C; all in the CS Auto). The entire procedure lasted about 1 month (Berod et al., 1981; Van Lookeren Campagne et al., 1991; Menco, 1995; Menco et al.,
1997).
EM: postembedding immunocytochemistry
Sections to be used for immunocytochemistry were
mounted on 300 mesh thin-bar hexagonal nickel Gilder
grids (EM Sciences, Fort Washington, PA). Blocking (2–3
hours) and antibody dilution was done with 0.1% acetylated bovine serum albumin (Ac-BSA; EM Sciences;
Leunissen, 1990) in Trizma-buffered saline, 10 mM Tris,
500 mM NaCl, pH 8.0 (TBS). Incubation with primary
antibodies was done overnight at 4°C without intermediary wash. Their binding was visualized with 10 or 15-nm
gold particles conjugated to secondary goat-anti-rabbit
(GAR) or rabbit-anti-goat (RAG) for polyclonal primary
antibodies and to goat-anti-mouse (GAM) for monoclonal
antibodies (EM Sciences). Gold colloids were diluted in 20
mM Tris, 150 mM NaCl, 0.1% Ac-BSA, pH 8.2, to an
optical density of about 0.10 at 520 nm (Menco et al.,
1994).
Identification of cells in sequential sections
Most of the primary antibodies used were generated in
the same species, making double labeling of these antibodies problematic. To avoid questions of secondary antibody cross-reactivity, immunocytochemistry for different
primary antibodies was carried out on adjacent sections.
Large composite micrographs were prepared of the same
region of epithelium in adjacent serial sections that had
been immunoreacted with antibodies to either Gi␣2 or Go␣.
The same was done for each of these subunits and TRP2.
Figure 6
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
This allowed careful examination of matched regions of
VNO neuroepithelium in sequential sections. The composites were delimited by the bars of the 300-mesh hexagonal
grids holding the sections. These areas could be up to 73
␮m in diameter, the maximum hole width of these grids
(EM Sciences Catalogue XIII, p. 61). Sets of composites
consisted of up to 80 individual micrographs (4 sections,
20 exposures). Low power magnifications (100⫻ to 200⫻)
served as a guide for matching of regions in the higher
power micrographs. Successive regions, labeled 1 through
9 and 19 and 20 (see Fig. 2I), are defined by the successive
occurrence of the VNO neuroepithelial surface in the open
grid areas. Lowest and highest numbers mark the transitional epithelium closest to the non-sensory VNO epithelium. The highest number, 20, is selected arbitrarily because it did not overlap in any of the sections with region
numbers counting from the other side, marked 1.
Controls
Antibody specificity for the G-proteins and TRP2 was
verified by preabsorption with an excess of corresponding antigen in immunocytochemical controls. Also, the
labeling pattern of each antibody was a positive control
for all others. Tissue controls were the non-sensory area
of the VNO, the sensory cilia and supporting cell microvilli of the MO, and the non-sensory cilia and microvilli of the nasal respiratory epithelium. Antibodies
to signaling proteins of MO olfactory cilia, applied to
VNO, served as additional tissue controls (Table 2, note
6). Negative controls consisted of PBS, and in the case of
polyclonal antibodies, preimmune serum, or normal
rabbit (NRS) or goat sera (NGS), all with the appropriate blocking solutions and all used instead of the primary antibodies.
Data analysis
Densities were counted in microvillar tufts apical to the
sensory cells from which the microvilli sprout, in such
tufts above neighboring sensory cells, and in the surrounding resin (Table 3). For this, a transparent plastic
sheet with a grid divided in cm2 squares was placed over
Fig. 6. Absence of IR in non-sensory VNO cells (A-C) and labeling
patterns in unfixed tissues (D). Cilia (large asterisk in A) and microvilli (small asterisk A) of VNO ciliated non-sensory cells and microvilli of cells in the transitional region to the non-sensory epithelium (arrow in B; region 1 in Fig. 2I) did not label with anti-TRP2
(1/10). The same was true for antibodies to the G-proteins. C: The
transitional region (near region 2 in Fig. 2I) contained some cells that
resemble MO receptor cells in their apices (triangle). However, the
cilia of these cells (large arrowhead) bound neither antibodies to VNO
nor those to MO signaling molecules (here: anti-Gi␣1-3, 1/12;) as was
true for apices (small asterisk) and microvilli (small arrowhead) of
VNO-supporting cells. VNO receptor cells (large asterisk) in this area
had Gi␣2(⫹) microvilli (arrow), but none had microvilli that were
Go␣(⫹). The apex from which the microvilli sprout also showed some
IR (large asterisk). D: Antibodies to Gi␣1,2 (1/10) bound to the microvilli of a VNO receptor cell (straight arrows) in unfixed and noncryoprotected tissues. The apex of the receptor cell (large asterisk) as
well as apices (small asterisk) and microvilli of surrounding supporting cells displayed no labeling. The border between the microvillous
surface and the feature-less resin (wide arrow) is an artifact caused by
slamming the tissue on the cold copper block used for cryofixation. The
gold particles in A,B were 15 nm across and those in C,D were 10 nm.
Scale bars ⫽ 1 ␮m.
477
each micrograph. Gold grains per cm2, recalculated per
␮m2, reflecting the intensity of labeling of each antibody in
each region, were statistically analyzed by one-way analyses of variance (ANOVAs) by using Statview 4.0 on a
Macintosh computer (Menco et al., 1997).
RESULTS
Verification of IR
For proper evaluation of the fine structural studies presented here, it was necessary to verify in immunoblots and
with LM IR in VNO tissues for several of the antibodies
(Tables 1, 2). Antibodies to Gi␣2 in the VNO that were used
by others (Shinohara et al., 1992; Jia and Halpern, 1996)
were not available to us. A commercially available monoclonal antibody to Gi␣2 (Li et al., 1995) did not work in our
study (Table 2). However, it labeled dendritic endings and
the microvilli of VNO receptor cells in another, preembedding, fine structural study (Matsuoka et al., 2001).
Therefore, to examine IR for Gi␣2, two antibodies were
used, one to Gi␣1,2 and one to Gi␣1-3 (Table 1), the latter
being used in the VNO by several others (Berghard and
Buck, 1996; Murphy et al., 2001). In the AOB, the anterior
region showed IR to Gi␣1,2, whereas the posterior portion
demonstrated IR to Go␣. The IR patterns for those antibodies agree with previous findings (Shinohara et al.,
1992; Halpern et al., 1995, 1998; Jia and Halpern, 1996;
Jia et al., 1997; Sugai et al., 1997). In contrast, antibodies
to Gi␣1-3 bound equally well to the posterior, Go␣(⫹), and
anterior, Gi␣1,2(⫹), regions of the AOB, indicating that the
Gi␣1-3 antibodies displayed cross-reactivity for Go␣ (Aoki
et al., 1992). This confirmed a suspicion based on EM
observations that suggested that the microvilli of all VNO
receptor cells immunoreacted with antibodies Gi␣1-3. To
avoid this problem of cross-reactivity, antibodies to Gi␣1,2
were subsequently used mainly to demonstrate IR for Gi␣2
in the VNO. TRP2 antibodies were only used for EM; blot
and LM IR of VNO epithelia for these antibodies is shown
elsewhere (Liman et al., 1999).
Immunoblots
Western blots of membrane preparations of VNO epithelia were tested for the presence of the ␣-subunits of the
Go and Gi proteins (Tables 1, 2, Fig. 1). Bands of appropriate molecular weights were observed for all antibodies
tested, except for those to Gi␣1. The band at 39 – 40 kDa is
consistent with the molecular weight of Go␣ (Fig. 1A).
Bands at molecular weights of 40 – 41 kDa are consistent
with the molecular weights of Gi␣2 and Gi␣3 (Figs. 1B,C;
Watson and Arkinstall, 1994; for Gi␣1-3 in the VNO, see
also Murphy et al., 2001). No band was obtained for Gi␣1
(Fig. 1D), indicating that Gi␣1 is not present in the VNO.
Thus, any Gi-type labeling in the LM and EM studies
below must be due to the presence of Gi␣2 or Gi␣3. Company specifications indicated that the Gi␣1-3 and the Gi␣1,2
antibodies label Gi␣2, Gi2 being the G-protein of the Gi(⫹)
VNO receptor cells (Berghard and Buck, 1996). This validated the use here of the anti-Gi␣1-3, but especially antiGi␣1,2 (the Gi␣1-3 antibodies were of limited use because of
the problems outlined above). The blots in Figures 1A-C
also show that more G-protein IR was associated with the
pellets than with supernatants. Extra bands in both pellets and supernatants for the antibodies to Gi␣1,2 and
Gi␣1-3 may reflect degradation or aggregation products or
Figure 7
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
nonspecific impurities present in the antibody preparations.
LM
IR for Gi␣1-3 (Figs. 2A,D), Gi␣1,2 (Figs. 2B,E), and Go␣
(Figs. 2C,F) was restricted to the epithelial surface and
axons (Berghard and Buck, 1996). Anti-Gi␣1,2 also labeled
cell apices in the non-sensory VNO area (Fig. 2E); immunoabsorption controls showed this to be nonspecific and
this labeling was not seen with EM. As seen in the immunoblots, anti-Gi␣1 showed no IR over control levels (Figs.
2G,H). Figure 2I serves as a map relating the LM survey
outlined here with the EM topographical study further on
in this paper (see Figs. 8 –10).
EM: the luminal area of the rat VNO
Although the ultrastructural morphology of the rat
VNO epithelial surface with conventional EM is well established (Vaccarezza et al., 1981; Mendoza and Szabó,
1988; Garossa and Coca, 1991; Johnson et al., 1993), we
highlight some aspects that help in the interpretation of
the fine structural IR data. Receptor cell apices are wider
and appear more electron lucent than those of neighboring
supporting cells. The apices of both cell types bear microvilli. Those of receptor cells are 0.03– 0.06 ␮m wide and
3–5 ␮m long (Table 4). They tie parallel to the epithelial
surface, and close to it. Supporting cell microvilli are
thicker (0.1– 0.2 ␮m) and shorter (2.5–3.5 ␮m), tie perpendicular to the receptor cell microvilli, and their tips extend
above the layer that contains the latter (Fig. 3, see also
Fig. 12).
EM: localization of signaling proteins
With EM, the affinity-purified antibodies (Table 1) to
Gi␣1,2 (Fig. 4A), Go␣ (Fig. 4B), and TRP2 (Fig. 5) showed
highly specific IR of the microvilli of VNO receptor cells.
Anti-TRP2 also shows some IR in apical membranes of the
receptor cell dendrites in between the microvilli (Fig. 5).
Otherwise, labeling in other parts of the receptor cell
apices was much lower or absent for all antibodies. Receptor cell axons, not further considered here, were also positive for Gi␣1,2, Gi␣1-3, and Go␣, although less so than the
microvilli. Axons were TRP2-(-).
Fig. 7. Immunoabsorption (A,B) and tissue controls (C-F). A:
VNO labeled with anti-Gi␣1,2 (1/10) preabsorbed with a 10-fold molar
excess of the antigenic peptide (A; Gi␣1,2-p) or with anti-TRP2 (1/10)
preabsorbed with a thousand-fold molar excess of the antigenic peptide (B; TRP2-p) suppressed dramatically IR of the receptor cell microvilli (arrows; compare with Figs. 4A and 5). The molecular weight
of the TRP2 peptide was 40 kDa and that of the antibody approximately 160 kDa (Liman et al., 1999). C,D: All MO epithelial surface
structures were TRP2(-) (1/10). This includes receptor cells and their
cilia (C) and the apex (asterisk in D) and microvilli (arrow in D) of a
microvillous cell that differed from regular supporting cells (D; Carr
et al., 1991; Menco, 1994). E,F: MO (E) and VNO epithelial surface (F)
that were immunoreacted with antibodies to the MO G-protein subunit Golf␣ (1/5). IR is prominent in distal parts of the MO cilia (arrow
in E) and is sparse and scattered in the VNO epithelial surface, also
at the level of receptor cell microvilli (arrow in F). In A-F: Large
asterisks, receptor cell apices; triangles, supporting cell apices;
serpentine-shaped arrows, supporting cell microvilli; small asterisks
in C and E, proximal parts of olfactory cilia. Gold particles were 10 nm
(A,E,F) and 15 nm (B,C,D) across. Scale bars ⫽ 1 ␮m.
479
IR was absent from the microvilli and cilia of almost all
other cell types examined. These include the microvilli of
VNO supporting cells (Figs. 4, 5; see also Figs. 8, 9) and of
minor populations of different-appearing VNO microvillous cells (not shown), cilia and microvilli of the VNO
non-sensory epithelium (Fig. 6A), the microvilli in the
transitional area between the sensory and non-sensory
epithelium (Fig. 6B), and nasal respiratory epithelia. The
transitional area contains some ciliated cells, the apices of
which resemble those of ciliated receptor cells of the MO.
In contrast to the microvilli of nearby VNO sensory cells
(see Fig. 8), the apices and cilia of the ciliated cells neither
showed IR for the VNO signaling molecules explored here
(Fig. 6C) nor IR for the signaling molecules of the MO
receptor cell cilia (Fig. 7E), such as Golf␣ and type III AC
(Menco et al., 1994; Table 2). Antibodies to the Gi subunits
labeled receptor cell microvilli in fixed (Figs. 4A, 6C, 8A,
9A,B; see also Fig. 11A) and unfixed freeze-substituted
tissues (Fig. 6D); those to Go␣ (Figs. 4B, 8B, 9C,D; see also
Fig. 11B) and TRP2 (Fig. 5; see also Figs. 11C,D) only
labeled microvilli in fixed tissues.
Omission of primary antibodies from the reaction mixture gave no labeling in either LM (Figs. 2G,H) or EM
preparations. Preabsorption of Gi␣1,2, Gi␣1-3, Go␣, and
TRP2 antibodies with their respective antigenic peptides
suppressed, both at LM and EM levels, IR to background,
as shown here for Gi␣1,2 (Fig. 7A) and TRP2 (Fig. 7B) in
VNO receptor cell microvilli (compare with Figs. 4A and 5,
respectively). Antibodies to Gi␣2 and TRP2 did not immunoreact with MO sensory cell cilia and the microvilli of
supporting and other MO microvillous cells (Figs. 7C,D).
Gi␣1 labeled diffusely MO epithelia (not shown; Sinnarajah et al., 1998). Go␣ IR was somewhat prominent in the
MO, but its labeling pattern was different from that seen
in the VNO. In the VNO, the microvilli of a subpopulation
of receptor cells were specifically Go␣(⫹). In the MO, the
apical cytoplasm of supporting cells, but not their microvilli, was labeled (Menco et al., 1994). None of the
antibodies to MO signal-transduction proteins labeled
VNO receptor cell microvilli. Besides antibodies to Golf␣
(Figs. 7E,F), these included antibodies to Gs␣, type III AC
(Menco et al., 1994), OCNC1 (Matsuzaki et al., 1999),
G␣q/11 (DellaCorte et al., 1996), and inositol 1,4,5trisphosphate (IP3) receptors (Cunningham et al., 1994;
Kalinoski et al., 1994; Table 2).
Several antibodies did not work well. Non-purified antibodies to the G-proteins and olfactory marker protein
(OMP; Table 2) labeled VNO, supporting cell microvilli
instead of receptor cell microvilli, as was the case with
NRS and NGS. Hence, this labeling pattern was considered to be nonspecific. Antibodies to other proteins believed to be involved in VNO signaling, type II AC
(Berghard and Buck, 1996), G␣q11, and IP3 receptors
(Wekesa and Anholt, 1997; Sasaki et al., 1999), gave negative or inconclusive (scattered labeling in no apparent
pattern) IR results (Table 2).
Epithelial topography
To demonstrate localization of tufts of Gi␣2(⫹) and
Go␣(⫹) microvilli belonging to nearby receptor cells along
the VNO sensory surface, two sets of serial sections are
included. Figure 8 shows such differential labeling for an
area near the transition from non-sensory to sensory epithelium (region 2, Fig. 2I). Figure 9 shows this for a more
centrally located area (region 7, Fig. 2I). The very initial
Fig. 8. Composites of serial sections, immunoreacted with antibodies to Gi␣1,2 (A; 1/5) and Go␣ (B,C; 1/10), in the VNO neuroepithelial surface in an area of transition to the non-sensory area of the VNO
(region 2 in Fig. 2I). The low power micrograph (B) presents a larger
area of the section depicted in C. Matching indicators mark the same
structures. iA-iE mark apices of individual cells with Gi␣1,2(⫹) microvilli and oA marks the one individual cell with Go␣(⫹) microvilli.
Sensory cell apices, including their membranes (e.g., the area parallel
to the short straight line of the apex of the cell labeled oA), displayed
much less labeling than the cells’ microvilli. Tufts of labeled microvilli
are marked with straight arrows, surrounding supporting cells are
marked with large arrowheads, and their unlabeled microvilli with
serpentine-shaped arrows. The gold particles were 10 nm across in A
and 15 nm in B,C. Scale bars ⫽ 1 ␮m in A,C, 10 ␮m in B.
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
Figure 8
(Continued)
481
B.Ph.M. MENCO ET AL.
Fig. 9 (Overleaf).
482
(Continued)
483
Figure 9
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
484
B.Ph.M. MENCO ET AL.
TABLE 3. Semiquantitative Evaluations on the Ultrastructural
Localization of Gi␣2 and Go␣ as Assessed by Densities of Gold Particles in
Microvillar Tufts Above VNO Sensory Cells That Contain the Particular
Antigen, Above Neighboring Cells That Do Not Contain the Antigen,
and in the Embedding Resin
Antibody to:
Gi␣2(⫹) cell
Go␣(⫹) cell
Resin
Gi␣1,2 (15 nm)1
8.5 ⫾ 4.9 (53)2
100%3
Labeled cell
13.2 ⫾ 6.0 (32)
100%
Labeled cell
3.7 ⫾ 2.8 (63)
39%
Neighboring cell
3.0 ⫾ 2.1 (42)
35%
Neighboring cell
5.8 ⫾ 2.6 (26)
44%
Neighboring cell
9.6 ⫾ 6.3 (67)
100%
Labeled cell
0.7 ⫾ 0.6 (17)
8%
Gi␣1,2 (10 nm)1
Go␣ (15 nm)
0.6 ⫾ 0.8 (8)
5%
0.5 ⫾ 0.5 (26)
5%
1
Fig. 10. Histogram showing that the transition zone between the
sensory and non-sensory VNO epithelium (regions 1-3 and 19-20 in
Fig. 2I) has significantly (P ⬍ 0.05; asterisks) more receptor cells with
Gi␣2(⫹) microvilli (dark gray) than with Go␣(⫹) microvilli (light gray).
More centrally, these numbers are about equal. Values in region 3
differed significantly (bullet, P ⬍ 0.05) from both, those with more and
those with fewer, labeled cells. The results are based on four animals.
Bars, standard errors.
part of the transition zone between the VNO sensory and
non-sensory epithelium had no cells that reacted with the
antibodies (Fig. 6B). IR only became apparent about halfway within transitional regions 1 or 20 (Fig. 2I), where the
microvilli of most receptor cells were Gi␣1,2(⫹). That is,
along a stretch of approximately 20 ␮m, the microvilli of
one of six cells were Go␣(⫹) (cell oA in Figs. 8A,C). In
contrast, more central regions had about equal numbers of
cells with Gi␣1,2(⫹) and Go␣(⫹) microvilli (Figs. 9A–E).
Along a 30 ␮m stretch, there were four cells with Gi␣1,2(⫹)
microvilli and three with Go␣(⫹) microvilli (Fig. 9E). Summarized over four animals, Figure 10 shows that in the
transition zone near the non-sensory VNO epithelium (regions 1–3, and 19 and 20 in Fig. 2I), significantly more
receptor cells had Gi␣2(⫹) microvilli than had Go␣(⫹) microvilli (P ⬍ 0.05). In the more central region, these numbers were about equal (regions 4 –9 in Fig. 2I). Total densities of all receptor cells expressing microvilli— both
Gi␣2(⫹) and Go␣(⫹)—were 200 –300 cells per mm or 4 to
9 ⫻ 106 cells per cm2 epithelium.
The immunocytochemical distinction in receptor cells is
not accompanied by differences in the morphological appearance of the apices of the cells. Apices of receptor cells
bearing Gi␣2(⫹) microvilli were indistinguishable from
Fig. 9 (Overleaf). Composites of serial sections, immunoreacted
with antibodies to Gi␣1,2 (A,B; 1/5) and Go␣ (C-E; 1/10), through a VNO
neuroepithelial surface area situated more centrally (region 7 in Fig.
2I) than the area shown in Figure 8. The low power micrograph (D)
presents a larger area of the section depicted in E. Matching indicators mark the same structures. iA, iB, and oA in A-C and E or iA-iD and
oA-oC in D mark the apices of individual cells with Gi␣1,2(⫹), respectively, Go␣(⫹) microvilli. These apices display much less labeling than
their microvilli. Absence of labeling includes apical membranes (e.g.,
the areas parallel to the short straight lines of the apices of the cells
labeled iB in A and B and those of the cells labeled oA in C and E).
Tufts of labeled microvilli are marked with straight arrows. Supporting cells are marked with large arrowheads, their unlabeled microvilli
with serpentine-shaped arrows. The gold particles were 10 nm (A,B)
and 15 nm across (C-E). Scale bars ⫽ 1 ␮m in A-C and E, 10 ␮m in D.
Primary antibodies to Gi␣1,2 were used to localize Gi␣2. Two sizes of gold grains were
used for the secondary antibodies, 10 and 15 nm, to localize Gi␣2.
2
Values ⫾ standard deviations (number of cells for which the values were determined
within parentheses) are given in density of gold grains per ␮m2 microvillous tuft above
a designated cell. Structure-devoid resin served as the control area for background.
Within rows, all values differed significantly (P ⬍ 0.0001). Absolute values within
columns cannot be compared, as each column depicts a different antibody.
3
Density values above the cells targeted by the antibodies are highest, 100%. Other
values in the same rows are given as a percentage of this value. In other words, in the
first row, the density of Gi␣2(⫹) gold grains apical to neighboring Go␣(⫹) cells is 35%
compared with that in microvillous regions apical to cells that express the Gi␣2(⫹)
protein. This spread is caused by microvillous tufts being wider than cell apices (see
Table 4, third column).
TABLE 4. Widths of Apices and of Microvillous Tufts of
Rat VNO Sensory Cells
Gi␣2(⫹) cells
Go␣(⫹) cells
Sensory cell
apices
Sensory cell
microvillous
tufts
Average widths of
the microvillous
tufts relative to
the average
diameters of the
apices of the cells
(percentages)
3.3 ⫾ 1.5 (134)1
3.2 ⫾ 1.2 (80)
5.4 ⫾ 2.1 (48)1,2
7.0 ⫾ 2.5 (38)
160%
219%
1
Values are given in ␮m ⫾ standard deviations, with the number of cells for which the
values were determined in parentheses. Within rows, values differed significantly (P ⬍
0.0001).
2
Widths of tufts are based on the length of microvilli of individual cells that could be
followed in single sections and on the extent of labeling in serial sections of receptor cell
microvilli when these cells were sufficiently spatially separated to identify their microvilli as originating from individual cells.
those bearing Go␣(⫹) microvilli. Features like widths of
the apices and of microvillar tufts and the presence of
centrioles inside the cells were the same for both types of
sensory cells. However, the microvilli of one cell extend
over the surface of neighboring cells, where labeling intensities are diluted in the microvillar tufts of these neighboring cells (Figs. 4, 8, and 9; Table 4). By using serial
sections and a semiquantitative analysis, we were able to
distinguish between cells that contribute to the microvillar labeling for Gi␣2 or Go␣, despite their morphological
similarities. Taking the labeling density of microvillar
tufts apical to the cell from which the microvilli sprout as
100% reveals that the labeling density of these microvilli,
when they extend into the microvillar tufts above apices of
neighboring sensory cells, is 30 –50% (Table 3).
In contrast to Gi␣1,2 and Go␣, which appeared to have
labeled the microvilli of two distinct types of receptor cells,
the microvilli of both of these receptor cells, thus those
with Gi␣1,2(⫹) and those with Go␣(⫹) microvilli, are
TRP2(⫹), also in the transition zone. Furthermore, the
microvilli of both cell types display a similar intensity of
labeling for TRP2 (Fig. 11).
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
485
Fig. 11. Two areas (A,C, and B,D) in VNO serial sections from the
same rat that had been immunoreacted with affinity-purified antibodies to Gi␣1,2 (A; 1/10), Go␣ (B; 1/10), and TRP2 channels (C, D; 1/10).
Microvilli in corresponding serial sections (arrows) of both Gi␣1,2(⫹)
(A) and Go␣(⫹) receptor cells (B) were also TRP2(⫹) (C and D, respec-
tively). Membranes of receptor cell apices (asterisks) reacted somewhat with the antibodies to TRP2, but not with those to the
G-proteins. Apices (triangle) and microvilli (serpentine-shaped arrows) of surrounding supporting cells did not label with any of the
antibodies. The gold particles were 15 nm across. Scale bars ⫽ 1 ␮m.
DISCUSSION
intramembranous particles, are alike (Breipohl et al.,
1982; Menco, 1992). This is true despite differences in the
cytoskeletal substructure (Vaccarezza et al., 1981) and the
length of these subcellular hairlets (rodent VNO receptor
cell microvilli are 5–10 ␮m long [this paper; Naguro and
Breipohl, 1982]; rodent MO receptor cell cilia are 50 – 60
␮m long [Seifert, 1970]). The similarity in the appearance
of the membranes of the two types of cellular hairlets
suggested that both structures subserve a similar function, i.e., the subcellular sites of chemosensory signal input. As there is virtually no other information on the
special properties of VNO receptor cell microvilli, the new
information may provide the strongest evidence to date
that these microvilli are adapted specifically for their chemoreceptive function. Consequently, the data provide cellular insight important to understand the complex physi-
This study demonstrates clearly that VNO receptor cell
microvilli are highly specialized for sensory signaling.
Other fine structural characterizations of VNO receptor
cells have shown that apices and microvilli of rat VNO
receptor cells contain Gi␣2 and Go␣ (Matsuoka et al., 2001).
However, this study shows that the microvilli of VNO
receptor cells displayed a much more prominent IR for
␣-subunits of the signaling G-proteins Gi2 and Go and for
TRP2 than any other component of the VNO receptor cell
apices (summarized in Fig. 12). This situation is comparable to the situation in the MO, where signaling molecules are especially abundant in the long distal parts of
receptor cell cilia (summarized in Menco, 1997a). It had
been established that the membrane appearances of MO
cilia and VNO microvilli, as reflected in the densities of
486
B.Ph.M. MENCO ET AL.
Mechanism of gating of TRP2
Fig. 12. Summary diagram of the major sites of localization of
VNO signal transduction components. IR for all signaling proteins:
Gi␣2 (visualized with antibodies to Gi␣1,2), Go␣, and TRP2 channels are
particularly concentrated in receptor cell microvilli. Scale bar ⫽ 1 ␮m.
ology and biochemistry of VNO signaling (Døving and
Trotier, 1998; Liman, 1996, 2001; Keverne, 1999; Dulac,
2000; Holy et al., 2000; Leinders-Zufall et al., 2000).
Cells with Gi␣2(ⴙ) and Go␣(ⴙ) microvilli
coexist within the VNO neuroepithelium
Until now, the pattern of the differential distributions of
Gi␣2(⫹) and Go␣(⫹) VNO receptor cells at the level of the
cells’ microvilli remained unclear. LM immunolabeling
patterns of rodent VNO receptor cells throughout these
cells led Jia and Halpern (1996) to postulate that there are
two types of these cells, Go␣(⫹) and Gi␣2(⫹). This was
confirmed by in situ hybridization (Berghard and Buck,
1996). Later studies demonstrated that these receptor cell
populations differ from each other in other important
chemical aspects. These differences include the nature of
the putative VNO odorant receptors that the cells express
(Herrada and Dulac, 1997; Matsunami and Buck, 1997;
Ryba and Tirindelli, 1997) and the nature of some other
molecules that may participate in VNO signaling, such as
phosphodiesterases (Lau and Cherry, 2000). The same
was true for several molecules that participate in the
development of the two populations (Kishimoto et al.,
1993; von Campenhausen et al., 1997; Halpern et al.,
1998). Use of serial sections yielded a sufficiently large
number of spatially separated cells now, to make the point
that the antibodies to Gi␣2 and Go␣ labeled microvilli of
different VNO receptor cells. The fact that in the transition zone to the non-sensory VNO epithelium, the VNO
epithelial surface contains primarily cells with Gi␣1,2(⫹)
microvilli helped to strengthen this case. These findings
are important to understand the putative roles of Gi␣2 and
Go␣ in VNO signaling.
TRP2 is a most suitable candidate for the current generating channel in all VNO receptor cells. In situ hybridization studies showed that Gi␣2(⫹) and Go␣(⫹) VNO receptor cells express TRP2 (Liman et al., 1999). This study
added that this is especially true at the sites of potential
input, receptor cell microvilli; microvilli of receptor cells
that are Gi␣2(⫹) and those that are Go␣(⫹) are TRP2(⫹).
TRP2 is a member of a class of ion channels that have been
suggested by some (Vannier et al., 1999) to mediate Ca2⫹
influx across the plasma membrane in response to release
of Ca2⫹ from intracellular stores. However, other researchers (Liman et al., 1999) have proposed that these
channels open in response to a phospholipid signaling
cascade in a Ca2⫹ store-independent manner. The ultrastructural data presented here show that TRP2 is most
abundant in sensory microvilli, structures that are unlikely to have an intracellular Ca2⫹-containing membranous compartment, such as endoplasmic reticulum.
Therefore, it is improbable that TRP2 is gated in response
to the release of Ca2⫹ from an intracellular store (Vannier
et al., 1999). Although Ca2⫹ stores may be present in the
dendritic apex, communicating at a distance to the TRP2
channels in the microvilli, it is more probable that TRP2 is
gated by a second messenger, as is true for the TRP
channels involved in the phototransduction cascade of
Drosophila melanogaster (Acharya et al., 1997; Chyb et
al., 1999). In the VNO receptor cell microvilli, this messenger may be generated by activation of chemosensory
receptors. Elevation of diacylglycerol (DAG) and/or IP3,
resulting from a PLC-catalyzed hydrolysis of phosphatidyl
inositol 4,5-bisphosphate, conceivably leads to opening of
TRP2 channels (Liman et al., 1999; Holy et al., 2000).
Other signaling proteins and other species
In rodents, there is evidence that type II (Berghard and
Buck, 1996) and/or type VI AC (Rössler et al., 2000) and
an OCNC2 channel (Berghard et al., 1996) also play a role
in VNO sensory signaling. It is unclear whether these
molecules mediate and/or modulate transduction together
with the molecules in the receptor cell microvilli, or
whether they are involved in other, e.g., non-sensory, processes. Also, the VNO of other species may, at least in
part, make use of a somewhat different array of signaling
molecules. For example, in the goat, all VNO signaling
tentatively involves Gi␣2 (Takigami et al., 2000), whereas
Gq␣ may play a role in the pig (Wekesa and Anholt, 1997).
Ciliated cells in the VNO and atypical
microvillous cells in the MO and VNO
The cilia of the sparse ciliated cells that resemble MO
receptor cells in their apices (Fig. 6C; Adams and
Wiekamp, 1984) and that are present in the transition
area between the sensory and the non-sensory regions of
the VNO did not immunoreact for any of the MO or VNO
signaling molecules. Therefore, these ciliated cells may be
the same as those found in the non-sensory VNO. Similarly, the microvilli of some atypical microvillous cells in
the VNO (not shown) and MO (Fig. 7D; Carr et al., 1991;
Menco, 1994) did not immunoreact with antibodies to
VNO or MO signaling proteins, making it unlikely that
these are VNO (or MO)-type receptor cells.
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
487
Topography
Conclusions
There is a differential distribution of receptor cells with
Gi␣2(⫹) and Go␣(⫹) microvilli along the VNO epithelial
surface (Fig. 10). This surface topographic patterning resembles somewhat the developmentally expressed topographic pattern in the MO (Menco and Jackson, 1997).
The latter parallels roughly four zones in which most
putative MO odorant receptors are located (Ressler et al.,
1993; Vassar et al., 1993). The current data, together with
results of in situ hybridization studies (Herrada and Dulac, 1997), suggest that three such zones may be present in
the VNO: the dorsal and ventral transitional areas with
mainly cells with Gi␣2(⫹) microvilli coincide roughly with
the areas where most proliferation is concentrated
(Berghard and Buck, 1996; Weiler et al., 1999). The
Gi␣2(⫹) cells may be those that are mainly mature in this
area. Alternatively, they may comprise a special population of cells in this region. A recent developmental study
suggests that the latter is the case indeed (Giacobini et al.,
2000). In the more central parts of the VNO neuroepithelium, where numbers of Gi␣2(⫹) and Go␣(⫹) cells are about
equal, our data agree with those of Matsuoka et al. (2001).
Although VNO receptor cell apices are about 1.5–2
times (Table 4) as wide as those of MO receptor cells,
apices of VNO supporting cells are narrower (Fig. 3). This
may be the reason why overall densities of VNO microvillous receptor cells, summated over Gi␣2(⫹) and Go␣(⫹)
ones, resemble those of MO ciliated receptor cells (Menco,
1983).
First, this study gave the most pertinent evidence to
date that VNO receptor cell microvilli are enriched specifically in the putative signaling molecules Gi␣2, Go␣, and
TRP2, providing important support for the role of these
molecules in VNO sensory transduction. Second, there are
two types of receptor cells. One of these has Gi␣2(⫹) microvilli, the other has Go␣(⫹) microvilli. Otherwise, these
receptor cells are morphologically indistinguishable in
their apices. Third, the microvilli of both receptor cell
types are TRP2(⫹). Fourth, a zonal distribution of these
receptor cell types also occurs. Many more cells with
Gi␣2(⫹) microvilli than cells with Go␣(⫹) microvilli are
present in the transition zone near the non-sensory VNO
epithelium.
Some technical and cautionary notes
Labeling restricted to VNO microvillar regions noted in
this study is consistent with some earlier LM studies
(Berghard and Buck, 1996; Murphy et al., 2001), including
observations on microvilli of singly dissociated VNO neurons, that demonstrated that these can be both Gi␣2(⫹)
and TRP2(⫹) (Liman et al., 1999). However, the new data
differed somewhat from those reported in other LM (Halpern et al., 1995; Jia and Halpern, 1996; Jia et al., 1997)
and EM studies (Matsuoka et al., 2001). This is because
the other investigators used antibodies to Go␣ and Gi␣2,
which labeled additional parts of VNO receptor cell apices.
The differences may be due to variances in tissue preparation and/or antibody source. The antibody-antigen interaction can be altered with different tissue preparation
conditions (Griffiths, 1993; Menco, 1995; Webster, 2000).
Therefore, one has to be careful when making assertions
on a single labeling condition. For example, besides the
differences noted above, we also encountered nonspecific
labeling (labeling of VNO supporting cell microvilli in the
case of non-purified antibodies, see also subscript 12 in
Table 2), cross-reactivity (antibodies to Gi␣1-3 also labeled
Go␣), and differential labeling under various fixation conditions (antibodies to Gi␣1,2 and Gi␣1-3 worked well in fixed
and unfixed tissues whereas those to Go␣ and TRP2 only
worked in fixed tissues). Finally, each gold particle could
have bound to more proteins and/or the gold particles
could not access all receptors. Actual receptor densities
could even be about 10 times higher than the density
values presented in Table 3 (Phillips and Bridgman,
1991). For that reason, the values in Table 3 merely serve
as a guide for minimum numbers of labeled proteins.
ACKNOWLEDGMENTS
We acknowledge the help of Drs. Q. Tian Wang and R.A.
Holmgren, Northwestern University, Department of Biochemistry, Molecular Biology and Cell Biology in the preparation of the immunoblots. Gene Minner is thanked for
photographic assistance.
LITERATURE CITED
Acharya JK, Jalink K, Hardy RW, Hartenstein V, Zuker CS. 1997. InsP3
receptor is essential for growth and differentiation but not for vision in
Drosophila. Neuron 18:881– 887.
Adams DR, Wiekamp MD. 1984. The canine vomeronasal organ. J Anat
138:771–787.
Anderson KT, Hansen A, Finger TE. 1999. Localization of olfactory-type
(Ors) and vomeronasal type (V2Rs) receptors in different olfactory
receptor neurons of goldfish. Chem Senses 24:593 (abstract 265).
Aoki C, Go C-G, Wu K, Siekevitz P. 1992. Light and electron microscopic
localization of ␣ subunits of GTP-binding proteins, Go and Gi, in the
cerebral cortex and hippocampus of rat brain. Brain Res 596:189 –201.
Bakalyar HA, Reed RR. 1990. Identification of a specialized adenylyl
cyclase that may mediate odorant detection. Science 250:1403–1406.
Berghard A, Buck LB. 1996. Sensory transduction in vomeronasal neurons: evidence for G␣o, G␣i2, and adenylyl cyclase II as major components of a pheromone signaling cascade. J Neurosci 16:909 –918.
Berghard A, Buck LB, Liman ER. 1996. Evidence for distinct signaling
mechanisms in two mammalian olfactory sense organs. Proc Natl Acad
Sci USA 93:2365–2369.
Berod A, Hartman, BK, Pujol JF. 1981. Importance of fixation in immunocytochemistry: use of formaldehyde solutions at variable pH for the
localization of tyrosine hydroxylase. J Histochem Cytochem 29:844 –
850.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72:248 –254.
Breipohl W, Mendoza AS, Miragall F. 1982. Freeze-fracturing studies on
the main and vomeronasal olfactory sensory epithelia in NMRI-mice.
In: Breipohl W, editor: Olfaction and endocrine regulation. London:
IRL Press. p 309 –322.
Buck LB. 2000. The molecular architecture of odor and pheromone sensing
in mammals. Cell 100:611– 618.
Carr VMcM, Farbman AI, Coletti LM, Morgan JI. 1991. Identification of a
new non-neuronal cell type in rat olfactory epithelium. Neuroscience
45:433– 449.
Cavaggioni A, Mucignat C, Tirindelli R. 1999. Pheromone signaling in the
mouse: role of urinary proteins and vomeronasal organ. Arch Ital Biol
137:193–200.
Chyb S, Raghu P, Hardie RC. 1999. Polyunsaturated fatty acids activate
the Drosophila light-sensitive channels TRP and TRPL. Nature 397:
255–259.
Cunningham AM, Ryugo DK, Sharp AH, Reed RR, Snyder SH, Ronnett
GV. 1994. Neuronal inositol 1,4,5-trisphosphate receptor localized to
the plasma membrane of olfactory cilia. Neuroscience 5:339 –352.
488
DellaCorte C, Restrepo D, Menco BPhM, Andreini I, Kalinoski DL. 1996.
G␣q/G␣11: immunolocalization in the neuroepithelium of the rat (Rattus
rattus) and the channel catfish (Ictalurus punctatus). Neuroscience
74:261–273.
Døving KB, Trotier D. 1998. Structure and function of the vomeronasal
organ. J Exp Biol 201:2912–2926.
Dulac C. 2000. Sensory coding of pheromone signals in mammals. Curr
Opin Neurobiol 10:511–518.
Garossa M, Coca S. 1991. Postnatal development of the vomeronasal epithelium in the rat: an ultrastructural study. J Morphol 208:257–269.
Giacobini P, Benedetto A, Tirindelli R, Fasolo A. (2000). Proliferation and
migration of receptor neurons in the vomeronasal organ of the adult
mouse. Dev Brain Res 123:33– 40.
Gilkey JC, editor. 1993. Freeze-substitution. Parts I and II. Microsc Res
Techn 24 (5 and 6).
Griffiths G. 1993. Fine structure immunocytochemistry. Berlin: SpringerVerlag.
Halpern M. 1987. The organization and function of the vomeronasal system. Annu Rev Neurosci 10:325–362.
Halpern M, Shnayder Shapiro L, Jia C. 1995. Differential localization of G
proteins in the opossum vomeronasal system. Brain Res 677:157–161.
Halpern M, Jia C, Shnayder Shapiro L. 1998. Segregated pathways in the
vomeronasal system. Microsc Res Techn 41:519 –520.
Harlow E, Lane D, editors. 1988. Antibodies. A laboratory manual. Cold
Spring Harbor, NY: Cold Spring Harbor Laboratories. p 478 –510.
Herrada G, Dulac C. 1997. A novel family of putative pheromone receptors
in mammals with a topographically organized and sexually dimorphic
distribution. Cell 90:763–773.
Hildebrand JG, Shepherd GM. 1997. Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Annu
Rev Neurosci 20:595– 631.
Holy TE, Dulac C, Meister M. 2000. Responses of vomeronasal neurons to
natural stimuli. Science 289:1569 –1572.
Inamura K, Matsumoto Y, Kashiwayanagi M, Kurihara K. 1999. Laminar
distribution of pheromone-receptive neurons in rat vomeronasal epithelium. J Physiol (Lond) 417:731–739.
Jia C, Halpern M. 1996. Subclasses of vomeronasal receptor neurons:
differential expression of G proteins (Gi␣2 and Go␣) and segregated
projections to the accessory olfactory bulb. Brain Res 719:117–128.
Jia C, Goldman G, Halpern M. 1997. Development of vomeronasal receptor
neuron subclasses and establishment of topographic projections to the
accessory olfactory bulb. Dev Brain Res 102:209 –216.
Johnson EW, Eller PM, Jafek BW. 1993. An immuno-electron microscopic
comparison of olfactory marker protein localization in the supranuclear
regions of the rat olfactory epithelium and vomeronasal organ neuroepithelium. Acta Otolaryngol (Stockh) 113:766 –771.
Johnston RE. 2000. Chemical communication and pheromones: the types of
chemical signals and the role of the vomeronasal system. In: Finger TE,
Silver WL, Restrepo D, editors. The neurobiology of taste and smell,
2nd edition. New York: Wiley-Liss. p 101–127.
Jones DT, Reed RR. 1989. Golf: an olfactory neuron specific-G protein
involved in odorant signal transduction. Science 244:790 –795.
Kalinoski DL, DellaCorte C, Menco BPhM, Restrepo D. 1994. Localization
of inositol 1,4,5-trisphosphate receptors in the olfactory neuroepithelium of the rat and channel catfish. Chem Senses 19:493 (abstract 137).
Karnovsky MJ. 1965. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27:137–138.
Keverne EB. 1999. The vomeronasal organ. Science 286:716 –720.
Kishimoto J, Keverne EB, Emson PC. 1993. Calretinin, calbindin-D28k
and parvalbumin-like immunoreactivity in mouse chemoreceptor neurons. Brain Res 610:325–329.
Krieger J, Schmitt A, Löbell D, Gudermann T, Schultz G, Breer H, Boekhoff I. 1999. Selective activation of G-protein subtypes in the vomeronasal organ upon stimulation with urine-derived compounds. J Biol
Chem 274:4655– 4662.
Lau YE, Cherry JA. 2000. Distribution of PDE4A and Go␣ immunoreactivity in the accessory olfactory system of the mouse. NeuroReport 11:
2732.
Leinders-Zufall T, Lane AP, Puche AC, Ma W, Novotny MV, Shipley MY,
Zufall F. 2000. Ultrasensitive pheromone detection by mammalian
vomeronasal neurons. Nature 405:792–796.
Leunissen JLM. 1990. Background suppression using Aurion BSA-C
and/or Tween-20威. Wageningen, The Netherlands: Aurion Newsletter
1.
B.Ph.M. MENCO ET AL.
Li X, Mumby SM, Greenwood A, Jope R. 1995. Pertussis toxin-sensitive
G-protein ␣-subunits: production of monoclonal antibodies and detection of differential increases on differentiation of PC12 and LA-N-5
cells. J Neurochem 64:1107–1117.
Liman ER. 1996. Pheromone transduction in the vomeronasal organ. Curr
Opin Neurobiol 6:487– 493.
Liman ER. 2001. Sex and the single neuron: pheromones excite. TINS
24:2–3.
Liman ER, Corey DP, Dulac C. 1999. Cloning and localization of TRP2, a
candidate transduction channel for mammalian pheromone reception.
Proc Natl Acad Sci USA 96:5791–5796.
Matsunami H, Buck LB. 1997. A multigene family encoding adverse array
of putative pheromone receptors in mammals. Cell 90:775–784.
Matsuoka M, Yoshida-Matsuoka J, Iwasaki N, Norita M, Costanzo RM,
Ichikawa M. 2001. Immunocytochemical study of Gi2␣ and Go␣ on the
epithelium surface of the rat vomeronasal organ. Chem Senses 26:161–
166.
Matsuzaki O, Bakin RE, Menco BPhM, Cai X, Ronnett GV. 1999. Immunolocalization of the olfactory cyclic nucleotide-gated channel subunit 1
(OCNC1) in normal and regenerating olfactory neuroepithelium. Neuroscience 94:131–140.
Menco BPhM. 1983. The ultrastructure of olfactory and nasal respiratory
epithelium surfaces. In: Reznik G, Stinson SF, editors. Nasal tumors in
animals and man. Vol. 1, Anatomy, physiology and epidemiology. Boca
Raton, FL: CRC Press. p 45–102.
Menco BPhM. 1992. Ultrastructural studies on membrane, cytoskeletal,
mucous and protective compartments in olfaction. Microsc Res Techn
22:215–224.
Menco BPhM. 1994. Ultrastructural aspects of olfactory transduction and
perireceptor events. Semin Cell Biol 5:11–24.
Menco BPhM. 1995. Freeze-fracture, deep-etch, and freeze-substitution
studies of olfactory epithelia, with special emphasis on immunocytochemical variables. Microsc Res Techn 32:337–356.
Menco BPhM. 1997a. Ultrastructural aspects of olfactory signaling. Chem
Senses 22:295–311.
Menco BPhM. 1997b. Ultrastructural localization of signaling proteins in
microvilli of the rat’s vomeronasal organ. Chem Senses 22:746 (abstract 358).
Menco BPhM, Jackson JE. 1997. A banded topography in the developing
rat’s olfactory epithelial surface. J Comp Neurol 388:293-306.
Menco BPhM, Morrison EE. 2002. Morphology of the mammalian olfactory
epithelium: form, fine structure, function, and pathology. In: Doty RL,
editor. Handbook of olfaction and gustation, 2nd edition. New York:
Marcel Dekker. In press.
Menco BPhM, Yankova MP. 1999. Ultrastructural localization of Gi␣2 and
Go␣ to microvilli of rat vomeronasal receptor cells. Chem Senses 24:94
(abstract 138).
Menco BPhM, Tekula FD, Farbman AI, Danho W. 1994. Developmental
expression of G-proteins and adenylyl cyclase in peripheral olfactory
systems: light microscopic and freeze-substitution electron microscopic
immunocytochemistry. J Neurocytol 23:708 –727.
Menco BPhM, Cunningham AM, Qasba P, Levy N, Reed RR. 1997. Putative odour receptors localize in cilia of olfactory receptor cells in rat and
mouse: a freeze-substitution ultrastructural study. J Neurocytol 26:
297–312 and 691–706 (erratum).
Mendoza AS, Szabó K. 1988. Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. II. Electron microscopy. Dev Brain Res 39:259 –268.
Meyer MR, Angele A, Kremmer E, Kaupp UB, Müller F. 2000. A cGMPsignaling pathway in a subset of olfactory sensory neurons. Proc Natl
Acad Sci USA 97:10595–10600.
Mishra RK. 1986. Central nervous system dopamine receptors. In: Boulton
AA, Baker GB, Hrdina PD, editors. Neuromethods 4, receptor binding.
Neuromethods series 1: neurochemistry. Clinton, NJ: Humana Press. p
23–54.
Mombaerts P. 2001. Vomeronasal receptors in mouse and human. Proc
AChemS 23:16 (abstract 61).
Murphy FA, Tucker K, Fadool DA. 2001. Sexual dimorphism and developmental expression of signal-transduction machinery in the vomeronasal organ. J Comp Neurol 432:61–74.
Naguro T, Breipohl W. 1982. The vomeronasal epithelia of the NMRI
mouse. A scanning electron-microscopic study. Cell Tissue Res 227:
519 –534.
Pantages E, Dulac C. 2000. A novel family of candidate pheromone receptors in mammals. Neuron 28:835– 845.
G-PROTEINS AND TRP2 IN VOMERONASAL MICROVILLI
Phillips TE, Boyne AF. 1984. Liquid nitrogen-based quick freezing: experiences with bounce-free delivery of cholinergic nerve terminals to a
metal surface. J Electron Microsc Techn 1:9 –29.
Phillips GW, Bridgman PC. (1991). Immunoelectron microscopy of acetylcholine receptors and 43 kD protein after rapid freezing, freezesubstitution, and low temperature embedding in Lowicryl K11M.
J Histochem Cytochem 39:625– 634.
Ressler KJ, Sullivan SL, Buck LB. 1993. A zonal organization of odorant
receptor gene expression in the olfactory epithelium. Cell 73:597– 609.
Reynolds ES. 1963. The use of lead citrate at high pH as an electronopaque stain in electron microscopy. J Cell Biol 17:208 –212.
Rössler P, Kroner C, Krieger J, Löbel D, Breer H, Boekhoff I. 2000. Cyclic
adenosine monophosphate signaling in the rat vomeronasal organ: role
of an adenylyl cyclase VI. Chem Senses 25:313–322.
Ryba NJP, Tirindelli R. 1997. A new multigene family of putative pheromone receptors. Neuron 19:371–379.
Sasaki K, Okamoto K, Inamura K, Tokumitsu Y, Kashiwayanagi M. 1999.
Inositol-1,4,5-trisphosphate accumulation induced by urinary pheromones in female rat vomeronasal epithelium. Brain Res 823:161–168.
Seifert K. 1970. Die Ultrastruktur des Riechepithels beim Makrosmatiker.
Eine elektronenmikroskopische Untersuchung. (The ultrastructure
of the olfactory epithelium of macrosomatics. An electron microscopic investigation. In: Bargmann W, Doerr E, editors. Normal and
pathological anatomy, Issue 21. Stuttgart: Georg Thieme Verlag).
Shinohara H, Asano T, Kato K. 1992. Differential localization of G-proteins
Gi and Go in the accessory olfactory bulb of the rat. J Neurosci 12:1275–
1279.
Sinnarajah S, Ezeh PI, Pathirana S, Moss AG, Morrison EE, Vodyanoy V.
1998. Involvement of Gi-protein in odorant-induced inhibition of adenylyl cyclase. FEBS Lett 426:377–380.
Sugai T, Sugitani M, Onoda N. 1997. Subdivisions of the guinea-pig accessory olfactory bulb revealed by the combined method with immunohistochemistry, electrophysiological and optical recordings. Neuroscience
79:871– 885.
Takigami S, Osada T, Yoshida-Matsuoka J, Matsuoka M, Mori Y, Ichikawa
M. 1999. The expressed localization of rat putative pheromone receptors. Neurosci Lett 272:115–118.
489
Takigami S, Mori Y, Ichikawa M. 2000. Projection pattern of vomeronasal
neurons to the accessory olfactory bulb in goats. Chem Senses 25:387–
393.
Tirindelli R, Mucignat-Caretta C, Ryba NJP. 1998. Molecular aspects of
pheromonal communication via the vomeronasal organ of mammals.
Trends Neurosci 21:482– 486.
Vaccarezza OL, Septa LN, Tramezzani JH. 1981. The vomeronasal organ
of the rat. J Anat 132:167–185.
Van Lookeren Campagne MB, Oestreicher B, Van der Krift TP, Gispen
WH, Verkleij AJ. 1991. Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural
localization of neural antigens. J Histochem Cytochem 39:1267–1279.
Vannier B, Peyton M, Boulay G, Brown D, Qin N, Jiang M, Zhu X,
Birnbaumer L. 1999. Mouse trp2, the homologue of the human trpc2
pseudogene, encodes mTrp2, a store depletion-activated capacitive
Ca2⫹ entry channel. Proc Natl Acad Sci USA 96:2060 –2064.
Vassar R, Ngai J, Axel R. 1993. Spatial segregation of odorant receptor
expression in the mammalian olfactory epithelium. Cell 74:309 –318.
von Campenhausen H, Yoshihara Y, Mori K. 1997. OCAM reveals segregated mitral/tufted cell pathways in developing accessory olfactory
bulb. NeuroReport 8:2607–2612.
Watson S, Arkinstall S. 1994. The G-protein linked receptor factsbook.
London: Academic Press.
Webster P. 2000. A commentary on immunolabeling. Microsc Today 00-9:
28 –34.
Weiler E, McCulloch MA, Farbman AI. 1999. Proliferation in the vomeronasal organ of the rat during postnatal development. Eur J Neurosci
11:700 –711.
Wekesa KS, Anholt RRH. 1997. Pheromone regulated production of
inositol(1,4,5)-trisphosphate in the mammalian vomeronasal organ.
Endocrinology 138:3497–3504.
Wekesa KS, Anholt RRH. 1999. Differential expression of G proteins in the
mouse olfactory epithelium. Brain Res 837:117–126.
Wu Y, Tirindelli R, Ryba NJP. 1996. Evidence for different chemosensory
signal transduction pathways in olfactory and vomeronasal neurons.
Biochem Biophys Res Commun 220:900 –904.