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 Previous Article | Next Article 
The Journal of Neuroscience, December 1, 1998, 18(23):9962-9976
Evidence for a Role of the Chemorepellent Semaphorin III and Its
Receptor Neuropilin-1 in the Regeneration of Primary Olfactory
Axons
R. Jeroen
Pasterkamp,
Fred
De
Winter,
Anthony J. G. D.
Holtmaat, and
Joost
Verhaagen
Graduate School for Neurosciences Amsterdam, Netherlands
Institute for Brain Research, 1105 AZ Amsterdam-ZO, The
Netherlands
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ABSTRACT |
To explore a role for chemorepulsive axon guidance mechanisms in
the regeneration of primary olfactory axons, we examined the expression
of the chemorepellent semaphorin III (sema III), its receptor
neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) during
regeneration of the olfactory system. In the intact olfactory system,
neuropilin-1 and CRMP-2 mRNA expression define a distinct population of
olfactory receptor neurons, corresponding to immature
(B-50/GAP-43-positive) and a subset of mature (olfactory marker
protein-positive) neurons located in the lower half of the
olfactory epithelium. Sema III mRNA is expressed in pial sheet cells
and in second-order olfactory neurons that are the target cells of
neuropilin-1-positive primary olfactory axons. These data suggest that
in the intact olfactory bulb sema III creates a molecular barrier,
which helps restrict ingrowing olfactory axons to the nerve and
glomerular layers of the bulb. Both axotomy of the primary olfactory
nerve and bulbectomy induce the formation of new olfactory receptor
neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema
III mRNA is transiently induced in cells at the site of the lesion.
These cells align regenerating bundles of olfactory axons. In contrast
to the transient appearance of sema III-positive cells at the
lesion site after axotomy, sema III-positive cells increase
progressively after bulbectomy, apparently preventing regenerating
neuropilin-1-positive nerve bundles from growing deeper into the
lesion area. The presence of sema III in scar tissue and the
concomitant expression of its receptor neuropilin-1 on regenerating
olfactory axons suggests that semaphorin-mediated chemorepulsive signal
transduction may contribute to the regenerative failure of these axons
after bulbectomy.
Key words:
CNS; CRMP; olfactory bulb; olfactory receptor neuron; neuropilin; plasticity; primary olfactory system; regeneration; semaphorin/collapsin
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INTRODUCTION |
The adult olfactory neuroepithelium
is a unique neural tissue, because it has retained its capacity to
replace dying neurons with new neurons formed by cell division from
stem cells in the basal region of the epithelium (for review, see
Graziadei and Monti Graziadei, 1978 ; Graziadei, 1990; Farbman, 1990,
1992). These new olfactory neurons extend axons penetrating the
cribriform plate and entering the CNS, where they continue to grow
through the growth-permissive nerve layer of the olfactory bulb until they have entered the glomerular neuropil, where they stop growing to
form synapses on the processes of second-order olfactory neurons (see
Fig. 1) (for review, see Farbman, 1992). The primary olfactory system
also has a remarkable capacity to recover from injury. After axotomy of
the primary olfactory nerve, new olfactory receptor neurons regenerate
into the CNS, establishing synaptic contacts with their target neurons
(Harding et al., 1977; Monti Graziadei and Graziadei, 1979; Graziadei
and Monti Graziadei, 1980; Doucette et al., 1983; Constanzo, 1985).
Removal of the olfactory bulb (bulbectomy) also induces neurogenesis.
However, in adult rodents, bulbectomy results in the formation of a
neural scar, which prevents regenerating olfactory fibers from reaching
the cortex (Monti Graziadei, 1983; Hendricks et al., 1994).
Cell adhesion and extracellular matrix (ECM) proteins are abundantly
expressed in the developing and mature olfactory system and help to
establish and maintain the complex connections between the olfactory
epithelium and the olfactory bulb (Miragall et al., 1988, 1989;
Doucette, 1990, 1996; Chung et al., 1991; Miragall and Dermietzel,
1992; Gonzalez et al., 1993; for review, see Mori, 1993; Gong and
Shipley, 1995, 1996; Treloar et al., 1996; Whitesides and LaMantia,
1996; Yoshihara and Mori, 1997; Yoshihara et al., 1997; Julliard and
Hartmann, 1998; Kafitz and Greer, 1998). In addition to these
growth-promoting factors, recent evidence suggests a role for
chemorepulsive proteins and their receptors in the patterning and
maintenance of the primary olfactory pathway (Giger et al., 1996;
Sheperd et al., 1996; Zhang et al., 1996; Kobayashi et al., 1997;
Livesey and Hunt, 1997; Williams-Hogarth et al., 1997; Yoshida et al.,
1997). Growth cones of cultured embryonic olfactory receptor neurons
collapse after exposure to the chemorepellent semaphorin
III(D)/collapsin-1 (sema III) (Kobayashi et al., 1997), a member of a
family of proteins, some of which function in repulsive axon guidance
(Kolodkin et al., 1992, 1993; Luo et al., 1993, 1995; Püschel et
al., 1995; Püschel, 1996). Developing olfactory receptor neurons
express neuropilin-1 (Satoda et al., 1995; Kawakami et al., 1996), a
sema III receptor (Feiner et al., 1997; He and Tessier-Lavigne, 1997;
Kolodkin et al., 1997; for review, see Kolodkin and Ginty, 1997), and
collapsin response mediator protein-2 (CRMP-2; also known as TOAD-64),
an intracellular protein mediating sema III-induced growth cone
collapse (Goshima et al., 1995; Minturn et al., 1995; Wang and
Strittmatter, 1996; Kamata et al., 1998). The embryonic spatiotemporal
expression patterns of sema III, neuropilin-1, and CRMP-2 suggest that
developing primary olfactory nerve fibers are instructed by sema III to
wait at or avoid particular cellular compartments of the olfactory pit
and telencephalic vesicle (Giger et al., 1996; Kobayashi et al.,
1997).
The regenerative response of the mature olfactory epithelium can be
viewed as a recapitulation of ontogeny. It is therefore conceivable
that semaphorins and their receptors also play a role in the
regeneration of primary olfactory axons. Here, we examined the
expression of sema III, neuropilin-1, and CRMP-2 after two types of
lesions of the olfactory pathway: bulbectomy and axotomy. We show that
after both lesions newly formed olfactory receptor neurons contain high
mRNA levels of the sema III receptor neuropilin-1 and CRMP-2.
Bulbectomy and axotomy, however, induce a striking differential
spatiotemporal expression of sema III mRNA at the site of the lesion.
After bulbectomy, sema III mRNA-positive cells fill the bulbar cavity
and completely surround regenerating bundles of neuropilin-1-positive
olfactory axons, apparently blocking their extension. In contrast,
after axotomy, sema III-positive cells show up transiently and define
channels through which regenerating olfactory nerve bundles find their
way to uninjured parts of the olfactory nerve layer. These observations
are consistent with the hypothesis that semaphorin III-neuropilin-1
signaling is involved in governing the regeneration of primary
olfactory neurons. The differential patterns of sema III expression, as
shown here after axotomy and bulbectomy, may be critical to the success
or failure of olfactory axon regeneration.
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MATERIALS AND METHODS |
Animals and surgical procedures
All surgical and animal care procedures were performed according
to the local guidelines of the Experimental Animal Care
Committee. Adult male Wistar rats (225-450 gm; Harlan CPB,
Zeist, The Netherlands) were housed in group cages and maintained on a
12 hr light/dark cycle with ad libitum access to food and water.
The anatomical relationships in the olfactory system are schematically
shown in Figure 1. Two procedures were
used to lesion the primary olfactory pathway: (1) olfactory bulbectomy,
i.e., destruction of distal axonal projections and synapses of primary olfactory neurons and their target cells; and (2) transection of the
primary olfactory nerve, i.e., axotomy of primary olfactory axons
between the cribriform plate and the olfactory bulb, a procedure that
causes no direct damage to the olfactory bulb. Rats were anesthetized
for aseptic surgery with Hypnorm (0.04 ml/100 gm, i.m.; Janssen
Pharmaceutical Ltd., Oxford, England) and Dormicum (0.08 ml/100 gm,
s.c.; Roche Nederland B.V., Mijdrecht, The Netherlands) and subjected
to surgery as described below. Buprenorphine hydrochloride (Temgesic)
(0.03 ml/100 gm, s.c.; Schering-Plow, Amstelveen, The Netherlands) was
given postoperatively.
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Figure 1.
Illustration of the anatomical relationships in
the primary olfactory system. The cell bodies of olfactory receptor
neurons are located in the olfactory epithelium of the nasal cavity and
project their axons through the cribriform plate into the olfactory
bulb glomeruli, where they terminate on the processes of second-order
olfactory neurons: the mitral, tufted, and periglomerular cells.
Throughout life, olfactory receptor neurons are replaced continuously
from a population of stem cells located in the basal region of the
epithelium. As a consequence, newly formed olfactory axons are
constantly being extended toward their targets in the main olfactory
bulb. To gain further insight into the molecular mechanisms underlying
axonal regeneration in the primary olfactory pathway, the expression of
the chemorepellent semaphorin III, its receptor neuropilin-1, and
CRMP-2 were investigated in the primary olfactory pathway after two
lesioning procedures: unilateral olfactory bulbectomy and unilateral
transection of the primary olfactory nerve. Injury to the primary
olfactory nerve results in degeneration and subsequent replacement of
olfactory receptor neurons. After transection of the primary olfactory
nerve between the cribriform plate and the olfactory bulb, newly formed
primary olfactory axons regenerate into the CNS, establishing synaptic
contacts with their targets in the olfactory bulb. Removal of the
olfactory bulb (bulbectomy) also induces neurogenesis. However, in
adult rodents, a neural scar prevents the regenerating primary
olfactory fibers from reaching undamaged areas, e.g., the frontal pole
of the cortex.
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Olfactory bulbectomy. The left olfactory bulb was exposed by
removing a square section of frontal bone covering the olfactory bulb
(±4 mm2), and the bulb was ablated by suction. Care
was taken not to damage the contralateral (right) olfactory bulb or the
frontal pole of the cortex. The square piece of frontal bone was
replaced to close the ablation cavity, and the skin was sutured.
Operated animals were allowed to recover and were killed at 3 (n = 4), 6 (n = 3), 10 (n = 5), 30 (n = 3), and 60 (n = 4) d after lesion. The extent of olfactory
bulbectomy was monitored by macroscopic observation and by histological analysis.
Transection of the primary olfactory nerve. To expose the
interface between the cribriform plate and the olfactory bulb, a groove
was drilled at 9.0 mm rostral to bregma in the left frontal bone
covering the olfactory bulb. A slightly bent needle was lowered between
the intracranial part of cribriform plate and the olfactory bulb, and
several deliberate side-to-side movements of the needle were used to
section the primary olfactory axons. This procedure resulted in
deafferentiation of the olfactory bulb, with reinnervation of the
olfactory glomeruli by new primary olfactory axons, which is in line
with previous observations (Graziadei and Monti Graziadei, 1980;
Doucette et al., 1983; Anders and Hurlock, 1996). Histological changes
observed at early postlesion time intervals (3 d) were used to confirm
the efficacy of the lesion procedure. Operated animals were allowed to
recover and were killed at 3 (n = 3), 6 (n = 3), 10 (n = 4), 30 (n = 3) and 60 (n = 3) d after lesion.
All animals with partial lesions or with lesion-induced damage in the
frontal pole of the cortex were discarded from this study (4 animals).
Unoperated age-matched control animals were killed at each of the above
indicated postoperative time intervals (n = 15).
Tissue preparation
In situ hybridization and combined in situ
hybridization-immunohistochemistry. At the appropriate
postoperative survival time, rats were deeply anesthetized with
Nembutal (0.125 ml/100 gm, i.p.; Sanofi Sante, Maassluis, The
Netherlands) and intracardially perfused with 100 ml of 0.9% NaCl,
followed by 300 ml of 4% paraformaldehyde (PFA) in 0.1 M
PBS, pH 7.4. After perfusion, olfactory epithelia and
olfactory bulbs were dissected out and post-fixed for 2.5 hr in 4% PFA
in 0.1 M PBS at 4°C. Decalcification of the olfactory epithelia with connected olfactory bulbs was performed overnight in a
solution containing 250 mM EDTA and 50 mM
phosphate buffer (PB), pH 7.5, at 4°C, followed by overnight
cryoprotection in 25% sucrose in 50 mM PB at 4°C. Tissue
blocks were embedded in Tissue-Tek (O.C.T. Compound 4583; Miles,
Elkhart, IN) and frozen in dry ice-cooled 2-methylbutane. Consecutive
horizontal or transversal cryostat sections (20 µm) were subjected to
in situ hybridization or double labeling combining
in situ hybridization with immunohistochemistry.
Immunohistochemistry. For single immunohistochemical
analysis [neuropilin-1 and olfactory marker protein (OMP)], rats were perfused with 100 ml of 0.9% NaCl, followed by 300 ml of periodate lysine paraformaldehyde fixative (2% PFA, 0.075 M
L-lysine, and 0.214% sodium metaperiodate) in 50 mM PB, pH 7.3. After overnight post-fixation in the same
fixative, brains were treated with EDTA to enhance tissue penetration,
cryoprotected, and frozen.
Reverse transcription-PCR cloning of rat CRMP-2
A cDNA mixture, synthesized by reverse transcription-PCR,
of total RNA from embryonic day 15 Wistar rat spinal cord served as a template for the PCR procedure. PCR primers were based on the cDNA
sequence of human CRMP-2 [Homo sapiens, European
Molecular Biology Laboratories Data bank HS172791 (Wang and
Strittmatter, 1996)]. Primers flanked the coding sequence and
contained EcoRI and XbaI restriction sites for
subcloning (sense primer 5'-CGGAATTCCACGCATCACGAGCG-3' and
antisense primer 5'-GCTCTAGACCAGGCTGGTGATGTTGGC-3',
EcoRI and XbaI sites are in italics). The
reaction was cycled five times in a cycle profile of 1 min at 94°C,
30 sec at 55°C, and 5 min at 74°C, followed by 33 times in a cycle
profile of 1 min at 94°C, 30 sec at 70°C, and 5 min at 74°C. The
amplified product was purified and subcloned in pBluescript KS(±)
vector (Stratagene, La Jolla, CA), using the EcoRI and
XbaI sites. The Sequenase 2.0 kit (Amersham, Cleveland, OH)
was used to confirm the CRMP-2 coding sequence.
In situ hybridization
Nonradioactive in situ hybridization was performed
using digoxigenin (DIG)-labeled cRNA probes transcribed from four
different cDNA templates: rat semaphorin(D)III/collapsin-1 cDNA
[entire coding region (Giger et al., 1996)], rat neuropilin-1 cDNA
[nucleotides 181-2593 of the coding region; a gift from Dr. A. L. Kolodkin, The Johns Hopkins University School of Medicine (Kolodkin
et al., 1997)], rat B-50/GAP-43 cDNA [entire coding region; a
gift from Dr. L. H. Schrama, Rudolf Magnus Institute for
Neurosciences, Utrecht, The Netherlands (Nielander et al., 1987)], and
rat CRMP-2 cDNA (nucleotides 1092-1780 of the coding sequence).
Briefly, DIG-labeled cRNA probes, message-complementary (antisense) or noncomplementary (sense), were generated by in vitro
transcription from completely linearized cDNA template, using the
appropriate RNA polymerases (T3, T7, SP6; Boehringer Mannheim,
Mannheim, Germany). To enhance tissue penetration and avoid nonspecific
background staining, the full-length cRNA probes were alkali-hydrolyzed
to an average length of 100-200 bases (Schaeren-Wiemers and
Gerfin-Moser, 1993).
Nonradioactive in situ hybridization was performed as
described by Giger et al. (1996), with minor modifications. In short, cryostat sections of 20 µm were cut at  20°C, thaw-mounted on Superfrost plus slides (Fisher Scientific, Den Bosch, The Netherlands), and post-fixed with 4% PFA in PBS for 5 min at room temperature (RT).
To enhance tissue penetration and decrease aspecific background staining, sections were reacted with proteinase K (10 µg/ml;
Boehringer Mannheim) in PBS containing 0.1% Triton X-100 for 10 min,
post-fixed for 20 min in 4% PFA in PBS, and acetylated with 0.25%
acetic anhydride in 0.1 M triethanolamine for 10 min, all
at RT. Subsequently, sections were prehybridized overnight at RT in
hybridization buffer (50% formamide, 5× Denhardt's solution, 5×
SSC, 250 µg/ml bakers yeast tRNA, and 500 µg/ml sheared and
heat-denatured herring sperm DNA). Hybridization was performed for 15 hr at 55°C (sema III and B-50/GAP-43) or 60°C (CRMP-2 and
neuropilin-1), using 200 ng/ml denatured DIG-labeled cRNA probe diluted
in hybridization buffer. After hybridization, sections were washed in
5× SSC for 5 min, 2× SSC for 1 min, 50% formamide containing 0.2×
SSC for 30 min, all at 55°C (sema III and B-50/GAP-43) or 60°C
(CRMP-2 and neuropilin-1), and adjusted to RT in 0.2× SSC for 5 min.
DIG-labeled RNA hybrids were detected with an anti-DIG Fab fragment
conjugated to alkaline phosphatase (Boehringer Mannheim) diluted 1:3000
in TBS, pH 7.5, for 3 hr at RT. Binding of
alkaline-phosphatase-labeled antibody was visualized by incubating the
sections in detection buffer (100 mM Tris, pH 9.5, 100 mM NaCl, and 5 mM MgCl2)
containing 240 µg/ml levamisole and color reagents of 300 µg/ml
nitro-bluetetrazolium chloride (Sigma, Deisenhofen, Germany) and 170 µg/ml 5-bromo-4-chloro-3-indolylphosphate (Sigma) for 14 hr at RT.
Neuropilin-1 antibody production
Anti-neuropilin-1 (AN-1) antibodies were produced as
described by Kolodkin et al. (1997). A fragment of rat neuropilin-1, corresponding to amino acids C583-I856, was cloned in the
BamHI and HindIII sites of the pQE30 vector
(Qiagen, Hilden, Germany), which was subsequently used to produce
6-histidine-tagged neuropilin-1 fragments in Escherichia
coli. These proteins were purified on a Ni-NTA-agarose
column (Qiagen, Hilden, Germany) according to specifications of
the manufacturer. Rabbits were immunized with ~0.5 mg of protein in
complete Freund's adjuvant and boosted two times in incomplete
Freund's adjuvant. AN-1 antibodies were affinity purified on a
neuropilin-1 protein immunosorbent column according to a method
described by Oestreicher et al. (1983). In short, neuropilin-1 protein
fragments were coupled to CNBr-activated Sepharose 4B in 0.1 M NaHCO3 and 0.5 M NaCl, pH 9.0, and subsequently washed with 1 M glycine, pH 8.0, 1 M NaCl in 0.1 M sodium acetate, pH 4.0, and 0.1 M sodium borate, pH 8.5. The Sepharose was packed in a
column, and 3 ml of serum was added. The column was washed with PBS,
and specifically bound antibodies were eluted with 100 mM
ammonium formate, pH 2.7. Eluents were neutralized with 1 M ammonia and concentrated by lyophilization.
Immunohistochemistry
Immunohistochemistry was performed according to standard
immunohistochemical procedures incorporating the
avidin-biotin-peroxidase complex, using 3,3'-diaminobenzidine
tetrachloride (DAB) as a chromophore. In some instances, in
situ hybridization was followed by immunohistochemistry.
Initially, in situ hybridization was combined with
immunofluorescence. Because of the dark purple in situ staining, fluorescent signals were not detectable in
double-stained olfactory receptor neurons. Therefore, in subsequent
double-labeling experiments, sites of primary antibody binding were
visualized with DAB.
Sections were washed in TBS containing 0.2% Triton X-100 (TBS-T) for
15 min at RT. B-50/GAP-43 was detected with affinity-purified polyclonal rabbit antibodies derived from antiserum #9527 [1:1000 dilution; a gift from Dr. L. H. Schrama (Oestreicher et
al., 1983)], OMP was detected by polyclonal goat antibodies
[antiserum #255, dilution 1:5000; a gift from Dr. F. L. Margolis,
University of Maryland School of Medicine (Keller and Margolis,
1975)], and neuropilin-1 was detected by affinity-purified polyclonal
rabbit antibodies from antiserum AN-1 (dilution 1:100). All primary
antisera were diluted in TBS-T containing 0.2% bovine serum albumin
(BSA) (Sigma), and sections were incubated in primary antiserum
overnight at 4°C. No immunostaining was detectable in control
sections in which the primary antibodies were replaced by TBS-T. After
three washes in TBS, sections were incubated with biotinylated goat anti-rabbit (1:100; B-50/GAP-43 and neuropilin-1) or biotinylated horse
anti-goat (1:100; OMP), all diluted in TBS-T containing 0.2% BSA, for
1 hr at RT. Then sections were washed three times in TBS and incubated
with avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector
Laboratories, Burlingame, CA) in TBS containing 0.25% gelatin and
0.5% Triton X-100, pH 7.5, for 1 hr at RT. After two washes in TBS and
a brief wash in 50 mM Tris-HCl, pH 7.6, sections were
reacted with a solution containing 0.035% DAB and 0.015% hydrogen
peroxide in Tris-HCl, pH 7.6, for 15 min at RT. The reaction was
terminated by several washes in Tris-HCl, pH 7.6, and sections were
mounted in glycerol.
To confirm the immature phenotype of regenerating olfactory receptor
neurons expressing neuropilin-1 or CRMP-2 mRNA, sections probed for
neuropilin-1 or CRMP-2 were stained for B-50/GAP-43 or OMP protein.
Although OMP-immunoreactivity was clearly visible in neuropilin-1- and
CRMP-2 mRNA-expressing olfactory receptor neurons, B-50/GAP-43 protein
was hardly detectable in somata of olfactory receptor neurons after the
in situ hybridization procedure (data not shown). The lack
of double staining might be attributable to the relatively low
B-50/GAP-43 protein levels in olfactory receptor somata (Verhaagen et
al., 1989; Meiri et al., 1991) or to destruction of the protein during
the in situ procedure.
Quantitative analysis
The number and position of olfactory receptor neurons expressing
B-50/GAP-43, CRMP-2, neuropilin-1 mRNAs, and OMP protein were
determined in unoperated age-matched controls and at different time
intervals after unilateral bulbectomy or transection of the primary
olfactory nerve (see Fig. 7). Sections were analyzed using a digitizer
(Calcomp 2000) and a Zeiss microscope (Axioskop) equipped with a
40× objective and 12.5× oculars. Per animal, the cells were
counted in three stretches (300 µm) of septal epithelium, which were
~100 µm apart. The relative position of each labeled cell in the
neuroepithelium was determined by measuring the ratio between the
distance of the cell to the basal lamina and the apical surface at that
point. The total number of counted cells at different positions in the
epithelium (in pieces of 10%; see Fig. 7) was determined using a
frequency analysis program (Quattro Pro 6.01; Novell).
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RESULTS |
In this study, the expression patterns of the genes encoding the
chemorepellent sema III, neuropilin-1, a sema III receptor, and the
intracellular CRMP-2 were examined by nonradioactive in situ
hybridization after olfactory bulbectomy or axotomy of the primary
olfactory nerve. Their expression profiles were compared with that of
established markers for immature (B-50/GAP-43) and mature (OMP)
olfactory receptor neurons (Farbman and Margolis, 1980; Monti Graziadei
et al., 1980; Miragall and Monti Graziadei, 1982; Margolis, 1985;
Danciger et al., 1989; Verhaagen et al., 1989, 1990; Meiri et al.,
1991; Schwob et al., 1992). The expression of neuropilin-1 was also
studied by immunohistochemistry.
The specificity of the in situ hybridization procedure was
inferred from the partially overlapping, but clearly distinct, distribution patterns of sema III, neuropilin-1, CRMP-2, and
B-50/GAP-43 mRNA. Sections subjected to the in situ
hybridization procedure, but with no probe added, or sections
hybridized with sense probe exhibited no hybridization signal.
Expression of neuropilin-1, CRMP-2, and sema III in the intact
adult olfactory system
Olfactory epithelium
In adult rats (16 to 18 weeks of age), neuropilin-1 mRNA and
CRMP-2 mRNA were observed in immature B-50/GAP-43-positive olfactory receptor neurons, and in a subpopulation of OMP-expressing olfactory receptor neurons located directly above the B-50/GAP-43-positive neurons (Fig. 2A-D;
see also Figs. 4A-C, 6A-D).
CRMP-2 mRNA expression was relatively abundant in immature olfactory
receptor neurons, and expression levels declined gradually in neurons
located more superficially (see Figs. 2B,
4B, 6B), whereas the intensity of the signal for neuropilin-1 mRNA was similar in B-50/GAP-43-positive immature and OMP-positive mature neurons (see Figs.
2A, 4A, 6A). Occasionally, patches of olfactory receptor neurons showing strong neuropilin-1 mRNA expression were intermingled with areas of cells displaying much lower hybridization signals (see Figs.
2A, 4A, 6A).
Computer-assisted analysis of the distribution of cell bodies expressing neuropilin-1 mRNA, CRMP-2 mRNA, B-50/GAP-43 mRNA, and OMP
protein demonstrated that in adult rats neuropilin-1 and CRMP-2 mRNA expression was confined to olfactory receptor neurons in the lower
60% of the epithelium and corresponds to immature B-50/GAP-43-positive and a subset of mature OMP-positive olfactory receptor neurons located
directly adjacent to the cohort of B-50/GAP-43-positive neurons (see
Fig. 7B). In young adult rats (7 to 8 weeks of age), the
cohort of neuropilin-1- and CRMP-2-positive neurons overlapped predominantly with immature B-50/GAP-43 neurons (see Fig.
7A). Neuronal perikarya in the olfactory epithelium
exhibited no immunoreactivity for neuropilin-1, consistent with
previous observations in tadpole (Satoda et al., 1995) and mouse
(Kawakami et al., 1996). Neuropilin-1 protein was detected in fascicles
of primary olfactory axons traversing the lamina propria.
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Figure 2.
Olfactory receptor neurons formed after bulbectomy
express neuropilin-1 and CRMP-2 mRNA. Rats subjected to unilateral
bulbectomy were allowed to recover for 3 (E-H), 10 (I-L), 30 (M-P), and 60 (Q-T)
d. Horizontal cryosections of septal olfactory epithelium from
unlesioned animals (CON, 16 weeks of age) and
bulbectomized animals were subjected to in situ
hybridization for neuropilin-1 mRNA (A,
E, I, M,
Q), CRMP-2 mRNA (B, F,
J, N, R), B-50/GAP-43 mRNA
(C, G, K,
O, S), and immunohistochemistry for OMP
protein (D, H, L,
P, T). In control epithelium,
neuropilin-1 mRNA (A) and CRMP-2 mRNA
(B) are expressed in olfactory receptor neurons
in the lower region of the olfactory epithelium, corresponding to
immature B-50/GAP-43 mRNA-expressing neurons (C)
and to a subset of mature OMP-positive neurons directly adjacent to the
immature neurons (D). Note that neuropilin-1 and
CRMP-2 signals are absent from sustentacular cells and stem cells. As a
result of bulbectomy, massive loss of mature OMP-expressing olfactory
receptor neurons has occurred. A few OMP-positive neurons remain
scattered throughout the ipsilateral epithelium (H,
L, P, T). The vast
majority of neurons in the bulbectomized epithelium are immature
B-50/GAP-43-positive (G, K,
O, S). After bulbectomy, neuropilin-1
(E, I, M,
Q) and CRMP-2 (F, J,
N, R) mRNA expression overlaps with the
cohort of immature B-50/GAP-43-expressing neurons. The
bulbectomy-induced changes in the mRNA expression for neuropilin-1 and
CRMP-2 persist up to at least 60 d after lesion (Q,
R). Scale bar, 55 µm.
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In situ hybridization on transverse sections along the
entire rostrocaudal axis of the olfactory epithelium revealed that neuropilin-1 mRNA and CRMP-2 mRNA were present throughout the entire
olfactory epithelium. Olfactory receptor neurons in the septum and in
all turbinates displayed neuropilin-1 and CRMP-2 mRNA expression (Fig.
3A,B).
Sema III hybridization signals were not observed in the olfactory
epithelium nor in other cellular compartments of the olfactory
turbinates of adult rats (data not shown).
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Figure 3.
Low-magnification overviews of the expression of
neuropilin-1 and CRMP-2 in the primary olfactory system. Low-power
photomicrographs of consecutive coronal sections of the olfactory
epithelium (A, B) and main olfactory bulb
(C, D). Sections of the adult rat
olfactory system were analyzed using in situ
hybridization for neuropilin-1 (A) and CRMP-2
(B) or immunohistochemistry for neuropilin-1
(C) and OMP (D). Note that
hybridization signals for neuropilin-1 and CRMP-2 are present
throughout the dorsoventral extent of the epithelium (A,
B). At the level of the olfactory bulb, however,
neuropilin-1 immunoreactivity is absent from glomeruli at the dorsal
and ventral boundaries of the olfactory bulb (C),
whereas OMP labels all glomeruli (D). Scale bar
(in D): A, B, 2 mm;
C, D, 900 µm.
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Olfactory bulb
Strong-to-moderate in situ hybridization signals for
sema III were present in the mitral and tufted cells, as shown
previously (Giger et al., 1998). Moderate-to-weak signals were observed
in subsets of periglomerular cells (Fig.
4G). Pial cells covering the
olfactory bulb and lining the caudal surface of the cribriform plate
were strongly labeled in young adult and adult rats (Fig. 4G). Immunolabeling of transversal and horizontal sections
of adult rat main olfactory bulb revealed that neuropilin-1-positive olfactory axons entered the olfactory nerve layer by passing through sema III-negative channels in the cribriform plate (Fig.
4G). Neuropilin-1-positive axons terminated in the glomeruli
of the olfactory bulb (Figs. 3C, 4G).
Neuropilin-1 immunoreactivity was confined to the rostrolateral and
caudal glomeruli of the main olfactory bulb. Moderate neuropilin-1
immunoreactivity was observed in most glomeruli, whereas a small number
of glomeruli was either very strongly labeled or unlabeled (Fig.
3C). The most rostral glomeruli and glomeruli in the dorsal
and ventral extent of the olfactory bulb lacked neuropilin-1 labeling
(Fig. 3C). As expected, OMP protein was detected in all
glomeruli (Fig. 3D).
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Figure 4.
A-F, Double labeling combining
in situ hybridization and immunohistochemistry to
examine the expression of neuropilin-1 mRNA and CRMP-2 mRNA in
OMP-positive neurons in the intact olfactory epithelium and after
bulbectomy. Horizontal sections of olfactory epithelium of unlesioned
animals (18 weeks of age) and bulbectomized animals (60 d after lesion)
were double stained for neuropilin-1 mRNA (A,
D), CRMP-2 mRNA (B, E), or
B-50/GAP-43 mRNA (C, F,
purple) and OMP protein (brown).
Sections probed for neuropilin-1 mRNA were only briefly stained for OMP
to allow detection of double-stained profiles. Note that in control
epithelium a subset of OMP-expressing mature neurons contains
neuropilin-1 and CRMP-2 mRNA (A-C), whereas
after bulbectomy, only a small number of neurons express both OMP and
neuropilin-1 mRNA or CRMP-2 mRNA
(D-F). G-K, Combined in
situ hybridization and immunohistochemistry on sections of the
olfactory bulb showing the expression of sema III mRNA in the intact
situation after bulbectomy or after axotomy and its relation to
neuropilin-1-positive olfactory axons. Sections were probed for sema
III mRNA (purple) and immunostained for
neuropilin-1 protein (brown). Solid
arrows point to sema III expression by non-neuronal cells.
G, Rostral is to the right. In control
olfactory bulb, hybridization signals for sema III are found in
non-neuronal cells in the pial sheet and in second-order olfactory
neurons. Note that neuropilin-1-positive fibers traverse the cribriform
plate through sema III-free spaces (open arrow) and
enter the olfactory nerve layer. H-K, Sections of the
lesion site at 10 and 60 d after bulbectomy (H,
J) or axotomy (I,
K). Rostral is to the top, and the
contralateral unlesioned side is to the right.
H, Non-neuronal cells expressing sema III mRNA surround
neuropilin-1-positive olfactory fiber bundles
(asterisks) in the lesion cavity at 10 d after
bulbectomy. Note that some fibers run through an opening in the rim of
sema III-positive cells into a sema III-free region of the scar
(long arrow). By 60 d, the bulbar cavity has been
invaded by numerous sema III mRNA-containing cells encapsulating
neuropilin-1-positive bundles of regenerating olfactory axons
(asterisk). In all animals examined, a single
encapsulated fiber bundle in the scar consistently lacking
immunoreactivity for neuropilin-1 was observed (circle).
This neuropilin-1-negative axon bundle might have arisen from olfactory
receptor neurons of the vomeronasal epithelium, because these neurons
do not express neuropilin-1. At 10 d after axotomy,
neuropilin-1-positive fibers are meandering between strings of sema
III-positive cells situated between the cribriform plate and the
unlesioned olfactory bulb (I). By 60 d, sema III signals in the lesion site have disappeared, and labeling
is again confined to the pial sheet (K).
cp, Cribriform plate; gl, glomerular
layer; onl, olfactory nerve layer. Scale bars: (in
F) A-F, 95 µm; (in
K) I, J,
K, 300 µm; (in K)
H, 125 µm.
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Expression of neuropilin-1, CRMP-2, and sema III after
unilateral bulbectomy
Olfactory epithelium
The changes in the expression of neuropilin-1 mRNA and CRMP-2 mRNA
after unilateral bulbectomy are illustrated in Figures 2, 4, and 7. On
the contralateral control side, both messengers were expressed in the
lower compartment of the epithelium. This expression pattern was
indistinguishable from that of unlesioned control rats (see Fig. 7,
compare B, G). Three days after bulbectomy, the
thickness of the epithelium at the lesioned side was reduced substantially, and the OMP-positive mature olfactory neurons had almost
completely disappeared (Fig. 2E-H). As has
been observed previously, the intensity of OMP immunoreactivity in the
few remaining OMP-positive ipsilateral mature olfactory receptor
neurons appeared greater than in neurons on the unoperated side (Monti
Graziadei, 1983; Verhaagen et al., 1990; Carr et al., 1998). In the
thin remaining layer of olfactory epithelium, moderate neuropilin-1 and
CRMP-2 mRNA expression was evident (Fig.
2E,F). At 10 and 30 d
after lesion, the thickness of the epithelium had increased as a result
of the formation of new olfactory receptor neurons. Most of these
neurons displayed an immature phenotype
(B-50/GAP-43-positive-OMP-negative) and expressed high levels of
neuropilin-1 and CRMP-2 mRNA (see Figs. 2I-P,
7C). At 60 d after lesion, the thickness of the
epithelium on the lesioned side had somewhat decreased compared with 10 and 30 d after lesion, but neuropilin-1 and CRMP-2 mRNA continued to be expressed throughout the population of immature olfactory neurons
(see Figs. 2Q-T, 7D). The phenotype of
neuropilin-1- and CRMP-2-positive regenerating olfactory receptor
neurons was confirmed by immunolabeling sections probed for
neuropilin-1 mRNA or CRMP-2 mRNA with OMP antibodies. This experiment
showed that only a small number of regenerating olfactory receptor
neurons expressed both neuropilin-1 mRNA or CRMP-2 mRNA and OMP protein
(Fig. 4D-F). Quantitative analysis showed an
increase in the relative number of neuropilin-1 mRNA- or CRMP-2
mRNA-expressing olfactory receptor neurons at 10 d after
bulbectomy compared with control epithelium (see Fig.
7A,C). At 60 d after lesion,
the number of olfactory neurons expressing the messengers for
neuropilin-1 or CRMP-2 had slightly decreased compared with 10 d
but was still higher than the number of olfactory receptor neurons
expressing neuropilin-1 or CRMP-2 in control epithelium (see Fig.
7B,D). At both 10 and 60 d
after bulbectomy, cohorts of neuropilin-1 mRNA- and CRMP-2 mRNA-expressing neurons displayed almost complete overlap with the
B-50/GAP-43 mRNA-containing neurons, thereby corroborating the
qualitative histological observations (see Fig.
7C,D).
At none of the postlesion time intervals was sema III mRNA expression
observed in either the ipsilateral or contralateral olfactory
epithelium (data not shown).
Olfactory bulb
After olfactory bulbectomy, a heterogeneous cellular scar
developed, occupying the entire bulbar cavity. At 10, 30, and 60 d
after lesion, prominent expression of sema III mRNA was present in an
increasing number of small non-neuronal cells of the scar (Figs.
4H,J,
5B,C).
Strings and patches of sema III mRNA-containing cells continuous with
the pial sheet covered the caudal surface of the cribriform plate. To
examine the relationship between sema III-positive cells and
regenerating olfactory axons in the scar, in situ
hybridization for sema III was combined with immunohistochemistry for
neuropilin-1 or B-50/GAP-43. In the unlesioned contralateral olfactory
bulb, neuropilin-1 and B-50/GAP-43 expression resembled control. At 3 and 6 d after bulbectomy, no regenerating B-50/GAP-43- or
neuropilin-1-positive fibers were visible in the bulbar cavity, suggesting that regenerating olfactory axons had not yet reached the
lesion site. At 10 d, neuropilin-1- and B-50/GAP-43-immunoreactive axons were observed directly adjacent to the cribriform plate and
penetrated the rostral part of the bulbar cavity. Regenerating olfactory receptor neurons elaborated axons into the bulbar cavity, avoiding the sema III-expressing cells. In addition, some fascicles of
regenerating olfactory axons were surrounded by patches and strings of
sema III-positive cells (Fig. 4H). At 30 and 60 d after bulbectomy, a progressive increase was observed in the number of sema III-positive cells. The bulbar cavity was entirely filled with
scar tissue containing multiple strings and patches of tightly packed
sema III-positive cells encapsulating the regenerating neuropilin-1-
and B-50/GAP-43-immunoreactive nerve bundles, apparently preventing
these fibers from extending further into the bulbar cavity (Figs.
4J, 5B,C). In
addition to multiple neuropilin-1-positive nerve bundles, we observed a
single encapsulated B-50/GAP-43 fiber bundle that was
neuropilin-1-negative in all animals examined (Fig.
4J). This neuropilin-1-negative axon bundle might
have arisen from olfactory receptor neurons of the vomeronasal
epithelium, because these neurons do not express neuropilin-1 (Satoda
et al., 1995).
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Figure 5.
Relationship between regenerating bundles of
olfactory axons and sema III mRNA expression in the injured olfactory
system. High-power photomicrographs showing horizontal sections of the
lesion site at 10 d after transection of the primary olfactory
nerve (A) or 60 d after bulbectomy
(B, C). Sections were probed for sema III
mRNA (purple) and immunolabeled subsequently for
neuropilin-1 (A) or B-50/GAP-43 protein
(B, C, brown). At 10 d after axotomy, neuropilin-1-positive olfactory axons grow through
sema III-free channels lined by strings of sema III mRNA-expressing
cells. These cells are continuous with the pial sheet and cover the
cribriform plate (A). At 60 d after
bulbectomy, bundles of regenerating olfactory axons
(asterisks) are tightly encapsulated by sema
III-positive cells (B, C). Scale bar, 170 µm.
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Expression of neuropilin-1, CRMP-2, and sema III after transection
of the primary olfactory nerve
Olfactory epithelium
The changes in the expression of neuropilin-1 mRNA and CRMP-2 mRNA
after transection of the primary olfactory nerve are illustrated in
Figures 6 and
7. As observed after olfactory
bulbectomy, transection of the primary olfactory nerve resulted in a
pronounced loss of mature OMP-positive olfactory receptor neurons and
an induction in the formation of new immature B-50/GAP-43-positive
neurons. At 10 and 30 d after transection, neuropilin-1 mRNA and
CRMP-2 mRNA were present in a more or less continuous band of immature cells (Fig. 6E-H). This response was similar
to that observed at 10 and 30 d after bulbectomy (for a
quantitative comparison, see Fig. 7C,E). At
60 d after transection, expression for neuropilin-1 and CRMP-2
resembled control expression patterns (Fig. 6, compare A,B, to
I,J), i.e., olfactory
receptor neurons in the lower compartment of the olfactory epithelium
expressed neuropilin-1 mRNA, whereas CRMP-2 mRNA was expressed in a
basal-to-apical gradient (Fig. 6I,J). Consistent with the
histological observations, quantitative measurements showed that the
distribution and relative numbers of neurons expressing neuropilin-1
and CRMP-2 mRNA at 60 d after lesion were very similar to control.
Noticeably, a somewhat larger cohort of OMP-expressing cells was
detected at 60 d after axotomy compared with age-matched control
(Fig. 7B,F).
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Figure 6.
Neuropilin-1 and CRMP-2 mRNA expression returns to
control during reconstitution of the olfactory epithelium after
axotomy. Rats subjected to primary olfactory nerve transection were
allowed to recover for 10 (E-H) and 60 (I-L) d. Horizontal cryosections of olfactory
epithelium from unlesioned animals (CON, 16 weeks of
age) and axotomized animals were subjected to in situ
hybridization for neuropilin-1 mRNA (A,
E, I), CRMP-2 mRNA
(B, F, J),
B-50/GAP-43 mRNA (C, G,
K), and immunohistochemistry for OMP protein
(D, H, L). In control
animals, neuropilin-1 and CRMP-2 mRNA-containing olfactory receptor
neurons correspond to cohorts of both immature B-50/GAP-43-positive and
mature OMP-positive neurons in the lower compartment of the epithelium
(A-D). At 10 d after axotomy, abundant mRNA
expression for neuropilin-1 and CRMP-2 is confined to immature
B-50/GAP-43 neurons occupying most of the epithelium
(E-H). The expression patterns for
neuropilin-1 and CRMP-2 are reminiscent of those seen at 10 d
after bulbectomy (Fig. 2I-L). By 60 d after
lesion, B-50/GAP-43 and OMP expression are very similar to control
patterns (K, L). At this postlesion time
interval, neuropilin-1- and CRMP-2 mRNA-positive neurons are again
confined to the lower region of the epithelium (I,
J). Scale bar, 55 µm.
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Figure 7.
Quantitative assessment of the distribution of
neuropilin-1, CRMP-2, B-50/GAP-43, and OMP-positive neurons in the
intact and regenerating olfactory epithelium. The number and
distribution of neuropilin-1 mRNA, CRMP-2 mRNA, B-50/GAP-43 mRNA, and
OMP-positive olfactory receptor neurons were determined at the left
side of the septal olfactory epithelium of unlesioned age-matched
control animals of 7-8 weeks of age (A), 16-18
weeks of age (B), and at 10 and 60 d after
bulbectomy (C, D) or transection of the
primary olfactory nerve (E, F).
In addition, at 60 d after bulbectomy, the
contralateral control side was analyzed (G). For
all groups, the number of animals analyzed was three. In young adult
animals (7-8 weeks of age), the population of neuropilin-1- and CRMP-2
mRNA-positive neurons overlap almost completely with B-50/GAP-43
mRNA-expressing neurons (A). In fully mature
animals (16-18 weeks of age), the cohorts of neuropilin-1- and
CRMP-2-expressing neurons overlap with B-50/GAP-43 immature neurons and
with OMP-positive mature neurons immediately on top of these immature
neurons (B). At 10 d after bulbectomy and
axotomy, cohorts of neuropilin-1 mRNA- and CRMP-2 mRNA-expressing
neurons display almost complete overlap with B-50/GAP-43
mRNA-containing neurons (C, E). At
60 d after bulbectomy, patterns of expression for neuropilin-1
mRNA and CRMP-2 mRNA resemble those seen at 10 d after lesioning,
although the total number of cells has slightly decreased
(D). The expression patterns in the contralateral
side of the epithelium (G) are indistinguishable
from those of unlesioned control animals (B). In
contrast to bulbectomy, at 60 d after axotomy, expression patterns
strongly resemble control (F). BX,
Bulbectomy; AX, axotomy.
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Olfactory bulb
At 3 and 6 d after olfactory nerve transection, the olfactory
bulb was almost devoid of neuropilin-1 and B-50/GAP-43
immunoreactivity. Nerve bundles in the lamina propria had shrunken and
contained neuropilin-1- and B-50/GAP-43-immunoreactive debris (data not shown). By 10 d, when the first olfactory axons had traversed the
cribriform plate and started to reinnervate the olfactory bulb,
regenerating olfactory axons were positive for neuropilin-1 and
B-50/GAP-43 (data not shown) (Figs. 4I,
5A). The relatively small lesion site contained sema
III-positive cells arranged in typical strings between the cribriform
plate and the olfactory bulb. Neuropilin-1- and
B-50/GAP-43-immunoreactive axons were lined by these strings of sema
III-positive cells, which accompanied them through the lesion site into
undamaged regions of the olfactory bulb (Figs. 4I,
5A). In contrast to the progressive increase of sema
III-positive cells after olfactory bulbectomy, sema III-positive cells
had primarily disappeared at 30 and 60 d after transection of the
olfactory nerve, and non-neuronal sema III mRNA expression was
restricted to cells at the caudal surface of the cribriform plate,
reminiscent of expression in control uninjured bulb (Fig. 4K). Expression of sema III mRNA in neurons of the
olfactory bulb was unchanged after axotomy (data not shown). Expression
patterns for neuropilin-1 and B-50/GAP-43 in the main olfactory bulb
had returned to control, except for some ectopic glomerular innervation in the rostral olfactory bulb (Fig. 4K).
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DISCUSSION |
The present results indicate that the expression patterns of
semaphorin III, neuropilin-1, and CRMP-2 are spatially and
temporally regulated during regeneration of the primary olfactory
pathway. In addition to previous studies exploring the role of
growth-promoting factors during regeneration of the olfactory nerve
(Liesi, 1985; Doucette, 1996), the current observations provide
evidence for chemorepulsive control of primary olfactory nerve
regeneration. In intact olfactory bulb, the ligand for neuropilin-1,
sema III, was expressed by non-neuronal cells at the caudal surface of
the cribriform plate by pial cells covering the lateral and medial aspect of the bulb and by second-order neurons in the olfactory bulb,
all in close proximity to neuropilin-1-positive olfactory axons (Fig.
8). After olfactory bulbectomy or axotomy
of the olfactory nerve, a recapitulation of developmental patterns of
neuropilin-1 and CRMP-2 mRNA expression was observed. Bulbectomy
induced extensive scar formation accompanied by the appearance of
numerous sema III mRNA-containing non-neuronal cells encapsulating
bundles of neuropilin-1-immunoreactive fibers, apparently arresting
their extension into deeper portions of the bulbar cavity (Fig. 8). In
contrast, after axotomy, transient sema III expression occurs in
strings and patches of cells lining neuropilin-1-immunoreactive olfactory axons, which appeared to grow uninhibited through sema III-free spaces to the intact nerve layer. Our results demonstrate, to
our knowledge for the first time, that injury to the CNS induces robust
expression of a chemorepulsive semaphorin, i.e., sema III, in the glial
scar.
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Figure 8.
Proposed roles of semaphorin III-neuropilin-1
signaling during regeneration of the primary olfactory pathway. Scheme
showing the intact (right) and lesioned
(left, bulbectomy) olfactory system. The results show a
complementary localization of sema III and its receptor neuropilin-1 in
both the intact and regenerating adult olfactory system. We propose
that sema III secreted by pial cells and second-order olfactory neurons
of the intact olfactory system helps to confine continuously ingrowing
neuropilin-1-positive olfactory axons to the olfactory nerve and
glomerular layers, thereby determining the gross pattern of innervation
of the olfactory bulb. After injury to the primary olfactory pathway,
sema III-positive non-neuronal cells in the lesion site are present in
close proximity to neuropilin-1-positive regenerating axons.
Interestingly, the differential spatiotemporal expression of sema III
after bulbectomy compared with axotomy appears to correlate to the
regenerative potential displayed after these two types of lesions. The
robust expression of sema III in scar tissue after bulbectomy and the
failure of olfactory axons expressing the sema III receptor
neuropilin-1 to regenerate across this scar suggests that the presence
of semaphorins, i.e., sema III, in CNS scar tissue contributes to the
regenerative failure of the injured CNS.
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Neuropilin-1 and CRMP-2 define a subpopulation of olfactory
receptor neurons
During development of the primary olfactory system, combinatorial
actions of cell adhesion molecules, ECM proteins, and odorant receptors
are governing the establishment of the highly organized connections
between the olfactory epithelium and the main olfactory bulb (Miragall
et al., 1989; Doucette, 1990, 1996; Chung et al., 1991; Miragall and
Dermietzel, 1992; Ressler et al., 1993; Vassar et al., 1993; Krull et
al., 1994; Strottmann et al., 1994; Gong and Shipley, 1995, 1996;
Mombaerts et al., 1996; Treloar et al., 1996; Whitesides and LaMantia,
1996; Yoshihara et al., 1997; Julliard and Hartmann, 1998). Recently,
patterning of developing olfactory axons has been suggested to involve
chemorepellents and their receptors (Giger et al., 1996; Sheperd et
al., 1996; Zhang et al., 1996; Kobayashi et al., 1997; Livesey and
Hunt, 1997; Williams-Hogarth et al., 1997; Yoshida et al., 1997).
Collapsin-1, the chicken homolog of sema III (Luo et al., 1993; Koppel
et al., 1997), induces growth cone collapse of cultured embryonic
olfactory receptor neurons (Kobayashi et al., 1997), which express
neuropilin-1 (Takagi et al., 1991) and CRMP-2, proteins required for
sema III-induced growth cone collapse (Goshima et al., 1995; Wang and
Strittmatter, 1996; Feiner et al., 1997; He and Tessier-Lavigne, 1997;
Kolodkin et al., 1997; Kamata et al., 1998). The sensitivity of
olfactory receptor neurons to sema III and the spatiotemporal
expression patterns of sema III, neuropilin-1, and CRMP-2 suggest that
sema III-neuropilin-1 signaling contributes to the guidance and target finding of developing primary olfactory axons.
In the adult olfactory neuroepithelium, neuropilin-1 and CRMP-2 are
expressed in differentiating B-50/GAP-43-positive neurons and in a
subset of OMP-positive neurons directly adjacent to the differentiating
neurons. Maturation of olfactory receptor neurons is accompanied by a
decline in the expression of B-50/GAP-43 and an induction of OMP. This
switch in gene expression coincides with the cessation of axonal growth
and the formation of synapses in the glomeruli of the olfactory bulb
(Farbman and Margolis, 1980; Miragall and Monti Graziadei, 1982;
Danciger et al., 1989; Verhaagen et al., 1989; Schwob et al., 1992).
The topography of neuropilin-1- and CRMP-2-positive cell bodies in the
olfactory epithelium suggests that the sema III receptor and CRMP-2 are expressed in immature olfactory receptor neurons, elaborating axons
into the bulb, as well as in young mature neurons that are establishing
connections on target cells in the bulb. Sema III may affect the
ongoing ingrowth of neuropilin-1-positive olfactory axons at at least
three sites in the intact adult olfactory bulb. First, as
neuropilin-1-positive primary olfactory axons traverse through holes in
the cribriform plate, sema III secreted from cells at the caudal
surface of the cribriform plate may form a chemorepulsive gradient,
instructing them to deflect from the cribriform plate into the deeper
portion of the olfactory nerve layer. Second, expression of sema III in
more caudal regions of the pial sheet indicates that sema III may
subserve a function in preventing olfactory axons from innervating the
contralateral bulb, thereby maintaining the strictly unilateral
projection of the primary olfactory nerve bundles (Farbman, 1992;
Shipley and Ennis, 1996). Finally, as olfactory fibers arrive in the
olfactory glomeruli, they cease growing and form synapses on
second-order neurons (Sheperd, 1972; Farbman, 1992; Shipley and Ennis,
1996). Continued expression of neuropilin-1 in axons entering the
glomeruli and the presence of its ligand sema III in periglomerular,
mitral, and tufted cells invites the speculation that target-derived
sema III serves as a signal inhibiting further extension of axon
endings into the deeper layers of the bulb. Interestingly, recent
findings show that morphological plasticity of synaptic boutons is
dependent on the relative balance between attractive and repulsive
factors (Winberg et al., 1998). Disturbance of the balance between
growth-promoting and growth-inhibiting forces by ectopic expression of
the growth-associated protein B-50/GAP-43 in mature olfactory receptor
neurons resulted in aberrant olfactory axon endings and some ectopic
primary olfactory projections into deeper layers of the olfactory bulb
(Holtmaat et al., 1995, 1997). In future studies, genetic
manipulation of sema III levels in vivo will be
important to further elucidate the role of this chemorepellent in
plasticity of the mature olfactory system.
Olfactory receptor neurons throughout the rostrocaudal and dorsoventral
extent of the neuroepithelium express neuropilin-1 mRNA, whereas in the
bulb, neuropilin-1 immunoreactivity is confined to the rostrolateral
and caudal glomeruli. Thus, expression of neuropilin-1 in the primary
olfactory system does not show the restricted spatial projection
observed for odorant receptor and ECM proteins (Buck and Axel, 1991;
Ressler et al., 1993; Vassar et al., 1993; Strottmann et al., 1994;
Mombaerts et al., 1996; Yoshihara et al., 1997). This is consistent
with observations in the tadpole (Satoda et al., 1995). Interestingly,
in the tadpole, primary olfactory axons containing no or low levels of
neuropilin-1 express another transmembrane protein, plexin (Ohta et
al., 1995; Satoda et al., 1995). The identification of one of the
plexins as a semaphorin receptor in the immune system (Comeau et al., 1998) suggests that sema III signaling in neuropilin-1-negative olfactory axons may be mediated by other semaphorin receptors, such as plexins.
Distinct patterns of expression of sema III after bulbectomy
and axotomy
Bulbectomy induces the formation of scar tissue, which constitutes
a barrier to the extension of regenerating olfactory axons through the
bulbar cavity into undamaged parts of the CNS, e.g., the frontal pole
of the cortex (Monti Graziadei, 1983; Hendricks et al., 1994). After
axotomy of the primary olfactory nerve, however, regenerating axons are
capable of reinnervating their glomerular targets. Interestingly, when
confronted with a transplanted optic nerve-derived glial scar,
axotomized olfactory axons fail to regenerate as well (Anders and
Hurlock, 1996). The failure of axons to regenerate through a scar, as
observed after bulbectomy, has been observed in other CNS lesion models
as well (Berry et al., 1983; Reier et al., 1983; Bovolenta et al.,
1992) and has been attributed, in part, to growth-inhibitory molecules,
including tenascin, proteoglycans, and myelin-derived glycoproteins
(Rudge and Silver, 1990; Snow et al., 1990; McKeon et al., 1991;
Laywell et al., 1992; Pindzola et al., 1993; Schwab et al., 1993;
Steindler, 1993; Levine, 1994; Mukhopadhyay et al., 1994; Brodkey et
al., 1995; Gates et al., 1996; Davies et al., 1997; Zhang et al.,
1997). The current results show that, in addition to these
growth-inhibitory molecules, high levels of sema III mRNA are present
in CNS scar tissue.
At 1 and 2 months after bulbectomy, large numbers of sema III
mRNA-containing cells occupy the scar and tightly encapsulate bundles
of regenerating axons expressing the sema III receptor neuropilin-1.
Preliminary observations suggest that the scar-related sema
III-positive cells are probably fibroblast-like cells of meningeal
origin (Pasterkamp et al., 1997; R. J. Pasterkamp and J. Verhaagen, unpublished observations). The sensitivity of developing olfactory receptor neurons to sema III (Kobayashi et al., 1997) and the
recapitulation of developmental patterns of neuropilin-1 and CRMP-2
expression after injury to the primary olfactory nerve suggest that
regenerating olfactory receptor neurons are sensitive to sema III as
they elaborate axons to the CNS. Increased or persistent expression of
neuropilin-1 and CRMP-2 after nerve injury is consistent with
observations in lesioned retinal ganglion, dorsal root ganglion, and
motor neurons (Fujisawa et al., 1995, Minturn et al., 1995; Pasterkamp
et al., 1998). It is tempting to speculate that sema III derived from
non-neuronal cells in the scar generates a chemorepulsive barrier in
the bulbar cavity that prevents extension of regenerating olfactory axons.
Interestingly, the sema III gene displays a differential spatiotemporal
pattern of expression after bulbectomy compared with axotomy. After
axotomy, sema III-positive cells are arranged in patches and strings
caudal to the cribriform plate. Bundles of regenerating axons grow
through sema III-free gaps in the lesion area to reach the intact nerve
layer. The growth-permissive properties of the olfactory ensheathing
cells (OEC) in the olfactory nerve layer are probably of critical
importance to the successful regeneration of olfactory nerve bundles
after axotomy (Doucette et al., 1983; Doucette, 1990; Ramón-Cueto
and Valverde, 1995). OEC produce a variety of molecules, including
CAMs, ECM components, and growth factors, supporting neurite outgrowth
(Liesi, 1985; Miragall et al., 1988; Miragall and Dermietzel, 1992;
Ramón-Cueto and Nieto-Sampedro, 1992; Ramón-Cueto et al.,
1993; Franceschini and Barnett, 1996; Kafitz and Greer, 1998). Another
beneficial effect related to OEC is their ability to penetrate glial
scar tissue, enabling regenerative sprouts to pass through the
nonpermissive lesion site (Ramón-Cueto et al., 1998). Noticeably,
transplantation of OEC in the injured spinal cord has resulted in
considerable regeneration across a lesion that would otherwise not
allow regeneration (Li et al., 1997; Ramón-Cueto et al., 1998).
Therefore, the removal of OEC as a result of bulbectomy may contribute
to the failure of regenerating olfactory axons to cross CNS scar
tissue. In contrast, the growth-permissive properties of OEC and the
transient expression of sema III in non-neuronal cells associated with
the lesion site after axotomy may, at least in part, account for the
successful regeneration of axotomized olfactory axons.
In summary, our results show a complementary localization of sema III
and its receptor neuropilin-1 in the intact and regenerating adult
olfactory system (Fig. 8). We propose that sema III in the pial sheet
and in second-order olfactory neurons of the intact olfactory system
helps to confine ingrowing neuropilin-1-positive olfactory axons to the
olfactory nerve and glomerular layers, thereby determining the gross
pattern of innervation of the olfactory bulb. After injury to the
primary olfactory pathway, sema III-positive non-neuronal cells appear
in close proximity to neuropilin-1-positive regenerating axons. The
differential spatiotemporal expression of sema III after bulbectomy
compared with axotomy correlates with the differential regenerative
potential observed after these lesions. Robust expression of the
chemorepellent sema III in scar tissue after bulbectomy and the failure
of neuropilin-1-expressing olfactory axons to regenerate across the
sema III-positive cells in the scar may indicate that this semaphorin
contributes to the growth-inhibitory nature of CNS scar tissue.
| |
FOOTNOTES |
Received Aug. 5, 1998; revised Sept. 8, 1998; accepted Sept. 10, 1998.
This work was supported by a Nederlandse Organisatie voor
Wetenschappel k Onderzcoek-Gebied Medische Wetenschappen
Pioneer Grant and grants from the Van Den Houten Fonds and the
Koninklke Nederlandse Akademie van Wetenschappen
Vernieuwingsfonds. We thank Gerben van der Meulen for his help
with the photographic work. We also thank Bob Baker and Guus Wolswijk
for careful review and discussion of this manuscript.
Correspondence should be addressed to Joost Verhaagen, Netherlands
Institute for Brain Research, Meibergdreef 33, 1105 AZ Amsterdam-ZO,
The Netherlands.
| |
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Top
Abstract
Introduction
