H . Yasuno et al. Brain Research 887 2000 53 –62
55
the respective cDNAs. These probes were labeled with drated in a graded ethanol series, cleared in xylene, and
35
S-deoxyadenosine triphosphate NEN, Boston, MA, coverslipped.
USA and terminal deoxynucleotidyl transferase Amer- sham, Buckinghamshire, UK, giving a specific activity of
2.4. Quantitative analysis
9 9
1.0310 –1.5310 cpm mg. Tissue sections were hybrid- ized after thawing, without any pretreatment, overnight at
Each experimental group consisted of six rats. For the in
5
428C in humidified boxes with 5310 c.p.m. of labeled situ hybridization histochemistry, measurements of the
probe per 100 ml of a mixture containing 43 SSC, 50 relative density of the areas of silver grains over selected
formamide, 0.12 M phosphate buffer, 13 Denhardt’s tissue profiles were performed. OMP mRNA and beta-
solution, 0.2 sodium dodecyl sulfate, 250 mg ml yeast tubulin mRNA levels in the OE were quantified using a
tRNA, 10 dextran sulfate, and 100 mM dithiothreitol. computerized image analysis system IBAS, Zeiss, Ger-
After hybridization, the sections were rinsed four times for many. At a magnification of 320 with bright-field
15 min each at 558C in 13 SSC, dipped into distilled illumination, OE were set such that only silver grains were
water, transferred through 60, 80, and 95 ethanol, and accurately discriminated from the background in the
then air dried. For autoradiography, the sections were lamina propria and read pixel-by-pixel by the computer.
coated with NTB-3 emulsion Kodak, Rochester, NY, We selected randomly 18 fields of view from each rat and
USA; diluted 1:1 with distilled water at 458C and exposed quantified the signal density. The signals for OMP and
for 2–3 weeks in light-tight boxes at 48C. After develop- beta-tubulin were expressed as a percentage of the signal
ment in D19 Kodak and fixation in 24 sodium thiosul- intensity of naive rats. Unpaired comparisons t-test were
fate, the sections were rinsed in distilled water, counter- used to determine if there were significant differences
stained with neutral red, dehydrated in a graded ethanol between groups. P , 0.05 two tail was considered to be
series, cleared in xylene, and coverslipped. statistically significant. All data points are expressed as the
mean6S.E.M. For immunohistochemistry, the number of PGP 9.5-
2.3. Immunohistochemistry labeled profiles per each field of view was counted and
averaged. Eighteen fields of view were selected randomly The sections were immunostained using the avidin–
from each rat. Unpaired comparisons t-test were used to biotin complex ABC with nickel ammonium sulfate
determine if there were significant differences between intensification. The primary antibody for PGP 9.5 poly-
groups. P , 0.05 two tail was considered to be statistical- clonal antibody Ultra Clone Limited, UK was diluted
ly significant. All data points are expressed as the 1:1500 with 0.1 M Tris–HCl buffered saline TBS, pH
mean6S.E.M. 7.5 containing 2 normal goat serum NGS. Anti-Trk A
polyclonal antibody Chemicon International, Temecula, CA, USA was diluted 1:250 and anti-Trk B polyclonal
3. Results
antibody Santa Cruz Biotechnology, Santa Cruz, CA, USA was diluted 1:20. Sections were incubated for 72 h
In situ hybridization histochemistry and immunohisto- at 48C in this solution. The sections were then rinsed with
chemistry were used to show the effect of exogenous NGF 0.1 M TBS, and then incubated in biotinylated goat anti-
and BDNF on the axotomized ORNs of rats. Bright-field rabbit IgG Vector diluted 1:200 in TBS containing 2
and dark-field autoradiography revealed, in naive rats, that NGS for 24 h at 48C. After rinsing with TBS, the sections
the hybridization signals for OMP mRNAs were concen- were incubated with ABC reagent Vector in TBS for 3 h
trated in the middle layer of the OE, indicating that the at 48C. The sections were again rinsed in 0.1 M TBS, then
mature ORNs express high levels of OMP mRNAs Fig. reacted in 0.05 diaminobenzidine tetrahydrochloride
1A,E. Five days after transection of the central branch of Sigma, St. Louis, MO, USA and 0.01 H O in 0.1 M
OE neurons, the OE showed significant degenerative
2 2
TBS containing 0.2 nickel ammonium sulfate for 5 min. changes which resulted in a reduced thickness of the
The sections were then rinsed in distilled water, dehy- epithelium Fig. 1B,F. The signal of OMP mRNA in
Fig. 1. See p. 56 Bright-field A, C, E, G and dark-field B, D, F, H photomicrographs showing the expression of OMP mRNA in OE. The naive OE expressed OMP mRNA abundantly in the middle layer A, B. The saline-treated control OE after axotomy for 5 days expressed almost no OMP mRNA
with reduced height of the epithelium C, D. The NGF-treated OE 5 days after axotomy expressed OMP mRNA moderately, but the thickness of the OE was reduced compared to the naive OE E, F. The BDNF-treated OE after axotomy contained only a weak signal for OMP mRNA G, H. White bars in
B, D, F, and G indicate the layer of the OE. Scale bar 50 mm.
Fig. 2. See p. 57 Bright-field A, C, E, G and dark-field B, D, F, H photomicrographs showing the expression of beta-tubulin mRNA in the OE. The naive OE expressed beta-tubulin mRNA in the under layer A, B. The saline-treated control OE 5 days after axotomy expressed almost no signal C, D.
The NGF-treated OE after axotomy expressed the same level of signals as the naive OE E, F. The BDNF-treated OE after axotomy showed a weak signal for beta-tubulin mRNA G, H. White bars in B, D, F, and G indicate the layer of the OE. Scale bar 50 mm.
56 H
H . Yasuno et al. Brain Research 887 2000 53 –62
57
58 H
control lesioned OE treated with saline decreased In order to examine the effect of neurotrophic factors on
dramatically compared to naive rats Fig. 1B,F. The OE the number of neurons in the neuroepithelium, immuno-
treated with NGF for 5 days after axotomy had a reduced histochemistry using anti-PGP 9.5, a neuronal cell marker,
height of the epithelium, as was the case for the control was employed on the axotomized OE materials. Many
lesioned OE, however the OE expressed moderate levels of neurons were labeled for PGP 9.5 at the base and in the
the signal for OMP mRNA Fig. 1C,G, which was more middle layer of the OE of naive rats Fig. 3A. Five days
dense than that of control lesioned OE that were treated after axotomy, the OE treated with saline control lesioned
with saline. In contrast, BDNF-treated OE after axotomy OE contained very few or almost no neurons labeled for
showed the same pattern of degeneration in the epithelium PGP 9.5, suggesting massive degeneration of ORNs by the
as the control lesioned OE; a reduced thickness and a very axotomy Fig. 3B. The application of NGF into the nasal
weak expression of OMP mRNA Fig. 1D,H. cavity also affected the neuronal number and resulted in
We also examined the expression in beta-tubulin mRNA the detection of a moderate number of profiles labeled for
on the same materials. Naive OE expressed beta-tubulin PGP 9.5 Fig. 3C. However, very few profiles were
mRNA in the lower third of the OE, suggesting its labeled for PGP 9.5 in BDNF-treated OE after axotomy
presence in immature ORNs Fig. 2A,E. Control lesioned Fig. 3D.
OE that were treated with saline and axotomized 5 days Fig. 4 shows the quantification of the relative signals for
prior had very few beta-tubulin mRNA signals Fig. 2B,F. OMP mRNA A, beta-tubulin mRNA B and the number
In NGF-treated OE, axotomized 5 days prior, dense signals of PGP 9.5-labeled profiles C in each OE. The level of
were observed at the same level as naive OE, despite the OMP mRNA in NGF-treated OE was significantly higher
decreased thickness of the epithelium Fig. 2C,G. How- than that in saline-treated OE P , 0.01, however BDNF
ever, BDNF-treated OE expressed only weak signals for treatment did not have any ameliorable effect on OMP
beta-tubulin mRNA Fig. 2D,H. mRNA expression. The same tendency appeared when the
Fig. 3. Location of PGP 9.5 immunostaining in the OE. The naive OE had numerous profiles labeled for PGP 9.5 A. The axotomized OE, treated with saline for 5 days, contained almost no profiles labeled for PGP 9.5 B. The NGF-treated OE after axotomy contained a moderate number of profiles
labeled for PGP 9.5 C. The BDNF-treated OE after axotomy contained few profiles labeled for PGP 9.5 D. Scale bar 50 mm.
H . Yasuno et al. Brain Research 887 2000 53 –62
59
Fig. 4. Effect of NGF and BDNF on OMP A and beta-tubulin B mRNA expression and the number of profiles labeled for PGP 9.5 C in ORNs. The signals for OMP and beta-tubulin mRNA were semi-quantified using a computerized image analysis system IBAS, Zeiss and are expressed as a
percentage of the signal intensity of naive rats. The number of profiles labeled for PGP 9.5 per unit area was counted. The NGF-treated OE had a significantly different level of OMP mRNA compared to saline-treated OE, but BDNF-treated OE was not significantly different from the saline-treated OE
A. The NGF-treated OE also had a significantly different level of beta-tubulin mRNA B and number of labeled profiles for PGP 9.5 compared to saline-treated OE C. P , 0.01.
effect of neurotrophins on the beta-tubulin mRNA was nerve was studied. We demonstrated that NGF has protec-
compared, i.e. NGF could reverse the down-regulation of tive effects on the expression of mRNA expression in both
beta-tubulin mRNA by axotomy, but BDNF could not mature and immature ORNs at the fifth day when NGF
reverse this effect. The NGF-treated OE had 75 of the was applied on axotomized OE for 5 days, but BDNF had
level of the beta-tubulin signal as compared with the naive no effect at a total dose of 10 mg. These data are consistent
OE, which suggests that the immature neurons in the lower with data that show that Trk A-IR was present in dendrites
epithelium were very responsive to NGF. In contrast, the and the axon bundle of ORNs in the olfactory mucosa in
number of NGF-treated OE neurons with PGP 9.5 labeling the naive rat and in the axotomized OE 5 days after injury.
was only 30 of the number of similarly labeled naive OE An estimate of the average lifetime of ORNs is 1 month
neurons; nevertheless, there were significantly more PGP in the rodent [21]. After transection of the olfactory nerve,
9.5-labeled NGF-treated OE neurons than in the saline- ORNs in the OE can fully mature and project afferent
treated OE group. axons directly back into the olfactory bulb after 1 month in
To help determine the mechanism underlying the re- the mouse. The day after transection, severe degeneration
sponsiveness to neurotrophins in axotomized ORNs, we of the ORNs was found, and there was an almost complete
examined the location of the receptors of NGF and BDNF, loss of OMP immunoreactive profiles for 6 days in pigeons
i.e. Trk A and Trk B, using immunohistochemistry. Naive [31]. This finding is consistent with our data that saline-
OE contained Trk A immunoreactive IR dendrites in the treated OE after axotomy for 5 days expressed almost no
top layer of the epithelium and in the axon bundle of OMP mRNA and beta-tubulin mRNA Fig. 1B,F. Two
ORNs in the lamina propria Fig. 5A. Trk B-IR was also weeks after transection, immature regenerating profiles
observed both in the axon bundle in the lamina propria and appear in the OE, and become mature in morphology after
in ORNs in the epithelium Fig. 5B. Five days after 4 weeks [18,30]. Further, olfactory-related behavior recov-
axotomy, Trk A-IR was similar to the naive OE in ers in 4 weeks [42].
dendrites and the axon bundle Fig. 5C. In the axotomized In the olfactory system, several studies have been
OE, the same pattern of labeling of Trk B-IR was observed carried out related to NGF. NGF is produced in the
as in the naive OE Fig. 5D. olfactory bulb [9,17], and is retrogradely transported to the
OE [29]. NGF present in the OE may modulate neuronal turnover [1]. The NGF receptor, Trk A, was found in
mature and regenerating ORNs [22,31]. Similarly, the low
4. Discussion affinity NGF receptor has been found in Schwann cells of
