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The Journal of Neuroscience, May 15, 1999, 19(10):4155-4168
Enhanced Neurotrophin-Induced Axon Growth in Myelinated Portions
of the CNS in Mice Lacking the p75 Neurotrophin Receptor
Gregory S.
Walsh1,
Karmen M.
Krol1,
Keith A.
Crutcher2, and
Michael D.
Kawaja1
1 Department of Anatomy and Cell Biology, Queen's
University, Kingston, Ontario, Canada K7L 3N6, and
2 Department of Neurosurgery, University of Cincinnati
Medical Center, Cincinnati, Ohio 45267-0515
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ABSTRACT |
Axonal growth in the adult mammalian CNS is limited because of
inhibitory influences of the glial environment and/or a lack of
growth-promoting molecules. Here, we investigate whether
supplementation of nerve growth factor (NGF) to the CNS during
postnatal development and into adulthood can support the growth of
sympathetic axons within myelinated portions of the maturing brain. We
have also asked whether p75NTR plays a role in this
NGF-induced axon growth. To address these questions we used two lines
of transgenic mice overexpressing NGF centrally, with or without
functional expression of p75NTR
(NGF/p75+/+ and NGF/p75 /
mice, respectively). Sympathetic axons invade the myelinated portions
of the cerebellum, beginning shortly before the second week of
postnatal life, in both lines of NGF transgenic mice. Despite the
presence of central myelin, these sympathetic axons continue to sprout
and increase in density between postnatal days 14 and 100, resulting in
a dense plexus of sympathetic fibers within this myelinated
environment. Surprisingly, the growth response of sympathetic fibers
into the cerebellar white matter of NGF/p75/
mice is enhanced, such that both the density and extent of axon ingrowth are increased, compared with age-matched
NGF/p75+/+ mice. These dissimilar growth responses
cannot be attributed to differences in cerebellar levels of NGF protein
or sympathetic neuron numbers between NGF/p75+/+ and
NGF/p75/ mice. Our data provide evidence
demonstrating that growth factors are capable of overcoming the
inhibitory influences of central myelin in the adult CNS and that
neutralization of the p75NTR may further enhance
this growth response.
Key words:
nerve growth factor; p75 neurotrophin receptor; myelin; axon growth; sympathetic; cerebellum
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INTRODUCTION |
One of the greatest obstacles that
hampers restoration of neural function after injury is the lack of
sustainable axon regrowth in myelinated structures of the adult
mammalian CNS. Myelin produced by oligodendrocytes in the brain
and spinal cord can inhibit axon growth both in vitro and
in vivo (Caroni and Schwab, 1988a ; Crutcher, 1989; Schnell
and Schwab, 1990; Schwab, 1990; Bregman et al., 1995; Lozano et al.,
1995; Schwab and Brösamle, 1997). Glial scars, which form shortly
after the integrity of the brain or spinal cord has been breached,
serve as both a physical (Reier and Houle, 1988; Reier et al., 1989)
and chemical (McKeon et al., 1991; Fawcett, 1994) barrier to axons
attempting to regrow after damage. It has also been suggested that an
inadequate supply of growth-promoting molecules may also contribute to
a lack of new axon extension in the damaged CNS (Schwartz et al.,
1989).
The neurotrophin family of growth factors, including nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), and
neurotrophin-3 (NT-3), has been shown to influence axon sprouting and
regeneration in the mammalian CNS. Menesini-Chen et al. (1978) reported that intraparenchymal infusions of NGF in the
neonatal rat brain promote the directional growth of
sympathetic axons toward the site of injection within yet-to-be
myelinated tracts. This neurotropism, however, could not be induced
through the myelinated tracts of adult animals. Sympathetic
fibers have been shown to grow into the NGF-rich, denervated
hippocampus of adult mammals, but this new growth is confined to the
gray matter (Crutcher et al., 1981; Crutcher and Chandler, 1985;
Crutcher and Marfurt, 1988). Recent investigations have demonstrated
that applications of BDNF and NT-3 can also stimulate the extension of
new axonal processes of CNS neurons through damaged areas of the brain
and spinal cord, but only over short distances and usually confined to
gray matter regions (Logan et al., 1994; Schnell et al., 1994; Mamounas
et al., 1995; Xu et al., 1995; Sawai et al., 1996; Bregman et al.,
1997; Grill et al., 1997; Ye and Houle, 1997; Schwab and Brösamle, 1997). Even when the inhibitory actions of central myelin are neutralized, new axons growing in response to neurotrophins prefer the gray matter as a more supportive microenvironment (Schwab and Brösamle, 1997).
Biological responses to NGF, including neurite outgrowth and axon
elongation, are mediated through binding to two transmembrane receptors. The trkA receptor, of the trk family of receptor
tyrosine kinases, is critical for initiating these stereotypical
actions of NGF (for review, see Klein, 1994; Green and Kaplan, 1995). The second receptor, the p75 neurotrophin receptor (NTR), may be
required to enhance trkA function, especially when NGF is in low
concentrations (Barker and Shooter, 1994; Hantzopoulos et al., 1994;
Verdi et al., 1994; Lachance et al., 1997). Consistent with this,
neurons from the trigeminal and superior cervical ganglia of
p75NTR-deficient mice show a shift to the right in
the NGF dose-response curve (Davies et al., 1993; Lee et al., 1994b),
a finding that may explain the perturbed patterns of sensory and
sympathetic innervation in p75 mutant mice (Lee et al., 1992, 1994a;
Kawaja, 1998). p75NTR can also initiate its own
signal transduction cascades (Dobrowsky et al., 1994; Carter et al.,
1996; Casaccia-Bonnefil et al., 1996), suggesting that
p75NTR intracellular signals may converge with those
generated by trkA to influence neurotrophin responses.
In a previous examination of transgenic mice overexpressing NGF in
astrocytes, we reported that postganglionic sympathetic fibers had
invaded the postnatal cerebellum (which had 50-fold higher NGF levels
than age-matched cerebella) and that this new growth was confined
predominantly to the myelinated tracts (Kawaja and Crutcher, 1997). It
is not certain whether this ingrowth of sympathetic axons is impeded by
the development and presence of myelin in the cerebellum of adult
transgenic mice. Thus, the objective of the present study was to
provide a quantitative assessment of this growth of sympathetic axons
into the developing cerebellum of NGF transgenic mice. To further
clarify the role of p75NTR in NGF-induced
sympathetic sprouting, we have also examined the postnatal growth of
sympathetic axons into the cerebellum of NGF transgenic mice, which
have a targeted deletion of the p75NTR gene.
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MATERIALS AND METHODS |
Animals. Three genotypically distinct strains of mice
were used in this investigation: (1) NGF/p75+/+
mice, which overexpress NGF centrally in astrocytes under control of
the promoter for glial fibrillary acidic protein (GFAP) and possess two
normal alleles for the p75NTR gene (Kawaja and
Crutcher, 1997); (2) NGF/p75/ mice, which
overexpress NGF in astrocytes and have a null mutation of
p75NTR (Coome et al., 1998); and (3) C57Bl/6 mice,
which are the background strain for both NGF/p75+/+
and NGF/p75/ mice. These two lines of NGF
transgenic mice have been bred to homozygosity. Because all littermates
carry the NGF transgene, we chose to use the background strain of mice
(i.e., C57Bl/6) as control animals. In the generation of this
transgenic strain of mice, we have shown that nontransgenic littermates
(like C57Bl/6 mice) display neither elevated levels of NGF in the
cerebellum nor an ingrowth of sympathetic axons into this foreign
target tissue (Kawaja and Crutcher, 1997). For developmental studies, animals from all three genotypes were killed on postnatal day 14 (P14), P28, P60, and P100. For experimental studies (see below), NGF
transgenic lines of mice at P60 and P97 were used, and all were
subsequently killed on P100. All animal procedures and surgical protocols were approved by the Queen's University Animal Care Committee.
Surgery. In the first experiment,
NGF/p75+/+ (n = 7) and
NGF/p75/ mice (n = 10) at P97
underwent unilateral sympathetic ganglionectomy. Animals were
anesthetized with the inhalant Metofane, and under sterile conditions
the left superior cervical ganglion (SCG) was surgically excised. After
recovering from the anesthesia, animals were assessed for ipsilateral
ptosis as confirmation of a successful sympathetic ganglionectomy.
Those animals displaying ptosis were allowed to survive for only 3 d after surgery; thus, this surgical procedure was referred to as an
acute ganglionectomy. On the third postoperative day (i.e.,
at P100), these animals were deeply anesthetized with sodium
pentobarbital (325 mg/kg, i.p.) and killed by transcardial perfusion.
In a second experiment, NGF/p75+/+
(n = 5) and NGF/p75/ mice
(n = 6) at P60 underwent unilateral sympathetic
ganglionectomy as described above. Those animals displaying ipsilateral
ptosis were allowed to survive for 40 d after surgery; thus, this
surgical procedure was referred to as a chronic
ganglionectomy. At P100, these animals were anesthetized with
sodium pentobarbital and killed by transcardial perfusion.
Immunohistochemistry. Mice were deeply anesthetized with
sodium pentobarbital and then perfused transcardially with a solution of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed, post-fixed for 2 hr in the same fixative, and
then immersed for three d in 30% phosphate-buffered sucrose. Brains
were sectioned coronally using a freezing microtome (40 µm
thickness), and sections were stored in cryoprotectant. To ensure that
immunostained tissues could be qualitatively and quantitatively compared between age-matched genotypes, all sections were processed under identical conditions, including buffer concentrations, antibody dilutions, temperature, and incubation and reaction times.
Free-floating sections of the hindbrain (with both the cerebellum and
brainstem) were initially treated in 0.3% hydrogen peroxide in 0.1 M Tris-buffered saline (TBS), pH 7.4, for 1 hr. Sections
were then incubated in 10% bovine serum albumin (BSA) and 0.25%
Triton X-100 in TBS for 1 hr. Endogenous avidin and biotin binding
sites were blocked in two successive steps (avidin-biotin blocking
kit; Vector Laboratories, Burlingame, CA). The sections were then
incubated for 48 hr at 4°C in one of the following primary IgGs:
sheep anti-rat tyrosine hydroxylase (TH) IgG (1:1000 dilution;
Chemicon, Temecula, CA), rat anti-bovine myelin basic protein (MBP) IgG
(1:2000; Chemicon), or rabbit anti-human GFAP IgG (1:1000; Chemicon).
Primary antibodies were diluted in a standard solution containing 3%
BSA and 0.25% Triton X-100 in TBS. All control sections were processed
in the absence of primary IgGs. After a rinse in TBS, the sections were incubated for 2 hr at room temperature in the standard solution containing one of the following biotinylated secondary antibodies (Vector Laboratories): rabbit anti-goat IgG (1:200; for TH
immunoreactivity), rabbit anti-rat IgG (1:500; for MBP
immunoreactivity), or goat anti-rabbit IgG (1:500; for GFAP
immunoreactivity). The sections were then rinsed and incubated in
avidin-biotin reaction complex (Vector Laboratories) for 2 hr at room
temperature and rinsed again. The sections were then reacted with a
solution containing 0.05% diaminobenzidine (DAB) tetrahydrochloride,
0.04% nickel chloride, and 0.015% hydrogen peroxide in 0.1 M TBS. The DAB reaction was terminated by washing the
sections in TBS. After this, sections were mounted on chrome
alum-gelatin-coated slides, dehydrated through a graded series of
ethanols, and coverslipped. All immunostained sections were viewed and
photographed under bright-field optics.
Electron microscopy. At P60, two
NGF/p75+/+ mice were deeply anesthetized with sodium
pentobarbital and perfused with a solution of 4% paraformaldehyde and
0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The
brains were cut on a vibratome (at 50 µm thickness), and sections of
cerebellum were stained immunohistochemically for TH (as described for
light microscopy), except that there was no hydrogen peroxide
pretreatment, nor was Triton X-100 used in the immunostaining. After
the DAB reaction, the sections were rinsed and post-fixed in 1% osmium
tetroxide in 0.1 M phosphate buffer, pH 7.4, for 2 hr at
room temperature. The sections were rinsed, dehydrated through a graded
series of methanols, cleared in propylene oxide, and embedded in a
mixture of Araldite and Epon. Ultrathin sections of the cerebellum were
cut on a Sorvall Ultramicrotome, and the sections were collected on
copper grids, stained with uranyl acetate and lead citrate, and viewed
and photographed in a Hitachi 7000 transmission electron microscope.
ELISA. At P100, C57Bl/6 (total n = 3), NGF/p75+/+ (total n = 9), and
NGF/p75/ (total n = 9) mice were
decapitated, and their cerebella were quickly removed for the
determination of NGF protein levels. Cerebella were taken from (1)
control, nonganglionectomized mice, (2) acutely ganglionectomized mice
(i.e., 3 d survival postoperatively), and (3) chronically
ganglionectomized mice (i.e., 40 d survival postoperatively). NGF
levels were not measured from ganglionectomized C57Bl/6 mice, because
no sympathetic axons are present in the CNS of these animals. The
cerebellum of each animal was divided into left (ipsilateral to a
ganglionectomy) and right (contralateral to a ganglionectomy) halves,
frozen in liquid nitrogen, and stored at  70°C. Coded tissue samples
were shipped on dry ice to Dr. Crutcher's laboratory (University of
Cincinnati Medical Center). Levels of NGF were determined using a
modified two-site ELISA that has previously been shown to be both
sensitive and specific for NGF (Saffran and Crutcher, 1990;
Crutcher et al., 1993). Because the tissues from
NGF/p75+/+ and NGF/p75/ mice
required additional dilutions to obtain accurate values for the very
high levels of NGF protein, all tissues assayed were identified only as
"transgenic" or "nontransgenic." In some cases, samples had to
be run again at higher dilution because of the high levels of NGF in
the tissue. The final values were calculated from the total dilution to
arrive at a concentration per wet weight of the initial sample. No
corrections were made for recovery. Results were tested for
significance by a Student's t test.
Quantitation of sympathetic sprouting in cerebellum. Coronal
sections of cerebellum stained immunohistochemically for TH were used
to measure the percent area occupied by TH-immunoreactive (TH-IR) axons
in the deep white matter (DWM) of C57Bl/6,
NGF/p75+/+, and NGF/p75/
mice. These measurements of axon density are expected to reflect the
degree of collateral growth by sympathetic fibers into the cerebella of
transgenic mice. Digitized images of brain sections were captured
directly from the light microscope with a 40× objective using a Sony
CCD color video camera. The density of TH-IR axons in the
cerebellar DWM was quantified using an image analysis software package
(BIOQUANT">Bioquant/TCW, R & M Biometrics). Video thresholding, a feature of the
software, was used to outline immunoreactive fibers on screen, and the
computer determined the area occupied by immunoreactive fibers. Percent
area was calculated by dividing the area of TH-IR axons by the area of
the DWM being measured. Measurements were taken from two randomly
selected regions of the cerebellar DWM per brain section (two sections
per animal). For developmental studies, a two-way ANOVA was used to
determine whether sympathetic axon density changed with age and
differed between genotypes. A Newman-Keuls multiple comparisons
post hoc analysis was used to test for differences
among ages within each genotype, and a Student's t test was
used for comparisons between genotypes at each age. For acutely and
chronically ganglionectomized animals, sympathetic axon density was
measured in both the ipsilateral and contralateral DWM of all animals
(as previously described). A two-way ANOVA was used to determine
whether sympathetic axon density in either the ipsilateral or
contralateral DWM differed between surgical protocols and differed
between genotypes. A post hoc Student's t
test was used for comparisons between groups. The data were plotted as
mean ± SD.
Morphometric analysis of the superior cervical ganglia.
C57Bl/6 (n = 4), NGF/p75+/+
(n = 3), and NGF/p75/
(n = 4) mice were deeply anesthetized with sodium
pentobarbital and perfused transcardially with a solution of 4%
paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate
buffer, pH 7.4. The SCGs were removed, post-fixed for 2 hr, and
immersed for 2 d in 30% phosphate-buffered sucrose. The ganglia
were then embedded in OCT (Miles, Elkhart, IN) and frozen in
2-methylbutane at 20°C. Serial sections through the entire ganglia
were cut on a cryostat at 10 µm thickness, thaw-mounted onto chrome
alum-gelatin-coated slides, stained for Nissl substance with thionin,
dehydrated through a graded series of ethanols, cleared, and
coverslipped for viewing under bright-field optics. Under
observer-blind conditions, all neuronal profiles displaying a prominent
nucleolus (or nucleoli) were counted on every fifth section through
each ganglion. The sampling frequency (50 µm) ensures that neurons
are not counted twice. Because NGF exposure increases the incidence of
split nucleoli in sympathetic neurons (Ruit et al., 1990), total neuron
counts were corrected for multiple split nucleoli, according to the
calculation of Coggeshall et al. (1984): N = n × [N(c.f.)/n(c.f.)], where N is the true number of sympathetic neurons, n is
the number of neurons displaying a prominent nucleolus (or nucleoli),
N(c.f.) is the number of neurons used to estimate the
correction factor, and n(c.f.) is the number of individual
nucleolar profiles found in these neurons that constitute
N(c.f.). To determine the correction factor
[N(c.f.)/n(c.f.)], one level from each ganglion
was selected, having between 71 and 140 neurons with a nucleolus (or
nucleoli). These data were tested for significance using a one-way
ANOVA with a post hoc Newman-Keuls test for
comparisons between groups. The data were represented as the mean ± SD.
mRNA expression of trkA within sympathetic neurons. At P60,
C57Bl/6 (n = 4), NGF/p75+/+
(n = 4), and NGF/p75/
(n = 4) mice were decapitated, and the right and left
SCGs (plus attached internal carotid arteries) were isolated and frozen
in liquid nitrogen. Trizol (Life Technologies, Gaithersburg, MD) was
used to isolate total RNA from the pooled tissues from each genotype.
One microgram of total RNA from each genotype was used to synthesize
cDNA (Superscript II preamplification system, Life Technologies). To be
certain that cDNA synthesis and amplification of gene products from all
three genotypes were possible, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH; a housekeeping gene product) was first assessed, then trkA.
Controls consisted of reactions conducted in either the absence of the
reverse transcriptase enzyme or the absence of template. DNAs encoding
for GAPDH and trkA were amplified with Taq polymerase using
primer-specific sequences for both gene products. For GAPDH, the primer
sequences were 5'-GTTGCCATCAATGACCCCTTCATTG-3' (5') and
5'-GCTTCACCACCTTCTTGATGTCATC-3' (3'). For trkA, the primer sequences
were 5'-GGTACCAGCTCTCCAACACTGAGG-3' (5') and
5'-CCAGAACGTCCAGGTAACTCGGTG-3' (3'). All reactions were conducted under
identical conditions (e.g., buffers, temperatures, and times) and
underwent 38 cycles. The amplified DNA products were separated in a
0.8% agarose gel and photographed.
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RESULTS |
NGF protein in the cerebellum of transgenic mice
In this study, two lines of GFAP-NGF transgenic mice were used,
one of which expresses functional p75NTR
(NGF/p75+/+) and the other of which has a null
mutation of the p75NTR gene
(NGF/p75/). Both strains of transgenic mice have
been shown to express the NGF transgene within the cerebellum as
determined by RT-PCR (Kawaja and Crutcher, 1997; Coome et al., 1998).
To confirm that these two strains of NGF transgenic mice possessed
equal levels of NGF protein, NGF levels in the cerebellum of C57Bl/6,
NGF/p75+/+, and NGF/p75/ mice
were measured using a two-site ELISA. In contrast to the low levels of
NGF protein found in the cerebellum of C57Bl/6 mice, the mean levels of
NGF protein measured in the cerebellum of NGF/p75+/+
and NGF/p75/ mice were 21- and 22-fold higher,
respectively (Fig. 1). No significant difference in the mean levels of NGF protein was detected between the
cerebellum of NGF/p75+/+ and
NGF/p75/ mice. These data are in agreement with
previous results (Coome et al., 1998).
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Figure 1.
Mean levels of NGF protein in the cerebellum of
C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice, as measured by a two-site ELISA.
The data are represented as fold differences relative to C57Bl/7 mice,
and error bars indicate SD. In contrast to the low levels of NGF
protein in the cerebellum of C57Bl/6 mice, levels of NGF protein are
21- and 22-fold higher in the cerebellum of age-matched
NGF/p75+/+ and NGF/p75/ mice,
respectively. No significant difference in cerebellar NGF levels is
detected between NGF/p75+/+ and
NGF/p75/ mice.
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Sympathetic axons invade the cerebellum
As revealed by TH immunohistochemistry, the cerebellum of adult
(P100) C57Bl/6 mice has a diffuse network of fine TH-IR fibers extending throughout the gray and white matter portions (Figs. 2A,
3A,B). In the cerebellar DWM,
these TH-IR axons are varicose and often observed coursing
perpendicular to the intrinsic cerebellar fibers. These TH-IR axons in
control C57Bl/6 mice comprise the normal innervation of the cerebellum
by neurons of the locus coeruleus, the principal
noradrenergic neurons of the CNS.
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Figure 2.
Coronal sections of cerebellum from C57Bl/6,
NGF/p75+/+, and NGF/p75/ mice
at P100, stained immunohistochemically for TH. In C57Bl/6 mice
(A), a diffuse network of TH-IR axons is evident
in both the cerebellar gray and white matter, representing the local
innervation by locus coeruleus neurons. In contrast to these
observations, the cerebella of both NGF/p75+/+
(B) and NGF/p75/
(C) mice display a robust ingrowth of TH-IR
axons. These dense plexuses of new TH-IR axons are confined
predominantly to the DWM layer and ICPs of the cerebellum and are only
occasionally seen extending into the gray matter layers of the
cerebellum. Qualitatively, the density of TH-IR axons appears increased
in the cerebellum of NGF/p75/ mice compared with
NGF/p75+/+ mice. The white matter portions of the
cerebellum of these mice, as outlined in the box in
A, are shown at two higher magnifications in Figure
3. Scale bar, 500 µm.
IV, Fourth ventricle.
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Figure 3.
Higher magnifications of the cerebellar DWM of
C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. Note the fine TH-IR locus
coeruleus axons, which often course perpendicular to the DWM fibers, in
control C57Bl/6 mice (A, B). In contrast, the DWM of
both NGF/p75+/+ (C, D) and
NGF/p75/ (E, F) mice
display a dramatic increase in TH-IR axons, which course approximately
in parallel with the intrinsic DWM fibers. Again note that the density
of TH-IR axons appears increased in the DWM of
NGF/p75/ mice relative to
NGF/p75+/+ mice. Scale bars: A, C, E,
150 µm; B, D, F, 50 µm.
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In agreement with our previous investigation (Kawaja and Crutcher,
1997), the cerebellum of NGF/p75+/+ mice, at P100,
possessed a marked increase in the number of TH-IR axons compared with
the cerebellum of C57Bl/6 mice (Figs. 2B, 3C,D). Interestingly, this profusion of new TH-IR axons was
localized predominantly to white matter areas of the cerebellum,
including the DWM, inferior cerebellar peduncle (ICP), and cerebellar
folia. These TH-IR axons rarely entered the deep cerebellar
nuclei and were only occasionally seen traversing the cerebellar
gray matter layers. At high magnification, these TH-IR axons were
varicose in nature and coursed mostly parallel to the intrinsic
myelinated fibers. There were also perivascular clusters of
immunostained axons in the DWM; such arrangements were not seen
in P100 C57Bl/6 mice. Our previous investigation with
NGF/p75+/+ mice revealed that these new TH-IR axons,
which invade the cerebellum, are postganglionic sympathetic fibers,
because unilateral removal of the SCG results in a dramatic loss of
these axons in the ipsilateral deep white matter (Kawaja and Crutcher,
1997; also see below). The cerebellum of
NGF/p75/ mice at P100 also possessed a similar
dense plexus of new TH-IR sympathetic axons (Figs. 2C,
3E,F). There was, however, a striking increase in the
density of TH-IR sympathetic axons in the DWM and ICP of
NGF/p75/ mice compared with age-matched
NGF/p75+/+ mice. There was no obvious increase in
TH-IR fibers in the gray matter. Although the reason why sympathetic
axons are confined to the white matter and do not extend into the
adjacent gray matter remains to be elucidated, possibilities include
(1) the presence of inhibitory factors in the gray matter, (2) physical
or geometrical constraints of the white matter, and (3) an increased
production of transgene NGF in the astrocytes of the white matter
relative to those of the gray matter.
In light of the finding that sympathetic axons form a dense plexus
within the cerebellum of adult NGF transgenic mice, it was of interest
to determine the developmental pattern of sympathetic ingrowth in these
animals. We have previously demonstrated that NGF production in the
cerebellum of NGF/p75+/+ mice begins shortly after
the day of birth, increases to 50-fold higher levels until the end of
the second postnatal week, and then slowly decreases to 20-fold higher
levels in the adult (Kawaja and Crutcher, 1997) (our unpublished
data). This pattern of NGF production parallels the ontogeny of GFAP
expression in the rodent CNS (Landry et al., 1990). The percent area
occupied by TH-IR axons in the cerebellar DWM of
NGF/p75+/+ and NGF/p75/ mice
was measured at various developmental ages. Statistical analysis of
TH-IR axon densities using a two-way ANOVA revealed that there were
significant main effects of age (F(3,22) = 201.6; p < 0.001) and genotype
(F(1,22) = 22.71; p < 0.001),
as well as significant interactions between age and genotype
(F(3,22) = 5.060; p < 0.01).
At P14, TH-IR sympathetic axons were already seen invading the
cerebellar DWM (Fig.
4A). In fact, the
percent area occupied by TH-IR axons from the locus coeruleus in the
DWM of P100 C57Bl/6 mice was low (4.1 ± 0.3%; n = 4), in comparison with that measured in P14
NGF/p75+/+ mice (12.8 ± 2.4%;
n = 4; p < 0.001, Student's
t test) (Fig. 5). On
examination of TH-immunostained sections of the cerebellum from
postnatal NGF/p75+/+ mice, it appeared as if the
density of TH-IR axons in the cerebellar DWM increased with age
(compare Figs. 4A,C,E, 3C). Quantitative analysis confirmed this trend, because the area occupied by TH-IR axons
in the DWM at P14 was increased by P28 (46.6 ± 1.5%;
n = 4; p < 0.001, Newman-Keuls test),
was similar between P28 and P60 (mean value, 45.9%; n = 2), and was increased again between P28 and P100 (65.8 ± 4.3%;
n = 4; p < 0.001) (Fig. 5). These
findings demonstrate that TH-IR sympathetic axons begin their growth
into the cerebellum of NGF/p75+/+ mice shortly
before P14 and continue to increase in density into adulthood.
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Figure 4.
Coronal sections of cerebellum from
NGF/p75+/+ (top row) and
NGF/p75/ (bottom row) mice at P14
(A, B), P28 (C, D), and P60 (E,
F), stained immunohistochemically for TH. TH-IR
sympathetic axons are already observed growing into the cerebellar DWM
by P14 in both NGF/p75+/+ and
NGF/p75/ mice. In both NGF transgenic lines of
mice, the density of TH-IR axons in the cerebellar DWM appears to
increase with age. It is also apparent that the density of TH-IR axons
is greater in the DWM of NGF/p75/ mice compared
with NGF/p75+/+ mice. Scale bar, 150 µm.
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Figure 5.
Quantitation of TH-IR axon density in the
cerebellar DWM of C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice at developmental time points.
There are significant increases in the percent area occupied by TH-IR
axons in the DWM of NGF/p75/ mice relative to
NGF/p75+/+ mice at both P14 and P100
(*p < 0.001). The density of TH-IR axons is lowest
in the DWM of P100 C57Bl/6 mice.
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Analysis of NGF/p75/ mice revealed that, similar
to NGF/p75+/+ mice, TH-IR sympathetic axons are
evident in the DWM by P14, and the density of these TH-IR axons
increases throughout the life of the animals (compare Figs.
4B,D,F, 3E). The percent area occupied by
TH-IR axons in the cerebellar DWM of NGF/p75/
mice increased dramatically between P14 and P28 (26.6 ± 4.4 vs 46.4 ± 6.4%; n = 4 for each; p < 0.001, Newman-Keuls test), was similar between P28 and P60
(49.7 ± 4.1%; n = 3; p < 0.05),
and was increased again between P60 and P100 (80.1 ± 5.9%;
n = 5; p < 0.001) (Fig. 5).
Remarkably, the density of TH-IR axons in the DWM of
NGF/p75/ mice was greater than that measured in
NGF/p75+/+ mice at both P14 and P100
(p < 0.01, Student's t test). Both lines of mice displayed similar densities of TH-IR axons at P28 (p = 0.9894, Student's t test), and
no statistical comparison could be made for P60. Taken together, our
data provide evidence that sympathetic axons, in the absence of
p75NTR, are capable of enhanced growth within the
NGF-rich cerebellum of transgenic mice during postnatal development and
into adulthood.
Axon growth occurs in myelinated portions of the cerebellum
One of the remarkable features of sympathetic growth into the
cerebellum of postnatal and adult transgenic mice is the topographical distribution of these aberrant fibers. In both
NGF/p75+/+ and NGF/p75/ mice,
the majority of TH-IR sympathetic axons remains confined to the DWM and
ICP, with few fibers extending into the adjacent gray matter layers. In
agreement with other investigations (Foran and Peterson, 1992; Hamano
et al., 1996), we were able to detect MBP, a major constituent of
central myelin, in the DWM and ICP of the cerebellum using
immunohistochemistry. As early as the second week of postnatal life, a
comparable intensity of MBP immunostaining was seen in both
NGF/p75+/+ and NGF/p75/ mice
(data not shown). This strong immunoreactivity was equally evident in
the myelinated portions of the cerebellum of P100
NGF/p75+/+ and NGF/p75/ mice.
The staining intensity for MBP in both lines of NGF transgenic mice was
comparable with that seen in the cerebellum of control C57Bl/6 mice.
These data reveal that the collateral growth of TH-IR sympathetic axons
into the cerebella of postnatal NGF/p75+/+ and
NGF/p75/ mice continues into adulthood, despite
the presence of central myelin within the DWM and ICP.
To assess which cellular substrates were used by these TH-IR
sympathetic axons, we examined the localization of TH immunoreactivity at the ultrastructural level in P100 NGF/p75+/+
mice. Small clusters of unmyelinated axons, some of which had immunoreactivity for TH, were found among the larger myelinated fibers
of the cerebellar DWM (Fig. 6). The
absence of a glial ensheathment of these unmyelinated TH-IR axons
indicates that Schwann cells do not migrate along with the sympathetic
axons invading the cerebellum. Rather, these TH-IR unmyelinated axons were seen immediately apposed to nonimmunoreactive unmyelinated axons
and myelinated axons alike. Although it is uncertain whether these
sympathetic fibers use intrinsic myelinated axons as substrates for
growth, it is evident that these peripheral fibers are capable of
continued growth within myelinated portions of the adult CNS.
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Figure 6.
Electron photomicrograph showing the localization
of TH immunoreactivity in the cerebellar deep white matter of adult
NGF/p75+/+ mice. TH-IR unmyelinated axons are seen
coursing among myelinated fibers of the cerebellum, often as part of a
small cluster of unmyelinated axons. TH-IR unmyelinated axons lack any
glial support and are often observed in close apposition to the surface
of myelinated axons and unmyelinated axons alike. Scale bar, 0.5 µm.
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Topography of sympathetic axons within the cerebellar DWM
To assess the extent to which sympathetic axons arising from the
right SCG invade both halves of the cerebellum,
NGF/p75+/+ and NGF/p75/ mice
underwent unilateral removal of the left SCG 3 d before P100. This
procedure allowed visualization of only those sympathetic axons arising
from the right SCG in both the ipsilateral and contralateral DWM. After
acute ganglionectomy in NGF/p75+/+ mice, the bulk of
TH-IR sympathetic axons from the contralateral SCG did not extend past
the cerebellar midline (Fig.
7A). In contrast, unilateral
SCG removal in NGF/p75/ mice revealed that many
TH-IR sympathetic axons of the contralateral SCG extended up to and
beyond the cerebellar midline, some of which continued into the upper
portion of the ipsilateral DWM (Fig. 7B).
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Figure 7.
Coronal sections of cerebellum from acutely
ganglionectomized NGF/p75+/+ and
NGF/p75/ mice, stained immunohistochemically for
TH. The unilateral removal of the SCG allows visualization of those
sympathetic fibers from the contralateral ganglion. In
NGF/p75+/+ mice (A), few TH-IR
axons from the contralateral SCG extend as far as the cerebellar
midline (arrow). In contrast, numerous contralateral
sympathetic axons in NGF/p75/ mice
(B) extend past the cerebellar midline
(arrow) and into the upper portions of the ipsilateral
DWM. Scale bar, 500 µm.
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We next made a detailed examination of sympathetic fiber density in the
ipsilateral and contralateral DWM of animals that had undergone either
an acute ganglionectomy or a chronic ganglionectomy. This was done to
determine (1) the degree to which sympathetic axons of the
contralateral SCG extend into the ipsilateral DWM and (2) whether this
unilateral sympathetic "denervation" of the cerebellum is followed
by collateral sprouting from the intact contralateral fibers.
Quantitation of sympathetic fiber density is shown in Figure
8. For all animals, with either an acute
or chronic ganglionectomy, the percent area occupied by TH-IR axons was
dramatically reduced in the ipsilateral DWM compared with the
contralateral DWM of the same animal (p < 0.001, Student's t test). Regardless of acute or chronic
ganglionectomy, the percent area occupied by TH-IR axons in the
contralateral (intact) DWM was higher in
NGF/p75/ mice relative to
NGF/p75+/+ mice (p < 0.001, two-way ANOVA; F(1,24) = 148.6), consistent with
our observations of sympathetic axon density in nonganglionectomized animals. Moreover, sympathetic axon density in the contralateral DWM
did not differ between acute and chronic ganglionectomy in either of
the NGF transgenic strains. Examination of the ipsilateral DWM revealed
that the density of sympathetic fibers was again higher in
NGF/p75/ mice relative to
NGF/p75+/+ mice (p < 0.001, two-way ANOVA; F(1,24) = 37.82), regardless of
the type of ganglionectomy. The finding that a small number of TH-IR
axons persisted in the ipsilateral DWM after acute ganglionectomy suggests that a minor proportion of contralateral (intact) SCG axons
normally project past the cerebellar midline and into the ipsilateral
DWM of both NGF transgenic strains. This proportion, however, is
greater in NGF/p75/ mice. Our data also indicate
that the effect of chronic ganglionectomy was not the same for
NGF/p75+/+ and NGF/p75/ mice.
Whereas no significant change in sympathetic fiber density was observed
in the ipsilateral DWM of NGF/p75+/+ mice between
acute and chronic ganglionectomy, sympathetic fiber density in the
ipsilateral DWM of NGF/p75/ mice was increased
after chronic ganglionectomy compared with acute ganglionectomy
(p < 0.01, two-way ANOVA; F(1,24) = 9.185). These findings suggest that unilateral removal of the SCG
results in the collateral sprouting of sympathetic axons of the
contralateral (intact) SCG within the myelinated portions of the adult
cerebellum in NGF/p75/ mice but not
NGF/p75+/+ mice.
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Figure 8.
Quantitation of sympathetic axon density in the
ipsilateral and contralateral DWM of both lines of transgenic mice
after acute and chronic ganglionectomy. Data for
NGF/p75+/+ mice (n = 7 acute and
5 chronic ganglionectomized animals) are represented by
boxes, and data for NGF/p75/ mice
(n = 10 acute and 6 chronic ganglionectomized
animals) are represented by circles. In all acutely and
chronically ganglionectomized animals, there is a significant decrease
in the percent area occupied by TH-IR axons in the ipsilateral DWM
relative to the contralateral DWM within the same experimental group
(p < 0.001). In the contralateral DWM, the
density of sympathetic axons is higher in
NGF/p75/ mice relative to
NGF/p75+/+ mice (p < 0.001), and these densities are not affected by chronic ganglionectomy.
Note that the density of sympathetic axons that persist in the
ipsilateral DWM, after either acute and chronic ganglionectomy, is also
higher in NGF/p75/ mice relative to
NGF/p75+/+ mice (p < 0.001). Importantly, sympathetic fiber density increases in the
ipsilateral DWM between acutely and chronically ganglionectomized
NGF/p75/ mice but not between acutely and
chronically ganglionectomized NGF/p75+/+ mice
(p < 0.01). All data are represented as
mean ± SD.
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Levels of NGF protein and reactive cerebellar astrocytes
Could the collateral sprouting of sympathetic axons observed in
the ipsilateral DWM of chronically ganglionectomized
NGF/p75/ mice be attributed to a selective
upregulation of NGF protein in the cerebella of
NGF/p75/ mice only? To address this possibility,
NGF levels were measured in the ipsilateral and contralateral halves of
the cerebellum of NGF/p75+/+ and
NGF/p75/ mice 3 and 40 d after unilateral
sympathetic ganglionectomy. For each animal, the level of NGF protein
in the ipsilateral cerebellum was plotted as the fold difference of NGF
levels measured in the contralateral half of the same animal (Fig.
9). In NGF/p75+/+
mice, the mean levels of NGF protein in the ipsilateral cerebellum were
increased relative to the contralateral cerebellum both 3 and 40 d
after ganglionectomy, but these increases were not statistically significant. In contrast, the mean level of NGF protein in the ipsilateral cerebellum of NGF/p75/ mice 3 d
after ganglionectomy was dramatically increased above that measured in
the contralateral cerebellum (p < 0.001, Student's t test). This upregulation in NGF protein in the
ipsilateral cerebellum appeared to be short-lived, because at 40 d
after ganglionectomy, the mean level of NGF protein in the ipsilateral
cerebellum was again not statistically different from that measured in
the contralateral cerebellum of NGF/p75/
mice.
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Figure 9.
Levels of NGF protein in the cerebellum of acutely
and chronically ganglionectomized NGF/p75+/+ and
NGF/p75/ mice (n = 3 animals
per experimental group), as measured by a two-site ELISA. Levels of NGF
in the ipsilateral half of the cerebellum are presented as fold
differences relative to the contralateral half from the same animal,
and error bars indicate SD. This analysis reveals a significant
increase in NGF levels only within the ipsilateral cerebellum of
NGF/p75/ mice after acute ganglionectomy
(*p < 0.001). In all other cases, no significant
differences in NGF levels are detected between ipsilateral and
contralateral cerebellar halves.
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What could account for this marked increase in NGF protein ipsilateral
to the SCG removal in acutely ganglionectomized
NGF/p75/ mice? Because the NGF transgene is
driven by the promoter for GFAP, we examined sections of cerebella
stained immunohistochemically for GFAP. Cerebellar sections from
nonganglionectomized C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice all possessed numerous
GFAP-positive astrocytes in the cerebellar DWM but fewer in the
adjacent gray matter layers. The staining intensity of GFAP
immunoreactivity appeared homogeneous throughout these structures and
comparable among genotypes (data not shown). In chronically
ganglionectomized NGF/p75+/+ and
NGF/p75/ mice, as well as acutely
ganglionectomized NGF/p75+/+ mice, a modest increase
in GFAP immunostaining was evident in the cerebellar white and gray
matter areas ipsilateral to the SCG removal. The ipsilateral cerebellum
of acutely ganglionectomized NGF/p75/ mice,
however, displayed the greatest increase in GFAP immunostaining, which
was evident in astrocytes of both cerebellar white and gray matter
areas. These observations provide an anatomical correlate of the
increased transgene production of NGF in these animals only. We
speculate that a greater gliotic reaction (which includes GFAP
upregulation) is triggered in the cerebella of
NGF/p75/ mice by the degeneration of relatively
more sympathetic fibers and, hence, upregulation of the NGF transgene.
Taken together, our data indicate that, at least in
NGF/p75/ mice, collateral sprouting of intact
(contralateral) sympathetic axons within myelinated portions of the
mature CNS can occur and is likely stimulated by a short-lived
upregulation of NGF protein. Because ganglionectomy failed to cause an
upregulation of NGF protein in NGF/p75+/+ mice, we
cannot exclude the possibility that sympathetic axons are capable of
collateral sprouting in the CNS of these mice.
Sympathetic neuron number
To exclude the possibility that this increased sympathetic
sprouting into the cerebellum was directly linked to increased sympathetic neuron survival in NGF/p75/ mice, we
determined the numbers of SCG neurons in adult animals. Specifically,
the SCGs from C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice were serially sectioned at 10 µm, and the number of neurons with a prominent nucleolus (or
nucleoli) were counted on every fifth section. Exposure to high levels
of NGF is known to increase the incidence of multiple nucleoli among
postganglionic sympathetic neurons (Ruit et al., 1990). Qualitative
assessment of the SCG revealed more sympathetic neurons with two to
four nucleolar fragments in NGF/p75+/+ and
NGF/p75/ mice than in C57Bl/6 mice (Fig.
10A). Quantitation of
the incidence of multiple nucleoli confirmed a significant increase in
the ratio of total nucleoli to SCG neurons counted among
NGF/p75+/+ mice (2.95 ± 0.13) and
NGF/p75/ mice (2.97 ± 0.12) relative to
C57Bl/6 mice (2.28 ± 0.06). In light of these observations, the
total SCG neuron counts were corrected for the presence of multiple
nucleoli, according to the method of Coggeshall et al. (1984). This
analysis demonstrated a statistically significant increase in the
numbers of sympathetic neurons of both NGF/p75+/+
mice (1789 ± 32; n = 3) and
NGF/p75/ mice (2045 ± 338;
n = 4) relative to control C57Bl/6 mice (1112 ± 168; n = 4; p < 0.05, Newman-Keuls
test) (Fig. 10B). No statistically significant
difference in the number of SCG neurons was detected between
NGF/p75+/+ and NGF/p75/ mice.
Thus, these finding indicate that the enhanced sprouting of sympathetic
axons in the cerebellum in NGF/p75/ mice is not
a reflection of increased survival of sympathetic neurons compared with
NGF/p75+/+ mice.
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Figure 10.
Morphological and neurochemical characteristics
of SCG from P60 C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. A, Nissl-stained
sections of SCG from C57Bl/6 (left),
NGF/p75+/+ (center), and
NGF/p75/ (right) mice reveal a
greater incidence of multiple nucleoli among somata of both NGF
transgenic lines of mice. B, Counts of neuronal profiles
in SCG of adult animals show a significant increase in the number of
SCG neurons in both NGF/p75+/+ and
NGF/p75/ mice compared with C57Bl/6 mice
(*p < 0.05). No significant difference was
detected in the number of SCG neurons between
NGF/p75+/+ and NGF/p75/ mice.
C, RT-PCR amplification of GAPDH and trkA in SCG taken
from P60 C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. GAPDH cDNA product (~700 bp) is
detectable in SCG from C57Bl/6 (lane 1),
NGF/p75+/+ (lane 2), and
NGF/p75/ (lane 3) mice. Likewise,
trkA cDNA product (~200 bp) is detectable in SCG from C57Bl/6
(lane 4), NGF/p75+/+
(lane 5), and NGF/p75/
(lane 6) mice. No trkA cDNA product is detectable
in the absence of RNA template (lane 7), and
GAPDH cDNA product is not detectable in the absence of the reverse
transcriptase enzyme (lane 8). L, 100 bp
ladder.
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trkA expression among sympathetic neurons
To exclude the possibility that differences in the degree of
sympathetic sprouting were attributable to a perturbed expression for
trkA mRNA, we used RT-PCR to ensure continued mRNA expression for trkA
in sympathetic neurons of the SCG (Fig. 10C). All three genotypes had detectable levels of the DNA products for trkA (and GAPDH), thereby revealing that the absence of p75NTR
expression did not affect the detection of trkA mRNA from the SCG of
NGF/p75/ mice; these data provide only a
qualitative assessment of the presence or absence of mRNA expression.
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DISCUSSION |
In the present study, elevated levels of NGF protein in the
developing CNS of transgenic mice coincides with the growth of sympathetic axons into the deep white matter portions of the cerebellum during the period of myelination. Despite the presence of central myelin, sympathetic axons continue to grow and increase in density within the cerebellar DWM as the animals mature into adulthood. Surprisingly, the growth of sympathetic axons within the myelinated portions of the NGF-rich cerebellum was greater in NGF transgenic mice
lacking expression of the p75NTR.
Axon growth within myelinated structures
It is generally accepted that the adult mammalian CNS is unable to
support axon growth over long distances. One of the factors influencing
the limited growth of axons is the presence of active growth-inhibitory
factors associated with oligodendrocytes and CNS myelin (Caroni and
Schwab, 1988a,b; Schwab and Caroni, 1988; Crutcher, 1989; Savio and
Schwab, 1990; for review, see Schwab et al., 1993). In support of this
notion, neutralization of the inhibitory properties of central myelin
allows both the regeneration and collateral sprouting of corticospinal
fibers (Savio and Schwab, 1990; Schnell and Schwab, 1990; Bregman et
al., 1995; Vanek et al., 1998). Another factor that may contribute to
the loss of plasticity in the CNS is the lack of appropriate
growth-promoting molecules (Schwartz et al., 1989). Menesini-Chen et
al. (1978) were the first to show that neurotrophins could promote axon
growth within the mammalian CNS. Infusions of NGF into the brainstem of
neonatal rats resulted in the ingrowth of sympathetic axons within
yet-to-be myelinated tracts of the CNS (Menesini-Chen et al., 1978).
However, similar infusions of NGF into the brainstem of adult animals
failed to elicit such ingrowth. Since this time, a number of studies
have demonstrated that local application of neurotrophins can enhance
sprouting responses of various CNS axons (Schnell et al., 1994;
Tuszynski et al., 1994; Xu et al., 1995; Sawai et al., 1996; Bregman et
al., 1997; Grill et al., 1997; Schwab and Brösamle, 1997; Ye and
Houle, 1997). In these experimental paradigms, however,
neurotrophin-induced axon sprouting remains primarily confined to
peripheral nerve grafts, transplants, and nonmyelinated portions of the
mature CNS. Our observation that NGF overexpression in the cerebellum
of postnatal transgenic mice elicits the growth of sympathetic axons
into the cerebellar white matter complements the findings of
Menesini-Chen et al. (1978). Our data further reveal that if NGF levels
remain elevated in the CNS as the animals mature, the growth of
sympathetic axons within myelinated tracts continues unabated into
adulthood. Importantly, Davies et al. (1997) recently showed that adult
neurons implanted into the adult CNS white matter are capable of
long-distance axonal growth. Using a microtransplantation technique
that minimizes glial scar formation, these authors demonstrated that
grafted neurons are capable of rapid and extensive growth of new axons through this myelinated environment. Taken together, our study and that
of Davies et al. (1997) provide evidence supporting the notion that
axonal growth in myelinated portions of the adult CNS is possible,
provided that there is an adequate supply of the appropriate neurotrophin.
An important issue is how sympathetic axons are able to increase in
density in the cerebellar DWM of NGF transgenic mice throughout life,
despite the presence of central myelin. At the ultrastructural level,
TH-IR axons are observed coursing through the cerebellar white matter
as part of a small cluster of unmyelinated axons. Thus, although TH-IR
unmyelinated axons are seen in contact with intrinsic myelinated
fibers, they are also in close apposition to other unmyelinated axons.
Our previous work with NGF/p75+/+ and
NGF/p75/ mice has demonstrated that unmyelinated
sensory axons, immunoreactive for the neuropeptide calcitonin
gene-related peptide, also invade the cerebellar DWM by the second
postnatal week (Kawaja et al., 1997; Coome et al., 1998). Moreover, in
both lines of NGF transgenic mice, these sensory axons are
immunoreactive for the glycoprotein L1 (Walsh et al., 1998), a cell
adhesion molecule that plays an important role in promoting axon
fasciculation (Fischer et al., 1986). On the basis of these findings,
we propose that sympathetic axons are capable of continued growth
within a myelinated environment by using other unmyelinated axons
as a substrate for growth. Such a postulate would predict that the
expression of cell surface molecules that promote axon-axon
fasciculation, such as L1, would be extremely important in allowing
neurotrophin-induced growth within myelinated environments of the
mature CNS.
Role of the p75NTR in the growth of
sympathetic axons
In the present study, we also used a hybrid line of mice, which
overexpresses NGF in astrocytes and has a null mutation in p75NTR (NGF/p75/ mice), to
further clarify the role of p75NTR in
neurotrophin-induced axon growth responses. Sympathetic axons invade
the myelinated cerebellar tracts of NGF/p75+/+ and
NGF/p75/ mice alike, both of which display
comparable levels of NGF protein. The most striking observation in
NGF/p75/ mice is that the magnitude of the
growth response by sympathetic axons within the cerebellum is greater
than that of NGF transgenic mice expressing functional
p75NTR. More specifically, the density of
sympathetic axons within the cerebellar DWM is increased, and
sympathetic axons extend further into the cerebellum of
NGF/p75/ mice compared with
NGF/p75+/+ mice.
How might the presence of p75NTR limit the growth of
sympathetic axons in an NGF-rich target? Because
p75NTR is not expressed by glial cells in the
cerebellar DWM, attenuation of axon growth must be a consequence of the
presence of p75NTR on sympathetic terminals. The
levels of p75NTR and trkA in sympathetic neurons
usually exist in a ratio of ~10:1 (Chao and Hempstead, 1995), despite
the fact that the trkA receptor is necessary for stereotypical NGF
responses, including neurite outgrowth (Loeb et al., 1991; Loeb and
Greene, 1993). Moreover, sympathetic neurons undergoing collateral
sprouting, in response to terminally derived NGF, upregulate only their
expression of p75NTR and not trkA (Miller et al.,
1994). Consistent with this, sympathetic neurons of the
NGF/p75+/+ mice have increased levels of expression
for p75NTR but not trkA (Coome and Kawaja, 1999). It
has been suggested that a selective upregulation in
p75NTR may represent an inhibitory feedback loop,
whereby the presence of more p75NTR at terminal
axons sequesters NGF away from the high-affinity receptor complex and
thus attenuates trkA-mediated NGF signaling (Miller et al., 1994). The
idea that p75NTR plays a role in the sequestration
of NGF on sympathetic terminals is supported by two lines of evidence.
First, examining the dissociation of 125I-NGF from distal
axons of sympathetic neurons in compartmented cultures, Ure and
Campenot (1997) reported that ~85% of axon-associated NGF is
surface-bound, only a portion of which is associated with very slow
dissociating (high affinity) sites. Second, sympathetic axons that
sprout into an NGF-rich tissue display NGF immunoreactivity (Yu and
Crutcher, 1995; Coome et al., 1998). This NGF immunostaining at
sympathetic terminal axons is dramatically reduced in the absence of
p75NTR (Coome et al., 1998). Another possible
outcome of p75NTR-mediated sequestration of NGF on
distal axons is that p75NTR-NGF receptor-ligand
complexes sterically interfere with axon-axon interactions mediated by
cell adhesion molecules. Although it is conceivable that factors that
inhibit fasciculation could reduce axonal growth responses, there is
little evidence supporting such a role for
p75NTR.
Finally, the possibility that p75NTR modulates NGF
signaling and collateral sprouting responses by directly activating an
intracellular pathway should not be ruled out. Recent studies, using
cell lines expressing p75NTR in the absence of
detectable levels of trkA, have demonstrated that ligand binding of
p75NTR can result in the activation and nuclear
translocation of the transcription factor NF- B (Carter et al.,
1996), enhancement of jun kinase activity (Casaccia-Bonnefil et al.,
1996), and generation of ceramide (Dobrowsky et al., 1994, 1995;
Casaccia-Bonnefil et al., 1996). In fact, increased levels of ceramide
in the distal neurites of sympathetic neurons perturbs new growth of
these fibers (de Chaves et al., 1997). Furthermore, BDNF-mediated
activation of p75NTR signaling leads to apoptosis
during the period of naturally occurring cell death among sympathetic
neurons (Bamji et al., 1998). That such a mechanism could account for
our observations of enhanced sympathetic sprouting in the absence of
p75NTR remains to be elucidated. Interestingly,
addition of BDNF has been shown to inhibit NGF-induced growth of
sensory axons (Kimpinski et al., 1997). This study, however, did not
examine whether this inhibition was a direct result of
p75NTR-mediated signaling. In addition, loading PC12
cells and embryonic sensory neurons with a peptide, identical to a
region of the cytoplasmic domain of p75NTR,
modulates NGF-induced neurite outgrowth in a manner that appears to be
mediated downstream of the ligand-receptor level (Dostaler et al.,
1996).
In sum, our data provide evidence demonstrating that supplementation of
NGF can elicit the growth of sympathetic axons within myelinated
environments of the adult mammalian brain. Furthermore, our results
indicate that the growth of sympathetic axons in response to elevated
levels of NGF is enhanced when the function of the p75NTR is neutralized. These findings of NGF-induced
growth of peripheral axons provide encouragement that similar
elongation of CNS axons in myelinated environments of the adult brain
is possible in response to other neurotrophins, such as BDNF and NT-3,
especially when p75NTR function is perturbed.
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FOOTNOTES |
Received Sept. 16, 1998; revised March 8, 1999; accepted March 9, 1999.
This work was supported by grants from the Botterell Foundation and
Advisory Research Committee at Queen's University (to M.D.K.) and
National Institutes of Health Grant NS17131 (to K.A.C.). G.S.W. was
supported by a studentship from the Rick Hansen Man in Motion
Foundation. We are grateful to Janet Elliott for the RT-PCR, Krissy
Klosowski for the ELISA determinations of NGF, Verna Norkum for the
cryostat and ultramicrotome sectioning, and Robert Temkin for the photography.
Correspondence should be addressed to Dr. Michael D. Kawaja, Department
of Anatomy and Cell Biology, Botterell Hall, 9th Floor, Queen's
University, Kingston, Ontario, Canada K7L 3N6.
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Copyright © 1999 Society for Neuroscience 0270-6474/99/19104155-14$05.00/0
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ABSTRACT
INTRODUCTION
