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The Journal of Neuroscience, January 1, 1999, 19(1):258-273
Absence of the p75 Neurotrophin Receptor Alters the Pattern of
Sympathosensory Sprouting in the Trigeminal Ganglia of Mice
Overexpressing Nerve Growth Factor
Gregory S.
Walsh,
Karmen M.
Krol, and
Michael D.
Kawaja
Department of Anatomy and Cell Biology, Queen's University,
Kingston, Ontario, Canada K7L 3N6
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ABSTRACT |
Sympathetic axons invade the trigeminal ganglia of mice
overexpressing nerve growth factor (NGF) (NGF/p75+/+
mice) and surround sensory neurons having intense NGF immunolabeling; the growth of these axons appears to be directional and specific (Walsh
and Kawaja, 1998 ). In this investigation, we provide new insight into
the neurochemical features and receptor requirements of this
sympathosensory sprouting. Using double-antigen immunohistochemistry, we demonstrate that virtually all (98%) trigeminal neurons that exhibit a sympathetic plexus are trk tyrosine kinase
receptor (trkA)-positive. In addition, the majority (86%) of
those neurons enveloped by sympathetic fibers is also calcitonin
gene-related peptide (CGRP)-positive; a smaller number of plexuses
(14%) surrounded other somata lacking this neuropeptide. Our results
show that sympathosensory interactions form primarily between
noradrenergic sympathetic efferents and the trkA/CGRP-expressing
sensory somata. To assess the contribution of the p75 neurotrophin
receptor (p75NTR) in sympathosensory sprouting, a
hybrid strain of mice was used that overexpresses NGF but lacks
p75NTR expression (NGF/p75 /
mice). The trigeminal ganglia of NGF/p75/ mice,
like those of NGF/p75+/+ mice, have increased levels
of NGF protein and display a concomitant ingrowth of sympathetic axons.
In contrast to the precise pattern of sprouting seen in the ganglia of
NGF/p75+/+ mice, sympathetic axons course randomly
throughout the ganglionic neuropil of NGF/p75/
mice, forming few perineuronal plexuses. Our results indicate that
p75NTR is not required to initiate or sustain the
growth of sympathetic axons into the NGF-rich trigeminal ganglia but
rather plays a role in regulating the directional patterns of axon growth.
Key words:
p75 neurotrophin receptor; transgenic; nerve growth
factor; axon growth; sympathetic; trigeminal
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INTRODUCTION |
Collateral sprouting of mature,
undamaged sympathetic axons is dependent on the availability of the
neurotrophin nerve growth factor (NGF). Tissues with augmented levels
of NGF, as a consequence of damage or disease (Donohue et al., 1989;
Aloe et al., 1992a, 1993; Zettler and Rush, 1993; Kapuscinski et al.,
1996), display an increased density of sympathetic fibers (Mangiarua
and Lee, 1990; Zettler et al., 1991; Aloe et al., 1992b; Falckh et al., 1992). Moreover, administration of NGF antibodies blocks the collateral sprouting response of sympathetic axons (Springer and Loy, 1985; Gloster and Diamond, 1992). A role for NGF in the guidance of elongating nerve fibers in vitro has been suggested
(Letourneau, 1978; Gundersen and Barrett, 1979; Gundersen, 1985), as
well as in vivo, because new growth of sympathetic axons
occurs toward natural and unnatural sites of increased NGF content
(Menesini-Chen et al., 1978; Edwards et al., 1989; Albers et al., 1994;
Hassankhani et al., 1995; Ma et al., 1995; Kawaja and Crutcher,
1997).
Injury to peripheral nerves elicits the sprouting of sympathetic fibers
into affected dorsal root ganglia (DRG) (McLachlan et al., 1993; Chung
et al., 1996; Zhou et al., 1996; Ramer and Bisby, 1997a), where they
form discrete projections to a subset of sensory somata and
subsequently envelop them in a perineuronal (basket-like) plexus of
fibers. These arborizations may represent the anatomical substrate for
the functional coupling between sympathetic and sensory neurons that
underlie the development of causalgia and sympathetically maintained
pain states that result from nerve injury (Richards, 1967; Devor, 1983;
Bonica, 1990). The molecular signal initiating this sympathetic
sprouting response is most likely NGF, because a similar pattern of
sprouting has been reported in the trigeminal ganglia of transgenic
mice overexpressing NGF in skin (Davis et al., 1994) and in glial cells
(Walsh and Kawaja, 1998), although recently leukemia inhibitory factor
has also been shown to elicit sympathetic sprouting and basket
formation in the intact DRG of adult rats (Thompson and Majithia,
1998).
The relative contributions of the NGF receptors in mediating axon
growth responses are not entirely clear. The trk tyrosine kinase receptor (trkA) is necessary and sufficient to confer NGF responsiveness (Koizumi et al., 1988; Loeb et al., 1991;
Ibáñez et al., 1992) and mediate stereotypical NGF
responses, such as neurite outgrowth (Loeb et al., 1991; Loeb and
Greene, 1993; Peng et al., 1995) and growth cone turning (Gallo et al.,
1997). The p75 neurotrophin receptor (p75NTR) was
originally reported to function as a positive modulator of trkA
activity (Benedetti et al., 1993; Barker and Shooter, 1994;
Hantzopoulos et al., 1994; Verdi et al., 1994; Lachance et al., 1997).
Recent experiments also indicate that p75NTR may
play an autonomous signaling role (Dobrowsky et al., 1994; Carter et
al., 1996; Casaccia-Bonnefil et al., 1996). Evidence is beginning to
emerge that p75NTR plays a role in axonal growth
responses. Disruption of NGF binding to p75NTR
reduces, but does not inhibit, growth cone turning (Gallo et al.,
1997), and a peptide analog of the cytoplasmic region of p75NTR modulates neurite outgrowth from PC12 cells
(Dostaler et al., 1996). Targeted deletion of the
p75NTR gene reduces neuronal sensitivity to NGF
(Davies et al., 1993; Lee et al., 1994b) and perturbs the developmental
innervation of selected peripheral targets by sympathetic axons (Lee et
al., 1994a; Kawaja, 1998). The role of p75NTR in the
collateral sprouting of mature sympathetic axons, however, is poorly understood.
Using transgenic mice that overexpress NGF among glial cells, we
recently demonstrated that sympathetic axons form dense pericellular plexuses with only those trigeminal neurons that stain
immunohistochemically for NGF (Walsh and Kawaja, 1998), indicating that
sympathetic fibers selectively target a subpopulation of sensory somata
that are NGF responsive. To provide added proof that this sprouting response is directional and specific, we have further characterized the
neurochemical phenotype of those trigeminal neurons that exhibit sympathetic arborizations. Our results reveal that the majority of
sympathetic plexuses forms around the trkA/calcitonin gene-related peptide (CGRP)-expressing population of sensory neurons in NGF transgenic mice. To investigate the role of p75NTR
in this sympathetic sprouting response, we used a new line of transgenic mice that overexpresses comparable levels of glial NGF but
lacks the functional expression of p75NTR (Coome et
al., 1998). Sympathetic sprouting in the trigeminal ganglia also occurs
in these hybrid mice, but the directional growth of these collateral
branches is perturbed in the absence of p75NTR
expression. Specifically, the incidence of sympathetic pericellular plexuses is markedly reduced, and those that do form are composed of
fewer fibers. Our data indicate that p75NTR plays a
role in enhancing the directional pattern of axon elongation but is not
required for the initiation of NGF-induced sympathetic sprouting.
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MATERIALS AND METHODS |
Animals and surgery. Five genotypically distinct
strains of mice were used in this investigation: (1)
NGF/p75+/+ mice, which overexpress NGF among glial
cells 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 possess both an overexpression of NGF among glial cells and
a null mutation of p75NTR (Coome et al., 1998), (3)
C57Bl/6 mice, which are the background strain for both
NGF/p75+/+ and NGF/p75/ mice,
(4) p75/ mice (Lee et al., 1992), and (5) BALB/c
mice, which are the background strain for both
NGF/p75/ and p75/ mice.
Adult (2-3 months of age) NGF/p75+/+ and
NGF/p75/ mice (n = 2 per
genotype) were anesthetized with the inhalant Metofane, and under
sterile conditions the left superior cervical ganglion (SCG) was
surgically removed. After a 4 d survival period, the animals were
deeply anesthetized with sodium pentobarbital (325 mg/kg, i.p.) and
killed by transcardial perfusion. The trigeminal ganglia were dissected
out and processed for immunohistochemistry (see below). All animal
procedures and surgical protocols were approved by the Queen's
University Animal Care Committee.
Enzyme-linked immunosorbent assay. The trigeminal ganglia
from adult C57Bl/6 (n = 5),
NGF/p75+/+ (n = 8), and
NGF/p75/ (n = 8) mice were
quickly removed after decapitation, frozen in liquid nitrogen, and
stored at  70°C. Samples were shipped on dry ice to Dr. Keith A. Crutcher (University of Cincinnati, OH) for determination of ganglionic
levels of NGF using a modified two-site ELISA. This assay has
previously been shown to be both sensitive and specific for NGF
(Saffran et al., 1989; Crutcher et al., 1993). The ELISAs were
performed without knowledge of the tissue source. Results were tested
for significance by a one-way ANOVA, and comparisons between groups
were made using a post hoc Newman-Keuls test. The data were
presented as mean total amount of NGF per ganglion (picograms of
NGF/ganglion), and error was represented as SD.
Tissue preparation. For immunohistochemistry, anesthetized
C57Bl/6, BALB/c, NGF/p75+/+,
NGF/p75/, and p75/ mice
were perfused transcardially with a solution of 4% paraformaldehyde in
0.1 M phosphate buffer, pH 7.4; to enhance immunostaining
for NGF in tissues, parabenzoquinone was routinely added to this
fixative to a final concentration of 0.2%. For neuronal counting,
anesthetized mice from all five genotypes were transcardially perfused
with a solution of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. All trigeminal ganglia were
dissected from the skulls, post-fixed for 2 hr, and immersed for 2 d in 30% phosphate-buffered sucrose. The ganglia were then embedded in
OTC (Miles Corporation, Elkhart, IN), frozen in 2-methylbutane at 20°C, and sectioned on a cryostat at 10 µm thickness. For immunohistochemistry, each slide had ganglia from C57Bl/6 (or BALB/c),
NGF/p75+/+, and NGF/p75/
mice, thereby ensuring equal exposure to all antibodies and allowing qualitative comparisons with respect to staining intensity. For neuron
counting, the ganglia were cut to completion, mounted on chrome alum
gelatin-coated slides, Nissl stained with thionin, dehydrated through a
graded series of ethanols, cleared, and coverslipped for viewing under
bright-field optics.
Immunohistochemistry. Sections of trigeminal ganglia
were initially treated in 0.3% hydrogen peroxide
(H2O2) in Tris-buffered saline (TBS),
pH 7.4, for 30 min. They 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 room
temperature in one of the following primary IgGs: sheep anti-rat
tyrosine hydroxylase (TH) IgG (1:1000 dilution; ], rabbit anti-synthetic trkA IgG (1:1000;
Chemicon). Primary antibodies were diluted in a standard
solution containing 3% BSA and 0.25% Triton X-100 in
TBS. For trkA immunohistochemistry, normal goat serum was
used in place of BSA for all standard solutions. All
control sections were processed in the absence of primary
IgGs. After a rinse in TBS, the sections were incubated in
the standard solution containing biotinylated rabbit
anti-goat IgG (1:200; Vector Laboratories; for TH
immunoreactivity) or biotinylated goat anti-rabbit IgG
(1:200; Vector Laboratories; for NGF, trkA, and CGRP
immunoreactivities) for 2 hr at room temperature. They
were then rinsed and incubated in avidin-biotin 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%
H2O2 in 0.1 M TBS. After the DAB reaction, sections were washed
in TBS, dehydrated through a graded series of ethanols,
coverslipped, viewed, and photographed under
bright-field optics.
Double-antigen immunohistochemistry. To investigate whether
TH-wrapped trigeminal neurons of NGF transgenic mice were trkA positive
and CGRP positive, double-immunolabeling for TH/trkA or TH/CGRP was
performed, using the method of Levey et al. (1986). Briefly, sections
were stained for the first antigen and reacted with DAB as the
chromagenic reagent, as described for single antigen immunohistochemistry. After the DAB reaction, sections were reacted in
0.3% H2O2 in TBS for 10 min to eliminate any
remaining peroxidase activity. Sections were then stained for the
second antigen as per single-antigen immunohistochemistry up to
and including the incubation in the avidin-biotin complex. Sections
were then rinsed in 0.01 M phosphate buffer, pH 6.6, for 15 min, and transferred to a solution containing 0.01% benzidine
dihydrochloride (BDHC) and 0.025% sodium nitroferricyanide in 0.01 M phosphate buffer, pH 6.6. After 10 min, the reaction was
initiated by adding H2O2 to a final
concentration of 0.005% in fresh BDHC solution. The reaction was
terminated after 5-10 min by rinsing with cold 0.01 M
phosphate buffer, pH 6.6, and then rapidly dehydrated through a series
of ethanols, coverslipped, viewed, and photographed under bright-field
optics. This technique yields a diffuse, brown-colored reaction product
for the first antigen, and a granular, blue-colored reaction product
for the second antigen.
Electron microscopy. For electron microscopy, anesthetized
NGF/p75+/+ mice (n = 2) and
NGF/p75/ mice (n = 2) were
perfused transcardially with a solution containing 4% paraformaldehyde
and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The trigeminal ganglia were dissected out, minced into smaller pieces,
and post-fixed in 1% osmium tetroxide in 0.1 M phosphate
buffer, pH 7.4, for 2 hr at room temperature. The tissues 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 tissues 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.
Quantitative analysis. For neuron counting, complete series
of Nissl-stained trigeminal ganglia were taken from C57Bl/6 mice (n = 3), BALB/c mice (n = 4),
NGF/p75+/+ mice (n = 4),
NGF/p75/ mice (n = 4), and
p75/ mice (n = 3). Under
observer-blind conditions, only those neurons containing a distinct
nucleolus (or nucleoli) were counted in sections 50 µm apart
throughout the entire ganglia. Then, images of the ganglia were
captured directly from the microscope at 40× objective using a Sony
CCD color video camera and imported into an image analysis software
package (Bioquant/TCW, R & M Biometrics, Nashville, Tennessee). The
perimeter of nuclear profiles of randomly selected trigeminal neurons
from each genotype were outlined manually using a computer mouse, and
the computer measured the nuclear area. Diameter was subsequently
determined on the assumption that the nuclei are approximately
circular. Results were tested for significance by a one-way ANOVA, and
comparisons between groups were made using a post hoc
Newman-Keuls test.
The proportion of TH-wrapped neurons that were trkA positive and CGRP
positive was determined from sections of trigeminal ganglia, taken from
NGF/p75+/+ mice, and double-immunostained for
TH/trkA and TH/CGRP, respectively. For TH/trkA-immunostained sections,
TH-wrapped neurons were sampled from six sections (50 µm apart) of
ganglia per animal (n = 3), for a total of 402 neurons;
for TH/CGRP-immunostained sections, TH-wrapped neurons were sampled
from four sections (100 µm apart) of ganglia per animal
(n = 3), for a total of 317 neurons. Neuronal profiles
with pericellular TH-immunostained axons were scored for the presence
or absence of trkA or CGRP immunoreactivity, and the data were
represented as a percentage of the total number of TH-wrapped neurons.
The cell size of trigeminal neurons surrounded by TH-IR axons
in NGF/p75+/+ mice was quantified. Images were
captured directly from the microscope at 40× objective using a Sony
CCD color video camera and imported into an image analysis software
package (as before). The perimeter of neuronal profiles with
pericellular TH-IR axons was outlined manually using a computer mouse,
and the computer measured the neuronal area. Diameter was subsequently
determined on the assumption that trigeminal neurons are approximately
circular. Perimeters of TH-wrapped neurons were traced from six
sections (100 µm apart) of ganglia per animal (n = 3), for a total of 160 neurons. To compare the cell size distribution
of TH-wrapped neurons with the cell size distribution of the total
trigeminal neuron population of C57Bl/6 and
NGF/p75+/+ mice, perimeters of randomly selected
Nissl-stained neurons (displaying a prominent nucleolus) were traced
from two sections (100 µm apart) of ganglia per animal
(n = 3), for a total of 823 and 554 neurons, respectively. Cell sizes were plotted as relative frequencies, and
statistical differences in the mean diameter of cell size distributions
were determined using a one-way ANOVA, and comparisons between groups
were made using a post hoc Newman-Keuls test.
Sections of trigeminal ganglia stained immunohistochemically for TH
were used to measure the area occupied by TH-IR axons within the
ganglionic fiber tracts of C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. The density of TH-IR axons in
the ganglionic fiber tracts was quantified on digitized images of
ganglia using image analysis software (as before). Video thresholding,
a feature of the software, was used to outline TH-IR fibers on screen,
and the computer determined the area occupied by immunoreactive fibers
within a defined region of interest. At least 20 measurements were made
in four sections (no less than 100 µm apart) of ganglia per animal
(n = 3 per genotype). Values were plotted as percentage
area, and error was represented as SD. Results were tested for
significance by a one-way ANOVA, and comparisons between groups were
made using a post-hoc Newman-Keuls test.
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RESULTS |
Specificity of sympathosensory projections
We have previously reported that trigeminal neurons in
NGF/p75+/+ mice stain more intensely for NGF than
neurons of control mice, a finding that indicates that these neurons
are internalizing and accumulating higher levels of NGF. Moreover,
sympathetic axons, which grow into the NGF-rich trigeminal ganglia of
NGF/p75+/+ mice, project only to those sensory
neurons displaying NGF immunostaining (Walsh and Kawaja, 1998). We
postulated that neurons exhibiting a TH-IR plexus must express the
high molecular weight neurotrophin receptor trkA, because NGF
responsiveness is dependent on expression of this receptor (Loeb et
al., 1991; Loeb and Greene, 1993). To confirm this hypothesis, we used
simultaneous double immunohistochemistry to demonstrate the coincidence
of TH-IR sympathetic plexuses with trkA-IR trigeminal somata in
sections of ganglia taken from NGF/p75+/+ mice.
Trigeminal neurons displaying a prominent TH-IR perineuronal plexus
were scored for the presence or absence of trkA immunoreactivity (Fig.
1A,B). This analysis
revealed that virtually all TH-IR perineuronal plexuses (98%; 394 of
402) surrounded neurons exhibiting trkA immunoreactivity; not all
trkA-positive trigeminal neurons, however, were surrounded by TH-IR
fibers. These results indicate that, in agreement with Davis et al.
(1998), sympathetic axons are attracted specifically to trkA-expressing
sensory neurons that appear to accumulate high levels of NGF within
their cell bodies.
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Figure 1.
A, B,
Sections of trigeminal ganglia from NGF/p75+/+ mice
double-immunostained for trkA (brown reaction product)
and TH (blue reaction product). Virtually all (98%)
sympathetic perineuronal plexuses are associated with trkA-positive
neurons (arrows). Not all trkA-positive somata, however,
exhibit a TH-IR perineuronal plexus.
C-H, Sections of trigeminal
ganglia from NGF/p75+/+ mice double-immunostained
for CGRP (blue reaction product) and TH
(brown reaction product). The majority (86%) of TH-IR
perineuronal plexuses is associated with CGRP-IR sensory neurons. Some
somata displaying a TH-IR perineuronal plexus are intensely
immunoreactive for CGRP (arrows), whereas others show
weak CGRP immunoreactivity (double arrows). A smaller
percentage (14%) of TH-IR perineuronal plexuses is associated with
somata displaying no detectable CGRP immunoreactivity (large
arrowhead). Scale bars, 50 µm.
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Averill et al. (1995) determined that the majority (92%) of
trkA-expressing DRG neurons co-express the neuropeptide CGRP in rats.
Thus, we reasoned that CGRP immunoreactivity would also identify those
trigeminal neurons exhibiting a TH-positive perineuronal plexus. The
proportion of TH-wrapped neurons showing CGRP immunoreactivity was
determined using sections of trigeminal ganglia of
NGF/p75+/+ mice double-immunostained for TH and
CGRP. Many sensory neurons surrounded by TH-IR fibers possessed
variable intensities of CGRP immunoreactivity (Fig. 1C-G);
not all CGRP-IR sensory neurons, however, were surrounded by
sympathetic fibers. Quantitative analysis revealed that most TH-IR
plexuses (86%; 273 of 317) were associated with sensory somata
immunopositive for CGRP; no distinction was made between those somata
possessing weak to strong immunolabeling for this neuropeptide.
Interestingly, a small but significant number of TH-IR perineuronal
plexuses (14%; 44 of 317) formed around sensory somata that displayed
no detectable immunoreactivity for CGRP (Fig. 1H).
These findings demonstrate that sympathetic axons selectively target
the trkA-positive/CGRP-positive population of trigeminal sensory
neurons, presumably those having unmyelinated axons and subserving
nociception (Lawson, 1992)
To further characterize the population of trigeminal neurons that
exhibit a TH-IR perineuronal plexus, we determined the size/frequency distribution of both the trigeminal neuron population and those neurons
enveloped by TH-IR axons in NGF/p75+/+ mice (Fig.
2). The diameter of trigeminal neurons of
C57Bl/6 mice ranged from 10 to 43 µm, with a mean diameter of
20.0 ± 5.0 µm. The diameter of trigeminal neurons of
NGF/p75+/+ mice ranged from 11 to 41 µm, with a
mean diameter of 23.0 ± 5.5 µm; the mean diameter of trigeminal
neurons of NGF/p75+/+ mice is significantly larger
than that of C57Bl/6 mice (p < 0.001; Newman-Keuls test), suggesting a neuronal hypertrophy in response to
elevated levels of NGF (see below). Neurons exhibiting a TH-IR perineuronal plexus predominantly had large diameters ranging from 22 to 50 µm, with a mean diameter of 35.1 ± 5.4 µm. This increase in the mean diameter of TH-wrapped neurons was found to be
statistically significant when compared with the mean diameter of the
whole trigeminal neuron population of NGF/p75+/+
mice (p < 0.001). It should be noted that the
diameters of TH-wrapped neurons presented here are likely a slight
overestimation of the true diameter, because the TH-IR fibers
surrounding these neurons obscured the edge of the perikaryon.
Nevertheless, the subset of sensory neurons that become enveloped by
sympathetic axons are among the largest neurons in the trigeminal
ganglia of NGF/p75+/+ mice.
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Figure 2.
Size histograms of the total trigeminal neuron
population from C57Bl/6 mice (top panel) and
NGF/p75+/+ mice (middle
panel), as well as those trigeminal neurons that are
surrounded by TH-IR fibers in NGF/p75+/+ mice
(bottom panel). Each bin width is 4 µm, and the
number assigned to each column represents the middle value for each
bin. Comparisons of these cell size distributions reveal that the mean
neuron size of C57Bl/6 mice is smaller than that of
NGF/p75+/+ mice (p < 0.001), and that TH-IR fibers preferentially envelop large-diameter
trigeminal neurons of NGF/p75+/+ mice
(p < 0.001).
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Altered pattern of sympathetic sprouting in the absence of
p75NTR expression
Previous work has demonstrated a role for
p75NTR in modulating the postnatal growth of
sympathetic axons, because selective innervation of some sympathetic
targets is perturbed in p75/ mice (Lee et al.,
1994a; Kawaja, 1998). To determine whether p75NTR
promotes axon growth among mature neurons, we examined the collateral sprouting response of sympathetic axons in trigeminal ganglia of
GFAP-NGF transgenic mice in the absence of p75NTR
expression (NGF/p75/ mice). This line of hybrid
mice, established through the selective interbreeding of
NGF/p75+/+ mice (the background strain of which is
C57Bl/6) and p75/ mice (the background strain of
which is BALB/c), exhibit no difference in the sites of NGF transgene
expression or levels of NGF protein in the CNS, as compared with
NGF/p75+/+ mice (Coome et al., 1998).
In C57Bl/6 and BALB/c mice (two wild-type mouse strains), a small
number of TH-IR axons was observed along the ganglionic capsule and
associated with blood vessels, forming perivascular networks (Fig.
3A,B). A sparse population of
TH-IR neuronal cell bodies was also observed, representing local
dopaminergic sensory neurons (Katz et al., 1983; Price and Mudge,
1983). As we have documented previously (Walsh and Kawaja, 1998),
numerous TH-IR varicose axons were found throughout the neuropil
regions of the ganglia from NGF/p75+/+ mice (Fig.
3C,E). These immunoreactive fibers, which appeared to enter
the ganglionic environment from the trigeminal nerve or by departing
from local blood vessels, did not project randomly among the sensory
somata. Rather, these fibers appeared to grow specifically toward a
subset of trigeminal somata. At high magnification, several TH-IR
fibers often converged on a single soma and commenced wrapping around
the perimeter of the perikaryon, thereby enveloping the soma in a
complete perineuronal plexus of noradrenergic fibers (Fig.
4A,C,E). After the
removal of the ipsilateral SCG, all TH-IR plexuses and the vast
majority of fibers were no longer evident, thus confirming that these
new fibers were sympathetic in origin.
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Figure 3.
Sections of trigeminal ganglia from C57Bl/6,
BALB/c, NGF/p75+/+, and
NGF/p75/ mice, stained immunohistochemically for
TH. The trigeminal ganglia of C57Bl/6 (A) and
BALB/c (B) mice possess TH-IR sympathetic fibers
forming perivascular plexuses (arrow), as well as a
small population of TH-immunopositive dopaminergic sensory neurons.
Many TH-IR fibers are seen in ganglia of NGF/p75+/+
mice, projecting to a subset of trigeminal somata and enveloping them
in a tight perineuronal plexus of fibers (C,
E). TH-IR fibers are also present in ganglia of
NGF/p75/ mice, but the pattern of sprouting
appears more random and TH-IR plexuses appear less prominent
(D, F). Scale bars:
A-D, 125 µm; E, F, 50 µm.
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Figure 4.
Examples of TH-IR perineuronal plexuses in
trigeminal ganglia from NGF/p75+/+ and
NGF/p75/ mice. In NGF/p75+/+
animals (A, C, E), several
TH-IR fibers often converge on the soma of individual sensory neurons,
enveloping the perimeter in a tight, dense plexus of fibers. In
NGF/p75/ mice (B,
D, F), TH-IR fibers randomly grow
within the neuropil, weaving around the cell bodies of sensory neurons,
sometimes in close contact with the somata. However, intense
perineuronal plexuses that tightly envelop the somata of
NGF/p75/ ganglia are rarely seen. Scale bar, 25 µm.
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Trigeminal ganglia of NGF/p75/ mice also
possessed a robust ingrowth of new TH-IR fibers. Similar to the TH-IR
fibers in ganglia of NGF/p75+/+ mice, TH-IR axons
in NGF/p75/ mice were varicose and were again
lost after ipsilateral SCG removal. What was most striking about the
appearance of TH-IR sympathetic axons in the ganglia of
NGF/p75/ mice was an impression of randomness,
or disorganization, in the pattern of sprouting (Fig.
3D,F). The directional growth of sympathetic axons
toward a subset of trigeminal sensory somata observed in
NGF/p75+/+ animals was not evident in the ganglia of
NGF/p75/ animals. Instead, TH-IR axons invading
the ganglionic neuropil of NGF/p75/ mice
appeared to weave in and around individual somata, rather than becoming
associated with one particular neuron cell body. It should be noted
that the formation of TH-IR perineuronal plexuses was not fully
inhibited, because a few TH-IR sympathetic fibers were observed to
envelop somata in the trigeminal ganglia of
NGF/p75/ mice (Fig.
4B,D,F). The morphology of these plexuses in
NGF/p75/ mice, however, was markedly different
from the perineuronal plexuses seen in NGF/p75+/+
mice: (1) a smaller number of TH-IR fibers appeared to contribute to
the perineuronal plexus, and (2) complete envelopment of the sensory
somata by TH-IR axons was rarely observed. Consistent with this, fewer
sympathetic perineuronal plexuses form in the DRG of
p75/ mice after peripheral nerve axotomy (Ramer
and Bisby, 1997b).
To confirm that there were indeed fewer sympathetic axons contributing
to the perineuronal plexuses in NGF/p75/ mice,
we examined trigeminal ganglia taken from both transgenic lines of mice
at the electron microscope level. Sensory neurons, examined in
NGF/p75+/+ and NGF/p75/ mice,
were closely apposed by the surrounding satellite cell processes. In
the ganglia of NGF/p75+/+ mice, a small population
of somata had bundles of unmyelinated fibers with axonal swellings
filled with clear vesicles immediately adjacent to the plasma membrane
of the perikaryon (Fig. 5A,B); the ultrastructural appearance of these axons intimately associated with a small number of cell bodies correlates with the observation of
TH-IR axons with varicosities surrounding a subset of trigeminal neurons at the light microscope level. These bundles of unmyelinated axonal profiles were evident around the entire surface of the cell
body, again resembling that appearance of TH-IR axons enveloping the
entire soma. On closer examination, nonmyelinating Schwann cell
processes were seen ensheathing these axons (Fig. 5A), in a
manner reminiscent of that seen in peripheral nerves. These bundles of
unmyelinated axons and their glial ensheathment were separate from the
outer satellite cell processes; these ultrastructural features of
sympathetic axons enveloping sensory somata concur with those reported
by Davis et al. (1994) and Chung et al. (1997). In the ganglia of
NGF/p75/ mice, such clusters of unmyelinated
axons within the interstitial space between neurons and satellite cells
were not observed (Fig. 5C,D). Rather, only a few single
unmyelinated axons were seen embedded within the processes of satellite
cells; axonal swellings were rarely observed. Thus, the observation
that TH-IR perineuronal plexuses in NGF/p75/
mice appeared "less intense" at the light microscope level is a
consequence of fewer unmyelinated axons contributing to the formation
of these plexuses, and not attributable to reduced levels of tyrosine
hydroxylase in p75NTR-deficient sympathetic axons.
Taken together, our findings indicate that the mechanism by which
sympathetic axons form perineuronal plexuses in the trigeminal ganglia
is perturbed in the absence of p75NTR. Examination
of trigeminal ganglia taken from NGF/p75/ mice,
double-immunostained for TH and CGRP, revealed that those few
perineuronal plexuses that do form are mostly associated with CGRP-positive trigeminal neurons (data not shown), similar to that seen
in NGF/p75+/+ mice. These data indicate that the
absence of p75NTR does not perturb the specificity
of sympathetic axons for a particular subset of trigeminal neurons, but
rather reduces the capability of sympathetic axons to locate the
appropriate trigeminal cells.
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Figure 5.
Electron photomicrographs of trigeminal
ganglia from NGF/p75+/+ and
NGF/p75/ mice. Sensory somata in
NGF/p75+/+ mice (A, B)
are surrounded by satellite cell processes closely apposed to their
outer cell membrane (S1). Some sensory cell
bodies (S2) have many clusters of small-diameter
unmyelinated axons (stars) immediately adjacent to their
plasma membrane. Occasionally, axonal swellings filled with clear
vesicles (Ax) are also seen among the bundles of axons.
These unmyelinated axons are ensheathed by nonmyelinating Schwann cells
(Sc). Sensory somata in NGF/p75/
mice (C, D) are likewise surrounded by
satellite cell processes closely apposed to their outer cell membrane
(S1 and S2). Although
small-diameter unmyelinated axons (stars) are also
associated with the sensory somata of NGF/p75/
mice, these fibers are fewer in number, do not occur in clusters, and
rarely display axonal swellings filled with clear vesicles.
Furthermore, these axons are not ensheathed by nonmyelinating Schwann
cells but rather appear to be embedded within the processes of
satellite cells. Arrowheads indicate intercellular space
between S1 and S2, which
is filled with collagen fibers. Scale bars, 1 µm.
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As mentioned previously, the trigeminal ganglia of both lines of NGF
transgenic mice have a greater density of TH-IR sympathetic fibers
than the ganglia of C57Bl/6 mice. Despite a reduced capability of
sympathetic axons projecting toward primary sensory neurons in
NGF/p75/ mice, there was an apparent increase in
the density of sympathetic sprouting in the fiber tracts of these
animals, in comparison to NGF/p75+/+ mice. As seen
in the neuropil, only a few TH-IR axons were evident in the fibers
tracts of C57Bl/6 mice (Fig.
6A); these likely
originated from the intrinsic dopaminergic population of trigeminal
neurons. The fiber tract portions of NGF/p75+/+ mice
displayed a marked increase in the number of TH-IR fibers (Fig.
6B), relative to the ganglia of C57Bl/6 mice. These
TH-IR sympathetic fibers were varicose in appearance and coursed in a
parallel arrangement to the intrinsic sensory fibers. The density of
TH-IR axons was further increased in the fiber tracts of the trigeminal ganglia taken from NGF/p75/ mice
(Fig. 6C). To confirm these increases in sympathetic
sprouting, we measured the percentage area occupied by TH-IR axons
within the fiber tract portions of trigeminal ganglia (Fig.
6D). This analysis revealed that the mean percentage
area occupied by sympathetic axons in the ganglia of
NGF/p75/ mice (9.6 ± 4.7%) was
significantly higher than that measured in the ganglia of
NGF/p75+/+ mice (3.8 ± 2.1%;
p < 0.001, Newman-Keuls test), which in turn was
significantly elevated over that seen in the ganglia of wild-type C57Bl/6 mice (0.3 ± 0.3%; p < 0.001).
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Figure 6.
Sections of trigeminal ganglia from C57Bl/6,
NGF/p75+/+, and NGF/p75/
mice, stained immunohistochemically for TH, and a bar graph depicting
the density of TH-IR axons in the ganglia of these three mouse
genotypes. In the trigeminal fiber tracts of C57Bl/6 mice
(A), few if any TH-IR axons are evident. In
marked contrast, the trigeminal fiber tracts of both
NGF/p75+/+ mice (B) and
NGF/p75/ mice (C) possess
numerous TH-IR axons coursing in parallel arrangement with the
intrinsic sensory fibers. Note the apparent increase in the number of
TH-IR axons in fiber tracts of NGF/p75/ mice
relative to NGF/p75+/+ mice. Scale bar, 100 µm.
Quantitation of the percentage area occupied by TH-IR axons in
trigeminal fiber tracts (D) confirms an increase
in the density of TH-IR axons in the fiber tracts of both
NGF/p75+/+ and NGF/p75/ mice,
relative to C57Bl/6 mice (*p < 0.001). The density
of TH-IR axons in the fiber tracts of NGF/p75/
mice is also significantly higher than that in
NGF/p75+/+ mice (+p < 0.001). Error bars represent SDs.
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Localization and detection of NGF in trigeminal ganglia
To localize NGF among sensory neurons, NGF immunostaining was
performed on slides having sections of trigeminal ganglia from each of
C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. In C57Bl/6 mice, few neurons
stained immunohistochemically for NGF, and the intensity of
immunostaining among these neurons was weak (Fig.
7A). This observation may
reflect a difficulty in immunohistochemical detection of the small
amount of NGF that is retrogradely transported by normal trigeminal
neurons. In contrast, the overexpression of NGF in
NGF/p75+/+ mice resulted in a dramatic increase in
the staining intensity of NGF among a subpopulation of neurons (Fig.
7B). It should be noted that NGF immunoreactivity in these
neurons is likely attributable to retrograde transport of NGF from
distal sites and not to endogenous production by the neurons themselves
(our unpublished data; in situ hybridization). In
NGF/p75/ mice, many trigeminal neurons also
possessed NGF immunoreactivity, but the intensity of immunostaining was
intermediate to that seen in the ganglia of C57Bl/6 and
NGF/p75+/+ mice (Fig. 7C). It may be
possible that the functional expression of p75NTR is
necessary for the proper immunolocalization of NGF within neuronal cell
bodies; this idea is supported by the fact that we have observed
similar reductions in NGF immunostaining among other NGF-responsive
neuronal populations (e.g., sympathetic neurons of the superior
cervical ganglion and cholinergic neurons of the medial septum; our
unpublished observations) in NGF/p75/
mice.
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Figure 7.
Sections of trigeminal ganglia from C57Bl/6,
NGF/p75+/+, and NGF/p75/
mice, stained immunohistochemically for NGF, and a bar graph depicting
the levels of NGF protein in the ganglia of these three mouse
genotypes. In C57Bl/6 mice (A), few NGF-positive
somata are detected. In contrast, many somata in trigeminal ganglia of
NGF/p75+/+ mice (B) display
strong immunostaining for NGF. Numerous somata in the trigeminal
ganglia of NGF/p75/ mice
(C) also display NGF immunoreactivity, but the
intensity of immunostaining is moderate as compared with that in
C57Bl/6 and NGF/p75+/+ mice. Scale bar, 50 µm.
Mean levels of NGF protein in the trigeminal ganglia of C57Bl/6
(n = 5), NGF/p75+/+
(n = 8), and NGF/p75/
(n = 8) mice were measured by a two-site ELISA
(D). This analysis reveals that trigeminal
ganglia of NGF/p75+/+ and
NGF/p75/ mice both possess significantly higher
levels of NGF protein than trigeminal ganglia of age-matched C57Bl/6
mice (*p < 0.01). The amount of NGF protein in
trigeminal ganglia of NGF/p75/ mice is
significantly reduced from that of NGF/p75+/+ mice
(+p < 0.001). Error bars represent
SDs.
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Previous work has demonstrated a correlation between NGF protein levels
and the density of sympathetic sprouting (Campenot, 1982; Isaacson et
al., 1997). Therefore we examined the levels of NGF protein in
trigeminal ganglia of C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice using a two-site ELISA (Fig.
7D). In contrast to the low levels of NGF protein detected
in the trigeminal ganglia of control C57Bl/6 mice (35.4 ± 5.2 pg
NGF/ganglion; n = 5), levels in the ganglia of
NGF/p75+/+ mice (269.4 ± 43.4;
n = 8) were significantly higher
(p < 0.01; Newman-Keuls test). Levels of NGF
protein in NGF/p75/ mice (121.9 ± 59.9;
n = 8) were also increased significantly above that in
C57Bl/6 mice (p < 0.01) but were significantly
lower than levels detected in NGF/p75+/+ mice
(p < 0.001). It is unlikely that this
discrepancy in NGF levels between both transgenic lines of mice is
caused by a decreased retrograde transport of NGF among p75-deficient
sensory neurons, because Curtis et al. (1995) reported that an absence
of p75NTR expression does not affect the transport
of NGF among DRG neurons in p75/ mice.
Alternative explanations may include differing rates of degradation of
NGF among sensory neurons and/or NGF uptake and retrograde transport
away from the ganglia by the greater number of sympathetic axons in the
ganglia of transgenic mice lacking p75NTR
expression. The most important observation, however, remains that
total ganglionic levels of NGF protein of both
NGF/p75+/+ and NGF/p75/ mice
are higher relative to C57Bl/6 mice.
Numbers of trigeminal ganglionic neurons
To ensure that differences in NGF levels are not attributed to
concomitant changes in the numbers of trigeminal ganglionic neurons, we
assessed the population sizes of C57Bl/6,
NGF/p75+/+, and NGF/p75/
mice; trigeminal ganglia from BALB/c and p75/
mice were also assessed as additional controls (Table
1). Because the average nuclear diameter
of trigeminal neurons from all five genotypes of mice did not exceed 16 µm, our sampling frequency of sections every 50 µm ensured that
neurons were not counted twice, and thus no corrections of neuron
numbers were made. Both C57Bl/6 and NGF/p75+/+ mice
(as well as BALB/c mice) had similar numbers of trigeminal neurons.
NGF/p75/ mice, however, possessed a 15%
reduction in the number of trigeminal neurons, in comparison to the
three aforementioned genotypes (p < 0.05;
Newman-Keuls test). A greater decrease in neuron number was observed
among p75/ mice, with a 30% reduction in
comparison to BALB/c (control) mice (p < 0.001). These results are in agreement with other investigations that
have suggested that p75/ mice exhibit a dramatic
loss of dorsal root sensory neurons (Lee et al., 1992; Bergmann et al.,
1997; Stucky and Koltzenburg, 1997). From these quantitative data it is
evident that NGF/p75/ mice display only a modest
reduction in neuron number, as compared with
NGF/p75+/+ mice, and this loss cannot solely account
for the discrepancies in ganglionic levels of NGF between the two
transgenic genotypes. Using unbiased quantitative methods,
NGF/p75+/+ and NGF/p75/ mice
display similar numbers of SCG neurons, both of which are increased in
comparison to that determined from C57Bl/6 mice (our unpublished data).
Thus, the discrepancies observed in the sprouting responses by
sympathetic axons in the trigeminal ganglia of
NGF/p75+/+ and NGF/p75/ mice
cannot be attributed to differences in neuron numbers of the SCG.
Neurochemical organization of the trigeminal ganglia
There are several lines of evidence that suggest that the
trkA-expressing subpopulation of sensory neurons plays a critical role
in the development of sympathosensory sprouting. First, this sprouting
response is correlated with a rise in NGF levels within sensory
ganglia, which, as our data suggests, occurs as a result of an
increased retrograde transport of NGF among trkA-expressing neurons.
Second, sympathetic axons selectively target the trkA-/CGRP-IR population of sensory neurons. Third, in NGF/p75+/+
mice, few if any sympathetic perineuronal plexuses form in the nodose
ganglia (our unpublished data), which contains only a small number of trkA-expressing visceral somata (Wetmore and Olson, 1995). To
exclude the possibility that the perturbed pattern of sympathetic
sprouting seen in NGF/p75/ mice is caused by a
selective loss of the trkA- and CGRP-expressing population of sensory
neurons, we examined trkA and CGRP immunohistochemistry in trigeminal
ganglia of C57Bl/6, NGF/p75+/+, and
NGF/p75/ mice. Many trkA-IR neurons of various
sizes were clearly evident in trigeminal ganglia of all mouse genotypes
(Fig. 8A,C,E).
Likewise, CGRP-IR trigeminal neurons were seen in ganglia of all mouse
genotypes, with a neuronal distribution similar to that of trkA (Fig.
8B,D,F). Qualitatively, the staining intensity
for both trkA and CGRP among trigeminal neurons appeared comparable in
NGF/p75+/+ and NGF/p75/ mice.
Thus, a lack of p75NTR expression markedly affects
the pattern and distribution of sympathetic sprouting, in the absence
of a selective loss of trkA- and CGRP-expressing neurons, that
population of trigeminal sensory neurons specifically targeted by the
invading sympathetic axons.
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Figure 8.
Sections of trigeminal ganglia from C57Bl/6,
NGF/p75+/+, and NGF/p75/ mice
stained immunohistochemically for trkA (A, C, E) and
CGRP (B, D, F). In C57Bl/6 mice, numerous somata
throughout the trigeminal ganglia possess moderate staining for both
trkA (A) and CGRP (B). In
comparison, sensory somata in NGF/p75+/+ mice
display an increased intensity of immunostaining for both trkA
(C) and CGRP (D).
NGF/p75/ mice also display strong immunostaining
for trkA (E) and CGRP (F).
Immunostaining for trkA and CGRP is evident in small- to medium-sized
trigeminal somata of C57Bl/6 mice, whereas immunostaining for trkA and
CGRP is seen in small- to large-sized somata of
NGF/p75+/+ and NGF/p75/ mice.
Scale bar, 50 µm.
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DISCUSSION |
Specificity of sympathosensory projections
The occurrence of sympathosensory sprouting has now been reported
in the spinal ganglia after peripheral nerve injury (McLachlan et al.,
1993; Chung et al., 1996; Zhou et al., 1996; Ramer and Bisby, 1997a)
and in the trigeminal ganglia as a consequence of transgenic
overexpression of NGF in the target tissues of sensory neurons (Davis
et al., 1994; Walsh and Kawaja, 1998). The presence of elevated NGF
levels in the spinal ganglia after sciatic nerve injury (Herzberg et
al., 1997) and in the undamaged trigeminal ganglia of NGF transgenic
mice (Davis et al., 1994; Walsh and Kawaja, 1998) suggests that
sympathosensory sprouting is an NGF-dependent phenomenon. In further
support of this idea, we have reported previously that sympathetic
axons invading the trigeminal ganglia of NGF/p75+/+
mice preferentially associate with a small number of sensory neurons
displaying NGF immunoreactivity (Walsh and Kawaja, 1998). This finding
also indicates that sympathetic axons specifically target that
subpopulation of neurons that retrogradely transport and accumulate
NGF: the trkA-expressing neurons. To confirm this hypothesis, we
determined that virtually all (98%) perineuronal plexuses of
sympathetic fibers are found surrounding trkA-IR sensory somata in
NGF/p75+/+ mice; these findings are in agreement
with those of Davis et al. (1998). The observation that sympathetic
axons are attracted exclusively toward trkA-expressing somata implies a
central role for these neurons in the mechanism underlying
sympathosensory sprouting. We speculate that trkA-expressing sensory
neurons, which bind and retrogradely transport target-derived NGF, are responsible for delivering NGF to the ganglionic environment, which in
turn initiates the directional ingrowth of sympathetic axons.
Our results further showed that the majority (86%) of sympathetic
plexuses in NGF/p75+/+ mice form around trigeminal
somata possessing CGRP immunoreactivity. The co-occurrence of trkA and
CGRP in most TH-wrapped neurons is not unexpected because these two
neural antigens are known to colocalize within the same subset of
sensory neurons (Verge et al., 1992; Averill et al., 1995), which are
typically small-diameter cells with unmyelinated axons, subserving
nociception (Lawson, 1992; Snider and McMahon, 1998). Because trkA- and
CGRP-expressing neurons are normally small in size, it is surprising
that those trigeminal neurons displaying sympathetic plexuses have
predominantly large diameters. As well, studies of nerve-injured rats
have demonstrated that sympathetic axons preferentially wrap
large-diameter DRG neurons (McLachlan et al., 1993; Chung et al.,
1996). One explanation that reconciles these data is that
large-diameter sensory neurons, which do not normally express trkA,
begin to express this receptor de novo, as has been proposed
to occur in adjuvant-induced models of inflammation (Woolf, 1996).
Alternatively, small-diameter sensory neurons, which normally express
trkA (McMahon et al., 1994), hypertrophy in response to the high levels
of NGF in NGF/p75+/+ mice. Support for this idea
comes from studies showing that NGF causes an increase in soma size
among trkA-expressing neuronal populations, both peripherally and
centrally (Gage et al., 1989; Higgins et al., 1989; Ruit et al., 1990;
Goodness et al., 1997). Furthermore, Chung et al. (1996) reported that
shortly after spinal nerve ligation, sympathetic axons associate with
small- and large-diameter somata, whereas at longer postoperative
periods, sympathetic fibers preferentially associate with only
large-diameter somata. These authors also proposed neuronal hypertrophy
as a possible explanation for their results. It seems likely,
therefore, that NGF-induced neuronal hypertrophy of small-diameter
neurons, rather than a phenotypic alteration, explains the presence of
trkA (and CGRP) within large-diameter sensory somata exhibiting a
perineuronal plexus of sympathetic fibers. Taken together, sympathetic
axons are attracted to sensory neurons, which all display a common
responsiveness to the neurotrophin NGF, and the majority produce the
neuropeptide CGRP.
Our results also show that a small but significant number (~14%) of
sympathetic plexuses are associated with sensory neurons lacking
detectable CGRP immunoreactivity. This raises the possibility that the
second population of trkA-IR neurons that attract sympathetic axons are
those that are normally referred to as "large light," having
myelinated axons presumably connected to mechanosensitive endings
(Lawson, 1992). In support of this notion, a subpopulation of
large-diameter sensory neurons in the undamaged DRG of rats (5% of the
total population) display high-affinity binding sites for NGF but no
CGRP immunoreactivity (Verge et al., 1989).
Role of p75NTR in sympathosensory sprouting
It is well documented that sympathetic axons sprout into tissues
displaying high concentrations of NGF, as a result of site-directed expression (Edwards et al., 1989; Albers et al., 1994; Hassankhani et
al., 1995; Kawaja and Crutcher, 1997) and damage- or disease-induced production (Crutcher, 1987; Aloe et al., 1992b; Zettler and Rush, 1993). Sympathetic axons ramify throughout these NGF-rich tissues giving the appearance of a diffuse network of fibers. Sympathetic axons
also invade the NGF-rich trigeminal ganglia of transgenic mice
overexpressing NGF, but unlike the random pattern of growth seen in
other NGF-rich tissues, sympathetic axons display a cellular specificity for certain neurons within sensory ganglia. In the absence
of p75NTR expression, we have shown that the bulk
growth of sympathetic axons can still occur into the NGF-rich
trigeminal ganglia of NGF transgenic mice, but the ability of
sympathetic axon collaterals to wrap individual neurons is perturbed.
How does the loss of p75NTR result in an alteration
of NGF-induced sympathosensory sprouting?
Because trkA-expressing sensory neurons have a central role in
sympathosensory sprouting, the alteration of sympathetic sprouting observed in NGF/p75/ mice could be an indirect
consequence of the role of p75NTR in the survival of
trkA-expressing neurons. Recent experiments with
p75NTR-deficient mice have suggested a dramatic loss
of sensory DRG neurons (Lee et al., 1992; Stucky and Koltzenburg,
1997). This cell loss, however, is apparently nonselective for
trkA-expressing sensory neurons (Bergmann et al., 1997). Quantitative
data from this investigation demonstrate that
NGF/p75/ mice have only a 15% reduction in the
number of trigeminal neurons relative to NGF/p75+/+
mice, suggesting that the overexpression of NGF can ameliorate the
survival of sensory neurons in mice lacking functional expression of
p75NTR. Furthermore, our results reveal a similar
distribution and staining intensity of trkA-immunoreactive neurons in
the trigeminal ganglia of NGF/p75+/+ and
NGF/p75/ mice. Thus, the loss of sensory neurons
attributable to the absence of p75NTR expression is
minimized in NGF/p75/ mice and is likely not a
determinant of the alteration in the sympathetic sprouting response.
Detection of NGF protein, using ELISA, reveals half the total level of
NGF in ganglia of NGF/p75/ mice as compared with
NGF/p75+/+ mice. Does a lack of
p75NTR affect the delivery of NGF to the ganglionic
environment by trkA-expressing trigeminal neurons?
p75NTR can increase the amount of NGF that becomes
bound to the trkA receptor, particularly at low ligand concentrations
(Barker and Shooter, 1994; Mahadeo et al., 1994), a function that may
enhance the initial binding and subsequent internalization of NGF at
distal sensory axons in normal target tissues. Such a role for
p75NTR on sensory axons in NGF transgenic mice may
be irrelevant because NGF levels are expected to be high near distal
axons of trkA-expressing sensory neurons [trigeminal axons invade the
cerebellum of NGF/p75+/+ and
NGF/p75/ mice, which both have 20-fold higher
NGF levels (Kawaja et al., 1997; Coome et al., 1998)]. Furthermore,
the retrograde transport of 125I-NGF in sensory neurons is
not affected in p75NTR-deficient mice (Curtis et
al., 1995). Thus, it is doubtful that the perturbed patterns of
sympathosensory sprouting in NGF/p75/ mice is a
consequence of an altered ability for the retrograde transport of NGF
in p75NTR-deficient sensory neurons.
It is most likely that p75NTR exerts its effects on
sympathosensory sprouting at the level of the sympathetic growth cone.
Sympathetic axons invading the ganglionic environment turn toward
NGF-IR sensory somata (Walsh and Kawaja, 1998); this pattern of
sprouting is reminiscent of growth cone turning responses to
substratum-bound NGF in vitro (Letourneau, 1978; Gallo et
al., 1997). Our finding that sympathetic pericellular plexuses form
less often in NGF/p75/ mice parallels the
observation that p75NTR function-blocking antibodies
reduce, but do not inhibit, the turning response of growth cones to
NGF-coated beads. Inhibitors of trkA signaling, however, totally block
this response (Gallo et al., 1997). Thus, p75NTR
appears to enhance NGF-induced local guidance of elongating axons. The
mechanism by which p75NTR acts to positively
modulate NGF-induced axonal morphogenesis most likely involves altering
the conformation of trkA into a high-affinity state through direct
receptor interactions (Mahadeo et al., 1994; Ross et al., 1996, 1998),
thereby enhancing trkA-mediated growth cone turning. The ability of
p75NTR to increase the NGF/trkA association rate
(Mahadeo et al., 1994) may be particularly important in allowing growth
cones to discriminate gradients of NGF, as may be present in the
trigeminal ganglia of NGF transgenic mice. p75NTR
expression on glial cells may also serve to enhance NGF gradients near
the site of NGF release, namely trkA-expressing sensory neurons. Recently, Zhou et al. (1996) reported that sympathetic pericellular plexuses in the DRG of nerve-injured rats were associated with p75NTR-IR glial cells. These authors proposed that
p75NTR may act as a presenting molecule for NGF to
nearby sympathetic sprouts (also see Taniuchi et al., 1986; Johnson et
al., 1988).
Last, our data show that despite lower levels of ganglionic NGF,
NGF/p75/ mice have a greater density of
sympathetic axons invading their trigeminal ganglia, as compared with
NGF/p75+/+ mice. These findings are not consistent
with previous investigations correlating an increased density of
sympathetic fibers with increasing concentrations of NGF (Korsching and
Thoenen, 1977; Campenot, 1982; Shelton and Reichardt, 1984; Isaacson et
al., 1997). The reason a greater density of sympathetic axons is
observed in NGF/p75/ mice is not clear. It is
unlikely that this increase in sympathetic sprouting is related to an
enhanced number of sympathetic neurons in
NGF/p75/ mice, because unbiased neuron counting
reveals comparable numbers of SCG neurons in both
NGF/p75+/+ and NGF/p75/ mice
(our unpublished observations). Rather, the coexpression of trkA and
p75NTR may attenuate NGF-induced, trkA-mediated axon
elongation by sympathetic neurons. In support of this, brain-derived
neurotrophic factor (BDNF) activation of p75NTR
reduces NGF-induced trkA tyrosine phosphorylation (MacPhee and Barker,
1997) and sympathetic neuron survival (Bamji et al., 1998) through
signaling pathways that involve ceramide production and c-jun
phosphorylation, respectively. In both experiments, however, BDNF
activation of p75NTR negatively modulated trkA
function only at low NGF concentrations, and hence, it is unclear
whether such a mechanism could account for the increased sprouting of
sympathetic axons in NGF-overexpressing mice that lack
p75NTR. It will be interesting to test this
hypothesis in other models of NGF-induced sprouting of sympathetic axons.
| |
FOOTNOTES |
Received July 29, 1998; revised Oct. 9, 1998; accepted Oct. 12, 1998.
This work was supported by grants from the Botterell Foundation and
Advisory Research Committee at Queen's University (M.D.K.). G.S.W. is
a recipient of a studentship awarded by the Rick Hansen Man in Motion
Foundation, and M.D.K. is a recipient of a Medical Research of Canada
Scholarship. Thanks are extended to Dr. Keith A. Crutcher for the ELISA
determinations of NGF (K.A.C. was supported by National Institutes of
Health Grant NS17131), to Ms. Verna Norkum for the cryostat sectioning
of the trigeminal ganglia, and to Mr. Bob 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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Abstract
Introduction
