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The Journal of Neuroscience, November 1, 1998, 18(21):8720-8729
Ciliary Neurotrophic Factor Receptor  in Spinal Motoneurons is
Regulated by Gonadal Hormones
Nancy G.
Forger1,
Christine K.
Wagner1,
Michael
Contois1,
Lynn
Bengston1, and
A. John
MacLennan2
1 Center for Neuroendocrine Studies and Department of
Psychology, University of Massachusetts, Amherst, Massachusetts 01003, and 2 Department of Neuroscience, University of Florida
Brain Institute, University of Florida College of Medicine,
Gainesville, Florida 32610
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ABSTRACT |
Ciliary neurotrophic factor receptor  (CNTFR) is the
ligand-binding component of the CNTF receptor. CNTFR expression is essential for the normal development of spinal motoneurons and is
required for the development of a sex difference in motoneuron number
in androgen-sensitive perineal motoneurons. We used immunocytochemistry to examine the expression and hormone regulation of CNTFR protein in
the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus
and retrodorsolateral nucleus of the lower lumbar spinal cord of
adult rats. CNTFR immunoreactivity (CNTFR-IR) was observed in the somata and dendrites of virtually all motoneurons. In all three
motor pools, the intensity of motoneuron soma labeling was greatest
among gonadally intact males and was reduced in females and
gonadectomized males. The density of CNTFR-IR in neuropil also
tended to be highest in intact males. Short-term (2 d) testosterone propionate treatment reversed the decline in the density of soma labeling in the SNB of castrated males but did not reverse any other
effects of castration. Long-term hormone treatment, achieved by
implanting males with testosterone capsules at the time of gonadectomy,
prevented the decline in soma labeling in all motor pools and partially
prevented the decline in neuropil label caused by castration. We
conclude that expression of CNTFR protein is androgen-regulated in
spinal motoneurons.
Key words:
motoneuron; androgen; ciliary neurotrophic factor
receptor; spinal cord; immunocytochemistry; hormone
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INTRODUCTION |
Ciliary neurotrophic factor (CNTF)
was first identified based on its ability to prevent the death of
parasympathetic neurons of the ciliary ganglion (Barbin et al., 1984 )
but, subsequently, has gained substantial attention as a potent
motoneuron trophic factor. CNTF can prevent the death of developing
motoneurons (Arakawa et al., 1990; Sendtner et al., 1990; Oppenheim et
al., 1991) and supports multiple innervation of striated muscles during
the period of synapse elimination (Jordan, 1996). In postweaning and
adult animals, administration of CNTF promotes motor nerve sprouting at
the neuromuscular junction (Gurney et al., 1992), enhances muscle fiber
reinnervation after nerve injury (Sahenk et al., 1994; Ulenkate et al.,
1994), and reduces motoneuron death in animal models of neuromuscular
disease (Sendtner et al., 1992; Mitsumoto et al., 1994; Ikeda et al.,
1995; Sagot et al., 1995). Deletion of the CNTF gene results in subtle
motor impairments in adult mice (Masu et al., 1993).
The actions of CNTF are mediated via a three-part receptor complex
consisting of a CNTF-binding component, CNTFR, and two signal
transducing components, gp130 and LIFR (Davis et al., 1993).
Expression of CNTFR is restricted primarily to cells of the nervous
system (Davis et al., 1991; Ip et al., 1993; MacLennan et al., 1994),
with motoneurons of adult rats expressing high levels of CNTFR
protein and mRNA (MacLennan et al., 1996; Lee et al., 1997b). Mice with
a targeted deletion of the CNTFR gene exhibit reduced motoneuron
number at birth and early postnatal mortality (DeChiara et al.,
1995).
In addition to expression of CNTFR, many brainstem and spinal
motoneurons express androgen receptors and are sensitive to androgens
in adulthood (Sar and Stumpf, 1977; Yu and McGinnis, 1986). For
example, androgens accelerate the rate of motor axon regeneration after
injury to the sciatic, facial, and hypoglossal nerves (Yu, 1982; Kujawa
and Jones, 1990; Kujawa et al., 1993, 1995). The sexually dimorphic
perineal motoneurons of the lower lumbar spinal cord exhibit
exceptional androgen sensitivity. The spinal nucleus of the
bulbocavernosus (SNB) and dorsolateral nucleus (DLN) of rodents
innervate striated muscles that attach to the base of the penis
(Schroder, 1980). SNB and DLN cell survival is dependent on androgens
during perinatal life and, as a result, males retain many more of these
motoneurons than do females (Breedlove and Arnold, 1980;
Nordeen et al., 1985; Sengelaub and Arnold, 1989). In adulthood
the soma size and dendritic extent of SNB and DLN motoneurons are
increased by androgens (Breedlove and Arnold, 1981; Kurz et al., 1986,
1991).
We have recently shown that CNTF mimics some effects of testosterone on
perineal motoneurons. For example, CNTF prevents the death of SNB
motoneurons in perinatal female rats (Forger et al., 1993). Moreover,
the normal sex difference in SNB cell number is absent in CNTFR
knock-out mice (Forger et al., 1997), suggesting that androgen cannot
rescue these motoneurons in animals lacking functional CNTF receptors.
The molecular mechanisms whereby androgens influence motoneuron
survival, soma size, dendritic extent, or speed of regeneration after
injury are not known but may involve hormonal modulation of the
expression of trophic factors and/or their receptors. We, therefore,
asked whether CNTFR expression is androgen-regulated in lumbar
motoneurons of adult rats.
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MATERIALS AND METHODS |
Animals
Sprague Dawley rats were purchased from Taconic (Germantown, NY)
and housed two per cage in a 14/10 light/dark cycle with food and water
available ad libitum. Gonadectomies and sham gonadectomies were performed via a single midline incision under Metofane
anesthesia.
Experiment 1
This experiment was designed to compare CNTFR
immunoreactivity (CNTFR-IR) in adult males and females and to
examine effects of gonadectomy and short-term testosterone replacement
on CNTFR-IR of males. Young adult male rats, 57-67 d of age, were
gonadectomized (Gdx) or sham-gonadectomized. Adult females of the same
age were unoperated. Twenty-six days after surgery half of the Gdx
males received two daily injections of testosterone propionate (TP; 500 µg/d) dissolved in 250 µl sesame oil. All other animals received two daily injections of the oil vehicle. Four treatment groups resulted: Sham males, Gdx + oil males, Gdx + 2 d TP males, and Females (n = 4 animals per group). Animals were
killed 24 hr after the last injection (4 weeks after surgery),
and immunocytochemistry for CNTFR was performed in four yoked runs,
each run examining tissue from one animal of each of the four
groups.
Experiment 2
Short-term (48 hr) testosterone replacement was ineffective in
reversing most effects of castration in experiment 1 (see below). Therefore, we next examined the effects of gonadectomy and long-term testosterone treatment on CNTFR-IR in motoneurons of adult males. Male Sprague Dawley rats, 72 d of age, were gonadectomized
(n = 12) or sham gonadectomized (n = 6). At the time of surgery each animal received a SILASTIC capsule
implanted subcutaneously at the nape of the neck. Half of the Gdx males
(Gdx + T) received capsules filled with crystalline testosterone
(Sigma, St. Louis, MO). The size of the tubing (45 mm length; 1.67 mm
inner diameter, 3.18 mm outer diameter) was chosen to provide plasma
testosterone levels in the high physiological range (Smith et al.,
1977). The remaining males received empty capsules of the same
size (Gdx + Blank and Sham groups). Capsules remained in place until
animals were killed at 7 weeks after surgery. Seminal vesicle weights were recorded at the time of killing as an indirect measure of recent
circulating androgen levels, and immunocytochemistry for CNTFR was
performed in two runs, with spinal cords of two animals from each group
in each run.
Immunocytochemistry
Animals were perfused with cold saline followed by 4%
paraformaldehyde. Spinal cords were removed and post-fixed in 4%
paraformaldehyde for 2 hr, then transferred to 15% sucrose/0.1
M phosphate buffer, pH 7.2, containing 2.5 mM
sodium azide overnight. Thirty micrometer coronal sections were cut on
a cryostat through the lumbosacral spinal cord, and every fifth section
(experiment 1) or every fourth section (experiment 2) was retained for
processing. To compare the expression of CNTFR in the lower lumbar
spinal cord with expression in other spinal regions the entire spinal
cords of several additional adult males were removed, and segments
(5-10 mm long) of the cervical, thoracic, and lumbar cord were
cryostat-sectioned in the horizontal plane.
Detection of CNTFR protein was performed by a modification of the
procedure described in MacLennan et al. (1996). Free-floating sections
were rinsed in PBS, and endogenous peroxidase activity was
quenched by an incubation in 0.3% H2O2 in
methanol for 30 min. Tissues were then rinsed in PBS and exposed to a
blocking solution [0.3% Triton X-100, 0.03% bovine serum albumin
(BSA), and 10% normal goat serum in PBS] for 1 hr, then incubated for 3 d at 4°C in a 1:200 dilution of affinity-purified polyclonal antiserum "3X" (MacLennan et al., 1996). Sections were sequentially washed, incubated for 1 hr with goat anti-rabbit biotinylated secondary
antibody, washed again, and incubated for 1 hr in an avidin-biotin-peroxidase conjugate (Vectastain Elite ABC kit, Vector
Laboratories, Burlingame, CA) and stained using 3,3'-diaminobenzidine as a substrate and nickel ammonium sulfate as an enhancer.
All label was abolished when the primary antiserum was omitted (data
not shown). Previously, MacLennan et al. (1996) used Western blotting
to demonstrate that the 3X antiserum selectively recognizes a
protein species in adult rat brain that migrates with an apparent
molecular weight of 78 kDa, consistent with the interpretation that
this antiserum is selective for the ~80 kDa CNTFR protein. In
addition, all 3X label in rat spinal cord and brain was eliminated when
the antiserum was preadsorbed with the immunizing peptide (MacLennan et
al., 1996).
Computer-aided image analysis
CNTFR-IR was quantified in the SNB, DLN, and
retrodorsolateral nucleus (RDLN) of lumbar spinal segments 5 and 6. SNB
and DLN motoneurons innervate the sexually dimorphic penile muscles (Schroder, 1980), whereas RDLN motoneurons primarily innervate intrinsic muscles of the foot that are present in both sexes
(Nicolopoulos-Stournaras and Iles, 1983).
All measurements were made by an investigator blind to the sex and
endocrine status of the animals. Images were captured with an Olympus
BH-2 microscope fitted with a CCD72 (Dage MTI, Michigan City,
MI) camera that was connected to a QuickCapture frame grabber board
(Data Translation Inc., Marlboro, MA) in a Macintosh IIfx computer.
NIH Image 1.57 software (W. Rasbaud, National Institutes of
Health, Bethesda, MD) was used to analyze captured images. The
"sharpen" function was applied once to all captured images before
quantification.
Selection of sections for analysis
To eliminate from analysis sections in which label was poor, or
in which one of the three cell groups (RDLN, DLN, or SNB) was absent,
we first identified those sections that had the greatest number of
pixels above threshold in the ventral horn. To perform this selection,
images were captured using a low-power objective (4×). Mean background
labeling and SD of background label were measured bilaterally in each
section in large oval areas (~1.24 × 105
µm2; 7500 pixels) in the gray matter medial to the
RDLN. The "density slice" function was then used to count all
pixels above threshold in the ventral horn, with threshold defined as
the mean background plus 2.5 times the SD of background density. Six
sections through the SNB region of each animal in experiment 1 and 10 sections through the SNB in animals from experiment 2 were initially
chosen. This represented 70 and 69% of all available sections through the SNB regions of the animals in experiments 1 and 2, respectively. All six sections of animals from experiment 1 were subjected to detailed quantitative analysis; 5 of the 10 identified sections of
animals in experiment 2 were randomly selected for further quantitative
analysis.
Three measures were made of each of the three motoneuron pools in the
selected sections. Images were captured using a 20× objective, and
sections from all animals within a given experiment were analyzed in a
single session, during which the microscope light intensity and camera
settings were kept constant.
Intensity of motoneuron soma labeling. From each
section three randomly chosen motoneuron somata in each cell group were
traced, and the density of label within the tracings (minus mean
background density in that section) was recorded. Optical density
measures were then used to classify labeled motoneurons on a four-point scale (light to very dark). Cell density scores from all motoneurons of
all animals in a given run were pooled, rank ordered, and separated by
quartile. Motoneurons with density scores among the lowest 25% of all
scores were arbitrarily classified as "light", whereas cells in the
second, third, and fourth quartiles were classified as "medium",
"dark", and "very dark", respectively. The conversion of
density readings to a nominal scale, and the subsequent analysis of the
resulting relative frequency distributions with nonparametric statistics avoided the necessity of making any assumptions about the
linearity of pixel density measures in our system.
Intensity of label in neuropil. Three randomly selected
areas of neuropil within each of the three motor nuclei were sampled from each section, and the density (minus background) was recorded. Neuropil scores were then pooled and classified as light, medium, dark,
or very dark, as above.
Total area covered by label. The outlines of the RDLN, DLN,
and SNB were traced, and the density slice function was used to count
all pixels above threshold in each nucleus. The DLN and RDLN were
analyzed unilaterally, whereas the SNB, which is located near the
midline, was measured bilaterally. Threshold was defined as above, with
background label determined in an oval area (~25,000 µm2; 38,150 pixels) in the gray matter between the
RDLN and DLN. The number of pixels above threshold in each motor pool
was summed across sections to obtain the total area covered by label
for each animal.
Cell counts
After the measures described above had been recorded, the
coverslips on tissues from animals in experiment 1 were soaked off, and
sections were counterstained with neutral red. The number of neutral
red-stained motoneuron profiles with a visible nucleolus was then
recorded for each motor pool. Mean nucleolus size was determined from
camera lucida tracings of ~20 motoneurons per pool per animal, and
was used to correct for split nucleoli by the method of Konigsmark
(1970). Because motoneuronal nucleoli are nearly spherical and are
small relative to the section thickness used, any bias introduced by
this count-correction procedure should be negligible (Clarke and
Oppenheim, 1995).
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Statistics |
The relative frequency distributions of intensity of motoneuron
soma labeling and intensity of label in neuropil were analyzed by
 2 tests for independence. A significant difference in
the overall 2 was followed by 2 tests
comparing each treatment group with the intact male group and
contrasting Gdx + oil (or Blank) males with Gdx + TP (or T) males.
Measures of the mean total area covered by label were analyzed by
separate one-way ANOVAs for each motor pool, and significant main
effects were followed by planned comparisons. Means are reported ± SEM.
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RESULTS |
Distribution of CNTFR immunoreactivity
Most CNTFR-IR in the spinal cord was localized to motor pools
(Fig. 1). CNTFR-IR was found in the
somata and dendrites of motoneurons throughout the spinal cord, and in
several cases labeled motoneuronal axons could be seen exiting the
ventral horn (Figs. 1, 2). A Nissl
counter stain revealed that virtually all motoneurons (>95%) were at
least lightly labeled, although the intensity of CNTFR-IR differed
between motor pools. The DLN of the lower lumbar spinal cord
consistently stood out as the most densely labeled cell group. In many
instances the DLN presented a striking pattern with intensely labeled,
punctate neuropil surrounding lighter motoneuron cell bodies (Fig.
1C). The SNB nucleus also tended to be intensely labeled
(Figs. 1B, 2A). Although we did not
systematically examine every segment of the spinal cord, we did sample
from the cervical, thoracic, and lumbar regions of the cord from
several animals. In all regions the motor pools were characterized by CNTFR-IR in somata surrounded by a sparse plexus of labeled neurites (Fig. 2), but in no other region did we observe a pattern comparable to
the very intense label characteristic of the DLN and SNB.
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Figure 1.
CNTFR immunoreactivity in the lower lumbar
spinal cord of adult male rats. A, Low magnification
view of a cross section through the spinal cord demonstrating
CNTFR-IR in the SNB, DLN, and RDLN motor pools. Scale bar, 200 µm.
B, D, Higher magnification of the SNB and
left RDLN of the section in A. C, High
magnification view of the DLN (not the same section or animal as in
A) showing the heavily labeled, punctate appearance of
the neuropil and the lighter label in motoneuron somata characteristic
of this nucleus. Scale bar in D, 50 µm (applies to
B-D). Arrows in
A and C indicate labeled axons exiting
the cord.
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Figure 2.
CNTFR immunoreactivity in horizontal sections
of the lumbar and thoracic spinal cord. A, Low
magnification view through the lower lumbar spinal cord.
Immunoreactivity is present in all motor columns but is especially
intense in the SNB and DLN. Scale bar, 600 µm. B,
Higher magnification view of the SNB region shown in A.
C-E, Upper thoracic, lower thoracic, and
upper lumbar spinal cord, respectively. Asterisks
indicate the midline in each case. Scale bars in
B-E, 200 µm.
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Experiment 1: four weeks of castration and short-term
testosterone replacement
Summary of results
CNTFR-IR was darker in the motoneuron somata of sham-operated
males than of females or gonadectomized males in all three motor pools.
Two days of testosterone treatment eliminated the difference in soma
labeling between sham-operated and gonadectomized males in the SNB but
not in the DLN or RDLN. CNTFR-IR in the neuropil was also more
intense in Sham males than in Gdx males in the SNB and RDLN. Two days
of TP treatment did not reverse the effects of gonadectomy on neuropil
labeling. These results are detailed below.
Intensity of CNTFR-IR in motoneuron somata
There was a significant difference across treatment groups in the
relative frequency distributions of soma labeling in all three motor
pools (Fig. 3; SNB:
2 = 28.4, p < 0.005; DLN:
2 = 53.4, p < 0.001; RDLN:
2 = 32.3, p < 0.001). SNB
motoneuron soma densities were shifted to significantly higher values
(darker somas) in Sham males than in Females or Gdx + oil males (p < 0.005 in both cases, Fig.
3A). The distribution of soma densities of gonadectomized
males receiving 2 d of testosterone (Gdx + 2d TP) was similar to
that of Sham males (p > 0.40) but also did not
differ significantly from that of Gdx + oil males
(p > 0.10).
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Figure 3.
Relative frequency distributions of the percent of
motoneuron somata with light, medium, dark, or very dark CNTFR-IR in
the animals of experiment 1. A, The distribution of
label intensities was shifted to higher (darker) values
in SNB motoneurons of Sham males than in Gdx + oil males or Females. B, C, DLN
and RDLN soma densities were higher in Sham males than
in all other groups.
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In the DLN, motoneuron soma density scores of Sham males were shifted
to darker values than those of all other groups
(p < 0.001 in each case, Fig. 3B).
This is especially clear when comparing the relative frequencies of the
most darkly labeled cells; >50% of the DLN cells of Sham males were
classified as very dark, whereas the relative frequency of very dark
cells was ~10% in all other groups. The Gdx + oil and Gdx + 2d TP
groups did not differ with respect to CNTFR-IR of DLN somata
(p > 0.20). RDLN somata of Sham males were
shifted to darker values than in any other group (p < 0.005 in each case, Fig. 3C),
and the two gonadectomized male groups did not differ on this measure
(p = 0.20).
Intensity of CNTFR-IR in neuropil
The relative frequency distributions of neuropil label intensity
differed significantly across treatment groups in the SNB (2 = 22.9; p < 0.001), DLN
(2 = 38.7, p < 0.001), and RDLN
(2 = 31.3, p < 0.001). Neuropil
densities in the SNB were shifted to higher values in Sham males than
in any of the other groups (p < 0.005 in each
case; Fig. 4A), and Gdx + 2d TP males did not differ from Gdx + oil males on this measure
(p > 0.50). DLN neuropil densities were also
higher in Sham males than in Females or Gdx + 2d TP males
(p < 0.005; Fig. 4B), but
Sham males and Gdx + oil males did not differ significantly
(p > 0.05). RDLN neuropil density scores in
Sham males were shifted to higher values than in Gdx + oil males or Gdx + 2d TP males (p < 0.001 in both cases; Fig. 4C) but did not differ from the distribution of scores in
Females (p > 0.50).
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Figure 4.
Relative frequency distributions of the intensity
of CNTFR-IR in the neuropil of animals in experiment 1. A, SNB neuropil densities were higher in Sham
males than in any other group. B, DLN neuropil
densities were higher in Sham males than in
Females or Gdx + 2d TP males. C, In the
RDLN, Sham males had higher neuropil densities than
Gdx + oil or Gdx + TP males, but neuropil
densities did not differ between Sham males and
Females.
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Total area covered by label
This measure ignores variations in density of label and simply
counts the number of pixels above a minimum background level in each
motor nucleus. There was no overall difference among the four
experimental groups in the amount of label in the RDLN or DLN (Table
1). Total area covered by label in the
SNB did differ by group (F = 10.03; p = 0.001) and was significantly lower in females than in all male groups
(p < 0.015 in each case). This difference may
simply reflect the much smaller number of SNB motoneurons in females
than in males. To test this assumption, we counted the total number of
motoneurons visible in the analyzed sections after neutral red
counterstaining. In accord with previous reports (Breedlove and Arnold,
1980; Jordan et al., 1982), we saw no sex difference in motoneuron
number in the RDLN (39.7 ± 2.2 vs 43.6 ± 4.0 for males and
females, respectively), an approximately twofold sex difference in the
DLN (37.2 ± 2.2 vs 18.7 ± 4.5), and a threefold sex
difference in the SNB (30.5 ± 2.1 vs 10.4 ± 2.6).
Experiment 2: seven weeks castration and long-term
TP replacement
Two days of TP replacement initiated 4 weeks after castration did
not reverse all effects of castration in experiment 1. We next examined
the effect of long-term testosterone replacement in castrates, starting
at the time of surgery. Seminal vesicle weights at time of killing
indicate that our testosterone capsules provided androgen levels to Gdx
animals that were at least as high as those in sham-Gdx males (Sham,
2173 ± 327 mg; Gdx + Blank, 103 ± 17 mg; Gdx + T, 2557 ± 48 mg).
Summary of results
CNTFR-IR was more intense in motoneuron somata of Sham and Gdx + T males than in Gdx + Blank males for all three cell groups (Fig.
5). Neuropil label was also more intense
in Sham than in Gdx + Blank males in all motor pools, and neuropil
label in Gdx + T animals was intermediate. These findings are detailed
below.
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Figure 5.
Photomicrographs depicting CNTFR-IR in the
lower lumbar spinal cord of Sham
(A), Gdx + Blank
(B), and Gdx + T
(C) males. Seven weeks of gonadectomy reduced the
intensity of CNTFR-IR in all motor pools, and this decrease was
prevented in males receiving testosterone capsules at the time of
gonadectomy. Scale bar in C, 200 µm (applies to
A-C).
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CNTFR-IR of motoneuron somata and neuropil
The pattern of results for motoneuron soma densities was identical
in the SNB, DLN, and RDLN (Figs. 5, 6).
In all cases there was an overall effect of treatment on the relative
frequency distributions of cell density measures (SNB:
2 = 29.5, p < 0.0005; DLN:
2 = 42.1, p < 0.0005; RDLN:
2 = 19.2, p < 0.005). Motoneuron
soma labeling was shifted to lower values in Gdx + Blank males than in
Sham males in each motor pool (SNB, p < 0.002; DLN,
p < 0.001; RDLN, p < 0.002).
Testosterone capsules fully prevented this decrease in castrated males;
Gdx + T males were not significantly different from the Sham group for
any motor pool, and soma densities were shifted to significantly higher
values in Gdx + T males than in Gdx + Blank males in each motor pool
(p < 0.002 in each case).
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Figure 6.
Relative frequency distributions of the percent of
motoneuron somata with light, medium, dark, or very dark CNTFR-IR in
the SNB (A), DLN (B), and
RDLN (C) in experiment 2. CNTFR-IR was shifted to lower scores in
motoneurons of Gdx + Blank males than in Sham males for
each motor pool. Long-term treatment of castrates with testosterone
(Gdx + T) prevented the decline in label in each
case.
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CNTFR-IR in neuropil also differed by treatment group (Fig.
7; SNB: 2 = 22.9, p < 0.001; DLN: 2 = 38.7, p < 0.0005; RDLN: 2 = 31.3, p < 0.0005). Compared to values in intact males,
neuropil densities were shifted to lower scores in gonadectomized males not given hormone replacement (p < 0.001 for
each of the three motor pools). Testosterone capsules only partially
prevented the decrease in neuropil labeling, however. Neuropil
densities were marginally higher in Gdx + T males than in Gdx + Blank
males in the SNB and RDLN (p = 0.051 and
p = 0.049, respectively) but not in the DLN
(p = 0.10). CNTFR-IR in the neuropil of Gdx + T and Sham males was equivalent in the SNB (p > 0.15) but was lower in Gdx + T males in the DLN and RDLN
(p < 0.001 in both cases).
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Figure 7.
Relative frequency distributions of intensity of
CNTFR-IR in the neuropil of animals in experiment 2. Neuropil label
was shifted to lower scores in the Gdx + Blank group
than in the Sham group in each motor pool. CNTFR-IR
in the neuropil of Gdx + T males was intermediate.
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Total area covered by label
There was an effect of treatment on the total area covered by
label in the SNB and DLN (F = 4.9, p < 0.05; F = 4.4, p < 0.05, respectively)
but not in the RDLN (F = 1.24, p > 0.30). A greater number of pixels were above threshold in the SNB and
DLN of Sham than of Gdx + Blank males (p < 0.05 in both cases). Sham and Gdx + T males did not differ on this measure
in any cell group (Table 2).
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DISCUSSION |
We find that spinal motoneurons express CNTFR in their somata,
dendrites, and axons, in confirmation of a previous report (MacLennan
et al., 1996). Thus, motoneurons may receive trophic support from CNTF
or from other CNTF-like molecules produced by multiple sources,
including neural afferents, target muscles, or the peripheral nerve.
CNTF itself is expressed at highest levels in Schwann cells of
peripheral nerve and does not appear to be made by muscles
(Stöckli et al., 1989). However, an additional ligand or ligands
for the CNTF receptor likely exist and regulate motoneuron number in
development (DeChiara et al., 1995; Shelton, 1996; Forger et al.,
1997).
The pattern of CNTFR-IR in the DLN was striking, especially with
respect to the very dense, often punctate neuropil staining. Previous
ultrastructural studies have reported an extensive bundle of tightly
packed dendrites running rostrocaudally through the DLN (Kerns and
Peters, 1974; Anderson et al., 1976). These dendrites are interspersed
among the motoneuronal somata and originate from the DLN motoneurons
themselves. It seems likely, therefore, that CNTFR-IR in the DLN
neuropil is caused primarily by CNTFR protein present on DLN
dendrites, which would have been cut in cross section in our coronal
sections. The labeling we observed in the SNB also matches previous
descriptions of the dendritic projections of SNB motoneurons (Kurz et
al., 1986; Rand and Breedlove, 1995), and horizontal sections did not
reveal any major input of CNTFR-immunoreactive fibers to the
SNB/DLN/RDLN area from higher or lower in the neuraxis (Fig.
2A). Taken together, we conclude that most of the
CNTFR-IR in the neuropil came from the dendrites of motoneurons
within each motor pool.
Castration of adult male rats caused a decrease in CNTFR-IR in the
somata of motoneurons of the SNB, DLN, and RDLN. Short-term (2 d)
testosterone treatment reversed the decline in soma labeling in the SNB
only, and long-term (7 weeks) testosterone replacement prevented the
decline in all three cell groups. Adult castration also results in a
reduction in motoneuron soma size in the SNB and, to a lesser extent,
the DLN (Breedlove and Arnold, 1981; Kurz et al., 1991). An effect on
soma size is not likely to have contributed to the changes we observed
in soma labeling, however. If the same number of CNTFR molecules
were distributed over smaller somata, one would expect the intensity of
label on somata to be increased in castrated males. Instead, the
opposite pattern was found, indicating a downregulation of CNTFR
protein after castration. Motoneuron soma labeling was also more
intense in intact males than in females in all three motor pools. The
lower circulating androgen levels in females than in males may result
in reduced expression of CNTFR in motoneurons of females.
Seven weeks of castration reduced the intensity of CNTFR-IR in the
neuropil of each motor pool. Within the RDLN, the reduction in neuropil
labeling occurred in the absence of any effect of castration on the
total area covered by label. That is, the total number of pixels above
background in the RDLN was equivalent in intact and gonadectomized
males, but the average intensity of label in the neuropil was reduced
by gonadectomy. We interpret this finding as a reduction in CNTFR-IR
per unit length of dendrite in the RDLN of castrated males. The
interpretation of effects of castration on neuropil labeling in the SNB
and DLN is less straightforward, however. We reason that the intensity
of label in neuropil will be influenced by both the amount of label per unit area of dendrite as well as the number of dendrites ramifying in a
given area. The dendritic arbors of SNB and DLN motoneurons are
reported to retract after castration, although there is some debate as
to whether the effect is large (Kurz et al., 1986, 1991) or rather
subtle (Sasaki and Arnold, 1991). We observed a significant reduction
in total area covered by label in the SNB and DLN of Gdx + Blank males
(Table 2), which may reflect an attenuation of dendritic trees after
castration. Thus, the decrease in intensity of label in the neuropil of
the SNB and DLN of Gdx + Blank males may be caused in part by a
reduction in the number of dendritic branches per unit area of
neuropil. It is worth noting, however, that even if all effects of
gonadectomy on intensity of label in neuropil and total area covered by
label are accounted for by a reduction in motoneuron size, the end
result would nevertheless be fewer CNTF receptors per motoneuron and,
presumably, reduced sensitivity to trophic factor support in
gonadectomized males.
Because testosterone itself is an androgen, the present results
demonstrate androgenic control of CNTFR-IR in spinal motoneurons. However, testosterone can be aromatized to estradiol, and at present we
cannot rule out the possibility that the observed effects of castration
and testosterone replacement were actually mediated by estrogenic
hormone metabolites. Resolving this issue would be of interest because
the three motor pools examined in this study all express androgen
receptors but do not exhibit estrogen-binding activity (Breedlove and
Arnold, 1980, 1983; Freeman et al., 1995; Jordan et al., 1997).
Identifying the active hormone metabolite or metabolites would,
therefore, help to resolve the question of whether effects of
testosterone on CNTFR-IR in spinal motoneurons is likely to be
mediated directly at the motoneurons themselves or indirectly via
hormone action on target muscles, afferents, or other
intermediaries.
Several previous studies have demonstrated that CNTFR mRNA levels in
the brain and spinal cord are altered after nervous system injury (Mata
et al., 1993; Rudge et al., 1994; Lee et al., 1997a,c; Oyesiku et al.,
1997). However, very little has been reported regarding factors that
regulate the expression of CNTFR under physiological, noninjury
conditions. By Northern blotting we recently found that adult
castration alters the level of CNTFR mRNA in the target muscles of
SNB motoneurons but has no effect on the abundance of CNTFR message
in homogenates of the whole lumbosacral spinal cord (Xu and Forger,
1998). Because many cell types are present in the cord, we could not
discern by Northern blotting whether CNTFR expression might be
androgen-regulated in specific subsets of cells. The present analysis
indicates that at least at the protein level, circulating androgen
levels do alter CNTFR expression in spinal motoneurons. This is true
not only for sexually dimorphic penile motoneurons (SNB, DLN), but also
for motoneurons innervating foot muscles (RDLN).
Previously, androgens have been shown to regulate the expression of
calcitonin gene-related peptide, cholecystokinin, -tubulin, -actin, and connexin mRNAs in SNB motoneurons (Popper et al., 1992;
Matsumoto et al., 1994, 1995). In addition, vasopressin-binding sites
on SNB motoneurons are downregulated by castration (Tribollet et al.,
1997). In each case, however, androgen manipulations had no effect on
gene expression in the RDLN, or in other motoneurons innervating
muscles that are not sexually dimorphic. Although most brainstem and
spinal motoneurons express androgen receptors (Sar and Stumpf, 1977; Yu
and McGinnis, 1986), as far as we are aware only choline
acetyltransferase mRNA has previously been shown to be regulated by
androgens in all motor pools examined (Blanco et al., 1997). We did not
quantify CNTFR expression in the few cervical and thoracic cords
examined in the present study. Nonetheless, our observation that
testosterone regulates CNTFR protein in the RDLN suggests that
androgen regulation of CNTFR protein may be a general property of
spinal motoneurons.
The present results are particularly interesting in light of the fact
that androgens have trophic effects on spinal and brainstem motoneurons. In addition to the actions of androgens on SNB and DLN
cell size discussed above, the rate of motor nerve regeneration is
enhanced by testosterone in adult rodents (Yu, 1982; Kujawa et al.,
1993, 1995). Testosterone also rescues SNB and DLN motoneurons from
developmental cell death in perinatal rats (Nordeen et al., 1985;
Sengelaub and Arnold, 1989) and attenuates motoneuron death after
axotomy of the laryngeal nerve in adult frogs (Pérez and Kelley,
1996). Finally, message for choline acetyltransferase (ChAT) is reduced
in spinal motoneurons of male rats by castration and is increased by
testosterone treatment (Blanco et al., 1997). These hormone effects are
remarkably similar to the actions of CNTF on peripheral nerve
regeneration, motoneuron survival, and motoneuron ChAT activity (Wong
et al., 1993; Forger et al., 1993; Kato and Lindsay, 1994; Sahenk et
al., 1994; Ulenkate et al., 1994). By upregulating CNTFR protein
expression in spinal motoneurons, testosterone presumably increases
motoneuron responsiveness to CNTFR ligands. This increased
responsivity to trophic support could underlie androgenic effects on
motoneuron survival, cell size, or axon regeneration.
| |
FOOTNOTES |
Received May 27, 1998; revised Aug. 20, 1998; accepted Aug. 20, 1998.
Supported by National Institutes of Health Grants NS35224, HD33044,
HD01188 and the Whitehall Foundation. We thank Karen Neitzel and Brenda
Devlin for technical assistance.
Correspondence should be addressed to Nancy G. Forger, Department of
Psychology, University of Massachusetts, Amherst, MA 01003-7710.
| |
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Abstract
Introduction
