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Nerve growth factor receptor trkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons

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THE JOURNAL OF COMPARATIVE NEUROLOGY 381:428–438 (1997)
Nerve Growth Factor Receptor TrkA
Is Down-Regulated During Postnatal
Development by a Subset of Dorsal
Root Ganglion Neurons
DEREK C. MOLLIVER AND WILLIAM D. SNIDER*
Center for the Study of Nervous System Injury, Department of Neurology,
Washington University School of Medicine, St. Louis, Missouri 63110
ABSTRACT
Nerve growth factor (NGF), signaling through its receptor tyrosine kinase, TrkA, is
required for the survival of all small and many intermediate-sized murine dorsal root ganglion
(DRG) neurons during development, accounting for 80% of the total DRG population.
Surprisingly, NGF/TrkA-dependent neurons include a large population that does not express
TrkA in adult mice (Silos-Santiago et al., 1995). This finding suggests two hypotheses:
Neurons lacking TrkA in the adult may express TrkA during development, or they may be
maintained through a paracrine mechanism by TrkA-expressing neurons. To determine
whether TrkA is expressed transiently by DRG neurons that lack the receptor in adulthood,
we examined the distribution of TrkA protein during development. We show here that TrkA
expression is strikingly developmentally regulated. Eighty percent of DRG neurons expressed
TrkA during embryogenesis and early postnatal life, whereas only 43% expressed TrkA at
postnatal day (P) 21. Because the period of TrkA down-regulation corresponds with a critical
period during which nociceptive phenotype can be altered by NGF (see Lewin and Mendell
[1993] Trends Neurosci. 16:353–359), we examined whether NGF modulates the downregulation of TrkA. Surprisingly, neither NGF deprivation nor augmentation altered the
extent of TrkA down-regulation. Our results demonstrate a novel form of regulation of
neurotrophin receptor expression that occurs late in development. All DRG neurons that
require NGF for survival express TrkA during embryogenesis, and many continue to express
TrkA during a postnatal period when neuronal phenotype is regulated by NGF. The
subsequent down-regulation of TrkA is likely to be importantly related to functional
distinctions among nociceptive neurons in maturity. J. Comp. Neurol. 381:428–438, 1997.
r 1997 Wiley-Liss, Inc.
Indexing terms: nerve growth factor; nociceptors; dorsal horn; regulation of phenotype;
neurotrophins
Early investigations into the actions of nerve growth
factor (NGF) determined that NGF supports the survival
of sympathetic neurons and neural crest-derived sensory
neurons during development (for review, see Thoenen and
Barde, 1980). Unlike sympathetic neurons, dorsal root
ganglion (DRG) neurons lose their dependence upon NGF
for survival during the first few days of postnatal life
(Gorin and Johnson, 1980; Lewin et al., 1992). However, in
the first 2 weeks of postnatal life, the specificity of unmyelinated and thinly myelinated DRG neurons for nociceptive stimuli is regulated by NGF (for review, see Lewin and
Mendell, 1993). In adulthood, approximately 40% of mature DRG neurons express the NGF receptor tyrosine
kinase, TrkA, suggesting that NGF continues to act upon a
r 1997 WILEY-LISS, INC.
subset of sensory neurons in maturity (Verge et al., 1992;
Averill et al., 1995; Molliver et al., 1995). Indeed, in adult
DRG neurons, NGF regulates the levels of expression of
substance P and calcitonin gene-related peptide (CGRP),
two neuropeptides associated with nociceptive transmis-
Contract grant sponsor: NINDS; Contract grant numbers: NS31768,
P01-NS17763.
*Correspondence to: W.D. Snider, M.D., Center for the Study of Nervous
System Injury, Department of Neurology, Box 8111, Washington University
School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110.
E-mail: sniderw@neuro.wustl.edu
Received 24 July 1996; Revised 18 October 1996; Accepted 27 October
1996
DOWN-REGULATION OF TrkA BY DRG NEURONS
sion (Lindsay and Harmar, 1989; Verge et al., 1995; for
review, see Hökfelt, 1991) that are highly colocalized with
TrkA (Averill et al., 1995; see also Verge et al., 1989, 1995).
Furthermore, administration of NGF causes thermal and
mechanical hyperalgesia, whereas depletion of NGF with
blocking antibodies results in a reduced sensitivity to
painful stimuli (see Lewin and Mendell, 1993). Thus, in
addition to regulating neuronal survival during embryonic
development and determining modality specificity in early
postnatal life, NGF regulates functional properties of
nociceptive sensory neurons in adulthood.
We have recently characterized a subset of small DRG
neurons that have morphological features associated with
nociceptive neurons but that do not express TrkA in
adulthood (Molliver et al., 1995; see also Averill et al.,
1995). Similar to TrkA-expressing neurons, these TrkA2
neurons are small in diameter, project to the superficial
dorsal horn, and presumably have cutaneous peripheral
axons (see Silverman and Kruger, 1988). However, the
small TrkA2 neurons differ from TrkA-expressing neurons
in that they lack substance P and CGRP, bind the Bandeiraea simplicifolia lectin I-B4 and are immunoreactive for
the D subunit of protein kinase C and the neurofilament
protein a-internexin (Molliver et al., 1995). These TrkA2
neurons presumably correspond to the 25–35% of DRG
neurons that do not express any member of the Trk family,
or the low-affinity neurotrophin receptor, p75 (McMahon
et al., 1994; Wright and Snider, 1995). Furthermore, small
TrkA1 and TrkA2 neurons project to different targets in
the superficial dorsal horn. Whereas TrkA-expressing neurons project to spinal cord lamina I and the dorsal portion
of lamina II (lamina IIo), small neurons lacking TrkA
project to the interior of lamina II (lamina IIi; Molliver et
al., 1995). Thus, several neurochemical and morphological
characteristics distinguish TrkA1 neurons from small neurons lacking TrkA.
Despite the restriction of TrkA expression to one subset
of small DRG neurons in adulthood, the elimination of
TrkA through gene targeting results in the loss of virtually
all small neurons (Silos-Santiago et al., 1995). This finding
suggests one of two possibilities: that all small DRG
neurons initially express TrkA, and the I-B4-binding neurons subsequently down-regulate TrkA expression, or that
I-B4 neurons never express TrkA but are dependent for
their survival on support from TrkA-expressing neurons
via a paracrine mechanism. Potential for paracrine interactions in the developing DRG is supported by findings that
brain-derived neurotrophin factor (BDNF) and neurotrophin 3 (NT-3) are expressed by many DRG neurons
during development (Ernfors et al., 1992; Schecterson and
Bothwell, 1992; Elkabes et al., 1994). To address this issue,
we examined the distribution of TrkA protein at selected
time points during embryonic and postnatal development.
In addition, we examined the ontogeny of CGRP and p75,
which are expressed by most or all mature TrkA1 neurons
(Verge et al., 1989, 1992; McMahon et al., 1994; Averill et
al., 1995; Wright and Snider, 1995), and binding of the
lectin I-B4, which selectively labels small TrkA2 neurons
(Averill et al., 1995; Molliver et al., 1995). Finally, in order
to investigate the possibility that NGF is involved in the
down-regulation of TrkA and in the differentiation of small
DRG neurons into I-B4-binding and CGRP-expressing
neuronal populations, we examined whether systemic
injections of NGF or antibodies to NGF during the first 2
429
TABLE 1. Primary Antisera1
Type
Rabbit anti-TrkA
Rabbit anti-p75
Rabbit anti-CGRP
Mouse antineurofilament
I-B4-biotin
I-B4-HRP
Dilution
Source
1:8,000
1:750
1:1,000
1:40
1:200
1:200
Louis Reichardt
Barbara Hempstead
Peninsula
Sigma
Sigma
Sigma
1The antisera and lectins used in this study are listed here with the working dilution
used and the source from which they were obtained. CGRP, calcitonin gene-related
peptide; I-B4-biotin, a biotinylated form of the lectin I-B4 from Bandeiraea simplicifolia;
I-B4-HRP, I-B4 conjugated to horseradish peroxidase (HRP).
weeks of postnatal life altered expression of TrkA, CGRP,
or I-B4 binding sites.
We show here that TrkA is expressed by 80% of DRG
neurons during embryogenesis and into postnatal development and is down-regulated in a subset of small neurons
during the 3 weeks after birth, well after the time at which
DRG neurons lose their dependence on NGF for survival.
We conclude that essentially all DRG neurons dependent
upon NGF/TrkA signaling for survival express TrkA during embryonic development. In contrast to TrkA, CGRP
exhibited a mature expression pattern by postnatal day (P)
1. Manipulation of postnatal NGF levels, which regulate
the differentiation of nociceptive phenotype, did not alter
the down-regulation of TrkA, nor did it alter the distribution of CGRP or I-B4 binding. The discrepancy between
TrkA and CGRP expression during development suggests
that these neurochemical features are regulated by different mechanisms. Finally, we suggest that the downregulation of TrkA contributes to the emergence of functionally distinct subsets of small DRG neurons.
MATERIALS AND METHODS
Tissue preparation
TrkA immunocytochemistry was performed in CF-1 mice
(Charles River, Wilmington, MA) n 5 3 for each developmental stage examined. Embryos at embryonic day (E) 13
and E15 were immersion fixed in 3% paraformaldehyde,
15% saturated picric acid in 0.1 M phosphate buffer, pH
7.4, for 2 hours. Postnatal day (P) 1, P7, P14, P21, and
adult mice were deeply anesthetized, transcardially perfused first with phosphate buffer and then with the fixative
described above, and postfixed for 2 hours. P1 animals
were anesthetized on ice; older animals were anesthetized
by sodium pentobarbital injection. In postnatal animals,
the spinal column was dissected out for cutting; embryos
were cut whole. All tissue was suspended overnight in 30%
sucrose after fixation. Tissue was then frozen in Optimal
Cutting Temperature (OCT) embedding medium (Baxter,
McGaw Park, IL), and 12 µm sections were cut on a
cryostat and stored at 220°C. All animal handling and
protocols were approved by the Animal Studies Committee
of Washington University.
Immunocytochemistry
Mounted sections were encircled with a Teflon coating by
using a Pap pen (Kyota International, Elk Grove Village,
IL) and were incubated for 1 hour in a blocking solution
consisting of Superblock buffer, a commercial blocking
buffer purchased from Pierce (Rockford, IL), 0.3% Triton
X-100 (Electron Microscope Sciences, Ft. Washington, PA),
1.0% porcine gelatin (Sigma), and 1.5% normal serum
430
D.C. MOLLIVER AND W.D. SNIDER
(Vector, Burlingame, CA). Table 1 lists the primary reagents used in this report. Primary and secondary antibodies were diluted in the same solution, diluted 1:1 with
Superblock buffer/1.5% normal serum. Sections were incubated in primary antibody overnight. They were then
washed three times for 5 minutes each with phosphatebuffered saline (PBS) and were placed in secondary antibody solution (donkey anti-rabbit IgG conjugated to the
indocarbocyanine dye Cy3; Jackson Immunoresearch Laboratories, West Grove, PA), washed three times for 5
minutes each in PBS, coverslipped in PBS, and then examined
under epifluorescence. For horseradish peroxidase-diaminobenzidine (HRP-DAB) visualization of antibody labeling, sections were removed from primary antibody, washed in PBS as
above, processed with a Vector Vectastain kit according to
instructions in the kit, then dehydrated, and coverslipped
with DPX. For a negative control, representative sections
were processed without a primary antibody.
Thirty-five millimeter slides of representative sections
for all procedures were taken on a Nikon Microphot FXA
microscope and digitized by using a Polaroid Sprintscan
connected to a Power Macintosh 9500. After creation of the
figures and adjustment for color, tone, and contrast, images were printed on a Tektronics dye-sublimation printer.
Lectin histochemistry
Sections were processed for lectin histochemistry by
substituting lectins for primary antibodies, as described
above, with the following modifications. Sections were
incubated overnight in I-B4 (Sigma) diluted to 10 µg/ml in
modified PBS containing 0.01 M MgCl2 and CaCl2, pH 6.8.
Slides received an additional 5 minute wash in 50 mM
Tris/1.8% NaCl after the primary incubation. Avidinylated
Cy3 (1:2,500) was diluted in the same buffer, and sections
were incubated for 1 hour. The high salt concentration
reduced background staining during avidin-biotin binding
(L. Slomianka, personal communication). After incubation
in the avidinylated reagent, the slides were washed in PBS
and incubated in the secondary antibody, as described
above. I-B4 conjugated to HRP was used for quantitation
of I-B4 histochemistry, which was followed by visualization with the avidin biotin complex (ABC)-DAB procedure,
as described above.
NGF perturbations
P1 mice were separated into three groups for injections.
The first group received 5 µg/g NGF in L15 medium
(purchased from Washington University Tissue Culture
Support Center, St. Louis, MO) once daily from P2 until
P14. The second group received 5 µl/g anti-NGF once daily
from P3 to P14, and the third group was given daily
injection of vehicle. All animals were killed by perfusion,
as described above, on P21. This procedure was performed
twice, for a total number of five mice for each group.
NGF was purified from mouse salivary glands (Boccini
and Angeletti, 1969). The titer of the goat anti-mouse NGF
antiserum was 80,000, which was determined as the
reciprocal of the highest dilution that blocked DRG neurite
outgrowth in vitro in the presence of 5 ng/ml NGF (Fenton,
1970; Ruit et al., 1992). Unlike DRG neurons, sympathetic
neurons do not lose dependence upon NGF for survival
after birth (Gorin and Johnson, 1980). Therefore, superior
cervical ganglia (SCGs) from two control, two NGFtreated, and two anti-NGF-treated P14 mice were com-
pared for a gross indication of the effectiveness of the
treatment protocol. SCGs from NGF-injected mice were
clearly larger than control SCGs, whereas SCGs from
anti-NGF-injected mice were drastically smaller (not
shown).
Quantification
To provide an estimate of the proportion of DRG neurons
expressing TrkA at each age examined, we calculated the
percentage of TrkA-IR neurons in selected sections of
DRG. Sections of lower lumbar ganglia used for quantification of TrkA immunoreactivity (IR) were counterstained
with cresyl violet prior to coverslipping and examined
under brightfield microscopy. Sections from E13, E15, and
P1 mice were examined with a 3100 water-immersion
objective; for older animals, a 350 water-immersion objective was used. Cells with clearly defined nuclei were
counted in multiple representative sections through the
ganglia and were considered positive if they contained a
clear deposition of cytoplasmic DAB reaction product
compared with background. For examination of the developmental time course of TrkA distribution, at least 1,000
cells were scored from each of three animals for each age
group: E15, P1, P7, P14, P21, and adult. The mean
percentage of positive neurons for each age group was
calculated, and the means were compared between groups.
An issue that must be addressed is whether our results
are accurate without stereological analysis. For example,
could the observed reduction in the percentage of TrkA-IR
neurons be the result of a differential increase in the size of
the nuclei of TrkA2 neurons over the first 3 postnatal
weeks? We consider this to be a highly unlikely possibility
for three reasons. First, at P1, TrkA-IR is excluded only
from large-diameter neurons, whereas, at P21, TrkA-IR is
excluded from a subset of small-diameter neurons as well.
Second, the selective loss of TrkA-IR from afferents in the
interior of lamina II is independent corroboration of the
down-regulation of TrkA in a specific subset of small
neurons (see Molliver et al., 1995). Finally, the percentage
of TrkA-IR neurons at P1 corresponds to the percentage of
TrkA-IR neurons in the adult plus the percentage of
neurons binding I-B4, providing further confirmation that
an identified subset of DRG neurons down-regulate TrkA.
Cross-sectional areas of TrkA-IR neurons in NGF- and
anti-NGF-treated animals were analyzed as a measure of
treatment effectiveness (see Rich et al., 1984). Sizefrequency histograms of cross-sectional areas of TrkA-IR
neurons from P21 NGF- and anti-NGF-treated animals
were compared to determine whether the treatments had
affected cell size. More than 300 neurons per treatment
group were counted in sections taken from each of the
animals treated. Quantification of TrkA-IR, CGRP-IR, and
I-B4-binding in treated animals was performed at P21, as
described above. The statistical significance of differences
between NGF and anti-NGF treated animals was determined by using a Student’s t-test.
RESULTS
TrkA is down-regulated by a subset
of DRG neurons after birth
TrkA immunocytochemistry resulted in dense cytoplasmic staining of DRG neurons (Fig. 1) as well as both
central and peripheral processes. At E13, the earliest time
DOWN-REGULATION OF TrkA BY DRG NEURONS
431
Fig. 1. TrkA protein is down-regulated during postnatal development. Shown here are representative sections from mice of different
ages stained for TrkA and counterstained to visualize negative neurons. Scale bars 5 40 µm in top and middle sections. Bottom two
sections are high-magnification micrographs of postnatal day (P) 1 and
P21 dorsal root ganglia (DRGs). Scale bars 5 20 µm in bottom sections.
At P1, the great majority of neurons (80%) are intensely TrkA-
immunoreactive (-IR). There is a progressive decrease in the number
of TrkA-IR neurons between P1 and P21, at which point the percentage of TrkA-containing neurons is similar to that in the adult.
High-magnification micrographs illustrate the large size of negative
neurons both at P1 and at P21. However, in contrast to P1, negative
neurons at P21 include many small-diameter neurons (arrows).
point examined, the majority of neurons in the DRG were
immunoreactive for TrkA (not shown). The widespread
distribution of TrkA staining in the DRG was maintained
between E13 and the day of birth. Quantification of
TrkA-IR neurons in lumbar ganglia at E15 revealed that
82% of all neurons in the DRG were immunopositive (Fig.
1). At this age, there was a clearly discernible range of
soma sizes, and the largest neurons were consistently
immunonegative for TrkA. After birth, there was a gradual,
progressive decrease in the number of DRG neurons
labeled (Figs. 1, 2). A distribution of TrkA-IR approximating that found in the adult was not seen until P21.
Because CGRP is highly colocalized with TrkA in adult
DRG (Averill et al., 1995), we determined whether the
extent of colocalization was similar during development.
Whereas the percentage of TrkA-IR neurons between E15
432
D.C. MOLLIVER AND W.D. SNIDER
Fig. 2. The percentage of TrkA-IR DRG neurons diminishes during
postnatal development. The percentage of TrkA-IR DRG neurons was
plotted against age to illustrate the gradual decrease in the proportion
of neurons expressing TrkA between embryonic day (E) 15 and
adulthood. Approximately 80% of DRG neurons are TrkA-IR between
E15 and P1, a proportion that decreases between P1 and P21 to adult
levels of roughly 40%. Vertical bars indicate S.E.M.
and P1 was twice that seen in the adult, DRG neuronal
CGRP-IR was visible in very few cells at E15 (not shown)
and increased to approximately adult numbers of neurons
by P1. Figure 3 shows that many small neurons were
clearly CGRP2 at P1. Thus, CGRP expression is restricted
to a subset of small and medium-sized neurons at an age
when TrkA is expressed by the great majority of DRG
neurons.
In the adult rat DRG, all neurons expressing TrkA also
express p75 (Verge et al., 1992; Wright and Snider, 1995).
We examined the distribution of p75 to determine whether
the low-affinity neurotrophin receptor showed an enhanced distribution during embryonic development similar to that of TrkA. Immunocytochemistry revealed that,
like CGRP-IR, p75-IR was more restricted than TrkA-IR at
P1, in that many small neurons did not contain p75-IR
(Fig. 3). In contrast to CGRP, p75 was also seen in large
neurons, consistent with its reported colocalization in the
adult with TrkA, TrkB, and partial colocalization with
TrkC (Wright and Snider, 1995).
In addition to examining markers of TrkA1 neurons, we
analyzed the development of I-B4 binding sites to determine whether they are expressed prior to the downregulation of TrkA. DRG neuronal binding by I-B4 was
first seen at P1, at which point it was very faint (not
shown). However, staining was sufficient to determine that
I-B4-binding labeled a subset of small neurons and that
the pattern was qualitatively similar to that seen in the
adult. Staining was more evident at P7 and, at P14,
Fig. 3. Calcitonin gene-related peptide (CGRP) and low-affinity
neurotrophin receptor (p75) are not expressed by all TrkA neurons at
P1. DRG sections from P1 mice were processed for fluorescence
immunohistochemistry for p75, TrkA, or CGRP, which are highly
colocalized in adulthood. This figure displays the clear discrepancy
between the large percentage of TrkA-IR neurons and the smaller
populations of CGRP-IR and p75-IR neurons. In addition to TrkAexpressing neurons, p75 labels a subset of large-diameter TrkCexpressing and TrkB-expressing neurons. Scale bars 5 40 µm.
DOWN-REGULATION OF TrkA BY DRG NEURONS
433
Fig. 4. Neurons down-regulating TrkA project to the interior of
lamina II. Sections of lumbar spinal cord stained for TrkA, neurofilament (NF), or p75 reveal the central target field of neurons downregulating TrkA. NF and p75 staining was used to identify the
boundaries of lamina II, the major target field of unmyelinated DRG
afferents. The white dots in the NF and p75 sections trace the lamina
II-III border. At P1, TrkA staining covers the entirety of laminae I and
II of the dorsal horn. TrkA staining in the dorsal horn retreats from the
ventral edge of lamina II as the percentage of TrkA1 neurons in the
ganglion decreases. At P14 , extensive TrkA label is seen only in lamina
I and at the dorsal edge of lamina II. Note that p75-IR avoids the
interior of lamina II, indicating that neurons projecting to this region
do not express p75. The comparison of TrkA-IR with neurofilament-IR
and p75-IR supports our interpretation that the major population of
neurons down-regulating TrkA is the Bandeiraea simplicifolia lectin
(I-B4)-binding population, which innervates the interior of lamina II
(see Molliver et al., 1995). Scale bar 5 40 µm.
appeared similar in intensity to the adult (not shown). The
presence of I-B4 binding at P1 suggests that these neurons
initially express both TrkA and lectin binding sites, as
essentially all small neurons express TrkA at this age.
While the percentage of DRG neurons expressing TrkA
decreased, there was a selective reduction in the ventral
extent of TrkA staining in lamina II (see Fig. 4). Between
P1 and P21, TrkA staining retreated from the ventral
border of lamina II until only afferents innervating lamina
I and the dorsalmost portion of lamina II were immunopositive. In addition, the overall density of staining for TrkA in
the dorsal horn decreased between birth and adulthood,
suggesting a reduction in the concentration of TrkA protein in central afferents after birth.
To verify that the intensely stained band of TrkA-IR
afferents seen in the dorsal horn at P1 was restricted to
lamina II, we determined the location of the lamina II–III
border histochemically. Antibodies to phosphorylated neurofilament triplet proteins label the large-caliber axons
destined to become myelinated but not the fine-caliber
unmyelinated C-fiber afferents (see Lawson, 1992). Because lamina II is primarily innervated by unmyelinated
axons in adult animals, whereas lamina III and IV are
innervated by large-caliber, low-threshold mechanoreceptors (for review, see Light, 1992), we used neurofilament-IR to discern the boundary of laminae II and III.
Comparison of TrkA and neurofilament staining at P1, as
Central projections of neurons
down-regulating TrkA
Both central and peripheral processes of DRG neurons
were robustly labeled by TrkA immunocytochemistry during embryonic and early postnatal development. The intense staining made it possible to examine developmental
changes in the innervation of the spinal cord by TrkA-IR
processes and, thus, identify the laminar target fields of
sensory neurons that down-regulate TrkA (Fig. 4). At P1,
TrkA-IR axons in the lumbar dorsal horn extended throughout the entire superficial dorsal horn but stopped abruptly
at the border of laminae II and III, with some staining in
lamina V. Occasional axons were visible projecting into the
lower laminae of the dorsal horn. These axons may be
collaterals projecting to lamina V or may indicate a small
population of TrkA-IR neurons with targets in laminae
III–IV. Because these axons are also visible at P21, they
probably do not arise from neurons down-regulating TrkA.
434
D.C. MOLLIVER AND W.D. SNIDER
TABLE 2. Neuronal Populations in NGF vs. Anti-NGF-Treated Mice1
Treatment
TrkA
NGF
Anti-NGF
CGRP
NGF
Anti-NGF
I-B4
NGF
Anti-NGF
% Labeled
43 6 4.20
41 6 2.44
38 6 3.80
36 6 2.90
39 6 1.04
39 6 1.99
1Data from the quantification of TrkA-IR, CGRP-IR, and I-B4 binding in nerve growth
factor (NGF) and anti-NGF-treated animals at postnatal day (P) 21. % Labeled,
percentage of positive neurons 6 S.E.M. There was no significant difference in the
distribution of TrkA, CGRP, or I-B4 between NGF and anti-NGF-treated mice (TrkA:
P 5 0.56; CGRP: P 5 0.36; I-B4: P 5 0.65). NGF, nerve growth factor; CGRP, calcitonin
gene-related peptide; I-B4, the lectin I-B4 from Bandeiraeia simplicifolia.
shown in Figure 4, indicates that the major band of intense
TrkA-IR in lamina II stops abruptly at the border of
laminae II and III.
CGRP-IR and p75-IR were examined in the spinal cord
to distinguish axons that express TrkA in the adult from
those expressing TrkA only during development (Fig. 4).
The adult pattern of CGRP-IR in dorsal horn laminae I,
IIo, and V was clearly established by P1 (not shown) and
did not extend into lamina IIi, as seen with TrkA staining
at that age. At P1, p75 staining revealed intensely labeled
afferents in laminae I, IIo, and III–IV (Fig. 4). However,
consistent with our hypothesis that the subset of small
neurons lacking p75 is identical to the I-B4-binding neuronal population, little or no p75 staining was seen in lamina
IIi, the primary target field of I-B4-binding neurons.
Comparison of p75 and TrkA staining in the dorsal horn
provides additional evidence that the majority of TrkA-IR
afferents do not penetrate ventral to lamina IIi at P1.
Furthermore, analysis of CGRP (not shown) and p75
staining in the dorsal horn indicated that TrkA-IR afferents projecting to lamina IIi at P1 contain neither CGRP
nor p75.
Perturbation of NGF levels does
not alter down-regulation of TrkA
To determine whether NGF is involved in the downregulation of TrkA expression, mice that had been injected
with either NGF or anti-NGF from P3 to P14 were
examined at P21 for changes in the distribution of TrkA-IR
in the DRG and dorsal horn. CGRP-IR and IB-4 binding
were also examined in these animals to evaluate the effect
of treatment on different subsets of neurons that express
TrkA early in postnatal life. The treatments, as expected,
had significant effects on the size of TrkA-IR neurons. The
average TrkA-IR neuronal area in NGF-treated animals
was 242.9 µm2 compared with 194.1 µm2 in anti-NGFtreated animals, a significant difference (P , 0.001; Students t-test). Size-frequency histograms revealed a shift to
larger areas of TrkA-IR neurons from animals treated with
NGF compared with anti-NGF (not shown). Thus, perturbation of NGF supplies had a significant biological effect
on DRG neurons in treated animals.
Quantification of the percentage of TrkA-IR DRG neurons in NGF- and anti-NGF-treated animals showed no
significant difference in the extent of TrkA staining between treatment groups (see Table 2), indicating that
manipulation of postnatal NGF levels does not alter the
extent of TrkA down-regulation. Likewise, the percentage
of neurons staining for CGRP or IB-4 binding was not
affected (Fig. 5). Furthermore, examination of the patterns
of staining in the dorsal horn for TrkA, CGRP, and I-B4
revealed that the laminar organization of central projections to the superficial dorsal horn was not altered by
systemic injections of NGF or anti-NGF (not shown).
Although we did not find an effect of NGF on any of the
parameters examined, we cannot absolutely exclude the
possibility of a transient effect of NGF on TrkA expression
that disappeared between P14 and P21.
DISCUSSION
Developmental regulation
of TrkA expression
Previous studies have demonstrated that NGF and TrkA
null mutant mice lose approximately 80% of the normal
complement of DRG neurons (Crowley et al., 1994; Smeyne
et al., 1994; Silos-Santiago et al., 1995). Thus, sensory
neuron loss in these animals is far more extensive than the
40% of DRG neurons that normally express TrkA in
maturity (Verge et al., 1992; Averill et al., 1995; Molliver et
al., 1995). We show that this apparent discrepancy is due
to the developmental down-regulation of TrkA expression.
Approximately 80% of DRG neurons express TrkA between
E15 and P1. The number of TrkA1 neurons diminishes
gradually, dropping to adult levels during the first 3 weeks
of postnatal life. Thus, our results suggest that 50% of
TrkA1 neurons down-regulate TrkA during postnatal development. The reduction in the percentage of TrkA-expressing neurons occurs for the most part well after P2, the age
at which DRG neurons lose their dependence upon NGF
for survival (Lewin et al., 1992), indicating that the TrkA
population is reduced through the down-regulation of
TrkA rather than through selective cell death. The population of neurons that down-regulates TrkA consists of small
neurons that bind the lectin I-B4, do not express CGRP or
p75, and project to lamina IIi of the dorsal horn. This
conclusion is supported by the progressive loss of TrkA-IR
from afferent axons in lamina IIi between P1 and P21.
Our results should be compared with previous in situ
hybridization studies of TrkA expression in the DRG
during development that yielded conflicting results. Several investigators have suggested, based on visual analysis, that most or all DRG neurons express TrkA during
early embryonic life (Ernfors et al., 1992; Schecterson and
Bothwell, 1992). However, percentages were not computed
in these studies. When we examined the percentage of
DRG neurons expressing TrkA during development by
using in situ hybridization in paraffin-embedded tissues
and excluded cells with low grain counts, we found that a
lower percentage (40–45%) of neurons expressed TrkA
than was reported in these earlier studies (Mu et al.,
1993). Our principal goal in that study was to achieve
optimal morphology in order to examine the relative sizes
of neurons expressing the different Trks. Paraffin-embedded tissue, as we pointed out, is thought to exhibit a
weaker signal in RNA hybridization studies than freshfrozen tissue (Tecott et al., 1987), and we considered it
plausible that we had failed to detect many TrkAexpressing neurons. We concluded that the determination
of absolute numbers and percentages of cells expressing
TrkA in embryonic life would require further investigation
(Mu et al., 1993). Antibodies against TrkA are now available, and we consider immunocytochemical analysis to be
more accurate in the quantification of embryonic neurons
DOWN-REGULATION OF TrkA BY DRG NEURONS
435
Fig. 5. The availability of nerve growth factor (NGF) does not
modulate the down-regulation of TrkA or neurochemical phenotype.
Representative sections of lumbar DRGs taken from P21 mice treated
with NGF or anti-NGF and reacted for TrkA immunocytochemistry or
lectin histochemistry. There was no apparent change in the percentages of TrkA or I-B4-binding neurons as a result of treatment. Arrows
indicate representative I-B41 neurons, and arrowheads indicate several large negative neurons. Scale bars 5 40 µm.
than autoradiographic in situ hybridization, because most
neurons in the embryonic DRG are very small and have
little cytoplasm, making it difficult to assign silver grains
to specific cells. In contrast, immunocytochemical staining
for TrkA filled positive cell bodies, allowing a more definitive identification of TrkA1 neurons. For these reasons, we
chose to use immunocytochemistry for the purpose of
quantification.
An important implication of these results is that virtually all neurons lost in the NGF and trkA null mutant mice
express TrkA during development. Thus, the expression of
TrkA by 80% of DRG neurons at P1 corresponds to the 80%
cell loss in the trkA(2/2) mouse. Furthermore, both major
populations of small neurons expressing TrkA during
development, CGRP-IR neurons and I-B4-binding neurons, are lost in the trkA null mutant (Silos-Santiago et al.,
1995). One might have expected that sensory neuron loss
in the trkA null mutant would have included more than
the TrkA population because of the potential for TrkA
neurons to provide a local source of neurotrophins within
the DRG. Both NT-3 and BDNF are widely expressed
within the ganglion during development (Ernfors et al.,
1992; Schecterson and Bothwell, 1992; Elkabes et al.,
1994), and TrkA-expressing neurons are known to express
BDNF in adulthood (Apfel et al., 1996). Apparently, however, NGF-unresponsive neurons do not require paracrine
support from TrkA-expressing neurons for survival during
embryogenesis.
It is not surprising that NGF supports the survival of
both I-B41 and CGRP-IR neuronal populations during
embryonic life, because both populations extensively innervate cutaneous tissues that express NGF as early as E11.5
(Davies et al., 1987; Silverman and Kruger, 1988; Wheeler
and Bothwell, 1992; White et al., 1996). What is surprising
is that I-B41 neurons cease expression of TrkA, whereas
CGRP-IR neurons continue to express TrkA throughout
life. Several other examples of developmental regulation of
neurotrophin receptor expression have been documented.
In the peripheral nervous system (PNS), sensory neurons
switch Trk expression and neurotrophin dependence between the times of neurogenesis and target innervation
(Buchman and Davies, 1993; Gaese et al., 1994; White et
al., 1996; Williams and Ebendal, 1995). This switch may
allow local sources of neurotrophins to support neurons
before they gain access to target-derived factors. In most
neuronal populations, expression of Trks persists into
adulthood, suggesting that neurotrophins continue to function in maturity (Merlio et al., 1992; Yan et al., 1993).
436
D.C. MOLLIVER AND W.D. SNIDER
However, expression of truncated TrkB and TrkC receptors increases after birth, and these noncatalytic isoforms
may restrict the signaling potential of neurotrophins in
adulthood in a region-specific manner (Ernfors et al., 1993;
Escandon et al., 1994; see also Biffo et al., 1995). Here, we
describe a novel form of developmental regulation of Trk
expression in which a population of neurotrophin-dependent neurons ceases expression of neurotrophin receptors
altogether after central and peripheral projections are
formed. As a result, many DRG neurons that are NGF
responsive during development are not subject to regulation by NGF in adulthood.
Functional implications
of TrkA down-regulation
The reduction in the percentage of TrkA1 neurons occurs
gradually over a period of 3 weeks after birth. The
persistence of widespread TrkA expression after the dependence on NGF for survival has passed suggests that there
may be a transient role for TrkA during early postnatal
life. Notably, the postnatal time course of TrkA downregulation coincides roughly with the critical period described by Mendell and colleagues during which injections
of NGF or anti-NGF can permanently alter the physiology
of DRG neurons with unmyelinated (C-fiber) and thinly
myelinated axons (Lewin et al., 1992; Lewin and Mendell,
1994). Neonatal NGF treatment recruits nonnociceptive
thermoreceptors and ‘‘mechanically insensitive’’ afferents
to a nociceptive phenotype, producing twice the control
number of C-fiber polymodal nociceptors (Lewin and Mendell, 1994). Conversely, treatment with anti-NGF altered
more than half of C-fiber polymodal nociceptors to an
abnormal low-threshold mechanoreceptor phenotype. The
gradual down-regulation of TrkA suggests that not just the
peptidergic neurons but also the I-B41 neurons are subject
to phenotypic regulation by NGF. Thus, during early
postnatal life, NGF may determine the stimulus specificity
of afferents that subsequently lose their NGF responsiveness.
Although differences in projection patterns and histochemical profiles between I-B4-binding neurons and CGRPexpressing neurons have been known for some time, there
has been debate about whether there are functional differences between these two populations (Silverman and
Kruger, 1988; Averill et al., 1995; Molliver et al., 1995; see
Hunt and Rossi, 1985; Hunt et al., 1992; Light, 1992).
Indeed, the action potential characteristics of C-fibers are
largely homogenous (Lawson et al., 1996), and at least 73%
of C-fibers in the rat DRG are polymodal nociceptors, too
large a population to be contained within the CGRPexpressing subset alone (Lynn and Carpenter, 1982). However, the ability to respond to NGF in the adult almost
certainly leads to functional distinctions between CGRP
and I-B4 populations. Up-regulation of expression of substance P and CGRP by NGF in the TrkA population is
likely to underlie central sensitization in hyperalgesia;
these peptides are also involved in the generation of the
peripheral inflammatory response (for reviews, see Basbaum and Levine, 1991; Hunt et al., 1992; Lewin and
Mendell, 1993). Furthermore, inflammation results in
increased levels of NGF both in the skin and in nerves
innervating the inflamed area (Woolf et al., 1994). Finally,
the ability to respond to certain noxious stimuli, such as
acid and the chemical irritant capsaicin, is regulated by
NGF (Bevan and Winter, 1995; McMahon et al., 1995),
suggesting a response mediated in the adult exclusively by
the TrkA population. Given these regulatory actions of
NGF, it is highly probable that the down-regulation of
TrkA by I-B4 neurons results in important differences in
the responses of I-B4-binding and peptidergic neurons to
certain classes of nociceptive stimuli.
It is important to acknowledge the possibility that I-B41
neurons are capable of responding to NGF through TrkA
expressed at levels below the limit of immunocytochemical
detection or via p75. However, several lines of evidence
suggest that these neurons are unresponsive to NGF.
First, in the adult, the distribution of TrkA mRNA (Verge
et al., 1992; McMahon et al., 1994; Wright and Snider,
1995) and the distribution of high-affinity NGF binding
sites (Verge et al., 1989) support the immunocytochemical
data in both percentages and cell sizes. Furthermore, in
situ hybridization analysis failed to detect mRNA for any
Trk family member or p75 in approximately 30% of adult
DRG neurons, a population that most likely corresponds to
the I-B4-binding neurons (McMahon et al., 1994; Wright
and Snider, 1995). The lack of p75-IR at P1 in many
TrkA-IR neurons or in lamina IIi supports the notion that
I-B4-binding neurons do not express p75 either in adulthood or during development. Finally, studies of the effects
of NGF on neuropeptide (Verge et al., 1995) and neurofilament (Verge et al., 1990) expression after injury in the
adult DRG found that administration of NGF produced
phenotypic changes only in neurons with high-affinity
NGF binding sites.
Developmental changes In TrkA
expression are not regulated by NGF
In view of the importance of NGF in the determination of
nociceptive phenotypes during postnatal development
(Lewin and Mendell, 1993; Mendell, 1995), it was of
interest to determine whether the postnatal downregulation of TrkA is also modulated by NGF. Therefore,
we examined the effects of postnatal NGF or anti-NGF
administration during the period of TrkA down-regulation
by DRG neurons. Perhaps surprisingly, the perturbation of
endogenous NGF supplies did not alter the extent of TrkA
down-regulation, indicating that the availability of NGF
does not determine which neurons down-regulate TrkA.
We also show that CGRP, I-B4, and p75, which discriminate between TrkA1 and TrkA2 neurons in the adult,
distinguish subsets of TrkA-IR neurons in neonates. The
essentially mature distribution of these markers at P1
indicates that their expression does not mature in concert
with TrkA expression. These results are in agreement with
a recent study indicating that the CGRP phenotype in rat
DRG neurons is determined before E15 (Robertson and
Hall, 1995). Furthermore, administration or depletion of
NGF did not alter the distribution of I-B4 binding or CGRP
IR, confirming previous findings that perturbation of
postnatal NGF supplies does not change the distribution of
CGRP (Lewin and Mendell, 1994). Thus, despite its importance in regulating physiological properties, NGF does not
appear to determine the neurochemical phenotype of TrkAexpressing neurons, as defined by NGF receptors, neuropeptides, and lectin binding sites.
Our results showing that NGF levels do not affect
expression of TrkA are consistent with other studies of
TrkA regulation in the peripheral nervous system, both at
early developmental stages and in adulthood. Studies of
the effects of NGF administration on sympathetic and
DOWN-REGULATION OF TrkA BY DRG NEURONS
DRG neurons both in vitro and in vivo provide evidence
that TrkA expression is not normally regulated by NGF
(Verge et al., 1992; Wyatt and Davies, 1993; Miller et al.,
1994), although NGF can partially rescue normal expression levels reduced by axotomy (Verge et al., 1992). Furthermore, in embryonic trigeminal ganglion neurons, the
marked increase in TrkA expression seen after target
innervation occurs normally in NGF null mutant mice
(Davies et al., 1995). Notably, however, several studies
have shown that NGF can transcriptionally up-regulate
TrkA in the central nervous system (CNS; Holtzman et al.,
1992; Gibbs and Pfaff, 1994). These findings suggest that
there are important differences in the regulation of TrkA
expression between the PNS and the CNS, although it is
not clear what the role of this differential regulation
might be.
CONCLUSIONS
Our findings have established that the great majority of
DRG neurons express TrkA throughout embryonic development. NGF/TrkA signaling acts at this time to support
neuronal survival (Crowley et al., 1994; Silos-Santiago et
al., 1995). Persistent widespread expression of TrkA in the
neonate after P2, when DRG neurons no longer require
NGF for survival, is consistent with the idea that NGF
regulates DRG neuronal phenotype during postnatal development (see Lewin and Mendell, 1993; Mendell, 1995).
However, the actions of NGF appear to be specific for
physiological aspects of phenotype, such as modality specificity, whereas neurochemical aspects of phenotype, such
as neuropeptide and neurotrophin receptor expression, are
not determined by NGF. Finally, in maturity, NGF/TrkA
signaling regulates functional properties of TrkA-expressing neurons, such as the levels of neuropeptide expression
and sensitivity to nociceptive stimuli (Lewin and Mendell,
1993). In contrast, I-B4 neurons are probably unresponsive to NGF in the adult. Thus, the down-regulation of
TrkA likely results in functional distinctions among subsets of small DRG neurons.
ACKNOWLEDGMENTS
We thank Dr. Louis F. Reichardt and Dr. Barbara
Hempstead for the generous gifts of their antibodies
against TrkA and p75 and Drs. Doug Wright and Fletcher
White for helpful input on the paper.
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