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The Journal of Neuroscience, November 15, 2001, 21(22):8873-8885
A Sensory Neuron Subpopulation with Unique Sequential Survival
Dependence on Nerve Growth Factor and Basic Fibroblast Growth Factor
during Development
Cristian G.
Acosta1,
Andrés R.
Fábrega1,
Daniel H.
Mascó2, and
Héctor S.
López1
1 Instituto de Investigación Médica
Mercedes y Martin Ferreyra, INIMEC-Consejo Nacional de Investigaciones
Científicas y Técnicas, (5000) Córdoba, Argentina,
and 2 Cátedra de Biología Celular, Facultad
Ciencias Exactas, Físicas y Naturales, Universidad Nacional de
Córdoba, (5000) Córdoba, Argentina
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ABSTRACT |
We characterized a subpopulation of dorsal root ganglion (DRG)
sensory neurons that were previously identified as preferential targets
of enkephalins. This group, termed P-neurons after their "pear"
shape, sequentially required nerve growth factor (NGF) and basic
fibroblast growth factor (bFGF) for survival in vitro during different developmental stages. Embryonic P-neurons required NGF, but not bFGF. NGF continued to promote their survival, although less potently, up to postnatal day 2 (P2). Conversely, at P5, they
needed bFGF but not NGF, with either factor having similar effects at
P2. This trophic switch was unique to that DRG neuronal group. In
addition, neither neurotrophin-3 (NT-3) nor brain-derived neurotrophic
factor influenced their survival during embryonic and postnatal stages,
respectively. The expression of NGF (Trk-A) and bFGF
(flg) receptors paralleled the switch in trophic
requirement. No single P-neuron appeared to coexpress both
Trk-A and flg. In contrast, all of them
coexpressed flg and substance P, providing a specific
marker of these cells. Immunosuppression of bFGF in newborn animals
greatly reduced their number, suggesting that the factor was required
in vivo. bFGF was present in the DRG and spinal cord, as
well as in skeletal muscle, the peripheral projection site of
P-neurons, as revealed by tracer DiIC183. The lack of requirement of NT-3 for survival and immunoreactivity for the neurofilament of 200 kDa distinguished them from muscle proprioceptors, suggesting that they are likely to be unmyelinated muscle fibers. Collectively, their properties indicate that P-neurons constitute a
distinct subpopulation of sensory neurons for which the function may be
modulated by enkephalins.
Key words:
dorsal root ganglion; NGF; bFGF; switch; survival; trophic dependence; rat; sensory neurons; subpopulation; muscle
innervation
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INTRODUCTION |
Several sensory neuron
subpopulations have been identified in the dorsal root ganglia (DRGs)
of mammals on the basis of anatomical, electrophysiological,
functional, cytochemical, and trophic criteria (Perl, 1992 ; McMahon et
al., 1994; Gilabert and McNaughton, 1997; Baudet et al., 2000). These
studies have greatly furthered our understanding of how sensory neurons
transmit specific sensory modalities, providing rich insights on
central issues such as the transmission of pain (Wood and Docherty,
1997; Caterina and Julius, 1999; Wood and Perl, 1999; Caterina et al.,
2000).
We have shown recently that activation of  -opioid receptors (DORs)
inhibited high-voltage-activated Ca2+
currents (HVACCs) in primary rat sensory neurons (Acosta and López, 1999), validating the cellular mechanism that is suspected to mediate the action of enkephalins and exogenous -opioid compounds on the transmission of normal and pain sensation (Dickenson et al.,
1987; Standifer et al., 1994). Acosta and López (1999) identified a neuronal subpopulation, referred to as P-neurons after their distinctive "pear-shaped" soma in vitro, in which the
frequency of DOR-mediated Ca2+ current
inhibition was remarkably high (75%) when compared with the other cell
types (18-35%). Those findings suggested that their function could be
preferentially modulated by enkephalins and prompted a characterization
of the properties of that subpopulation to clearly determine whether
they represent a distinct group. P-neurons are of medium size and
express substance P (SP), properties that have been useful for
classifying primary sensory neurons (Harper and Lawson, 1985; Cardenas
et al., 1995; Gilabert and McNaughton, 1997). However, further
refinement is needed because several subpopulations share such
properties. We hypothesize that P-neurons are likely to constitute a
previously uncharacterized group because both their unmistakable shape
in vitro and preferential coupling of DOR to their HVACCs
have been noted only recently (Acosta and López, 1999).
Taking advantage of their distinct morphology to unequivocally identify
P-neurons in culture, this study determined unique characteristics of
that cell group concerning trophic requirements during development,
anatomical projections, and cytochemical features. Most distinctively,
P-neurons sequentially required nerve growth factor (NGF) and basic
fibroblast growth factor (bFGF) for survival along embryonic and
postnatal stages. The need of specific trophic factors has provided a
powerful criterion to define distinct subpopulations of sensory neurons
and infer their possible physiological role (Levi-Montalcini and
Angeletti, 1968; Kucera et al., 1995; Lewin and Barde, 1996; Davies,
1997). In particular, the switch of neurotrophic requirements has been
recognized recently, underscoring a developmental complexity that goes
beyond the classical view of target-derived trophic support (Birren et
al., 1993; Molliver and Snider, 1997; Enokido et al., 1999; Baudet et
al., 2000; Enomoto et al., 2000). P-neurons also displayed a
recognizable cytochemical pattern and supplied sensory innervation to
skeletal muscle. These data strongly indicate that they constitute a
subpopulation of sensory neurons with distinctive developmental and
cellular characteristics, in addition to the previously reported high
sensitivity to enkephalins (Acosta and López, 1999).
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MATERIALS AND METHODS |
Cell culture. All procedures were in accordance with
the Guide for the Care and Use of Laboratory Animals of the
Society for Neuroscience. Sensory neurons from DRGs of rat embryos or
newborn rats (up to 7 d old) were isolated as described previously
(Acosta and López, 1999). Briefly, embryonic or postnatal DRGs
were enzymatically dissociated by incubating the tissue for 15-30 min
at 37°C with 0.125% trypsin and 0.625% collagenase, or 0.25%
trypsin and 1.25% collagenase, respectively. The enzymatic activity
was halted by adding 1 ml of Eagle minimal essential medium
supplemented with 10% fetal bovine serum (MEM10). After centrifugation
at 2000 rpm for 5 min, the pellet was resuspended in MEM10 containing
different trophic factors or the compound K252a at the concentrations
specified in Results. A final step of cell dissociation was performed
mechanically by passing the material through Pasteur pipettes of
increasingly smaller tip diameters. Approximately 70 µl of the cell
suspension were plated on coverslips coated with 0.25% collagen and
0.05% poly-D-lysine. Embryonic day 18 (E18)
cultures were grown on poly-D-lysine 1 mg/ml
alone (~300 ng/mm2) because the neurons
showed some tendency to detach from the mixed substrate. No differences
were found in the survival of postnatal neurons grown in any of those
substrates. Plating cell density was standardized, using a Neubauer
chamber, to ~104 cells per milliliter.
The coverslips were placed in an incubator (36°C, 5%
CO2) for 1-2 hr to allow for cell adhesion.
Then, MEM10 alone or supplemented with trophic factors or K252a was
added to the culture dishes containing the coverslips until reaching a
volume of ~2 ml. The cultures were kept in those conditions for 24 hr
to permit the stabilization of neuronal number and morphological phenotypes. Then, we performed the first neuronal counting, which was
defined as the initial condition in all survival assays. Immediately after the first counting, the MEM10 was completely replaced by defined
media N2 alone (control groups) or supplemented with trophic factors or
K252a. Half of the media was replaced every 48 hr thereafter, but a
fresh aliquot of trophic factors or K252a was added daily to the media.
The cultures consisted of a mixed population of neuronal and
non-neuronal cells. Two consecutive applications of 5-10
µM  -arabinocytofuranoside (at days 2 and
3) were used to eliminate dividing fibroblasts. In some cultures the
dissociated ganglia were passed throughout a 20% Percoll gradient by
centrifugation at 2500 rpm for 6-8 min to reduce the fibroblast
population. Penicillin-streptomycin (150 U/150 µg per milliliter,
respectively) was always included in the media. The following
definitions were used in this study: E0 was defined as the day of
mating, embryonic age was defined relative to E0, postnatal day 0 (P0)
was the day of birth, and postnatal age was defined with respect to P0.
Evaluation of neuronal survival. Neuronal survival was
assessed in cultures grown on etched grid coverslips from Bellco Glass (Vineland, NJ); alphanumeric coordinates on the coverslips allowed counting the cells within an identified region of the culture. A
minimum of 500 neurons were counted in each replication of the experiments. Taking advantage of their relatively small fractional contribution to the culture population, the survival of each individual P-neuron was followed and recorded. Survival was estimated as the
average percentage (±SEM) of cells remaining alive relative to their
number at the initial condition. The neurons were counted daily until
no P-neurons remained in the culture. The culture density at the time
of the first neuronal counting, estimated using the coverslip etched
grid, already reflected the survival-promoting effect of the different
factors at embryonic or postnatal stages. The survival of P-neurons
showed no correlation with the observed density. For example, similar
survival rates after 90 hr in vitro were obtained with bFGF,
despite a threefold difference in density after the first 24 hr in
culture. Dying cells were recognized on the basis of signs such as
pyknosis, shrinkage and fragmentation, membrane disruption, loss of
adhesion to the substratum or complete lysis. Because, as noted above,
the morphological integrity of each individual P-neuron was recorded,
the chances of mistakenly counting a live cell as dead, or vice versa,
were negligible. P-neurons never became round over the entire duration
of the experiment. Very few neurons identified as round at the time of
the first counting became pear-shaped by the time of the second
counting (48 hr after plating), keeping that phenotype thereafter, a
change that was unrelated to the presence of any of the trophic
factors. Those cells were counted as P-neurons and might have caused,
at the most, an overestimation of survival of 10%. The number of replications of the experiments is stated in Results or Figure legends.
The results were analyzed statistically using a two-way ANOVA test
[treatment × days in vitro (DIV)]. The percentage of
P-neuron survival was the dependent variable, and the repeated measures were provided by the survival percentage at each consecutive DIV. The
interaction between factors was evaluated with multiple comparison tests (post hoc analysis with the Tukey test;
p = 0.05). For E18, P2, and P5, the interaction values
were F(6,18) = 12.63, p < 0.0001; F(6,18) = 12.97, p < 0.0001; and
F(8,24) = 5.69, p < 0.0001, respectively.
Immunocytochemistry. The cells were withdrawn from the
incubator 24 hr after plating and washed for 5 min with PBS, then fixed with 4% paraformaldehyde-sucrose for 20 min at 37°C and washed again for 5 min with PBS. In experiments detecting endogenous proteins
(bFGF, substance P, neurofilament 200 kDa, -tubulin isoform III),
the cells were permeabilized with 0.2% Triton X-100 for 5 min, and
nonspecific binding sites were blocked with 5% bovine serum albumin
(BSA) for 1 hr. Surface antigen detection (Trk-A and
flg) did not use permeabilization. The coverslips were covered with primary antibodies, usually overnight at 4°C, in 1% BSA
at the dilutions indicated in Results. The primary antibodies were
subsequently washed three times with PBS, and the cells were incubated
at room temperature for 1 hr with the corresponding secondary
antibodies, labeled with fluorescein isothyocianate (FiTC) or
rhodamine. Finally, the cells were washed three times, and the
coverslips were mounted on glasses with FluorSave (Calbiochem, La
Jolla, CA). In double-staining experiments, the described procedure was
repeated after the incubation with the second primary antibody. Images
were acquired using an epifluorescence microscope (Axiovert TM-31;
Zeiss, Oberkochen, Germany) and recorded on optical disk.
A series of preliminary experiments established the appropriate
dilutions of primary antibodies as those allowing a clear distinction
from the background and causing no labeling of cell types (neurons or
other) that were known to be negative for the tested antigen. The
antibodies against Trk-A ( -Trk-A),
flg (-flg), and bFGF
(-bFGF) were tested with Western immunoblots
following standard protocols (Cáceres et al., 1992) and found to
specifically label the bands corresponding to the molecular weight of
their target proteins (see Figs. 5, bottom panel,
6B, 9A). The proteins for Western blotting
were obtained from tissues of E20, P1, or P5 rats (as indicated in the
corresponding figure legends), homogenized at 4°C in Ripa 1×.
Samples were centrifuged at 14,000 rpm for 15 min; the supernatant was
recovered, centrifuged again, and kept at  20°C. The proteins were
quantified using colorimetric methods. In all cases, 20 µg of
proteins were seeded in each lane. Although a 1:200 dilution of
-Trk-A labeled a single band corresponding to
Trk-A (~135 kDa), it only moderately labeled cultured
embryonic neurons at a 1:20 dilution, a result attributed to disruption of the trypsin-sensitive extracellular loop of Trk-A
recognized by the antibody after the enzymatic treatment required for
the DRG dissociation. We used -Trk-A at a 1:50 dilution
because it yielded the same number of labeled neurons as lower
dilutions, with a minor loss of fluorescence intensity. The
-flg produced satisfactory labeling in the range
1:400-1:1000.
Identification of targets innervated by P-neurons. To
determine the peripheral tissue innervated by P-neurons, we injected several possible targets with the lipophilic fluorescent neuronal tracer DiIC18(3) (DiI). A single peripheral site
was injected per animal. After being selectively taken up by neuronal
termini present in the region of application, this compound diffuses
along the cell membrane without crossing to other neurons (Honig and Hume, 1989). Its detection in cultured P-neurons was used to
qualitatively specify their peripheral target. To this end, the DRGs
were dissected and cultured on
poly-D-lysine-covered glass in low-volume
videomicroscopy chambers 3 d after the injections, a time at which
the dye presumably reached the somata of sensory neurons according to
its diffusion rate on lipid membranes in vivo (6-7 mm/d). A
very small volume of DiI at low concentration (2-5 µl of 1% DiI in
N,N-dimethylformamide) was injected into skin, subcutaneous
tissue, skeletal muscle, or joints with a Gilmont microsyringe. This
protocol minimized the possibility of dye diffusion to neighboring
tissues, so that the chances of assigning labeled P-neurons to a wrong
target were negligible. Because this injection protocol might miss a
sparse cutaneous innervation by P-neurons, we also evaluated this
possibility by injecting several subcutaneous sites of a rat with
larger volumes of DiI at high concentration (six injections of 10 µl
each, 3% DiI).
Treatment with antibody against bFGF in vivo. For
3 d, rats of age P2 received daily injections of 100 µl of
-bFGF diluted 1:10 in PBS. The control group was injected
similarly with PBS alone. At P6, DRG sensory neurons from test and
control groups were isolated, and all neurons present in the culture
were counted 24 hr after plating to evaluate the number of P-neurons in
treated animals and controls. The serum of PBS- or antibody-injected
animals was examined with the technique of dot immunoblots to assess
whether -bFGF reached the bloodstream after its
intraperitoneal injection. The serum was obtained 1 or 12 hr after an
injection of PBS or antibody. To separate the serum, the blood from
injected animals was collected in heparinized Eppendorf tubes (50 U/ml), incubated for 1 hr at 37°C, and kept overnight at 4°C. Then,
samples were centrifuged at 10,000 rpm for 10 min at 4°C, and the
supernatant was collected and centrifuged again. The new supernatant
(serum, ~100 µl) was kept at 20°C until used. To detect the
-bFGF, 10 µl dots containing 250 ng of pure human bFGF
were added to a nitrocellulose membrane, dried, and allowed to bind to
the paper for 1 hr. This resulted in a final concentration of bFGF of
~25 µg/ml, considered optimal for this technique. Then, the
nitrocellulose membranes were washed three times with TBS for 5 min and
blocked overnight at 4°C with a 5% milk suspension in TBS plus
0.05% Tween 20 (TBST). On the following day, the membranes were
incubated for 18 hr at 4°C with the sera or with -bFGF
diluted in TBST. Finally, the membranes were washed several times with
TBST and incubated with a biotinylated mouse anti-rabbit IgG (1:400)
for 1 hr at room temperature, which would then conjugate the rabbit
-bFGF present in the serum of injected animals. After
three washouts with TBST, the membranes were exposed to a
peroxidase-extravidin complex (1:2000) for 30 min at room temperature.
The Enhanced Chemiluminescence reaction kit was used to detect the
antibody. Exposure time was 20-30 sec.
Electrophysiology. Na+,
K+, and Ba2+
currents were recorded using the whole-cell configuration of the
patch-clamp technique (Hamill et al., 1981). Appropriate external and
pipette solutions for each current type were used as described
elsewhere (Acosta and López, 1999; Everill and Kocsis, 1999).
Antibodies and reagents. The neuronal tracer
DiIC18(3) was obtained from Molecular Probes
(Eugene, OR). Rat tail collagen type I was from Biomedical Technologies
(Stoughton, MA), and the enzymes used for tissue dissociation were from
Worthington (Lakewood, NJ). All trophic factors and K252a were from
Alomone Labs (Jerusalem, Israel). The rabbit (Rb) polyclonal anti-human
bFGF (affinity purified, raised in rabbit against a peptide
corresponding to amino acids 3-17 mapping within the N-terminal region
of human bFGF precursor), Rb polyclonal anti-rat FGF receptor 1 (affinity purified, raised in rabbit against a peptide corresponding to amino acids 808-822 mapping within the C-terminal region of human FGFR-1), and mouse monoclonal anti-TrkA (clone 6G10) were from Research
Diagnostics (Flanders, NJ). The monoclonal anti-bovine bFGF antibody
was from Chemicon (Temecula, CA). The antibody against substance P was
from Sera-Lab (commercialized by Accurate Chemical & Scientific
Corp.,Westbury, NY). The monoclonal mouse anti- tubulin isotype III
(clone SDL.3D10), polyclonal Rb anti-neurofilament 200, and all other
reagents were obtained from Sigma (St. Louis, MO). Stock solutions of
trophic factors were prepared in sterile water; aliquots were
maintained at 70°C for no more than 3 months. Stock solution of
K252a was prepared in cell culture-tested DMSO to yield a 1:2000
dilution of the organic solvent in the culture medium. No adverse
effects on neurons have been reported for this concentration of DMSO.
The photosensitive K252a was stored in a light-proof container at
20°C until used, and the dishes treated with this compound were
protected from light.
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RESULTS |
Subpopulation of P-neurons in culture
The distinct pear-shaped soma of the sensory neurons, which for
that reason we have designated P-neurons, allowed different independent
observers to readily and consistently identify them in DRG primary
cultures (Fig. 1A). The
other subpopulations were classified according to the diameter of their
round somata into small (<15 µm), medium (15-26 µm), and large
(>26 µm) neurons (Perl, 1992; Gilabert and McNaughton, 1997). By
size, P-neurons corresponded to the medium size group. Inspection of a
large number of cultures established from rats of increasing ages
(E18-P9), at 1 d intervals, showed that they contributed 4-7%
of the total population in vitro throughout that period,
with the larger percentages at postnatal stages. They might represent a
subset of a larger subpopulation, of which only a fraction assumes a
pear-shaped soma. It was this feature, however, that allowed their
unequivocal identification as a separate group. They exhibited
characteristics of healthy neurons such as the development of axons
after several days in vitro, as shown by labeling the axonal
protein class-III -tubulin (Fig. 1B) (Ferreira and
Caceres, 1992), and a normal expression of currents through several
voltage-dependent ion channels (Na+,
K+, and Ca2+)
(Fig. 1C). P-neurons were present already from the moment of cell plating, as well as in cultures grown on different substrates such
as laminin (10 µg/ml), fibronectin (15 µg/ml), and collagen (1 µg/mm2) (data not shown). Thus, their
peculiar shape, which exhibited little change in long-term cultures (up
to 14 d), did not appear to reflect either cell injury or abnormal
cell function.
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Figure 1.
Morphological phenotype and ion currents of
"pear"-shaped sensory neurons (P-neurons) in culture.
A, Typical P-neuron (arrow) in a DRG
culture from a rat embryo (E18). Phase contrast photograph after 8 DIV.
For comparison, round sensory neurons were included in the view field
(bottom left). B, Labeling of the soma
(sharp arrowhead) and axon (triangle
arrowhead) of a P-neuron with an antibody against the class III
isoform of -tubulin. Culture from a rat of age P5 after 2 DIV,
supplemented with bFGF (10 ng/ml). Scale bar indicates 20 µm in
A and 30 µm in B. C,
Expression of voltage-activated ion currents in three different
postnatal P-neurons. The left, middle,
and right panels show whole cell currents through
K+, Ca2+ plus
Na+, and Ca2+ channels,
respectively, recorded under voltage clamp. The permeant ions were
K+, Ca2+ plus
Na+, and Ba2+, and the current
was activated with voltage pulses to 0-10 mV from holding potentials
of 70 or 80 mV.
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Requirement of NGF and bFGF for the survival of P-neurons
during development
Distinct subpopulations of sensory neurons require for survival
during development one or more specific trophic factors
(Levi-Montalcini and Angeletti, 1968; Ruit et al., 1992; Kucera et al.,
1995; Lewin and Barde, 1996; Davies, 1997). To characterize the
properties of P-neurons, we examined whether they depended on specific
trophic factors during embryonic and early postnatal development by
evaluating their survival in cultures established from animals of
increasing ages (E18-P5), supplemented with NGF, bFGF, neurotrophin-3
(NT-3), or brain-derived neurotrophic factor (BDNF). The results were compared with the survival rates of round neurons in the same cultures.
The survival of P-neurons depended on NGF and bFGF, but not on NT-3 or
BDNF. The plots of Figure 2A illustrate the survival of P-neurons over 4-5 DIV in defined media alone (control), or supplemented with NGF or bFGF. The requirement of NGF and bFGF occurred
in a sequential fashion during development. At E18, NGF (50 ng/ml)
promoted the survival of 35.6 ± 0.5% of P-neurons at 4 DIV (Fig.
2A, left),
whereas none survived in defined media alone or with bFGF (10 ng/ml).
The difference between the survival observed with NGF and any of the
other treatments (control or bFGF) was statistically significant over
the entire period assayed (2-4 DIV). Conversely, at P5 63.5 ± 3.9 and 55.3 ± 5.3% of P-neurons survived with bFGF at 4 and 5 DIV, respectively, whereas the effect of NGF was barely above control
(Fig. 2A, right). The effect of bFGF was
statistically significant with respect to both NGF and control. NGF and
bFGF had similar effects at P2 (Fig. 2A,
middle), with ~50% of P-neurons surviving after 3 DIV in
the presence of either factor alone, an effect that differed
statistically from the virtual lack of survival observed in control
conditions. At P3, bFGF promoted considerably more survival than NGF
(44.2 ± 8.2 and 15.8 ± 2.2%, respectively, at 3 DIV). The
modest survival that was observed with nonsupplemented media after 3 DIV at P5 (25.5 ± 5.1%) most likely reflected the progressive
drop in trophic factor requirements of sensory neurons after birth
(Levi-Montalcini and Angeletti, 1968). The unmistakable morphology of
P-neurons, together with the alphanumeric-coded grid of the coverslips
onto which the cells were plated, allowed us to individually follow each P-neuron over several DIV, assuring that the observations corresponded to that single subpopulation of sensory neurons.
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Figure 2.
Time course of the survival of
P-neurons in cultures supplemented with NGF or bFGF from animals of
different ages. A, Average percentage (±SEM) of
surviving P-neurons as a function of DIV, relative to their number at 1 DIV (taken as 100%), in cultures established from rats of ages E18,
P2, and P5. The cultures were maintained in defined media alone
(squares) or defined media supplemented with 50 ng/ml
NGF (white circles) or 10 ng/ml bFGF (black
circles). Each plot contains data from three experiments. NGF
but not bFGF promoted the survival of E18 neurons (left
panel). The reversed result was obtained with P5 neurons
(right panel). At P2 (middle
panel), the factors had similar effects. Analysis of the
data at 2-5 DIV (ANOVA, and Tukey post hoc test;
p = 0.05) indicated highly significant
statistical differences (asterisks) between the NGF
treatment and bFGF treatment or control (E18), between
NGF or bFGF treatment and control (P2), and between bFGF
treatment and NGF or control (P5). B,
Dose-response curve of the average survival (±SD) of P-neurons from
P5 animals (estimated at 3 DIV) as a function of bFGF concentration
(n = 2). C, The survival-promoting
effect of NGF (50 ng/ml) on E20 P-neurons was completely blocked by
Trk-A kinase activity inhibitor K252a (50 nM) (n = 2). The bars
indicate the percentage of survival (±SD) after 4 DIV in defined media
alone (C) or supplemented with NGF
(N), K252a (K), or both
(N + K) at the concentrations
specified above. K252a prevents the effect of NGF in a statistically
significant way (see asterisk).
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The concentrations of NGF and bFGF used in these experiments were those
that optimally promote survival as shown elsewhere for NGF (Ruit et
al., 1992) and here for bFGF (Fig. 2B). In agreement with other reports in neurons, 5-10 ng/ml bFGF was the most effective concentration, with smaller and larger concentrations being ineffective and detrimental, respectively (Schmidt and Kater, 1993; Abe and Saito,
2000). The alkaloid K252a (50 nM), an inhibitor
of the tyrosine kinase pathway that is activated by the high-affinity receptor of NGF (Trk-A) (Koizumi et al., 1988), prevented
the effect of that neurotrophin on the survival of E20 P-neurons (Fig. 2C). Dose-response data showed that 50 nM K252a inhibits 80% of Trk-A kinase
activity, fully blocks the Trk-A-mediated differentiation of
PC12 cells, and has negligible effects on other tyrosine kinase receptors (Berg et al., 1992). Our results are thus consistent with NGF
acting through the signaling pathway involving Trk-A and
tyrosine kinase activity, and not through its low-affinity receptor p75
(Lewin and Barde, 1996) or other tyrosine kinase pathways. The effect
of NGF most likely reflected its well known ability to prevent
embryonic sensory neurons from entering programmed cell death (Vogel,
1993; Yao and Cooper, 1995). Postnatal P-neurons deprived of bFGF
underwent nuclear changes typically associated with apoptosis (data not shown).
Figure 3 (left) summarizes all
of our data on the extent and time course of the dependence of
P-neurons on NGF and bFGF as a function of the age of the animal at the
time of the isolation of sensory neurons (E18-P5). The data points
indicate the average percentage (±SEM) of surviving neurons after 3 DIV in control media alone or supplemented with NGF alone, bFGF alone,
or both factors. Both factors together promoted a survival rate similar to that achieved individually by the most effective factor at any given
age. The switchover of survival dependence, defined as the day at which
the survival-promoting effects of NGF and bFGF were similar, occurred
at P2. The gradual increase in survival rate over P3-P5 with any
factor alone or combined was similar to that observed in control
cultures (Fig. 3, left) and was assumed to reflect the
decrease in trophic factor requirements after birth. To show more
clearly the switch in trophic factor requirement, the background
survival that was obtained in the absence of factors was subtracted as
follows. In each experiment, the control values (no treatment) were
subtracted from the treatment values. Then, the data were averaged
(Fig. 3, right).
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Figure 3.
Switch in the requirement of P-neurons
from NGF to bFGF at different developmental stages.
Left, average percentage (±SEM) of survival of
P-neurons after 3 DIV, relative to their initial number, in cultures
established from animals of different embryonic and postnatal ages,
maintained in defined media alone (control, squares),
supplemented with NGF (50 ng/ml; white circles), bFGF
(10 ng/ml; black circles), or both NGF + bFGF (50 and 10 ng/ml, respectively; triangles). Right,
same data as in left, but subtracting in each experiment
the percentage of survival in control conditions (defined media alone)
to make the trophic switch clearer. The switch of trophic dependence
occurred at P2, defined as the day at which NGF and bFGF had similar
effects (arrow). The survival obtained with both factors
was not larger than that observed with the most effective factor
at a given age, although it appeared to approach the sum of NGF and
bFGF effects at E18 and P5. The survival-promoting effects of the
trophic factors were statistically significant, as indicated in Figure
2. For better visualization of the trophic switch, statistically
significant differences were not labeled with asterisks in these
summary plots.
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The sequential requirement of NGF and bFGF was unique
to P-neurons
Only P-neurons required NGF prenatally and bFGF postnatally. None
of the other sensory subpopulations (round small, medium, and large)
depended on those factors in a sequential fashion. However, NGF or bFGF
did affect the survival of embryonic or postnatal round sensory neurons
in the way illustrated in Figure
4A. We focused on the
survival of round neurons in cultures that were obtained from E18
and P5 animals because the switch in trophic dependence of P-neurons
was most obvious at those ages. Classifying E18 round neurons into
small, medium, and large groups was uncertain because their size in
culture changed considerably with trophic factors at that early stage,
and the size differences in the first two groups were subtle.
Therefore, E18 round neurons were lumped together into a single class
("round"). P5 round neurons were readily sorted into small, medium,
and large categories. As expected from the literature (Silos-Santiago
et al., 1995), NGF greatly enhanced, in a statistically significant
manner, the survival of round E18 neurons (70.1 ± 14.5% at 3 DIV) when compared with control cultures (14.3 ± 8.3% at 3 DIV)
(Fig. 4A, left). In contrast to its lack
of effect on postnatal P-neurons, NGF also enhanced the survival of P5
round neurons, with equal effectiveness among categories (between 76 and 84% at 3 DIV), although the increase in survival over the control
was smaller because of the expected drop in the requirement of trophic
factors of postnatal sensory neurons (Fig. 4A,
right) (see Ehrhard and Otten, 1994; Wewetzer et al., 1999).
bFGF promoted the survival of E18 round neurons but had no effect on
the survival of small, medium, or large P5 round neurons, as
illustrated in Figure 4A. In the presence of bFGF,
30.2 ± 9.7% of embryonic round neurons survived after 3 DIV,
compared with 14.3 ± 8.3% in control cultures, and ~55% of postnatal round neurons survived after 3 DIV regardless of the presence
of the factor in the culture. Thus, the pattern of bFGF effect on round
neurons clearly differed from that observed with P-neurons. NT-3 caused
some increase in the survival of E18 round neurons and small P5 round
neurons. The former was an expected result because NT-3 is essential
for the survival of round neurons that will supply proprioceptive
afferents to skeletal muscle spindles at ~E17-E18 (Ernfors et al.,
1994). BDNF appeared toxic to large P5 round neurons, in agreement with
other reports (Koh et al., 1995) and references therein (Fig.
4B, right). Neither NT-3 (10 ng/ml) nor
BDNF (50 ng/ml) had any observable effect on the survival of embryonic
or postnatal P-neurons (Fig. 4B).
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Figure 4.
Differential effects of NGF, bFGF, NT-3,
and BDNF on the survival of round and P-neurons of the DRG in culture.
The experiments were performed at developmental stages at which the
differences in the requirements for NGF or bFGF by P-neurons are most
salient (E18 and P5) and show that the
switch of survival requirements from NGF to bFGF was restricted to
P-neurons. A, Average percentage (±SEM) survival of E18
and P5 round neurons after 3 DIV (open bars), relative
to their number at 1 DIV (taken as 100%), in the presence of 50 ng/ml
NGF or 10 ng/ml bFGF. Round neurons were lumped into a single group
(R) at E18 and subclassified into small
(S; <15 µm), medium (M; 15-26 µm),
and large (L; >26 µm) neurons at P5. P-neuron data
were included for comparison (hatched bars). The control
group received no trophic factors. NGF dramatically enhanced the
survival of E18 round and P-neurons (3 replications). The effect of NGF
on P5 round neurons of any size group was mild, but larger than that
observed on P5 P-neurons. bFGF promoted the survival of E18 round
neurons, but not of P5 round neurons. B, Percentage
survival of E18 and P5 P-neurons and round neurons after 3 DIV with
NT-3 (10 ng/ml) or BDNF (50 ng/ml). NT-3 had no effect on the survival
of E18 or P5 P-neurons, and slightly enhanced that of round neurons
(E18 and small P5). BDNF was rather detrimental to P-neurons and large
P5 round neurons (2 replications). Statistically significant
differences between a given treatment and the control are labeled with
* (P-neurons data) or ** (round neurons data) (ANOVA; Tukey post
hoc test; p = 0.05).
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Expression of NGF and bFGF receptors in embryonic and
postnatal P-neurons
We studied by immunofluorescence microscopy the expression in
P-neurons of Trk-A and the high-affinity bFGF receptor
(flg or FGF-receptor 1) before, during, and after the
switch in trophic requirements. These experiments addressed the
important point that if Trk-A and flg are to
mediate the survival-promoting effect of NGF and bFGF, respectively,
then they must be present in the membrane of P-neurons at the
appropriate times during development. Figure
5 illustrates representative examples of
the expression of Trk-A and flg immunoreactivity
in P-neurons, as observed at the ages E18, P2, and P6. In all cases,
the immunolabeling experiments were performed after 1 DIV. The
expression of Trk-A or flg qualitatively mirrored
the change in the requirements for their corresponding ligands (three
experimental replications). Thus, during the embryonic period in which
only NGF enhanced survival (E18-E20), all P-neurons (n = 21 cells) expressed Trk-A, but none expressed
flg (Fig. 5A,B). At P2,
the age at which NGF and bFGF had similar survival promoting effects, a
group of P-neurons expressed Trk-A (n = 12 cells), whereas a different set expressed flg
(n = 14 cells) (Fig.
5C-F). We never observed coexpression of
Trk-A and flg in any given neuron or lack of
expression of either receptor. However, we cannot rule out the
possibility that lower levels of expression of those receptors, although functional, could be below the detection sensitivity of our
immunolabeling technique. In fact, some degree of undetected coexpression might explain, at least partially, why the survival that
was obtained with both NGF and bFGF during P2 did not approached the
sum of the survivals obtained with those factors separately. At P5-P6,
a stage at which only bFGF promoted survival, we detected expression of
flg but not Trk-A (n = 23 cells,
from four experiments) (Fig. 5G,H). The
mouse -Trk-A and rabbit -flg were used at
1:50 and 1:500 dilutions, respectively.
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Figure 5.
Expression of Trk-A and
flg in P-neurons at different ages. Detection of
Trk-A-like (left column) and
flg-like (right column) immunoreactivity
as revealed with mouse -Trk-A (1:50) and rabbit
-flg (1:500) antibodies, respectively, in cultures
established from animals of ages shown on the left. Each
row corresponds to a same view field but with the filter
selected for FiTC (left) or rhodamine
(right), which labeled the secondary antibodies
(arrowheads indicate P-neurons). All experiments were
performed 24 hr after plating the neurons. E18 P-neurons were
immunoreactive for Trk-A (A) but
not flg (B). At the age
corresponding to the switchover of trophic requirement
(P2), a set of P-neurons displayed Trk-A
immunoreactivity (C) and a different set
displayed flg immunoreactivity
(F). No P-neurons were immunoreactive for both or
for none of the proteins (D, E). P6
P-neurons were immunoreactive for flg (H) but not
Trk-A (G). Scale bar (shown in
G): A, B, 18 µm;
C, D, 20 µm; E,
F, 15 µm; and G, H, 30 µm. Bottom, Western blots assaying the expression of
Trk-A (left) and flg
(right) in different tissues with the antibodies used
for neuronal immunolabeling. The lanes correspond to
spinal cord (1), DRG (2),
skeletal muscle (3), kidney
(4), and brain (5) in
left panel, and to DRG (1), lung
(2), skeletal muscle (3),
spinal cord (4), heart (5),
and brain (6) in right panel (20 µg of tissue proteins
seeded in each lane). The monoclonal
-Trk-A labeled a single band that corresponded to the
molecular weight (MW) of Trk-A (~140 kDa). The rabbit
-flg labeled a single band corresponding to the
estimated MW of FGFR-1 (~90 kDa) in all tissues, except in skeletal
muscle. The extra bands in skeletal muscle presumably
reflected the antibody reaction with the C-terminal epitope of
truncated forms of FGFR-1 expressed in that tissue (Templeton and
Hauschka, 1992).
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Expression of Trk-A or flg was not restricted to
P-neurons. A number of studies have shown expression of the
Trk-A mRNA and the protein in several types of rat sensory
neurons (Mu et al., 1993; McMahon et al., 1994; White et al., 1996).
Similarly, these neurons have been reported to express flg
(Grothe and Wewetzer, 1996). In accord with those reports, we found
Trk-A immunoreactivity in round embryonic neurons and small
and medium round postnatal neurons, and we found flg
immunoreactivity in medium and large cells (Fig.
5B,G). Coexpression of both
receptors appeared very infrequently in round neurons (an example is
shown in Fig. 5G,H). No switch in the
expression from Trk-A to flg was observed in round neurons, although these experiments could not completely rule out
that possibility.
Western blot data indicated that the antibodies specifically
reacted with their targets. Thus, the monoclonal -Trk-A
labeled a single band of ~140 kDa in spinal cord, DRG, and whole
brain and failed to react with proteins from muscle and kidney (Fig. 5,
bottom left), in agreement with the expression of Trk-A in those tissues (Lomen-Hoerth and Shooter, 1995; Wheeler et al., 1998).
The polyclonal -flg labeled a single band of ~90 kDa in DRG, lung,
spinal cord, heart, and brain (Fig. 5, bottom right), as
expected from the literature (Perderiset et al., 1992; Hughes and Hall,
1993; Sugi et al., 1995). The presence of more than one band in
skeletal muscle most probably reflected the antibody reaction with the
C-terminal epitope of truncated forms of FGFR-1 that was present in
that tissue (Templeton and Hauschka, 1992).
Requirement of bFGF by postnatal P-neurons
in vivo
To investigate whether the survival promoting effect of bFGF on
P-neurons that was observed in vitro also occurred in
vivo, postnatal rats received daily intraperitoneal injections of
an antibody against bFGF (-bFGF) during the days
P2-P5 aimed at sequestering the factor that P-neurons needed from P2
onward. This strategy has been successfully used for studying the
dependence of sensory neurons on NGF and other trophic factors (Carroll
et al., 1992; Ruit et al., 1992) or NT-3 (Oakley et al., 1995; Zhou and
Rush, 1995a; Lefcort et al., 1996). We compared the number of P-neurons
in DRG cultures obtained from injected animals with their number in
control cultures obtained from PBS-injected animals. If P-neurons did
require bFGF in vivo, then there should be a deficit of
those cells in cultures of antibody-injected animals. In agreement with
this expectation, there was a substantial reduction in the number of
P-neurons found in cultures obtained from antibody-injected animals,
with a 2.4-fold reduction of their total number (Fig. 6A). The detection by
immunoblot of circulating antibody shortly after its intraperitoneal
injection gave support to our assumption that it reached the peripheral
nervous system through the bloodstream (Fig. 6B). In
addition, in cultures of antibody-injected animals, there was a marked
reduction in the number of fibroblasts and glial cells, which were
ubiquitous in cultures from control or PBS-injected animals. This
further indicated that the circulating antibody effectively neutralized
the endogenous bFGF, because those cell types require that factor for
survival (Vescovi et al., 1993). Representative photographs of cultures
from control and Ab-injected animals are shown in Figure 6,
C and D. In addition to the reduced number of
P-neurons and non-neuronal cells, the culture of Ab-treated animals
differed from the typical cultures in several respects. The neurons had
a grainy profile and tended to form clusters. In agreement with the
lack of effect of bFGF on postnatal round neurons (Fig.
4A), their number did not seem to be significantly
reduced in antibody-injected animals. However, they had thinner nerve
fibers at the time of the DRG isolation, and therefore we cannot
exclude more subtle effects perhaps attributable to the loss of
non-neuronal cells (Jessen and Mirsky, 1999) and references
therein.
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Figure 6.
Effect in vivo of an antibody
against -bFGF on the number of P-neurons during the
early postnatal period. A, The proportion of P-neurons
in culture was 2.4-fold smaller in -bFGF-injected
animals (1.3%) as compared with controls (3.17%). Number of P-neurons
and round neurons counted were 45 of 3458 and 78 of 2463, respectively.
B, Immunodot blots showing that the
-bFGF can be found in rat serum 1 hr
(first row), but not 12 hr (second
row), after its intraperitoneal administration. Each
dot illustrates the result of treating a substrate of
pure human bFGF (250 ng in 10 µl) with -bFGF (1:50)
as positive control, or serum (without dilution) from animals treated
with -bFGF for 3 d plus one extra dosis 1 hr
before bleeding (S-Ab), PBS for 3 d plus one dosis
of -bFGF 1 hr before bleeding
(S-PBS-Ab), PBS for 3 d (PBS), or
from untreated control animals (NT). The
immunodot blot reaction was revealed with a secondary antibody. The
reaction reliably detected circulating -bFGF 1 hr
after the last antibody injection but not after 12 hr. The antibody
-bFGF did not cross-react with mNGF 7S (25 ng/µl)
(third row). C, D,
Representative Nomarski-interference photographs of cultures obtained
from rats that received one injection per day of PBS (100 µl)
(C) or -bFGF (100 µl, 1:10)
(D) during postnatal days 2-4. The number of
non-neuronal cells that depend on the supply of bFGF (glia and
fibroblasts) was greatly reduced after the antibody treatment.
White arrowhead, P-neuron; black
arrowheads, round neuron; white angles,
non-neuronal cells (fibroblasts or glia). At a 1:50 dilution, the
antibody injection produced a minor reduction in the number of
P-neurons. Scale bar, 50 µm. Cultures were plated in video microscopy
chambers and kept in MEM10 for 24 hr, and the media was replaced by PBS
for image acquisition 24 hr later. Throughout the experiment, the
cultures were maintained at 37°C and supplemented with 10 ng/ml bFGF
to support the survival of P-neurons.
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Innervation of peripheral tissues by P-neurons
To further characterize the P-neuron sensory subpopulation, we
studied to which peripheral tissues they provide afferent innervation. Several potential targets, such as skin, subcutaneous tissue, skeletal
muscle, and joints were injected in vivo with the intensely fluorescent lipophilic neuroanatomical tracer DiI, which is selectively taken up by nerve cell terminals and diffuses centrally along the
neuronal membranes (Schroeder and McCleskey, 1993). If P-neurons peripheral terminals selectively innervate any of the above targets, then the tracer should eventually reach the somata of P-neurons only
after the injection of the specific target(s).
Inspection of DRG cultures prepared 2-3 d after DiI administration
into a single peripheral site revealed labeled P-neurons only after
skeletal muscle dye injections. Of 20 labeled neurons found in that
case, seven were P-neurons (of an estimated population of 100 P-neurons), a reasonable yield of our minimum dye injection protocol
(see Materials and Methods) (Fig.
7A). Single injections into
skin (n = 6), subcutaneous tissue (n = 13), or joints (n = 10) only labeled round neurons.
Injections of a higher concentration of DiI into a large fraction of
the subcutaneous tissue (~30-40%) confirmed the lack of innervation
of that tissue by P-neurons (Fig. 7B). As expected, that dye
application yielded a larger number of labeled round neurons (Fig.
7C). In these experiments, the phenotype of P-neurons was
accurately identified under phase contrast before obtaining the images,
as illustrated in Figure 7.
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Figure 7.
Peripheral target innervated by P-neurons as
revealed by the retrograde transport of the lipophilic fluorescent
neuronal tracer DiI (1%) injected into several tissues of newborn rats
(P2). Cultures of DRG were examined for fluorescently labeled neurons
(arrowheads) 3-4 d after a single injection of DiI.
A, Phase contrast Nomarski photograph
(left) and patchy pattern of DiI labeling
(right) of the same P-neuron after the injection of a
single 2 µl of DiI in the main muscle mass of both hindlegs.
B, Dye injections into skin or subcutaneous targets
never labeled P-neurons. Phase contrast photographs of two P-neurons
(left, arrowheads) lacking DiI labeling
(right, arrows) after large DiI
injections at high concentration into several cutaneous sites of a rat.
C, Round sensory neurons from the same animal in B labeled
with DiI. Round labeled neurons (arrowheads) were also
observed after injecting a very small volume (3 µl) of a low
concentration of DiI into a single cutaneous site (data not shown).
D, Photographs of the same view field after the
treatment with antibodies against the -tubulin isotype III
(top) and the 200 kDa neurofilament protein
(bottom). P-neurons showed no immunoreactivity for the
200 kDa neurofilament (arrows), whereas some round
neurons were labeled. All neurons were positive for the -tubulin
isotype III, which clearly delineated the soma shape. Scale bars:
A, D, 20 µm; B, 18 µm;
C, 15 µm. The data were replicated three to four
times.
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Sensory innervation of skeletal muscle includes large myelinated
fast-conducting and unmyelinated slow-conducting fibers (Zhou and Rush,
1995b). Our data suggest that P-neurons correspond to the second group.
First, P-neurons showed no immunoreactivity for 200 kDa protein subunit
of neurofilaments, an exclusive marker of large myelinated
fast-conducting sensory neurons (Lawson et al., 1984) (Fig.
7D). As expected, the antibody clearly labeled the large
round neurons present in the same cultures. Second, the survival of
embryonic fast-conducting myelinated muscle afferents (proprioceptors
innervating muscle spindles) both in vivo and in
vitro require NT-3 (Hory-Lee et al., 1993; Oakley et al., 1995, 1997; Wright et al., 1997), a neurotrophin that had no effect on the
survival of P-neurons (Fig. 4B). Lastly, the size of
proprioceptors is among the largest of sensory neurons, whereas
P-neurons fall into the medium size group.
Coexpression of substance P and other markers in P-neurons
We have previously shown that the somata of postnatal neurite-free
P-neurons in culture display immunoreactivity for SP (Acosta and
López, 1999), a peptide that has been involved in the
transmission of nociceptive stimuli (Holland and Goldstein, 1990).
Here, we have confirmed and extended those results. Every single
postnatal P-neuron present in the culture was immunoreactive for SP
(n = 50, replicated at least in eight assays). As is
well known, SP immunoreactivity can be found in other sensory neuron
subpopulations, particularly in small round neurons (O'Brien et al.,
1989; Nothias et al., 1993). Unlike P-neurons, however, only a fraction
of those subpopulations expressed the peptide (data not shown).
Interestingly, two replications of double-labeling experiments showed
that whenever embryonic P-neurons were immunoreactive for
Trk-A, they lacked SP immunoreactivity (n = 12) (Fig.
8A,B).
In contrast, SP and flg strictly coexpressed in postnatal
P-neurons (n = 8) (Fig. 8C,D).
Thus, in addition to their unique requirement of NGF and bFGF, this
one-to-one correlation between flg and SP expression was a
unique marker further typifying these cells. Of the round neurons that
expressed SP, only a minor fraction (~7%; 2 of 29 neurons) showed
immunoreactivity for flg (Fig.
8E,F). Conversely, round
neurons expressing flg lacked immunoreactivity for SP (data not shown).
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Figure 8.
Substance P expression strictly paralleled the
expression of flg in cultured P-neurons, as illustrated
by double-labeling immunofluorescence experiments. Left
column shows examples of Trk-A (top
row) or flg (other rows)
immunoreactive neurons, and right column shows examples
of substance P (SP) immunoreactive neurons. The right
and left panels in each row show the same
view field. At E18 (top row), all P-neurons
(arrowheads) expressed Trk-A
(A) and none expressed SP immunoreactivity
(B) (n = 12). A large number
of round neurons were also immunoreactive for Trk-A
(data not shown). At this stage there was no detectable immunolabeling
for flg in P-neurons. At P2 (middle row),
the P-neurons that were immunoreactive for flg
(C) were also immunoreactive for SP
(D) (n = 8). In contrast,
those negative for flg were also negative for SP (data
not shown). Only flg-positive P-neurons contained
substance P. In non-P-neurons, coexpression of flg and
SP was infrequent (~7%). An example from P5 animals is shown in
E and F. Arrowheads
indicate round neuron somata that coexpressed flg and
SP, out of 29 round neurons present in the field. Arrows
indicate axons that showed SP, but not flg,
immunoreactivity. In all cases, we used a rat monoclonal antibody
against SP at a dilution of 1:20. Scale bars: A,
B, 15 µm; C, D, 20 µm;
E, F, 50 µm.
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Potential sources of bFGF
Like other primary sensory neurons, the cell bodies of P-neurons
are located within the DRG itself; their pseudounipolar axons project
centrally and peripherally to the spinal cord and, as shown before, to
skeletal muscle, respectively. Assessing whether the sites with which
P-neuron somas or fibers anatomically relate (DRG, spinal cord,
skeletal muscle) are potential sources of bFGF is important to
hypothesize on how the switch in trophic dependence might correlate with developmental events, such as the establishment of
innervation. The presence of bFGF was determined in the relevant peripheral tissues using Western blots at different developmental stages (E20, P1, and P5) (Fig.
9A). bFGF was present in
skeletal muscle as well as in the spinal cord and the DRG at all ages
tested. Interestingly, the highest expression of the factor in muscle coincided with the time at which all P-neurons became bFGF-dependent. A
more indirect evidence of the bFGF presence in the tissues examined above was obtained from a set of separate immunocytochemical
experiments that were performed during the postnatal stage at which
that factor promoted survival (P3-P6). The representative data of
Figure 9 show that neurons and glial cells of the DRG (Fig.
9B,D) and spinal cord (Fig.
9E), as well as skeletal muscle fibers (Fig. 9C),
were immunoreactive for bFGF. In sharp contrast, P-neuron themselves or
fibroblasts completely lacked immunolabeling (Fig.
9B,D). These data indicate that
P-neurons may potentially obtain bFGF from several types of cells. Our
data show that the factor is available in sites reachable by P-neurons
at the time they become bFGF-dependent.
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Figure 9.
Expression of bFGF immunoreactivity at sites with
which P-neurons relate anatomically, revealing potential sources of
bFGF at the time it was required for P-neuron survival. All tissues
were isolated from animals of age P3-P5. A, Western
blot showing the expression pattern of bFGF in skeletal muscle
(SM), spinal cord (SC), and
DRG at ages indicated under the corresponding
lanes. Labeled bands of ~18 kDa
(arrow), the MW of bFGF, indicate expression of that
protein in all of those tissues. The locations of 22 and 78 kDa MW
markers in a gel run in parallel are indicated by
arrowheads and corresponding MW numbers.
B, DRG cultured cells. Some round neurons expressed bFGF
immunoreactivity. In contrast, P-neurons themselves were always
negative (arrow). C, At P3, 90% of
skeletal muscle fibers, the presumptive innervation target of P-neurons
according to our data (Fig. 7), were clearly positive for bFGF. A
representative example is shown here. D, Glial cells
(presumptive type-2 astrocytes, identified with double staining with an
antibody against -GFAP) (data not shown) from the same ganglia used
in A were also immunolabeled for bFGF. E,
Photograph of a bFGF-positive neuron from the spinal cord. Experiments
were performed using a mouse monoclonal antibody
(-bFGF) at 1:200 dilution. Scale bars:
B, 40 µm; C, 20 µm;
D, 100 µm; E, 15 µm.
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DISCUSSION |
Distinguishing a group of sensory neurons as a distinct,
homogenous subpopulation usually requires determining several
characteristics of the cells, with their specific requirements of
trophic factors during development being a major defining criterion
(Cameron et al., 1992; Perl, 1992; Snider, 1994; Lewin and Barde,
1996). We report a set of properties of the cell group that we have
referred to as P-neurons, which, together with our previous study
(Acosta and López, 1999), indicate that they constitute a
distinct subpopulation of the rat DRG. A defining feature of P-neurons
was their requirement of NGF prenatally and bFGF postnatally for
survival during development, because no other type of sensory neurons
studied here or elsewhere depended on that specific sequence of trophic
factors (Vescovi et al., 1993; Grothe and Wewetzer, 1996; Ogilvie et
al., 2000). Originally reported by Buchman and Davies (1993), this
pattern of trophic dependence has been increasingly recognized in
sensory (Molliver et al., 1997) and other neuronal types (Davies,
1994). For instance, mouse trigeminal neurons prenatally switch from BDNF-NT-3 to NGF (Buchman and Davies, 1993), and IB4-positive mouse
sensory neurons switch from NGF to GDNF early after birth (Molliver et
al., 1997). The need of more than a trophic factor, either sequentially
or simultaneously, has also been strongly implied by work done in null
mutants for neurotrophins or their receptors (White et al., 1996; Liebl
et al., 1997). P-neurons specifically express Trk-A (E18) or
flg (P5) at the ages at which their respective ligands act
as sole survival factors, a correlation suggesting that the membrane
exchange of the relevant receptors may be linked to the regulation of
the trophic switch in vivo, as hypothesized elsewhere
(Hashino et al., 1999; Baudet et al., 2000). It is at present unclear
to what extent NGF and bFGF act on overlapping groups of P-neurons.
Like most developing DRG neurons, embryonic P-neurons depend on NGF
(Levi-Montalcini and Angeletti, 1968; Kucera et al., 1995; Lewin and
Barde, 1996) and become independent of it shortly after birth. However,
they have not been previously identified as a specific group depleted
after prenatal NGF deprivation. This treatment causes the loss of most
small diameter neurons, presumably peptidergic, and mediating
nociceptive functions in the adult (Davies et al., 1987; Lewin and
Mendell, 1993; Snider, 1994). From our data, P-neurons do not appear to
be contemplated in that group. The former represent ~70% of the
total population, are claimed to express SP or CGRP, and mostly
innervate skin (for review, see Snider, 1994), whereas the medium size
P-neurons contribute a relatively small fraction, have a different
morphological phenotype in culture, do not express SP whenever they
express Trk-A, and innervate skeletal muscle but not
cutaneous tissue (see below). This last finding is especially interesting in view of the fact that embryonic NGF-dependent neurons are assumed to include most nociceptors, despite being presently unclear whether muscle nociceptors require that neurotrophin (Lewin and
Barde, 1996; Snider and McMahon, 1998). With regard to SP expression in
P-neurons, it was detected at times that closely agree with previous
findings in the rat DRG (Hall et al., 1997).
This is the first report, as far as we are aware, showing a clear
survival-promoting effect of bFGF on peripheral sensory neurons.
Previous studies reported a minor effect, if any, on the survival of
chick DRG neurons (Eckenstein et al., 1990; Oppenheim et al., 1992),
whereas it clearly acts as survival factor on central neurons (Beck et
al., 1993; Grothe and Wewetzer, 1996). Most commonly, bFGF has been
found to be a powerful mitogen and differentiating factor (Birren and
Anderson, 1990; Birren et al., 1993; Vescovi et al., 1993; Vaccarino et
al., 1999). A strong indication that bFGF is required in
vivo for the survival of P-neurons is the large reduction in the
number of those cells in newborn animals injected with the antibody
against that factor. In particular, our result rules out that the
dependence on bFGF observed in vitro results from the
axotomy caused by tissue dissociation (Ji et al., 1995).
Our data fit only partially into the classical view that the survival
of primary sensory neurons during development depends on the obtention
of specific target-derived trophic factors by their growing axons
(Levi-Montalcini and Angeletti, 1968; Lewin and Barde, 1996). P-neurons
depend on NGF mostly before the development of target innervation
by growing sensory axons (Reynolds et al., 1991; Coggeshall et al.,
1994) (but see Mirnics and Koerber, 1995). Their requirement of NGF
better correlates with the period of naturally occurring death, which
peaks between E15 and E19, and is over just after birth. This conforms
to the more recent notion that NGF is required by embryonic sensory
neurons during the period of massive neuronal death, supplied by
sources other than the peripheral targets, before their innervation
(Coggeshall et al., 1994; Davies, 1997; Wetts and Vaughn, 1998).
Accordingly, Trk genes (receptors for neurotrophins) are
expressed early in development (Mu et al., 1993). Although we do not
know whether P-neurons require NGF before E18, the factor appears to
regulate their survival over a relatively extended time (E18-P2). One
possible early source of trophic factors is the spinal cord (Snider et
al., 1992; Fitzgerald et al., 1993; Coggeshall et al., 1994). Unlike
NGF, the onset of bFGF effect occurs at an age in which sensory axons
have reached skeletal muscle fibers, a target of P-neurons innervation
(see below) (Coggeshall et al., 1994). Consistent with the idea of target-derived factor, bFGF is present in skeletal muscle at P3-P4, although it is also found in neuronal and glial cells of the spinal cord and the DRG, as shown by others (Moore et al., 1991; Ji et al.,
1995; Grothe et al., 1997).
The notion of trophic switch implies that exactly the same cells
initially requiring some trophic factor subsequently need another. No
conclusive proof of this has been possible for two main reasons (Birren
et al., 1993; Molliver et al., 1997; Enokido et al., 1999; Enomoto et
al., 2000): (1) the neurons requiring each trophic factor might be
generated in different waves of neurogenesis when the switch occurs
very early in development, and (2) the lack of a property, or of a
marker uniquely identifying a single subpopulation of living neurons.
Although this study does not show a trophic switch in a single neuron,
it leaves little room for an alternative explanation. Both NGF and bFGF
requirements occurred after the termination of neurogenesis, and the
phenotype of P-neurons allowed their unequivocal identification in
culture. Moreover, the stable proportion of P-neurons over ages
E18-P5, together with the specific effects of NGF and bFGF at E18 and P5, respectively, would imply that if the switch occurred in different groups, then NGF-dependent P-neurons must differentiate into round cells postnatally, whereas a matching number of separate, round neurons
must differentiate into bFGF-dependent P-neurons. This speculation and
the additional requirement that those two unrelated subpopulations
should display a coordinated change in their sensitivity to the
survival factors (Fig. 3) seem highly unreasonable.
Additional features distinguish the group of P-neurons. They project to
skeletal muscle but not skin or joints. They are, however, different
from the proprioceptors innervating muscle spindles. These are large,
express the 200 kDa neurofilament but not peptides, and require NT-3
for survival during embryonic life (Hory-Lee et al., 1993; Kucera et
al., 1995; Zhou and Rush, 1995b). P-neurons are smaller, contain SP but
not the 200 kDa neurofilament, and do not require NT-3. Thus, they are
likely to fall into the group of unmyelinated muscle afferents
(Abrahams, 1986; Mense, 1996), although we cannot exclude projections
to visceral targets. Interestingly, and consistent with our data, chick
embryonic muscle sensory neurons require NGF (Hory-Lee et al., 1993),
and adult muscle afferents only rarely express Trk-A
(McMahon et al., 1994). The expression of the pain-related peptide SP
in P-neurons merits some comments. First, the coexpression of
flg and SP is a specific marker of postnatal P-neurons.
Second, it suggests that they might have a nociceptive function in view
of the fact that that all SP-containing sensory neurons have been
reported to respond to noxious stimuli, and mostly project to deep,
noncutaneous peripheral targets (Levine et al., 1993; Zheng and Lawson,
1994; Lawson et al., 1997). P-neurons might represent a subset of a
larger subpopulation, of which only a fraction adopts a pear shape, and
perhaps included in some group of sensory afferents previously studied.
Nonetheless, this is the first study that characterizes them as a
specific group. On the basis of this report and our previous work, we
hypothesize that P-neurons represent nociceptors of skeletal muscle for
which function could be strongly regulated by enkephalins (Acosta and López, 1999). Alternatively, they might correspond to fine
afferents involved in SP-mediated, circulatory reflexes (Kniffeki et
al., 1981; Wilson and Hand, 1997).
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ABSTRACT
INTRODUCTION
