The Journal of Neuroscience, April 15, 1998, 18(8):3059–3072
A Distinct Subgroup of Small DRG Cells Express GDNF Receptor
Components and GDNF Is Protective for These Neurons after
Nerve Injury
David L. H. Bennett,1 Gregory J. Michael,2 Navin Ramachandran,1 John B. Munson,3 Sharon Averill,2
Qiao Yan,4 Stephen B. McMahon,1 and John V. Priestley2
Department of Physiology, United Medical and Dental Schools (St. Thomas’ Campus), London, SE1 7EH, United
Kingdom, 2Department of Anatomy, Queen Mary and Westfield College, London, E1 4NS, United Kingdom, 3Department
of Neuroscience, University of Florida College of Medicine, Gainsville, Florida 32610, and 4Department of Neuroscience,
Amgen Inc., Thousand Oaks, California 91320
1
Several lines of evidence suggest that neurotrophin administration may be of some therapeutic benefit in the treatment of
peripheral neuropathy. However, a third of sensory neurons do
not express receptors for the neurotrophins. These neurons are
of small diameter and can be identified by the binding of the
lectin IB4 and the expression of the enzyme thiamine monophosphatase (TMP). Here we show that these neurons express
the receptor components for glial-derived neurotrophic factor
(GDNF) signaling (RET, GFRa-1, and GFRa-2). In lumbar dorsal
root ganglia, virtually all IB4-labeled cells express RET mRNA,
and the majority of these cells (79%) also express GFRa-1,
GFRa-2, or GFRa-1 plus GFRa-2.
GDNF, but not nerve growth factor (NGF), can prevent several
axotomy-induced changes in these neurons, including the
downregulation of IB4 binding, TMP activity, and somatostatin
expression. GDNF also prevents the slowing of conduction
velocity that normally occurs after axotomy in a population of
small diameter DRG cells and the A-fiber sprouting into lamina
II of the dorsal horn. GDNF therefore may be useful in the
treatment of peripheral neuropathies and may protect peripheral neurons that are refractory to neurotrophin treatment.
In the adult animal, specific dorsal root ganglion (DRG) cell
populations require particular neurotrophins for their phenotypic
maintenance (Verge et al., 1996). The trk receptors in general are
expressed in a nonoverlapping manner by sensory neurons in
combination with the low-affinity neurotrophin receptor p75
(Wright and Snider, 1995). Large diameter DRG cells mostly
possess myelinated axons and respond principally to low threshold stimuli. These neurons express trkB or trkC or both (McMahon et al., 1994). Small diameter DRG cells, in contrast, have
unmyelinated axons and are principally nociceptors and thermoceptors. Half of this group (40% of total DRG cells) constitutively synthesize neuropeptides and express trkA (Averill et al.,
1995; Molliver et al., 1995). The other half of the small diameter
DRG cells (35% of total DRG cells) possess cell surface glycoconjugates that can be identified by binding of the lectin Isolectin
B4 from Griffonia simplicifolia (I B4) (Silverman and Kruger,
1990). They also express the enzyme thiamine monophosphatase
(TM P). During development these cells are dependent on NGF
for survival (Silos-Santiago et al., 1995). During the postnatal
period, however, these cells downregulate trkA expression (Bennett et al., 1996a; Molliver and Snider, 1997). It is this population
that in the adult does not express detectable levels of the lowaffinity neurotrophin receptor p75 nor any known trk receptor
(McMahon et al., 1994; Averill et al., 1995; Molliver et al., 1995;
Wright and Snider, 1995). In this study we have examined the
possibility that GDNF exerts a trophic action on these neurons.
GDNF is a member of the transforming growth factor-b
(TGF-b) superfamily (Lin et al., 1993) and is related to neurturin
(Kotzbauer et al., 1996). GDNF has been demonstrated to have
potent survival-promoting effects on midbrain dopaminergic neurons (Beck et al., 1995; Bowenkamp et al., 1995) and motoneurons
(Henderson et al., 1994; Oppenheim et al., 1995; Yan et al., 1995).
There is growing evidence that GDNF can have a trophic action
on sensory neurons. In GDNF-deficient mice there is a significant
reduction in the number of spinal sensory neurons (Moore et al.,
1996). During the late embryonic and postnatal period, the survival of a subpopulation of DRG cells is supported by this factor
in vitro (Buj-Bello et al., 1995), and those neurons that are
supported are IB4 binding (Molliver et al., 1997). GDNF can also
prevent the death of axotomized developing sensory neurons in
vivo (Matheson et al., 1997).
The receptor for GDNF is thought to be a complex of GFRa-1
(Jing et al., 1996, Treanor et al., 1996; GFRa Nomenclature
Committee, 1997), which acts as a ligand binding domain, and
RET, which acts as the signal transducing domain (Durbec et al.,
1996, Trupp et al., 1996). Neurturin also appears to use RET for
signaling, but operates via another GPI-linked binding protein
termed GFRa-2 (Baloh et al., 1997; Buj-Bello et al., 1997; GFRa
Nomenclature Committee, 1997; K lein et al., 1997). GDNF may
Received Dec. 8, 1997; revised Jan. 23, 1998; accepted Jan. 28, 1998.
This work was f unded by the Medical Research Council of Great Britain.
D.L.H.B. is supported by the Special Trustees of Guy’s and St. Thomas’ Hospitals.
We acknowledge the expert technical assistance of C. Abel and S. Hamilton. We also
thank Genentech for the provision of rhNGF and Amgen for the provision of
rhGDN F; H. S. Phillips and R. D. K lein for the provision of the GFRa-2 sequence;
and Dr. D. O. Clary, Dr. T. Görcs, and Professor J. M. Polak for the provision of the
trkA, somatostatin, and CGRP antisera, respectively.
D.L.H.B. and G.J.M. contributed equally to this work.
Correspondence should be addressed to Professor S. B. McMahon, Department of
Physiology, St. Thomas’ Hospital Medical School, Lambeth Palace Road, L ondon
SE1 7EH, UK.
Copyright © 1998 Society for Neuroscience 0270-6474/98/183059-14$05.00/0
Key words: IB4; trkA; RET; somatostatin; GFRa-1; GFRa-2;
axotomy; C-fibers; nociception; pain; sprouting; spinal cord
3060 J. Neurosci., April 15, 1998, 18(8):3059–3072
also be able to act via GFRa-2, particularly in the presence of
RET (Sanicola et al., 1997). In this study, we have examined the
expression of these GDNF receptor subunits within adult sensory
neurons.
One means of studying the trophic requirements of different
subgroups of sensory neurons has been to determine to what
extent injury-induced changes can be reversed by the administration of exogenous trophic factors (Verge et al., 1995, 1996). The
second aim of the present work was to investigate the efficacy of
GDNF in reversing such axotomy-induced changes in adult sensory neurons.
MATERIALS AND METHODS
Animal surger y. Adult male Wistar rats underwent unilateral sciatic nerve
section combined with an intrathecal inf usion of recombinant human
GDN F (rhGDN F), rhNGF or control buffer. The sciatic nerve was
exposed under pentobarbitone anesthesia (40 mg / kg, i.p., with sterile
precautions) and ligated 20 mm distal to the obturator tendon. Concurrently a small laminectomy was performed between L6 and S1 vertebrae,
and the dura was cut. A SIL ASTIC tube of 0.6 mm outer diameter was
introduced intrathecally so that its tip lay at the level of the lumbar
enlargement of the spinal cord. The intrathecal tubing was attached to an
Alzet miniosmotic pump (type 2002; Alzet, Alza Corporation, Palo
Alto, CA) delivering at a rate of 0.5 ml / hr. Animals received either a
control inf usion (n 5 4; saline with rat serum albumin, 1 mg /ml) or this
vehicle plus rhGDN F (n 5 4 at 12 mg /d; n 5 3 at 1.2 mg /d) or rhNGF
(n 5 3 at 12 mg /d; n 5 3 at 1.2 mg /d). Another group of animals
underwent axotomy with either control inf usion (n 5 4) or GDN F
treatment (n 5 4 at 12 mg /d; n 5 3 at 1.2 mg /d). T welve days later the left
sciatic nerve was re-exposed and injected with 4 ml of 1% B-subunit of
cholera toxin (C TB) (List Biological Labs, C ampbell, CA) in distilled
water, using a micropipette glued to a Hamilton syringe. Animals were
perf used with heparinized saline followed by 4% paraformaldehyde 14 d
after sciatic section. Another group of normal animals (n 5 5) was
labeled with C TB in the same manner but did not undergo sciatic
axotomy. After perf usion the left and right L4 and L5 DRGs were
removed as well as L3–L6 segments of the spinal cord. Pins were placed
in the right side of the spinal cord at the border between L3/ L4, L4/ L5,
and L5/ L6 to ease identification of the spinal levels during analysis.
Tissues were post-fixed in 4% paraformaldehyde for 2 hr, after which
they were transferred to 15% sucrose overnight. One group of animals
(n 5 4) was perf used in the same manner and used for in situ hybridization. Another group of naive animals (n 5 3) was used to provide
control values for I B4, somatostatin, calcitonin gene-related peptide
(CGRP), and TM P staining.
Electrophysiological analysis was performed on five different groups of
animals: normal intact animals (n 5 4); animals in which the tibial nerve
had been cut and tied 2 weeks previously and with an intrathecal cannula
delivering 1 mg /ml normal rat serum albumin in saline at 12 ml /d (n 5 5);
animals with tibial nerve axotomy and continuous intrathecal delivery of
rhGDN F (12 mg /d; n 5 3), rhNGF (12 mg /d; n 5 3), or rhGDN F and
rhNGF (12 mg /d each; n 5 4). This surgery was performed under
pentobarbitone anesthesia and with sterile precautions.
Staining procedures. Sections of DRG and spinal cord were cut at a
thickness of 15 and 20 mm, respectively. Sections of DRG were cut
serially onto slides so that each slide contained an ordered series of
sections throughout the ganglia, at a separation of at least 150 mm
between sections. When the spinal cord was cut, every fifth section was
mounted serially onto slides. Every slide therefore had a series of sections
through the L4 and L5 region of spinal cord at a separation of at least 800
mm between sections. For immunostaining, primary antisera were rabbit
anti-CGRP (1:2000, gift of Professor J. M. Polak), rabbit antisomatostatin (1:2000, gift of Dr. T. Görcs), goat anti-C TB (1:2000, List),
and biotinylated I B4 (10 mg /ml, Sigma). Secondary antisera were FI TC or TRI TC -conjugated anti-rabbit or anti-goat IgG (1:200, Jackson labs)
or FI TC -conjugated E xtr-Avidin (1:200, Sigma). Histochemistry for
TM P was also performed as described previously (McMahon, 1986).
Analyses of colocalization of RET immunoreactivity with other DRG
products were performed on 8 mm cryostat sections using dual-labeling
immunofluorescence. The production and staining characteristics of the
RET antiserum have already been described (Molliver et al., 1997). RET
immunostaining was combined sequentially with markers described
above as well as sheep anti-CGRP (1:2000, Affiniti) and anti-rabbit trkA
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
(Averill et al., 1995). Indirect tyramide signal amplification (TSA, New
England Nuclear) was used for the first reaction when staining with two
rabbit antisera as described previously (Michael et al., 1997). The lack of
cross-reactivity is thought to be caused by the fact that the primary
antiserum is highly diluted compared with when it is used in indirect
immunofluorescence without amplification (e.g., trkA 1:100,000 vs
1:4000), and therefore the second series reactions do not detect it. This
was verified by a lack of staining in control single-labeled preparations
using indirect immunofluorescence and the antiserum dilutions used
for TSA.
For combined fluorescence histochemistry with in situ hybridization,
cryostat sections (6 – 8 mm) were cut and thaw-mounted onto Superfrost
Plus slides (BDH Chemicals, Poole, UK). Immunocytochemistry and /or
lectin binding histochemistry was performed before in situ hybridization
(Michael and Priestley, 1996b; Michael et al., 1997). Sections were
incubated for 40 – 48 hr at room temperature with trkA antibody (4
mg /ml), N52 monoclonal antibody to phosphorylated heav y chain neurofilament (1:400, Sigma), or biotinylated I B4 (10 mg /ml) diluted in
diethylpyrocarbonate (DEPC)-treated PBS containing 0.2% Triton
X-100, 0.1% sodium azide, 0.5 mM dithiothreitol, and 100 U/ml RNasin
(Promega, Madison, W I). Lectin binding buffer also contained 0.1 mM
MnC l2 , 0.1 mM MgC l2 , and 0.1 mM C aC l2. Sections were washed in
DEPC PBS and incubated for 4 hr in tetramethyl rhodamine isothiocyanate (TRI TC)-conjugated secondary antibodies (1:200, Jackson Laboratory, Bar Harbor, M E) or fluorescein isothiocyanate (FI TC)-conjugated
E xtr-Avidin (1:200, Sigma) diluted in the same buffer without added
divalent cations. After additional washes in DEPC PBS, sections were
processed through prehybridization steps, hybridized to 35S-dATP endlabeled oligonucleotides, and washed as described previously (Michael
and Priestley, 1996a). Slides were dipped in autoradiographic emulsion
(Amersham, Arlington Heights, IL) and developed after 4 – 6 weeks.
After the slides were coverslipped with PBS glycerol (1:3 containing
2.5% 1,4-diazobicyclo-(2,2,2)-octane), fluorescent labeling and silver
grains were visualized using epifluorescence microscopy combined with
either epipolarized illumination or dark-field illumination. The oligonucleotides used for probes were complementary to nucleotides 996 –1029
of the rat GFRa-1 sequence (Jing et al., 1996) and nucleotides 161–194
of the rat RET sequence (C anzian et al., 1995), and for GFRa-2 the
oligonucleotide sequence cctggactgatgtttgtcgtgagctctgtgaagc was used
(K lein et al., 1997).
Controls for the specificity of in situ hybridization included adding a
100-fold excess of unlabeled oligonucleotide to hybridization buffer,
which effectively competed all specific binding of radiolabeled probe. Use
of the GFRa-1, GFRa-2, and RET to label sections of rat brain yielded
patterns of hybridization identical to reported patterns (Trupp et al.,
1997) (our unpublished observations). All probes were synthesized to be
of the same size and G1C base content and produced reproducible and
characteristic patterns of labeling.
Image anal ysis. After in situ hybridization, cells that had silver grains
over the cell cytoplasm at least five times background were counted as
positive. For quantitation of in situ hybridization, counts of cells labeled
for RET, GFR-a1, and GFRa-2 and coexpressed cell markers were
conducted on ganglia from at least four animals, with separation between
analyzed sections being at least 100 mm. At least 1500 cells were counted
for each probe/peptide combination. For counts of the percentage of cell
profiles expressing CGRP, somatostatin, I B4 binding, and TM P activity
after different treatments, six sections were randomly selected for each
marker for each animal in each group. In each section the total number
of cell profiles was counted using dark-field illumination, and then the
number of positively stained cell profiles was counted.
For image analysis of I B4, TM P, CGRP, and C TB staining within the
dorsal horn, four randomly selected sections of L4/5 spinal cord were
used from each animal. T wo sections were selected from L4 and two
sections were selected from L5 to ensure that the analysis was not biased
toward one region of the lumbar enlargement. For analysis of I B4,
CGRP, and TM P staining, images of spinal cord sections were captured
directly off the microscope at 253 objective magnification using a Grundig FA87 digital camera with integrating framestore. The image was then
thresholded to a set level to reveal the labeling. Four boxes of size 27 3
27 mm were placed over lamina II of the axotomized side (within the
sciatic territory) and in equivalent positions on the contralateral (i.e.,
intact sciatic) side of the sections. The area occupied by labeled terminals
was then calculated for each box. A similar method was used for analysis
of C TB staining, but in this case the four boxes were placed over lamina
III of the sciatic-labeled territory and four were placed dorsal to the
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
J. Neurosci., April 15, 1998, 18(8):3059–3072 3061
lamina III boxes in lamina II outer. The area occupied by C TB-stained
terminals within each of these boxes was then calculated.
This image analysis system was similarly used for cell size distribution
analyses of RET, GFRa-1, and GFRa-2. Images of DRG sections were
captured directly off the microscope as described above. C ell profiles
were outlined using a hand-held mouse from which the cell area was
calculated. For the RET analysis 1128 profiles were drawn, for the
GFRa-1 analysis 990 profiles were drawn, and for the GFRa-2 analysis
567 profiles were drawn. C ell size distribution analyses were also performed on normal L4/5 ganglia (n 5 4 animals), axotomized ganglia (n 5
4 animals), and ganglia that had undergone axotomy in combination with
GDN F treatment (12 mg /d; n 5 4). T welve sections that had been cut at
a thickness of 15 mm and stained with toluidine blue were selected for
each animal. The selected section was then divided into quadrants, and
all the profiles within a randomly selected quadrant were outlined. In
total 2033 profiles were drawn in the normal group, 2256 in the axotomy
group, and 1875 in the axotomy plus GDN F group.
Trophic factor effects on electrophysiolog ical properties of a xotomized
C-fibers. As an independent measure of the efficacy of trophic factors, we
studied the electrophysiological properties of damaged C -fibers. The
conduction velocity (C V) distribution of C -fibers projecting through the
tibial nerve was measured in urethane-anesthetized animals (1.25 gm / kg,
i.p.) in terminal experiments. Fine strands of the L5 dorsal root were
dissected and mounted on recording electrodes. The tibial nerve was
continuously electrically stimulated at 2 Hz with square wave current
pulses: 5 mA, 1 msec. The evoked activity on the root filament was
amplified and filtered by conventional means, and the averages of 64 –128
responses were constructed (see Fig. 9). In these averages it was possible
to determine the latency of individual fibers. All fibers conducting at less
than 2 m /sec were included in analysis. T ypically 3–10 C -fibers were
found in each strand. A sample of approximately 50 individual C -fibers
was measured in each animal, from which the conduction velocity distribution was computed. Distributions from three to five animals in each
experimental group were averaged and plotted as cumulative sums (e.g.,
see Fig. 9). The distributions of C Vs were statistically compared using
the Kolmogorov– Smirnov test.
RESULTS
GDNF receptor expression within sensory neurons
Abundant labeling for RET, GFRa-1, and GFRa-2 mRNAs was
observed in lumbar DRG cells (Fig. 1), with 64 6 4.4, 40.6 6 1.5,
and 32.8 6 1.0% of DRG cell profiles labeled, respectively. Cells
of all sizes showed labeling, but GFRa-2 and RET mRNAs were
expressed by proportionally more small and intermediate-sized
cells (Figs. 1, 2).
To identif y the cell types that were labeled, in situ hybridization was combined with immunocytochemistry for markers that
are widely used to characterize three main DRG subpopulations
(Averill et al., 1995). Strikingly, a high level of expression of all
three GDNF receptor components (RET, GFRa-1, and GFRa-2)
was found in cells labeled with the lectin IB4 (Fig. 2, Table 1), a
marker for small neurons that do not express any of the trk
receptors (Averill et al., 1995; Molliver et al., 1995). In the case of
RET, virtually all IB4 cells express RET mRNA (95%), and the
IB4 cells account for a very high percentage of the RET population (79%) (Table 1). In contrast to RET, GFRa-1 and GFRa-2
mRNAs are each expressed in only approximately half of the
IB4 cells (46 and 55%, respectively) (Table 1). To determine
whether GFRa-1 and GFRa-2 are expressed by the same IB4
cells, serial sections were triple-labeled for trkA, IB4, and
GFRa-1 or GFRa-2 mRNAs (Fig. 3). This analysis revealed
that the RET/ IB4 cells can be subdivided into four, roughly
equally sized subgroups, based on their expression of the GFRa
subunits: GFRa-1 alone (21% of IB4 cells), GFRa-2 alone
(28%), both GFRa-1 and GFRa-2 (30%), and neither GFRa-1
nor GFRa-2 (21%).
In contrast to the high expression of GDNF receptor components in the IB4 cells, expression was low in the second subpopu-
Figure 1. C ell size distribution of DRG cell profiles positively and
negatively labeled for RET, GFRa-1, and GFRa-2 within L4/5 dorsal root
ganglia. RET and GFRa-2 are present predominantly in small and intermediate diameter DRG cell profiles but are also present in some large
diameter DRG cell profiles. GFRa-1 is more evenly distributed through
the whole cell size spectrum.
lation of DRG cells, namely the trkA immunoreactive cells.
GFRa-2 mRNA was observed in very few trkA immunoreactive
cells (3%) (Table 1), and although RET mRNA was expressed by
a significant number of trkA cells (28%) (Table 1), they belonged
to a group that also showed IB4 labeling (Table 1). The majority
of trkA cells do not show IB4 labeling (Averill et al., 1995;
Michael et al., 1997) and did not express either RET or GFRa-2
3062 J. Neurosci., April 15, 1998, 18(8):3059–3072
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
Figure 2. E xpression of RET, GFRa-1, and GFRa-2 in I B4-labeled DRG cells. In situ hybridization for RET ( a), GFRa-1 ( c), or GFRa-2 ( e) was
combined with I B4 labeling (b, d, f ). a and b show that many I B4 cells express RET (arrows indicate double-labeled cells). A similar pattern is seen in
c and d and in e and f in relation to GFRa-1 and GFRa-2, except that the GFRa components are expressed in a smaller proportion of I B4 cells. Long
arrows indicate I B4-labeled cells that express GFRa-1 or GFRa-2, whereas short open arrows indicate I B4 cells that do not express GFRa-1 or GFRa-2.
Scale bar, 50 mm.
mRNAs (Table 1). However, a small number of these cells do
express GFRa-1 mRNA (Table 1). The GDNF receptor components were also expressed in the third subpopulation of DRG
cells, namely large neurons that can be identified by labeling
with anti-neurofilament antisera such as N52. GFRa-1 and RET
mRNAs are expressed by a significant number of N52 immunoreactive cells (40 and 33%, respectively) (Table 1), but GFRa-2 is
virtually absent (only 5% of N52 cells).
To f urther study the pattern of RET expression, a polyclonal
antiserum to RET was used (Molliver et al., 1997). Staining
of L4/5 DRG sections (Fig. 4) revealed immunoreactivity in a
population of DRG cells similar to that labeled by in situ
hybridization. Thus 72% of L4/5 DRG cell profiles were RET
immunoreactive, and of these the majority were also I B4labeled (96% of I B4 cells were RET immunoreactive) (Fig. 4).
Only 27 and 30%, respectively, of RET immunoreactive cells
showed immunoreactivity for trkA or for the neuropeptide
CGRP (Fig. 4). The distribution of RET immunoreactivity in
the lumbar enlargement of the spinal cord was also studied.
RET immunoreactive terminals were present principally in
lamina IIi (Fig. 4), the same region in which I B4 labeling is
observed (Fig. 4). It was interesting, given that some large
diameter DRG cells express RET (see above), that clear labeling for RET immunoreactive terminals was not observed in the
regions of the spinal cord where these neurons terminate, i.e., the
deep dorsal horn or the ventral horn.
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
J. Neurosci., April 15, 1998, 18(8):3059–3072 3063
Table 1. Percentage of DRG neurons co-expressing immunoreactivity for trkA, IB4, trkA 1 IB4, or N52 and in situ hybridization signal for RET,
GFRa-1, and GFRa-2 mRNAs
RET mRNA
TrkA
I B4
TrkA 1 I B4
N52
GFRa-1 mRNA
GFRa-2 mRNA
% RET
expressing other
% other
expressing RET
% GFRa-1
expressing other
% other
expressing GFRa-1
% GFRa-2
expressing other
% other
expressing GFRa-2
15.0 6 0.8
78.7 6 1.1
13.5 6 2.2
25.8 6 3.3
27.7 6 2.2
95.3 6 0.3
77.0 6 8.4
33.0 6 2.0
17.9 6 1.2
49.5 6 1.0
2.5 6 0.5
40.0 6 3.8
18.0 6 1.0
46.0 6 3.6
12.7 6 1.5
40.0 6 2.0
2.8 6 0.4
78.3 6 1.8
2.1 6 0.6
16.7 6 1.7
2.9 6 0.4
54.5 6 0.6
9.2 6 2.3
5.0 6 0.4
Figure 3. GFRa-1 and GFRa-2 expression in I B4 cells. GFRa-1 and GFRa-2 are coexpressed in one group of I B4 cells, expressed separately in other
groups, and not expressed at all in a fourth group. Serial sections are shown triple-labeled for trkA (a, d), I B4 (b, e), and either GFRa-1 ( c) or GFRa-2
( f ) mRNAs. A I B4 cell expressing only GFRa-1 is identified by an arrow. An arrowhead indicates a I B4 cell that expresses only GFRa-2. The double
arrow indicates a I B4 cell that expresses both GFRa-1 and GFRa-2. Note that none of these cells are trkA immunoreactive. The star indicates a I B4
cell that expresses neither GFRa-1 nor GFRa-2. This cell is also trkA immunoreactive. Also shown is a large cell (asterisk) that expresses both GFRa-1
and GFRa-2. It is not I B4-labeled or trkA immunoreactive. Scale bar, 50 mm.
GDNF reverses axotomy-induced changes in the
IB4-binding population of sensory neurons
To investigate the trophic effects of GDNF on sensory neurons,
the ability of GDNF and NGF to reverse axotomy-related
changes in different populations of sensory neurons was compared. Two different doses of these factors were used: a low dose
of 1.2 mg /d and a high dose of 12 mg /d administered continuously
over 14 d (these doses were based on our own and others previous
findings) (Bennett et al., 1996b, Verge et al., 1995), and because
these proteins are being administered in vivo, these doses are
much higher than would be considered appropriate in vitro.
There were marked phenotypic changes in the IB4-binding
population of small diameter DRG cells after 2 weeks of axotomy, including a reduction in the percentage of DRG cell profiles that bind IB4, from ;40% to ,20% (Fig. 5, Table 2).
Intrathecal application of low-dose GDNF significantly increased
the number of DRG cell profiles binding IB4 after axotomy ( p ,
0.001; unpaired t test), and the high-dose GDNF treatment was
even more effective ( p , 0.001; unpaired t test) (Fig. 5, Table 2).
In contrast, intrathecal application of NGF (at either dose) had
no significant effect on this marker (Fig. 5, Table 2). TM P is an
enzyme present principally in the IB4-binding “trk-less” population of DRG cells. A histochemical reaction was used to reveal
TM P activity within DRG cells, and this produced a black
reaction product within the cytoplasm of cells. Axotomy led to a
large reduction in the proportion of DRG cell profiles that expressed TM P activity (Fig. 5, Table 2). Intrathecal administration
of GDNF at a low dose produced a significant increase in the
proportion of cell profiles expressing TM P activity compared
with no treatment ( p , 0.05; unpaired t test) (Table 2). Intrathecal administration of GDNF at a high dose after axotomy was
even more effective ( p , 0.001; unpaired t test) (Fig. 5, Table 2)
and restored TM P activity to a level not significantly different
from normal. Intrathecal administration of NGF (at either dose)
3064 J. Neurosci., April 15, 1998, 18(8):3059–3072
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
Figure 4. Colocalization of RET immunoreactivity with neurochemical markers in DRG cells and spinal cord. a–f, Dual labeling showing RET
immunofluorescence (a, c, e) combined with I B4 labeling ( b), trkA immunofluorescence ( d), and CGRP immunofluorescence ( f ) in DRG cells. Arrows
indicate extensive colocalization of RET and I B4 in small diameter cells (a, b). Note that all I B4 cells show RET immunoreactivity. However, several
RET positive cells do not bind I B4 (asterisks). RET labeling is not evident in many trkA cells (c, d). Asterisks denote trkA cells that are not co-labeled
for RET. Similarly, few CGRP-expressing DRG cells are RET immunoreactive (e, f ). Asterisks indicate cells that do not express RET but are labeled
for CGRP. The arrow indicates a cell that is dual-labeled. g–j, Low-magnification ( g, i) and high-magnification (h, j) micrographs showing RET
immunofluorescence ( g, h) and I B4 (i, j) double labeling in the dorsal horn of the spinal cord. Labeling is most intense in inner lamina II. Arrows in
h and j indicate individual double-labeled axons. Scale bars (shown in f ): a–f, 50 mm; (shown in i): g, i, 100 mm; (shown in j): h, j, 30 mm.
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
J. Neurosci., April 15, 1998, 18(8):3059–3072 3065
Figure 5. Histochemistry for TM P (a–d), I B4 labeling (e–h), and CGRP immunofluorescence (i–l ) in dorsal root ganglia of control animals (a, e, i),
animals with unilateral sciatic nerve section (b, f, j), animals with unilateral sciatic nerve section combined with intrathecal GDNF treatment (12 mg/d)
(c, g, k), and animals with unilateral sciatic nerve section combined with intrathecal NGF treatment (12 mg/d) (d, h, l ). Sciatic nerve section causes a loss
of TM P ( b) and I B4 ( f ) labeling, which is prevented by GDNF treatment (c, g) but not by NGF (d, h). In contrast, the loss of CGRP staining caused
by sciatic nerve section ( j ) is prevented by NGF ( l ) but not by GDNF ( k). Scale bar, 50 mm. CTR L, Control; AXOT, axotomized.
was ineffective in restoring the proportion of DRG cell profiles
expressing TM P activity after axotomy (Fig. 5, Table 2). Somatostatin is a neuropeptide expressed in a subgroup of trk-less
DRG cells (Kashiba et al., 1996); its expression drops after
axotomy (Table 2). Administration of GDNF at a low dose
produced a small nonsignificant increase in the number of DRG
cell profiles expressing somatostatin after axotomy. Intrathecal
administration of GDNF at a high dose prevented this axotomyinduced change ( p , 0.05; unpaired t test; compared with axo-
tomy alone) (Table 2). Intrathecal administration of NGF was
ineffective in preventing this change (Table 2).
These effects are in contrast to those seen in the other population of small diameter DRG cells, those that express the trkA
receptor and the neuropeptide CGRP. CGRP expression in DRG
cell profiles fell markedly after axotomy, from ;40% of cell
profiles to ;25%. Intrathecal application of NGF at a low dose
could partially prevent this reduction (Table 2). Intrathecal application of NGF at a high dose prevented this reduction ( p ,
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
3066 J. Neurosci., April 15, 1998, 18(8):3059–3072
Table 2. The percentage of profiles stained for IB4, TMP, CGRP, or SOM in naive animals or after axotomy alone or axotomy in combination with
treatment with GDNF (1.2 or 12 mg/d) or NGF (1.2 or 12 mg/d)
I B4
TM P
CGRP
SOM
Naive
Axotomized
Axotomized 1
GDNF 1.2
Axotomized 1
GDNF 12
Axotomized 1
NGF 1.2
Axotomized 1
NGF 12
41.1 6 0.7% (7498)
36.6 6 0.7% (5446)
40.3 6 0.5% (8394)
5.4 6 0.1% (6756)
19.1 6 1.0% (8400)
19.6 6 4.7% (8440)
24.5 6 4.3% (6243)
2.6 6 0.26% (5560)
33.7 6 0.9% (7664)
28.7 6 2.4% (7653)
19.3 6 0.6% (8401)
3.5 6 0.2% (7983)
36.0 6 0.6% (5878)
38.6 6 0.9% (7751)
26.8 6 1.3% (6732)
4.8 6 0.41% (5569)
20.7 6 1.7% (6626)
20.4 6 0.5% (5979)
34.6 6 0.9% (6331)
2.0 6 0.03% (6570)
22.7 6 2.7% (5999)
20.2 6 0.3% (5657)
38.8 6 0.9% (5380)
2.3 6 0.1% (5177)
The numbers in parentheses represent the total number of profiles counted. SOM, Somatostatin.
0.05; unpaired t test) (Fig. 5, Table 2). Administration of GDNF
(at a low dose or a high dose) had no significant effect on the
proportion of DRG cell profiles expressing CGRP after axotomy
(Fig. 5, Table 2).
A size analysis was performed to ensure that changes in DRG
cell profile counts after different interventions did not occur as a
consequence of alterations in cell size. The mean cell profile size
in L4/5 ganglia in normal animals was 477 6 28 mm 2 (n 5 4), after
2 week sciatic axotomy it was 449 638 mm 2 (n 5 4), and after
sciatic axotomy and GDNF treatment (12 mg /d; n 5 3) it was 494
633 mm 2. There was no significant difference between these
groups ( p . 0.2; unpaired t test).
Thus, NGF and GDNF had complementary actions in their
ability to rescue phenotypic changes in trk- and non-trkexpressing small DRG neurons, respectively. We did not find any
significant effects of GDNF on the percentage of DRG cells
expressing IB4 and TM P in the normal DRG (data not shown).
GDNF reverses a number of axotomy-induced changes
within the dorsal horn of the spinal cord
The alterations seen in DRG cell bodies were also reflected in
changes within the dorsal horn after axotomy. TM P activity and
IB4 binding were normally present within lamina IIi of the dorsal
horn, the same region in which RET immunoreactive terminals
were present (Fig. 4). After 2 weeks of axotomy, TM P activity
was virtually absent from the sciatic projection territory of the
dorsal horn. IB4 binding was also markedly reduced (Figs. 6, 7a).
Quantitative image analysis demonstrated that continuous intrathecal administration of GDNF at a low dose had a significant
effect on TM P activity and IB4 binding after axotomy ( p , 0.05;
unpaired t test; compared with no treatment) (Figs. 6, 7a). Administration of GDNF at a high dose was more effective and
could almost completely restore TM P activity and IB4 binding
levels within the dorsal horn ( p , 0.001, unpaired t test, comparing GDNF treatment with axotomy alone; p . 0.2, compared
with intact) (Figs. 6, 7a). Administration of NGF at a low dose
had no significant effect on IB4 binding after axotomy and produced only a slight increase in TM P activity after axotomy ( p ,
0.05; unpaired t test; comparing NGF treatment with axotomy
alone) (Figs. 6, 7a). NGF administration at a high dose had a
small but significant effect in restoring TM P activity and IB4
binding (by ;10 –15%, p , 0.05; unpaired t test comparing NGF
treatment with axotomy alone) (Figs. 6, 7a). The effect of NGF on
TM P activity within the dorsal horn was much less than that of
GDNF ( p , 0.001; comparing GDNF with NGF treatment at
both high and low doses).
CGRP immunoreactive terminals are normally present within
laminae I and II of the dorsal horn, the region in which trkAexpressing DRG cells terminate (Averill et al., 1995; Molliver et
al., 1995). After axotomy there was ;60% reduction in CGRP
immunoreactivity within the sciatic termination territory (Fig.
7a), as determined by image analysis. Intrathecal treatment with
NGF at either a low or high dose largely prevented this change
( p , 0.001; unpaired t test; comparing NGF treatment at either
dose to no treatment) (Fig. 7a). GDNF treatment at a low dose
had no significant effect on CGRP levels after axotomy ( p . 0.05;
unpaired t test). GDNF treatment at a high dose had a small
(;10%) but significant rescue effect on CGRP levels ( p , 0.05;
unpaired t test; comparing GDNF treatment to no treatment).
The cholera toxin B subunit (C TB) binds to the GM1 receptor,
which is selectively expressed by myelinated sensory afferents.
CTB undergoes transganglionic transport by these afferents and
so can be used to study A-fiber terminations within the dorsal
horn of the spinal cord. In normal animals (Fig. 8), CTB-labeled
terminals were present within lamina I and the deep laminae of
the spinal cord (laminae III–IV). There were some terminals
present in lamina IIi but very few labeled fibers were present in
lamina IIo. Image analysis demonstrated that the ratio of labeling
in lamina IIo to lamina III was extremely low (0.003 6 0.0006)
(Fig. 6b). Two weeks after axotomy, CTB was present throughout
lamina II, including lamina IIo, and there was also more intense
labeling within lamina I (Fig. 8). This change is accepted to
indicate A-fiber sprouting into the superficial laminae (Woolf et
al., 1992, 1995; Bennett et al., 1996b). There was a significant
increase in the ratio of labeling within lamina IIo to lamina III (to
0.631 6 0.07 after axotomy; p , 0.01; unpaired t test) (Fig. 7b). In
animals that had received a 2 week intrathecal infusion of GDNF
at a low dose, the A-fiber sprouting within lamina II was largely
prevented (Fig. 8). Very few CTB-labeled terminals were present
within lamina IIo. Quantitative image analysis demonstrated that
the ratio of CTB staining between lamina IIo and lamina III after
this treatment was 0.016 6 0.002 ( p , 0.01; unpaired t test
compared with no treatment) (Fig. 7b). Treatment with GDNF at
the higher dose was even more effective (the ratio of CTB
staining between lamina IIo and lamina III was 0.007 6 0.003,
which was not significantly different from that seen in normal
intact animals). The difference between untreated and GDNFtreated axotomized animals was highly significant ( p , 0.001;
unpaired t test) (Fig. 7b). The profuse CTB labeling that normally occurs within lamina I after axotomy also appeared to be
prevented by GDNF treatment.
Trophic factor effects on electrophysiological
properties of axotomized C-fibers
Axotomy produces a conduction velocity slowing in C-fibers; we
investigated the efficacy of GDNF and NGF in reversing this
change. The conduction velocity (CV) distribution of C-fibers
projecting through the normal tibial nerve was unimodal, with a
mean of 0.86 6 0.06 m /sec. After two weeks of axotomy, velocity
was slowed to a mean of 0.68 6 0.03 m /sec, and this was significant ( p , 0.01; unpaired t test). This slowing was also apparent
as a leftward shift in the cumulative sum plots of CV of units (Fig.
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
J. Neurosci., April 15, 1998, 18(8):3059–3072 3067
Figure 6. Histochemistry for TM P at the level of L4
in the dorsal horn of animals with unilateral sciatic
nerve section ( a), animals with unilateral sciatic nerve
section combined with high (12 mg/d) ( b) and low (1.2
mg/d) ( c) dose intrathecal GDNF treatment, and animals with unilateral sciatic nerve section combined
with high (12 mg/d) ( d) and low (1.2 mg/d) ( e) dose
intrathecal NGF. Sciatic nerve section causes a loss of
TM P in the sciatic termination territory within lamina
IIi (demonstrated by arrows). GDNF treatment at either dose is effective at preventing this loss (b, c),
whereas NGF is much less effective at either dose used
(d, e). Scale bar, 100 mm. Axot., Axotomized.
9), and this shift was also statistically significant ( p , 0.01;
Kolmogorov–Smirnov). Intrathecal provision of GDNF, at 12
mg /d, throughout the 2 week period of axotomy, partially and
significantly prevented this slowing (Fig. 9) ( p , 0.05; Kolmogorov–Smirnov). The slowest conducting C-fibers were especially
rescued by GDNF. NGF at 12 mg /d also had a partial and
significant effect in preventing axotomy-induced slowing (Fig. 9)
( p , 0.05; Kolmogorov–Smirnov), although in this case C-fibers
throughout the CV distribution were more equally affected, suggesting that GDNF and NGF do not affect the same population
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
3068 J. Neurosci., April 15, 1998, 18(8):3059–3072
of afferents. Consistent with this suggestion, intrathecal provision
of both NGF and GDNF, at 12 mg /d each, produced the greatest
rescue of C-fiber CV. In fact, in this case neither the CV distribution nor the mean CV (0.83 6 0.03 m /sec) differed significantly
from that seen in intact animals ( p . 0.05; Kolmogorov–Smirnov
and unpaired t test, respectively). Thus, these results provide an
independent measure of the ability of GDNF and NGF, delivered
by this route and at this dose, to produce a near-complete reversal
of axotomy effects in C-fibers.
DISCUSSION
The principal conclusion of this work is that GDNF is a trophic
factor for a substantial subgroup of adult primary sensory neurons
that are neurotrophin independent. This conclusion is based on
two lines of investigation: the localization of receptor components and the selective rescue effects of exogenous GDNF on
axotomized sensory neurons, as discussed below.
GDNF receptor component expression within
sensory neurons
Figure 7. a, The ratio of the area occupied by I B4, TM P, or CGRP
stained terminals within lamina II of the dorsal horn of the spinal cord on
the axotomized side versus the normal side in animals that have undergone axotomy (n 5 4) or axotomy in combination with an intrathecal
infusion of GDNF at a dose of either 1.2 mg /d (n 5 3) or 12 mg /d (n 5
4) or NGF at a dose of either 1.2 mg /d (n 5 3) or 12 mg /d (n 5 3). GDNF
at a dose of 12 mg/d almost completely prevented the axotomy-induced
reduction in staining intensity of I B4 and TM P ( p , 0.001; unpaired t
test; comparing GDNF with no treatment after axotomy). The lower dose
of GDNF (1.2 mg/d) also had a significant effect in preventing the
axotomy-induced reduction in staining intensity of these markers but was
less effective than the higher dose. The high dose GDNF had a small but
significant effect in preventing the axotomy-induced reduction in CGRP
staining ( p , 0.05; unpaired t test). NGF could almost completely prevent
the axotomy-induced reduction in CGRP staining ( p , 0.001; unpaired t
test; comparing NGF with no treatment after axotomy). NGF at 12 mg /d
had a small but significant effect on the axotomy-induced reduction in I B4
and TM P expression ( p , 0.05; unpaired t test). b, The ratio of the area
occupied by C TB-labeled terminals in lamina II compared with lamina III
of the dorsal horn in normal (n 5 5), axotomized (n 5 4), and axotomy
1 GDNF (Axot. GDNF ) 1.2 mg /d (n 5 3) and 12 mg /d (n 5 4) animals.
Note that there is a significant increase in labeling in lamina II after
axotomy ( p , 0.01; unpaired t test), which is almost completely prevented
by treatment with GDNF at the higher dose. GDNF treatment at the low
dose also had a significant effect ( p , 0.01 compared with no treatment;
unpaired t test) but was less effective than the higher dose.
The signal transducing domain of the GDNF receptor RET was
found to be present in 60 –70% of DRG cell profiles. GFRa-1 and
GFRa-2, the ligand binding domains for GDNF and neurturin,
had a more restricted distribution (they were expressed in 45 and
33% of DRG cell profiles, respectively). GDNF receptor components were strikingly expressed by the IB4 binding, trk-less population of DRG cells. Almost all IB4 cells express RET. Approximately 50% of IB4 cells also express GFRa-1. This implies that
50% of IB4 cells coexpress both receptor components and are
therefore likely to be highly sensitive to GDNF. The other 50%
of the IB4 cells express RET, apparently in the absence of
GFRa-1. GDNF has been reported by some authors to be able to
activate RET in the absence of GFRa-1 and also to be able to act
via the related neurturin receptor component GFRa-2 (Baloh et
al., 1997; Buj-Bello et al., 1997; K lein et al., 1997; Sanicola et al.,
1997). We found that this receptor component was also highly
localized within the IB4-binding population of DRG cells. In
some cells it was coexpressed with GFRa-1 and in others it was
expressed independently of GFRa-1. The majority (80%) of
IB4-binding DRG cells expressed either one or both ligand
binding components. Therefore GDNF may be able to act on a
larger population of IB4 cells than just those that express
GFRa-1. These findings also suggest that a proportion of the
IB4-binding population of DRG cells are likely to be responsive
to neurturin. RET immunoreactive terminals were found to
project principally to lamina IIi of the dorsal horn of the spinal
cord, the same lamina in which the IB4-binding DRG cells
terminate. In contrast to the IB4 cells, the other population of
small diameter DRG cells (those that express trkA) generally lack
RET, GFRa-1, and GFRa-2. Any coexpression is largely accounted for by the known overlap between trkA and IB4 (18% of
trkA cells also bind IB4) (Averill et al., 1995). A significant
number of large diameter DRG cells (as revealed by staining with
N52) express GFRa-1 or RET or both, suggesting that these cells
may also be responsive to GDNF.
Neuroprotective effects of GDNF on sensory neurons
The receptor localization data discussed above shows that a large
proportion of the “neurotrophin-independent” population of
small diameter DRG cells express receptor components for
GDNF. Markers that can be used to define this population
include binding of the lectin IB4 and the enzyme TM P. A small
subset of these cells also expresses the neuropeptide somatosta-
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
J. Neurosci., April 15, 1998, 18(8):3059–3072 3069
Figure 8. Transport of C TB to the dorsal horn of the spinal cord at the level of L3 (a–c), L4 (d–f ), and L5 ( g–i) after sciatic nerve label in control
(CTR L) animals (a, d, g), animals that have undergone axotomy (AXOT ) (b, e, h), and animals that have undergone axotomy combined with GDNF
(AXOT1GDNF ) treatment (12 mg/d) (c, f, i). In the normal animal, C TB-labeled terminals are present in lamina I and the deeper laminae of the dorsal
horn (III–IV) but are excluded from lamina II (a, d, g). After axotomy, C TB-labeled terminals appear in lamina II (denoted by asterisks), and there is
also more dense labeling of axon bundles within lamina I (arrows in b, e, h). These axotomy-related changes are prevented by treatment with GDNF,
where the C TB labeling pattern appears the same as control, and this is seen consistently throughout L3–L5 (c, f, i). Scale bar, 100 mm.
tin. In this study we directly examined whether the IB4-binding
population of sensory neurons would be responsive to GDNF (as
compared with NGF) after axotomy. Intrathecal delivery was
used, which we have previously demonstrated to be effective at
delivering trophic factors to sensory neurons (Bennett et al.,
1996b; Michael et al., 1997).
After axotomy we found that at both doses used, GDNF was
much more effective than NGF at restoring IB4 binding, TM P
staining, and somatostatin expression within both the DRG and
the dorsal horn of the spinal cord. Conversely, NGF was much
more effective than GDNF at restoring CGRP expression within
the DRG and dorsal horn after axotomy. These findings complement those on receptor distribution. NGF has selective effects on
the CGRP-expressing population of DRG cells, whereas conversely, GDNF has selective actions on the IB4-binding (i.e.,
neurotrophin-independent) population of cells. The known overlap between these markers (Averill et al., 1995) probably accounts
for the limited nonselective effects seen. Our electrophysiological
results provide an independent means of assessing the trophic
effects of GDNF and NGF on small diameter DRG cells (Cfibers). GDNF and NGF appeared to prevent conduction velocity
slowing after axotomy in distinct populations of C-fibers, and
importantly, the actions of these factors when administered together were additive. We have shown previously that conduction
velocity slowing in large sensory neurons, induced by axotomy, is
only marginally affected by intrathecal GDNF treatment (Munson and McMahon, 1997).
We have also demonstrated that GDNF could prevent the
axotomy-induced A-fiber sprouting into lamina II of the dorsal
horn. This may represent a direct effect of GDNF on large DRG
neurons or it may occur as a consequence of the rescue effect of
GDNF on IB4-binding small diameter DRG cells. There is now
a body of evidence suggesting that degenerative atrophy of
C-fiber terminals within lamina II after axotomy (Knyihar-Csillik
et al., 1987) is critically important for A-fibers sprouting into this
region. This evidence derives from the fact that C-fibers in the
sciatic nerve have a more restricted mediolateral and rostrocaudal
distribution than sciatic A-fibers, and the sprouting of A-fibers
occurs only in the termination region of axotomized C-fibers
(Woolf et al., 1995). Furthermore, capsaicin, which selectively
damages C-fibers, can induce A-fiber sprouting (Mannion et al.,
1996). We have shown previously that NGF can prevent A-fiber
sprouting (Bennett et al., 1996b), and the demonstration here that
GDNF is also effective is likely to represent the “rescue” of the
3070 J. Neurosci., April 15, 1998, 18(8):3059–3072
Figure 9. The conduction velocity (C V) of C -fibers projecting through
the tibial nerve was measured by stimulation of that nerve electrically and
recording and averaging activity in fine strands of the L5 dorsal root. a
shows a representative recording from an animal in which the tibial nerve
had been cut and tied 2 weeks previously; the animal was treated continuously with intrathecal GDNF and NGF (each at 12 mg/d). Arrows show
examples of individual C -fiber potentials occurring in response to the
stimulation. b, Cumulative sum plots showing the average C V distributions constructed from groups of animals receiving different treatment
(n 5 3–5 animals per group). Error bars show SEM. Note that axotomy
results in a significant slowing of C -fibers (seen as a leftward shift in the
Qsum plots), and both NGF and GDNF partially prevent this slowing.
IB4-binding population of small diameter afferents after axotomy. These results are interesting in that, as we have demonstrated here, NGF and GDNF support largely separate populations of C-fibers and yet either can prevent the sprouting response
after axotomy.
Functional implications of GDNF effects
Our data indicate that GDN F has a potent and selective effect
on the I B4-binding population of DRG cells, and similar
selectivity has recently been observed in vitro (Molliver et al.,
1997; Leclere et al., 1998). The I B4 population of primary
afferents are primarily small in diameter. Given that 80 –90%
of C -fibers are nociceptors (Lynn and C arpenter, 1982; Kress
et al., 1992), I B4-binding DRG cells must be principally nociceptive in f unction (Willis and Coggeshall, 1991). These neurons are capsaicin sensitive (Fitzgerald, 1983) and possess free
nerve endings in various tissues, including skin, muscle, joint,
and viscera. It has recently been shown that this group of
sensory neurons selectively expresses the purinergic receptor
P2X3 (Vulchanova et al., 1996). This receptor is thought to be
important in mediating the nociceptive actions of ATP (Cook
et al., 1997). The sensitivity of this population to GDN F
suggests that this factor may be important in the development
and maintenance of pain-signaling systems.
One important question is whether GDNF is normally required for the phenotypic maintenance of the IB4 population of
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
DRG cells or whether the rescue effects on these cells reflect a
purely pharmacological action. GDNF is produced by peripheral
targets (Trupp et al., 1995) and may normally be available to these
afferents. It is unknown, as yet, whether neurturin also has actions
on the IB4-binding population of DRG cells.
It is interesting that the IB4-binding population of DRG cells
develops such marked sensitivity to GDNF during postnatal
development. During embryonic development these neurons are
dependent for survival on NGF and are absent in animals that
lack trkA (Silos-Santiago et al., 1995). The developmental regulation of RET has been studied by Molliver et al. (1997) in the
mouse. RET was only clearly seen in DRG cells from embryonic
day 15. E xpression increased in IB4 cells during the late embryonic and early postnatal period and reached the adult levels by
approximately postnatal day 7. At the time RET is reaching its
peak, the same neurons downregulate trkA and lose their NGF
sensitivity (Bennett et al., 1996a; Molliver and Snider, 1997).
Molliver et al. (1997) also reported a pattern of GFRa-1/RET
distribution similar to what we report here.
Nerve injury may result in abnormalities of sensation and
importantly the generation of a chronic pain state in both animals
and man. One important mechanism for these changes is the
impaired retrograde transport of trophic factors after nerve injury (McMahon and Bennett, 1997). As a consequence of nerve
injury, there is a large upregulation of GDNF expression within
the damaged nerve (Trupp et al., 1995), as has been shown
previously for NGF (Lindholm et al., 1987). E xpression of
GFRa-1 has also been reported to increase in injured nerves
(Baloh et al., 1997; Trupp et al., 1997), and this may act to present
GDNF to regenerating neurons. Our results imply that the upregulation of GDNF expression in damaged nerves is not sufficient to prevent axotomy-induced changes.
The restitution of IB4 binding, TM P activity, and somatostatin
expression by GDNF after axotomy may indicate a general beneficial action of GDNF in normalizing the properties of damaged
sensory neurons. These anatomical findings were supported by
the evidence that GDNF can partially prevent the conduction
velocity slowing that occurs in a population of C-fibers after
axotomy. The A-fiber sprouting that occurs after axotomy has
previously been implicated in the generation of some aspects of
neuropathic pain. The suggestion is that when the large diameter,
low-threshold mechanoreceptive A-fibers form synapses in lamina II with presumed pain-signaling postsynaptic dorsal horn
systems, this provides an explanation for the condition of allodynia, or touch-evoked pain, that is frequently seen in neuropathic pain patients (Woolf et al., 1992).
Behavioral data suggest that GDNF can exert trophic effects on
IB4 binding nociceptive afferents without altering their responses
to acute noxious thermal and mechanical stimuli (D. L. H. Bennett and S. B. McMahon, unpublished observations). This is in
marked contrast to the actions of NGF, which acts on the other
major group of nociceptors, those expressing trkA. NGF acutely
or chronically administered to animals and man can produce pain
and hyperalgesia (Lewin et al., 1993; Petty et al., 1994; Woolf et
al., 1994; Andreev et al., 1995). Because many forms of neuropathy in man affect small diameter nociceptive afferents, our results suggest that GDNF may be of some use in the treatment of
these conditions. In particular, we would predict that GDNF may
add to, rather than simply substitute for, the effects of NGF in the
treatment of neuropathies.
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
REFERENCES
Andreev NY, Dimitrieva N, Koltzenburg M, McMahon SB (1995) Peripheral administration of nerve growth factor in the adult rat produces
a thermal hyperalgesia that requires the presence of sympathetic postganglionic neurones. Pain 63:109 –115.
Averill S, McMahon SB, C lary SB, Reichardt LF, Priestley JVP (1995)
Immunocytochemical localization of trkA receptors in chemically identified subgroups of adult rat sensory neurons. Eur J Neurosci
7:1484 –1494.
Baloh RH, Tansey MG, Golden JP, Creedon DJ, Heukeroth RO, Keck
CL, Z imonjic DB, Popescu NC, Johnson EM, Milbrandt J (1997)
TrnR2, a novel receptor that mediates neurturin and GDNF signaling
through Ret. Neuron 18:793– 802.
Beck KD, Valverde J, Alexi T (1995) Mesencephalic dopaminergic neurons protected by GDNF from axotomy-induced degeneration in the
adult brain. Nature 373:339 –341.
Bennett DL H, Averill S, C lary DO, Priestley JV, McMahon SB (1996a)
Postnatal changes in the expression of the trkA high affinity NGF
receptor in primary sensory neurons. Eur J Neurosci 8:2204 –2208.
Bennett DL H, French J, Priestley JVP, McMahon SB (1996b) NGF but
not NT-3 or BDNF prevents the A fiber sprouting into lamina II of the
spinal cord that occurs following axotomy. Mol C ell Neurosci
8:211–220.
Bowenkamp KE, Hoffman AF, Gerhardt GA, Henry M A, Biddle P T,
Hoffer BJ, Granholm ACE (1995) Glial cell line-derived neurotrophic
factor supports survival of injured midbrain dopaminergic neurons.
J Comp Neurol 355:479 – 489.
Buj-Bello A, Buchman V L, Horton A, Rosenthal A, Davies AM (1995)
GDNF is an age-specific survival factor for sensory and autonomic
neurons. Neuron 15:821– 828.
Buj-Bello A, Adu J, Pinon LGP, Horton A, Thompson J, Rosenthal A,
Chinchetru M, Buchman V, Davies A (1997) Neurturin responsiveness requires a GPI-linked receptor and Ret receptor tyrosine kinase.
Nature 387:721–724.
C anzian F, Ushijima T, Nagao M, Matera I, Romeo G, C eccherini I
(1995) Genetic mapping of the RET protooncogene on rat chromosome 4. Mamm Genome 6:433– 435.
Cook SP, Vulchanova L, Hargreaves KM, Elde R, McCleskey EW (1997)
Distinct ATP receptors on pain-sensing and stretch sensing neurons.
Nature 387:505–508.
Durbec P, Marcos-Gutierrez C V, Kilkenny C, Grigoriou M, Wartiowaaria K, Suvanto P, Smioth D, Ponder B, Costantini F, Saarma M,
Sariola H, Pachnis V (1996) GDNF signalling through the Ret receptor tyrosine kinase. Nature 381:789 –793.
Fitzgerald M (1983) C apsaicin and sensory neurons: a review. Pain
15:109 –130.
GFRa Nomenclature Committee (1997) Nomenclature of GPI linked
receptors for the GDNF ligand family. Neuron 19:485.
Henderson CE, Phillips HS, Pollock RA, Avies A, Lemeulle C, Armanini
M, Simmons L, Moffet B, Vandlen R (1994) GDNF: a potent survival
factor for motoneurons present in peripheral nerve and muscle. Science
266:1062–1064.
Jing S, Wen D, Yu Y, Holst PL, Luo Y, Fang M, Tamir R, Antonio L, Hu
Z, Cupples R, Louis JC, Hu S, Altrock BW, Fox GM (1996) GDNFinduced activation of the ret protein tyrosine kinase is mediated by
GDNFR-alpha, a novel receptor for GDNF. C ell 85:1113–1124.
Kashiba
H,
Ueda
Y,
Senba
E (1996) Coexpression
of
preprotachykinin-A, alpha-calcitonin gene-related peptide, somatostatin, and neurotrophin receptor family messenger RNAs in rat dorsal
root ganglion neurons. Neuroscience 70:179 –189.
Klein RD, Sherman D, Ho W H, Stone D, Bennett G, Moffat B, Vandlen
R, Simmons L, Gu Q, Hongo J, Devaux B, Poulsen K, Armanini M,
Nozaki C, Asai N, Goddard A, Phillips H, Henderson C, Takahashi M,
Rosenthal A (1997) A GPI-linked protein that interacts with RET to
form a candidate neurturin receptor. Nature 387:717–721.
Knyihar-Csillik E, Rakic P, C sillik B (1987) Transganglionic degenerative atrophy in the substantia gelatinosa of the spinal cord after peripheral nerve transection in rhesus monkeys. C ell Tissue Res 247:599 – 604.
Kotzbauer P T, Lampe PA, Heukeroth RO, Golden JP, Creedon DJ,
Johnson EM, Milbrandt JD (1996) Neurturin a relative of glial-cellline-derived neurotrophic factor. Nature 6608:467– 470.
Kress M, Koltzenburg M, Reeh PW, Handwerker HO (1992) Responsiveness and functional attributes of electrically localized terminals of
cutaneous C -fibres in vivo and in vitro. J Neurophysiol 68:581–595.
Leclere PG, Ekstrom P, Edstrom A, Priestley JV, Averill S, Tonge DA
J. Neurosci., April 15, 1998, 18(8):3059–3072 3071
(1998) Effects of glial cell line-derived neurotrophic factor on axonal
growth and apoptosis in adult mammalian sensory neurons in vitro.
Neuroscience 82:545–558.
Lewin GR, Ritter AM, Mendell LM (1993) Nerve growth factorinduced hyperalgesia in the neonatal and adult rat. J Neurosci
230:2136 –2148.
Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F (1993) GDNF: a
glial cell line-derived neurotrophic factor for midbrain dopaminergic
neurons. Science 260:1130 –1132.
Lindholm D, Heumann R, Meyer M, Thoenen H (1987) Interleukin-1
regulates synthesis of nerve growth factor in non-neuronal cells rat
sciatic nerve. Nature 330:658 – 659.
Lynn B, C arpenter SE (1982) Primary afferent units from the hairy skin
of the rat hind limb. Brain Res 238:29 – 43.
Mannion RJ, Doubell TP, Coggeshall RE, Woolf C J (1996) Collateral
sprouting of uninjured primary afferent A-fibers into the superficial
dorsal horn of the adult rat spinal cord after topical capsaicin treatment
of the sciatic nerve. J Neurosci 16:5189 –5195.
Matheson CR, Garnahan J, Urich JL, Bocangel D, Z hang TJ, Yan Q
(1997) Glial cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor for sensory neurons: comparison with the effects of the
neurotrophins. J Neurobiol 32:22–32.
McMahon SB (1986) The localization of fluoride-resistant acid phosphatase (FR AP) in the pelvic nerves and sacral spinal cord of rats.
Neurosci Lett 64:305–310.
McMahon SB, Bennett DL H (1997) Growth factors and pain. In: Handbook of pharmacol 130 (Dickenson A, Besson J-M), pp 135–157. Berlin:
Springer.
McMahon SB, Armanini M P, Ling LH, Phillips HS (1994) E xpression
and coexpression of Trk receptors in subpopulations of adult primary
sensory neurons projecting to identified peripheral targets. Neuron
12:1161–1171.
Michael GJ, Priestley JV (1996a) E xpression of trkA and p75 nerve
growth factor receptors in the adrenal gland. NeuroReport
7:1617–1622.
Michael, GJ, Priestley JV (1996b) Combined immunocytochemistry and
in situ hybridization. In: In situ hybridization techniques for the brain
(Henderson Z, ed), pp 111–118. New York: Wiley.
Michael GJ, Averill S, Nitkunan A, Rattray M, Bennett DL H, Yan Q,
Priestley JV (1997) Nerve growth factor treatment increases brainderived neurotrophic factor selectively in trkA-expressing dorsal root
ganglion cells and in their central terminations within the spinal cord.
J Neurosci 17:8476 – 8490.
Molliver DC, Snider W D (1997) Nerve growth factor receptor trkA is
down-regulated during postnatal development by a subset of dorsal root
ganglion neurons. J Comp Neurol 381:428 – 438.
Molliver DC, Radeke MJ, Feinstein SC, Snider W D (1995) Presence or
absence of trkA protein distinguishes subsets of small sensory neurons
with unique cytochemical characteristics and dorsal horn projections.
J Comp Neurol 361:404 – 416.
Molliver DC, Wright DE, Leitner ML, Parsadanian AS, Doster K, Wen
D, Yan Q, Snider W D (1997) I B4-binding nociceptors switch from
NGF to GDNF dependence in early postnatal life. Neuron 19:849 – 861.
Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips HS,
Reichart LF, Ryan AM, C arver-Moore K, Rosenthal A (1996) Renal
and neuronal abnormalities in mice lacking GDNF. Nature 382:76 –79.
Munson JB, McMahon SB (1997) Effects of GDNF on axotomised sensory and motor neurons in adult rats. Eur J Neurosci 9:1126 –1129.
Oppenheim RW, Houenou LJ, Johnson JE, Lin L-F H, Li L, Lo AC,
Newsome AL, Prevette DM, Wang S (1995) Developing motor neurons rescued from programmed and axotomy-induced cell death by
GDNF. Nature 373:344 –346.
Petty BG, Cornblath DR, Adornato BT, Chaudhry V, Flexner C, Wachsman M, Sinicropi D, Burton LE, Peroutka SJ (1994) The effect of
systemically administered recombinant human nerve growth factor in
healthy human subjects. Ann Neurol 36:244 –246.
Sanicola M, Hession C, Worley P, C armillo P, Ehrenfels C, Walus L,
Robinson S, Jaworski G, Wei H, Tizard R, Whitty A, Pepinsky R, C ate
R (1997) Glial cell line-derived neurotrophic factor-dependent RET
activation can be mediated by two different cell-surface accessory
proteins. Proc Natl Acad Sci USA 94:6238 – 6243.
Silos-Santiago I, Molliver DC, Ozaki S, Smeyne RJ, Fagan AM, Barbacid
M, Snider W D (1995) Non-TrkA-expressing small DRG neurons are
lost in TrkA deficient mice. J Neurosci 15:5929 –5942.
Silverman JD, Kruger L (1990) Selective neuronal glycoconjugate ex-
3072 J. Neurosci., April 15, 1998, 18(8):3059–3072
pression in sensory and autonomic ganglia: relation of lectin reactivity
to peptide and enzyme markers. J Neurocytol 19:789 – 801.
Treanor JJ, Goodman L, de Sauvage F, Stone DM, Poulsen KT, Beck CD,
Gray C, Armanini M P, Pollock RA, Hefti F, Phillips HS, Goddard A,
Moore MW, Buj-Bello A, Davies AM, Asai N, Takahashi M, Vandlen
R, Henderson CE, Rosenthal A (1996) Characterization of a multicomponent receptor for GDNF. Nature 382:80 – 83.
Trupp M, Ryden M, Jornvall H, Funakoshi H, Timmusk T, Arenas E,
Ibanez CF (1995) Peripheral expression and biological activities of
GDNF, a new neurotrophic factor for avian and mammalian peripheral
neurons. J C ell Biol 130:137–148.
Trupp M, Arenas E, Fainzilber M, Nilsson A-S, Sieber B-A, Grigoriou M,
Kilkenny C, Salazar-Grueso E, Pachnis V, Arumae U, Sariola H,
Saarma M, Ibanez CF (1996) Functional receptor for GDNF encoded
by the c-ret proto-oncogene. Nature 381:785–788.
Trupp M, Belluardo N, Funakoshi H, Ibanez CF (1997) Complementary
and overlapping expression of glial cell line-derived neurotrophic factor
(GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates
multiple mechanisms of trophic actions in the adult rat C NS. J Neurosci
17:3554 –3567.
Verge V M, Richardson PM, Wiesenfeld-Hallin Z, Hokfelt T (1995)
Differential influence of nerve growth factor on neuropeptide expression in vivo: a novel role in peptide suppression in adult sensory
neurons. J Neurosci 15:2081–2096.
Bennett et al. • GDNF Is Trophic for IB4-Binding DRG Cells
Verge V M, Gratto KA, Karchewski LA, Richardson PM (1996) Neurotrophins and nerve injury in the adult. Philos Trans R Soc Lond B Biol
Sci 351:423– 430.
Vulchanova L, Riedl M, Shuster S, Wang J, Buell G, Surprenant A, North
RA, Elde R, (1996) Immunohistochemical localization of the P2X3
receptor subunit in rat dorsal root ganglion (DRG) neurons. Soc
Neurosci Abstr 22:1810.
Willis W D, Coggeshall RE (1991) Sensory mechanisms of the spinal
cord, Ed 2. New York: Plenum.
Woolf C J, Shortland P, Coggeshall RE (1992) Peripheral nerve injury
triggers central sprouting of myelinated afferents. Nature 355:75–78.
Woolf C J, Safieh-Garabedian B, Ma QP, Crilly P, Winter J (1994) Nerve
growth factor contributes to the generation of inflammatory sensory
hypersensitivity. Neuroscience 62:327–331.
Woolf C J, Shortland P, Reynolds M, Ridings J, Doubell T, Coggeshall
RE (1995) Reorganization of central terminals of myelinated primary
afferents in the rat dorsal horn following peripheral axotomy. J Comp
Neurol 360:121–134.
Wright DE, Snider W D (1995) Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia.
J Comp Neurol 351:329 –338.
Yan Q, Matheson C, Lopez OT (1995) In vivo neurotrophic effects of
GDNF on neonatal and adult facial motor neurons. Nature 373:
341–344.