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Neuronal Phenotype and Tyrosine Kinase Receptor Expression in Cocultures of Dorsal Root Ganglion and Skeletal Muscle Cells.

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THE ANATOMICAL RECORD 292:107–112 (2009)
Neuronal Phenotype and Tyrosine
Kinase Receptor Expression in
Cocultures of Dorsal Root Ganglion and
Skeletal Muscle Cells
LIHONG WANG,1 ZHEN LIU,1 HUAXIANG LIU,2 YU WAN,3
HUAIJING WANG,1 AND ZHENZHONG LI1*
1
Department of Anatomy, Shandong University School of Medicine, Jinan, China
2
Department of Rheumatology, Shandong University Qilu Hospital, Jinan, China
3
Key Lab of Medical Neurobiology in Universities of Shandong Province, Shandong
University School of Medicine, Jinan, China
ABSTRACT
The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons
are two major phenotypical classes in dorsal root ganglion (DRG). Tyrosine kinase receptor (Trk)A, TrkB, and TrkC are three members of the
Trk family which may be relevant to neuronal phenotypes. Whether target skeletal muscle cells generate their expression remains unclear. Neurons containing substance P (SP), calcitonin gene-related peptide (CGRP),
neurofilament 200 (NF-200), TrkA, TrkB, and TrkC were quantified using
immunohistochemistry in rat DRG neuronal cultures and cocultures of
DRG neurons and skeletal muscle cells. The percentage of NF-200 and
TrkC-expressing neurons in cocultures of DRG neurons and skeletal muscle cells was significantly higher, 26.86% 6 3.17% (NF-200) and 27.74% 6
3.63% (TrkC) compared with 20.92% 6 1.98% (NF-200) and 16.70% 6
3.68% (TrkC) in DRG cultures; whereas the percentage of SP, CGRP,
TrkA, and TrkB-expressing neurons was not changed significantly by the
addition of target skeletal muscle cells. Thus, target skeletal muscle cells
may influence neurofilament-phenotype and TrkC receptor but not neuropeptide-phenotype and TrkA and TrkB receptors. Anat Rec, 292:107–
112, 2009. Ó 2008 Wiley-Liss, Inc.
Key words: tyrosine kinase receptor; substance P; calcitonin
gene-related peptide; neurofilament; phenotype;
neuron; dorsal root ganglion
Targets of neuronal innervation play a vital role in
regulating the survival and differentiation of innervating neurotrophin-responsive neurons (Howe et al., 2001).
However, it is still largely unclear whether target skeletal muscle cells influence dorsal root ganglion (DRG)
neuronal phenotyes and tyrosine kinase receptors (Trk)
expression.
The neuropeptide-immunoreactive (IR) and neurofilament-IR neurons are two major phenotypical classes in
DRG. Neuropeptide-IR neurons are considered to be
with unmyelinated or thinly myelinated nociceptive
afferents which are considered to innervate skin and viscera. Neurotransmitter expression in neurons is regulated by innervated target tissue and intrinsic neuronal
Ó 2008 WILEY-LISS, INC.
Lihong Wang and Zhen Liu contributed equally to this
article.
Grant sponsor: National Natural Science Foundation of
China; Grant number: 30770888; Grant sponsor: Natural Science Foundation of Shandong Province of China; Grant number: Z2006D05; Grant sponsor: Foundation for Excellent Young
Scientists in Shandong Province of China; Grant number:
02BS091.
*Correspondence to: Zhenzhong Li, Department of Anatomy,
Shandong University School of Medicine, Jinan 250012, China.
E-mail: zli@sdu.edu.cn
Received 23 June 2008; Accepted 15 July 2008
DOI 10.1002/ar.20777
Published online 2 December 2008 in Wiley InterScience (www.
interscience.wiley.com).
108
WANG ET AL.
properties (Braun et al., 1996). Cultured neurons
expressed neuropeptides with a time course and in proportions similar to those in vivo (Hall et al., 1997). Neurofilament-IR neurons typically have myelinated axons
which are considered to innervate muscle spindle. Neurofilament-IR neurons are also present in DRG cell cultures (Hall et al., 1997). It has been identified that DRG
neurons express three Trk receptors (TrkA, TrkB, and
TrkC) that only minimally overlap. TrkA-expressing
neurons are mostly small cells with unmyelinated axons
and are thought to innervate nociceptors and thermoreceptors. TrkC-expressing neurons are mostly large cells
with myelinated axons and likely receive input from peripheral mechanoreceptors. TrkB-expressing cells vary
from small to large and are generally medium sized and
are likely to have myelinated axons (Wright and Snider,
1995). The activation of Trk receptors seems to be essential in most cases for the biological effects of neurotrophins (Barbacid, 1994).
Target tissues are essential for the maintenance of the
function of neurons and nerve–muscle communication.
In the absence of limb-derived trophic signals, the
affected neurons fail to survive. Limb-bud targets removal results in the loss of spinal sensory neurons that
normally innervate the limbs in the chick embryo. Sensory neurons lost in the absence of target-derived
trophic support should be rescued by treatment with
muscle extract or neurotrophic factors which are known
to be required for the survival of these neurons (Snider,
1994; Caldero et al., 1998). Extracellular application of
skeletal myosin II to embryonic sensory neurons
resulted in increasing axon formation and branching in
each neuron in vitro (Silver and Gallo, 2005). It has
been shown that the increase in excitability of skeletal
muscle is regulated by the excitatory afferents in the
spinal cord-DRG-skeletal muscle organotypic coculture
system (Streit et al., 1991).
There is some evidence for phenotypic plasticity in
sensory neurons (Hall et al., 1997). Outgrowing sensory
neurons appear to have more flexibility in their pathway
and target choices than do motoneurons in vivo (Wang
and Scott, 1999). However, much less is known about
the influence of target muscle cells on DRG neuronal
phenotyes and Trk receptors. Here, we have established
a neuromuscular coculture model of DRG neurons and
skeletal muscle cells to test what extent to the expression of substance P (SP), calcitonin gene-related peptide
(CGRP), neurofilament 200 (NF-200), TrkA, TrkB, and
TrkC in DRG neurons in the presence of target skeletal
muscle cells when compared with that in DRG culture
in the absence of skeletal muscle cells.
MATERIALS AND METHODS
Cell Cultures
DRG cell culture preparations utilized embryonic 12.5day-old Wistar rats maintained in the Experimental Animal Center at Shandong University of China. Under
aseptic conditions, the bilateral DRG was removed from
each embryo, placed in culture medium, and minced into
fragments 0.5 mm in diameter, digested with 0.25%
trypsin (Sigma) in D-Hanks solution at 378C for 10 min,
centrifuged, and triturated in growth media supplemented with 5% fetal bovine serum (Gibco). Dissociated
DRG cells were plated at a density of 23105 cells/mL in
6-well clusters (Costar, Corning, NY) which would
contain 24 mm diameter coverslips precoated with polyL-lysine (0.1 mg/mL) and then incubated at 378C in a 5%
CO2 incubator.
Skeletal muscle cell culture preparations utilized newborn Wistar rats maintained in the Experimental Animal Center at Shandong University of China. Skeletal
muscle cell cultures were prepared separately from, but
simultaneously with, the primary cultures of DRG cells.
Under aseptic conditions and using the newborn rat,
skeletal muscle was removed from the hind limb of each
animal, minced with fine dissecting scissors into fragments 0.5 mm in diameter, digested with 0.25% trypsin (Sigma) in D-Hanks solution at 378C for 20 min, centrifuged, and triturated in growth media supplemented
with 5% fetal bovine serum (Gibco). Isolated skeletal
muscle cells were plated at a density of 23105 cells/mL
in flasks (Costar, Corning, NY) and then incubated at
378C in a 5% CO2 incubator for 2 hr to remove nonskeletal muscle cells.
At this time, the neuromuscular cocultures were prepared as follows. The skeletal muscle cell suspension
was transferred from the flasks to the 6-well clusters
which would contain dissociated DRG cells. Cultures of
dissociated DRG cells alone were maintained continuously in culture media. All these culture preparations
were incubated at 378C in a 5% CO2 incubator for 6
days with media change every 2 days.
The composition of the culture media is DMEM/F-12
(1:1) supplemented with 10% fetal bovine serum, 2% B27 supplement (Gibco), L-glutamine (0.1 mg/mL, Sigma),
insulin (0.25 mg/mL, Sigma), penicillin (100 U/mL), and
streptomycin (100 mg/mL).
Double Fluorescent Labeling of MAP2 and SP,
CGRP, NF-200, TrkA, TrkB, or TrkC
At 6 days of culture age, DRG cultures and neuromuscular cocultures were processed for double immunofluorescent labeling of microtubule associated protein 2
(MAP2) and SP, CGRP, NF-200, TrkA, TrkB, or TrkC.
The cells on coverslips were rinsed quickly one time in
0.1 mol/L Sorenson’s phosphate buffer to remove media.
The cells were fixed in 4% paraformaldehyde, pH 7.4,
for 20 min at 48C. After washing in 0.1 mol/L phosphate
buffer saline (PBS) for three times, the cells were
blocked by 2% normal goat serum in 0.1% Triton PBS to
block nonspecific sites and permeates cells. The samples
were incubated with rabbit polyclonal anti-SP, CGRP,
NF-200, TrkA, TrkB, or TrkC (1:500) overnight at 48C,
respectively. After washing in 0.1 mol/L PBS three
times, the samples were incubated by goat anti-rabbit
conjugated to Cy3 (1:200) for 45 min in dark. After
washing for three times in 0.1 mol/L PBS, the cells were
incubated with mouse monoclonal anti-MAP2 (1:200) for
60 min in dark. After washing three times in 0.1 mol/L
PBS, the cells were incubated with goat anti-mouse conjugated to Cy2 (1:200) for 45 min in dark. After washing
in 0.1 mol/L PBS, the cells were coverslipped immediately with Vectashield anti-fade mounting media (Vecto
Laboratories, Inc.) and stored at 48C until observation
by fluorescent microscope.
NEURONAL PHENOTYPE AND TYROSINE KINASE RECEPTOR EXPRESSION
Quantitative Analysis of the Percentage
of Neuropeptide-, NF-200-, and
Trk-expressing Neurons
SP-IR, CGRP-IR, NF-200-IR, TrkA-IR, TrkB-IR, or
TrkC-IR neurons were observed under a fluorescent
microscope (Nikon) with 203 objective lens. SP-IR,
CGRP-IR, NF-200-IR, TrkA-IR, TrkB-IR, or TrkC-IR
neurons in five visual fields in the central part of each
coverslip were counted as the positive neurons in each
sample. The number of total neurons (MAP2-IR) were
also counted in the same visual field. Then, the percentage of SP-IR, CGRP-IR, NF-200-IR, TrkA-IR, TrkB-IR,
or TrkC-IR neurons would be obtained.
Statistical Analysis
Data are expressed as mean 6 SD. Statistical analysis
was evaluated with SPSS software by one-way ANOVA
followed by the Student-Newman-Keuls test for significance to compare the differences among various groups.
Significance was accepted at P < 0.05.
RESULTS
The Effects of Skeletal Muscle Cell on DRG
Neuronal Phenotyes
To test the effects of skeletal muscle cells on SP,
CGRP, and NF-200 expression in DRG neurons, DRG
cells were cultured for 6 days with or without target
skeletal muscle cells and processed for double fluorescent labeling of MAP2 and SP, CGRP, or NF-200, and
then, DRG neurons containing SP, CGRP, and NF-200
were quantified. 117 SP-IR, 110 CGRP-IR, and 192 NF200-IR neurons are in 914, 1121, and 910 total DRG
neurons, respectively, in the absence of skeletal muscle
cells. 130 SP-IR, 114 CGRP-IR, and 279 NF-200-IR neurons are in 947, 1066, and 1053 total DRG neurons,
respectively, in the presence of skeletal muscle cells. The
target skeletal muscle cells could promote NF-200
expression but not neuropeptides. 26.86% 6 3.17% of
DRG neurons expressed NF-200 in neuromuscular cocultures which is higher than that in DRG cultures alone
(20.92% 6 1.98% NF-200-expressing neurons of total
cells). The percentage of SP-IR (13.63% 6 1.85%) and
CGRP-IR (11.00% 6 3.93%) neurons in neuromuscular
cocultures is not changed significantly as compared with
that in DRG cultures alone (12.65% 6 2.38% SP-IR and
10.2% 6 5.87% CGRP-IR neurons) (Fig. 1).
The Effects of Skeletal Muscle Cell on Trk
Expression in DRG Neurons
To test the effects of skeletal muscle cells on Trk
receptors expression in DRG neurons, DRG cells were
cultured for 6 days with or without target skeletal muscle cells and processed for double fluorescent labeling of
MAP2 and TrkA, TrkB, or TrkC, and then, TrkA-IR,
TrkB-IR, or TrkC-IR DRG neurons were quantified. 232
TrkA-IR, 95 TrkB-IR, and 158 TrkC-IR neurons are in
910, 905, and 933 total DRG neurons, respectively, in
the absence of skeletal muscle cells. 271 TrkA-IR, 111
TrkB-IR, and 269 TrkC-IR neurons are in 974, 949, and
962 total DRG neurons, respectively, in the presence of
skeletal muscle cells. The target skeletal muscle cells
109
could promote TrkC expression but not TrkA and TrkB.
DRG neurons (27.74% 63.63 %) expressed TrkC in neuromuscular cocultures which is higher than that in DRG
cultures alone (16.7% 6 3.68% TrkC-expressing neurons
of total cells). The percentage of TrkA-IR (27.75% 6
1.91%) and TrkB-IR (11.61% 6 1.88%) neurons in neuromuscular cocultures is not changed significantly when
compared with that in DRG cultures alone (25.30% 6
3.00% TrkA-IR and 10.31% 6 3.09% TrkB-IR neurons)
(Fig. 2).
DISCUSSION
The aim of this study was to approach the question of
neuronal phenotyes and Trk receptors expressing dependence on target skeletal muscle cells during development. As previous reports, multiple appropriate sensory
neuron phenotypes arise in a regulated fashion in cultured neurons isolated before target connections have
formed, and some candidate target tissues can modulate
that intrinsic expression pattern (Hall et al., 1997). The
identification of the cell types that express the Trk
receptors is of particular interest because neurons subserving specific sensory modalities depend on distinct
neurotrophins for survival during development (Stephens et al., 2005). In this study, we found that: (1) the
percentage of DRG neurons contained neurofilament
increased significantly in neuronmuscular cocultures of
DRG neurons and target skeletal muscle cells as compared with that in DRG neuronal cultures, whereas the
percentage of DRG neurons contained SP or CGRP in
neuronmuscular cocultures was not changed significantly as compared with that in DRG cultures and (2)
the percentage of TrkC-expressing DRG neurons
increased significantly in neuronmuscular cocultures of
DRG neurons and target skeletal muscle cells when
compared with that in DRG neuronal cultures, whereas
the percentage of TrkA- or TrkB-expressing DRG neurons in neuronmuscular cocultures was not changed significantly when compared with that in DRG cultures.
It has been shown that the developmental regulation
of sensory neurons containing CGRP that predominantly
contact visceral and cutaneous peripheral target end
organs in vivo (Hall et al., 1997), whereas abundant
neurofilament expression is characteristic of large neurons that innervate muscle spindles (Lawson et al.,
1984; Perry et al., 1991). The results in this study provide an important demonstration that neurofilamentphenotype but not neuropeptide-phenotype could be
regulated by the presence of target skeletal muscle cells.
These results are consistent with that SP-IR and CGRPIR neurons did not require an intact DRG or connections
with other tissues to regulate neuropeptides often
expressed by nociceptive neurons (Hall et al., 1997). In
the present experiment, DRG was dissected out from
embryonic rats on embryonic days 12.5 (E12.5) before
neuropeptides appear in DRG neurons (Hall et al.,
1997). At E12.5, many DRG neuron precursors are
undergoing their final mitoses, and most have not
extended peripheral processes (Lawson et al., 1984;
Mirnics and Koerber, 1995). These results implicated
that some neurons in the embryonic DRG seem to be
intrinsically specified to later express SP and CGRP.
Neurofilament-IR neurons typically have myelinated
axons which are considered to innervate muscle spindle
110
WANG ET AL.
Fig. 1. Panels A and B are double-fluorescent labeling of MAP2
and NF-200 in DRG neurons in absence or presence of skeletal muscle cells at 6 days of culture age (Scale bar 5 50 mm). Panel A is DRG
cultures in absence of skeletal muscle cells (A1: NF-200; A2: MAP2;
A3: overlay of A1 and A2). Panel B is cocultures of DRG neurons and
skeletal muscle cells (B1: NF-200; B2: MAP2; B3: overlay of B1 and
B2). Panel C is the percentage of SP-, CGRP-, and NF-200-expressing DRG neurons in absence or presence of skeletal muscle cells at
6 days of culture age (n 5 5). *P < 0.01.
(Hall et al., 1997). In DRG and skeletal muscle fibers cocultures, peripheral neurites of DRG neurons coiling
around regenerated muscle fibers correspond to that
part of the sensory muscle spindle apparatus which
developed in vivo (Spenger et al., 1991). Our results are
in agreement with the earlier previous reports. Neurofilament expression is dependent on, at least in part, the
presence of target skeletal muscle cells.
Activation of receptor trkA seems important for the
survival of cutaneous and visceral afferents, whereas
trkC may be more important for muscle afferents (HoryLee et al., 1993; Crowley et al., 1994; Klein et al., 1994;
McMahon et al., 1994; Smeyne et al., 1994; Hall et al.,
1997). It has been suggested that the distribution of
TrkC is necessary for the maintenance of the DRG proprioceptive neurons during embryonic development in
mice (Stephens et al., 2005). TrkC-expressing neurons
are mostly large cells (Wright and Snider, 1995) which
are in a direct contact with the skeletal muscle target
(Stephens et al., 2005). In this study, the percentage of
TrkA- and TrkB-expressing DRG neurons observed in
both DRG cultures and neuromuscular cocultures is similar to that in DRG in vivo (Snider, 1994; Wright and
Snider, 1995). The percentage of TrkC-expressing DRG
neurons in DRG neuronal cultures observed in this
study is lower than that in DRG in vivo (Snider, 1994;
Stephens et al., 2005). In neuromuscular cocultures of
DRG neurons and target skeletal muscle cells, the percentage of TrkC-expressing DRG neurons is similar to
that in DRG in vivo (Wright and Snider, 1995) suggested
that TrkC expression is partially dependent on the presence of target skeletal muscle cells.
NEURONAL PHENOTYPE AND TYROSINE KINASE RECEPTOR EXPRESSION
111
Fig. 2. Panels A and B are double fluorescent labeling of MAP2
and TrkC in DRG neurons in absence or presence of skeletal muscle
cells at 6 days of culture age (Scale bar 5 50 mm). Panel A is DRG
cultures in absence of skeletal muscle cells (A1: TrkC; A2: MAP2; A3:
overlay of A1 and A2). Panel B is cocultures of DRG neurons and skel-
etal muscle cells (B1: TrkC; B2: MAP2; B3: overlay of B1 and B2).
Panel C is the percentage of TrkA-, TrkB-, and TrkC-expressing DRG
neurons in absence or presence of skeletal muscle cells at 6 days of
culture age (n 5 5). *P < 0.01.
In conclusion, our data suggest that target skeletal
muscle cells are important for maintenance neurofilament phenotype but not neuropeptide phenotype and
maintenance TrkC but not TrkA and TrkB expression in
cultured DRG neurons. The expression of neurofilament
and TrkC is regulated by target skeletal muscle cells is
consistent with the close relationship between neurofilament- or TrkC-expressing DRG neurons and target skeletal muscle cells in vivo. Additional studies are necessary to clarify the mechanisms of interactions between
neurofilament- or TrkC-expressing DRG neurons and
target skeletal muscle cells.
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