TRPA1 Is Functionally Expressed Primarily by IB4-
Binding, Non-Peptidergic Mouse and Rat Sensory
Marie E. Barabas., Elena A. Kossyreva., Cheryl L. Stucky*
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America
Subpopulations of somatosensory neurons are characterized by functional properties and expression of receptor proteins
and surface markers. CGRP expression and IB4-binding are commonly used to define peptidergic and non-peptidergic
subpopulations. TRPA1 is a polymodal, plasma membrane ion channel that contributes to mechanical and cold
hypersensitivity during tissue injury, making it a key target for pain therapeutics. Some studies have shown that TRPA1 is
predominantly expressed by peptidergic sensory neurons, but others indicate that TRPA1 is expressed extensively within
non-peptidergic, IB4-binding neurons. We used FURA-2 calcium imaging to define the functional distribution of TRPA1
among peptidergic and non-peptidergic adult mouse (C57BL/6J) DRG neurons. Approximately 80% of all small-diameter
(,27 mm) neurons from lumbar 1–6 DRGs that responded to TRPA1 agonists allyl isothiocyanate (AITC; 79%) or
cinnamaldehyde (84%) were IB4-positive. Retrograde labeling via plantar hind paw injection of WGA-Alexafluor594 showed
similarly that most (81%) cutaneous neurons responding to TRPA1 agonists were IB4-positive. Additionally, we cultured DRG
neurons from a novel CGRP-GFP mouse where GFP expression is driven by the CGRPa promoter, enabling identification of
CGRP-expressing live neurons. Interestingly, 78% of TRPA1-responsive neurons were CGRP-negative. Co-labeling with IB4
revealed that the majority (66%) of TRPA1 agonist responders were IB4-positive but CGRP-negative. Among TRPA1-null
DRGs, few small neurons (2–4%) responded to either TRPA1 agonist, indicating that both cinnamaldehyde and AITC
specifically target TRPA1. Additionally, few large neurons ($27 mm diameter) responded to AITC (6%) or cinnamaldehyde
(4%), confirming that most large-diameter somata lack functional TRPA1. Comparison of mouse and rat DRGs showed that
the majority of TRPA1-responsive neurons in both species were IB4-positive. Together, these data demonstrate that TRPA1
is functionally expressed primarily in the IB4-positive, CGRP-negative subpopulation of small lumbar DRG neurons from
rodents. Thus, IB4 binding is a better indicator than neuropeptides for TRPA1 expression.
Citation: Barabas ME, Kossyreva EA, Stucky CL (2012) TRPA1 Is Functionally Expressed Primarily by IB4-Binding, Non-Peptidergic Mouse and Rat Sensory
Neurons. PLoS ONE 7(10): e47988. doi:10.1371/journal.pone.0047988
Editor: Bradley Taylor, University of Kentucky Medical Center, United States of America
Received August 17, 2012; Accepted September 19, 2012; Published October 25, 2012
Copyright: ? 2012 Barabas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Supported by National Institutes of Health grants NS40538 and NS070711 to CLS. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Sensory nerve terminals detect peripheral stimuli in order to
discern touch, temperature and pain. These neurons make up a
considerably heterogeneous population that can be classified into
subgroups based on peripheral targets, functional properties and
central projections[1–3]. Larger-diameter neurons ($27 um) tend
to have myelinated axons, as with Ab and Ad fibers in vivo, whereas
smaller-diameter neurons (,27 mm) tend to have unmyelinated, C
fiber axons in vivo . Calcitonin Gene-Related Peptide (CGRP)
expression and isolectin B4 (IB4) binding are two common
histochemical markers used to define subpopulations of small-
diameter neurons[1–4]. CGRP is expressed by peptidergic sensory
neurons[3–6], whereas IB4 binds to a-D-galactose carbohydrate
residues typically expressed on the plasma membrane of non-
peptidergic neurons[4,7–9]. In peripheral skin targets, IB4-binding
neurons terminate superficially between keratinocytes within the
epidermis, whereas peptidergic neurons terminate in deeper layers
of the epidermis and dermis . Centrally, peptidergic neurons
terminate in the outer laminae (I and outer II) of the spinal dorsal
horn and target spinal neurons that transmit nociceptive
information to the thalamus or brainstem nuclei, regions that
mediate the sensory discriminative aspects of pain[10–16]. In
contrast, the non-peptidergic, IB4-binding population primarily
terminates on interneurons within the inner lamina II of the spinal
cord and target interneurons that express PKCc[10,10–14,17].
Rostrally, the input from IB4-binding neurons ultimately projects
to brain areas including the amygdala and hypothalamus, regions
involved in affective components of pain . Therefore, it is
reasonable to hypothesize that specific receptors that transduce
environmental or endogenous stimuli would distribute preferen-
tially between peptidergic and non-peptidergic subpopulations in
order to provide selective input to these diverse neural pain
The Transient Receptor Potential Ankyrin 1 (TRPA1) channel
has been the focus of intense interest for its role in inflammatory
nociception[18–21] and its potential function in transduction of
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e47988
mechanical and cold signals[20,22–25]. Previous investigations of
the distribution of TRPA1 among peptidergic and non-peptidergic
(IB4-positive) neurons have largely employed in situ hybridization
and immunohistochemical techniques, which have produced
disparate results[18,21,26–28]. Some studies indicate greater
TRPA1 expressionin peptidergic,
[18,21,26,27], while others found more TRPA1 in non-peptider-
gic, IB4-binding neurons[27–29]. For example, Story and
colleagues  found TRPA1 mRNA in 3.6% of dorsal root
ganglia (DRG) neurons from adult rat, and a majority of these
TRPA1-positive cells expressed CGRP. On the other hand,
Caspani and colleagues  reported that 28% of lumbar 3–6
DRG neurons from adult mouse contain TRPA1 mRNA but only
2–3% of these neurons were CGRP-positive. Discrepancies
between studies may have resulted from the inherent limitations
of in situ hybridization and immunohistochemistry techniques. For
in situ hybridization, the presence of mRNA does not always
accurately predict that the respective protein will be expressed, as
RNA can have a high turnover rate and become degraded prior to
translation. Immunohistochemistry can sometimes lead to false-
positives due to non-specific binding of antibodies or false-
negatives due to lower sensitivity of antibodies. Further, receptors
might be retained in internal organelles and not functionally
expressed at the cell membrane, as has been shown for TRPA1
. The sensitivity of both mRNA and antibody staining
approaches depends on the thresholds established for positive
versus negative cells.
Calcium imaging combined with live cell markers is a useful
approach to determine functional expression of receptors.
However, inconsistent results have also been reported for the
TRPA1 agonists, allyl isothiocyanate (AITC) and cinnamaldehyde
(CINN). Investigators report rates as low as 3–7% [19,31] to as
high as 30%  for response to 100 mM CINN. Reports for
AITC vary in kind, with 18 to 45% of neurons responding to
50 mM AITC [27,31]. The discrepant results may be due to the
wide range of agonists concentrations and duration of application
used in these studies [19,21,21,32], the use of different species
(mouse versus rat), or the duration neurons are maintained in
Therefore, we set out to resolve the discrepancies in functional
TRPA1 expression in identified populations of mouse and rat
DRG neurons by using ratiometric calcium imaging and internally
consistent parameters. We used lumbar 1–6 DRG ganglia because
somata within these DRGs project to skin areas typically probed
by behavior assays, such as the plantar hind paw. For both mouse
and rat, we used similar isolation protocols, culture duration and
media. We did not routinely add exogenous growth factors to the
media because adult DRG neurons do not require growth factors
for survival , and growth factors, such as nerve growth factor
(NGF), have been shown to increase the functional expression of
TRP channels, including TRPA1 [34,35]. We identified popula-
tions of C fiber-type, small-diameter neurons in live cultures by
using IB4-FITC as well as a novel CGRP-GFP mouse where GFP
is expressed in CGRPa-expressing neurons. Under these condi-
tions, functional TRPA1 is predominantly found in small-diameter
neurons that are IB4-positive and CGRP-negative.
Materials and Methods
Cinnamaldehyde (CINN) and allyl isothiocyanate (AITC) were
purchased from Sigma. The same lot was used throughout all
experiments. Stock solutions of 100 mM AITC and CINN were
made in ethanol and working solutions were prepared in
extracellular buffer every 4–5 hours during experiments. Cells
were superfused with CINN for 3 min or with AITC for 1 min.
Unless otherwise specified, experiments were conducted on
male C57BL/6J mice, ages 2–4 months (purchased from Jackson
Laboratories). One experiment utilized TRPA12/2(KO) mice of
both sexes (2–12 months old) in which the exons essential for the
Trpa1 gene function were deleted . These TRPA1 KO mice
were back-crossed to the C57BL/6J background for over 10
generations. Other experiments utilized CGRP-GFP+/2(CGRP-
GFP) mice, a gift from Mark Zylka  (males, 3–4 months old),
and these mice were also created on a C57BL/6J background.
Male Sprague-Dawley rats were purchased from Jackson Labo-
ratories, ages 3–6 months old. All experimental procedures were
approved by the Institutional Animal Care and Use Committee of
the Medical College of Wisconsin.
Mice were briefly anesthetized with isoflurane (Midwest
Veterinary Supply) via inhalation and euthanized by decapitation.
Lumbar (L) dorsal root ganglia (DRG) 1–6 were isolated
bilaterally, unless otherwise specified, and placed into 1 ml Hank’s
Balanced Salt Solution (Gibco). After DRG extraction, 1 ml HBSS
was replaced with Dulbecco’s Modified Eagle’s Medium/Ham’s
nutrient mixture F-12 (DMEM/Hams-F12; Gibco). The ganglia
were incubated at 37uC and 5% CO2with 1 mg/ml collagenase
Type IV (Sigma) for 40 min, followed by incubation with 0.05%
trypsin (Sigma) for 45 min. We used the same protocol for
culturing rat DRG neurons except that 2 mg/ml collagenase Type
IV (Sigma) was used. Ganglia were washed and resuspended in
complete cell medium (see below), then dissociated into single
somata via trituration through a P200 pipette tip. The neurons
were plated onto laminin-coated glass coverslips and incubated for
2 hours at 37uC and 5% CO2to allow adherence. Coverslips were
then flooded with complete cell medium consisting of DMEM/
Hams-F12, 10% heat-inactivated horse serum, 2 mM L-gluta-
mine, 0.8% D-glucose, 100 units penicillin and 100 mg/ml
streptomycin. Unless noted, no exogenous growth factors were
added. For experiments that indicate the inclusion of nerve growth
factor (NGF) in the growth media, 100 ng/ml NGF was added to
the complete media and incubated with the cells overnight. Most
calcium imaging experiments were performed 18–24 hr after cells
were plated, with the exception of the experiments specifically
conducted at earlier time points (4.5–8.5 hrs and 10.5–14.5 hrs) as
Retrograde Labeling of Cutaneous Neurons
The medial center aspect of the plantar hind paw of C57BL/6J
male mice was injected with 20 mL 1% WGA-Alexafluor594 in
sterile saline (Invitrogen). One week after injection, the ipsilateral
lumbar 3–5 DRG neurons were cultured as described above. Our
prior studies show that in C57BL/6J mice, virtually all labeling
from medial plantar hind paw injections occurs in L3-5 DRG
ganglia . Cells that fluoresced clearly above background
fluorescence levels were targeted for calcium imaging experiments.
Calcium Imaging and Analysis
Calcium imaging was performed using dual-wavelength fluo-
rescent calcium indicator FURA-2AM (Invitrogen). Isolated DRG
neurons were loaded with 2.5 mL/ml FURA-2AM in extracellular
buffer containing 2% BSA for 45 min at room temperature,
followed by a 30 min wash in extracellular buffer. The extracel-
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
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Figure 1. TRPA1 agonists allyl isothiocyante and cinnamaldehyde elicit calcium responses with different latencies in DRG neurons.
A. Representative traces of individual, dissociated lumbar 1–6 dorsal root ganglia (DRG) neurons from C57BL/6J wild-type mice during FURA-2
calcium imaging. Separate neurons were tested with 100 mM allyl isothiocyanate (AITC; 1 min; top trace) or cinnamaldehyde (CINN; 3 min; bottom
trace) at 6 ml/min. Note that the latency to peak amplitude for CINN responses was longer than that for AITC, whereas responses to 50 mM K+ (30 s)
occurred almost immediately after the start of superfusion. B. Average latency from the start of superfusion to maximum amplitude of response for
AITC and CINN (3 culture preparations from 5–6 animals for each group). The latency between onset of superfusion and peak response for CINN was
significantly longer than that for AITC (p,0.0001; t-test). C–D. Concentration-response curves for AITC (C) and CINN (D) for percentage of responders
(left two panels) and peak amplitude of calcium responses (right panel). Neurons that exhibited a $20% increase in FURA ratio from baseline during
agonist superfusion were considered ‘‘responsive’’ (3 cultures from 5–6 animals for each group; AITC: 446 total neurons; CINN: 281 total neurons). All
neurons were tested with only one concentration of one agonist. For percentage of responders, the EC50 for AITC was 38.5 mM and that for CINN was
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
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lular buffer was composed of (in mM): 150 NaCl, 10 HEPES, 8
glucose, 5.6 KCl, 2 CaCl2and 1 MgCl2(pH 7.4, 32063 mOsm).
Coverslips with loaded cells were mounted onto a perfusion
chamber and superfused with buffer at a constant rate of 6 ml/
min using an AutoMate pressurized perfusion system. A 4-way
manifold system with a 2 cm bath inlet provided immediate
release of buffer and chemical agents into the superfusion
chamber. This superfusion system allowed no dead space and
minimized air bubbles in the lines. Identical tubing was used for
each input line. Experiments were conducted at room temperature
(23u61uC). Fluorescence images were captured with a cooled
CCD camera (CoolSNAP FX; Photometrics, Tucson, AZ).
Metafluor imaging software was utilized in order to detect and
analyze intracellular calcium changes throughout the experiment
(Molecular Devices, Sunnyvale, CA). A $20% increase in
intracellular calcium from baseline was considered a response.
All neurons were tested with only one concentration of one
agonist. At the end of each protocol, 50 mM KCl solution was
applied to depolarize neurons, thereby allowing for identification
of viable neurons from non-neuronal cells or non-functioning
neurons. Neuronal viability of non-responsive cells was confirmed
by response to 50 mM KCl. For mouse, neurons were considered
‘‘small’’ if the average somata diameter was less than 27 mm. Rat
neurons were considered ‘‘small’’ if the average somata diameter
was less than 30 mm.
Upon completion of imaging protocol, cells were incubated with
10 mg/ml isolectin B4 conjugated to fluorescein isothiocyanate
(IB4-FITC; Sigma) for 10 min and then washed with extracellular
buffer and visualized using FITC filters. Neurons were considered
to be IB4-positive neurons if they had a complete ring of FITC
stain around the soma perimeter.
Determination of CGRP-GFP-positive Cells
Small diameter cells from the CGRP-GFP2/+mice were
separated into CGRP-positive and –negative subgroups. Cells
were deemed CGRP-positive if their GFP fluorescence was $1
standard deviation above background fluorescence. Background
fluorescence was determined by fluorescence levels of wild-type
neurons from the CGRP-GFP mouse line that did not express
GFP (fluorescence due to autofluorescence only).
TRPA1 Agonists AITC and CINN Elicit Calcium Responses
with Different Latencies in Adult Mouse DRG Neurons
Allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) are
commonly-used exogenous agonists of TRPA1 [19,21,21,26,32].
Figure 1A shows responses of mouse lumbar 1–6 dorsal root
ganglia (DRG) neurons to AITC or CINN (both 100 mM). AITC
induced an increase in intracellular calcium, reaching a peak
within 5862 sec from the onset of perfusion onto cells (Fig. 1B).
Alternatively, CINN-evoked excitation was preceded by a longer
delay with calcium peak occurring after 17562 sec (Fig. 1B). This
difference in response latency was not due to imperfections in our
superfusion system since 50 mM potassium induced an immediate
response for every application (Fig. 1A). The superfusion rate for
all chemicals was consistently 6 ml/min, and switching stimuli to
buffer only did not elicit a change in intracellular calcium,
indicating that the stimulus-induced calcium increases were not
due to mechanical stimulation. Responses to 50 mM potassium
also served as a positive control for neuronal viability. Due to the
differences in latency to response for CINN and AITC, we
superfused cells with CINN for 3 min and with AITC for 1 min
(both 100 mM) to ensure we included all responders in our
We performed concentration-response curves for each agonist.
A $20% increase in FURA 340/380 ratio over baseline during
agonist superfusion was considered a response, and neurons were
tested with one concentration of only one agonist as depicted in
Figure 1A. For percentage of neurons activated, the EC50 for
AITC was 38.5 mM whereas that for CINN was 60.2 mM. (Fig. 1C,
D). Both compounds activated a maximum percentage of neurons
at approximately 300 mM. We defined the magnitude of response
as the amplitude of the increase in Fura-2 ratio from baseline
evoked by each stimulus. For magnitude of response, the EC50 for
AITC was 115.3 mM, whereas that for CINN was 97.5 mM.
Next we determined the size distribution of the somata of
lumbar DRG neurons that respond to AITC and CINN. While a
number of studies indicate that large neurons do not express
TRPA1 immunoreactivity[24,28,38–40], prior data from our
laboratory has shown that the terminals of some cutaneous Ab
fiber low-threshold neurons express TRPA1 protein, and me-
chanical responsiveness of several types of cutaneous Ab and Ad
fibers are altered in global TRPA1-null mice . However, here
we found that very few neurons greater than 27 mm responded to
either AITC (6.3%; 2/32) or CINN (3.6%; 5/140; Figs. 2A, B)
and therefore, we focused the rest of the experiments on small-
diameter (,27 mm) neurons.
To determine whether AITC and CINN at a near maximal
concentration (both 100 mM) are specific agonists for TRPA1, we
tested responses in small-diameter lumbar DRG neurons from
global TRPA1-null mice . AITC evoked calcium increases in
only 4.3% (7/162) of small neurons, whereas CINN elicited
responses in just 2% (4/193) of the neurons (Fisher’s exact,
p=0.2386, n.s.). These data indicate that both AITC and CINN
at 100 mM selectively target the TRPA1 channel protein on
isolated DRG somata. Since CINN elicited slightly fewer
responses in TRPA1-null neurons, we used CINN for subsequent
experiments where only one TRPA1 agonist was utilized.
TRPA1 is Functionally Expressed Predominantly by Non-
peptidergic, IB4-binding Neurons
To determine which populations of small-diameter neurons
typically respond to TRPA1 agonists, we incubated lumbar
DRG neurons from adult mouse with IB4 conjugated to FITC
(IB4-FITC) after exposure to AITC or CINN. Examples of
neurons stained with IB4-FITC are shown in Figure 3A. IB4-
positive neurons were identified by a complete halo of FITC
fluorescence around the perimeter of the soma. Approximately
50% of all small neurons from mouse lumbar ganglia stained
positively for IB4 (56%; 339/601). Among all small neurons
tested, 66% (158/241) responded to 100 mM AITC and 42%
(155/366) responded to 100 mM CINN. Next, we sorted the
small-diameter neurons into IB4-positive and IB4-negative.
Among all IB4-positive neurons, 79% (125/159) responded to
AITC and 72% (129/180) responded to CINN (Fig. 3B).
Conversely, among all IB4 negative neurons, only 40% (33/82)
responded to AITC and 14% (25/180) responded to CINN
60.2 mM (both calculated from 10–1000 mM data). For peak response amplitude, the EC50 for AITC was 115.3 mM (calculated from 10–300 mM data)
and that for CINN was 97.5 mM (calculated from 10–1000 mM data).
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
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(Fig. 3B). Overall, approximately 80% of neurons responding to
either AITC (79%; 125/158) or CINN (84%; 129/154) were
IB4 positive. In addition, the IB4-positive responsive neurons
exhibited a greater peak magnitude to both AITC and CINN
than did the IB4-negative responsive neurons (Fig. 3C). These
findings were consistent across ten independent experiments (ten
separate animals and culture preparations). Thus, TRPA1
appears to be functionally expressed mainly by IB4-positive
small-diameter neurons in adult mice.
To confirm our findings using an alternate label, we used
CGRP as a marker for the peptidergic subgroup in neurons
cultured from a novel CGRP-GFP mouse line in which GFP
expression is driven by the CGRPa promoter  (Fig. 4A). In this
mouse strain, the majority (89%) of all neurons that were
immunoreactive for a CGRP antibody (which recognizes both a
and b CGRP isoforms) were CGRPa-GFP-positive . Likewise,
the majority (68%) of CGRPa-GFP-positive neurons bound the
CGRP antibody . The finding that not all CGRPa-GFP-
Figure 2. Cell size distributions of adult mouse and rat DRG neurons responding to TRPA1 agonists. Distribution of somata diameters
(1 mm bins) of lumbar DRG neurons in culture preparations from mouse or rat. Number of neurons responding to AITC or CINN (both 100 mM) are
shown in grey bar portion, non-responsive neurons are shown in white bar portion, and total cells in each size bin are reflected by the top of the bar
(sum of the grey and white bars). A. Mouse lumbar 1–6 DRG neurons tested with 100 mM AITC (3 cultures from 6 animals; 271 total neurons; 160
responders; 59% responders). B. Mouse lumbar 1–6 DRG neurons tested with 100 mM CINN (7 cultures from 7 animals; 493 total neurons; 160
responders; 32% responders). C. Mouse neurons labeled via retrograde tracer injected into the medial plantar hind paw. All neurons were taken from
lumbar 3–5 DRGs ipsilateral to injection. Only labeled neurons were used for recordings. Thus all neurons in the graph were labeled from the hind
paw plantar skin. Neurons were tested with 100 mM CINN (5 cultures from 10 animals; 131 total neurons; 43 responders; 33% responders). D. Rat
lumbar 1–6 DRG neurons tested with 100 mM CINN (3 cultures from 3 animals; 312 total neurons; 80 responders; 26% responders).
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
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positive neurons expressed CGRP immunoreactivity was likely
due to greater sensitivity and detection of the GFP marker than the
CGRP antibody . First we found that there was no difference
between the overall percentage of small neurons that responded to
100 mM CINN or mean amplitude of responses between neurons
from CGRP-GFP mice (44%; 109/248; mean response amplitude
173614%) compared to those from wild-type (C57BL/6J) mice
(42%; 155/366; mean response amplitude 15569%), indicating
that the mouse strains, which are both on a C57BL/6J
background, express similar levels of TRPA1 at the lumbar
DRG level. Significantly more CGRP-negative neurons (48%; 85/
176) responded to CINN compared to CGRP-positive neurons
(33%; 24/72; Fig. 3B). Among small neurons that responded to
CINN, 78% (85/109) were CGRP-negative and only 22% (24/
109) were CGRP-positive. However, there was no difference in the
average amplitude of response to CINN in CGRP-negative and
CGRP-positive responders (Fig. 3C). The CGRP-positive re-
sponders exhibited a trend for larger responses, but this was not
Next, to determine the overlap in IB4 binding and CGRP
expression in small neurons, we used IB4 staining with a red
fluorophore (IB4-AlexaFluor594) together with the CGRP-GFP
Surprisingly, among the CGRP-positive neurons, 50% (36/72)
were co-labeled for IB4 (Fig. 4B). There was a significantly
greater percentage ofIB4-positive/CGRP-negative
combinations (Fig. 4C). In fact, the majority of neurons that
responded to CINN were IB4-positive and CGRP-negative
(66%; 72/109). IB4-negative/CGRP-negative neurons exhibited
significantly smaller response amplitudes to CINN than IB4-
positive/CGRP-positive neurons (Fig. 3C). Togther, these data
indicate that TRPA1 is functionally expressed primarily by IB4-
positive/CGRP-negative neurons in mouse. These data also
suggest that IB4 staining is a better predictor of neurons that
are likely to respond to TRPA1 agonists than is CGRP
Figure 3. TRPA1 is functionally expressed predominantly by IB4-positive and CGRP-negative neurons. A. Representative brightfield
(left) and FITC (right) images of lumbar 1–6 DRG neurons from C57BL/6J wild-type mouse stained with IB4-FITC (60x objective). IB4-positive neurons
were defined by a halo of FITC-labeling around the entire perimeter of the somata of small-diameter (,27 mm) neurons. The red arrow indicates an
IB4-positive neuron, whereas the white arrow indicates an IB4-negative neuron. B. Percentage of small-diameter neurons responding to TRPA1
agonists (all 100 mM) defined by IB4-binding or CGRP expression. A greater percentage of IB4-positive neurons responded to TRPA1 agonists than the
percentage of IB4-negative neurons (overall effect: Chi square p,0.0001; ***p,0.0001). Likewise, a greater percentage of CGRP-negative neurons
responded to CINN than CGRP-positive neurons (*p,0.0350 Fisher’s exact). CGRP data were generated from 3 animals in 2 cultures. C. Amplitude of
responses to TRPA1 agonists (all 100 mM) of small-diameter DRG neurons defined by IB4-binding or CGRP expression. IB4-positive neurons responded
with a greater peak average amplitude than IB4-negative neurons (overall effect: ANOVA p,0.0001; *p,0.05, Tukey). There was no difference
between CGRP-positive and CGRP-negative peak amplitudes.
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Plantar Skin-projecting Neurons Express TRPA1 Primarily
in IB4-positive Neurons
Lumbar DRG neurons are considerably heterogeneous with
respect to their peripheral target projections, which include skin,
muscle, bone, tendons and visceral organs. Many of the non-
peptidergic, IB4-binding or MrgprD-positive neurons innervate
superficial skin [3,11,12,14,42]. Because the plantar skin of the
hind paw is a common region examined in behavioral assays and
is an area used for ex vivo skin-nerve electrophysiological
preparations, we retrograde-labeled DRG somata that project to
this skin region with WGA-Alexafluor594. After 7 days, neurons
from ipsilateral lumbar 3–5 ganglia were dissociated and cultured.
Neurons that were strongly labeled with distinct, bright red
fluorescence were imaged. The mean diameter of retrograde-
labeled (cutaneous) neurons was smaller (20.860.5 mm) than those
of mixed lumbar 1–6 DRG cultures (23.760.2 mm; compare
Figs. 2B, C; t-test, p,0.0001). The difference in cell sizes was
expected since most afferents innervating the superficial skin
correspond to non-peptidergic (IB4-positive), small-diameter
neurons [3,9,11,12,43]. Similar to lumbar DRG neurons with
mixed peripheral targets, 38% (42/112) of all small-diameter
cutaneous neurons responded to 100 mM CINN. Additionally, the
mean response amplitudes were similar between cutaneous
(159.4616.9; n=42) and mixed target responders (155.768.7;
n=154). As in our results with mixed target DRG cultures,
significantly more IB4-positive cutaneous neurons (50%, 34/68)
responded to CINN than IB4-negative neurons (18%, 8/44;
Fisher’s exact, p=0.0007). Out of all cutaneous neurons
responding to CINN, 81% (34/42) were IB4-positive. IB4-positive
cutaneous neurons responded with an average amplitude
(171619%) that was not different than IB4-negative neurons
(110633%; t-test, p=0.1588). These data indicate that TRPA1 is
functionally present in neurons that innervate plantar hind paw
skin and, as with DRG somata with mixed peripheral targets, most
cutaneous neurons that express functional TRPA1 are IB4-
Figure 4. TRPA1 is functionally expressed mainly by neurons that are both IB4-positive and CGRP-negative. A. Representative
brightfield (left) and FITC (right) images of lumbar 1–6 DRG neurons from the CGRP-GFP+/2mouse strain (20x objective). CGRP-positive neurons were
identified by GFP fluorescence levels that were three standard deviations above the average autofluorescence levels of DRG neurons from CGRP-
GFP+/+wild-type mice. The green arrows indicate CGRP-positive neurons. B. Distribution of both CGRP expression and IB4 binding among all small-
diameter lumbar 1–6 DRG neurons (248 total neurons). A total of 29% of small neurons were CGRP positive. Note that 50% of these (14.5%) were also
IB4-positive. C. Percentage of small diameter neurons responding to 100 mM CINN defined by IB4 and CGRP labeling. A greater percentage of IB4-
positive/CGRP-negative neurons responded to CINN than the percentage of responders with other staining combinations (overall effect: Chi square
p,0.0001; ***p,0.0001, **p=0.0019, Fisher’s exact for 2-group comparisons). The majority of responders (66%; 72/109) were IB4-positive and CGRP-
negative. D. Amplitude of responses to 100 mM CINN grouped by CGRP and IB4 labeling. The responses of IB4-positive/CGRP-positive were
significantly greater than those for IB4-negative/CGRP-negative neurons (overall effect: ANOVA p=0.0250; *p,0.05 Tukey post hoc test).
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
PLOS ONE | www.plosone.org7 October 2012 | Volume 7 | Issue 10 | e47988
The Pattern of TRPA1 Expression Differs between Mouse
and Rat DRG Neurons
A frequent question is whether rat and mouse DRG neurons
are similar. An optimal approach to compare species is to use
identical techniques in the same laboratory to determine whether
there are inter-species differences in the distribution of TRPA1 in
sensory neurons. Therefore, we conducted calcium imaging
experiments on lumbar 1–6 DRG neurons isolated from rat. The
same culturing techniques and media were used, except twice the
concentration of collagenase was used for chemical dissociation
for rat DRGs than for mice (see Methods). We found a number
of differences between rat and mouse sensory neurons. First, as
expected, the distribution of soma diameters for all lumbar DRG
neurons is larger in rat than mouse (Figs. 2D, B). The range of
cell sizes for rat was 15–47 mm (median: 25.8 mm), whereas the
size range for mouse neurons was 12–41 mm (median: 22.5 mm).
Further, the size of neurons responding to CINN was signifi-
cantly larger in rat than mouse (Figs. 2D, B). The rat neurons
that responded to CINN had a mean soma diameter of
24.460.4 mm (median: 24 mm; n=80), whereas the mean
diameter of CINN responders from mouse was 20.660.2 mm
(median: 20.4 mm; n=160; t-test, p,0.0001). Based on the size
of CINN responders in rat, we considered small-diameter
neurons to be those less than 30 mm in diameter, compared to
27 mm in mouse. Mouse and rat neuronal populations had
different percentages of responders and mean response ampli-
tudes to CINN (Figs. 5A, B). Fewer total small-diameter neurons
from rat responded to CINN, but the responsive neurons in rat
had greater response amplitudes than those from mouse (Figs. 5A,
B). In contrast to mouse where a larger percentage of IB4-
positive neurons functionally express TRPA1 than the percentage
of IB4-negative neurons, the percentage of IB4-positive and IB4-
negative neurons responding to CINN was similar for rat
(Fig. 5C). However, of the responding neurons in rat, the
majority (68%; 51/75) were IB4-positive. The peak magnitude
response for IB4-positive and IB4-negative neurons in rat was
also similar (Fig. 5D). Interestingly, these differences between
mouse and rat did not appear to result from fewer small rat
neurons labeled with IB4 since significantly more small neurons
from rat were IB4-positive compared to mouse (Fig. 5E;
p,0.0001 Fisher’s exact). Despite these details, the overall data
indicate that most neurons that functionally express TRPA1 are
IB4-positive in rat as in mice.
TRPA1 Function and Distribution Changes with Duration
Time in culture is another factor that may potentially affect
response to TRPA1 agonists. We therefore recorded from
mouse neurons at 4.5–8.5 hr, 10.5–14.5 hr, and 18–24 hr after
plating. As the culture duration progressed, we found that a
greater percentage of small-diameter neurons responded to
specifically among IB4-positive neurons, and functional TRPA1
levels stabilized by 10.5 hr after the neurons were plated
(Fig. 6A). Although there were fewer IB4-positive neurons
responding to CINN at the earliest time point (4.5–8.5 hr), we
still found significantly more IB4-positive neurons with func-
tional TRPA1 than IB4-negative neurons (Fig. 6A). The
response amplitudes also increased as culture duration pro-
gressed, and this effect was specific to IB4-positive neurons
(Fig. 6B). We also observed greater response amplitudes for IB4-
positive than for the IB4-negative responders at 18–24 hrs, but
not at earlier time points (Fig. 6B). There was no difference in
the percentage of neurons that stained positively for IB4 at any
time point (Fig. 6C), suggesting that the increase in IB4-positive
CINN-responsive neurons is not likely due to changes in IB4-
Exogenous NGF has No Effect on Distribution of TRPA1
In all of the previous experiments, we did not use exogenous
growth factors in our culture media since nerve growth factor
(NGF) can alter responsiveness of a number of TRP channels,
including TRPA1 [34,35,44], and adult DRG neurons do not
require growth factors such as NGF to survive in culture .
However, this is a factor that varies between studies and might
affect expression of TRPA1. Therefore, we investigated the
influence of exogenous NGF (100 ng/ml, overnight) on the
functional distribution of TRPA1 in vitro (DRGs from 3 mice in
3 separate cultures). We found that significantly more total small
neurons cultured with NGF responded to 100 mM CINN (53%;
115/216) than those cultured without NGF (42%; 155/366;
Fisher’s exact, p=0.0126). Response amplitudes, however, were
not affected (155.768.7% without NGF; 155.6610.4% with
NGF). When grouped by IB4 binding, we found that significantly
more IB4-positive neurons (79%; 93/118) responded to CINN
than IB4-negative neurons (22%; 22/98) after exposure to NGF.
These results were not different than our results without NGF
exposure (IB4-positive neurons: 72%; 129/180; IB4-negative
neurons: 14%; 25/180). Further, IB4-positive neurons treated
with NGF overnight still exhibited greater peak response
174.1611.5% n=93; IB4-negative: 77.5615.5% n=22; t-test
p=0.0002). These responses were not different from untreated
74.1612% n=25). Therefore, although more neurons function-
ally express TRPA1 after treatment with exogenous NGF, the
distribution of functional TRPA1 is not affected. Even with NGF
treatment, more IB4-positive neurons functionally express TRPA1
than IB4-negative neurons.
Here we set out to resolve the contrasting claims regarding the
populations of DRG neurons that express functional TRPA1
channels. Using standardized conditions, our results demonstrate
that the majority of DRG neurons from mouse that respond to
TRPA1 agonists are small-diameter, IB4-positive and CGRP-
negative. Similarly in rat, most neurons that functionally express
TRPA1 were IB4-positive. Very few large-diameter neurons from
either species responded to TRPA1 agonists. In addition, we found
that culture duration affects TRPA1 function in that responsive-
ness to TRPA1 agonists increases specifically in IB4-positive
neurons despite the lack of added exogenous growth factors.
TRPA1 Functional Expression Correlates Extensively with
IB4 Binding and Less with CGRP Expression
Contrary to a number of previous studies using in situ
hybridization and immunochemistry that reported high TRPA1
expression among peptidergic,
[18,21,26,27], we consistently found that the majority of small-
diameter neurons that respond to TRPA1 agonists are IB4-
binding and CGRP-negative. Our results agree with those of
Hjerling-Leffler and colleagues , Kim and colleagues  and
Caspani and colleagues  who reported high TRPA1 expres-
sion in IB4-positive neurons from mouse and rat. Disparities on
this matter appear to be due to the markers used in each study.
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
PLOS ONE | www.plosone.org8October 2012 | Volume 7 | Issue 10 | e47988
Those studies employing IB4 binding have all found high overlap
between TRPA1 and IB4 binding, whereas studies that examined
only CGRP or Substance P expression concluded that TRPA1 is
expressed by peptidergic neurons. Our study is the first to employ
both IB4 binding and CGRP expression in live neurons that are
tested with TRPA1 agonists. We find that IB4 binding is a better
Figure 5. The pattern of functional TRPA1 expression differs between mouse and rat DRG neurons. A. Percentage of small-diameter
mouse and rat DRG neurons responding to 100 mM CINN. Small-diameter neurons for mouse were defined as less than 27 mm, whereas those for rat
were defined as less than 30 mm in soma diameter. Significantly more small-diameter mouse neurons responded to CINN than rat neurons
(***p=0.0096, Fisher’s exact). B. Peak amplitude of responses for mouse and rat small-diameter neurons to 100 mM CINN. Rat neurons had a greater
response amplitude to CINN than did mouse neurons (***p=0.0039, t-test). C. Percentage of mouse and rat neurons responding to 100 mM CINN
defined by IB4 staining. For mouse, significantly more IB4-positive neurons responded than IB4-negative neurons (left bars). However, for rat there
was no difference between IB4-positive and IB4-negative neurons (overall effect: Chi square p,0.0001; ***p,0.0001, Fisher’s exact). Nonetheless,
among the rat neurons that responded to CINN, the majority were IB4-positive (68%; 51/75). The mouse data set (left two bars) is the same data as
previously shown in Fig 3B (middle two bars). D. Amplitude of responses to 100 mM CINN of mouse and rat neurons defined by IB4 binding. In mouse,
the IB4-positive neurons responded with greater amplitudes than IB4-negative neurons. In contrast to mouse, there was no difference between the
amplitudes of IB4-positive and IB4-negative neurons from rat (overall effect: ANOVA p,0.0001; *p,0.01, Tukey post hoc test). The mouse data (left
two bars) is the same as shown in Fig 3C (middle two bars). E. Distribution of IB4 binding among all small-diameter neurons from mouse and rat.
Significantly more neurons were IB4-positive in rat than in mouse (p,0.0001, Fisher’s exact).
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
PLOS ONE | www.plosone.org9 October 2012 | Volume 7 | Issue 10 | e47988
indicator of TRPA1 expression than is CGRP expression in
mouse. Surprisingly, we found that half of the CGRP-positive
neurons in mouse bind IB4. Therefore, CGRP and IB4 do not
label exclusive populations in mouse lumbar DRGs and caution
should be exercised when using IB4 to identify ‘‘non-peptidergic’’
As in mice, the majority of TRPA1-responsive neurons in rats
were IB4-positive. However, while 80% of IB4-positive mouse
neurons responded to CINN, only 30% of IB4-positive neurons
from rat responded. This difference was not due to a lower IB4
binding as more neurons were IB4-positive in rat than mouse. The
difference is likely due in part to a lower overall responsiveness to
TRPA1 agonists in rat.
IB4 Positive Neurons and TRPA1 both Play Key Roles in
Mechanical Pain Behavior
Accumulating evidence suggests that IB4-binding neurons play
particular roles in somatosensation and pain. First, in non-injured
skin, Mrgprd-positive afferents, which comprise approximately
90% of IB4 positive cutaneous afferents, are critical for
transduction of noxious mechanical, but not thermal, stimuli
. Second, IB4-binding neurons are especially subject to
plasticity after tissue injury. They become sensitized to mechanical
stimuli in a chronic model of sickle cell disease pain .
Additionally, mechanical hypersensitivity after nerve injury and
muscle inflammation depend on IB4-positive nerve fibers [45,47].
IB4-positive neurons have been shown to play a key role in
hyperalgesic priming[48–50], whereby the inflammatory media-
Figure 6. TRPA1 function in IB4-positive neurons increases with duration in culture. A. Percentage of small-diameter L1-6 DRG neurons
from adult mouse responding to 100 mM CINN defined by IB4 binding and duration between plating and imaging cells (5–7 cultures prepared from
5–7 animals). The percentage of IB4-positive responders significantly increased between the 4.5–8.5 hr and the 10.5–14.5 hr time points after plating
(overall effect: Chi square p,0.0001; ***p,0.0001, Fisher’s exact). At the earliest time point tested (4.5–8.5 hrs), although there was a smaller
percentage of IB4-positive neurons responding to CINN than at the later time points, there were still significantly more IB4-positive than IB4-negative
neurons responding at this time (**p=0.0005, Fisher’s exact). In contrast, there was no difference in IB4-negative responders across any of the time
points. The data set for 18–24 hrs (right two bars) is the same as that shown in Fig 3B (middle two bars). B. Average amplitude of responses to
100 mM CINN defined by IB4 binding and duration between plating and imaging neurons. The amplitude of responses for IB4-positive neurons
increases between 8.5 and 18 hrs (overall effect: ANOVA p,0.0001; *p,0.05, Tukey post hoc test). There was no overall difference in the amplitude of
responses for IB4-negative responders. C. Distribution of IB4 staining among all small-diameter neurons defined by duration between plating and
imaging cells. There was no significant change in IB4 binding across any of the time points tested (Chi square p=0.0735). The data set for 18–23 hrs
(right bar) is the same as shown in Fig 5E (left bar).
TRPA1 in IB4-Binding, Non-Peptidergic Neurons
PLOS ONE | www.plosone.org10 October 2012 | Volume 7 | Issue 10 | e47988
tors NGF and prostaglandin E2(PGE2) initiate activation and
membrane translocation of PKCe selectively in IB4-positive
nociceptors[50–54]. Hyperalgesic priming in IB4-positive noci-
ceptors has been proposed to mediate the transition from acute to
chronic pain [50,54]. Our finding that TRPA1 expression
increases selectively on IB4-positive neurons with time in culture
is consistent with the idea that dissociation of DRGs may simulate
a type of ‘‘injury’’ that DRG neurons recover from over time.
The finding that functional TRPA1 is preferentially expressed
by IB4-positive neurons suggests that TRPA1 may mediate
specific contributions of IB4-positive neurons to mechanical pain.
Growing evidence suggests that TRPA1 plays an integral role in
both the development and maintenance of inflammatory mechan-
ical hyperalgesia [18,40,55,56], and TRPA1 inhibition at the
peripheral terminal reverses the mechanical sensitization of C
fibers after inflammation . Thus, the TRPA1-expressing IB4-
positive neurons may be particularly important for conveying
mechanical pain from skin. TRPA1 is a promising target for
therapeutic intervention of inflammatory pain, and therapies
targeting TRPA1 on IB4-positive neurons may reduce mechanical
hyperalgesia without abolishing normal tactile acuity for patients
that suffer from inflammatory pain.
The authors thank Dr. Mark Zylka for the generous gift of CGRP-GFP
mice and Dr. Quinn Hogan for critical comments on the manuscript.
Conceived and designed the experiments: EAK MEB CLS. Performed the
experiments: EAK MEB. Analyzed the data: EAK MEB CLS. Contributed
reagents/materials/analysis tools: CLS. Wrote the paper: MEB CLS.
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