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RESEARCH ARTI CLE

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Acupuncture-Stimulated Activation of Sensory
Neurons
*
Min-Hee Kim, Yang-Chun Park, Uk Namgung*
Department of Oriental Medicine, Daejeon University, Daejeon, Republic of Korea
Available online Jun 16, 2012
Received: Dec 22, 2011
Revised: Mar 9, 2012
Accepted: Mar 16, 2012
KEYWORDS
acupuncture;
electroacupuncture;
GAP-43;
sensory neuron;
vagus nerve;
zusanli
Abstract
Acupuncture is one of the key therapeutics in clinical oriental medicine, and recent
studies using experimental animals have begun to provide the pathophysiological basis
for the efcacy of acupuncture. Here, we investigated neuronal responses in rodent
models given acupuncture stimulation. In both mice and rats, acupuncture stimulation
at zusanli (ST36) generated an increased expression of axonal growth-associated protein
(GAP-43) in the sensory neurons of the dorsal root ganglion (DRG). Electroacupuncture
stimulation at ST36 in rats induced GAP-43 mRNA and protein expression in DRG neurons
at the levels of lumbar 4 and 5. Stimulation on a non-acupuncture site as a sham control
induced GAP-43 expression as well, but the induction level was lower than it was with
acupuncture. We further found that acupuncture stimulation upregulated phospho-
Erk1/2 signals in DRG neurons. Electroacupuncture stimulation induced c-Fos expression
in the neurons of the dorsal motor nucleus of the vagus nerve (DMV), which was identied
by retrograde tracing. These data suggest that acupuncture stimulation may generate
physiological effects on the autonomic nervous system via the activation of a somatosen-
sory pathway.
1. Introduction
Acupuncture is one of the key therapeutics in clinical oriental
medicine and its efcacy has become more widely accepted
worldwide than before. According to the philosophical theory
in oriental medicine, acupuncture stimulation relieves the
dysfunctional blockage of meridian channels through which
qi, the life energy, ows. A growing body of evidence shows
that acupuncture stimulation can engender pathophysiolog-
ical consequences which alleviate disease symptoms for
*
Declaration of any source of nancial income: None.
* Corresponding author. Department of Oriental Medicine, Daejeon University, 96-3 Yongun-dong, Daejeon 300-716, Republic of Korea.
E-mail: unamgung@dju.kr
Copyright 2012, International Pharmacopuncture Institute
http://dx.doi.org/10.1016/j.jams.2012.05.002
Available online at www.sciencedirect.com
Journal of Acupuncture and Meridian Studies
j our nal homepage: www. j ams- kpi . com
J Acupunct Meridian Stud 2012;5(4):148e155
several organs, including those in the endocrine, immune and
nervous systems [1,2].
Several lines of theoretical and experimental studies,
including brain imaging studies, implicate that the nervous
system may be involved in transmitting acupuncture signals
into the target organ, in which traditional nerve-reex
theory, gate control theory, and neurotransmitter release
theory are considered [2,3]. Most widely demonstrated is
the pain relief, in which the acupuncture stimulation
regulates the neuronal pathway responsible for pain
suppression [4]. Emerging evidence reveals that acupunc-
ture therapy is effective for treating disorders such as
gastritis, vasomotor syndrome in breast cancer patients,
and neuronal death as shown in Parkinsons animal model
[5e7]. A recent study showed that stimulation at the
zusanli (ST36) acupuncture point (acupoint) in mice induces
purinergic receptor activation, which inhibits the trans-
mission of pain signals to the brain [8]. Interestingly, puri-
nergic receptor activation in the sciatic nerve increases the
synthesis of axonal growth-associated protein (GAP-43) in
dorsal root ganglion (DRG) sensory neurons [9]. GAP-43 is
the neural-specic protein known to play a role in neuronal
development and activity-dependent synaptic plasticity
[10,11]. However, whether and/or how acupuncture stim-
ulation can activate a neuronal pathway resulting in phys-
iological consequences is largely unknown. In some studies,
acupuncture stimulations on non-meridian, as well as
meridian, sites are similarly effective, raising the issue of
the physiological identity of the acupoint and the placebo
effects [12,13].
Here, we investigated the effects of acupuncture
stimulation on nervous system activation in mice and rats.
In order to determine acupoint-specic neuronal
responses, we examined the effects of sham acupuncture
at the same time, and both classic needle acupuncture
and electroacupuncture were employed. Our data show
that acupoint stimulation generates neuronal responses in
terms of increased expression of GAP-43 and Erk1/2 acti-
vation in DRG sensory neurons and induction of c-Fos
expression in neurons of the dorsal vagal complex (DVC)
area.
2. Materials and methods
2.1. Materials
Sprague-Dawley rats (male, 200e250 g, Dae Han Biolink,
Eumseong-gun, Chungcheongbuk-do, Korea) and albino ICR
mice (male, 20e25 g Dae Han Biolink, Eumseong-gun,
Chungcheongbuk-do, Korea) were maintained in an animal
room with regulated temperature (22

C), 60% humidity, and

a 12-hour light and 12-hour dark cycle. Animals were
allowed to eat commercial chow (Dae Han Biolink) and
drink water ad libitum. All protocols involving live and
postoperative animal care were approved by the Daejeon
University Institutional Animal Use and Care Committee,
and were in accordance with the Animal-Use Statement and
Ethics Committee Approval Statement for Animal Experi-
ments provided by Daejeon University (Daejeon, Korea).
2.2. Acupuncture stimulation
Acupuncture needles (0.20 7 mm) were purchased from
Haeng Lim Seo Won Acuneedle Company (Seoul, Korea).
Rats and mice were anesthetized with 80 mg/kg of ket-
amine and 5 mg/kg of xylazine, and acupuncture therapy
was performed similarly as described [4,8]. In mice, the
needle was inserted in the zusanli point (ST36; 3e4 mm
below and laterally 1e2 mm for the midline knee to a depth
of 2e3 mm). For rats, the needle was inserted in the cor-
responding ST36 (6e7 mm below and laterally 2e3 mm to
a depth of 4e5 mm). For sham stimulations, the needle was
inserted at the points 5 mm and 10 mm lateral to ST36 in
mice and rats, respectively. These locations are known to
be free from classical acupoints. Acupuncture needles were
inserted for 30 minutes, with a slow rotation every 5
minutes. Animals were subjected to acupuncture therapy
for 3 days and sacriced at day 4. Electroacupuncture was
performed at the ST36 point by using the same kinds of
needles as above. After the needle had been inserted into
the shaven skin, electric stimulation was given at 1 Hz,
using an electroacupuncture stimulator (Pulse Generator
PG-306, Suzuki Iryoki, Oita-Shi, Japan) for 30 minutes, with
the lowest and the intermediate modes of intensity for
mice and rats, respectively. Stimulation was given once per
day for 3 days, and the animals were sacriced 30 minutes
or 24 hours after the last acupuncture treatments.
2.3. Retrograde tracing and c-Fos analysis
Mice were anesthetized with ketamine and xylazine, and
uorogold (FG; 2 mL of 5% saline, Molecular Probes, Eugene,
OR, USA) was injected into the exposed vagus nerve at the
cervical level by using a Hamilton syringe (Innovative Labor
System, Stutzerbach, Germany). Some animals were sub-
jected to sciatic nerve injury immediately after FG injec-
tion, and some other animals were given
electroacupuncture on the 5
th
day after FG injection for 3
days thereafter and were sacriced 30 minutes after the
last acupuncture. All animals were sacriced 7 days after
FG injection, and coronal brain sections (30 mm sections)
were collected. Fluorescence images of FG-labeled neurons
in the brain sections were captured and analyzed by using
Photoshop software. The brain sections on the slides were
subjected to immunouorescence staining with anti-c-Fos
antibody (Santa Cruz Biotech, Santa Cruz, CA, USA 1:400).
2.4. Immunouorescence staining
DRG tissues were embedded and frozen at -20

C. Sections
(20 mm thickness) were cut on a cryostat and mounted on
positively charged slides. Immunouorescence staining was
performed as described previously [14]. Briey, sections
were xed with 4% paraformaldehyde and 4% sucrose in PBS
at room temperature for 40 minutes, permeabilized with
0.5% Nonidet P-40 in phosphate-buffered saline (PBS), and
blocked with 2.5% horse serum and 2.5% bovine serum
albumin for 4 hours at room temperature. Sections were
incubated with anti-GAP-43 antibody (1:400, Santa Cruz
Biotechnology, Santa Cruz, CA, USA), anti-phospho-Erk1/2
antibody (1:400; Cell Signaling, Danvers, MA, USA), or anti-
Neuronal activation by using acupuncture 149
bIII-tubulin antibody (1:200, TUJ1, Covance, Princeton, New
Jersey, USA), after which they were incubated with
rhodamine-labeled goat anti-rabbit secondary antibody
(1:400, Molecular Probes, Eugene, OR, USA) or uorescein-
labeled sheep anti-mouse antibody (1:400, Molecular
Probes) in 2.5% horse serum and 2.5% bovine serum albumin
for 1 hour at room temperature and cover-slipped with
gelatin mount medium. We included control sections treated
withsecondary antibody alone. Incases whenthenonspecic
signals were high, the data were excluded for further anal-
ysis. Sections were viewed with a uorescence microscope
(Nikon, Tokyo, Japan) and the images were captured using
a Nikon camera. The merged images were produced by using
the layer blending mode options of the Adobe Photoshop.
2.5. RT-PCR
Total RNA was isolated from the DRGs at lumbar levels 4 and
5 by using Easy-BLUE reagent (Intron, Sungnam, Korea).
cDNA was prepared using 2 mg of total RNA as a template for
a reverse transcription (RT) reaction with MMLV reverse
transcriptase (Promega, Madison, WI, USA) and random
primer (Bioneer, Daejeon, Korea) for 1 hr at 37

C. For PCR
amplication of GAP-43 and actin cDNA, the RT reaction
was diluted 4-fold in H
2
O, and 5 mL of cDNA in 80 mL of total
reaction volume was used for PCR with Taq DNA polymerase
(Takara, Ohtsu, Japan). For quantitative comparison of
GAP-43 mRNA expression among samples, 30 cycles of
amplication was optimal for both GAP-43 and actin RT-
PCR. The primer sequences used for PCR were forward
primer (5
0
-GATGCAGCCCCAGCCACCAG-3
0
) and reverse
primer (5
0
-TCAGGTGGGGGCAACGTGGA-3
0
) for GAP-43
mRNA, and forward primer (5
0
-CACACTGTGCCCATCTATGA-
3
0
) and reverse primer (5
0
-TACGGATGTCAA CGTCACAC-3
0
)
for actin mRNA. The amplied DNA sizes were 454 bp and
409 bp for GAP-43 and actin, respectively. PCR-amplied
DNA products for individual samples were analyzed on
agarose gels. The images of these gels were transferred to
Photoshop images and quantied using the i-Solution soft-
ware (Image & Microscope Technology, Daejeon, Korea).
2.6. Statistical analysis
Data were presented as mean standard error of mean
(sem). The mean numbers of data in individual groups were
compared by using the one-way ANOVA test followed by the
Tukey test (SPSS computer software version 12.0), and
statistically signicant differences were reported as
*p <0.05, **p <0.01, or ***p <0.001.
3. Results
To determine whether acupuncture stimulation on ST36
triggers neuronal responses, we investigated GAP-43 mRNA
expression by using RT-PCR in the DRG sensory neurons of
mice. The basal level of GAP-43 mRNA was found in the
intact tissue, and its level was slightly elevated by sham
acupuncture stimulation and was signicantly increased by
ST36 acupuncture (Fig. 1A). A quantitative comparison of
GAP-43 mRNA between the acupuncture and the sham
groups showed GAP-43 expression to be 34% higher in the
acupuncture group; however, the difference was not
statistically signicant. A robust increase in GAP-43 mRNA
was caused by injury stimulation on the nerve. Immuno-
uorescence staining showed that GAP-43 signals were
detected in the DRG sensory neurons of intact mice and
that the overall signal intensities in the sham, acupuncture,
and injury groups were elevated in a proportional manner
as mRNA increased (Fig. 1B).
To examine whether GAP-43 expression was similarly
induced in the rat model, we gave needle stimulation to
Figure 1 Regulation of GAP-43 expression in mice given
acupuncture stimulation. (A) RT-PCR of GAP-43 mRNA in DRG
tissues. Mice were subjected to acupuncture stimulation by
inserting the needle on ST36 (ACU) and to sham stimulation and
sciatic nerve injury (SNI). A DRG from an intact animal was
used as the control. (Upper) Representative data on RT-PCR for
GAP-43 and actin as an internal loading control. (Bottom)
Quantication of the band intensity in a ratio of GAP-43 to
actin (number of independent experiments Z4). Data denote
mean sem (**p <0.01, ***p <0.001 vs. intact group). (B)
Immunouorescence staining of GAP-43 of the DRG of animal
groups with different treatments. Scale bar: 200 mm.
150 M.-H. Kim et al.
ST36, and GAP-43 expression was investigated in the DRG.
As shown in Fig. 2A, both acupuncture and sham stimula-
tions generated signicant increases in GAP-43 mRNA
expression compared to the intact control, but the effects
were more signicant for the acupuncture group than for
the sham group (Fig. 2A). Immunouorescence staining
showed more intense protein signals in animal groups given
acupuncture stimulation and nerve injury than in the intact
control (Fig. 2B). We noted that more neurons in the
acupuncture-stimulated groups were GAP-43-positive than
in the intact control group.
In rodents, sensory neurons for the sciatic nerves are
distributed in the DRGs at lumbar levels 4e6. To localize
the DRG neurons specically responding to ST36 and to
examine the reproducibility of acupuncture effects, we
performed electroacupuncture at ST36 in the rats and
analyzed GAP-43 expressions in the DRGs at lumbar levels 4
and 5 separately. In both DRGs, GAP-43 mRNA was
increased by sham stimulation, but further increased by
acupuncture stimulation (Figs. 3A and 3B). Immunouo-
rescence staining revealed that among the different prep-
arations, consistent GAP-43 signals were found in the DRG
neurons at lumbar level 4 in the acupuncture group, and
a similar pattern was also observed in the sham group,
although the overall protein level was lower than it was in
the acupuncture group (Fig. 3C). In the intact animals, GAP-
43 signals were much weaker than they were in the
acupuncture group.
We further investigated whether electroacupuncture
stimulation activated phospho-Erk1/2 signals in the rat DRG
neurons. As shown in Fig. 4A, basal levels of phospho-Erk1/
2 signals were observed in the intact group, and in the sham
control, some neurons showed moderately elevated signals.
More intense signals were found in both the acupuncture
and the sciatic nerve injury groups. Phospho-Erk1/2 signals
were co-localized with neuronal marker protein bIII-tubulin
(Fig. 4B), indicating that acupuncture stimulation induced
Erk1/2 activation as well as GAP-43 in DRG sensory neurons.
To explore the possibility that acupuncture stimulation
on ST36 had any neurophysiological interaction with the
autonomic nervous system, we investigated the activation
of neurons in the DVC of the mouse brain where the vagal
afferent and efferent bers communicate with the visceral
organs. Electroacupuncture on ST36 and sciatic nerve injury
induced c-Fos signals over the DMV and the nucleus of the
solitary tract (NST) (Fig. 5AeC). Retrograde labeling of DMV
neurons after the injection of FG into the peripheral vagus
nerve revealed that some, but not all, of the c-Fos signals
were co-localized with FG-labeled DMV neurons (Fig. 5B).
Additionally, weak c-Fos signals were scattered over the
NST area after electroacupuncture stimulation (Fig. 5C).
4. Discussion
A growing body of evidence shows that acupuncture stim-
ulation in an experimental animal generates pathophysio-
logical responses, which may reect a clinical correlation
to acupuncture therapy. However, a mechanistic basis on
how the acupuncture generates physiological responses is
not known, although the philosophical theory states that
acupoint stimulation invigorates the meridian ow of qi
thus balancing yin-yang. As an initial step to elucidate the
potential role of the nervous system in mediating
acupuncture stimulation, we examined the responsiveness
of DRG sensory neurons following ST36 stimulation in mice
and rats, where all of the peripheral somatosensory inputs
are transmitted to the spinal cord via DRG sensory neurons.
Our data show that acupuncture stimulation clearly induces
signals of GAP-43 and phospho-Erk1/2 in DRG neurons. To
characterize acupuncture-specic responsiveness, we
analyzed DRG neuronal responses to sham acupuncture in
parallel. Our data show that a certain level of neuronal
response to sham stimulation is generated, although the
extent of the responsiveness is weaker than that of
acupuncture stimulation. We further demonstrated that
acupuncture-mediated ascending signals induced c-Fos
Figure 2 GAP-43 expression in rat DRG after acupuncture
stimulation. (A) RT-PCR analysis of GAP-43 mRNA in the DRG.
Animals were treated with acupuncture stimulation by insert-
ing needles on ST36 (ACU) and with sham stimulation and
sciatic nerve injury (SNI). (Upper) Representative data on RT-
PCR for GAP-43 and actin as an internal loading control.
(Bottom) Quantication of the band intensity in a ratio of GAP-
43 to actin (number of independent experiments Z4). Data
denote mean sem (*p <0.05, **p <0.01 vs. intact group). (B)
Immunouorescence staining of GAP-43 in the DRG of animal
groups with different treatments. Scale bar: 100 mm.
Neuronal activation by using acupuncture 151
signals in the DVC, some of which were localized to the DMV
neurons.
As the primary target molecule determining
acupuncture-specic neuronal responses, we selected GAP-
43. GAP-43 is expressed in developing neurons and in some
adult neurons which are involved in activity-dependent
synaptic plasticity [11]. Possibly, GAP-43 is most clearly
induced at the gene expression level after peripheral nerve
injury. Injuries on either peripheral nerves or central
nervous system axons generate retrograde signals which are
transmitted into the cell body and trigger regenerative
responses in the cell nucleus [15]. Purinergic receptor
(P2Y
2
) activation by ATP-gS injection in the sciatic nerve
has been reported to upregulate GAP-43 expression in DRG
sensory neurons [9]. Interestingly, acupuncture stimulation
on ST36 in mice has induced focal increases in ATP, ADP,
AMP, and adenosine, as well as adenosine A1 receptor
activation, which is known to generate antinociceptive
action [8]. Thus, we speculate that purines released by
ST36 stimulation may initiate retrograde signaling events
such as GAP-43 production in the soma.
Our data showed that GAP-43 mRNA expression was
increased by 130% and 30% by ST36 acupuncture in mice and
rats, respectively. There were some increases caused by
sham stimulation, but the induction levels were consis-
tently lower than those caused by acupuncture, implying
Figure 3 GAP-43 expression in the rat DRG after electroacupuncture. RT-PCR analyses of GAP-43 mRNA in the DRG at (A) lumbar
level 4 and (B) lumbar 5. Animals were treated with electroacupuncture stimulation by inserting the needle on ST36 (EACU) and
with sham stimulation and sciatic nerve injury (SNI). (Upper) Representative data on RT-PCR for GAP-43 and actin as an internal
loading control. (Bottom) Quantication of the band intensity in a ratio of GAP-43 to actin (number of independent exper-
iments Z4). Data denote mean sem (*p <0.05, **p <0.01, ***p <0.001 vs. intact group). (C) Electroacupuncture at ST36 (EACU)
and sham stimulation were given to individual animals (#1 and #2 per experimental group) and these animals were used for the
GAP-43 analyses of the DRG at lumbar levels 4 and 5 by immunouorescence staining. Scale bar: 100 mm.
152 M.-H. Kim et al.
acupuncture-specic effects. When we applied an alter-
native stimulation paradigm with electroacupuncture to
the same acupoint, overall responses in terms of GAP-43
induction were similar to those of conventional acupunc-
ture in the rats; GAP-43 mRNA levels were comparable
between the DRGs at lumbar levels 4 and 5, although the
induction was more consistent in the DRG at lumbar level 4.
Although further studies are required to conrm whether
GAP-43 is a reliable indicator for acupuncture-specic
neuronal responses, our experimental approach to
exploring two rodent species has provided convincing
evidence that nervous system activation appears to
mediate acupuncture stimulation.
As another indicator of neuronal responses, Erk1/2
activation was investigated. Phospho-Erk1/2 is known to be
increased in response to diverse external stimulations and
to be associated with activation of neurons in culture and
in vivo systems [16]. At this moment, whether GAP-43 and
phospho-Erk1/2 are functionally linked to each other in
DRG neurons is not known. Presumably, a retrograde signal
of phospho-Erk1/2 may induce cell-body responses,
including GAP-43 expression in the nucleus [17], which is
consistent with pharmacological demonstration of Erk1/2-
dependent GAP-43 regulation [9].
How can acupuncture-mediated somatic nerve activa-
tion lead to physiological and pathological consequences
for visceral organs? While pathological responses such as
gastric motility changes following acupuncture support the
notion that vagus nerve activation may be one of the major
targets for acupuncture stimulation [18,19], underlying
neurophysiological mechanisms are still elusive. Previous
studies showed that the peripheral somatosensory signals
could be integrated into the DVC neuronal circuits
composed of the NST, the DMV, and the area postrema
[20e22]. Our study shows that c-Fos signals after electro-
acupuncture at ST36 were detected in the DMV and the NST,
but the signal was much stronger in the DMV neurons as
identied by FG dye retrogradely traced from the periph-
eral vagus nerve. The distribution patterns of c-Fos in the
NST and the DMV neurons are somewhat different from
those in the previous reports [23e25], which may reect
variations in the experimental paradigms, including the
stimulation protocol. NST neurons receive visceral afferent
signals via the solitary tract and communicate with
numerous ascending and descending neural pathways
between the vagus nerve and the brain. The DMV integrates
ascending somatosensory inputs, as well as NST neurons,
and relays visceral outputs to internal organs [26]. Previous
studies reported the generation of an evoked potential in
the NST area or the neurons in the rostro ventrolateral
medulla (RVLM) following electric stimulation on the
periphery [20,27]. Interestingly, acupuncture at ST25 was
Figure 4 Phospho-Erk1/2 signals in rat DRG after electroacupuncture. (A) After acupuncture treatments, phospho-Erk1/2 signals
were analyzed by immunouorescence staining in the DRG of individual animals. (B) Double immunouorescence staining of DRG
sections with anti-phospho-Erk1/2 antibody and anti-bIII-tubulin antibody (TUJ1). The merged image shows that phospho-Erk1/2
and bIII-tubulin signals are highly colocalized (in yellow). The images in (B) are representatives from DRG sections prepared
from the rats given electroacupuncture. Scale bar: 100 mm.
Neuronal activation by using acupuncture 153
reported to increase c-Fos signals in RVLM [23]. Thus,
acupuncture-mediated somatosensory signals may affect
DMV neurons via indirect neuronal circuits in the lower
brain stem areas. In summary, we found that acupuncture
stimulation on ST36 activates DRG sensory neurons and DMV
neurons. Future studies on characterizing the neuronal
circuitry in the DVC that innervates vagal efferents are
critical in understanding the pathophysiological bases for
acupuncture-specic neural mechanisms.
Acknowledgment
This work was supported by Daejeon University Research
Fund.
References
1. Mayer DJ. Acupuncture: an evidence-based review of the
clinical literature. Annu Rev Med. 2000;51:49e63.
2. Perlow BW. Acupuncture: its theory and use in general prac-
tice. Proc R Soc Med. 1973;66:426e428.
3. Dhond RP, Yeh C, Park K, Kettner N, Napadow V. Acupuncture
modulates resting state connectivity in default and sensori-
motor brain networks. Pain. 2008;136:407e418.
4. Zhao ZQ. Neural mechanism underlying acupuncture analgesia.
Prog Neurobiol. 2008;85:355e375.
5. Lux G, Hagel J, Backer P, Backer G, Vogl R, Ruppin H, et al.
Acupuncture inhibits vagal gastric acid secretion stimulated by
sham feeding in healthy subjects. Gut. 1994;35:1026e1029.
6. Walker EM, Rodriguez AI, Kohn B, Ball RM, Pegg J, Pocock JR,
et al. Acupuncture versus venlafaxine for the management of
vasomotor symptoms in patients with hormone receptor-
positive breast cancer: a randomized controlled trial. J Clin
Oncol. 2010;28:634e640.
7. Park HJ, Lim S, Joo WS, Yin CS, Lee HS, Lee HJ, et al.
Acupuncture prevents 6-hydroxydopamine-induced neuronal
death in the nigrostriatal dopaminergic system in the rat Par-
kinsons disease model. Exp Neurol. 2003;180:93e98.
8. Goldman N, Chen M, Fujita T, Xu Q, Peng W, Liu W, et al.
Adenosine A1 receptors mediate local anti-nociceptive effects
of acupuncture. Nat Neurosci. 2010;13:883e888.
9. Arthur DB, Akassoglou K, Insel PA. P2Y2 receptor activates
nerve growth factor/TrkA signaling to enhance neuronal
differentiation. Proc Natl Acad Sci U S A. 2005;102:
19138e19143.
10. Aigner L, Arber S, Kapfhammer JP, Laux T, Schneider C,
Botteri F, et al. Overexpression of the neural growth-
Figure 5 Co-localization of c-Fos signals with FG-labeled DMV motor neurons in mice after electroacupuncture. (A) Hematoxylin
and eosin staining of coronal brain sections. Fluorescence images of FG-labeled neurons and c-Fos signals in the (B) DMV and the (C)
NST areas. While FG-labeled neurons by retrograde tracing were observed exclusively in the DMV, but not the NST, areas, c-Fos
signals were seen in both the DMV and the NST areas. AP Zarea postrema; NST Znucleus of the solitary tract; DMV Zdorsal motor
nucleus of the vagus nerve; CBL Zcerebellum. The scale bars in (A) and (B) represent 300 mm and 100 mm, respectively.
154 M.-H. Kim et al.
associated protein GAP-43 induces nerve sprouting in the adult
nervous system of transgenic mice. Cell. 1995;83:269e278.
11. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant
of neuronal development and plasticity. Trends Neurosci.
1997;20:84e91.
12. Schneider A, Enck P, Streitberger K, Weiland C, Bagheri S,
Witte S, et al. Acupuncture treatment in irritable bowel
syndrome. Gut. 2006;55:649e654.
13. Ernst E. Acupunctureea critical analysis. J Intern Med. 2006;
259:125e137.
14. Han IS, Seo TB, Kim KH, Yoon JH, Yoon SJ, Namgung U. Cdc2-
mediated Schwann cell migration during peripheral nerve
regeneration. J Cell Sci. 2007;120:246e255.
15. Skene JH. Axonal growth-associated proteins. Annu Rev Neu-
rosci. 1989;12:127e156.
16. Segal RA, Greenberg ME. Intracellular signaling pathways
activated by neurotrophic factors. Annu Rev Neurosci. 1996;
19:463e489.
17. Hanz S, Fainzilber M. Retrograde signaling in injured ner-
veethe axon reaction revisited. J Neurochem. 2006;99:13e19.
18. Iwa M, Matsushima M, Nakade Y, Pappas TN, Fujimiya M,
Takahashi T. Electroacupuncture at ST-36 accelerates colonic
motility and transit in freely moving conscious rats. Am J
Physiol Gastrointest Liver Physiol. 2006;290:G285eG292.
19. Yin J, Chen J, Chen JD. Ameliorating effects and mechanisms
of electroacupuncture on gastric dysrhythmia, delayed
emptying, and impaired accommodation in diabetic rats. Am J
Physiol Gastrointest Liver Physiol. 2010;298:G563eG570.
20. Zagon A. Sciatic and vagal sensory inputs converge onto non-
baroreceptive neurones of the rostral ventrolateral medulla.
Brain Res. 2001;896:64e68.
21. Zagon A. Does the vagus nerve mediate the sixth sense? Trends
Neurosci. 2001;24:671e673.
22. Potts JT, Paton JF, Mitchell JH, Garry MG, Kline G, Anguelov PT,
et al. Contraction-sensitive skeletal muscle afferents inhibit
arterial baroreceptor signalling in the nucleus of the solitary
tract: role of intrinsic GABA interneurons. Neuroscience. 2003;
119:201e214.
23. Iwa M, Tateiwa M, Sakita M, Fujimiya M, Takahashi T.
Anatomical evidence of regional specic effects of acupunc-
ture on gastric motor function in rats. Auton Neurosci. 2007;
137:67e76.
24. Liu JH, Li J, Yan J, Chang XR, Cui RF, He JF, et al. Expression of
c-fos in the nucleus of the solitary tract following electro-
acupuncture at facial acupoints and gastric distension in rats.
Neurosci Lett. 2004;366:215e219.
25. Kim SK, Kim J, Woo HS, Jeong H, Lee H, Min BI, et al. Elec-
troacupuncture induces Fos expression in the nucleus tractus
solitarius via cholecystokinin A receptor signaling in rats.
Neurol Res. 2010;32:116e119.
26. Travagli RA, Hermann GE, Browning KN, Rogers RC. Brainstem
circuits regulating gastric function. Annu Rev Physiol. 2006;68:
279e305.
27. Person RJ. Somatic and vagal afferent convergence on solitary
tract neurons in cat: electrophysiological characteristics.
Neuroscience. 1989;30:283e295.
Neuronal activation by using acupuncture 155