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ArticleTitle Depressing effect of electroacupuncture on the spinal non-painful sensory input of the rat
Article Sub-Title
Article CopyRight Springer-Verlag Berlin Heidelberg
(This will be the copyright line in the final PDF)
Journal Name Experimental Brain Research
Corresponding Author Family Name Quiroz-Gonzlez
Particle
Given Name Salvador
Suffix
Division Departamento de Acupuntura y Rehabilitacin
Organization Universidad Estatal del Valle de Ecatepec
Address Av. Central s/n, Esq. Leona Vicario, Col. Valle de Anhuac, Secc. A,
Ecatepec, Estado de Mxico, CP 55210, Mexico
Email sqg20@yahoo.com.mx
Author Family Name Segura-Alegra
Particle
Given Name Bertha
Suffix
Division Facultad de Estudios Superiores, FES Iztacala
Organization UNAM
Address Mexico, CP 54090, Mexico
Email bsegura@campus.iztacala.unam.mx
Author Family Name Jimnez-Estrada
Particle
Given Name Ismael
Suffix
Division Departamento de Fisiologa, Biofsica y Neurociencias
Organization Centro de Investigacin y, Estudios Avanzados del IPN
Address Av. Instituto Politcnico Nacional 2508, Col. San Pedro Zacatenco, AP.
14-740, Mexico, DF, CP 07000, Mexico
Email ijimenez@fisio.cinvestav.mx
Schedule
Received 13 February 2014
Revised
Accepted 12 April 2014
Abstract The aim of this study was to explore the effect of electroacupuncture (EA) applied in the Zusanli (ST36) and
Sanyinjiao (SP6) points on the N1 component of the cord dorsum potential (CDP) evoked by electrical
stimulation of the sural nerve (SU) in the rat. The experiments were performed in 44 Wistar rats (250300 g)
anesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg). A bilateral laminectomy was performed to
expose the L3 to S2 segments of the spinal cord. The SU nerve was exposed and placed on pairs of hook
electrodes for electrical stimulation. The N1-CDPs were recorded with three silver-ball electrodes located on
the dorsal surface of the L5 to S1 segments. Ipsilateral high and low EA stimulation (100, 2 Hz, 6 mA, 30 min)
induced a considerable reduction in the amplitude (45 5.6, 41 6.2 %) of the N1-CDP recorded at the L6
segmental level. Recovery of the N1-CDP amplitude occurred approximately 13 s after EA. Sectioning of
the saphenous and superficial peroneal nerves reduced the depressing effect provoked by the EA stimulation
(18.7 1.3, 27 3.8 %). Similarly, sectioning of the posterior and anterior tibial, deep peroneal and
gastrocnemius nerves partially reduced the effect provoked by EA (11 1.5, 9.8 1.1, 12.6 1.9 %).
Intravenous picrotoxin (1 mg/kg) also reduced the action of low and high EA (23 4.8, 27 5.2 %). It is
suggested that EA stimulation depresses non-painful sensory pathways through the activation of specific
inhibitory pathways that receive modulatory actions from other sensory and muscle afferent inputs in the rat
spinal cord.
Keywords (separated by '-') Electroacupuncture - Cord dorsum potentials - Sural nerve - Spinal cord
Footnote Information
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Journal : Large 221 Dispatch : 19-4-2014 Pages : 9
Article No : 3965 LE TYPESET
MS Code : EBR-14-0106 CP DISK
1 3
Exp Brain Res
DOI 10.1007/s00221-014-3965-2
RESEARCH ARTICLE
Depressing effect of electroacupuncture on the spinal non-painful
sensory input of the rat
Salvador Quiroz-Gonzlez Bertha Segura-Alegra
Ismael Jimnez-Estrada
Received: 13 February 2014 / Accepted: 12 April 2014
Springer-Verlag Berlin Heidelberg 2014
stimulation (18.7 1.3, 27 3.8 %). Similarly, sectioning
of the posterior and anterior tibial, deep peroneal and gas-
trocnemius nerves partially reduced the effect provoked by
EA (11 1.5, 9.8 1.1, 12.6 1.9 %). Intravenous picro-
toxin (1 mg/kg) also reduced the action of low and high EA
(23 4.8, 27 5.2 %). It is suggested that EA stimulation
depresses non-painful sensory pathways through the activa-
tion of specic inhibitory pathways that receive modulatory
actions from other sensory and muscle afferent inputs in
the rat spinal cord.
Keywords Electroacupuncture Cord dorsum potentials
Sural nerve Spinal cord
Introduction
Acupuncture is a therapeutic modality that emerged from
Traditional Chinese Medicine. The World Health Organi-
zation recommends the use of acupuncture for the treat-
ment of a wide variety of diseases (Zhang et al. 2014; Yin
et al. 2010; Barnes et al. 2008). A novel contemporary
form of acupuncture, electrical stimulation of acupuncture
points (APs), also known as electroacupuncture (EA), has
been widely used in both clinical and experimental stud-
ies (Vickers et al. 2012; Zhao 2008). Several studies have
demonstrated that EA exerts an analgesic effect on neuro-
pathic pain in rat models (Lau et al. 2008; Kim et al. 2004;
Huang et al. 2004; Hwang et al. 2002) and relieves acute or
chronic inammatory pain (Kim et al. 2006; Zhang et al.
2004). There has been particular interest in determining the
mechanisms involved in the antinociceptive effect of EA
(Zhao 2008; Leung 2012). Acupuncture analgesia is a clear
manifestation of modulatory processes occurring at differ-
ent levels of the central nervous system (Zhang et al. 2014;
Abstract The aim of this study was to explore the effect
of electroacupuncture (EA) applied in the Zusanli (ST36)
and Sanyinjiao (SP6) points on the N1 component of the
cord dorsum potential (CDP) evoked by electrical stimu-
lation of the sural nerve (SU) in the rat. The experiments
were performed in 44 Wistar rats (250300 g) anesthe-
tized with ketamine (100 mg/kg) and xylazine (2 mg/kg).
A bilateral laminectomy was performed to expose the
L3 to S2 segments of the spinal cord. The SU nerve was
exposed and placed on pairs of hook electrodes for elec-
trical stimulation. The N1-CDPs were recorded with three
silver-ball electrodes located on the dorsal surface of the
L5 to S1 segments. Ipsilateral high and low EA stimu-
lation (100, 2 Hz, 6 mA, 30 min) induced a considerable
reduction in the amplitude (45 5.6, 41 6.2 %) of the
N1-CDP recorded at the L6 segmental level. Recovery of
the N1-CDP amplitude occurred approximately 13 s after
EA. Sectioning of the saphenous and supercial peroneal
nerves reduced the depressing effect provoked by the EA
S. Quiroz-Gonzlez (*)
Departamento de Acupuntura y Rehabilitacin, Universidad
Estatal del Valle de Ecatepec, Av. Central s/n, Esq. Leona Vicario,
Col. Valle de Anhuac, Secc. A, CP 55210 Ecatepec,
Estado de Mxico, Mexico
e-mail: sqg20@yahoo.com.mx
B. Segura-Alegra
Facultad de Estudios Superiores, FES Iztacala, UNAM,
CP 54090 Mexico, Mexico
e-mail: bsegura@campus.iztacala.unam.mx
I. Jimnez-Estrada
Departamento de Fisiologa, Biofsica y Neurociencias, Centro
de Investigacin y, Estudios Avanzados del IPN, Av. Instituto
Politcnico Nacional 2508, Col. San Pedro Zacatenco,
AP. 14-740, CP 07000 Mexico, DF, Mexico
e-mail: ijimenez@sio.cinvestav.mx
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Quiroz-Gonzlez et al. 2014; Zhao 2008). The rst conver-
gence of impulses originating from pain sites and acupoints
occurs in the spinal dorsal horn, where the neuronal noci-
ceptive responses appear to be depressed by both pre- and
post-synaptic inhibition during EA stimulation (Zhao 2008;
Li et al. 1993). Although the effectiveness of EA for pain
control has been demonstrated experimentally in animal
models, little is known about the possible effects exerted by
EA on the cutaneous non-painful sensory input in the spi-
nal cord. Electrical stimulation of cutaneous nerves induces
the activation of sensory neurons located in the dorsal
horn of the spinal cord (Bernhard 1953; Willis et al. 1973;
Coombs et al. 1956), which produces cord dorsum poten-
tials (CDPs) consisting of clearly dened components. The
rst observed component in the CDPs is the afferent volley
(AV), which is caused by the electrical activation of low-
threshold afferent bers (Coombs et al. 1956). This is fol-
lowed by one negative component (named N1-CDP), which
is generated by the monosynaptic activation of the dorsal
horn neurons via the A afferents or non-painful bers in
the cutaneous nerves (Bernhard 1953; Willis et al. 1973;
Coombs et al. 1956). Subsequently, a long-lasting positive
component occurs, named the P wave, which is ascribed to
the current ows generated during the presynaptic depolari-
zation of afferent bers (PAD) and presynaptic inhibition
(Rudomin and Schmidt 1999). The present study aimed
to analyze the effect of EA stimulation on the non-painful
sensory neurons at the level of the spinal cord. To achieve
this goal, we recorded the N1-CDP evoked by cutaneous
SU nerve stimulation and during EA stimulation. In a sec-
ond series of experiments, we evaluated how this effect is
modied by sectioning cutaneous and muscular nerves.
Because it is amply recognize that GABAergic mechanism
is involved in the modulation of primary afferent depolari-
zation (PAD) of cutaneous and muscular nerves, we ana-
lyzed the possible effect of picrotoxin (PTX) on the actions
of the EA. Some of these observations have been published
previously in abstract form (Quiroz-Gonzalez et al. 2013).
Methods
Animals
Male Wistar rats (n = 44) weighing 250300 g (8
10 weeks old) that were obtained from the animal house
of our institution were used. All animals had free access to
water and were housed under identical environmental con-
ditions of light and dark cycles (12:12 h) and temperature
(2224 C). All experiments were carried out in accordance
with the guidelines of the Mexican Ofcial Norm (NOM-
062-ZOO-1999) and in accordance with the National Insti-
tutes of Health Guide for the Care and Use of Laboratory
Animals (NIH Publication No. 8023, revised in 1978). The
study protocol was approved by the institutional bioethical
committee for Care and Handling of Laboratory Animals
(Protocol 0267-05, CINVESTAV).
Surgical procedures
Initially, the animals were anesthetized with an intraperito-
neal injection of a mixed solution of ketamine (100 mg/kg)
and xylazine (2 mg/kg). This injection was supplemented
every hour by additional doses of ketamine (50 mg/kg),
applied in the same low abdominal region to ensure that an
adequate level of anesthesia, dened as the absence of with-
drawal reexes. We used ketamine as an analgesic because
it has been demonstrated that it did not signicantly reduce
the response of spinal dorsal horn neurons to low-threshold
afferent inputs in the intact animal, but in contrast suppress
noxiously evoked activity of wide dynamic range neurons
(Collins 1986). Subsequently, the femoral vein was exposed
and cannulated for the administration of a solution of pic-
rotoxin (PTX, 1 mg/kg of body weight; Quirz-Gonzalez
et al. 2012). A bilateral laminectomy was performed in the
lumbosacral enlargement (from the L4 to S1 segments) of
the spinal cord. Several nerves of the right hind leg were
carefully exposed and prepared for stimulation and/or
sectioning: the main branch of the sural (SU), supercial
peroneus (SP), tibial (TA), saphenous (SA) deep peroneus
(DP) and gastrocnemius (GS) nerves and the rats were then
secured in a stereotaxic frame. The surgical procedure for
sural nerve dissection on the hind leg was carefully made
to avoid the possible damage of the Zusanli (ST36) and
Sanyinjiao (SP6) acupoints. The animals body temperature
was monitored using a thermal probe located in the back
muscles and connected to an automatic feedback control
unit and a heating blanket to maintain the animals body
temperature at 37 C. The skin aps and muscle around the
exposed tissues were raised and tied to a metal stereotaxic
frame to form a pool, which was lled with warm mineral
oil to prevent the tissues from drying.
SU nerve stimulation
The central end of the sural (SU) nerve in the left hind
limb was mounted on a pair of stimulating silver hooks
connected to an isolated-current-pulse generator (Isoex
D 4030), and the CDPs were produced in all experiments
by single square-current pulses (0.05 ms duration; at 1 Hz)
of graded intensity. The pulses were monitored by record-
ing the voltage drops across a resistor (1,000 ) that was
placed in the current return path. The electrical threshold
(1 T) was established as the minimum stimulus strength
(usually between 0.1 and 0.13 mA) necessary to cause a
discernible CDP response on the surface of the spinal cord.
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Electroacupuncture stimulation
Pairs of stainless-steel acupuncture needles (0.25 mm in
diameter and 5 mm in length) were inserted perpendicu-
larly at a depth of 3 mm at the Zusanli (ST36, 5 mm lateral
to the anterior tubercle of the tibia) and Sanyinjiao acu-
points (SP6, 3 mm proximal to the medial malleolus, at the
posterior border of the tibia). These acupoints are tradition-
ally used in acupuncture-induced analgesia for the treat-
ment of several pain syndromes (Zhang et al. 2003; Huang
et al. 2002). To disclose the region-specic effect of EA,
we stimulate two non-acupoints (NAP) nearby to ST36 and
SP6, the rst located 2 mm lateral to ST36 and the second
3 mm posterior to SP6. The transpositional method (Yin
et al. 2008) for rats was used to determine the acupoint
locations. The needles were connected to an isolated-cur-
rent- pulse generator (Isoex D 4030), and square trains of
high or low frequency (100 or 2 Hz, respectively) and vari-
able strength current pulses (16 mA, 0.1 ms) were applied
in the acupoints located both contralateral and ipsilateral of
the sural nerve stimulation for a total of 30 min.
CDP recording
Chlorinated silver-ball electrodes were placed on the dorsal
surface of several segments in the lumbosacral spinal cord
(L3S2) to record the CDPs (Fig. 1), and the corresponding
reference silver electrodes were inserted into the adjacent
paravertebral musculature. Each recording pair of elec-
trodes was connected to an individual low-noise, high-gain
differential amplier (Grass, model P511; band-pass lters
were set at 0.310 kHz). The resulting recordings were dig-
itized, averaged (n = 40 samples at 1 Hz), and stored in a
digital computer using a specially designed software (built
in the Lab-VIEW environment). The peak amplitude of the
N1-CDPs was measured and subsequently analyzed.
Data analysis
All statistical analyses were performed using the Graph-
Pad Prism (version 4) software. Data were expressed as the
mean standard deviation. A two-way analysis of variance
test for multiple comparisons followed by a Bonferroni cor-
rection was used to determine the differences in the ampli-
tudes of the N1-CDPs produced by SU nerve stimulation
and those produced by EA stimulation on ST36 and SP6.
Repeated measure ANOVA followed by Newman-Keuls
posthoc test was used to determine the differences between
the amplitude of control SU N1-CDPS and the amplitude
of the conditioned SU nerve responses evoked during EA
stimulation. The differences were considered signicant at
p < 0.05.
Results
CDPs provoked by SU nerve and EA stimulation
Single electrical pulses applied to the SU nerve (34 T,
at 1 Hz) produced N1-CDPs of a relatively large amplitude
(Fig. 1a). The largest N1-CDP occurred at the L6-segmen-
tal level and gradually decreased in amplitude in the adja-
cent rostro-caudal spinal segments (L5, L4 and S1, respec-
tively, in Figs. 1a, 2a, 3a). Similar recordings were obtained
in 6 other experiments. This rostro-caudal distribution of
the CDPs agreed well with previous reports (Gonzlez et al.
2011). Single electrical current pulses applied to the ST36
and SP6 acupoints (34 T, at 1 Hz) produced N1-CDPs
with largest amplitude at the L5 and L6 segmental levels,
decreasing in amplitude in L4 and S1 spinal segments. The
N1-CDPs produced by SU nerve stimulation showed a sim-
ilar rostro-caudal distribution than those produced by EA
applied to ST36 and SP6, but they are smaller in amplitude
(p < 0.05; Fig. 1a, b).
Fig. 1 Experimental arrangement for the N1-CDP recording: a aver-
age N1-CDP produced by SU nerve stimulation and EA stimulation
recorded at L4 to S1 spinal cord segments. b Graphs illustrating the
longitudinal distribution on the spinal segments of the N1-CDP aver-
ages amplitudes (n = 7) produced by SU nerve and EA stimulation at
ST36 and SP6. (*p < 0.05)
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Effect of high frequency EA stimulation on the amplitude
of the N1-CDP
The N1-CDP recorded on the surface of the L5 to S1 seg-
ments gradually decreased in amplitude when the high
frequency (100 Hz) EA stimulation strength was progres-
sively increased (from 1 to 6 mA; Fig. 2a, b). The maximal
depression of the N1-CDP (45 5.6 %) was observed at
the L6 segment (Figs. 2a, b, 3a, b) and occurred immedi-
ately upon and during EA stimulation (30 min). In most
Fig. 2 Inhibition of the
N1-CDP by high (100 Hz) EA
stimulation on ST36 and SP6:
a average N1-CDP produced
by SU nerve stimulation and
recorded in several spinal
segments (L5S1) before EA
(control) and during ipsilateral
or contralateral EA stimulation
at different stimulus intensities
(16 and 6 mA, respectively).
b Graphs illustrating the mean
reduction (n = 11) in the
N1-CDP component recorded
in the S1L5 spinal segments
during a 100 Hz ipsilateral EA
stimulation. Asterisks indicate
signicant differences between
N1-CDP responses produced by
SU nerve stimulation before EA
and during high EA stimulation
(*p < 0.05 and **p < 0.01)
Fig. 3 Time course of inhibi-
tory effect of EA on N1-CDP
component: a CDP recording
showing that after EA, there
was a recovery of the N1-CDP
component produced by SU
nerve stimulation in several
spinal segments (S1L5).
b Graphs illustrating the
changes in the percent ampli-
tude of the N1-CDPs produced
before (control), during and
after EA. Asterisks indicate
signicant differences between
N1-CDP responses produced
by SU nerve stimulation before
EA and during posthigh EA
stimulation (*p < 0.05 and
**p < 0.01)
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cases, the increment in intensity of EA stimulation (above
6 mA) did not increase the magnitude of the depression at
the different segments analyzed. Contralateral EA stimu-
lation (6 mA) and NAP stimulation had no effect on the
N1-CDP (Fig. 2a, b). After removal of the EA stimulus,
recovery of depressive actions on the N1-CDP occurred
within approximately 12 s (Fig. 3a, b).
Effect of low frequency EA stimulation on the amplitude
of the N1-CDP
30 min of low frequency EA stimulation (2 Hz) induce the
reduction in amplitude of the N1-CDP (41 6.2 %), but
only when the N1-CDP caused by EA stimulation occurred
at 100 ms before the N1-CDP produced by SU nerve stimu-
lation (Fig. 4B, C, D). No statistical differences were found
when the N1-CDP caused by EA stimulation occurred after
N1-CDP produced by SU nerve stimulation (Fig 4E, F).
Effect of nerve sectioning on the depressive action of EA
Sectioning the sensory saphenous and supercial pero-
neal nerves (Fig. 5a, b) induced a signicant reduc-
tion in the depressing effect provoked by EA stimulation
on the SU-evoked N1-CDP (18.7 1.3 %, n = 7 and
27 3.8 %, n = 7, respectively). Meanwhile, section-
ing of the tibial, deep peroneal and gastrocnemius nerves
(Fig. 5a, b) reduced the depressing effect provoked by EA
on the N1-CDPs but to a lesser extent (11 1.5 %, n = 7;
9.8 1.1 %, n = 7; and 12.6 1.9 %, n = 7, respectively).
The effect of PTX on the depressive action of EA
In order to analyze the possible GABAergic mechanism on
the depressive actions of EA on the SU-evoked N1-CDP,
systemic injection of a GABA
A
antagonist, picrotoxin
(PTX, 1 mg/kg) was delivered. As shown in Fig. 6D, F, the
application of PTX reduces the depressive actions of low
frequency EA (23 4.8 %, n = 7), as compared with con-
trol recording (Fig. 6A, F). Similar reductions were found
(27 5.2 %, n = 7) on the depressive actions of high fre-
quency EA stimulation (Fig. 6E, F). Intravenous saline
vehicle does not produced any effect on the EA conditioned
depression of the SU-evoked N1-CDP.
Discussion
It is well established that the N1-CDP generated by the
electrical stimulation of cutaneous nerves is produced by
the monosynaptic activation of dorsal horn neurons located
in the Rexeds laminae III to VI via A afferent bers or
low-threshold cutaneous afferent bers (Bernhard 1953;
Willis et al. 1973; Coombs et al. 1956). In our study, stimu-
lation of the sensory SU nerve and EA at the ST36 and SP6
acupoints provoked N1-CDPs that were simultaneously
Fig. 4 Inhibition of the SU N1-CDP by low (2 Hz) EA stimula-
tion applied on the ST36 and SP6 acupoints: a averaged N1-CDP
(n = 16 recordings) produced by SU nerve stimulation and recorded
in L6 spinal segment before EA stimulation, b during EA (EA-CDP)
applied 70 ms previously to the stimulus of the SU nerve, c 40 ms, d
20 ms, and e 30 ms after SU-evoked N1-CDP (ASU-CDP). f Graphs
illustrate averaged (SD) percent reduction values of the N1-CDP
component recorded on the L6 spinal segment in nine animals, during
a 2 Hz ipsilateral EA stimulation. Asterisks indicate signicant differ-
ences between N1-CDP responses produced by SU nerve stimulation
before EA and during low EA stimulation (*p < 0.05 and **p < 0.01)
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Fig. 5 Reduction in the
effect of EA at 100 Hz on the
N1-CDP component by section-
ing nerves: a CDPs recording
showing the effect of nerve sec-
tioning on the depressive action
evoked in N1-CDP by high EA
stimulation (100 Hz).
b Graphs illustrating the
changes in the percent ampli-
tude of the N1-CDPs produced
by EA and abolished by nerve
sectioning (n = 7 animals per
experimental procedure). SP
supercial peroneous; SA saphe-
nous; TA tibial; GS gastrocne-
mius soleus nerves. Asterisks
indicate signicant differences
between control N1-CDP
responses evoked by SU nerve
stimulation and during EA
stimulation before and after the
sectioning of cutaneous and/or
muscular nerves (*p < 0.05 and
**p < 0.01)
Fig. 6 Effect of picrotoxin (PTX; 1 mg/kg) on the depressive effect
of EA on the N1-CDP: a averaged N1-CDP produced by SU nerve
stimulation and recorded in L6 spinal segment before EA stimula-
tion, b during low frequency EA (2 Hz) applied 40 ms previously to
the stimulus of the SU nerve, c with high frequency EA (100 Hz),
d The effect of intravenous injection of PTX on the low frequency
EA conditioned depression of the SU-evoked N1-CDP, e PTX under
high frequency EA conditioned depression, f, g low or high frequency
EA+ vehicle intravenous administration, h graphs illustrate averaged
(SD) percent reduction values of the N1-CDP component recorded
on the L6 spinal segment, during a 2 Hz EA stimulation (7 animals),
100 Hz EA stimulation (7 animals) intravenous PTX (7 animals),
and saline vehicle control (3 animals). Asterisks indicate signi-
cant differences between N1-CDP responses produced by SU nerve
stimulation before EA, during EA stimulation and intravenous PTX
(*p < 0.05 and **p < 0.01)
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recorded on several segments of the lumbosacral enlarge-
ment (L4 to S1). The SU nerve evoked N1-CDP with the
largest was recorded on the L6 segment, and it is consist-
ent with those reported in other studies (Willis et al. 1973;
Gonzlez et al. 2011). Meanwhile, the largest N1-CDPs
evoked by EA were recorded at the L5L6 spinal segments.
These observations may suggest that the sensory inputs
activated by sural nerve and EA acupoint stimulation prob-
ably share spinal pathways which probably interact synap-
tically at the spinal cord level. According to the later, we
also found that conditioning low and high frequency EA
stimulation depressed the N1-CDPs produced by SU nerve
stimulation. In contrast, EA stimulation on non-acupoint
sites does not evoke signicant changes in the SU nerve
evoked N1-CDP, suggesting a specic acupoint effect of
EA. It is proposed that EA reduces the activation of dorsal
horn neurons provoked by low-threshold cutaneous afferent
bers by the activation of specic sensory pathways in the
spinal dorsal horn of the rat.
Transmission of non-nociceptive and nociceptive infor-
mation via primary afferents is modulated at the rst spinal
relay by highly complex processes (Rudomin and Schmidt
1999; Le Bars 2002; Besson and Chaouch 1987). It has
been accepted that electrical stimulation of primary affer-
ent bers effectively modulates the synaptic efcacy of
the same and/or other afferent bers in the spinal cord (De
LaTorre et al. 2009; Rudomin and Hernandez 2008). Elec-
trophysiological evidences have shown that electrical stim-
ulation of A bers may depress nociceptive activation of
spinal dorsal horn neurons for short periods of time (Chung
et al. 1984a, b). Moreover, both brief electrical stimulation
of afferent C-bers and prolonged high frequency burst
stimulation of the sciatic nerve at A ber strength produce
long-term depression (LTD) of C-ber-evoked eld poten-
tials (Liu et al. 1998). In addition, LTD of synaptic trans-
mission in substantia gelatinosa neurons can be induced
by low frequency stimulation of primary A-afferent bers
(Sandkuhler et al. 1997).
Electroacupuncture stimulation also provokes consider-
able changes in the neuronal activity evoked by peripheral
nerve stimulation. Kim et al. (2011) showed that EA pro-
duced a signicant reversal of enhanced evoked responses
of the deep dorsal horn (lamina IVVII) neurons as well as
after discharges developed in ankle-sprained rats. The EA-
induced inhibition lasted for at least 30 min after the ter-
mination of EA. In other study, it was found that 2 Hz EA
induce LTD in the C-ber-evoked eld potentials recorded
within the spinal dorsal horn of rats with neuropathic pain.
In contrast, 100 Hz EA-induced long-term potentiation
(LTP) but LTD in control rats (Xing et al. 2007).
In the present study, we found a signicant reduction in
the amplitude of the N1-CDPs evoked by SU nerve stimu-
lation during ipsilateral EA stimulation (46 mA) in several
spinal segments, particularly at the L6 segment of the rat
spinal cord. The ST36 acupoint receives sensory innerva-
tion from saphenous, supercial peroneal, and lateral sural
cutaneous nerves and motor innervation from the deep per-
oneal and anterior tibialis nerves (Zhou et al. 2010), while
the SP6 acupoint receives sensory innervation from the
saphenous, sural and medial crural nerves and motor inner-
vation from the tibial nerve (Zhou et al. 2010). The spinal
projection of these nerves showed a considerable overlap,
particularly at the L5L6 segmental level, even though they
innervate different hind limb areas (Maslany et al. 1992;
Panneton et al. 2005). It thus seems reasonable to expect
that during EA stimulation, the sensory and motor inner-
vation of the acupoints are activated and that the highest
effect of EA is observed at the L6 segment.
It is known that the acupuncture effect may occur in a
gradual manner and last for a long period of time (Zhao
2008; Leung 2012). Several lines of evidence suggest that
neurotransmitters and endogenous opioids are involved in
the depressive effect of EA on spinal nociceptive neurons
and that they participate in the analgesic effect of acupunc-
ture (Zhao 2008; Leung 2012). In the present study, the
depressive effect of EA on the N1-CDP component showed
a fast onset (<1 min) and continued during EA stimulation
and after the removal of the stimulation for a relative short
period of time (12 s). According to the latter, it could be
proposed that the effect of EA on nociceptive and non-
nociceptive pathways may be mediated by different spinal
mechanisms.
It is well established that the N1-CDP could be
depressed by the conditioning stimulation of sensory
peripheral nerves and such effect is mainly attributed
to PAD and presynaptic inhibition through GABAergic
mechanism (Lidierth 2006; Quirz-Gonzalez et al. 2012).
This kind of inhibition had its highest effect at 2030 ms
and last until 100 ms (Rudomin and Schmidt 1999; Lidi-
erth 2006; Quirz-Gonzalez et al. 2012). We found in
this study that the depressive effect of low frequency EA
occurred when the N1-CDP produced by SU nerve stim-
ulation appear between 5 and 90 ms after the occurrence
of the N1-CDP evoked by EA stimulation, with the high-
est effect between 20 and 40 ms, this interval of depression
and the time of the highest effect are quite similar to the
time course of the presynaptic depolarization of afferent
bers and presynaptic inhibition. This observation is rein-
forced by the antagonist effect of Picrotoxin (PTX) on the
depression exerted by EA on cutaneous spinal responses.
In concordance with this several evidences, we proposed
that presynaptic mechanism could participate in the depres-
sive actions of acupuncture. It is also shown that PTX
not abolish the effect of EA, the remaining PTX-resistant
depression of the N1-CDP may be ascribe to the activation
of non-GABAergic mechanism, most likely glycinergic
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pathways (Rudomin and Schmidt 1999) or including to the
accumulation of potassium ions in the spinal cord (Kremer
and Lev-Tov 1998).
We also found that the depressive effects evoked by EA
were partially abolished by the sectioning of cutaneous and
muscle nerves that innervated the ST36 and SP6 acupoints.
The changes produced by sectioning of cutaneous nerves
were larger than those produced by the section of muscle
nerves. According to this evidence, it could be suggested
that specic heterosynaptic inhibitory pathways receiving
sensory and muscular inputs could be involved in the effect
of EA on low-threshold sensory pathways.
It is well known that the N1-CDP is produced by a
groups of sensory neurons that receive cutaneous large
A, afferent bers (Bernhard 1953). These spinal neurons
located in the laminae III-VI are responsible to transmit
ne touch, vibration, propiocepcion to supraspinal cent-
ers (Willis et al. 1973). It may be suggest that EA at low
or high frequency affects the transmission of these differ-
ent sensory modalities at the spinal cord level and could
have some implications in the process of the information
in the somatosensory cortex. Several lines of evidence
have hypothesized that large diameter sensory bers play a
major role in the pathogenesis of some types of neuropathic
pain (Devor 2009; Campero et al. 1998). Devor (2009)
has showed that dorsal root ganglion A afferents, which
normally signals touch and vibration, change their electri-
cal and neurotransmitter characteristics when they are sec-
tioned (axotomized). Such condition seems to switch the
sensory input of A afferents from non-painful to painful
signals (phenotypic switching), triggering, and maintain-
ing central sensitization.
Since it has been shown that EA stimulation exerts anal-
gesic and antinociceptive effects by modulating the activ-
ity of spinal dorsal horn neurons and the experimental
evidence obtained in this study indicates that high and low
frequency EA stimulation also affect low-threshold non-
painful sensory pathways at the spinal cord level in the rat,
it could be proposed that the depression of low-threshold
cutaneous pathways is involved in the reduction in neuro-
pathic pain produced by EA stimulation. However, further
studies are necessary to analyze this possibility by analyz-
ing the effect of EA on low-threshold sensory pathways in
an animal model of neuropathic pain.
In conclusion, the present study showed that EA stim-
ulation depressed non-painful sensory spinal pathways
through the activation of specic inhibitory pathways that
receive modulatory actions from sensory and muscle affer-
ent inputs in the rat spinal cord.
Acknowledgments We thank American Journal Experts for edit-
ing the English of this text, Jos Carlos Guadarrama Olmos for tech-
nical assistance and to Enrique Velazquez and Porrio Reyes for
their programming assistance. This work was partially supported
by fellowships granted to I. Jimnez-Estrada and B. Segura-Alegra
from the Sistema Nacional de Investigadores. S. Quiroz-Gonzalez
was partially supported by PROMEP (No. 103.5-13-6729) and
SNI-CONACYT.
References
Barnes PM, Bloom B, Nahin RL (2008) Complementary and alter-
native medicine use among adults and children. Natl Health Stat
Report 10:123
Baron R, Saguer M (1993) Postherpetic neuralgia. Are C-nociceptors
involved in signaling and maintenance of tactile allodynia? Brain
116:14771496
Bernhard CG (1953) The spinal cord potentials in leads form the cord
dorsum in relation to peripheral source of afferent stimulation.
Acta Physiol Scand 29:129
Besson JM, Chaouch A (1987) Peripheral and spinal mechanisms of
nociception. Physiol Rev 67:67186
Campero M, Serra J, Marchettini P, Ochoa JL (1998) Impulse gen-
eration and autoexcitation in single myelinated afferent bers in
patients with peripheral neuropathy and positive sensory symp-
toms. Muscle Nerve 21:16611667
Chung JM, Fang ZR, Hori Y, Lee KH, Endo K, Willis WD (1984a)
Prolongated inhibition of primate spinothalamic tracks cells by
peripheral nerve stimulation. Pain 19:259275
Chung JM, Lee KH, Hori Y, Endo K, Willis WD (1984b) Factors
inuencing peripheral stimulation produced inhibition of primate
spinothalamic tracs cells. Pain 19:277293
Collins JG (1986) Effects of ketamine on low intensity tactile sensory
input are not dependent upon a spinal site of action. Anesth Analg
65(11):11231129
Coombs JS, Curtis DR, Landgren S (1956) Spinal cord potentials
generated by impulses in muscle and cutaneous afferent bers. J
Neurophysiol 19:452467
De LaTorre S, Rojas-Piloni G, Martnez-Lorenzana G, Rodrguez-
Jimnez J, Villanueva L, Conds-Lara M (2009) Paraventricular
oxytocinergic hypothalamic prevention or interruption of long-
term potentiation in dorsal horn nociceptive neurons: electro-
physiological and behavioral evidence. Pain 144:320328
Devor M (2009) Ectopic discharge in A afferents as a source of neu-
ropathic pain. Exp Brain Res 196:115128
Eccles JC, Schmidt RF, Willis WD (1963) Depolarization of the central
terminals of cutaneous afferent bers. J Neurophysiol 26:646661
Gonzlez SQ, Alegra BS, Olmos JC, Jimnez-Estrada I (2011) Effect
of chronic undernourishment on the cord dorsum potentials and
the primary afferent depolarization evoked by cutaneous nerves
in the rat spinal cord. Brain Res Bull 85:6874
Huang C, Wang Y, Han JS, Wan Y (2002) Characteristics of elec-
troacupuncture-induced analgesia in mice: variation with
strain, frequency, intensity and opioid involvement. Brain Res
945(1):2025
Huang C, Li HT, Shi YS, Han JS, Wan Y (2004) Ketamine potentiates
the effect of electroacupuncture on mechanical allodynia in a rat
model of neuropathic pain. Neurosci Lett 368:327331
Hwang BG, Min BI, Kim JH, Na HS, Park DS (2002) Effects of elec-
troacupuncture on the mechanical allodynia in the rat model of
neuropathic pain. Neurosci Lett 320:4952
Jimnez I, Rudomin P, Solodkin M (1987) Mechanisms involved in
the depolarization of cutaneous afferents produced by segmen-
tal and descending inputs in the cat spinal cord. Exp Brain Res
69:195207
Kerr FW, Wilson PR, Nijensohn DE (1978) Acupuncture reduces
the trigeminal evoked response in decerebrate cats. Exp Neurol
61:8495
AQ7
AQ8
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384
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405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
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437
438
439
440
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458
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460
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464
465
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477
478
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485
486
487
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489
490
491
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493
494
495
496
497
A
u
t
h
o
r

P
r
o
o
f
U
N
C
O
R
R
E
C
T
E
D

P
R
O
O
F
Journal : Large 221 Dispatch : 19-4-2014 Pages : 9
Article No : 3965 LE TYPESET
MS Code : EBR-14-0106 CP DISK
Exp Brain Res
1 3
Kim JH, Min BI, Na HS, Park DS (2004) Relieving effects of elec-
troacupuncture on mechanical allodynia in neuropathic pain
model of inferior caudal trunk injury in rat: mediation by spinal
opioid receptors. Brain Res 998:230236
Kim HW, Roh DH, Yoon SY, Kang SY, Kwon YB, Han HJ, Lee HJ,
Choi SM, Ryu YH, Beitz AJ, Lee JH (2006) The anti-inamma-
tory effects of low- and high-frequency electroacupuncture are
mediated by peripheral opioids in a mouse air pouch inamma-
tion model. J Altern Complement Med 12:3944
Kremer E, Lev-Tov A (1998) GABA-receptor-independent dorsal root
afferents depolarization in the neonatal rat spinal cord. J Neuro-
physiol 79:25812592
Lau WK, Chan WK, Zhang JL, Yung KK, Zhang HQ (2008) Electroa-
cupuncture inhibits cyclooxygenase-2 up regulation in rat spinal
cord after spinal nerve ligation. Neuroscience 155:463468
Le Bars D (2002) The whole body receptive eld of dorsal horn mul-
tireceptive neurons. Brain Res Rev 40:2944
Leung L (2012) Neurophysiological basis of acupuncture-induced
analgesiaan updated review. J Acupunct Meridian Stud
5:261270
Li CY, Zhu LX, Li WM, Ji CF (1993) Relationship between presyn-
aptic depolarization and effect of acupuncture, -aminobutyric
acid, opioid peptide substance P. Acupunct Res 18:178182
Lidierth M (2006) Local and diffuse mechanisms of primary afferent
depolarization and presynaptic inhibition in the rat spinal cord. J
Physiol 576:309327
Liu XG, Morton CR, Azkue JJ, Zimmermann M, Sandkuhler J (1998)
Long-term depression of C-bre-evoked spinal eld potentials by
stimulation of primary afferent A-bres in the adult rat. Eur J
Neurosci 10:30693075
Maslany S, Crockett DP, Egger MD (1992) Organization of cutane-
ous primary afferent bers projecting to the dorsal horn in the rat:
WGA-HRP versus B-HRP. Brain Res 569:123135
Ochoa JL (1994) Pain mechanisms in neuropathy. Curr Opin Neurol
7:407414
Panneton WM, Gan Q, Juric R (2005) The central termination of sen-
sory bers from nerves to the gastrocnemius muscle of the rat.
Neuroscience 134:175187
Quiroz-Gonzalez S, Guadarrama Olmos J, Segura Alegra B, Jimenez
Estrada I (2013) Depressing effect of electroacupuncture on the
N1 component of the cord dorsum potential in the rat spinal cord.
Abstr Soc Neurosci S-3601
Quiroz-Gonzlez S, Segura-Alegra B, Guadarrama-Olmos JC, Jim-
nez-Estrada I (2014) Cord dorsum potentials evoked by electroa-
cupuncture applied to the hind limbs of rats. J Acupunct Meridian
Stud 7:2532
Quirz-Gonzalez S, Segura-Alegra B, Olmos JC, Jimnez-Estrada I
(2012) The effect of chronic undernourishment on the synaptic
depression of cutaneous pathways in the rat spinal cord. Brain
Res Bull 89:97101
Rudomin P (2007) In search of lost presynaptic inhibition. Exp Brain
Res 196:139151
Rudomin P, Hernandez E (2008) Changes in synaptic effectiveness of
myelinated joint afferents during capsaicin-induced inammation
of the footpad in the anesthetized cat. Exp Brain Res 187:7184
Rudomin P, Schmidt R (1999) Presynaptic inhibition in the vertebrate
spinal cord revisited. Exp Brain Res 129:137
Sandkuhler J, Chen JG, Cheng G, Randic M (1997) Low-Frequency
stimulation of afferent A-Fibers induces long term depression at
primary afferent synapses with substantia gelatinosa neurons in
the rat. J Neurosci 17:64836491
Vickers AJ, Cronin AM, Maschino AC, Lewith G, MacPherson H,
Foster NE et al (2012) Acupuncture for chronic pain: individual
patient data meta-analysis. Arch Intern Med 172:14441453
Willis WD, Weir MA, Skinner RD, Bryan RN (1973) Differen-
tial distribution of spinal cord eld potentials. Exp Brain Res
17:169176
Xing GG, Liu FY, Qu XX, Han JS, Wan Y (2007) Long-term syn-
aptic plasticity in the spinal dorsal horn and its modulation by
electroacupuncture in rats with neuropathic pain. Exp Neurol
208:323332
Yin CS, Jeong HS, Park HJ, Baik Y, Yoon MH, Choi CB, Koh HG
(2008) A proposed transpositional acupoint system in a mouse
and rat model. Res Vet Sci 84:159165
Yin CS, Shim BS, Lee H, Choi SH (2010) Acupuncture in accom-
plishing health for all. Neurol Res 32:1821
Zhang YQ, Ji GC, Wu GC, Zhao ZQ (2003) Kynurenic acid enhances
electroacupuncture analgesia in normal and carrageenan-injected
rats. Brain Res 966:300307
Zhang RX, Lao L, Wang L, Liu B, Wang X, Ren K, Berman BM
(2004) Involvement of opioid receptors in electroacupuncture-
produced anti-hyperalgesia in rats with peripheral inammation.
Brain Res 1020:1217
Zhang R, Lao L, Ren K, Berman BM (2014) Mechanisms of acu-
puncture-electroacupuncture on persistent pain. Anesthesiology
120:482503
Zhao ZQ (2008) Neural mechanism underlying acupuncture analge-
sia. Prog Neurobiol 85:355375
Zhou F, Huang D, Xia Y (2010) Neuroanatomic basis of acupuncture
points. In: Xia Y, Cao XD, Wu GC, Cheng JS (eds) Acupunc-
ture therapy for neurological diseases: a neurobiological view.
Springer, Berlin, pp 3280
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