Group Title: Molecular Pain 2005, 1:4
Title: A P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord
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Title: A P2X receptor-mediated nociceptive afferent pathway to lamina I of the spinal cord
Series Title: Molecular Pain 2005, 1:4
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Gu JG
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Molecular Pain BioMed



Research

A P2X receptor-mediated nociceptive afferent pathway to lamina I
of the spinal cord
Meng Chen and Jianguo G Gu*


Address: Department of Oral and Maxillofacial Surgery, McKnight Brain Institute and College of Dentistry, University of Florida, Gainesville,
Florida, 32610, USA
Email: Meng Chen mgchen@dental.ufl.edu; Jianguo G Gu* jgu@dental.ufl.edu
* Corresponding author


Published: 7 January 2005
Molecular Pain 2005, 1:4 doi:10.1 186/1744-8069-1-4


Received: 24 November 2004
Accepted: 17 January 2005


This article is available from: http://www.molecularpain.com/content/ 1/1/4
2005 Chen and Gu; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Of the six lamina regions in the dorsal horn of the spinal cord, lamina I is a major sensory region
involved in nociceptive transmission under both physiological and pathological conditions. While
P2X receptors have been shown to be involved in nociception, it remains unknown if P2X
receptors are involved in nociceptive transmission to lamina I neurons. Using rat spinal cord slice
preparations and patch-clamp recordings, we have demonstrated that the excitatory synaptic
transmission between primary afferent fibers and lamina I neurons is significantly affected by ATP
and a,P-methylene-ATP. The synaptic effects of them include the increases of the frequency of both
miniature excitatory postsynaptic currents (mEPSCs) and spontaneous EPSCs (sEPSCs), and
decreases of evoked EPSCs (eEPSCs). These effects were blocked by pyridoxalphosphate-6-
azophenyl-2', 4'-disulfonic acid (PPADS, 10 rIM) and suramin (30 rIM). In the neurons for which ATP
and a,P-methylene-ATP had effects on mEPSCs, sEPSCs and eEPSCs, capsaicin produced similar
synaptic effects. Our results indicate that P2X receptors are expressed on many afferent fibers that
directly synapse to lamina I neurons. Furthermore, these P2X receptor-expressing afferent fibers
are capsaicin-sensitive nociceptive afferents. Thus, this study reveals a P2X receptor-mediated
nociceptive afferent pathway to lamina I of the spinal cord and provides a new insight into the
nociceptive functions of P2X receptors.


Background
Spinal cord dorsal horn, the first central site for sensory
processing, is divided into structurally and functionally
distinct lamina regions [1]. Lamina I, or marginal zone of
the spinal cord dorsal horn, is a critical region in nocicep-
tive transmission. Different from other lamina regions,
many lamina I neurons receive nociceptive inputs and
directly relay the nociceptive information through ascend-
ing pathways to the brain [1,2]. Nociceptive transmission
through lamina I and their modulation there have impor-
tant implications in both physiological and pathological
pain conditions [2,3]. Thus, a chemical mediator may


have significant influence on pain conditions if it has an
effect on this nociceptive pathway.

Extracellular ATP is a chemical mediator that has multiple
effects in different tissues including nervous systems [4].
ATP is involved in sensory signaling at peripheral sites [5-
7] and synaptic modulation at central sites in the somatic
sensory system [8,9]. At the periphery, ATP may directly
stimulate nociceptive afferent fibers through the activa-
tion of P2 receptors, resulting in nociceptive inputs to the
dorsal horn of the spinal cord or the equivalent sensory
structure in the brain [6,10]. At the central sites in the


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dorsal horn of the spinal cord, the inner part of lamina II
(lamina IIi) may be a region where ATP-induced sensory
inputs are transmitted. This is based on the strong P2X3
receptor-immunoreactivity in this region [11] as well as
previous electrophysiological evidence [12,13]. Lamina V
may be another CNS site where ATP-sensitive inputs are
transmitted. Many neurons in lamina V region receive
sensory inputs with a wide dynamic range including both
nociceptive and non-nociception signals [1]. Previous
studies have shown that ATP modulates synaptic trans-
mission to lamina V of the dorsal horn. The synaptic mod-
ulation is mediated by the activation of P2X receptors and
these P2X receptors are localized at the presynaptic termi-
nals of afferent fibers innervating lamina V neurons. The
ATP-sensitive afferent terminals to lamina V neurons were
found to be capsaicin-insensitive [14].

ATP and its receptors (P2 receptors) have been shown to
be involved in inflammatory and neuropathic pain condi-
tions [15,16]. P2 receptors may be therapeutic targets for
the management of pathological pain conditions [17,18].
Although the significance of lamina I neurons in inflam-
matory and neuropathic pain conditions have been well
recognized [19,20], it is currently not known whether
lamina I is one of the CNS sites for transmitting ATP-sen-
sitive nociceptive inputs. This issue is important for
understanding how ATP and its receptors are involved in
nociceptive transmission under physiological and patho-
logical conditions. This critical information has not been
provided in the previous studies, which is mainly due to
the difficulty in making electrophysiological recordings
from lamina I neurons [2,3]. In the present study, we per-
formed patch-clamp recordings from lamina I neurons
and studied effects of ATP and its analog apmeATP on
excitatory synaptic transmission to lamina I neurons. We
demonstrated, for the first time, a P2X receptor-mediated
nociceptive pathway to the lamina I of the spinal cord
dorsal horn.

Results
Effects of ATP and aflmeATP on miniature EPSCs of
lamina I neurons
We performed patch-clamp recordings from lamina I neu-
rons in spinal cord slice preparations (Figure 1A). Under
40X objective with IR-DIC microscopic system, white
matter and gray matter of the dorsal horn could be distin-
guished. All recordings (Figure 1A) were made from dor-
sal horn neurons whose somas were either within or
adjacent to the white matter (within a distance of 30 gim).

We first studied whether lamina I neurons directly
received synaptic inputs from ATP-sensitive presynaptic
terminals. This was done by testing the effects of ATP (100
gM) on mEPSCs recorded from lamina I neurons. Minia-
ture EPSCs were recorded in the presence of 10 mM lido-


caine. The use of the high concentration of lidocaine was
to block both TIX-sensitive and TIX-resistant Na+ chan-
nels so that the effects of ATP reflected its direct action at
the synaptic sites of the recorded neurons [21]. In this
study, basal levels of mEPSCs were first recorded for 10
min. Miniature EPSC frequency and mEPSC amplitude at
basal levels were served as controls, which had a variation
within + 10% in the same cells. We defined an increase of
mEPSC frequency and amplitude to be more than 120%
of control following ATP application, provided they
returned to basal levels after washout of ATP. Bath appli-
cation of 100 iM ATP for 2 min increased mEPSC fre-
quency in 13 out of 30 lamina I neurons recorded (Figure
1B,D). Miniature EPSC frequency of these 13 cells
increased to 210 + 22% of control, from 0.56 + 0.11 Hz at
the basal level to 1.08 + 0.19 Hz following the application
of 100 gtM ATP (n = 13, P < 0.01, Figure ID). Miniature
EPSC frequency in all these 13 cells returned to basal lev-
els 10 min after washout of ATP. In the cells for which
mEPSC frequency was increased, the amplitude of mEP-
SCs was not significantly changed following ATP applica-
tion (Figure 1D; 118 7% control, 11 1 pA in control
and 12.0 0.9 pA following ATP application, n = 13, P >
0.05). For the remaining 17 cells, neither mEPSC fre-
quency nor mEPSC amplitude was increased following
ATP application (not shown). In all the cells recorded, we
did not observe any direct whole-cell inward current dur-
ing ATP application, a result consistent with previous
studies in tissue slice preparations. In the above experi-
ments and the experiments described below, 100 iM ATP
was applied in the presence of 10 iM ARL67156, an ecto-
ATPase inhibitor that was used to prevent ATP metabo-
lism [14].

We tested apmeATP, a metabolically stable ATP analog,
on mEPSCs recorded from lamina I neurons. Similar to
ATP, application of 100 iM apmeATP increased mEPSC
frequency in 14 out of 35 lamina I neurons (Figure 1C,D).
Of these 14 cells, mEPSC frequency increased to 195 +
25% of control (n = 14, P < 0.01, Figure ID). Miniature
EPSC amplitude in these cells was not significantly differ-
ent from the control (Figure 1D; 102 2% control, n = 14,
P > 0.05). In the above experiments, apmeATP did not
directly evoke whole-cell inward currents in any cell.
These results provided electrophysiological evidence indi-
cating that the presynaptic terminals contacting the
recorded lamina I neurons are ATP-sensitive/apmeATP-
sensitive. The expression of P2 receptors at these presyn-
aptic terminals is suggested.

To test whether ATP-sensitive presynaptic terminals that
contacted lamina I neurons were potentially related to
nociceptive transmission, we tested capsaicin sensitivity
in 8 cells that had responses to acmeATP and ATP. Capsa-
icin was used because it selectively actives a subset of


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A

b--~


B Control




100 gIM ATP



I0 pA
100ms

C
Control





100 iM cameATP


S1.0
0.8
o
a 0.6
Sa.4

F 0.2
O 0


0 5 10 15 20 25
Inter-event Intervals (s)


1.0------
0.8
0.6
0.4 ---- Control
100 M ap3meATP
0.2

0 20 40 60 80 100
Inter-event Intervals (s)


100 ms


* Frequency
o Amplitude


m -requency
o Amplitude


Figure I
Effects of ATP and apmeATP on mEPSCs recorded from lamina I neurons of rat spinal cord slice preparations
A. The image on the left shows a spinal cord slice section viewed under a 4X objective. An electrode is shown to the left side
of the tissue section. The tip of the electrode is in lamina I. Scale bar: 100 pm. The image in the middle shows the tissue section
viewed under a 40X objective with the IR-DIC system. Near the center of the field is a lamina I neuron (arrow indicated) that
is patched with an electrode. The cell is on the border between white matter and gray matter. White matter (W), lamina I (I),
and lamina II (11) are indicated. Lamina I region is outlined with two dash lines. Scale bar: 10 pm. The drawing on the right indi-
cates the locations of all neurons recorded in this study. B. Representative traces show mEPSCs at the basal level (control) and
following the application of 100 pM ATP. The graph on the right shows cumulative probability histogram of inter-event inter-
vals in the same neuron. The inter-event intervals, which reflect the changes of mEPSC frequency, were significantly shifted fol-
lowing the application of 100 pM ATP (P < 0.01, Kolmogorov-Smirnov test). C. The experiment was the same as B except 100
apmeATP was tested. The inter-event intervals were significantly shifted following the application of 100 pM apmeATP (P <
0.01, Kolmogorov-Smirnov test). D. The bar graph shows pooled results from experiments represented in B and C. Both ATP
(n = 13) and apmeATP (n = 14) increased mEPSC frequency without affecting mEPSC amplitude. E. Effects of capsaicin (2 pM)
on mEPSCs in 8 cells that were tested for ATP and apmeATP in D. The scale is in logarithm. Data represent Mean SEM, *p
< 0.05, **p < 0.01, compared with controls, paired Student's t-test.




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A 100 pM ATP

I - mil


-


nr


S50 pA
Smin

----- -


50 pA
100ms


100 gM ATP
-W 100 100 pM ATP

I 601 kIN


< 0
0 200 400 600 0 200 400 600
Time (sec) Time (sec)


m Frequency
600 ,, Amplitude
500

400
300

200

100 -
00 --- --- ---


j 4


m Frequency
n Amplitude


100- ---

10-

1V
Control


Capsaicin


Figure 2
Effects of ATP and apmeATP on sEPSCs of lamina I neurons A. The trace on the tope panel shows sEPSCs recorded
from a lamina I neuron before and following the application of 100 gIM ATP. Two small portions, one before ATP and the other
following ATP application (arrows indicated), are shown at the expanded scale. Two histograms below the traces show time
courses of sEPSC frequency (left) and amplitude (right) in the same cell. Time bin is 20 sec. B. The bar graph shows pooled
results of the effects of ATP (100 IM, n = 44), 10 gIM apmeATP (n = 8), and 100 gIM apmeATP (n = 26) on sEPSC frequency
(filled bars) and amplitude (open bars). Data were normalized by the basal levels. C. The bar graph shows effects of capsaicin
on sEPSCs in 6 neurons that were tested in B with 100 IM ATP and 100 gIM apmeATP.



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nociceptive afferent fibers that express VR1 receptors. We
found that all of the 8 apmeATP-responsive neurons also
responded to capsaicin (2 iM) with a large increase of
mEPSC frequency (Figure 1E; 0.4 0.1 Hz in control vs 18
+ 7 Hz following capsaicin, n = 8, P < 0.05). Miniature
EPSC amplitude was also found to be increased by capsa-
icin (10.8 1.5 pA in control vs 15.8 + 2.2 following cap-
saicin, n = 8, P < 0.05), which was most likely due to the
high mEPSC frequency that produced temporal peak sum-
mation. There was no direct whole-cell inward current
that was observed during capsaicin application.

Effects of ATP and its analogs on spontaneous EPSCs
recorded from lamina I neurons
We tested effects of P2 receptor agonists on spontaneous
EPSCs (sEPSCs). In these experiments, Na+ channels were
not blocked so that action potentials were permitted to be
generated. This allowed us to observe a more global effect
of P2 receptor agonists on synaptic activity in lamina I. Of
106 lamina I neurons that were recorded, 44 cells showed
increases in sEPSC frequency after application of 100 iM
ATP (Figure 2A,B). The changes of sEPSC frequency in
these 44 cells were 474 + 70% of control (Figure 2B; 0.50
+ 0.09 Hz in control vs 1.98 + 0.30 Hz following ATP, n =
44, P < 0.01). sEPSC amplitude was also significantly
increased (Figure 2B; 157 14% of control, 13.4 1.0 pA
in control vs 19.8 2.3 pA following ATP, n = 44, P <
0.01,).

Similar response was observed when apmeATP was
tested. Of 47 cells tested with 100 iM apmeATP, 26 of
them showed increases in sEPSC frequency and ampli-
tude. The change of sEPSC frequency was 358 + 67% of
control (Figure 2B, n = 26, P < 0.01,). The change of sEPSC
amplitude was 163 + 23% of control (Figure 2B, n = 26, P
< 0.05). At a lower concentration of 10 iM apmeATP, 8
out of 19 cells showed increases of sEPSC frequency and
the change of sEPSC frequency in these cells was 224 +
62% of control (Figure 2B, n = 8, P < 0.05).

We further tested effects of capsaicin on sEPSCs in 6 cells
that showed the increases of sEPSC by apmeATP and ATP.
All 6 cells showed significant increases of sEPSC frequency
following the application of 2 gM capsaicin (Figure 2C;
0.55 + 0.18 Hz in control vs 22.1 + 7.8 Hz following
capsaicin, n = 6). sEPSC amplitude was also found to be
significantly increased by 2 iM capsaicin (Figure 2C; 14.6
+ 4.9 pA in control vs 22.5 + 3.5 pA following capsaicin, n
=6).

Effects of P2 antagonists on ATP-induced increases of
sEPSCs
We tested the effects of PPADS and suramin on ATP-
induced increases of sEPSCs (Figure 3A,B). Of 5 cells for
which 100 gtM ATP increased sEPSC frequency to 512


127% of basal levels in the absence of PPADS, sEPSC fre-
quency was 155 + 52% of the basal levels following 100
gtM ATP in the presence of 10 gtM PPADS (n = 4), and was
117 + 17% of basal levels following 100 gtM ATP in the
presence of 30 iM suramin (n = 3). Similarly, apmeATP-
induced increases of sEPSCs in 3 cells (294 + 78% of basal
sEPSC frequency) were abolished in the presence of 10
ItM PPADS (117 14% of basal sEPSC frequency). These
results suggested that P2 receptors are involved in the
increases of spontaneous excitatory synaptic activity to
lamina I neurons.

While ATP-induced increases of sEPSCs were abolished by
both PPADS and suramin, ATP still could increase sEPSCs
in the presence of reactive blue 2, a P2Y receptor
antagonist. As shown in Figure 3C, in 11 cells showing
increases of sEPSCs in response to 100 gtM ATP (354 +
85% of basal levels, P < 0.05), 100 gtM ATP increased the
sEPSC frequency to 221 + 49% of basal levels when 100
iM RB-2 was present (P < 0.05, compared with basal lev-
els, Figure 3C). Taken together, these results suggest that
P2X receptors are involved in ATP-induced increases of
sEPSCs.

Effects of ATP and a/meATP on synaptic inputs from
primary afferent fibers to lamina I neurons
To further demonstrate that ATP-sensitive terminals are
derived from primary afferent fibers, we studied effects of
ATP and apmeATP on synaptic inputs elicited by stimula-
tion of dorsal root (i.e. primary afferent fibers). Dorsal
root stimuli resulted in the evoked EPSCs (eEPSCs)
recorded from lamina I neurons. Monosynaptic eEPSCs
showed large variations in their latency among different
cells (Figure 4A). The conduction velocity of afferent
inputs, calculated from the latency of eEPSCs and the
length of the dorsal roots, was from 0.33 to 4 m/s (1.1
0.11 m/s, n = 57, Figure 4A), in the range of C-fiber and
A6-fiber conduction velocity at these ages of rats [22]. Syn-
aptic failure rates were low before the application of ATP
and apmeATP. However, following the application of 100
gtM ATP or 100 gtM apmeATP, there was a significant
increase in synaptic failure rates and a significant decrease
of the averaged eEPSC amplitude in many lamina I neu-
rons (Figure 4B to 4E). Of 13 cells tested with 100 gtM ATP
in the presence of 10 gtM ARL67156 and 2 mM caffeine, 9
of them showed increases in synaptic failure rates and
decreases in the averaged eEPSC amplitude (Figure 4B,D).
The failure rates were 8 + 3% in control and increased to
78 + 7% following 100 gtM ATP applications (n = 9, P <
0.01); the averaged eEPSC amplitude was 35 9 pA in
control and reduced to 12 4 pA following 100 gtM ATP
application (24 7% of control, n = 9, P < 0.01). Of 29
cells tested with 100 iM apmeATP, 18 of them showed
depression of the averaged amplitude of eEPSCs, accom-
panied with the increases of failure rates (Figure 4C,E).


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ATP



ATP ATP
Suramin PPADS




0 200 400 600 1000 1200 1400 2200 2400 2600 4400 4600 4800 (s)


600
o
c 500
0
0 400
O
o 300
o 200
O
L 100--
0
40


C?


',9 \
C3


NO


S100 M ATP
S10 100 gM RB2
C8

U-
04

S 0 100200 400500600(s)
0 100 200 300 400 500 600(s)


* Frequency
o[ Amplitude
*


Control ATP ATP/RB2


Figure 3
Block of ATP-induced synaptic responses by P2 antagonists A. The histogram shows the effects of ATP on sEPSC fre-
quency in a lamina I neuron in the absence and presence of suramin (30 IM) or PPADS (10 IM). Suramin and PPADS were
pre-applied for 10 min and were present during the recording. After 30 min washout of antagonists, 100 gIM ATP was applied
again for a test of recovery. B. Summarized data show effects of ATP on sEPSC frequency (filled bars) and amplitude (open
bars) in the absence (n = 5) and presence of PPADS (10 IM, n = 4) and suramin (30 IM, n = 3). C. Histogram on the left shows
a time course of ATP-induced increase of sEPSC frequency in a lamina I neuron in the presence of 100 IM reactive blue
2(RB2). Bar graph on the right is a summary that shows the effects of 100 gIM ATP on the sEPSCs in the absence and presence
of 100 gM RB2 (n = II).



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The failure rates were 12 + 5% in control and increased to
53 + 10% following 100 iM &ameATP applications (n =
18); the eEPSC amplitude was reduced to 35 + 7% of con-
trol following 100 tiM actmeATP application (n = 18, p <
0.01).

The suppression of eEPSCs could be due to a direct action
on primary afferent axons whose terminals synapse to
lamina I neurons or due to an effect indirectly through
GABAergic presynaptic inhibition. To exclude the latter
possibility, we tested the effects of 100 gM ATP and 100
gM &ameATP on eEPSCs in the presence of the GABA-A
receptor inhibitor bicuculline (20 iM). We found that
both ATP and apmeATP still could suppress eEPSCs (Fig-
ure 5). Of 7 cells tested with 100 gtM ATP in the presence
of bicuculline, 5 of them showed suppression of eEPSCs.
Synaptic failure rates increased from 26 + 6% before ATP
to 68 + 15% following 100 iM ATP (n = 5, p < 0.01, Figure
5B). The averaged eEPSC amplitude was also significantly
decreased (Figure 5C). Of 14 cells tested with 100 iM
apmeATP in the presence of bicuculline, 6 of them
showed depression of eEPSCs. For these 6 cells, synaptic
failure rates were increased from 27 + 10% before
apmeATP to 50 + 15% following the application of 100
gM &ameATP (n = 6, p < 0.05, Figure 5B), the averaged
eEPSC amplitude decreased to 33% + 9% of control
following 100 iM &ameATP (n = 6, p < 0.01, Figure 5C).
These results suggest that the suppression of primary affer-
ent synaptic inputs to lamina I neurons was due to a direct
action of ATP or apmeATP on primary afferent fibers.

If P2X receptors were involved in the suppression of eEP-
SCs, the suppression should be abolished in the presence
of P2X antagonists. We tested the effects of PPADS on
acmeATP-induced suppression ofeEPSCs (Figure 6). Of 4
cells for which 100 iM &ameATP suppressed eEPSC
amplitude to 31 + 11% of control (n = 4, P < 0.05) in the
absence of PPADS, apmeATP did not significantly sup-
press eEPSC amplitude when 10 gtM PPADS was present
(82 + 7% of control, n = 4, Figure 6C). Thus, P2X receptors
are expressed on primary afferent fibers that contact lam-
ina I neurons.

To study whether ATP-sensitive afferent fibers that syn-
apsed to lamina I neurons were nociceptive afferent fibers,
we tested capsaicin sensitivity of the evoked eEPSCs. Of
the 6 lamina I neurons that showed eEPSC suppression by
ATP and apmeATP (Figure 7A), all of them also showed
eEPSC suppression following the application of 2 iM cap-
saicin (Figure 7B,C, D). The synaptic failure rates of these
6 cells were 12 + 6% before capsaicin and increased to 65
+ 16% (n = 6, p < 0.05) following the application of 2 gtM
capsaicin. The averaged amplitude of eEPSCs was 46 + 17
pA before capsaicin and reduced to 18 + 10 pA following
2 iM capsaicin (n = 6, p < 0.01). Of these six recordings,


the afferent conduction velocities were less than 1 m/s
(from 0.5 to 0.8 m/s) in 4 cells and more than 1 m/s (1.1
and 1.5 m/s) in 2 cells. These results suggest that ATP-sen-
sitive afferent fibers that synapse to lamina I neurons are
nociceptive afferent fibers.

Discussion
In the present study we have shown that ATP and its ana-
log apmeATP produce profound effects on excitatory syn-
aptic transmission to lamina I neurons of the spinal cord
dorsal horn. Pharmacological tests indicate that P2X
receptors are involved in the synaptic responses and that
P2X receptors are expressed on many capsaicin-sensitive
afferent fibers innervating lamina I neurons. In view of the
significance of lamina I in transmitting nociceptive signals
and in the development of pathological pain conditions
[2,3], our findings provide a new insight into P2X receptor
functions in physiological and pathological pain condi-
tions [16,17,23].

In this study, the presynaptic actions of ATP and
apmeATP were evidenced electrophysiologically by their
effects on mEPSCs and eEPSCs [21]. Both ATP and
apmeATP increased mEPSC frequency without affecting
mEPSC amplitude; both ATP and apmeATP also
increased synaptic failure rates of eEPSCs. The involve-
ment of P2X receptors is supported by several lines of
pharmacological evidence. First, apmeATP is a selective
agonist to P2X receptors [24] and its effects on mEPSCs,
sEPSCs and eEPSCs were similar to ATP in this study. Sec-
ond, at low micromolar concentrations, PPADS is a selec-
tive antagonist for P2X receptors [24]; we found that
PPADS at 10 gM abolished synaptic effects of ATP and
apmeATP. Third, ATP and apmeATP still could induce
synaptic responses in the presence of the P2Y receptor
antagonist RB2. Fourth, the potential complication by the
ATP metabolite adenosine was excluded with the use of
caffeine to block adenosine receptors and the used of
ARL67156 to prevent ATP metabolism [14].

We have found that in the same lamina I neurons for
which ATP and apmeATP had synaptic effects, capsaicin
produced similar responses. For example, in the lamina I
cells for which apmeATP increased synaptic failure rates
of eEPSCs, capsaicin also enhanced synaptic failure rates
in these cells. These findings suggest that many ATP- and
apmeATP-sensitive presynaptic terminals to lamina I
neurons were derived from capsaicin-sensitive nociceptive
afferent fibers. Our previous study showed that P2X recep-
tor-expressing afferent fibers innervating lamina V neu-
rons were capsaicin-insensitive A6-fibers [14]. In the
present study, we have found that P2X receptor-expressing
afferent fibers to lamina I are capsaicin-sensitive and
could be either A6 or C-fibers. Thus, the P2X-expressing
afferent fibers are distinct in their lamina distribution and


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225
C-fiber 2 -
S1 -


50 pA D
A 0.0 0.5 1.0 1.5 2.0 2.5 30 3.5 4.0 4.5
20 ms Conducting Velocity ([ms)


Control


100 pM ATP


100 iM apmeATP Recover


S00 pA
10 ms
100 [M apmeATP Recover


S50 pA
I rns


co 0
0^ c<


Control ATP a3meATP


Figure 4
Suppression of eEPSCs by ATP and apmeATP A. The trace on the left shows an averaged eEPSC recorded from a lam-
ina I neuron that received monosynaptic inputs from A6-afferent fibers. The averaged eEPSC was obtained from eEPSCs elic-
ited by 10 sweeps of stimuli. The trace on the right is an averaged eEPSC from a lamina I neuron that received monosynaptic
inputs from C-fibers. The graph shows numbers of lamina I neurons recorded for all the eEPSC experiments in this study and
their corresponding afferent conduction velocity (n = 57). B. Top panel shows superimposed eEPSCs following 10 sweeps of
stimuli in normal bath solution (control), following the application of 100 pM ATP, and washout of ATP (recovery). The aver-
aged eEPSCs from 10 sweeps of stimuli are shown in the bottom panel. C. Similar to B except apmeATP was tested in a dif-
ferent lamina I neuron. D. The bar graph summarizes the increases of synaptic failure rates by 100 pM ATP (black bar, n = 9)
and 100 piM apmATP (gray bar, n = 18). E. A summary of the reduction of average eEPSC amplitude by 100 pM ATP (black
bar, n = 9) and 100 piM apmATP (gray bar, n = 18).




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AS-fiber


Control





Control


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Control


100 pM ATP


120

100'

80

60'

40'


A r ~,A


Recover





50 pA


20 ms











**


o e Control ATP apmeATP





Figure 5
Suppression of eEPSCs by ATP and apmeATP in the presence of bicuculline A. The traces show eEPSCs recorded
from a lamina I neurons under the following conditions: in bath solution with 20 IM bicuculline (control), following the applica-
tion of 100 pM ATP in the presence of 20 pM bicuculline, and washout of ATP (recovery). In each set of a test, eEPSCs were
elicited by 10 sweeps of stimuli. B. The bar graph shows a pooled result of increases in synaptic failures by ATP (100 piM, n =
5) and apmeATP (100 pM, n = 6). C. A summary shows the decreases of average eEPSCs by 100 piM ATP (n = 5) and 100 pM
apmeATP (n = 6). All recordings were made from lamina I neurons in the presence of 20 pM bicuculline.


in their capsaicin sensitivity. The lamina I innervation and
the capsaicin sensitivity of these ATP-sensitive afferent fib-
ers strongly suggest the nociceptive function of these P2X
receptors.

The synaptic failures of eEPSCs by ATP and apmeATP
could be due to conduction block following the activation


of P2X receptors on primary afferent axons. Alternatively,
the increase of synaptic failure rates was caused by presy-
naptic inhibition through GABAergic inhibitory interneu-
rons [25]. This latter possibility, however, is discounted
because ATP and apmeATP still increased the failure rates
of the evoked EPSCs in the presence of bicuculline to
block GABA-A receptors. These results further support the


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0 I







http://www.molecularpain.com/content/1//4


Control


100 pM apmeATP


Recover


Control/10 pM PPADS






100 pM 3apmeATP/10 pM PPADS


rV_


Recover


50pA _
10ms


120
a)
~~ so-
* 100
-9-
E 2 80

C) 0

0 s 40
| -
oa)
1 20-


Control apmeATP apmeATP/PPADS



Figure 6
Block of apmeATP-induced suppression of eEPSCs by PPADS A. The three traces on the top, middle and bottom
panels show eEPSCs recorded from a lamina I neuron in normal bath solution (Control), during the application of 100 IM
apmeATP and after washout of apmeATP (Recover), respectively. B. The same neuron shown A was tested again in the pres-
ence of 10 piM PPADS. In both A and B, a trace of eEPSC was obtained from an average of eEPSCs evoked by 10 sweeps of
stimuli. C. Pooled results (n = 4) show effects of 100 piM apmeATP on eEPSC amplitude in the absence and presence of 10 ipM
PPADS. Data represent percent of control values, and eEPSCs in control is scaled as 100%.


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Control


Control


100 gM apmeATP




S... Recover

Recover


1 20 pA
20 ms


2 gM Capsaicin






Recover



I 20 pA
20 ms


"o
2 120
- 1 100
80
, 60
40
D- 20
a


Control Capsaicin


Control Capsaicin


Figure 7
Capsaicin-sensitivity of uameATP-sensitive primary afferents to lamina I A. Example recordings from a lamina I
neuron show eEPSCs following 10 sweeps of stimuli in normal bath solution (control), following the application of 100 iM
apmeATP, and washout of the drug (recovery). B. Capsaicin (2 iM) was tested in the same neuron as shown in A. C. The bar
graph summarizes capsaicin-induced synaptic failures in 6 lamina I neurons that received ATP/lameATP-sensitive primary
afferent inputs. D. A summary shows capsaicin-induced reduction of averaged eEPSC amplitude (n = 6). All neurons were
recorded from lamina I that received ATP/lameATP-sensitive primary afferent inputs.


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idea that P2X receptors are expressed on primary afferent
fibers innervating lamina I neurons. The synaptic failure
of eEPSCs following P2X receptor activation does not
discount the involvement of P2X receptors in transmitting
nociceptive signals to lamina I regions. In opposite, it sug-
gests that P2X receptor action may directly depolarize pri-
mary afferent fibers to initiate nociceptive signals, which
are then transmitted to lamina neurons. Consistent with
this idea, out results showed that both frequency and
amplitude of sEPSCs were significantly increased follow-
ing the application of ATP or apmeATP.

Of more than 10 subtypes of functional P2X receptors
identified so far, low concentrations of apmeATP selec-
tively activate those that contain P2X1 or P2X3 subunits
[24]. Thus, the sensitivity to 10 jiM apmeATP in our study
narrows down the possible P2X receptor subtypes [26].
The sensitivity to the block by 10 jiM PPADS and 30 jiM
suramin in our study may allow us to further exclude a
number of P2 receptors including P2Y receptors, homo-
meric P2X4 receptors, homomeric P2X6 receptors, and
homomeric P2X7 receptors [26]. Previous studies have
shown the involvement of P2X3 receptors in nociception
[7]. However, because P2X3-expressing afferent terminals
are restricted in the inner layer of lamina II [11], it is less
likely that the functional P2X receptors in our study are
P2X3-containing subtypes. The P2X receptor subtype(s)
involved in the nociceptive pathway shown in this study
may not be easily identified due to the lack of selective
agonists and antagonists for most of P2X receptor sub-
types and due to the possible presence of more P2X recep-
tor subtypes. The answer to this critical issue in future
studies may provide new therapeutic targets for pain
management.

Methods
Spinal cord slice preparation
Principles of laboratory animal care (NIH publication No.
86-23, revised 1985) were followed in all the experiments
described in the present study. Spinal cord slice prepara-
tions and patch-clamp recordings were described in
details in our previous studies [14]. In brief, Sprague Daw-
ley rats at the postnatal age of 10-21 days were used.
Transverse spinal cord slices were prepared from lumbar
enlargement of the spinal cords. Two types of spinal cord
slice sections were made. One type was in the thickness of
400 jim without dorsal root, and was used for studying
mEPSCs and sEPSCs. The other type was in the thickness
of 600 jim with attached dorsal roots, and was use for
experiments testing eEPSCs. The length of the dorsal roots
was in a range of 8-12 mm. The spinal cord slices were
maintained in a basket submerged in ~200 ml Krebs solu-
tion (24 C). The Krebs solution contained (in mM): NaCl
117, KC1 3.6, CaC12 2.5, MgCl2 1.2, NaH2PO4 1.2,


NaHCO3 25 and glucose 11; the solution was saturated
with 95 % 02 and 5% CO2 and had pH of 7.3.

Patch-clamp recordings
In each experiment a spinal cord slice was transferred to a
0.5 ml recording chamber and placed on the stage of an
upright microscope. The microscope was equipped with
an infrared differential interference contrast (IR-DIC) sys-
tem. The spinal cord slice was superfused with the Krebs'
solution flowing at 10 ml/min at room temperature
(24 C); the Krebs' solution was equilibrated with 95% 02
and 5% CO2. Lamina I, the marginal region of the dorsal
horn, was identified under the microscope. Individual
neurons were identified with a 40x water immersion
objective. Whole-cell patch-clamp recordings were made
from lamina I neurons with electrodes filled with an inter-
nal solution containing (in mM): 135K-gluconate, 5KC1,
0.5CaCl2, 2MgCl2, 5EGTA, and 5HEPES; the pH of the
solution was adjusted to 7.3 with NaOH. The resistances
of the electrodes were ~5 MQ when filled with the internal
solution. The access resistance was below 30 MQ and was
not compensated. Signals were amplified and filtered at 2
kHz (Axopatch 200B) and sampled at 5 kHz using
pCLAMP 7.0 (Axon instruments). EPSCs were recorded
with cells held at -60 mV. The holding potential was close
to the reversal potential for GABAA and glycine receptors
under the experimental conditions so that the outward
IPSCs were minimized and usually undetectable.

Spontaneous EPSCs were recorded in the normal Krebs'
bath solution, and mEPSCs were recorded in the presence
of 10 mM lidocaine. To record eEPSCs, stimuli were
applied to a dorsal root with a suction electrode.
Stimulation intensities were 60-150 iA for A6 fiber and
200-800 iA for C-fibers; stimulation duration was always
0.1 msec. Evoked EPSCs were usually elicited by 10
sweeps of stimuli at a time interval of 5 s, which were used
to obtain the average of eEPSCs. Monosynaptic eEPSCs
were judged by a constant latency to the repeated stimuli.
Conduction velocity was calculated from the latency of
eEPSCs and the length of dorsal roots.

Effects of P2 agonists and capsaicin on mEPSCs, sEPSCs
and eEPSCs were tested using ATP (100 jiM), a,P-methyl-
ene-ATP (afmeATP, 10 and 100 jiM), and 2 jiM capsaicin.
When ATP was tested, bath solution always contained the
ecto-ATP inhibitor ARL6 7156 (10 jiM), and the adenosine
receptor antagonist caffeine (2 mM) was also present. P2
antagonists including pyridoxalphosphate-6-azophenyl-
2',4'-disulfonic acid (PPADS, 10 jiM), suramin (30 jiM)
and reactive blue 2 (RB2, 100 jiM) were used in this study.
When the antagonists were tested, they were first pre-
applied for 10 min, and then were co-applied with P2 ago-
nists. The time intervals for multiple applications of test-
ing compounds were 20 min. All compounds were


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applied via the bath solution. All compounds were pur-
chased from Sigma (St. Louis, MO).

Data analysis
Synaptic events including sEPSCs and mEPSCs were ana-
lyzed using Mini Analysis Program (Jaejin software,
Anderson Place, GA) with criteria being the same as previ-
ously described [21,14]. In analyzing the changes of
mEPSC frequency or sEPSC frequency following the bath
application of P2 agonists, frequency time courses before
and after P2 agonists were first constructed with time bin
of 10 s. Then the average response in continuous 6 bins
(60 s) around the peak was used to calculate the changes
in reference to the basal level (control). Cells were
assigned to be responsive to a test compound when there
were more than 20% increases in mEPSC frequency or
sEPSC frequency as previously described [21]. For eEPSCs,
eEPSCs were elicited by 10 sweeps of stimuli, which were
used for calculating the averaged amplitude of eEPSC.
Cells were considered to be responsive to a testing com-
pound when there were more than 20% increases in the
averaged amplitude of eEPSCs. Unless otherwise indi-
cated, data were presented as mean + SEM. Paired Stu-
dent's t-tests were used for statistical comparison, and
significance was considered at the level of the p < 0.05.


Competing interests
The authors) declare that they have no competing
interests.


Acknowledgments
This work was supported by a grant NS38254 to J.G.G.

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