Content uploaded by Kazuyuki Murase
Author content
All content in this area was uploaded by Kazuyuki Murase on Mar 18, 2016
Content may be subject to copyright.
BioMed Central
Page 1 of 10
(page number not for citation purposes)
Molecular Pain
Open Access
Research
Effect of excitatory and inhibitory agents and a glial inhibitor on
optically-recorded primary-afferent excitation
Hiroshi Ikeda*, Takaki Kiritoshi and Kazuyuki Murase
Address: Department of Human and Artificial Intelligence Systems, Graduate School of Engineering, and Research and Education Program for Life
Science, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan
Email: Hiroshi Ikeda* - ikeda@synapse.his.fukui-u.ac.jp; Takaki Kiritoshi - kiritoshi@synapse.his.fukui-u.ac.jp;
Kazuyuki Murase - murase@synapse.his.fukui-u.ac.jp
* Corresponding author
Abstract
The effects of GABA, excitatory amino-acid receptors antagonists and a glial metabolism inhibitor
on primary-afferent excitation in the spinal dorsal horn were studied by imaging the presynaptic
excitation of high-threshold afferents in cord slices from young rats with a voltage-sensitive dye.
Primary afferent fibers and terminals were anterogradely labeled with a voltage-sensitive dye from
the dorsal root attached to the spinal cord slice. Single-pulse stimulation of C fiber-activating
strength to the dorsal root elicited compound action potential-like optical responses in the
superficial dorsal horn. The evoked presynaptic excitation was increased by the GABAA receptor
antagonists picrotoxin and bicuculline, by glutamate receptor antagonists D-AP5 and CNQX, and
by the glial metabolism inhibitor mono-fluoroacetic acid (MFA). The increase in presynaptic
excitation by picrotoxin was inhibited in the presence of D-AP5, CNQX and MFA. Presynaptic
modulation in the central terminal of fine primary afferents by excitatory and inhibitory amino acids
may represent a mechanism that regulates the transmission of pain.
Introduction
The sensory information which arrives at the central ter-
minals of sensory neurons in the spinal dorsal horn is reg-
ulated by presynaptic inhibition. The reduction in
amplitude of propagated action potentials as a result of
primary afferent depolarization (PAD) is thought to be a
mechanism of presynaptic inhibition [for review, see [1]].
Early studies suggested that γ-aminobutyric acid (GABA)
receptors at primary afferent terminals contribute to pres-
ynaptic inhibition. Pharmacological studies demon-
strated a contribution of GABAA receptors to the induction
of PAD in large primary afferents [for review see [2]], and
that the GABAA receptor antagonists picrotoxin and bicu-
culline reduce PAD [3,4]. The possible presence of PAD in
fine myelinated and unmyelinated primary afferent fibers
has also been reported indirectly by measuring their
antidromic activation thresholds [5-7], and by showing
the depolarization of small-diameter dorsal root ganglion
cells by GABA [8]. Recent studies report the possible con-
tribution of excitatory amino-acid (EAA) receptors to PAD
in fine primary afferent fibers by exogenous activation of
presynaptic AMPA, kainite and NMDA receptors [9-11].
Although neurons neighboring afferent terminals had
been thought to be a source of neurotransmitters which
regulate presynaptic excitation, glial cells around afferent
terminals have been proposed to be a source of these neu-
rotransmitters [12,13]. It is reported that release of neuro-
transmitter from the presynaptic terminal not only
Published: 26 September 2008
Molecular Pain 2008, 4:39 doi:10.1186/1744-8069-4-39
Received: 25 July 2008
Accepted: 26 September 2008
This article is available from: http://www.molecularpain.com/content/4/1/39
© 2008 Ikeda et al; 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.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 2 of 10
(page number not for citation purposes)
stimulates the postsynaptic neuron but also activates the
perisynaptic glial cells [13,14]. The activated glial cells, in
turn, release neurotransmitters such as glutamate and/or
ATP [12,14,15]. It is thought that these neurotransmitters
can directly stimulate the postsynaptic neuron and can
feed back onto the presynaptic terminal either to enhance
or to depress further release of neurotransmitter [12,13].
Recently, we succeeded in recording the presynaptic exci-
tation of fine afferents in a slice preparation of spinal dor-
sal horn by staining primary afferent fibers anterogradely
from the dorsal root with a voltage-sensitive dye [16]. In
the present study, using optical imaging along with vari-
ous pharmacological agents, we examined the effects of
glutamate receptors, GABAA receptor antagonists and a
glial metabolic inhibitor on optically-recorded presynap-
tic excitation. Some of the results described here have
been published in abstract form [17].
Figure 1
Increase in net neuronal excitation in the spinal dorsal horn following application of picrotoxinFigure 1
Increase in net neuronal excitation in the spinal dor-
sal horn following application of picrotoxin. A, Optical
responses elicited by a high-intensity single-pulse stimulation
(a current pulse of 2.0 mA with a duration of 0.5 ms) to the
dorsal root in the control condition (left image) and in the
presence of picrotoxin (right image) 4.8 ms after stimulation.
Images were taken from the area indicated by the red square
in the photo of the transverse slice. The percent change in
light absorption is depicted using simulated color as
described in the color bar. B, Spatial distributions of the opti-
cal responses in the control (thin lines) and picrotoxin (bold
lines) conditions along three dorso-ventral lines, a-c, in the
photo of a transverse slice indicated in A. Horizontal bars
indicate the area of the substantia gelatinosa (SG) identified
visually. C, Spatially averaged time courses of responses in
control (thin line) and picrotoxin (bold line) conditions. The
time courses were obtained in the dorsal horn from the area
indicated by the filled white square in the photo of a trans-
verse slice indicated in A. D, Optical responses elicited by a
low-intensity single-pulse stimulation (a current pulse of 1.0
mA with a duration of 0.5 ms) to the dorsal root in control
(left image) and picrotoxin (right image) conditions 4.8 ms
after stimulation. Images were taken from the area indicated
by the red square indicated in A. The percent change in the
light absorption is depicted using simulated color as
described in the color bar. E, Spatially averaged time courses
of responses in control (thin line) and picrotoxin (thick line)
conditions. The time courses were obtained in the dorsal
horn from the area indicated by the filled white rectangle
indicated in A.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 3 of 10
(page number not for citation purposes)
Results
Effect of picrotoxin on afferent-induced excitation in the
dorsal horn
Fig. 1A shows an example of optically recorded neuronal
excitation elicited by high-intensity, single-pulse stimula-
tion of the dorsal root (current pulse of 2.0 mA with a
duration of 0.5 ms), which activates both the A and C fib-
ers in the dorsal root, in a slice stained with a voltage-sen-
sitive dye. As we have reported previously [16,18], dorsal-
root stimulation induced prolonged neuronal excitation
(> 100 ms) in lamina I-III of the spinal dorsal horn (Fig.
1C).
Bath application of a GABAA receptor antagonist, picro-
toxin (100 μM), increased the optically-recorded net neu-
ronal excitation, a sum of pre- and postsynaptic
excitations (Fig. 1A–C, 129 ± 6%, p < 0.01, n = 9). The
neuronal excitation induced by low-intensity stimulation
(current pulse of 1.0 mA with a duration of 0.5 ms), which
activates mainly A fibers in the dorsal root [18], was
slightly increased by picrotoxin (Fig. 1D, E, 104 ± 5%, n =
5).
The effect of capsaicin treatment
To confirm that picrotoxin is effective on the neuronal
excitation induced by C-fiber activity, we depleted most
C-fiber inputs by neonatal capsaicin treatment [19] and
then examined the effects of picrotoxin on the optical
response. In slices taken from these capsaicin-treated rats,
the magnitude of neuronal excitation evoked by high-
intensity stimulation in the superficial dorsal horn was
significantly smaller than that of normal rats (Fig. 2A, B, p
< 0.05 at 2.0 mA, 2.5 mA, and 3 mA, n = 5). These results
indicate that capsaicin-treated rats lacked capsaicin-sensi-
tive C-fibers [20]. No significant potentiation was
Figure 2
0 03%.
10 ms
0.5 mA
1.0 mA
1.5 mA
2.0 mA
Control
0 03%.
10 ms
Picrotoxin
0.5 1.0 1.5 2.0 3.0
2.5
0.02
0.04
0.00
Intensity of stimulation (mA)
Normal
Capsaicin
A
B
C
Responce (%)
0.06
*
**
The effect of picrotoxin on neuronal excitation in slices taken from capsaicin-treated ratsFigure 2
The effect of picrotoxin on neuronal excitation in
slices taken from capsaicin-treated rats. A, Examples of
optical response time courses in substantia gelatinosa of spi-
nal dorsal horn elicited by various intensities of single-pulse
stimulations to the dorsal root in a slice taken from a capsai-
cin-treated rat. The arrowhead indicates the time when stim-
ulation was applied. B, The mean amplitude of optical
responses elicited by the stimulation pulses shown in A in
slices taken from normal rats (open circles) and capsaicin-
treated rats (filled circles). C, Examples of time courses of
optical responses in the substantia gelatinosa of the spinal
dorsal horn elicited by a high-intensity single-pulse stimula-
tion to the dorsal root in a slice taken from a capsaicin-
treated rat in control (thin line) and picrotoxin (thick line)
conditions. The arrowhead indicates the time when stimula-
tion was applied. * P < 0.05.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 4 of 10
(page number not for citation purposes)
observed by picrotoxin in slices taken from the capsaicin-
treated rats (Fig. 2C, 101 ± 3%, n = 9).
Effect of picrotoxin on presynaptic excitation
We then stained only the primary afferent fibers for 3
hours via anterograde application of the voltage-sensitive
dye in the suction pipette used for dorsal root stimulation.
Dorsal root stimulation induced short-lasting (< 5 ms),
action-potential or compound action potential-like opti-
cal signals in the spinal dorsal horn (thin trace in Fig. 3A)
[16]. When the same slice was perfused with the solution
containing the voltage-sensitive dye for 20 min, a pro-
longed component appeared in the optical response (bold
trace in Fig. 3A), as was seen in the net excitation
described in the previous section.
The presynaptic excitation induced by high-intensity stim-
ulation, but not by low-intensity stimulation, was
increased in the presence of picrotoxin (Fig. 3B &3C high-
intensity: 125 ± 2%, p < 0.01, low-intensity: 83 ± 3%, n =
4). The presynaptic excitation induced by high-intensity
stimulation was also increased in the presence of bicucul-
line (2 μM) (132 ± 7%, p < 0.01, n = 3).
Effect of excitatory amino acid antagonists on presynaptic
excitation
We next examined the effects of excitatory amino acid
antagonists on presynaptic excitation induced by high-
intensity stimulation. Application of the non-NMDA
glutamate receptor antagonist 6-cyano-7-nitroquinoxa-
line-2,3-dione (CNQX, 10 μM) alone increased presynap-
tic excitation (Fig. 4A &4D, 116 ± 2%, p < 0.01, n = 4).
Application of the NMDA-receptor antagonist D-2-
amino-5-phosphonovaleric acid (D-AP5, 50 μM) together
with CNQX produced a larger increase (Fig. 4B &4D, 125
± 5%, p < 0.01, n = 4).
Effect of picrotoxin in the presence of excitatory amino
acid antagonists on presynaptic excitation
We further examined the effects of picrotoxin in the pres-
ence of excitatory amino acid antagonists on presynaptic
excitation induced by high-intensity stimulation. In slices
treated with both CNQX (10 μM) and D-AP5 (50 μM), the
picrotoxin-induced increase in neuronal excitation was
not augmented, but was smaller than that in control slices
(Fig. 5A &5C, 114 ± 3%, p < 0.01, n = 4).
Figure 3
10 ms
0.01 %
A
B
2mA
1mA
Control
Picrotoxin
20 ms
0.02 %
Stained from dorsal root
Stained additionally from bath
net
pre
0
10
20
30
40
Responce (% control)
C
*
*
The increase in presynaptic excitation in the spinal dorsal horn by picrotoxinFigure 3
The increase in presynaptic excitation in the spinal
dorsal horn by picrotoxin. A, Spatially averaged time
course of the optical response elicited by a high-intensity sin-
gle-pulse stimulation to the dorsal root in a slice stained
anterogradely with a voltage-sensitive dye applied to the dor-
sal root (thin line), and the time course obtained from the
same slice after bath application of the voltage-sensitive dye
(thick line). These spatially averaged time courses were
obtained in 16 × 16 pixels in the substantia gelatinosa. B, Spa-
tially averaged time courses of optical responses in slices
stained anterogradely with a voltage-sensitive dye. The upper
and lower traces indicate the responses in the substantia
gelatinosa of spinal dorsal horn by a high and a low-intensity
stimulation in control (thin line) and picrotoxin (thick line)
conditions, respectively. C, The bar graph summarizes the
picrotoxin-induced increases in the net neuronal excitation
(net), presynaptic excitation (pre).
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 5 of 10
(page number not for citation purposes)
Effect of glial metabolic inhibitor
Mono-fluoroacetic acid (MFA) is known to block glial
metabolism [21]. Therefore, we next examined the effects
of MFA on presynaptic excitation in the presence of EAA
antagonists and picrotoxin.
MFA (5 mM) alone, and MFA together with D-AP5 and
CNQX, increased the presynaptic excitation slightly (MFA
alone: Fig. 4D, 112 ± 3%, p < 0.01, n = 3, MFA, D-AP5 &
CNQX: Fig. 4C &4D, 130 ± 9%, p < 0.01, n = 4). MFA (5
mM), in addition to D-AP5 and CNQX, completely
blocked the increase in neuronal excitation induced by
picrotoxin (Fig. 5B &5C, 102 ± 3%, n = 4).
Discussion
In this study, using optical imaging with a voltage-sensi-
tive dye, we showed that net neuronal excitation evoked
by dorsal root stimulation of C fiber-activating strength
was potentiated by picrotoxin. We then recorded the exci-
tation of only the presynaptic elements by anterograde
staining via the dorsal root, and showed that it was also
potentiated by picrotoxin and bicuculline. Application of
CNQX alone potentiated the presynaptic excitation
evoked by dorsal root stimulation. Application of CNQX
and D-AP5 also potentiated the presynaptic excitation.
The potentiation of presynaptic excitation by picrotoxin
was inhibited by D-AP5 and CNQX. MFA alone potenti-
ated slightly. Application of MFA together with D-AP5
and CNQX completely blocked the potentiation of presy-
naptic excitation by picrotoxin.
Effect of picrotoxin on net neuronal excitation
Bath application of picrotoxin potentiated the net neuro-
nal excitation in lamina I-III of the dorsal horn evoked by
high-intensity dorsal root stimulation. We have previ-
Figure 4
D-AP5+CNQX+MFA
D-AP5+CNQX
CNQX
0
10
20
30
40
D-AP5+CNQX
D-AP5+CNQX+MFA
A
B
C
D
Responce (% control)
0.02 %
5ms
CNQX
0.02 %
5ms
0.02 %
5ms
MFA
*
*
*
*
The effect of glutamate receptor antagonists and a glial metabolism inhibitor on the increase of neuronal excitation by picrotoxinFigure 4
The effect of glutamate receptor antagonists and a
glial metabolism inhibitor on the increase of neuro-
nal excitation by picrotoxin. A, Spatially averaged time
courses of presynaptic excitation in the substantia gelatinosa
of spinal dorsal horn elicited by a high-intensity single-pulse
stimulation to the dorsal root in the presence of D-AP5 and
CNQX (thin line), and after adding picrotoxin (bold line). B,
Spatially averaged time courses of the optical response in the
substantia gelatinosa of spinal dorsal horn elicited by a high-
intensity single-pulse stimulation to the dorsal root in the
presence of D-AP5, CNQX and MFA (thin line), and after
adding picrotoxin (bold line). C, The bar graph summarizes
the picrotoxin-induced increases in the presynaptic excita-
tion without D-AP5 and CNQX (w/o D-AP5+CNQX) in the
presence of D-AP5 and CNQX (D-AP5+CNQX), and presy-
naptic excitation in the presence of D-AP5, CNQX and MFA
(D-AP5+CNQX+MFA) elicited by high-intensity stimulation.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 6 of 10
(page number not for citation purposes)
ously shown that net excitation consists of early-presynap-
tic and delayed-postsynaptic components, and that the
presynaptic excitation of A-fiber origin is much less than
that of C-fiber origin [18]. In this study, in addition, we
showed that the neuronal excitation elicited by high-
intensity stimulation is weak in slices taken from neonatal
capsaicin-treated rats that had lost their behavioral
response to noxious stimulation, presumably due to the
loss of their C-fibers [19]. Therefore, the neuronal excita-
tion evoked by high-intensity stimulation mainly reflects
the response to noxious stimuli. Under normal condi-
tions, therefore, the nociceptive information in the super-
ficial dorsal horn is persistently depressed via GABAA
receptors.
Picrotoxin was more effective in neuronal excitation in
slices taken from normal rats than from capsaicin-treated
rats. These results suggest that the effects of picrotoxin
observed in this study mainly reflect its effect on C-fibers.
However, we can not separate the neuronal excitation
induced by A-fibers from that by C-fibers only. We have
shown that the neuronal excitation induced by the activa-
tion of large-diameter fibers is very small [18]. Therefore,
we were unable to clarify whether or not the optically-
recorded neuronal excitation induced by large-diameter
fibers is potentiated by picrotoxin. There are many
reports, in addition, demonstrating that GABAA receptors
are expressed not only at central terminals of primary
afferent fibers but also in dorsal horn neurons and that
blocking GABAA receptors evokes excitation of dorsal
horn neurons. Thus, it is expected that applying picro-
toxin might also affect dorsal horn neuron excitability
resulting from the blockade of GABAA receptors on dorsal
horn neurons. Therefore, it is puzzling that the potentia-
tion by picrotoxin was not observed in capsaicin-treated
spinal cord slices.
Figure 5
Picrotoxin in D-AP5+CNQX+MFA
Picrotoxin in D-AP5+CNQX
5ms
0.01 %
5ms
0.01 %
w/o D-AP5+CNQX
D-AP5+CNQX
D-AP5+CNQX+ MFA
0
10
20
30
A
B
C
Responce (% control)
*
*
The effect of glutamate receptor antagonists and a glial metabolism inhibitor on presynaptic excitationFigure 5
The effect of glutamate receptor antagonists and a
glial metabolism inhibitor on presynaptic excitation.
A, Spatially averaged time course of presynaptic excitation in
the substantia gelatinosa in spinal dorsal horn elicited by high-
intensity stimulation to the dorsal root in a control slice (thin
line), and that after the application of CNQX (bold line). B,
Spatially averaged time courses of presynaptic excitation
before (thin) and after (thick) the application of D-AP5 and
CNQX. C, Spatially averaged time courses of presynaptic
excitation before (thin line) and after (bold line) the applica-
tion of D-AP5 and CNQX and MFA. D, The bar graph sum-
marizes the increases in presynaptic excitation induced by
the application of CNQX, by D-AP5 and CNQX, and by D-
AP5, CNQX, and MFA.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 7 of 10
(page number not for citation purposes)
Effect of picrotoxin on presynaptic excitation
In this study, neuronal excitation of just the presynaptic
elements was recorded by anterogradely staining with a
voltage-sensitive dye applied via the dorsal root. This pre-
synaptic excitation evoked by high-intensity dorsal root
stimulation was not decreased by the application of the
EAA antagonists, D-AP5 and CNQX. The anterograde
staining, therefore, successfully labeled only presynaptic
elements that consist of primary afferents and their termi-
nals, but not postsynaptic neurons. Although it is impos-
sible to measure the actual membrane potential values by
the imaging system, it is highly likely that the evoked exci-
tation represents compound action potentials in primary
afferent fibers and/or terminals, because of its short dura-
tion.
Picrotoxin potentiated the evoked presynaptic excitation.
This finding confirms that, under normal conditions, the
generation of action potentials in primary afferents in the
superficial dorsal horn is persistently inhibited via GABAA
receptors.
Effect of EAA antagonists on presynaptic excitation
The potentiation of presynaptic excitation was also
observed by the application of EAA antagonists. It is
reported that the receptors for EAA exist on primary affer-
ent terminals, and that the activation of these receptors
inhibits transmitter release from the terminals [9-11].
Therefore, the effect of EAA antagonists on presynaptic
excitation may be due to the blockage of such EAA recep-
tors on primary-afferent terminals. Alternatively, the
action of EAA antagonists on postsynaptic GABAergic
interneurons might have caused the EAA effect. In immu-
nocytochemical studies, it was shown that GABAergic
interneurons around primary afferent terminals make
axoaxonic or dendroaxonic synapses in the superficial
laminae of the dorsal horn [22-24]. In this study, the
potentiation of presynaptic excitation by picrotoxin was
not observed in the presence of D-AP5 and CNQX that
inhibit excitatory synaptic transmission from primary
afferents to postsynaptic neurons. These results favor the
possibility that EAA antagonists inhibit the activity of
GABAergic interneurons resulting in less release of GABA
that acts on primary afferents. GABAergic interneurons
thus might be the source for inhibition of presynaptic
excitation, at least in part.
Since we applied glutamate receptor antagonists into the
bath, activities of both postsynaptic excitatory neurons
and GABAergic inhibitory neurons could be blocked
unselectively, and there is no way to inhibit only one of
the others, release of GABA and release of excitatory
amino acids. It is therefore impossible to conclude which
of the mechanisms takes the primary role at present. The
possible inhibition via excitatory neurons, in addition to
GABAergic inhibition, thus needs to be clarified in future
with different techniques. It is also unclear whether the
presynaptic inhibition observed in this study is due to the
tonic neurotransmitter release or feedback responses from
the activation of interneurons. The use of paired stimuli
might provide the answer, by measuring the attenuation
of the response to the second stimulus.
Contribution of glial cells to presynaptic inhibition
The glial metabolism inhibitor MFA, together with the
glutamate receptor antagonists D-AP5 and CNQX, com-
pletely blocked the picrotoxin-induced increase in presyn-
aptic excitation. This result indicates that glial cells also
contribute to GABAergic presynaptic inhibition. Several
studies in culture [25,26] and in slices of the olfactory
bulb [27] revealed that GABA or GABA-like substances
can be secreted from glial cells, and they suggested that
GABA released from glial cells is a source of tonic inhibi-
tion. Furthermore, in the spinal dorsal horn, it has been
suggested that nociceptive transmission is inhibited by
tonic GABA release [28,29]. Therefore, although there is
no evidence that glial cells in the spinal dorsal horn can
secret GABA, it is possible that nociceptive information in
the spinal dorsal horn is inhibited by tonic release of
GABA from glial cells. Thus, we still have to investigate
whether the glial cells in the spinal dorsal horn can also
Possible mechanism of presynaptic inhibition in primary affer-ent terminals by GABAFigure 6
Possible mechanism of presynaptic inhibition in pri-
mary afferent terminals by GABA. Bath application of
bicuculline (Bic) and/or picrotoxin (Pic) increases presynaptic
excitation by blocking GABA receptors (GABA-R) on affer-
ent terminals. MFA blocks the GABAergic action partially,
inhibiting glial cells and leaving the neuronal path intact.
Blocking glutamate receptors with D-AP5 and CNQX poten-
tiates the presynaptic excitation by inhibiting the activity of
GABAergic neurons and the presynaptic glutaminergic
action. Further addition of MFA together with D-AP5 and
CNQX removes all the inhibitory actions to the presynaptic
terminals.
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 8 of 10
(page number not for citation purposes)
produce and release GABA. In addition, because MFA may
inhibit other glial functions, we also need to examine the
effects of other glial functions on presynaptic inhibition.
Experiments with other doses of MFA and/or other glial
inhibitors are also necessary to confirm.
Conclusion
We demonstrated directly with an optical method that
extracellular application of GABA receptor antagonists,
glutamate receptor antagonists, and a glial metabolism
inhibitor increases presynaptic excitation in the superfi-
cial dorsal horn. The increase in presynaptic excitation by
picrotoxin was inhibited in the presence of glutamate
receptor antagonists, and an inhibitor of glial metabo-
lism. These results suggest that primary afferent terminals
are inhibited by GABA release from GABAergic inhibitory
neurons and also from glial cells (Fig. 6). Numerous
reports suggest the inhibition of nociceptive information
by GABA in the spinal dorsal horn. It is also reported that
activation of GABAA receptors in the primary afferent ter-
minals produces PAD [for review, see [2]], and that PAD
reduces the amplitude of propagated action potentials in
primary afferent terminals, thereby reducing neurotrans-
mitter release [30]. In the processing of nociceptive infor-
mation flow, PAD has been considered as a mechanism of
the 'gate control' theory suggested by Melzack and Wall
[31]. It is also suggested that PAD is a mechanism for allo-
dynia and hyperalgesia in chronic pain [32]. Further stud-
ies may clearly confirm such pain control mechanisms by
directly recording afferent excitation with an optical
method, overcoming the difficulties encountered in previ-
ous studies.
Methods
Preparation
All animal studies were undertaken according to protocols
approved by the university animal ethics committee. All
efforts were made to minimize the number of animals
used and their suffering. Eighteen- to 25-day-old Wister
rats were anaesthetized by diethyl ether. Following lami-
nectomy, the spinal cord was excised and several trans-
verse slices (500 μm thick) with attached dorsal root were
prepared from the lumbosacral enlargement. The animals
were then sacrificed by an overdose of ether. Preparation
for optical imaging of the gross neuronal excitation of
afferent fibers has been described in detail [18]. In short,
each slice was stained in a bath filled with the voltage-sen-
sitive absorption dye, RH-482 (0.1 mg/ml, 20 min) and
set in a submersion-type chamber (0.2 ml) on an inverted
microscope (IMT, Olympus, Tokyo) equipped with a 150
W halogen lamp. A dorsal root of the slice was suctioned
into a glass pipette from which stimulus current was
applied. As we have reported [18], a single current pulse
of 2 mA with a duration of 0.5 ms activated both A and C
fibers evoking an intense optical signal in stained slices,
while no signal was observed in unstained slices [33,34].
For optical imaging of excitation in primary afferent fib-
ers, an unstained slice was set in the chamber, and the dor-
sal root was suctioned into a pipette filled with the
voltage-sensitive dye (0.1 mg/ml) [16]. In response to the
C-fiber-activating dorsal root stimulation, the optical
response became observable after 3 hours of staining, but
not within 1 to 2 hours. The staining period was thus fixed
at 3 hours. The voltage-sensitive dye in the pipette was
then washed out, and the dorsal root was re-suctioned
into the pipette containing no dye.
Slices were perfused with Ringer's solution containing (in
mM): 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4
CaCl2, 26 NaHCO3, 0.2 thiourea, 0.2 ascorbic acid, and
10 glucose (oxygenated with 95% O2 and 5% CO2) at
room temperature (23 ± 2°C). The same solution was
used for the suction pipette.
Optical recording
As described in Ikeda et al. [18], the light absorption
change, at a wavelength of 700 ± 32 nm, in a 0.83 mm ×
0.83 mm area of the dorsal horn was recorded by a Del-
talon 1700 imaging system (Fuji Film Co., Tokyo) with
128 × 128 pixel photo sensors at a frame rate of 0.6 ms.
The dorsal root was stimulated via a glass suction elec-
trode. Eight single pulses were given at a constant interval
of 15 s. Starting 10 ms before each stimulus, the image
sensor took 128 consecutive frames of the light-absorp-
tion images at a sampling interval of 0.6 ms. A reference
frame, which was taken immediately before each series of
128 frames, was subtracted from the subsequent frames.
Eight series of such difference images were averaged and
stored in the system memory. We determined the initial
frame by averaging the first 15 frames of the difference
image and then subtracting this average from each of the
128 frames of the image data on a pixel-by-pixel basis to
eliminate the effects of noise contained in the reference
frame. The ratio image was then calculated by dividing the
image data by the reference frame.
As we have reported [33,34], by stimulation of 5 – 20 Hz
for 1 s or longer to the dorsal root, a slow intrinsic optical
signal with a duration of 1 – 2 minutes was elicited, but
not by single-pulse stimulation. Therefore, the optical sig-
nals induced by single stimulation presented in this study
primarily reflected the changes produced by the voltage-
sensitive dye, presumably by the cellular electrical activi-
ties.
Neonatal capsaicin treatment
Postnatal day 2 rats were anaesthetized with diethyl ether
and injected subcutaneously at the dorsal cervix with a
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 9 of 10
(page number not for citation purposes)
capsaicin solution (50 mg/kg). Three weeks after the injec-
tion, rats were tested for 1 min on a hot plate (65°C).
While normal, untreated rats raised and licked their feet
within 10 s, successfully treated rats did not react for at
least 1 min.
Drugs
The RH-482 (NK-3630) dye was obtained from Nippon
Kanko Shikiso (Okayama, Japan). The D-2-amino-5-
phosphonovaleric acid, capsaicin, and monofluoroacetic
acid, were from Sigma (St. Louis, MO). 6-cyano-7-nitro-
quinoxaline-2,3-dione, picrotoxin and bicuculline meth-
ochloride were from Tocris Cookson Ltd. (Bristol, UK).
Statistics
Results were expressed as means ± SE. Paired Student's t-
tests or non-parametric ANOVA (Tukey-Kramer test) were
used to examine statistical differences.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HI and KM participated in the conception, design, and
interpretation of the study. HI carried out the experi-
ments, performed the data analysis. All authors wrote the
manuscript.
Acknowledgements
This work was supported by a grants from the Japanese Society for the Pro-
motion of Sciences, Research and Education Program for Life Science at
University of Fukui, Uehara Memorial Foundation, Yazaki Memorial Foun-
dation for Science and Technology, and University of Fukui.
References
1. Willis WD, Coggeshall RE: Sensory Mechanisms of the Spinal
Cord. Third edition. New York: Plenum; 2004.
2. Willis WD: Dorsal root potentials and dorsal root reflexes: a
double-edged sword. Exp Brain Res 1999, 124:395-421.
3. Levy RA, Anderson EG: The effect of the GABA antagonists
bicuculline and picrotoxin on primary afferent terminal
excitability. Brain Res 1972, 43:171-180.
4. Quevedo J, Eguibar JR, Jimenez I, Rudomin P: Differential action of
(-)-baclofen on the primary afferent depolarization pro-
duced by segmental and descending inputs. Exp Brain Res 1992,
91:29-45.
5. Calvillo O, Madrid J, Rudomin P: Presynaptic depolarization of
unmyelinated primary afferent fibers in the spinal cord of
the cat. Neuroscience 1982, 7:1389-1409.
6. Fitzgerald M, Woolf CJ: Effects of cutaneous nerve and intrasp-
inal conditioning of C-fibre afferent terminal excitability in
decerebrate spinal rats. J Physiol 1981, 318:25-39.
7. Lin Q, Zou X, Willis WD: Aδ and C primary afferents convey
dorsal root reflexes after intradermal injection of capsaicin
in rats. J Neurophysiol 2000, 84:2695-2698.
8. Desarmenien M, Santangelo F, Loeffler JP, Feltz P: Comparative
study of GABA-mediated depolarizations of lumbar Aδ and
C primary afferent neurones of the rat. Exp Brain Res 1984,
54:521-528.
9. Kerchner GA, Wilding TJ, Li P, Zhuo M, Huettner JE: Presynaptic
kainate receptors regulate spinal sensory transmission. J
Neurosci 2001, 21:59-66.
10. Lee CJ, Bardoni R, Tong CK, Engelman HS, Joseph DJ, Magherini PC,
MacDermott AB: Functional expression of AMPA receptors on
central terminals of rat dorsal root ganglion neurons and
presynaptic inhibition of glutamate release. Neuron 2002,
35:135-146.
11. Bardoni R, Torsney C, Tong CK, Prandini M, MacDermott AB: Pre-
synaptic NMDA receptors modulate glutamate release from
primary sensory neurons in rat spinal cord dorsal horn. J Neu-
rosci 2004, 24:2774-2781.
12. Araque A, Perea G: Glial modulation of synaptic transmission
in culture. Glia 2004, 47:241-248.
13. Newman EA: New roles for astrocytes: regulation of synaptic
transmission. Trends Neurosci 2003, 26:536-542.
14. Volterra A, Meldolesi J: Astrocytes, from brain glue to commu-
nication elements: the revolution continues. Nat Rev Neurosci
2005, 6:626-640.
15. Bowser DN, Khakh BS: ATP excites interneurons and astro-
cytes to increase synaptic inhibition in neuronal networks. J
Neurosci 2004, 24:8606-8620.
16. Ikeda H, Murase K: Glial nitric oxide-mediated long-term pre-
synaptic facilitation revealed by optical imaging in rat spinal
dorsal horn. J Neurosci 2004, 24:9888-9896.
17. Kusudo K, Ikeda H, Murase K: Optical analyses of GABAergic
presynaptic inhibition in spinal dorsal horn. Japanese J Physiol
2006:238.
18. Ikeda H, Ryu PD, Park JB, Tanifuji M, Asai T, Murase K: Optical
responses evoked by single-pulse stimulation to the dorsal
root in the rat spinal dorsal horn in slice. Brain Res 1998,
812:81-90.
19. Yang K, Furue H, Fujita T, Kumamoto E, Yoshimura M: Alterations
in primary afferent input to substantia gelatinosa of adult rat
spinal cord after neonatal capsaicin treatment. J Neurosci Res
2003, 74:928-933.
20. Kusudo K, Ikeda H, Murase K: Depression of presynaptic excita-
tion by the activation of vanilloid receptor 1 in the rat spinal
dorsal horn revealed by optical imaging. Mol Pain 2006, 2:8.
21. Hülsmann S, Oku Y, Zhang W, Richter DW: Metabolic coupling
between glia and neurons is necessary for maintaining respi-
ratory activity in transverse medullary slices of neonatal
mouse. Eur J Neurosci 2000, 12:856-862.
22. Alvarez FJ, Kavookjian AM, Light AR: Synaptic interactions
between GABA-immunoreactive profiles and the terminals
of functionally defined myelinated nociceptors in the mon-
key and cat spinal cord. J Neurosci 1992, 12:2901-2917.
23. Bernardi PS, Valtschanoff JG, Weinberg RJ, Schmidt HH, Rustioni A:
Synaptic interactions between primary afferent terminals
and GABA and nitric oxide-synthesizing neurons in superfi-
cial laminae of the rat spinal cord. J Neurosci 1995,
15:1363-1371.
24. Todd AJ, Lochhead V: GABA-like immunoreactivity in type I
glomeruli of rat substantia gelatinosa. Brain Res 1990,
23:171-174.
25. Jow F, Chiu D, Lim HK, Novak T, Lin S: Production of GABA by
cultured hippocampal glial cells. Neurochem Int 2004,
45:273-283.
26. Liu QY, Schaffner AE, Chang YH, Maric D, Barker JL: Persistent
activation of GABA(A) receptor/Cl(-) channels by astrocyte-
derived GABA in cultured embryonic rat hippocampal neu-
rons. J Neurophysiol 2000, 84:1392-1403.
27. Kozlov AS, Angulo MC, Audinat E, Charpak S: Target cell-specific
modulation of neuronal activity by astrocytes. Proc Natl Acad
Sci USA 2006, 103:10058-10063.
28. Ataka T, Gu JG: Relationship between tonic inhibitory currents
and phasic inhibitory activity in the spinal cord lamina II
region of adult mice. Mol Pain 2006, 2:36.
29. Cronin JN, Bradbury EJ, Lidierth M: Laminar distribution of
GABAA- and glycine-receptor mediated tonic inhibition in
the dorsal horn of the rat lumbar spinal cord: effects of picro-
toxin and strychnine on expression of Fos-like immunoreac-
tivity. Pain 2004, 112:156-163.
30. Katz B, Miledi R: A study of synaptic transmission in the
absence of nerve impulses. J Physiol 1967, 192:407-436.
31. Melzack R, Wall PD: Pain mechanisms: a new theory. Science
1965, 150:971-979.
32. Cervero F, Laird JM: Mechanisms of touch-evoked pain (allody-
nia): a new model. Pain 1996, 68:13-23.
Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Molecular Pain 2008, 4:39 http://www.molecularpain.com/content/4/1/39
Page 10 of 10
(page number not for citation purposes)
33. Murase K, Saka T, Terao S, Ikeda H, Asai T: Slow intrinsic optical
signals in the rat spinal dorsal horn in slice. Neuroreport 1998,
9:3663-3667.
34. Asai T, Kusudo K, Ikeda H, Takenoshita M, Murase K: Intrinsic opti-
cal signals in the dorsal horn of rat spinal cord slices elicited
by brief repetitive stimulation. Eur J Neurosci 2002,
15:1737-1746.
