Group Title: Molecular Pain 2005, 1:20
Title: Substance P-driven feed-forward inhibitory activity in the mammalian spinal cord
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Title: Substance P-driven feed-forward inhibitory activity in the mammalian spinal cord
Series Title: Molecular Pain 2005, 1:20
Physical Description: Archival
Creator: Nakatsuka T
Chen M
Takeda D
King C
Ling J
Xing H
Ataka T
Vierck C
Yezierski R
Gu JG
Publication Date: 38532
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Bibliographic ID: UF00100284
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

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Molecular Pain BioMedCent


Research

Substance P-driven feed-forward inhibitory activity in the


mammalian spinal cord
Terumasa Nakatsukat1,2, Meng Chent1,2, Daisuke Takedat1,2,
Christopher King1,3, Jennifer Ling1,2, Hong Xing1,2, Toyofumi Ataka1,2,
Charles Vierck1,4,5, Robert Yezierskil,3,5 and Jianguo G Gu*1,2,4,5


Address: 'McKnight Brain Institute, University of Florida, Gainesville, Florida 32610, USA, 2Department of Oral & Maxillofacial Surgery and
Diagnostic Sciences, College of Dentistry, University of Florida, Gainesville, Florida 32610, USA, 3Department of Orthodontics, College of
Dentistry, University of Florida, Gainesville, Florida 32610, USA, 4Department of Neuroscience, College of Medicine, University of Florida,
Gainesville, Florida 32610, USA and 5Comprehensive Center for Pain Research, University of Florida, Gainesville, Florida 32610, USA
Email: Terumasa Nakatsuka nakatsuk@cc.saga-u.ac.jp; Meng Chen mgchen@dental.ufl.edu; Daisuke Takeda dtakeda@dental.ufl.edu;
Christopher King cking@dental.ufl.edu; Jennifer Ling jling@dental.ufl.edu; Hong Xing hxing@dental.ufl.edu;
Toyofumi Ataka tataka@dental.ufl.edu; Charles Vierck vierck@mbi.ufl.edu; Robert Yezierski ryezierski@dental.ufl.edu;
Jianguo G Gu* jgu@dental.ufl.edu
* Corresponding author tEqual contributors



Published: 29 June 2005 Received: 06 May 2005
Molecular Pain 2005, 1:20 doi:10.1 186/1744-8069-1-20 Accepted: 29 June 2005
This article is available from: http://www.molecularpain.com/content/1/1/20
2005 Nakatsuka 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.



Abstract
In mammals, somatosensory input activates feedback and feed-forward inhibitory circuits within
the spinal cord dorsal horn to modulate sensory processing and thereby affecting sensory
perception by the brain. Conventionally, feedback and feed-forward inhibitory activity evoked by
somatosensory input to the dorsal horn is believed to be driven by glutamate, the principle
excitatory neurotransmitter in primary afferent fibers. Substance P (SP), the prototypic
neuropeptide released from primary afferent fibers to the dorsal horn, is regarded as a pain
substance in the mammalian somatosensory system due to its action on nociceptive projection
neurons. Here we report that endogenous SP drives a novel form of feed-forward inhibitory
activity in the dorsal horn. The SP-driven feed-forward inhibitory activity is long-lasting and has a
temporal phase distinct from glutamate-driven feed-forward inhibitory activity. Compromising SP-
driven feed-forward inhibitory activity results in behavioral sensitization. Our findings reveal a
fundamental role of SP in recruiting inhibitory activity for sensory processing, which may have
important therapeutic implications in treating pathological pain conditions using SP receptors as
targets.



Feedback/feed-forward inhibitory modulation driven by substance for decades [5-7], as supported by studies,
glutamate has been well studied in the dorsal horn of the including chemical ablation of lamina I neurons express-
spinal cord [1-3]. Little is know whether feedback/feed- ing the SP receptors [8], genetic disruption of the genes
forward inhibitory active may be driven in a glutamate- encoding substance P [9] and its receptors [10]. The noci-
independent manner. A number of neuropeptides includ- ceptive function of SP is mainly attributed to the activa-
ing substance P (SP) are also released from nociceptive tion of NK1 receptors (NK1R) that are expressed on
primary afferent fibers [4]. SP has been regarded as a pain nociceptive projection neurons located in lamina I of the

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dorsal horn [8,11,12]. It is unknown whether endog-
enously released SP can directly drive, in a glutamate-
independent manner, inhibitory activity within the spinal
cord to control nociceptive responses.

We performed patch-clamp recordings from dorsal horn
neurons in lamina V (Figure la), a region important for
nociceptive transmission and modulation [1,2]. When
primary afferent fibers (dorsal roots) were briefly stimu-
lated electrically (500 pA, 5 stimuli in 2.5 sec), EPSCs
(excitatory postsynaptic currents) were recorded from
lamina V neurons (Figure Ib). All EPSCs were blocked by
ionotropic glutamate receptor antagonists 20 [tM CNQX
plus 50 [tM APV (Figure Ib) or 3 mM kynurenic acid [3].
Brief stimulation of primary afferent fibers also evoked
IPSCs (inhibitory postsynaptic currents). These immedi-
ate IPSCs (Figure Ic top) were driven by glutamatergic
synaptic input, or glutamate-driven feed-forward inhibi-
tory activity, because they were completely abolished in
the presence of CNQX plus APV (Figure Ic bottom). How-
ever, when prolonged stimulation was applied (500 pA,
20 Hz, 1 min), a robust and long-lasting increase of IPSC
frequency and amplitude was recorded in the presence of
CNQX plus APV (n = 5, Figure 1d-f) or kyurenic acid (KA,
n = 7, Figure 2c). These results revealed a feed-forward
inhibitory (FFI) pathway not driven by glutamate.

We used capsaicin, the active ingredient of hot chili pep-
pers, to stimulate primary afferent fibers. Capsaicin is
widely used as a natural stimulant for studying nocicep-
tion. It excites nociceptive primary afferent fibers to
release glutamate and neuropeptides including substance
P through activation of TRPV1 receptors [13-15]. Capsai-
cin (2 [tM) produced a robust and long-lasting increase in
IPSC frequency and amplitude in the presence of 3 mM
kynurenic acid (Figure Ig-j). The capsaicin effects were
similar in the presence of kynurenic acid or other gluta-
mate receptor antagonists (Additional file: 1, Figure la-
c), indicating that the effects were unlikely due to an
incomplete block of glutamate-driven FFI. Inhibitory neu-
rons in lamina V use both GABA and glycine as co-trans-
mitters [16], and increases of IPSCs by capsaicin were
completely abolished in the presence of 20 [tM bicucul-
line and 2 [tM strychnine (n = 8).

It is unknown whether, transmitters, other than glutamate
released from primary afferent fibers can directly drive
inhibitory circuitry in the spinal cord. If a transmitter can
drive FFI, exogenous application should increase inhibi-
tory activity. We examined neuropeptides thought to be
released from primary afferent fibers. Galanin (300 nM),
NPY (neuropeptide Y, 1 [tM), somatostatin (2 [tM), and
CGRP calcitoninn gene-related peptide, 0.5 [tM) were
tested, but none increased IPSCs (Figure 2a). However, SP
significantly increased inhibitory activity under condi-


tions when ionotropic glutamate receptors were blocked;
SP increased IPSC frequency to ~350% of control (Figure
2a, n = 6) and amplitude to ~200% of control (n = 6).

If endogenously SP drives FFI following capsaicin stimu-
lation, SP receptor antagonists should attenuate FFI. APTL
(D-Argl, D-Pro2, D-Trp7,9, Leull]-Substance P, 10 [tM),
a neurokinin receptor antagonist, substantially blocked
capsaicin-induced increases in IPSCs (Figure 2b). L-
733,060 (2 [tM) and L-732,138 (100 [tM), two NK1 recep-
tor (NK1R) antagonists, also inhibited capsaicin-induced
increases in IPSCs. NK3 receptors are expressed in the dor-
sal horn, but SB22200 (2 [tM), a NK3 receptor antagonist,
did not significantly attenuate capsaicin-induced
increases of IPSCs. Similar to capsaicin stimulation, we
found that FFI elicited by electrical stimulation was largely
abolished by the NK1R antagonist ATPL (Figure 2c). These
results suggest that endogenous SP drives inhibitory
activity.

NK1Rs couple with either the pertussis toxin (PTX)-insen-
sitive Gq/G11 family [17] or PTX-sensitive Gi/Go family
depending on cell types [18,19]. To elucidate which type
of G-proteins was involved in SP-driven FFI, PTX was
tested. We found that capsaicin-induced increases in
inhibitory synaptic activity were completely abolished
when spinal cord slices were pretreated with PTX (Figure
2b). Capsaicin-induced increases of inhibitory synaptic
activity were also completely blocked in the presence of
NEM (N-Ethylmaleimide), a Gi/Go protein inhibitor (Fig-
ure 2b). Thus, PTX-sensitive G-protein is involved in SP-
driven FFI.

To confirm the involvement of NK1Rs, we used spinal
cord slice preparations obtained from both wild type
(NK1R+/+) and NK1R knockout mice (NK1R-/-). While
capsaicin increased IPSCs in NK1R+/+ mice, it had no effect
in NK1R-/- mice (Figure 2d). Consistent with this result, SP
(1 [tM) did not increase IPSCs in NK1R-/- mice, but did
substantially increase IPSCs in NK1R+/+ mice (Figure 2d,
Additional file: 1, Figure 2a,b). Thus, endogenous SP
released from primary afferent fibers drives inhibitory
activity (SP-driven FFI).

Possible cellular mechanisms of SP-driven FFI include i)
direct excitation of inhibitory neurons; ii) via intermedi-
ate steps; and/or iii) through synaptic modulation. If
NK1Rs are expressed on dorsal horn inhibitory interneu-
rons [20], SP may directly excite inhibitory neurons. To
test this possibility, we used dorsal horn neuron cultures
made from GIN mice, a strain of transgenic mice that
express EGFP (enhanced green fluorescent protein) under
control of a promoter for GAD67 [21]. In GIN mice,
almost all EGFP neurons examined in the dorsal horn are
inhibitory neurons [22]. As shown in Figure 2e, SP (100


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Figure I
Feed-forward inhibitory activity in the absence of glutamatergic driving force. a, Rat spinal cord slice with attached
dorsal root. A portion of the root is sucked into a stimulation electrode. Recordings were made in lamina V. b, Five consecu-
tive traces show EPSCs evoked by electrical stimulation (top). The EPSCs were abolished in the presence of 20 [tM CNQX
plus 50 [tM APV (bottom). Vh = -60 mV. c, In the same cell, stimulation evoked IPSCs (top), which were abolished in the pres-
ence of 20 [tM CNQX and 50 [tM APV (bottom). Vh = -10 mV. d, In the same cell, trains of stimulation (20 Hz for I min)
increased IPSCs in the presence of 20 [tM CNQX plus 50 [tM APV. Top trace was IPSCs recorded before and after electrical
stimulation. The bottom 3 traces are at an expanded scale. Vh = -10 mV. e&f, Time course of IPSC frequency (e) and amplitude
(f). Horizontal bars indicate stimulation. Overall, at peak responses, IPSC frequency increased to 376 47% of control (n = 5,
P < 0.05); IPSC amplitude increased to 228 74% of control (n = 5, P < 0.05). Similar results were also obtained in the pres-
ence of 3 mM kynurenic acid (see Figure 2c). g-j, Capsaicin-induced increases in inhibitory activity in the absence of glutama-
tergic driving force. g, The top trace is a continuous recording of IPSCs from a rat lamina V neuron before and following the
application of 2 [tM capsaicin in the presence of 3 mM kynurenic acid. The bottom 2 traces are at an expanded scale. h, The
time course of IPSC frequency in (g). bin width: 10s. i&j, Capsaicin-induced increases in IPSC frequency (i) and amplitude (j)
recorded from 6 rat lamina V neurons in the presence of 3 mM kynurenic acid.



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Figure 2
Feed-forward inhibitory activity driven by SP through NKI receptor activation. a, Effects of exogenously applied
neuropeptides on IPSCs recorded in rat lamina V neurons. Neuropeptides tested include galanin (0.3 [tM, n = 4), somatostatin
(2 [tM, n = 4), NPY (I [M, n = 4), CGRP (0.5 [tM, n = 5), and substance P (I [tM, n = 6). b, Antagonism of capsaicin-induced
increases in inhibitory activity in rat lamina V neurons. Capsaicin was applied in the presence of neurokinin receptor antago-
nists APTL (10 [M, n = 5), L-733, 060 (2 LM, n = 4), L-732,138 (100 [M, n = 5), SB222200 (2 LM, n = 8), and the Gi/o protein
blockers pertussis toxin (2 tgg/ml, n = 4) and NEM (100 [M, n = 5). c, Antagonism of electrical stimulation-induced increases in
inhibitory activity by the SP antagonist APTL (10 [M, n = 7). Recordings were from rat lamina V neurons. d, Effects of capsaicin
and SP on IPSCs in NKI R+/ mice (n = 8 for capsaicin, n = 10 for SP) and NKI R-7- mice (n = 21 for cap, n = II for SP). Experi-
ments were performed in the presence of 3 mM kynurenic acid (a-d) or 20 [M CNQX plus 50 [M APV (some experiments in
a&b). e, Images show a cultured GIN mice EGFP neuron (arrow indicated) before (left) and after loading the Ca2+ indicator
Fluor-3 (middle), and following application of 100 nM SP (Right). The experiment was performed in the presence of 500 nM
TTX and 3 mM kynurenic acid. Similar results were obtained from 22 EGFP neurons. f, The florescence image shows a spinal
cord slice obtained from a GIN mouse. An EGFP neuron in lamina V is indicated by a small box and enlarged in a bigger box. g,
Non-adaptive action potential firing induced by depolarizing current (50 pA) in the EGFP neuron. h, Application of I [tM SP
produced a prolonged depolarization and action potential firings (top) in the same cell. The dotted line (bottom) shows, at
expanded scale, the membrane depolarization (action potentials are omitted for clarity). Kynurenic acid (3 mM) was present
throughout the experiments (n = 7).



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nM) increased intracellular Ca2+ in ~30% (23/77) of EGFP
neurons tested in the presence of 500 TIX and 3 mM
kynurenic acid. We determined whether EGFP neurons in
lamina V responded to SP using spinal cord slices pre-
pared from GIN mice (Figure 2f). Most EGFP neurons
recorded (64%) showed non-adaptive action potential fir-
ing in response to membrane depolarization (Figure 2g).
Of 22 EGFP neurons examined, 7 (~30%) responded to 1
[tM SP with prolonged membrane depolarization (5 1
mV, n = 7) and action potential firing (Figure 2h). These
results suggest that a cellular mechanism of SP-driven FFI
is direct excitation of inhibitory interneurons by SP.

We found that SP (Additional file: 1, Figure 3a-c) and
capsaicin (n = 12) had no effect on mIPSCs. SP also did
not affect paired-pulse eIPSC ratio and corresponding
eIPSC ratio (Additional file: 1, Figure 3d-f). These results
suggest that SP/NK1R-mediated increases of IPSCs repre-
sent feed-forward neuronal activity rather than pre- or
post-synaptic modulation at inhibitory synaptic junction
sites.

We evaluated the extent SP-driving inhibitory activity con-
tributes to the total inhibitory activity under normal con-
ditions, i.e. without blocking glutamate-driven FFI. We
also compared temporal phases between SP-driven FFI
and glutamate-driven FFI. In NK1R+/+ mice, IPSC fre-
quency and amplitude were increased after trains of elec-
trical stimulation (Figure 3a,c,d), similar to the results
when glutamatergic driving force was blocked (Figure la-
f & Figure 2c). In contrast, in NK1R-/- mice, IPSCs were not
significantly changed after the same trains of stimulation
(Figure 3b,c,d). We examined IPSCs during electrical stim-
ulation and found that, in both NK1R+/+ and NK1R-/-
mice, IPSCs were elicited pulse-by-pulse immediately fol-
lowing each stimulus. These immediate IPSCs represented
glutamate-driven FFI because they could be blocked by
ionotropic glutamate receptor antagonists (see Figure Ic).
Since the pulse-by-pulse inhibitory activity was seen in
both NK1R+/+ and NK1R-/- mice, but the long-lasting
increases in IPSCs after trains of stimulation were only
observed in NK1R+/+ mice, it suggests that the latter is
driven by substance P through NK1R activation. Similar to
electrical stimulation, a large and long-lasting increase in
inhibitory synaptic activity was observed in NK1R+/+ mice
but not in NK1R-/- mice after capsaicin stimulation in the
absence of ionotropic glutamate receptor antagonists (Fig-
ure 3e). Thus, SP-driven FFI and glutamate-driven FFI
have distinct temporal phases.

One physiological role of SP-driven FFI may be to balance
neuronal activity and counteract SP-mediated nociceptive
responses in the dorsal horn. To examine this potential
physiological function, a behavioral model was used to
see if blockade of SP-driven FFI, using an NK1R antago-


nist, causes behavioral sensitization to nociceptive stim-
uli. However, an NK1R antagonist will also block SP-
mediated nociceptive response, thus interfering with the
observation of a functional change following blockade of
SP-driven FFI. To solve this complication, we chemically
ablated NK1R-expressing neurons in the superficial lam-
ina (Figure 4a&b); most of these neurons are nociceptive
projection neurons responsible for SP-mediated nocicep-
tion [8] and SP-evoked descending modulation [23].
Ablating NK1R-expressing neurons in the superficial lam-
ina was achieved by intrathecally applying substance P-
conjugated saporin (SP-SAP) [8], a targeted toxin for
NK1R-expressing neurons (Figure 4a&b). In these
animals, NK1R-expressing neurons in deep laminae
remain intact or less affected [8,12,24]. To verify that SP-
driven FFI remains intact, we used spinal cord slices pre-
pared from SP-SAP treated animals and made recordings
from lamina V neurons. Capsaicin was found to increase
IPSCs to a similar degree in animals with (Figure 4c) or
without SP-SAP treatment (Figure Ig-j, Supplementary
Figure 1), indicating that the ablation did not affect SP-
driven FFI in lamina V.

SP-SAP treated animals were used to access if the SP-
driven FFI plays a role in controlling nociceptive behavio-
ral responses. Reflexive lick/guard (L/G) responses to
nociceptive heat stimuli at 44.5 C [24] were determined.
Both the control and SP-SAP groups showed similar base-
line responses to noxious stimuli (Figure 4d) [8]. Control
rats showed behavioral sensitization following applica-
tion of capsaicin cream to the planter surface, but a sub-
stantial attenuation of behavioral sensitization was
observed in parallel experiments carried out in SP-SAP
animals [8]. To examine whether the NK1-expressing neu-
rons in deeper laminae of SP-SAP animals may intrinsi-
cally control behavioral responses to nociceptive heat
stimuli, the behavioral responses were determined follow-
ing blockade of NK1Rs by its antagonist CP-96,345 (36
nmol). Nociceptive reflexes showed sensitization when
CP-96,345 was applied in SP-SAP animals, but behavioral
hypersensitivity was attenuated by CP-96,345 in control
animals (Figure 4d). The opposite effects of NK1R antag-
onists between normal and SP-SAP animals indicate a
dual function of NK1Rs in nociceptive processing in vivo.
The behavioral sensitization by the NK1R antagonist in
SP-SAP animals revealed a role of SP-driven FFI in control-
ling nociceptive responses.

SP-driven FFI is a novel sensory processing mechanism.
The unique feature is its temporal phase that extends long
time after stimulation. This is distinct from glutamate-
driven feedback/feed-forward inhibitory activity. Com-
promising SP-driven FFI can result in sensory hypersensi-
tivity, providing implications in sensory pathology and
therapeutics that targets neurokinin system [8,12].


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NK1 R+/+ Stim




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Figure 3
SP-driven inhibitory activity under conditions when glutamatergic driving force is intact. All experiments were
performed in bath solution without glutamate receptor antagonists. a, A continuous recording of IPSCs from a lamina V neu-
ron of a NKI R+/+ mouse. Four traces (bottom) show, at an expanded scale, the IPSCs before, during, I sec after, and 9 min
after trains of stimulation. The trace during stimulation is at a more expanded scale to show pulse-by-pulse elPSCs. b, Same as
a except the experiment was performed on a NKI R-7- mouse. The pulse-by-pulse elPSCs (second trace of lower panel) were
similar to those of NKI R+/+ mice, but ISPCs returned to the basal level immediately after termination of the train stimulation
(third trace of lower panel). c, Time course of IPSC frequency (top) and amplitude (bottom). IPSCs during stimulation are not
included. d, Peak IPSC frequency and amplitude after trains of stimulation in NKI R+'+ (n = 6) and NKI R-'- mice (n = 8). In a-d,
stimulation was applied at intensity of 500 ptA and a frequency of 20 Hz. e, Capsaicin-induced changes of IPSCs in lamina V neu-
rons of NKI R+/+ mice (n = 6) and NKI R-7- mice (n = 16).






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Figure 4
Assessment of the role of SP-driven inhibitory activity in behavioral responses to noxious stimuli. a&b, Micro-
graphs show NKI receptor immunoreactivity in the lamina I region of a normal rat (a) and 14 days following intrathecal appli-
cation of SP-SAP (b). c, Capsaicin-induced increases of IPSCs recorded from lamina V neurons of SP-SAP treated rats (n = 5).
d, The first set of bars show baseline of reflexive lick/guard response to heat stimuli at 44.5oC in normal (open bar, n = 8) and
SP-SAP rats (solid bar, n = 8). The second set of bars show sensitization of behavioral responses by capsaicin in normal rats (n
= 8) and attenuation of the behavioral response in SP-SAP rats (n = 8). The third set of bars show that the NKI receptor
antagonist CP97 attenuated behavioral responses in normal rats (n = 8) but sensitize behavioral response in SP-SAP rats (n = 8)
(see Additional file: I).


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Methods
Electrophysiology recordings were performed on lamina
V neurons in transverse spinal cord slices prepared from
rats, NK1R+/+ and NK1R-/- mice, and GIN mice. Sprague
Dawley rats were used at the age of 35 7 days. Balb/c
NK1R knockout mice (NK1R-/-) and GIN mice [21]
(Jackson Laboratory) were used at the age of 21-35 days.
Transverse slices were sectioned (600 |tm in thickness)
from spinal cord L5 segments of these animals [25]. In
each experiment, a spinal cord slice was transferred to a
recording chamber. The slice was superfused with a bath
solution containing (in mM) 117NaC1, 3.6KC1, 2.5CaCl2,
1.2MgCl2, 1.2NaH2PO4, 25NaHCO3, and 11glucose,
equilibrated with 95% 02 and 5% CO2, pH 7.35, 24C.
For voltage-clamp recordings, electrodes (~5 MQ) were
filled with a solution containing (in mM): Cs2SO4 110,
CaC12 0.5, MgCl2 2, Tea-Cl 5, EGTA 5, HEPES 5, pH 7.2.
For current-clamp recordings, electrodes were filled with a
solution containing (in mM): potassium gluconate 120,
KC1 20, MgCl2 2, Na2ATP 2, NaGTP 0.5, HEPES 20, EGTA
0.5, pH 7.2. In experiments to determine EPSCs, cells were
held at -60 mV. When IPSCs were recorded, cells were
held at -10 mV. Unless otherwise indicated, IPSCs were
recorded in the presence of 3 mM kynurenic acid. Minia-
ture IPSCs (mIPSCs) were recorded in the presence of 500
nM TIX.

To stimulate primary afferent fibers, capsaicin (2 [M) was
bath applied for 1 min. Capsaicin-induced increases in
inhibitory activity were characterized pharmacologically
with APTL (D-Argl, D-Pro2, D-Trp7,9, Leull ]-Substance
P, 10 [tM), L-733, 060 (2 pM), L-732,138 (100 pM),
SB222200 (2 [M), pertussis toxin (PTX, 2 [tg/ml), NEM
(N-Ethylmaleimide; 100 mM). Except for PTX, all com-
pounds were applied through bath solution; all antago-
nists and blockers were pre-applied for 10 min. In
experiments using PTX, spinal cord splices were pretreated
with 2 [tg/ml PTX for 2-4 hours.

To elicit feed-forward inhibitory activity by electrical stim-
ulation, dorsal roots were stimulated electrically through
a suction electrode. Stimulation was applied at an inten-
sity of ~500 pA and pulse duration of 100 [tsec. Unless
otherwise indicated, stimulation was applied in a train of
pulses that had a frequency of 20 Hz and duration of 1
min. Recordings were performed in the bath solution con-
taining (in mM) 117 NaC1, 3.6 KC1, 4 CaCl2, 0.5 MgCl2,
1.2 NaH2PO4, 25 NaHCO3, 11 glucose, equilibrated with
95% 02 and 5% CO2.

To examine whether SP had effects on evoked IPSCs,
paired-pulse evoked IPSCs were examined before and fol-
lowing application of 1 [M SP. Paired-pulse evoked IPSCs
were elicited by focal stimulation in lamina V near the
recorded neurons. Stimuli were applied at the intensity of


50-150 pA, pulse duration of 100 [is, and paired-pulse
interval of 100 ms. The interval between two sets of
paired-pulses was 10 s.

Calcium Imaging was performed on dorsal horn neuron
cultures (5-7 days) made from neonatal GIN mice [26].
Cells were perfused with bath solution containing (in
mM): 150 NaC1, 5 KC1, 2 MgCl2, 2 CaCl2, 10 glucose, 10
HEPES, pH 7.4; 500 nM TTX and 3 mM kynurenic acid.
EGFP neurons were first identified and an image was
taken. Cells were then loaded with the Ca2+ indicator
Fluo-3 on the stage of microscope. Subsequently, calcium
imaging was performed [27], the effect of SP (100 nM) on
EGFP neurons was tested.

To chemically ablating NK1R-expressing lamina I neurons
with SP-SAP [8,24], a 32 g catheter was inserted into the
lumbosacral subarachnoid space (L6-S1) of adult rats
(250-300 g) [28] and SP-SAP (300 ng, substance P-conju-
gated saporin, Advanced Targeting System) was injected
through the catheter to the lumbar enlargement. Fourteen
days after this procedure, animals were used for in vitro
electrophysiological recordings or in vivo behavioral tests.
Controls were animals after sham operation.

Behavioral tests were performed on 8 SP-SAP treated ani-
mals and 8 control animals. Reflexive lick/guard
responses were assessed in two consecutive ten-minute tri-
als involving 36.0C (pre-test) trial and then a 44.5C
(test) [24,29]. Lick responses were defined as a stereo-
typed lifting of the hindlimb followed by holding and
licking the hindpaw. Guard responses were defined as an
exaggerated raising of the hindlimb. Peripheral sensitiza-
tion of behavioral responses was induced by application
of capsaicin cream (1%) to the planter surface of one
hindpaw. Reflexive responses were assessed three hours
after application. To test the effects on behavioral
responses following blockade of NK1 receptors, CP-
96,345 (36 nmol), an NK1 antagonist was applied
through the catheter 10 min before behavioral tests.

NK1 receptor immunostaining was performed after
behavioral tests to confirm the effective removal of NK1 R-
expressing lamina I neurons in SP-SAP treated animals.
NK1R immunostaining was performed using a polyclonal
anti-NK1R serum (1:3000) on a series of sections (100 tm
in thickness) cut from L5 of the spinal cord.

Analysis of synaptic events, including threshold setting
and peak identification criteria, were performed according
to a method previously described [26]. For calcium imag-
ing experiments, responsive neurons are defined as AF/Fo
> 20%. The duration of behavioral responses were col-
lected by custom software (EVENTLOG) across testing ses-
sions for all rats [24,29]. Unless otherwise indicated, data


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Molecular Pain 2005, 1:20








http://www.molecularpain.com/content/1/1/20


represent Mean SEM, p < 0.05, student-t test. Statistical
analysis of behavioral responses was performed by
ANOVA, followed by Newman-Keuls post-tests.


Additional material


Additional file 1
Substance P-driven inhibitory activity in the mammalian spinal
cord
Substance P-driven inhibitory activity in the mammalian spinal cord
Click here for file
[http://www.biomedcentral.com/content/supplementary/1744-
8069-1-20-Sl.doc]


Acknowledgements
We thank Drs. A. MacDermott, MW. Salter, L. Wang, and M. Zhuo for
comments on an earlyversion of the manuscript,J. Palmer for his assistance
in behavioral testing. This work was supported by National Science Foun-
dation Grant 0237317 (J.G.G) and National Institute of Health Grant
NS38254 (J.G.G).

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