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The Journal of Neuroscience, February 15, 2002, 22(4):1228-1237
Activation of Central Terminal Vanilloid Receptor-1 Receptors and
  -Methylene-ATP-Sensitive P2X Receptors Reveals a Converged
Synaptic Activity onto the Deep Dorsal Horn Neurons of the Spinal
Cord
Terumasa
Nakatsuka1,
Hidemasa
Furue2,
Megumu
Yoshimura2, and
Jianguo G.
Gu1
1 McKnight Brain Institute of the University of Florida
and Division of Neuroscience, Department of Oral Surgery, College of
Dentistry, University of Florida, Gainesville, Florida 32610, and
2 Department of Integrative Physiology, Graduate School of
Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka, 812-8582 Japan
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ABSTRACT |
Using a spinal cord slice preparation and patch-clamp recordings
from spinal cord dorsal horn neurons, we examined excitatory and
inhibitory circuits connecting to lamina V neurons after the activation
of afferent central terminal vanilloid receptor-1 (VR1) receptors and
P2X receptors. We found that single neurons in lamina V often received
excitatory inputs from two chemically defined afferent pathways. One of
these pathways was polysynaptic from capsaicin-sensitive afferent
terminals. In this pathway the capsaicin-sensitive afferent input first
activated interneurons in superficial laminas, and then the
excitatory activity was transmitted onto lamina V neurons.
The second excitatory input was monosynaptic from
  m-ATP-sensitive/capsaicin-insensitive afferent terminals. Both
capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive pathways also recruited polysynaptic inhibitory inputs to lamina V
neurons. Furthermore, we demonstrated that simultaneous activation of
both capsaicin-sensitive afferent pathways and
m-ATP-sensitive/capsaicin-insensitive pathways could generate a
temporal summation of excitatory inputs onto single lamina V neurons.
These convergent pathways may provide a mechanism of sensory
integration for two chemically defined sensory inputs and may have
implications in different sensory states.
Key words:
capsaicin; VR1 receptors; ATP; ,-methylene-ATP; P2X receptors; EPSCs; IPSCs; spinal cord slice
preparation
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INTRODUCTION |
The capsaicin vanilloid receptor-1
receptor (VR1) is a ligand-gated cation channel that functions as a
sensor for heat, protons, and endogenous VR1 ligands (Caterina et al.,
1997 ; Kress and Zeilhofer, 1999; Zygmunt et al., 1999; Hwang et al.,
2000; Smart et al., 2000). VR1 is expressed on a subpopulation of
primary afferent neurons that have small diameter and are potentially
nociceptive sensory neurons (Jancso et al., 1977; Holzer, 1991;
Caterina et al., 1997; Guo et al., 1999). At peripheral nerve endings
VR1 activation may be associated with the generation of burning pain sensation (Szolcsanyi, 1977; Holzer, 1991; Tominaga et al., 1998). VR1
plays an essential role in tissue injury- and inflammation-induced thermal hyperalgesia (Caterina et al., 2000; Davis et al., 2000). In
addition to their expression at peripheral nerve endings, VR1 immunoreactivity (VR1-ir) also is found on the central terminals of nociceptive afferent fibers (Guo et al., 1999).
P2X receptors (P2X) are a family of cation channels gated by
extracellular ATP (Jahr and Jessell, 1983; Krishtal et al., 1983). To
date, seven P2X subunits (P2X1 to
P2X7) have been cloned (North and Surprenant,
2000; Khakh et al., 2001), and six (P2X1 to
P2X6) are expressed on primary sensory neurons
(Collo et al., 1996; Vulchanova et al., 1996, 1997, 1998; Xiang et al.,
1998; C. Li et al., 1999; Novakovic et al., 1999). Activation of
P2X at peripheral sensory nerve endings may initiate sensory impulses
and may be associated with nociceptive and non-nociceptive sensory
signals (Burnstock and Wood, 1996; Cook et al., 1997; Sawynok and Reid, 1997; Dowd et al., 1998; Cockayne et al., 2000; Souslova et al., 2000;
Tsuda et al., 2000). Immunochemical studies have shown the presence of
P2X subunits on the central terminals of primary afferents (Guo et al.,
1999) and have shown that presynaptic P2X could modulate sensory
synaptic transmission (Gu and MacDermott, 1997; Li et al., 1998).
Dorsal root ganglion (DRG) neurons expressing VR1 usually also express
P2X3 receptors (Guo et al., 1999; Ueno et al.,
1999; Wood, 2000). Therefore, capsaicin-sensitive afferent fibers are usually ATP-sensitive as well (C. Li et al., 1999; Ueno et al., 1999; Petruska et al., 2000). Based on the immunoreactivity for VR1 and
P2X3 (Guo et al., 1999), sensory signals induced
by the activation of VR1 or P2X3 should be
conveyed primarily to the superficial laminas of the spinal cord.
However, many DRG neurons express P2X, but not VR1 (C. Li et al.,
1999; Ueno et al., 1999; Petruska et al., 2000). These primary
sensory neurons are ATP-sensitive/capsaicin-insensitive (Tsuda et al.,
2000). For ATP-sensitive/capsaicin-insensitive afferents, many of their
central terminals synapse directly on deep dorsal horn (DH) neurons in
lamina V (Nakatsuka and Gu, 2001). Thus, capsaicin-sensitive and
ATP-sensitive/capsaicin-insensitive afferents are two chemically
distinct populations of sensory fibers.
Within the neuronal network of the DH, synaptic interactions of
different sensory inputs may play a role in sensory phenomena such as
hyperalgesia and allodynia. The sensory interactions could be involved
in capsaicin-induced receptive field expansion and secondary
hyperalgesia (Simone et al., 1989a; LaMotte et al., 1991; Pedersen et
al., 1996). Here, we examined synaptic pathways of capsaicin-sensitive
inputs and m-ATP-sensitive/capsaicin-insensitive inputs and their
interactions in the DH neuronal network.
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MATERIALS AND METHODS |
Tissue preparation. Transverse spinal cord slices
(500 µm in thickness) were prepared from L5 spinal cords of rats at
the postnatal age of 11-21 d (Nakatsuka et al., 2000). Some slices were prepared from adult rats at ages between 7 and 9 weeks as described previously (Yoshimura and Jessell, 1990). Spontaneous EPSCs
(sEPSCs), miniature EPSCs (mEPSCs), spontaneous IPSCs (sIPSCs), and
miniature IPSCs (mIPSCs) were recorded from slices without dorsal roots
attached. Evoked EPSCs (eEPSCs) were recorded from slices with L5
dorsal roots attached. The length of the dorsal roots was in the range
of 4-15 mm. In each experiment a spinal cord slice was transferred to
a recording chamber and placed on the stage of an upright microscope
equipped with an infrared differential interference contrast (IR-DIC)
system. The volume of the recording chamber was ~0.5 ml. Lamina
regions were identified with a 10× objective. Recordings from spinal
slices of postnatal rats were performed under visual guidance;
individual neurons could be identified with a 40× objective. A
blind-patch technique was used for recordings in spinal cord slice
preparations from adult rats (Nakatsuka et al., 2000). The spinal cord
slice was superfused with Krebs' solution flowing at 10 ml/min at room
temperature (22°C). The Krebs' solution contained (in
mM): 117 NaCl, 3.6 KCl, 2.5 CaCl2,
1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose; the solution was
equilibrated with 95% O2/5%
CO2, and the pH of the saturated solution was
7.4.
Patch-clamp recordings. Whole-cell patch-clamp recordings
were made from DH neurons with electrodes filled with an internal solution containing (in mM): 135 K+-gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, and
5 HEPES; the pH of the solution was adjusted to 7.3 with NaOH, and the
osmolarity was adjusted to 320 mOsm with sucrose. The resistances of
the electrodes were ~5 M when filled with the internal solution. The junction potentials of the electrodes were not corrected. The
access resistance was below 20 M and was not compensated. Signals
were amplified and filtered at 2 kHz (Axopatch 200B) and sampled at 5 kHz. When EPSCs were recorded, the cells were held at  60 mV. At this
holding potential the outward IPSCs were minimized and usually
undetectable. sEPSCs were recorded in the absence of tetrodotoxin
(TTX), and mEPSCs in the presence of 0.5 µM TTX, 20 µM bicuculline, and 2 µM strychnine were
present in the bath solution in some experiments. When IPSCs were
recorded, the cells were held at 10 mV. This holding potential was
close to the reversal potential for glutamate receptors. Therefore, the
inward EPSCs were minimized and usually undetectable. sIPSCs were
recorded in the absence of TTX and mIPSCs in the presence of 0.5 µM TTX. In some experiments for the recordings of sIPSCs
and mIPSCs, 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX) and 50 µM
D-()-2-amino-5-phosphonopentanoic acid
(D-APV) also were present in the bath solution. In
experiments designed to record sEPSCs and sIPSCs simultaneously, the
cells were held at 45 mV. At this holding potential the sEPSCs were inward currents and the sIPSCs were outward currents.
,-Methylene-ATP (m-ATP; 100 µM), capsaicin (2 µM), and other testing compounds were applied via the
bath solution. The intervals for multiple applications of testing
compounds were 20 min. Analyses of sEPSCs, mEPSCs, sIPSCs, and mIPSCs
were performed as described previously (Gu and MacDermott, 1997) with
the commercial software Mini Analysis (Synaptosoft, Decatur, GA;
http://www.synaptosoft.com). Cells were assigned to be
responsive to the testing compounds when there were >20% increases in
the frequency of EPSCs or IPSCs. To record eEPSCs, we applied stimuli
(30-120 µA, 0.1 msec for A fiber; 200-800 µA, 0.1 msec for
C-fiber) to a dorsal root with a suction electrode, and we judged
monosynaptic connection by constant latency of eEPSCs (Nakatsuka et
al., 2000). Conduction velocity was calculated on the basis of the
latency of eEPSCs and the length of the dorsal root. In experiments to
determine action potential firing on postsynaptic neurons, DH neurons
in lamina V were under current-clamp configuration.
VR1 immunostaining. Spinal cord slice sections (250 µm in
thickness) were obtained in the same way as those sections for
electrophysiology recordings. They were put in a 35 mm Petri dish and
fixed with 4% paraformaldehyde (PFA; in PBS buffer solution) for 12 hr
at 4°C. The slices were transferred into a solution containing 4% PFA and 0.4% Triton X-100 and incubated at 4°C for 2 hr. They were
washed three times with PBS and then mounted onto glass slides and
allowed to air dry. Then they were encircled with hydrophobic resin
(PAP Pen, The Binding Site). Slices were incubated for 1 hr in a
solution of 1:30 normal goat serum in PBS with 0.4% Triton X-100
(GS-PBS-T) to block nonspecific antibody binding. Slices were incubated
with a polyclonal guinea pig anti-VR1 receptor antibody (1:2000;
Neuromics, Minneapolis, MN) overnight at 4°C. After a rinse with 1%
goat serum in PBS solution three times, for 20 min each time, the
slices were incubated further for 3 hr at room temperature with a
secondary antibody. The secondary antibody (1:100 in 1% goat serum PBS
solution) was a goat anti-guinea pig IgG conjugated with AlexaFluor 594 (Molecular Probes, Eugene, OR). Slices were washed three more times
with 1% goat serum in PBS solution. After a glycerol-based
anti-photobleach medium was applied, the slices were covered with
coverslips. Sections were viewed by a fluorescent microscope (Olympus
IX-70), and images were captured with a digital camera.
-Methylene-ATP, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic
acid (PPADS), capsaicin, bicuculline, strychnine, and capsazepine were
purchased from Sigma (St. Louis, MO). CNQX, D-APV, and TTX were purchased from Tocris Cookson (St. Louis, MO). Unless otherwise indicated, the data represent the mean ± SEM. Paired Student's t tests were used for statistical comparison, and
significance was considered at the p < 0.05 level.
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RESULTS |
Spreading excitatory activity of capsaicin-sensitive inputs to
deep laminas
In the spinal cord slice preparation, the central terminals of
primary afferent fibers remain in the slice sections and are functionally intact for synaptic transmission, although the dorsal roots are removed (P. Li et al., 1999; Nakatsuka et al., 2000). In this study synaptic activity in the lamina V neurons was examined. When capsaicin (2 µM) was bath applied for 1 min, there
was a large increase in the sEPSC frequency in all 18 neurons that were recorded (Fig. 1A). The
sEPSC frequency was 382 ± 55% of control (n = 18; p < 0.05) after capsaicin application. The effects
of capsaicin lasted for >2 min. Capsaicin-induced increases of sEPSC frequency (495 ± 81% of control, n = 6) were
abolished in the presence of the VR1 receptor antagonist 10 µM capsazepine (101 ± 4%,
n = 6; Fig. 1B). CNQX (10 µM), a non-NMDA receptor antagonist, completely
blocked sEPSCs (n = 6; Fig. 1C). When
similar experiments were performed and recordings were made from
neurons in lamina III-IV, four neurons of seven recordings also showed
increases in sEPSC frequency. The remaining three cells showed few
changes after capsaicin application (data not shown). Thus,
capsaicin-sensitive inputs can transmit excitatory activity to deep
laminas, especially to lamina V.
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Figure 1.
Capsaicin-induced excitatory synaptic activity in
lamina V neurons. A, Capsaicin produced a large increase
in sEPSC frequency in lamina V neurons. Two sample traces
(left) show sEPSCs recorded from a lamina V neuron
before (Control) and after the application of 2 µM capsaicin for 1 min (Cap). The time
course of the changes in sEPSC frequency is shown in the histogram
(middle). The graph on the right shows
the pooled results from 18 lamina V neurons. The capsaicin effect on
each cell was obtained from a time course histogram of sEPSC frequency
and is the averaged response in 30 sec around the peak value.
B, VR1 receptor antagonist capsazepine (10 µM) abolished the effects of capsaicin. Three sample
traces (left) show sEPSCs before
(Control) and after the application of 2 µM capsaicin (Cap) and after the
application of 2 µM capsaicin in the presence of 10 µM capsazepine (Cap/Capz). The time course
of sEPSC frequency is shown in the histogram (right).
Similar results were obtained in five other neurons. Capsazepine was
preapplied for 10 min. C, The sEPSCs were blocked
completely by 20 µM CNQX (n = 6).
D, Capsaicin produced little effect on mEPSC frequency
in lamina V neurons. Experiments were performed in the presence of 0.5 µM TTX. Two sets of sample traces show mEPSCs before
(Control/TTX) and after the application of 2 µM capsaicin (Cap/TTX). The graph
on the right shows the pooled results from 21 cells. In
all of the experiments capsaicin was applied for 1 min. The intervals
for multiple applications of testing compounds were 20 min in all of
the experiments.
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TTX has been used widely to block active interneuronal transmission;
any polysynaptic transmission between DH neurons should be blocked by
0.5 µM TTX (Gu et al., 1996; Gu and MacDermott, 1997). In
sharp contrast to the above capsaicin effects in the absence of TTX, in
the presence of 0.5 µM TTX the capsaicin did not produce
any increase in the frequency of mEPSCs in 20 of 21 cells that were
recorded in lamina V (Fig. 1D). These results strongly suggest that most capsaicin-sensitive inputs to lamina V
neurons are transmitted polysynaptically and that few
capsaicin-sensitive terminals synapse monosynaptically on lamina V neurons.
To determine whether, in the absence of TTX, capsaicin-induced
excitatory synaptic activity in lamina V was via interneurons in the
superficial laminas, we removed superficial laminas (Fig. 2A, left)
and then performed experiments in the same way as those shown in Figure
1A. Under this condition only a few neurons (3 of 15 cells; Fig. 2A, right) in lamina V showed
increases of sEPSC frequency by 2 µM capsaicin.
This result is in sharp contrast to the recordings of sEPSCs from
intact slices in the absence of TTX. Figure 2B is a
summary comparing the percentage of lamina V neurons responsive to
capsaicin applications in the three different experimental conditions
that have been described above.
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Figure 2.
Removal of superficial laminas abolishes the
capsaicin-induced activity in lamina V neurons. A, The
photomicrograph (left) shows a spinal cord slice with
one side of the superficial laminas removed. A part of a patch
electrode is seen also, and the electrode tip is inside the tissue
~70 µm from the surface. The location of the electrode tip is in
lamina V. The graph on the right shows sEPSC frequency
recorded from lamina V neurons in such preparations after a 1 min
application of 2 µM capsaicin (n = 15). B, A summary comparing the percentage of lamina V
neurons that had increased sEPSCs by capsaicin under three conditions:
the intact spinal cord slice in the absence of TTX
(Intact/no TTX; n = 18), the
intact spinal cord slice in the presence of TTX
(Intact/TTX; n = 21), and the spinal
cord slice with one side of the superficial laminas removed and in the
absence of TTX (No superficial laminae/no TTX;
n = 15). Data are from Figures
1A,D, 2A.
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To determine whether, in the superficial laminas, capsaicin-sensitive
central terminals and DH neurons were connected monosynaptically, we
determined the effects of capsaicin on mEPSCs in lamina II neurons in
the presence of 0.5 µM TTX. After the application of 2 µM capsaicin, 14 of 16 cells showed increases in the
mEPSC frequency (Fig. 3A,B);
the overall changes of mEPSC frequency were 460 ± 60% of control
(n = 16; p < 0.05). This result
provided electrophysiological evidence that capsaicin-sensitive central
terminals synapse monosynaptically on superficial DH neurons.
Consistent with this conclusion, immunostaining with an antibody
against VR1 receptors showed that VR1-ir was distributed mainly in the
superficial laminas (laminas I and II; Fig. 3C). The results
from Figures 1 to 3C support a major capsaicin-sensitive pathway that relays excitatory activity to lamina V neurons (Fig. 3D).
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Figure 3.
Monosynaptic connections between
capsaicin-sensitive afferent terminals and superficial lamina neurons.
A, Capsaicin produced a large increase in mEPSC
frequency in lamina II neurons. Two sample traces show mEPSCs before
(Control) and after the application of 2 µM capsaicin in the presence of 0.5 µM TTX.
B, Pooled results show that 2 µM capsaicin
produced increases in mEPSC frequency in most lamina II neurons that
were tested (14 of 16 cells). C, Fluorescent microscopic
image of VR1 receptor immunoreactivity in a spinal cord section. Strong
VR1-ir was observed in superficial laminas (n = 8).
D, A summary of the proposed capsaicin-sensitive pathway
based on the results from Figures 1-3C.
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-Methylene-ATP-sensitive/capsaicin-insensitive inputs to
lamina V neurons
A previous study in our laboratory (Nakatsuka and Gu, 2001) has
indicated that many afferent central terminals to lamina V neurons
express m-ATP-sensitive P2X receptors and that these terminals
appear to be capsaicin-insensitive. The monosynaptic connection of
these terminals to lamina V neurons and their capsaicin sensitivity
were confirmed in the present study. As shown in Figure 4, 100 µM m-ATP
increased mEPSC frequency in a lamina V neuron in the presence of 0.5 µM TTX. The effect lasted for >3 min with a 1 min
m-ATP application (Fig. 4C), suggesting that a
nondesensitizing m-ATP-sensitive type of P2X receptor
mediated the responses. Of 28 lamina V neurons that were recorded, 27 cells showed such a response to 100 µM
m-ATP. The overall changes of mEPSC frequency were 391 ± 56% of control (n = 28; p < 0.05).
The m-ATP-induced increases of mEPSC frequency were abolished
(n = 6; Fig. 4A) in the presence of
the P2X receptor antagonist PPADS (10 µM). CNQX (20 µM) completely blocked mEPSCs in the cells
for which 100 µM m-ATP increased mEPSC
frequency (Fig. 4B; n = 6). Because
few capsaicin-sensitive terminals monosynaptically connected with lamina V neurons (Fig. 1D), most
m-ATP-sensitive terminals that synapse directly on lamina V
neurons should be m-ATP-sensitive/capsaicin-insensitive terminals. This was confirmed by testing the effects of both capsaicin and m-ATP on mEPSC frequency in the same lamina V neurons in the
presence of 0.5 µM TTX (Fig. 4C). As
demonstrated in Figure 4C, capsaicin did not produce any
change in mEPSC frequency in a cell for which m-ATP produced a
large increase in mEPSC frequency (see also Fig.
5A,B). Figure
4D schematically illustrates the major monosynaptic
m-ATP-sensitive/capsaicin-insensitive pathway to lamina V
neurons, based on the effects of m-ATP and capsaicin on mEPSC
frequency recorded from lamina V neurons (see also Nakatsuka and Gu,
2001).
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Figure 4.
Monosynaptic connections between
m-ATP-sensitive terminals and lamina V neurons. A,
Activation of m-ATP-sensitive P2X receptors produced increases in
mEPSC frequency in lamina V neurons. Three sets of traces show mEPSCs
before (Control; left) and after the
application of 100 µM m-ATP (middle)
and after the application of 100 µM m-ATP in the
presence of 10 µM PPADS (right). The mEPSC
frequency became 391 ± 56% of control (n = 28) after the application of 100 µM m-ATP. The
effects of m-ATP were abolished completely in the presence of 10 µM PPADS (n = 6). B,
The sEPSCs were blocked completely by 20 µM CNQX
(n = 6). C, The histogram shows that
100 µM m-ATP produced an increase in mEPSC
frequency in a lamina V neuron and that 2 µM capsaicin
had no effect in the same neuron. Similar results were observed in 15 other cells. D, The diagram illustrates the presence of
an m-ATP-sensitive/capsaicin-insensitive pathway to lamina V
neurons (see also Nakatsuka and Gu, 2001).
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Figure 5.
Synaptic convergence of excitatory inputs from
capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive
pathways. A, The histogram shows the effects of
capsaicin and m-ATP on the frequency of mEPSCs and sEPSCs in the
same neuron. Capsaicin (2 µM) did not induce any change
in mEPSC frequency. In the same neuron m-ATP (100 µM) induced an increase in mEPSC frequency. After the
washout of TTX both capsaicin and m-ATP increased the sEPSC
frequency. B, Summary of the changes in the frequency
and amplitude of mEPSCs in the experiments represented in
A (n = 16). Only m-ATP induced
increases of mEPSC frequency in lamina V neurons. C,
Summary of the changes in the frequency and amplitude of sEPSCs in the
experiments represented in A (n = 14). sEPSC frequency was increased by capsaicin and by m-ATP in
the absence of TTX. The data represent the mean ± SEM;
*p < 0.05, paired Student's t
tests.
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Synaptic convergence of excitatory inputs from capsaicin-sensitive
and m-ATP-sensitive/capsaicin-insensitive pathways
To determine whether excitatory inputs from capsaicin-sensitive
terminals and m-ATP-sensitive/capsaicin-insensitive terminals converged onto single lamina V neurons, we recorded mEPSCs and sEPSCs
from the same neurons after the sequential applications of capsaicin
and m-ATP. As shown in Figure 5A, m-ATP (100 µM), but not capsaicin (2 µM), produced increases of mEPSC frequency in
the presence of 0.5 µM TTX. However, in the
same neuron in the absence of TTX, not only m-ATP but also
capsaicin produced large increases of sEPSC frequency (Fig.
5A). Figure 5B summarizes the changes in the
frequency of mEPSCs after the sequential applications of capsaicin and
m-ATP in the same neurons in the presence of TTX. Capsaicin (2 µM) did not have any effect on mEPSC frequency (n = 16). However, mEPSC frequency was increased to
447 ± 106% of control (n = 16; p < 0.05; Fig. 5B) by 100 µM
m-ATP in the same neurons. After the washout of TTX the effects
of capsaicin (2 µM) and m-ATP (100 µM) were determined in 14 of the above 16 cells. The results are summarized in Figure 5C. In contrast to mEPSCs, after the washout of TTX the sEPSC frequency was increased substantially by capsaicin in all 14 cells that were tested. The overall changes were 465 ± 81% of control (n = 14; p < 0.05; Fig. 5C). Similar to the
effects of m-ATP on mEPSCs, m-ATP also produced increases
of sEPSC frequency (408 ± 68% of control, n = 14; p < 0.05). In addition to the changes in sEPSC
frequency, there was also a slight but significant increase in the
sEPSC amplitude by capsaicin and m-ATP (Fig. 5C).
These results indicate the convergence of two excitatory inputs from
capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive
pathways to lamina V neurons (see Fig. 10).
Young rats were used in the above experiments (ages between 11 and
21 d). We also performed experiments in spinal cord slices obtained from adult rats (ages between 7 and 9 weeks) to determine whether similar results could be obtained in mature animals. In six
lamina V cells that were tested in the presence 0.5 µM
TTX, 100 µM m-ATP increased mEPSC frequency in all
six cells (170 ± 13% of control; p < 0.05; data
not shown). Of these cells, four cells were tested with 2 µM capsaicin, and none of them showed increases
of mEPSC frequency (104 ± 5% of control). However, when TTX was
not present, 2 µM capsaicin increased sEPSC
frequency (283 ± 102% of control, n = 4;
p < 0.05; data not shown). These results are similar
to those obtained in young rats.
Recruitment of inhibitory pathways
Both capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive inputs also recruited
inhibitory inputs onto lamina V neurons (Fig.
6). The recruitment of inhibitory inputs
was determined by measuring sIPSCs when lamina V neurons were held at
10 mV. The inhibitory inputs produced outward postsynaptic currents
under this condition (Fig. 6A). The frequency of
sIPSCs was increased after the application of 2 µM capsaicin in all 12 cells that were recorded
(Fig. 6A,C). In the same neurons the frequency of
sIPSCs also was increased after the application of 100 µM m-ATP (Fig. 6B,C).
The sIPSCs were blocked completely in the presence of 20 µM bicuculline plus 2 µM strychnine (Fig. 6A,B),
indicating that the sIPSCs were mediated by GABAergic/glycinergic
inputs from DH inhibitory interneurons. Figure 6C summarizes
the changes in the frequency and amplitude of sIPSCs after the
sequential applications of 2 µM capsaicin and
100 µM m-ATP in the same neurons. sIPSC frequency was 388 ± 51% of control (n = 12;
p < 0.05) after capsaicin application and 441 ± 83% of control (n = 12; p < 0.05)
after m-ATP application. sIPSC amplitude also was increased
slightly after the applications of m-ATP and capsaicin. When
experiments were performed in the presence of 0.5 µM TTX to block active interneuronal transmission (Fig. 6D), both mIPSC frequency and
amplitude were not changed after the application of 2 µM capsaicin (n = 8) or 100 µM m-ATP (n = 8). These
results indicate that both capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways recruit inhibitory synaptic circuitry and that the recruited inhibitory inputs can converge onto the same lamina V neurons.
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Figure 6.
Recruitment of inhibitory circuitry after the
activation of central terminal VR1 receptors or P2X receptors.
A, Capsaicin induced an increase in sIPSC frequency.
Three sets of sample traces show sIPSCs recorded from a lamina V neuron
in control (left), after the application 2 µM capsaicin alone (middle), and after the
application of 2 µM capsaicin in the presence of both 20 µM bicuculline and 2 µM strychnine
(right). B, The m-ATP induced an
increase of sIPSC frequency. Experiments were similar to those in
A except that 100 µM m-ATP was
tested. The recordings in A and B were
performed on the same neuron. C, Summary of the
increases in sIPSC frequency induced by 2 µM capsaicin or
100 µM m-ATP in the same neurons
(filled bars; n = 12). The
changes of sIPSC amplitude are shown also (open bars;
n = 12). The experiments were performed in the
absence of TTX. D, Capsaicin and
m-ATP had no effect on mIPSCs. Of the 12 cells tested in
C, eight cells also were tested in the presence of 0.5 µM TTX. The frequency (filled bars)
and amplitude (open bars) of mEPSCs were not affected by
2 µM capsaicin or 100 µM m-ATP
(n = 8). In all recordings the cells were held at
10 mV. The data represent the mean ± SEM; *p < 0.05; paired Student's t tests.
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Synaptic convergence of excitatory and inhibitory inputs to the
same lamina V neurons after the activation of capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways
To determine whether both excitatory and inhibitory inputs
converge on the same lamina V neurons, we recorded sEPSCs and sIPSCs simultaneously, and we examined the effects of capsaicin as well as
m-ATP in the same lamina V neurons. The simultaneous recordings of sEPSCs and sIPSCs were performed with cells held at 45 mV. At this
holding potential the glutamatergic sEPSCs were inward currents and
GABAergic/glycinergic sIPSCs were outward currents (Fig.
7A). As shown by the sample
traces in Figure 7A, the increases in the frequency of both
sEPSCs and sIPSCs were observed clearly under this simultaneous
recording condition after the applications of m-ATP (100 µM) and capsaicin (2 µM). The sEPSC frequency increased to 347 ± 97% of control (p < 0.05; n = 6), and the sIPSC frequency increased to 447 ± 186% of control
(p < 0.05; n = 6) after the
application of 2 µM capsaicin (Fig.
7B). In the same neurons m-ATP (100 µM) increased the sEPSC frequency to 363 ± 68% of control (p < 0.05; n = 6) and increased the sIPSC frequency to 372 ± 107% of control
(p < 0.05; n = 6; Fig.
7B). Based on the results from Figures 1 to 7, a schematic
diagram in Figure 10 summarizes the major pathways involved in the
excitatory and inhibitory convergence of capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways.
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Figure 7.
Synaptic convergence of both excitatory and
inhibitory inputs on the same lamina V neurons. A,
Sample traces show simultaneous recordings of both sEPSCs and sIPSCs in
a lamina V neuron in normal bath solution
(Control), after the application of 100 µM m-ATP, and after the application of 2 µM capsaicin in a lamina V neuron. The cell was voltage
clamped at 45 mV. At this holding potential both the sEPSCs (inward
currents) and sIPSCs (outward currents) could be detected
simultaneously. B, A summary shows the increases of both
sEPSC frequency and sIPSC frequency in experiments
(n = 6) as represented in A. The
data represent the mean ± SEM; *p < 0.05, paired Student's t test.
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Temporal summation in lamina V neurons
Because capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways displayed
convergent excitatory and inhibitory activities onto lamina V neurons,
we determined whether the presence of two excitatory inputs could
overcome the inhibitory influence, resulting in hyperactivity in lamina
V neurons. Under current-clamp configuration the occurrence of action
potential spikes was measured after the applications of 2 µM capsaicin alone, 100 µM m-ATP alone, or both of them together. Some action potential spikes were
observed for brief periods when capsaicin or m-ATP was applied
separately (Fig. 8A,B).
However, when capsaicin and m-ATP were coapplied, a larger
increase in spike frequency occurred, lasting up to 10 min after a 2 min application of capsaicin plus m-ATP (n = 4;
Fig. 8A,B). These results suggest that the convergent excitatory inputs from capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways may produce a
nonlinear summation and long-lasting enhancement of lamina V
activity.
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Figure 8.
Temporal summation and action potential spikes
after simultaneous activation of both capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways. A,
Three traces show the action potential spikes after the application of
2 µM capsaicin alone (top), 100 µM m-ATP alone (middle), and both of
them together (bottom). B, Time course of
action potential spikes after m-ATP alone, capsaicin alone, and
both of them together (n = 4). Resting membrane
potentials were 68 ± 7 mV. Action potential spikes are
truncated to show some sEPSPs. Each data point in
B represents the number of action potentials in a period
of 30 sec.
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Dorsal root stimulation-evoked synaptic convergence of sensory
inputs from capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive afferent fibers
Dorsal root stimulation evoked monosynaptic A-fiber eEPSCs with or
without polysynaptic eEPSCs in lamina V neurons. The occurrence of
polysynaptic eEPSCs often depended on stimulation intensity. As
shown in Figure 9, at A-fiber
stimulation intensity (100 µA, 0.1 msec) a lamina V neuron showed
only monosynaptic A-like eEPSCs (Fig. 9A). When
stimulation intensity increased to C-fiber intensity (500 µA, 0.1 msec) (Nakatsuka et al., 2000), dorsal root stimulation resulted in
monosynaptic eEPSCs followed by polysynaptic eEPSCs in the same lamina
V neuron (Fig. 9B). Similar results were obtained in 16 other cells. At C-fiber stimulation the conduction velocity for the
initial monosynaptic eEPSCs was 2.8 ± 0.2 m/sec (2.0-5.1 m/sec;
n = 17), within the A-fiber conduction range. The
subsequent polysynaptic eEPSCs were separated either completely (Fig.
9B) or partially (Fig. 9C) from the monosynaptic
A-like eEPSCs, depending on the length of the dorsal roots.
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Figure 9.
Evoked monosynaptic and polysynaptic excitatory
inputs to lamina V neurons and their sensitivity to capsaicin.
A, Four sample traces, aligned at the time of each
stimulus, represent eEPSCs (indicated by I)
obtained in a lamina V neuron after dorsal root stimulation (4 stimuli)
at A-fiber stimulation intensity (~100 µA, 0.1 msec). Note that
there is no change in the latency of eEPSCs, indicating monosynaptic
transmission. B, eEPSCs were elicited from the same
neuron in A after dorsal root stimulation (4 stimuli) at
C-fiber stimulus intensity (~500 µA, 0.1 msec). Two groups of
eEPSCs were observed. In the first group (indicated by
I), eEPSCs occurred with the same latency as
those shown in A, and the afferent conduction velocity
was 5.3 m/sec. In the second group (indicated by
II), eEPSCs had a longer latency.
Arrows point to the changes in the latency of the group
II eEPSCs, indicating polysynaptic transmission. The apparent
conduction velocity for these polysynaptic inputs was  0.6 m/sec.
Conduction velocity is calculated on the basis of eEPSC latency and the
length of the attached dorsal root. In the spinal cord slice used in
A and B, the attached root was extra long
(15 mm). C, D, An example shows monosynaptic A-like
eEPSCs (I) and polysynaptic eEPSCs
(II) recorded from a lamina V neuron in normal
bath solution (C) and after the bath application
of 2 µM capsaicin for 10 min (D).
The afferent conduction velocity was 2.1 m/sec for the monosynaptic
inputs that generated monosynaptic A-like eEPSCs. The apparent
conduction velocity was 0.46 m/sec for the polysynaptic inputs. The
length of the dorsal root in this experiment was 4 mm.
E, A summary of the results (n = 4)
from experiments represented in C and D.
The amplitude of polysynaptic eEPSCs was determined after subtracting
the extrapolated decay component of the previous monosynaptic
eEPSCs.
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The monosynaptic A-like inputs and the polysynaptic inputs to
the same lamina V neurons were examined further for their
sensitivity to capsaicin. Dorsal root stimulation was applied at
C-fiber stimulus intensity to elicit monosynaptic A-like eEPSCs and
polysynaptic eEPSCs first (Fig. 9C), and then the effects of
2 µM capsaicin on these eEPSCs were determined
(Fig. 9D). In four lamina V neurons that were tested, the
monosynaptic A-like eEPSCs were not affected significantly by
capsaicin (224 ± 35 pA in control vs 229 ± 32 pA in the
presence of capsaicin; Fig. 9C-E). However, the
polysynaptic eEPSCs were sensitive to capsaicin in all four cells that
were tested (Fig. 9C-E); the average amplitude of
polysynaptic eEPSCs was decreased significantly from 156 ± 45 pA
in the control condition to 24 ± 10 pA in the presence of 2 µM capsaicin. Together with our recent study
that showed m-ATP sensitivity of monosynaptic A-like eEPSCs in
almost all lamina V neurons that were recorded (Nakatsuka and Gu,
2001), these results suggest that sensory impulses elicited at high
stimulation intensity may result in the convergence of synaptic inputs
from capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive afferent fibers to lamina V neurons.
| |
DISCUSSION |
We have studied putative synaptic circuits involved in the
transmission of capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive inputs in the spinal cord
by activation of central terminal VR1 receptors and
m-ATP-sensitive P2X receptors. Capsaicin-sensitive inputs
initially are transmitted from primary afferent terminals to the
superficial laminas. The excitatory activity of capsaicin-sensitive inputs is transformed further to both excitatory and inhibitory signals
in the dorsal horn and is spread into lamina V neurons. Furthermore,
polysynaptic capsaicin-sensitive inputs converge with monosynaptic
m-ATP-sensitive/capsaicin-insensitive inputs onto lamina V
neurons (Fig. 10). This convergence
produces temporal summation in a single lamina V neuron.
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Figure 10.
Schematic illustration of a proposed synaptic
convergence of capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive sensory pathways onto
lamina V neurons. The diagram shows two excitatory sensory pathways.
One is from m-ATP-sensitive/capsaicin-insensitive afferent
central terminals. These terminals make monosynaptic excitatory
synapses onto lamina V neurons (1). The other
pathway is from capsaicin-sensitive afferent central terminals. These
terminals first synapse onto interneurons in superficial lamina
(2). Then the excitatory inputs are relayed to
lamina V neurons (3). The site 1
synapse is sensitive to m-ATP but insensitive to capsaicin. The
site 2 synapse is sensitive to capsaicin. Because most
capsaicin-sensitive afferent neurons express rapidly desensitizing P2X
receptors (C. Li et al.; 1999; Ueno et al., 1999; Nakatsuka and
Gu, 2001), capsaicin-sensitive terminals also may be sensitive to
m-ATP, i.e., capsaicin-sensitive/m-ATP-sensitive terminals.
The diagram also shows the recruitment of inhibitory pathways mediated
by GABAergic/glycinergic interneurons (light gray).
Other pathways may be present and are not shown in the diagram.
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Spreading excitatory activity of capsaicin-sensitive inputs from
the superficial laminas to lamina V neurons
VR1-ir has been observed mainly in the superficial laminas (see
also Tominaga et al., 1998; Guo et al., 1999). In the present study the
mEPSC frequency was increased when recordings were made from lamina II
neurons. Because mEPSCs were measured in the presence of TTX, the
active interneuronal transmission among DH neurons was prevented (Gu et
al., 1996). Therefore, monosynaptic connections between
capsaicin-sensitive fibers and lamina II neurons were revealed
electrophysiologically. In contrast to the recordings in lamina II, few
lamina V neurons showed increased mEPSC frequency by capsaicin. This
indicates that few capsaicin-sensitive afferent fibers have
monosynaptic connections with lamina V neurons. However, when sEPSCs
were examined in the absence of TTX, capsaicin produced an increase in
sEPSC frequency. This result suggests that capsaicin-sensitive inputs
could be transmitted to lamina V polysynaptically. The spread of
capsaicin-sensitive activity to deep laminas was mediated by
interneurons in the superficial laminas. This is supported further by
the results that capsaicin-induced increases of sEPSCs in lamina V
neurons were abolished after the removal of the superficial laminas.
These results indicate that there are extensive neuronal interactions
between functionally distinct spinal cord laminar regions. It has been
assumed that neurons in the superficial laminas interact with cells in
the deep DH. Consistent with this assumption, anatomical evidence
showed synaptic-like contacts between superficial and deep DH neurons
(Ritz and Greenspan, 1985; Light and Kavookjian, 1988). However, this
assumption has been questioned because of the lack of strong
electrophysiological evidence (Willis and Coggeshall, 1991). Our
results indicate that superficial and deep laminas, the two
functionally distinct sensory regions in the DH, have extensive signal
transmission when VR1 receptors on primary afferent fibers are activated.
The VR1 receptor has been shown to be a sensor for nociceptive heat
stimuli (Caterina et al., 1997, 2000; Davis et al., 2000). The
widespread neuronal activity after the activation of VR1 receptors suggests that capsaicin-sensitive inputs have profound effects on
spinal cord neuronal activity not only in superficial laminas but also
in deep laminas. The widespread neuronal activity after the activation
of VR1 receptors may be a mechanism for the development of secondary
hyperalgesia after intradermal capsaicin injections or burning injury
(Simone et al., 1989b; LaMotte et al., 1991; Pedersen et al., 1996) and
for VR-1 receptor-mediated thermal allodynia after tissue inflammation
(Caterina et al., 2000; Davis et al., 2000).
-Methylene-ATP-sensitive/capsaicin-insensitive inputs to
lamina V neurons
The increase of mEPSC frequency by m-ATP in lamina V neurons
indicates the presence of m-ATP-sensitive P2X receptors on the
presynaptic terminals to lamina V neurons. We have demonstrated previously that m-ATP-sensitive terminals on lamina V neurons were derived mainly from A fibers and that activation of P2X receptors on those terminals not only increased spontaneous glutamate release from those central terminals but also potentiated the evoked
EPSCs (Nakatsuka and Gu, 2001). The present work, together with our
previous results (Nakatsuka and Gu, 2001), also indicates that
m-ATP-sensitive terminals to lamina V neurons are
capsaicin-insensitive. The presence of
ATP-sensitive/capsaicin-insensitive sensory neurons has been shown
previously in acutely dissociated DRG neurons (C. Li et al.,
1999; Ueno et al., 1999; Petruska et al., 2000; Tsuda et al.,
2000). It remains to be shown whether
m-ATP-sensitive/capsaicin-insensitive afferent fibers represent
all or part of ATP-sensitive/capsaicin-insensitive afferent fibers.
Functionally, it has been shown that P2X receptors on
m-ATP-sensitive/capsaicin-insensitive neurons play a role in
mechanical allodynia (Tsuda et al., 2000).
Synaptic convergence and summation of capsaicin-sensitive input and
m-ATP-sensitive/capsaicin-insensitive input onto lamina V
neurons
We have found that the two chemically distinct sensory inputs,
capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive inputs, synaptically converge onto lamina V neurons. Lamina V neurons
of the spinal cord DH are known to receive a variety of sensory inputs,
including nociceptive and non-nociceptive inputs (Willis and
Coggeshall, 1991; Woolf, 1994). Sensory convergence has been observed
in many lamina V neurons after electrical or physical stimuli (Mendell,
1966; Wagman and Price, 1969; McMahon and Morrison, 1982; Takahashi and
Yokota, 1983; Alarcon and Cervero, 1990). Our study shows that two
chemically defined sensory inputs synaptically converge onto lamina V
neurons. The demonstration of the synaptic convergence of
capsaicin-sensitive input and the m-ATP-sensitive/capsaicin-insensitive input should help us to understand further the functional roles of VR1 receptors and a subtype
of P2X receptors in sensory transmission.
The synaptic convergence shown here includes both excitatory and
inhibitory pathways. The recruitment of inhibitory inputs during the
activation of capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive pathways may provide a
negative feedback mechanism. The recruitment of inhibitory pathways
after the application of m-ATP was less likely because of the
direct activation of P2X receptors on DH interneurons. Previously, two
studies showed that ATP increased the release of GABA and glycine from
a subpopulation of DH neurons (Hugel and Schlichter, 2000; Rhee et al.,
2000). However, in the same studies the effects were not produced by
m-ATP, indicating the absence of m-ATP-sensitive P2X
receptors on inhibitory DH interneurons in postnatal rats.
Consistently, we did not find significant changes of mIPSC frequency by
m-ATP. Thus, the increase of sIPSC frequency by m-ATP is
attributable to the recruitment of inhibitory DH interneurons (Fig.
7).
Convergent excitatory inputs may overcome inhibitory controls and
produce a nonlinear temporal summation, resulting in long-lasting increases of action potential firing in lamina V neurons (Fig. 8).
Another possible contribution to the increased activity in lamina V
during the coapplication of m-ATP and capsaicin (Fig. 8A,B) is the synergistic effect of m-ATP and
capsaicin on capsaicin-sensitive terminals. However,
capsaicin-sensitive DRG neurons usually only express the rapidly
desensitizing P2X receptors (Guo et al., 1999; C. Li et al.,
1999; Petruska et al., 2000; Nakatsuka and Gu, 2001), and these
receptors only have transient synaptic effects when activated
(Labrakakis et al., 2000). Therefore, it is less likely that
m-ATP action on capsaicin-sensitive terminals plays a significant role in the long-lasting temporal summation. However, long-lasting synaptic effects of m-ATP have been observed for the
m-ATP-sensitive/capsaicin-insensitive terminals to lamina V
neurons (Fig. 4C; see also Nakatsuka and Gu, 2001). Thus the
long-lasting increases of action potential firing in lamina V neurons
after the coapplication of m-ATP and capsaicin (Fig. 8) should
represent mainly the temporal summation of excitatory inputs from
capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive inputs.
Dorsal root stimulation-evoked synaptic convergence of sensory
inputs from capsaicin-sensitive and
m-ATP-sensitive/capsaicin-insensitive afferent fibers
We have shown that monosynaptic A-like eEPSCs and polysynaptic
eEPSCs converge on the same lamina V neurons after dorsal root
stimulation at C-fiber intensity. By testing their capsaicin sensitivity, we have shown that the monosynaptic A-like inputs are
insensitive to capsaicin, but most polysynaptic afferent inputs are
sensitive to capsaicin (Fig. 9). These results extend our findings on
the basis of the recordings of sEPSCs and mEPSCs. Together with our
previous findings that the evoked monosynaptic A-like eEPSCs were
sensitive to m-ATP (Nakatsuka and Gu, 2001), our results suggest
that sensory impulses carried by capsaicin-sensitive fibers and
m-ATP-sensitive/capsaicin-insensitive fibers can converge on the
same lamina V neurons.
Capsaicin-sensitive polysynaptic inputs were evident by the suppression
of polysynaptic eEPSCs in the presence of capsaicin. Consistently,
monosynaptic capsaicin-sensitive eEPSCs in lamina II were blocked by
capsaicin (Yang et al., 1999), potentially because of conduction block
(Urban and Dray, 1993; Yang et al., 1999). We found that polysynaptic
eEPSCs were not abolished completely by capsaicin. This may suggest
that some capsaicin-insensitive inputs also converge polysynaptically
on lamina V neurons. The lack of capsaicin sensitivity for monosynaptic
A-like eEPSCs was observed in all four cells that were tested in
lamina V, suggesting that A-afferent fibers to these neurons were
capsaicin-insensitive. However, our results do not exclude the presence
of capsaicin-sensitive A-fibers (Urban and Dray, 1993; Ringkamp et
al., 2001). Capsaicin-sensitive fibers appear to be rare in lamina V,
based on VR1-ir and our mEPSC experiments. Thus, capsaicin-sensitive
A-fibers, if present in a large number, may be located mainly in the
superficial laminas.
Implications
Simultaneous activation of different sensory pathways by
endogenous ligands for VR1 receptors and P2X receptors may be common during tissue injury, inflammation, and other pathological conditions because chemical mediators such as protons, ATP, and ADP may be released under these conditions. Thus the putative excitatory summation
of capsaicin-sensitive and m-ATP-sensitive/capsaicin-insensitive inputs may occur, which may lead to the development of hyperactivity in
deep DH neurons. However, convergent inhibitory inputs may play an
important role in preventing such hyperactivity. The balance among
these excitatory and inhibitory circuits may have important physiological function. A shift of the balance may represent
state-dependent sensory processing.
| |
FOOTNOTES |
Received Aug. 28, 2001; revised Nov. 29, 2001; accepted Nov. 29, 2001.
This work was supported by National Institutes of Health Grant NS38254
(J.G.G), by Office of Naval Research Grant N00014-1-0188 (J.G.G), and by a Human Frontier Science Program grant (M.Y). We thank
A. MacDermott, S. Siegelbaum, D. Price, B. Cooper, and R. Yezierski for
providing thoughtful comments on this manuscript. We appreciate J. X. Ling for general assistance during this work.
Correspondence should be addressed to Jianguo G. Gu, McKnight Brain
Institute of the University of Florida, University of Florida, Box
100416, Gainesville, FL 32610. E-mail: jgu{at}dental.ufl.edu.
| |
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