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The Journal of Neuroscience, September 1, 2001, 21(17):6522-6531
ATP P2X Receptor-Mediated Enhancement of Glutamate Release and
Evoked EPSCs in Dorsal Horn Neurons of the Rat Spinal Cord
Terumasa
Nakatsuka and
Jianguo G.
Gu
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
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ABSTRACT |
Presynaptic ATP P2X receptors have been proposed to play a role in
modulating glutamate release from the first sensory synapse in the
spinal cord. Using spinal cord slice preparations and patch-clamp recordings from dorsal horn neurons in lamina V of the rat spinal cord,
we showed that the activation of P2X receptors by  , -methylene-ATP (m-ATP) resulted in a large increase in the frequency of
spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs). The increases
in mEPSC frequency by m-ATP were not blocked by the
Ca2+ channel blocker, 30 µM
La3+, but were abolished in a bath solution when
Ca2+ was omitted. The increases in mEPSC frequency
by m-ATP were blocked completely by the P2 receptor
antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS)
at 10 µM. Furthermore, the EPSCs evoked by dorsal root
stimulation were potentiated by m-ATP as well as by the ecto-ATPase inhibitor ARL67156 and were depressed in the presence of P2
receptor antagonists PPADS (10 µM) and suramin (5 µM). The effects of these compounds on the evoked EPSCs
were associated with the changes in glutamate release probability of
primary afferent central terminals. Our results indicate that
m-ATP-sensitive P2X receptors play a significant role in
modulating excitatory sensory synaptic transmission in the spinal cord,
and the potential role of endogenous ATP is suggested.
Key words:
ATP; purinergic receptors; EPSCs; glutamate release; primary afferent fibers; patch-clamp technique; spinal cord slice
preparation
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INTRODUCTION |
ATP P2X receptors, a family of
nonselective cation channels gated by extracellular ATP (Jahr and
Jessell, 1983 ; Krishtal et al., 1983; Khakh et al., 2001), may play an
important role in somatic sensory transmission. P2X receptors are
highly expressed on different functional types of primary sensory
neurons (Vulchanova et al., 1997; Xiang et al., 1998; Guo et al., 1999;
Li et al., 1999; Petruska et al., 2000). Immunocytochemical evidence
shows the presence of different types of P2X receptors in both
peripheral and central terminals of primary afferent fibers (Vulchanova
et al., 1996, 1998; Guo et al., 1999; Novakovic et al., 1999).
Activation of P2X receptors at peripheral sensory nerve endings may
initiate sensory impulses and may be associated with nociceptive and
non-nociceptive sensory signals (Cook et al., 1997; Sawynok and Reid,
1997; Dowd et al., 1998; Tsuda et al., 2000). At central terminals P2X
receptors have been proposed to play a role in modulating glutamate
release from sensory synapses, based on a previous study that used a
coculture system of dorsal root ganglion (DRG) and dorsal horn (DH)
neurons (Gu and MacDermott, 1997). Consistent with this hypothesis, ATP has been shown to have presynaptic effects in lamina II of the DH (Li
and Perl, 1995), and activating presynaptic P2X receptors could have
facilitative effects on sensory spinal transmission to lamina II of the
DH (Li et al., 1998). Khakh and Henderson (1998) also have demonstrated
a presynaptic P2X receptor-mediated facilitation of glutamate release
at synapses in the brainstem. The role of endogenous ATP in modulating
glutamate release from sensory afferent terminals was indicated (Khakh
and Henderson, 1998; Li et al., 1998). However, it is currently not
clear whether the central terminals of primary afferent fibers
connecting to deep lamina DH neurons also express P2X receptors at
presynaptic sites and, if so, whether the activation of these receptors
may facilitate glutamate release.
To date, at least seven P2X subunits (P2X1 to
P2X7) have been cloned (for review, see North and
Surprenant, 2000). Except for P2X6, all P2X subunits can
form functional homomeric receptors in different heterologous
expression systems (Khakh et al., 2001). Among those homomers,
P2X1 and P2X3 receptors mediate a rapidly desensitizing response to both ATP and the agonist
,-methylene-ATP (m-ATP). The other five homomers mediate a
nondesensitizing response to ATP and are essentially insensitive to
m-ATP (Khakh et al., 2001). Coexpression of different P2X
receptor subunits results in several functional heteromers, including
P2X1+5, P2X2+3, P2X2+6, and
P2X4+6 (Lewis et al., 1995; Le et al., 1998, 1999; Torres
et al., 1998; Haines et al., 1999; King et al., 2000). Of those
heteromers, P2X2+6 receptors are insensitive to m-ATP
(King et al., 2000). Thus, m-ATP makes some functional P2X
receptors pharmacologically distinguishable from others.
Of the seven cloned P2X receptor subunits, six (P2X1 to
P2X6) are expressed in primary sensory neurons (Collo et
al., 1996; Vulchanova et al., 1996, 1997, 1998; Xiang et al., 1998). In
the spinal cord the mRNAs for P2X1, P2X2,
P2X4, P2X5, and P2X6 subunits have
been found; mRNAs for P2X2, P2X4, and
P2X6 are the most abundant (Collo et al., 1996).
Immunochemical studies on spinal cord sections with antibodies against
P2X1, P2X2, and P2X3 subunits have
shown the expression of these subunits on the primary afferent central terminals (Vulchanova et al., 1996, 1997, 1998; Guo et al., 1999). Other P2X receptor subunits also may be expressed at the primary afferent central terminals in the spinal cord. In the present study we
have explored the functions of P2X receptors at some sensory synapses
in deep laminas (lamina V) of the spinal cord.
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MATERIALS AND METHODS |
Spinal cord slice preparation. Transverse spinal cord
slices (500 µm in thickness) were prepared from L5 spinal cords of
rats at postnatal age 11-21 d, as described previously (Nakatsuka et al., 2000). Unless otherwise indicated, slices without dorsal roots
attached were used for recordings of spontaneous EPSCs (sEPSCs) and
miniature EPSCs (mEPSCs); slices with attached L5 dorsal roots were
used for evoked EPSCs (eEPSCs). A spinal cord slice was transferred to
a recording chamber (~0.5 ml) and placed on the stage of an upright
infrared-differential interference contrast (IR-DIC) microscope. The
slice was superfused (10 ml/min) with Krebs' solution containing (in
mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose in 95% O2/5%
CO2, pH 7.3, at 22°C.
Preparation of acutely dissociated DRG neurons. Dorsal root
ganglia were removed from rats (age of 4-7 weeks) and placed in a
35°C bath solution containing dispase (neutral protease, 5 mg/ml; Boehringer Mannheim, Indianapolis, IN) and collagenase (2 mg/ml; Sigma
type 1, St. Louis, MO). After 1 hr of incubation the DRGs were
triturated to dissociate the neurons. The dissociated cells were plated
on coverslips precoated with poly-L-lysine and allowed to
adhere for 1 hr before recording.
Preparation of cultured DRG neurons. Dorsal root ganglia
were isolated from rat embryos aged 16 d (E16) in
utero, exposed to 0.25% trypsin for 20 min, and dissociated. The
dissociated primary sensory neurons were plated on glass coverslips
previously prepared with a monolayer of rat cortical astrocytes. At the
time of plating, 2.5S NGF (10 ng/ml) and 5-fluoro-2'-deoxyuridine (10 µM) were added, and 2.5S NGF was added once
every week when the cells were fed with fresh media. The cultures of
2-3 weeks were used for the experiments. Principles of
Laboratory Animal Care (National Institutes of Health publication
86-23; revised, 1985) was followed in all of the tissue preparation
procedures described above.
Patch-clamp recordings from spinal cord slices. Lamina
regions were identified under a 10× objective, and individual neurons were identified with a 40× objective under IR-DIC microscope. Whole-cell patch-clamp recordings were made from DH neurons with electrodes (~5 M ) filled with a solution containing (in
mM) 135 K+-gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, and 5 HEPES. Signals were amplified and filtered at 2 kHz (Axopatch 200B, Axon Instruments, Foster City, CA) and sampled at 5 kHz. Cells were held at  60 mV,
which was close to the reversal potential for GABAA and
glycine receptors under the experimental conditions. At this holding
potential the outward IPSCs were minimized and usually were
undetectable. sEPSCs were recorded in the absence of TTX. mEPSCs were
recorded in the presence of 0.5 µM TTX (the term mEPSCs
is used for simplicity; "sEPSCs in the presence of TTX" may be a
more suitable term). In some experiments mEPSCs were recorded in the
presence of 20 µM bicuculline and 2 µM
strychnine in bath solution. m-ATP, capsaicin, and other testing
compounds described in Results were applied via bath solution. In some
experiments the effects of 100 µM ATP on sEPSC frequency
were tested in the presence of 50 µM ARL67156 plus 2 mM caffeine. The application intervals for testing
compounds were 20 min. At this interval the agonist responses were
reproducible. Analyses of sEPSCs and mEPSCs were performed as described
previously (Gu and MacDermott, 1997).
To record eEPSCs from lamina V, we applied a stimulus (~120
µA, 0.1 msec) to a dorsal root with a suction electrode. Monosynaptic connection was judged by constant latency of the eEPSCs when multiple stimuli were applied (Nakatsuka et al., 2000). Most lamina V neurons that we recorded had monosynaptic connections with primary afferent fibers (93% from 28 recordings) in our slice preparations. The stimulation condition usually yielded few synaptic failures. In the
experiments to determine synaptic potentiation of eEPSCs by P2X
receptor activation, we first identified monosynaptic eEPSCs and then
gradually reduced the intensity (Malinow and Tsien, 1990) to a level at
which some synaptic failures (30-70%) occurred. For higher
sensitivity the cells with higher synaptic failure rates were assigned
for tests with m-ATP or ARL67156, and the cells with lower
failure rates were tested with P2X receptor antagonists. Synaptic
failure was judged by current levels in the range of current noise
baseline of Gaussian distribution (±3 pA). One to three cells were
recorded from a slice of each rat.
Patch-clamp recordings from acutely dissociated or cultured DRG
neurons. The coverslips with dissociated DRG neurons were mounted
in a 0.5 ml recording chamber and placed on the stage of an Olympus
IX70 microscope. Cells were perfused continuously with bath solution
(22°C) flowing at 1 ml/min. The bath solution contained (in
mM) 150 NaCl, 5 KCl, 2 MgCl2, 2 CaCl2, 10 glucose, and 10 HEPES, pH-adjusted to 7.4 with
NaOH; osmolarity was adjusted to 320 mOsm with sucrose. Cells were
voltage clamped at 70 mV in the whole-cell configuration. Signals
were amplified and filtered at 2 kHz (Axopatch 200B) and sampled at 5 kHz. The recording electrode internal solution contained (in
mM) 120 KCl, 5 Na2-ATP, 0.4 Na2-GTP, 5 EGTA, 2.25 CaCl2, 5 MgCl2, and 20 HEPES, pH-adjusted to 7.4 by KOH, with an
osmolarity of 315-325 mOsm. Recording electrode resistance was between
2.0 and 5.0 M. m-ATP (10 µM) or capsaicin (1 µM) was applied rapidly to neurons through a glass tube
(inner diameter, ~500 µm) positioned 1.0 mm away from the cell, and
each was applied for 2 sec. The gravity-driven solution flow was
controlled electronically by solenoid valves and triggered from a computer.
The potential nonspecific effects of
pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on action
potentials and Na+ and Ca2+
channel activity were examined in cultured DRG neurons. Conditions, including the bath solution and electrode internal solutions for patch-clamp recordings from cultured DRG neurons, were similar to the
recordings from acutely dissociated DRG neurons. Under current clamp
the action potentials were evoked by current steps of 600 pA for 4 msec. The tests were performed both in normal bath solution and after a
10 min continuous perfusion of 50 µM PPADS in the bath
solution. To examine the potential nonspecific effects of PPADS on
Na+ and Ca2+ channel activity, we
held the cells at 80 mV under perforated voltage-clamp configuration,
and the electrode internal solution contained Cs+
(Gu et al., 1996). Inward currents with Na+ and
Ca2+ channel components (Gu and MacDermott, 1997)
were evoked by voltage steps to +10 mV for 200 msec. The tests were
performed both in normal bath solution and after a 10 min continuous
perfusion of 50 µM PPADS in the bath solution.
ATP, m-ATP, ARL67156, PPADS, suramin, capsaicin, caffeine,
bicuculline, strychnine, and LaCl3 were purchased from
Sigma. CNQX and TTX were purchased from Tocris (St. Louis, MO). Unless otherwise indicated, data represent 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 |
P2X receptor-mediated enhancement of spontaneous
glutamate release
We performed patch-clamp recordings from DH neurons in lamina V of
spinal cord slices. Under IR-DIC microscopy lamina regions in the
dorsal horn were identified first with a 10× objective (Fig.
1A), and then
individual neurons in lamina V were visualized under a 40× objective
(Fig. 1B). To test whether the activation of P2X
receptors may release glutamate from afferent central terminals and/or
DH interneuron terminals onto lamina V DH neurons, we bath-applied m-ATP (100 µM), a metabolic stable ATP
analog and selective P2X receptor agonist, to determine its effects on
the frequency of sEPSCs. Application of m-ATP (100 µM) produced a large increase in sEPSC
frequency (Fig. 1C,D) in most lamina V neurons (Fig. 1E). Of 23 cells that were recorded, 21 cells showed
substantial increases in sEPSC frequency, and only two cells showed no
increase after m-ATP (Fig. 1E). The overall
changes of sEPSC frequency were 355 ± 58% of control
(p < 0.05, paired t test;
n = 23). The effects lasted for ~200 sec (202 ± 12 sec; n = 21) after a 60 sec m-ATP application
(Fig. 1D). Figure 1E shows the
changes of sEPSC frequency in all of the 23 lamina V neurons that were tested after m-ATP application. When m-ATP was applied
repeatedly at 10 min intervals, it produced similar increases in sEPSC
frequency (n = 10) (Fig. 1F).
However, when 100 µM ATP was applied for 60 sec, sEPSC frequency showed either a biphasic change with initially a
transient increase (<20 sec) followed by a depression phase (n = 3) (Fig.
2A) or had only a
depression phase (n = 5). This was most likely
attributable to the rapid metabolism of ATP and the effects of its
metabolite adenosine. To test this possibility, we next performed
experiments in the presence of the ecto-ATPase inhibitor ARL67156 (50 µM) and the adenosine receptor antagonist caffeine (2 mM) (Salter et al., 1993; Westfall et
al., 1996). Under this condition a 60 sec application of ATP (100 µM) produced a nearly threefold increase of
sEPSC frequency (270 ± 30%; p < 0.05;
n = 10) (Fig. 2B) that lasted for
~120 sec (123 ± 6 sec), a result similar to the effects of
m-ATP on sEPSC frequency. In addition to the increases of sEPSC
frequency, ATP also produced small inward currents (11 ± 7 pA) in
2 of 10 recorded neurons (Fig. 2C). Because the use of ATP
may have potentially more complications than the use of m-ATP
(see Discussion), we used m-ATP as the P2X receptor agonist in
the remaining study.
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Figure 1.
Effects of m-ATP on sEPSC frequency recorded
from lamina V dorsal horn neurons. A, B, Spinal cord
slice preparation viewed under an IR-DIC microscope. Lamina regions
were identified under 10× objective (A). A part
of a patch electrode is seen in A also. The electrode
tip is inside the tissue ~70 µm from the surface, and its lamina
location is indicated by a box. A neuron in the
boxed region can be seen under 40×
objective (B, center of the field). The
patch electrode is to the left. C, Sample
traces of sEPSCs recorded from a lamina V neuron in normal bath
solution (Control, top two traces) and
after bath application of 100 µM m-ATP
(bottom two traces). D, Time course of
the increases in sEPSC frequency after bath application of m-ATP.
The time of m-ATP application is indicated by a horizontal
bar. Time bin is 10 sec. E, Results from 23 lamina V neurons. The change in sEPSC frequency after m-ATP is
expressed as the percentage of the control EPSC frequency in
logarithmic scale. Each filled circle represents the
response recorded from a cell. F, Effects on sEPSC
frequency by two repeated applications of 100 µM
m-ATP. Similar results were obtained in the other nine
cells.
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Figure 2.
Inhibition of ATP metabolism by ARL67156 and the
effects of ATP on sEPSC frequency. A, Bath application
of 100 µM ATP produced a biphasic change in sEPSC
frequency, with a transient increase followed by a depression phase in
a lamina V neuron. In the same cell 100 µM m-ATP
produced prolonged increases in sEPSC frequency. Of eight cells that
were tested as shown in A, three showed biphasic
responses and five cells had only the depression phase.
B, ATP (100 µM) produced a prolonged
increase of sEPSC frequency in the presence of the ecto-ATPase
inhibitor ARL67156 (50 µM). Traces on the
right are sample traces of sEPSCs in normal bath
solution (Control) and after the application of
100 µM ATP in the presence of 50 µM
ARL67156. Similar results were obtained in the other nine cells.
C, ATP (100 µM) also induced inward
whole-cell currents in some DH neurons in the presence of 50 µM ARL67156. Of 10 cells that were recorded, inward
whole-cell currents were undetectable (top trace) in
eight cells but were detected (bottom trace) in two
cells. In the experiments shown in B and
C, the bath solution also contained 2 mM
caffeine.
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We tested whether m-ATP-sensitive terminals were connected
monosynaptically with most lamina V neurons. This was done by determining the effects of m-ATP on sEPSCs in the presence of 500 nM TTX (mEPSCs will be used for simplicity in the following parts). The presence of TTX blocks active interneuronal transmission between DH neurons. Under this condition the bath application of 100 µM m-ATP for 60 sec still significantly increased
mEPSC frequency (Fig. 3A,B) in
most neurons that were recorded (Fig. 3C). Of 23 cells that
were recorded, 22 of them showed large increases in mEPSC frequency,
and one cell had little change (Fig. 3C). The overall
changes of mEPSC frequency were 382 ± 49% of control (p < 0.05, paired t test;
n = 23). The effects lasted for ~170 sec (174 ± 13 sec; n = 22). Of those cells showing the increases in mEPSC frequency by m-ATP, 19 of them also were tested with capsaicin (2 µM). mEPSC frequency was not
affected by a 60 sec capsaicin application (n = 19)
(Fig. 3B,D).
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Figure 3.
Effects of m-ATP on mEPSC frequency recorded
from lamina V dorsal horn neurons. A, The top
trace shows mEPSCs recorded from a lamina V neuron before and
after the application of 100 µM m-ATP. The
bottom traces show, at an expanded time scale, the
mEPSCs before (left three traces) and after
(right three traces) m-ATP application.
B, Histogram shows the time course and degree of the
increases in mEPSC frequency after 100 µM m-ATP. It
also shows, in the same recording, that 2 µM capsaicin
did not have an effect on mEPSCs. C, Results from 23 lamina V neurons show that most lamina V neurons responded to
m-ATP with an increase in mEPSC frequency. D, Of
the 23 cells in C, 19 neurons were tested with 2 µM capsaicin, and little change in mEPSC frequency was
observed. In all experiments, m-ATP and capsaicin were applied
for 60 sec. The time bin is 10 sec in the histogram.
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To determine whether Ca2+ entry through P2X
receptors at presynaptic terminals may contribute directly to the
increases in spontaneous glutamate release, we preapplied 30 µM La3+ to block the potential
Ca2+ entry through voltage-gated
Ca2+ channels (Gu and MacDermott, 1997). Under this
condition and in the presence of 500 nM TTX, 100 µM m-ATP still produced a large increase in the
frequency of mEPSCs (273 ± 20% of control; p < 0.05; n = 4) (Fig.
4Aa). On the other
hand, when experiments were performed in a 0 Ca2+
bath solution (Ca2+ was omitted from normal bath
solution), m-ATP did not produce a significant increase of mEPSC
frequency (117 ± 16% of control; n = 5) (Fig.
4Ab). Whereas m-ATP increased mEPSC frequency
in almost all lamina V neurons, mEPSC amplitude remained within 99 ± 2% of control (n = 26) (Fig. 4B).
We tested whether the increase of mEPSCs by m-ATP could be
blocked by the P2X receptor antagonist PPADS (10 µM). In five cells 10 µM m-ATP-induced significant increases of
mEPSCs (261 ± 25% of control; p < 0.05) in
normal bath solution; in these five cells, when 10 µM PPADS was present, the effects of
m-ATP on mEPSC frequency were abolished completely (99 ± 4% of control) (Fig. 4C). CNQX (10 µM), a non-NMDA receptor antagonist, was tested
on those cells for which m-ATP induced increases in the frequency
of mEPSCs or sEPSCs. CNQX completely blocked mEPSCs (n = 5) (Fig. 4D) as well as sEPSCs (data not shown; n = 5). These results suggest that P2X receptors are
present at glutamatergic terminals monosynaptically contacting lamina V
neurons.
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Figure 4.
Characterization of m-ATP-induced increases
of mEPSC frequency. Aa, All experiments were performed
in the continuous presence of 30 µM
La3+ to block voltage-gated Ca2+
channels. Two sample traces on the left represent mEPSCs
from a lamina V neuron before (Control, top
trace) and after the bath application of 100 µM
m-ATP (bottom). Histogram on the
right shows the time course and degree of the increases
in mEPSC frequency in the presence of 30 µM
La3+. Similar results were obtained in three other
cells. Ab, Sample traces (left) and
frequency histogram (right) show little change of mEPSC
frequency after the application of 100 µM m-ATP in
the 0 Ca2+ both solution. Similar results were
obtained in the other four cells. B, Histograms of mEPSC
frequency (left) and amplitude (right)
indicate that m-ATP (100 µM) increased the
frequency, but not the amplitude, of mEPSCs. C, The
increase of mEPSC frequency by m-ATP was blocked by 10 µM PPADS (n = 5). D,
mEPSCs were inhibited completely by 10 µM CNQX
(n = 5).
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Whole-cell currents directly evoked by m-ATP in
sensory neurons
To explore whether m-ATP-sensitive terminals were derived
mainly from the central terminals of primary afferent fibers that remained in the spinal sections or mainly from terminals of DH interneurons, we determined the expression of m-ATP-sensitive P2X
receptors on DH neurons and DRG neurons. This was done by examining
m-ATP-evoked whole-cell currents from DH or DRG neurons. In 101 spinal cord DH neurons, 55 in lamina V and 46 in lamina II, 100 µM m-ATP did not induce any detectable whole-cell
current directly (Fig. 5A). We
next directly examined m-ATP-evoked whole-cell currents in
acutely dissociated DRG neurons. Of 20 cells that were recorded, six of
them were large DRG neurons (diameter, >50 µm) and had no response
to 10 µM m-ATP (data not shown). Thirteen cells were small-to-medium DRG neurons and had responses to the application of 10 µM m-ATP. Of the 13 m-ATP-sensitive DRG neurons, six (cell size 35-45 µm) showed
m-ATP-evoked P2X currents that had nondesensitizing current
components (Fig. 5B, left). The amplitude of
nondesensitizing components was 560 ± 120 pA (n = 6). Interestingly, all of those six cells did not have a measurable
response to 1 µM capsaicin (Fig. 5B,
right). For the other seven m-ATP-sensitive DRG
neurons (cell size 20-30 µm), the m-ATP-evoked currents
(830 ± 230 pA; n = 7; peak currents) showed rapid
and complete desensitization during a 2 sec m-ATP application
(Fig. 5C, left). In this type of
m-ATP-sensitive neurons, 1 µM capsaicin
also evoked inward currents (Fig. 5C, right).
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Figure 5.
Examination of m-ATP-evoked, P2X
receptor-mediated, whole-cell currents on DH and DRG neurons.
A, The recording is from a DH neuron in lamina V in a
slice preparation. The m-ATP application is indicated with a
horizontal bar above the recording trace. Holding
current is 20 pA, with the cell being held at 70 mV. Bath solution
contained (in µM) 20 CNQX, 50 APV, 20 bicuculline, and 2 strychnine. m-ATP (100 µM) did not evoke any
detectable inward current directly. Similar results were obtained from
the other 100 DH neurons in slices. Among those recordings, 55 cells
were from lamina V, and the remaining cells were from lamina II.
B, An inward current with a large nondesensitizing
component was evoked by 10 µM m-ATP in an acutely
dissociated DRG neuron (left trace). The initial
desensitizing phase is truncated. The steady-state current component
was sustained during the 2 sec m-ATP application. The
right trace shows that in the same neuron 1 µM capsaicin did not induce any detectable current.
Similar results were obtained in five other DRG neurons.
C, A rapidly desensitizing current (left)
was evoked by 10 µM m-ATP in a DRG neuron. In the
same neuron, 1 µM capsaicin evoked an inward current
(right). The desensitization to m-ATP was complete
and reached baseline in ~500 msec during the 2 sec m-ATP
application. Similar results were obtained in six other DRG
neurons.
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m-ATP-sensitive synapses and A-afferent fiber
central terminals
We recorded eEPSCs to determine the properties of primary afferent
terminals that synapsed with lamina V neurons, using spinal cord slices
with attached dorsal roots (Fig.
6A). In the same neurons we also examined the effects of m-ATP on the sEPSC
frequency. In 12 lamina V cells that showed increases in sEPSC
frequency by 100 µM m-ATP (306 ± 39% of control) (Fig. 6B), stimulation of the dorsal
root always evoked EPSCs with monosynaptic A-afferent properties (Fig.
6C). Conduction velocities of afferent fibers for those
recordings were 2.3 ± 0.2 m/sec (n = 12), within
the range of A -afferent fiber conduction velocities for the same age
group of rats (Nakatsuka et al., 2000). Latency of eEPSCs after
repeated stimulation at different stimulation frequencies was constant
in each recording (Fig. 6C). One cell that showed monosynaptic A-afferent eEPSCs had no response to 100 µM m-ATP. Of those 12 cells as shown in
Figure 6, eight of them were tested with the bath application of
capsaicin (2 µM). Little change in mEPSC
frequency was observed in those cells (100 ± 2% of control). The
association of m-ATP-sensitive terminals and A-afferent terminals was also evident in the experiments below (see Fig. 8).
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Figure 6.
m-ATP-sensitive terminals and monosynaptic
A-afferent EPSCs recorded in lamina V neurons. A, The
image shows a spinal cord slice with an attached dorsal root. A suction
electrode for root stimulation is shown on the right. A
part of the root was sucked into the stimulation electrode.
B, Sample traces show the increase of sEPSC frequency by
100 µM m-ATP in a lamina V neuron.
C, In the same neuron as in B, dorsal
root stimulation at 0.2 Hz (left) and 20 Hz
(right) elicited monosynaptic A-afferent eEPSCs.
Repeated stimulation (20 times) at either frequency did not change the
latency of eEPSCs. The same results were obtained in 11 other neurons
in lamina V. Conduction velocity is 2.3 ± 0.2 m/sec
(n = 12), within A-afferent fiber range.
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P2X receptor-mediated enhancement of glutamate release in
responding to the stimulation of A-primary afferent fibers
ATP could be released in DH regions (Salter et al., 1993; Bardoni
et al., 1997; Li et al., 1998). If released, will endogenous ATP
increase glutamate release from m-ATP-sensitive terminals onto
lamina V neurons? Repetitive stimulation (20 times at 20 Hz, 1 sec
duration) was applied to a dorsal root at A-fiber stimulation intensity (~120 µA, 0.1 msec). At the end of this repetitive
stimulation there was a brief increase in sEPSC frequency that lasted
for ~5 sec. The sEPSC frequency after the stimulation (post-stim) was
260 ± 23% (n = 11) (Fig.
7A,B) of sEPSC frequency
before the stimulation (pre-stim). When the same repetitive stimulation
was conducted in the presence of 10 µM PPADS,
post-stim sEPSC frequency was 171 ± 23% (n = 7)
of pre-stim sEPSC frequency, significantly lower than the post-stim
sEPSC frequency in normal bath solution (Fig. 7A,B). Similar
experiments also were performed in the presence of 5 µM suramin. The post-stim sEPSC frequency was
187 ± 33% (n = 4; not illustrated as a figure)
of pre-stim sEPSC frequency in the presence of suramin, significantly
lower than the post-stim sEPSC frequency in normal bath solution. The
effects of PPADS or suramin at the concentrations that were used were
unlikely to be nonspecific actions on glutamatergic transmission
because pre-stim sEPSC frequency was not affected in the presence of 10 µM PPADS (n = 4) (Fig.
7A,B) or 5 µM suramin (data not
shown). There was also no change in the basal frequency and amplitude of sEPSCs by 10 µM PPADS (frequency, 101 ± 3% of control; amplitude, 99 ± 1% of control;
n = 8) (Fig. 7C). We performed an analysis of the amplitude of individual electrically evoked EPSCs during the
train of dorsal root stimulation under control conditions and in the
presence of PPADS. Similar to the finding by Li et al. (1998), we found
that there were changes in the paired pulse ratio in the presence of 10 µM PPADS. The paired pulse ratio was 78 ± 16% under control conditions and 192 ± 38% in the presence of
PPADS (n = 4; not illustrated as a figure). These
results suggested that the effects of PPADS were on the presynaptic
sites (Li et al., 1998).
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Figure 7.
Effects of P2X antagonists on sEPSCs
after repetitive stimulation to afferent fibers. Aa,
Top two traces show sEPSCs recorded from a lamina V
neuron before and after repetitive stimulation (20 times at 20 Hz, 1 sec duration) in normal bath solution. Bottom two traces
show, in the same cell in the presence of 10 µM PPADS,
the sEPSCs before and after the same repetitive stimulation.
Stimulation intensity was similar to that in Figure 6C.
Ab, An example shows responses of three trials in the
same cell. The Post-Stim effects were tested three times
in normal bath solution and three times in the presence of PPADS. The
interval of each test was 2 min. The number of sEPSCs was counted for 5 sec before and immediately after the finish of the repetitive
stimulation. Similar results were obtained in two other cells.
B, A summary of the changes of sEPSC frequency before
and after the repetitive stimulation in normal bath solution
(open bars) and in 10 µM PPADS
(filled bars; n = 7 cells).
PPADS significantly reduced the stimulation-induced enhancement of
sEPSC frequency (p < 0.05).
C, The frequency and amplitude of basal sEPSCs were not
changed after a 10 min perfusion of 10 µM PPADS
(n = 8). D, Two traces show action
potentials elicited by current steps in a DRG neuron in normal bath
solution (left) and after the perfusion of 50 µM PPADS for 10 min. Similar results were obtained in
five other cells. E, Two traces show currents elicited
by voltage steps from 80 to +10 mV in a DRG neuron in normal bath
solution (left) and after a 10 min perfusion of 50 µM PPADS (right). The initial current
component with rapid decay phase is attributable mainly to
Na+ channels, and the subsequent steady-state
current component represents mainly voltage-gated
Ca2+ channel activity (Gu and MacDermott, 1997). No
change was observed after 50 µM PPADS; similar results
were obtained in five other cells. Recordings in E were
performed with perforated patch-clamp technique;
Cs+-internal electrode solution was used (Gu et al.,
1996).
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Because some sEPSCs might be generated by action potentials and
involved in Ca2+ entry through voltage-gated
Ca2+ channels, we determined whether PPADS had
effects on the size and shape of action potentials as well as
Na+ and Ca2+ channel activity in
cultured DRG neurons (Fig. 7D,E). The size and shape of
action potentials elicited by a current step showed identically in
normal bath solution and after a 10 min perfusion of cells with bath
solution containing 50 µM PPADS
(n = 6) (Fig. 7D). Furthermore, the inward
currents of both the Na+ channel component and
Ca2+ component (Gu and MacDermott, 1997) were also
identical in normal bath solution and in the bath solution containing
50 µM PPADS (n = 6) (Fig.
7E).
Activation of other presynaptic ligand-gated cation channels has been
shown to result in either potentiation or depression of the evoked
EPSCs at glutamatergic synapses in the brain (MacDermott et al., 1999).
Will activation of m-ATP-sensitive P2X receptors result in
synaptic potentiation of A-afferent glutamatergic transmission to
lamina V neurons? Stimuli were applied at a reduced intensity (see
Materials and Methods) to a dorsal root, and monosynaptic A-eEPSCs
and synaptic failures occurred randomly (Fig.
8Aa,
bottom). Glutamate release likely was occurring at a few
afferent central terminals during this stimulation protocol, as was
evident by a much smaller amplitude of eEPSC (compare with eEPSC size
in Fig. 6C). After the application of 1 µM m-ATP for 5 min, the synaptic failure
rates that followed the same stimulation protocol were decreased
significantly (Fig. 8Ab, bottom), and the
mean amplitude of eEPSCs was increased significantly (Fig.
8Ab, top). When the ecto-ATPase inhibitor
ARL67156 (10 µM for 5 min) was used to prevent
the breakdown of endogenous ATP, we also observed effects similar to
m-ATP (Fig. 8B). Figure 8C
summarizes the effects of m-ATP (n = 4) and
ARL67156 (n = 4) on synaptic failure rates and mean
amplitudes and shows significant decreases of failure rates (Fig.
8Ca) and increases in mean eEPSC amplitude (Fig.
8Cb). On the other hand, in the presence of 10 µM PPADS or 5 µM
suramin the failure rates increased significantly (Fig.
8D).
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Figure 8.
P2X receptor-mediated potentiation of evoked
EPSCs. Aa, The bottom panel shows
consecutive eEPSCs and synaptic failures recorded on a lamina V neuron
in control. The mean amplitude is shown on the top.
Basal noise level was within ± 2.5 pA around the current level of
baseline. Ab, Synaptic failures (bottom)
were decreased and mean amplitude (top) was increased
after the bath application of 1 µM m-ATP.
B, The experiment was the same as that in
A, except that the effects of 10 µM
ARL67156 were tested. The conduction velocity was 4 m/sec in
A and 2 m/sec in B. C, A
summary of the decrease of failure rates (Ca) and the
increase of mean eEPSC amplitude (Cb) by 1 µM m-ATP (n = 4) and 10 µM ARL67156 (n = 4).
D, A summary of the increase of failure rates after the
application of 10 µM PPADS (n = 4)
and 5 µM suramin (n = 4). In all
experiments eEPSCs were induced by dorsal root stimulation (see
Materials and Methods). Data represent the mean ± SEM;
*p < 0.05, paired Wilcoxon test.
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DISCUSSION |
In the present study we have provided the electrophysiological
evidence for the presence of presynaptic P2X receptors at the central
terminals of primary afferent fibers connecting to lamina V neurons of
the spinal cord dorsal horn. Our results indicate that the activation
of these receptors can result in a robust increase of spontaneous
glutamate release, as evidenced by a large increase in the frequency of
sEPSCs and mEPSCs. Furthermore, we have provided evidence suggesting
that P2X receptor activation can modulate the evoked glutamate release
after the stimulation of primary afferent fibers. Our results also
suggest that endogenously released ATP, associated with primary sensory
fiber stimulation, may enhance spontaneous and evoked glutamate release.
P2X receptors on primary afferent central terminals versus P2X
receptors on DH neurons
We have shown that m-ATP-sensitive terminals extensively
synapse to lamina V neurons. Many of those terminals might be derived from primary afferent fibers. This is consistent with the presence of
m-ATP-sensitive P2X receptors on DRG neurons. It is supported further by our findings that the eEPSCs after stimulation of primary afferent fibers were potentiated by m-ATP and ARL67156 and
depressed by PPADS and suramin. The effects of those purine compounds
were at the central terminals of primary afferent fibers, because the potentiation of eEPSCs was associated with the increase of presynaptic release probability (Fig. 8).
There is a possibility that some m-ATP-sensitive terminals may be
derived from DH interneurons. Previously, it was reported that
presynaptic P2X receptors were expressed on inhibitory DH neurons;
their activation resulted in the release of GABA or glycine (Hugel and
Schlichter, 2000; Rhee et al., 2000). Interestingly, presynaptic P2X
receptors on inhibitory DH neurons in those studies were found to be
m-ATP-insensitive. It will be very interesting to know whether
P2X receptors are expressed at presynaptic terminals of excitatory
DH neurons. In attempting to determine whether m-ATP-induced increases in mEPSC frequency might be attributable partially to its
direct action on DH neurons, we examined the possible expression of
functional m-ATP-sensitive P2X receptors in DH neurons. However, after we tested >100 DH neurons, 100 µM m-ATP did
not evoke any detectable whole-cell current on those neurons. This
result may suggest that m-ATP-sensitive DH neurons, if present,
will be a very small population. This suggestion is also supported by a
previous study with acutely dissociated DH neurons (Bardoni et al.,
1997). However, we still should not rule out completely the possible
presence of m-ATP-sensitive P2X receptors at presynaptic terminals of glutamatergic DH neurons.
In this study we also have performed some experiments by using ATP. It
appears that, before exogenously applied ATP reached the recorded
neurons (usually inside slices ~70 µm from the surface of the
tissue), there had been substantial ATP metabolism that primarily
decreased actual ATP concentrations around the recorded cells. However,
the ecto-ATPase inhibitor ARL67156 effectively might prevent ATP
metabolism in the spinal cord slice. ATP may have complicated effects
in the slice preparations, not only because of its metabolism but also
because of its direct effects on some DH neurons (see also Salter et
al., 1993; Li and Perl, 1995; Bardoni et al., 1997; Li et al.,
1998).
m-ATP-sensitive terminals and P2X receptor subtypes
Although primary afferent neurons express six P2X subunits (Collo
et al., 1996; Xiang et al., 1998), the spinal cord distribution of P2X
receptor-expressing afferent terminals has not been well characterized.
Immunoreactivity to P2X1, P2X2, and
P2X3 subunits is present at primary afferent terminals in
superficial laminas (Vulchanova et al., 1996, 1997). The
m-ATP-sensitive synapses shown in this study could be located in
deep laminas, but they also could be located in the superficial laminas
because deep lamina neurons extend their dendrites to superficial
laminas (Ritz and Greenspan, 1985). P2X3 subunits are
expressed extensively at primary afferent terminals innervating inner
lamina II, raising a possibility that our m-ATP-sensitive
terminals are derived from P2X3 receptor-expressing
terminals. However, this possibility may be inconsistent with the
finding that our m-ATP-sensitive afferent terminals are
capsaicin-insensitive and are not from C-afferent fibers. Nevertheless,
~20% of primary afferent neurons did not coexpress P2X3
subunits and capsaicin VR1 receptors (Guo et al., 1999). Furthermore,
P2X3 subunits might not be expressed exclusively on
unmyelinated, IB4-positive sensory neurons as previously thought
(Petruska et al., 2000). Thus, we cannot rule out the possibility that
P2X3-expressing afferent terminals synapse to lamina V
dendrites. If P2X3-expressing afferent terminals were involved in our study, the subtypes of P2X receptors that mediated the
augmented glutamate release would most likely be heteromeric P2X2+3 receptors. Heteromeric P2X2+3
receptors respond to m-ATP with weak desensitization and probably
are expressed on some DRG neurons (Lewis et al., 1995; Burgard et al.,
1999; North and Surprenant, 2000). We showed that m-ATP produced
a sustained increase in spontaneous glutamate release with little or
weak desensitization, which appears to be consistent with some properties of heteromeric P2X2+3 receptors. However, it has been shown recently that heteromeric P2X1+5 receptors and heteromeric P2X4+6 receptors in heterologous expression
systems also respond to m-ATP with a weakly desensitizing current
component (Le et al., 1998, 1999; Torres et al., 1998; Haines et al.,
1999). A more recent study has provided first evidence suggesting that functional heteromeric P2X1+5 receptors are expressed in native tissues (Surprenant et al., 2000). Thus, these two heteromeric P2X receptors, if expressed on primary afferent fibers, may account for
the effects of m-ATP shown in this study.
Future studies with subtype-selective P2X receptor antagonists such as
TNP-ATP (North and Surprenant, 2000; Surprenant et al., 2000; Khakh et
al., 2001) may help to reveal the subtype(s) of P2X receptors mediating
the augment of glutamate release onto lamina V neurons.
Primary afferent type(s) of m-ATP-sensitive terminals
We have provided evidence suggesting that some
m-ATP-sensitive terminals may be derived from A-afferent
fibers. The results that A-eEPSCs can be potentiated after P2X
receptor activation directly support this point. Few C-afferent fibers
have monosynaptic connections with lamina V neurons (Willis and
Coggeshall, 1991), suggesting that the m-ATP-sensitive terminals
in our study were unlikely to be derived from C-afferent fibers.
However, there is a possibility that some A/A fibers also may
express m-ATP-sensitive P2X receptors, because lamina V neurons
receive input from large primary afferents as well (Brown, 1982). In
our study the stimulation of dorsal root with intensity sufficient to
activate A/A fibers did not evoke A/A monosynaptic
responses in lamina V neurons. Similar results have been reported
(Yoshimura et al., 1992). This is thought to be attributable to the
impairment of large myelinated fibers in the transverse spinal slice
preparations because of their descending and ascending path before
terminating in lamina V (Brown, 1982). Nevertheless, the involvement of
A/A-afferent terminals in m-ATP-induced glutamate release
is discounted by our finding that large-diameter (>50 µm) DRG
neurons had little response to m-ATP or ATP (see also Li et al.,
1999). A-afferent terminals to lamina V are known to carry
nociceptive and non-nociceptive sensory information (Willis and
Coggeshall, 1991). We have shown that most A-afferent terminals to
the lamina V region were m-ATP-sensitive/capsaicin-insensitive. This raises a good possibility that at least some nociceptive A-afferent fibers may express m-ATP-sensitive P2X receptors at
their central terminals. One possible origin of the
m-ATP-sensitive central terminals could be from
A-mechanoreceptors of keen joints because the
A-mechanoreceptors were found to be
m-ATP-sensitive/capsaicin-insensitive (Dowd et al., 1998).
The potential origin of endogenous ATP
Our results suggest that endogenous ATP may be released in the
dorsal horn regions during dorsal root stimulation. The simplest interpretation of our results is that ATP was released from primary afferent terminals. The endogenously released ATP then acted on presynaptic P2X receptors on the primary afferent terminals, which in
turn enhanced glutamate release probability (Figs. 7, 8). The possible
release of ATP from primary afferent central terminals was proposed
first by Holton and Holton (1954). Using spinal cord synaptosomal
preparations, White et al. (1985) demonstrated the release of ATP by
high K+ and suggested that ATP may be released from
both primary afferent terminals and dorsal horn interneurons.
Consistently, with the use of electrophysiological approaches, ATP was
suggested to be released in the spinal cord dorsal horn after focal
electric stimulation (Bardoni et al., 1997; Li et al., 1998). It was
suggested that the endogenously released ATP might function as a fast
synaptic transmitter (Bardoni et al., 1997) or modulator (Li et al.,
1998). Jo and Schlichter (1999) showed that ATP could be released from many dorsal horn interneurons. It also has been shown that glutamate can evoke the release of ATP from astrocytes (Queiroz et al., 1999).
Thus, in addition to the potential release of endogenous ATP from
primary afferent terminals, there is also a possibility that ATP is
released from postsynaptic dorsal horn neurons and/or surrounding
astrocytes as a consequence of glutamate receptor action on these
cells. If the latter hypothesis is true, then ATP is a retrograde
signaling molecule in the sensory synapses.
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FOOTNOTES |
Received March 29, 2001; revised June 20, 2001; accepted June 21, 2001.
This work was supported by National Institutes of Health Grant NS38254
and Office of Naval Research Grant N00014-01-1-0188 (J.G.G.). We thank
A. MacDermott, S. Siegelbaum, D. Price, and B. Cooper for providing
thoughtful comments on this manuscript. We appreciate J. Ling for
general assistance during this work.
Correspondence should be addressed to Jianguo G. Gu, McKnight Brain
Institute of the University of Florida and Division of Neuroscience,
Department of Oral Surgery, College of Dentistry, University of
Florida, 1600 SW Archer Road, Gainesville, FL 32610. E-mail:
jgu{at}dental.ufl.edu.
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TOP
ABSTRACT
INTRODUCTION
