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The Journal of Neuroscience, March 15, 2000, 20(6):2121-2130
Presynaptic P2X Receptors Facilitate Inhibitory GABAergic
Transmission between Cultured Rat Spinal Cord Dorsal Horn
Neurons
Sylvain
Hugel and
Rémy
Schlichter
Laboratoire de Neurophysiologie Cellulaire et Intégrée,
Unité Mixte de Recherche 7519-Centre National de la Recherche
Scientifique, Université Louis Pasteur, 67084 Strasbourg Cedex,
France
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ABSTRACT |
The superficial layers of the spinal cord dorsal horn (DH) express
P2X2, P2X4, and P2X6 subunits entering into the formation of ionotropic
(P2X) receptors for ATP. Using a culture system of laminae
I-III from neonatal rat DH, we show that ATP induced a fast
nonselective cation current in 38% of the neurons (postsynaptic effect). ATP also increased the frequency of miniature IPSCs
(mIPSCs) mediated by GABAA receptors or by glycine
receptors in 22 and 9%, respectively, of the neurons tested
(presynaptic effect) but had no effect on glutamatergic transmission.
The presynaptic effect of ATP on GABAergic transmission was not
significantly affected by thapsigargin (1 µM) but was
completely dependent on Ca2+ influx. Presynaptic and
postsynaptic effects were inhibited by suramin,
pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, and reactive blue
and were not reproduced by uridine 5'-triphosphate (UTP) or adenosine
5'-O-(2-thiodiphosphate) (ADP- -S), suggesting the implication of
ionotropic P2X rather than of metabotropic P2Y receptors.
 -methylene-ATP (100 µM) did not reproduce the effects of ATP. ATP reversibly increased the amplitude of electrically evoked GABAergic IPSCs and reduced paired-pulse inhibition or facilitation without affecting IPSC kinetics. This effect was preferentially, but not exclusively, observed in neurons coreleasing ATP and GABA. We conclude that in cultured DH neurons, the effects of
ATP are mediated by P2X receptors having a pharmacological profile
dominated by the P2X2 subunit. The presynaptic receptors might underlie
a modulatory action of ATP on a subset of GABAergic interneurons
involved in the spinal processing of nociceptive information.
Key words:
dorsal horn; spinal cord; nociception; purine; GABAA receptor; inhibitory postsynaptic current; IPSC; synaptic transmission; presynaptic modulation
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INTRODUCTION |
ATP plays an important role in cell
metabolism but acts also as a neurotransmitter (Surprenant et al.,
1995 ; Burnstock, 1997; MacDermott et al., 1999). The fast ATP-mediated
excitatory synaptic component involves the activation of ionotropic P2X
receptors, but ATP can also activate G-protein-coupled metabotropic
(P2Y) receptors to alter the excitability of postsynaptic neurons
(Burnstock, 1997; North and Barnard, 1997; Ralevic and Burnstock,
1998). Moreover, ATP can be coreleased with a classical
neurotransmitter at peripheral as well as central synapses (Burnstock,
1997; Jo and Schlichter, 1999).
To date seven distinct P2X subunits (P2X1-P2X7) have been cloned, six
of which (P2X1-P2X6) are expressed in the CNS (Collo et al.,
1996; Vulchanova et al., 1997; Le et al., 1998b; Kanjhan et al., 1999).
In situ hybridization studies have indicated that P2X4 and
P2X6 subunits, and to a lesser extent P2X2, are widely distributed
throughout the CNS (Collo et al., 1996) and could therefore underlie
the fast excitatory membrane effects of ATP. However, the existence of
fast P2X receptor-mediated synaptic transmission has been demonstrated
only in a relatively limited number of CNS structures: the medial
habenula (Edwards et al., 1992), the locus coeruleus (Nieber et al.,
1997), the spinal cord (Bardoni et al., 1997; Jo and Schlichter, 1999),
and the hippocampus (Pankratov et al., 1998). Moreover,
immunocytochemical studies have shown that P2X subunits are present on
the soma and the dendrites of postsynaptic neurons as well as on
presynaptic terminals (Vulchanova et al., 1997; Le et al., 1998b;
Kanjhan et al., 1999), raising the possibility that ATP could modulate
synaptic transmission at CNS synapses by both postsynaptic and
presynaptic actions.
The dorsal horn (DH) of the spinal cord gray matter is involved in the
processing of nociceptive (painful) messages (Millan, 1999), and ATP
was shown to play an important role in the modulation of nociceptive
messages at the spinal level (Salter et al., 1993). In fact, purines
exert complex effects in the DH because ATP excites DH neurons (Jahr
and Jessell, 1983; Li and Perl, 1995), whereas adenosine, which results
from the extracellular degradation of ATP by ectonucleotidases
(Zimmermann, 1994, 1996), has inhibitory effects (Salter et al., 1993).
Postsynaptic ionotropic receptors are known to underlie fast synaptic
transmission in the nervous system, and at the presynaptic level such
ionotropic receptors are also likely to modulate the release of
neurotransmitters (MacDermott et al., 1999). P2X receptors are involved
in ATP-mediated facilitation of glutamate release from primary sensory
afferents in the spinal cord (Li and Perl, 1995; Gu and MacDermott,
1997) and in the brain stem (Khakh and Henderson, 1998). Yet, there has
been no report concerning a presynaptic P2X receptor-mediated
modulatory effect on synaptic transmission between CNS neurons. We have
recently shown the existence of a close relationship between ATP and
GABA in cultured laminae I-III DH neurons (Jo and Schlichter, 1999), and the present study, was aimed at searching for possible presynaptic effects of ATP in particular on GABAergic synaptic transmission.
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MATERIALS AND METHODS |
Tissue culture. The technique for preparing primary
cultures of spinal dorsal horn neurons was described in detail
elsewhere (Jo et al., 1998a,b). Briefly, after decapitation of 3- to
4-d-old Wistar rats under deep diethyl-ether anesthesia, a laminectomy was performed, and the dorsal third of the spinal cord was cut with a
razor blade. The tissue fragments were digested enzymatically for 45 min at 37°C with papain (20 U/ml, Sigma, St. Louis, MO) in oxygenated
divalent-free Earle's balanced salt solution (EBSS, Life Technologies,
Gaithersburg, MD). The enzymatic digestion was stopped by adding 3 ml
EBSS containing bovine serum albumin (1 mg/ml; Sigma), trypsin
inhibitor (10 mg/ml; Sigma), and DNase (0.01%; Sigma), and a
mechanical dissociation was performed with a 1 ml plastic pipette. The
homogenate was deposited on top of 4 ml of a solution of composition
similar to that described above, except that the concentration of
bovine serum albumin was increased to 10 mg/ml. After centrifugation (5 min at 500 rpm), the supernatant was removed and replaced with 5 ml of
culture medium the composition of which was the following: MEM-
(Life Technologies), fetal calf serum (5% vol/vol; Life Technologies),
heat-inactivated horse serum (5% vol/vol; Life Technologies),
penicillin and streptomycin (50 IU/ml for each; Life Technologies),
transferrin (10 mg/ml; Sigma), insulin (5 mg/ml; Sigma), putrescine
(100 nM; Sigma), and progesterone (20 nM;
Sigma). After trituration with a fire-polished Pasteur pipette, the
cells were plated on 35 mm collagen-coated plastic culture dishes in
the central compartment, which was delimited by a small (internal
diameter 15 mm) circular glass ring. This ring was glued onto the
bottom of the dish with paraffin wax and could easily be removed before
electrophysiological experiments. Cultures were maintained in a
water-saturated atmosphere (95% air, 5% CO2) at
37°C until use (10-15 d). Two days after the cells were seeded,
cytosine arabinoside (10 µM) was added to the culture medium for 24 hr to reduce glial proliferation.
Electrophysiological recordings. Experiments were performed
at room temperature (20-22°C) after 10-15 d in culture. Patch-clamp recordings were made with the perforated patch-clamp technique using
amphotericin B as the pore-forming agent (Rae et al., 1991) with an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA) and
low-resistance (3-4 M ) electrodes. The external solution contained
(in mM): NaCl 135, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 5, and glucose 10, pH 7.35. The
external solution also contained 6-cyano-7-nitroquinoxaline-2,3-dione
(CNQX, 10 µM), D-amino-phosphonovaleric acid
(D-APV, 30 µM), and strychnine (1 µM) to block fast glutamatergic and glycinergic synaptic
transmissions and tetrodotoxin (TTX, 0.5 µM), which
blocks Na+-dependent action potential
propagation, to record GABAergic miniature IPSCs (mIPSCs) in isolation.
When testing the effect of ATP on glutamatergic or glycinergic
transmissions, CNQX and APV or strychnine were omitted from the
external medium and bicuculline (10-20 µM) was added. In
some experiments, in which we wanted to prevent an ATP-mediated
depolarization of the presynaptic terminals, all external NaCl was
replaced by an equimolar amount of
N-methyl-D-glucamine chloride
(NMDG-Cl). When the extracellular CaCl2
concentration was reduced to 0.3 mM, the
MgCl2 concentration was increased to 5 mM. The pipette was first filled at its tip with
a solution containing (in mM): CsCl 125, CaCl2 5, MgCl2 2, HEPES 10, and EGTA 10, pH 7.35, and then backfilled with the same solution
containing amphotericin B (150 µg/ml). The amphotericin B (Sigma)
stock solution (30 mg/ml) was prepared in dimethyl sulfoxide (DMSO)
just before the recording session. Voltage and current traces were
stored digitally on a videotape recorder (sampling rate 20 kHz) and/or on a personal computer after being filtered at 5 kHz by the Axopatch 200A. Acquisition and analysis were performed with Axograph 3 (Axon
Instruments) and more recently with the program SYNAPSE (developed and
kindly supplied by Dr. Y. De Koninck, McGill University, Montreal,
Canada). General principles and details on the analysis of synaptic
currents can be found elsewhere (Poisbeau et al., 1996; Jo et al.,
1998a). The analysis of miniature synaptic currents was performed on
sequences with at least 300 events each. This corresponded typically to
2 min of recording in control conditions and the complete sequence
(~30 sec) during the application of P2X receptor agonists. All mIPSCs
analyzed were selected manually on the basis of their rapid rise times
(RTs) and exponential decay kinetics.
Electrical stimulation. To study the effect of ATP on action
potential-evoked release of GABA, we stimulated electrically (extracellular stimulation) the cell body of a visually identified neuron establishing an apparent physical contact with the neuron from which we recorded. The stimulation procedure was identical to that
described previously for the same culture preparation (Jo et al.,
1998a; Jo and Schlichter, 1999). Stimulation was performed with short
pairs (interval 400 msec) of stimuli (0.1 msec in duration) delivered
at 0.1 Hz. Their amplitude was between  10 and 20 V. In these
experiments, ECl was fixed at 90 mV
by replacing 146 mM CsCl with 75 mM
Cs2SO4 in the pipette
solution, whereas the equilibrium potential for cations
(ECations) remained at 0 mV. TTX was
omitted from the extracellular medium to allow action potential
generation and propagation in the presynaptic neuron. Under such
conditions it was possible to determine whether the neuron stimulated
electrically was coreleasing ATP and GABA or if it was releasing GABA
alone (Jo and Schlichter, 1999) because P2X receptor-mediated inward
EPSCs could be recorded in isolation at a holding potential (HP) of
90 mV, whereas GABAergic IPSCs were recorded in isolation at an HP of
0 mV. We routinely first verified the presence or absence of
electrically evoked P2X receptor-mediated inward EPSCs at an HP of 90
mV before setting the HP at 0 mV and testing the effect of ATP on
GABAergic electrically evoked IPSCs (eIPSCs). For analysis, we averaged
on one hand 10-20 traces recorded under control or wash-out conditions
and on the other hand the 10 traces recorded during the application of
ATP (100 µM), which lasted 100 sec. The
paired-pulse inhibition or facilitation ratio (R) was
determined as R = (I2 I1) · 100/I1,
where R is expressed as percentage,
I1 is the amplitude of the first
eIPSC, and I2 is the amplitude of the
second eIPSC. R had a negative value in the case of
paired-pulse inhibition and a positive value in case of paired-pulse
facilitation. We first determined R under control conditions
and then during the application of ATP (100 µM). The change in paired-pulse ratio was
calculated as follows for each neuron tested: change in paired-pulse
ratio (in %) = (R1 R2) · 100/R1,
with R1 and
R2 representing the paired-pulse ratios under control conditions and in the presence of ATP (100 µM), respectively.
Drugs and application of substances. All substances
were prepared as 1000 times concentrated stock solutions.
Bicuculline (Sigma), strychnine (Sigma), TTX (Latoxan), ATP (Sigma),
adenosine 5'-O-(3-thiotriphosphate) (ATP- -S; Sigma), UTP (Sigma),
,-methylene ATP (,-me-ATP; Sigma), ADP (Sigma), adenosine
5'-O-(2-thiodiphosphate) (ADP--S; Sigma), suramin (Sigma),
pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS; Tocris
Cookson, distributed by Bioblock), reactive blue (Sigma), and
isoguvacine (Peninsula Laboratories, Belmont, CA) were prepared in
distilled water, and D-APV (30 mM; Sigma) was
prepared in NaOH (1 M). All these stock solutions were
stored at 20°C. CNQX (Tocris Cookson, distributed by Bioblock) and
thapsigargin (Sigma) were prepared in DMSO and stored at 4°C.
The substances to be tested were dissolved at their final
concentrations in extracellular solution just before the recording session. ATP antagonists, with the exception of reactive blue, were
applied by bath perfusion, and ATP agonists were applied by local
perfusion using a U-tube. In the case of the antagonists suramin and
PPADS, the same concentration of antagonist was also included in the
U-tube solution along with the agonist to be applied locally. The
antagonist reactive blue was coapplied with ATP agonists by means of
the U-tube in the absence of reactive blue in the bath solution. In
electrical stimulation experiments, all substances were applied by bath
perfusion at rate of 2-3 ml/min, which allowed relatively rapid
application of substances because the recordings were performed in a
central chamber (of the culture dish) having a total volume of 250 µl.
All statistical results are given as mean ± SEM.
Statistical differences between results were determined using
Student's t test or one-way ANOVA (Origin, Microcal
software) by setting the confidence interval at 0.05. Differences in
cumulative probability distributions were determined by a
Kolmogorov-Smirnov test (program written and kindly supplied by Dr.
Jean-Luc Rodeau, Strasbourg).
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RESULTS |
In an initial phase of this work, we tested the effect of ATP
(30-100 µM) in the absence of any antagonist of
ionotropic glutamate, glycine, or GABA receptors. Under these
conditions we noticed that ATP induced a fast membrane current
characteristic of the activation of ionotropic receptors or increased
the frequency of spontaneously occurring synaptic events or both. The
latter had relatively slow deactivation time constants (35-40 msec)
that are characteristic of GABAA
receptor-mediated IPSCs in our preparation (Jo and Schlichter, 1999)
(see below). However, to assess the specificity of the facilitatory
action of ATP on GABAergic transmission, we tested its effect on
pharmacologically isolated glutamatergic and glycinergic transmissions.
All experiments were performed in the presence of TTX (0.5 µM) to record miniature synaptic currents in isolation
and to avoid the activation of polysynaptic pathways.
Glutamatergic transmission
Glutamatergic miniature EPSCs (mEPSCs) were recorded in the
presence of strychnine (1 µM) and bicuculline (10 µM). We recorded from 20 neurons displaying glutamatergic
mEPSCs. In all cases, ATP (100 µM) failed to increase the
frequency of mEPSCs, indicating that ATP did not modulate
presynaptically glutamate release between cultured DH neurons.
Glycinergic transmission
Glycinergic mIPSCs were recorded in 23 neurons in the presence of
CNQX (10 µM), D-APV (30 µM),
and bicuculline (10 µM). ATP (100 µM)
increased the frequency of glycinergic mIPSCs in 2 of 23 cases, i.e.,
9% of the cells tested. This result suggested that ATP was likely to
facilitate presynaptically glycine release from a small subset of
glycinergic synapses. The rarity of these ATP-sensitive glycinergic
synapses, at least in our culture system, precluded a detailed
pharmacological study on the subtypes of ATP receptors involved in this
modulatory phenomenon.
It appeared, however, that the effect of ATP was more frequently
observed on GABAergic transmission (see below), and we decided to study
this point in more detail.
Dual effect of ATP on cultured dorsal horn neurons
All experiments described below were performed in the presence of
blockers of glutamatergic and glycinergic transmissions (CNQX 10 µM, D-APV 30 µM, strychnine 1 µM) and in the presence of 0.5 µM TTX
(except in electrical stimulation experiments). The HP was 60 mV if
not stated otherwise.
Postsynaptic effect of ATP
ATP (30-100 µM) induced a fast inward current
(rise-time  1 sec) in 60 of 156 neurons (38%). This response was
accompanied by an increase in membrane noise that recovered quickly
after cessation of ATP application (Fig.
1A). Moreover, the ATP
response displayed little desensitization as indicated by similar
amplitudes of membrane currents when ATP applications were repeated at
short (<5-10 sec) intervals or by the limited attenuation (typically <30%) of the current during prolonged ( 10 sec) ATP applications (Fig. 1A). The mean amplitude of the peak currents
induced by ATP (100 µM) at a holding potential
of 60 mV was 39 ± 41 pA (n = 41, range 5-175
pA).
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Figure 1.
Presynaptic and postsynaptic effects of ATP in
cultured DH neurons. In this and all other figures, recordings were
made in the presence of CNQX (10 µM), D-APV
(30 µM), and strychnine (1 µM) in the
extracellular medium to block fast glutamatergic and glycinergic
synaptic transmissions. TTX (0.5 µM) was also present to
record GABAergic mIPSCs. A, Local applications of ATP
induced fast inward currents in the postsynaptic neuron. Two successive
and close (10 sec interval) ATP (100 µM) applications
elicited currents of similar amplitudes. During a long-lasting (5 sec)
application of ATP, the current decayed only partially and slowly,
indicating the absence of significant desensitization.
B, Current-voltage (I-V)
relationship of the ATP (100 µM)-induced current obtained
by applying a 2 sec lasting monotonic voltage ramp from 100 mV to +30
mV. The reversal potential was close to 0 mV, and the
I-V relationship showed marked inward
rectification at negative membrane potentials. C1,
Application of ATP (100 µM) reversibly increased the
frequency of mIPSCs recorded as fast downward deflections of the
current trace. C2, Coexistence of presynaptic and
postsynaptic effects of ATP. In some cells, ATP (100 µM)
induced both an inward current and an increase in mIPSC frequency. All
traces were recorded at an HP of 60 mV. In A,
C1, and C2, the horizontal black
bars represent the duration of ATP application.
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As illustrated in Figure 1B, the reversal potential
of the ATP-gated current was close to 0 mV, a value that was compatible with the activation of a nonselective cation conductance (Jo and Schlichter, 1999). The I-V relationship showed
marked inward rectification at negative membrane potentials, a
phenomenon that is classically observed with ionotropic ATP-gated
receptors (P2X receptors) as well as with other calcium permeable
ligand-gated channels (Jonas and Burnashev, 1995; Burnashev, 1996).
Presynaptic effect of ATP
Local application of ATP (30-100 µM) increased the
frequency of GABAergic mIPSCs in 33 of 149 neurons (22%) recorded,
indicating the existence of a facilitatory effect of ATP on synaptic
GABA release. Two types of effects of ATP were observed (Fig.
1C). In 58% of the cases (19 of 33 neurons) ATP simply
increased the frequency of mIPSCs in a reversible manner, whereas in
the remaining 42% of the neurons (14 of 33), the presynaptic
facilitation of GABA release was associated with a tonic fast
postsynaptic inward current that had the same characteristics as that
of the postsynaptic ATP-induced current described in the preceding
section (i.e., a rise-time 1 sec). The mean increase in frequency of
synaptic currents was of 101 ± 36% (n = 8),
287 ± 185% (n = 7), and 280 ± 205%
(n = 18) for 30, 50, and 100 µM
ATP, respectively, an increase of 100%, meaning a doubling of the
initial frequency. The values for the three concentrations tested were
not significantly different (one-way ANOVA, p > 0.05).
When all results were pooled the mean increase in frequency of mIPSCs
was 254 ± 197% (n = 33, range 32-686%).
As in the case of the postsynaptic current induced by ATP, the increase
in frequency of mIPSCs induced by ATP was reproducible during multiple
successive applications (Fig.
2A). This was an important prerequisite particularly when trying to assess the effect of
an agonist or an antagonist (see below) on the ATP-induced change in
synaptic current frequency. Furthermore, we confirmed that the
miniature synaptic events that were stimulated presynaptically by ATP
were GABAergic mIPSCs because they were reversibly blocked by the
GABAA receptor antagonist bicuculline (Fig.
2B). When ATP triggered important increases in
synaptic current frequency, we often recorded a slowly developing
inward current (Fig. 2) that was attributable to the temporal summation
of GABAergic mIPSCs as demonstrated by the fact that both mIPSCs and
the slow current were reversibly blocked by bicuculline (Fig.
2B). In addition to its bicuculline sensitivity, this
current was also characterized by a very slow rise time ( 1 sec),
which was clearly distinct from that of the fast currents induced by
ATP acting at postsynaptic ionotropic receptors (e.g., compare with
Fig. 1D).
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Figure 2.
ATP increased the frequency of GABAergic mIPSCs in
a reproducible manner. A, Two successive ATP (100 µM) applications spaced by a 2 min interval induced a
similar (reproducible) increase in mIPSC frequency. B,
When coapplied with bicuculline (10 µM), a selective
antagonist of GABAA receptors, ATP (100 µM)
no longer increased the frequency of synaptic events, indicating that
ATP increased the frequency of GABAA receptor-mediated
mIPSCs. The inward current developing slowly during the application of
ATP was caused by the temporal summation of mIPSCs but not by a direct
activation of postsynaptic ATP receptors, as demonstrated by its
blockade by bicuculline. HP, 60 mV.
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Pharmacological properties of postsynaptic and presynaptic effects
of ATP
Effects of agonists
As illustrated in Figure
3A, the postsynaptic current
induced by ATP was reproduced by the nonhydrolyzable ATP analog
ATP--S in all cells tested (n = 11). When tested at
the same concentration (100 µM), the amplitude
of the ATP--S current amounted on average 70 ± 18%
(n = 11) of that induced by ATP. In contrast,
,-me-ATP (100 µM), an agonist at certain
homomeric and heteromeric P2X receptors (Collo et al., 1996; North and
Barnard, 1997; Le et al., 1998a; Ralevic and Burnstock, 1998; Khakh et
al., 1999b), did not ever induce any response in neurons in which ATP
or ATP--S triggered an inward current (n = 9). At a
concentration of 10 µM (n = 11), ADP had no effect on cells in which ATP (10 µM) induced an inward current. At 100 µM, ADP triggered no current in three of four
cells tested and induced a current that represented 67% of that
elicited by ATP (100 µM) in the remaining cell.
Neither ADP--S (50 µM; n = 5), a P2Y1 receptor agonist (Ralevic and Burnstock, 1998), nor UTP (100 µM; n = 6) triggered any
response in cells responding to ATP with an inward current.
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Figure 3.
Effects of P2X and P2Y receptor agonists on
cultured DH neurons displaying a postsynaptic (A)
or presynaptic (B) response. A,
ATP (100 µM) induced an inward current that was partially
reproduced by the same concentration of ATP--S, a nonhydrolyzable
analog of ATP. ATP (10 µM), ADP (10 µM), or
ADP--S (50 µM), an agonist at P2Y1 receptors, had no
effect in the same cell. B, ATP (100 µM)
increased the frequency of GABAergic mIPSCs. This effect was mimicked
by ATP--S (100 µM) and to a lesser extent by a lower
concentration of ATP (10 µM) or ADP (10 µM). ADP--S (50 µM) was without effect.
For each panel (column), all recordings were from the same cell. HP,
60 mV.
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Similar observations were made for the presynaptic effect of ATP (Fig.
3B). The response to ATP (100 µM)
was always mimicked by ATP--S (100 µM),
which induced a 118 ± 55% (n = 6) increase (i.e., a doubling) in mIPSC frequency. The effect of ATP--S amounted to 89 ± 38% of that of ATP (n = 5). In cells in
which ATP or ATP--S increased mIPSCs frequency, ,-me-ATP (100 µM; n = 4), ADP--S (50 µM; n = 6), and UTP (100 µM; n = 4) failed to induce any
change in mIPSC frequency. In 5 of 13 neurons (38.5%), ATP (10 µM) induced an increase in mIPSC frequency, and
ADP (10 µM) had no effect in the same cells. In
five other cells (38.5%), ADP (10 µM) had a
stimulatory effect on mIPSC frequency that represented on average 55 ± 28% of the effect of ATP (10 µM).
Finally, in three cells (23%), ADP (10 µM)
elicited a greater effect than ATP (10 µM). The
ADP response represented 199 ± 48% (n = 3) of
the ATP response. We never observed any effect of ADP in cells that did
not respond to ATP (n = 14). ADP--S (50 µM) did not reproduce the effect of ADP (10 µM) in three of three cells tested.
Effects of antagonists
We compared the effects of the three classic P2X receptor
antagonists, suramin, reactive blue, and PPADS (Ralevic and Burnstock, 1998) on the postsynaptic and presynaptic effects of ATP. Because of
the slow onset kinetics of P2 receptor blockade by suramin and PPADS,
these two antagonists were allowed to equilibrate in the extracellular
medium before being coapplied with ATP. In contrast, reactive blue
blocks P2X receptors rapidly, and we tested its effect during
coapplication with ATP in the absence of reactive blue in bath (Le et
al., 1998a). The results obtained are summarized in Figure
4.
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Figure 4.
Effects of P2X receptor antagonists on
postsynaptic and presynaptic actions of ATP. Different concentrations
of suramin (black bars), PPADS (white
bar), and reactive blue 2 (hatched bars) were
tested on postsynaptic (A) and presynaptic
(B) responses to ATP (100 µM). The
histogram represents the percentage of inhibition by each
antagonist of the control response to ATP, which was determined as the
amplitude of the membrane current (POSTSYNAPTIC
EFFECT) or the increase in frequency of mIPSCs
(PRESYNAPTIC EFFECT) induced by ATP (100 µM) in the absence of antagonists. Error bars represent
SEM. HP, 60 mV.
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At the postsynaptic level, the current induced by ATP (100 µM) was inhibited in an apparently dose-dependent manner
by suramin and reactive blue. The percentages of inhibition were of
18 ± 19% (n = 5, range 0-50%) and 64 ± 28% (n = 5, range 20-100%) for 1 and 30 µM suramin, respectively, and of 32 ± 27% (n = 6, 0-70%) and 84 ± 20%
(n = 4, range 50-100%) for 1 and 10 µM reactive blue. PPADS was tested at a single
concentration of 50 µM and inhibited the ATP
response by 87 ± 19% (n = 3, range
60-100%).
At the presynaptic level, the use of these antagonists was more
delicate because they tended to block directly GABAergic mIPSCs by a
mechanism independent of P2 receptors that was attributable to a direct
inhibition of GABAA receptors (Ralevic and
Burnstock, 1998; Jo and Schlichter, 1999). Therefore, we decided to
test the effect of lower concentrations of these antagonists on the presynaptic response to ATP to avoid nonspecific effects as much as
possible. We first verified the effect of such low concentrations of P2
receptor antagonists on the postsynaptic whole-cell
Cl current induced by local application
of a nonsaturating concentration (20 µM) of the selective
GABAA receptor agonist isoguvacine. Suramin (1 µM) and PPADS (5 µM) had no effect on the
amplitude of the isoguvacine current (n = 5), and
reactive blue (10 µM) inhibited the
GABAA receptor-gated current by 21 ± 10%
(n = 7, range 0-33%). As illustrated in Figure
4B, at concentrations as low as 1 or 5 µM, these antagonists potently inhibited the
presynaptic effect of ATP but had no detectable effect on mIPSC
characteristics or frequency on their own. The mean percentages
of inhibition of the ATP effect were 85 ± 13% (n = 5, range 65-100%), 66 ± 22% (n = 5, range
40-100%), and 100% (n = 4) for suramin (1 µM), reactive blue (1 µM), and PPADS (5 µM),
respectively. Perfusion of suramin (1 µM,
n = 5) or PPADS (5 µM,
n = 4) alone did not significantly modify the basal
frequency of mIPSCs (t test, p > 0.05),
suggesting the absence of a tonic P2X receptor-mediated facilitatory
component on GABA release under the prevailing experimental conditions.
Taken together, our results indicate that the postsynaptic and
presynaptic ATP receptors detected in our study had a similar pharmacological profile that was consistent with the involvement of P2X
receptors in the observed effects.
ATP does not alter mIPSC kinetics
The major effect of ATP or ATP--S on GABAergic synaptic
transmission was to increase mIPSC frequency, indicating a presynaptic site of action (Fig.
5A-C). As
mentioned above, the mean increase in frequency was 257 ± 197%
(n = 33). To determine whether a postsynaptic modulation of mIPSCs was also involved, we analyzed in more detail the
amplitude distribution and the kinetic characteristics (rise time,
deactivation time constant) of GABAergic mIPSCs before and during
application of ATP (100 µM). In this analysis
we considered only cells in which ATP induced an increase in mIPSC
frequency without inducing an inward current at the postsynaptic level, because the development of such a current was always associated with an
increase in membrane noise that could interfere with the detection of
mIPSC and the analysis of their kinetic characteristics. As shown in
Figure 5D, the amplitude distribution of mIPSCs was similar
under control conditions or in the presence of ATP (Kolmogorov-Smirnov test). This was true in all cases examined (n = 9).
Similarly, the RTs and monoexponential deactivation time constants
( ) of mIPSCs were unaffected by ATP (Fig. 5E). The values
for RTs were 1.88 ± 0.43 msec and 1.85 ± 0.40 msec
(n = 8) in the absence and presence of ATP (100 µM), respectively (t test,
p > 0.05). Similarly, the values of were of
33.1 ± 3.9 msec and 35.3 ± 4.0 msec (n = 8)
in the absence and presence of ATP (100 µM)
respectively (t test, p > 0.05).
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Figure 5.
ATP increased the frequency but did not alter the
amplitude or the kinetic characteristics of GABAergic mIPSCs.
A, B, In a neuron displaying mIPSCs under
control conditions (A), application of ATP (100 µM) markedly increased the frequency of these synaptic
currents (B). C, Cumulative
probability histogram of time intervals between successive mIPSCs,
before and during ATP (100 µM) application. ATP shifted
the distribution of interevent intervals to the left, indicating an
increase in mIPSC frequency. D, Cumulative probability
histogram of mIPSC amplitudes. Both distributions match perfectly and
were not significantly different (Kolmogorov-Smirnov test), indicating
that ATP did not modify the amplitude of mIPSCs. E, ATP
(100 µM) had no effect on mIPSCs decay kinetics. Averaged
traces from 10 events recorded under control conditions (left
trace) or during application of 100 µM ATP
(right trace) are represented. is the value of the
time constant of the single exponential fit represented as the
solid line superimposed on each trace. All data are from
the same neuron. HP, 60 mV.
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Calcium dependence of the presynaptic effect of ATP
We next determined the contribution of intracellular versus
extracellular Ca2+ to the presynaptic
stimulatory effect of ATP on mIPSC frequency.
Intracellular Ca2+ stores
To assess the role of intracellular
Ca2+ stores, we tested the effect of
thapsigargin, which blocks the Ca2+ pump
of the endoplasmic reticulum and therefore prevents the refilling of
intracellular Ca2+ stores. In particular,
thapsigargin was shown to block the ATP-induced elevation of free
intracellular Ca2+ concentration mediated
by the activation of P2Y receptors in dorsal horn astrocytes (Salter
and Hicks, 1994, 1995). We first elicited control responses to ATP (100 µM) before superfusing the cells with thapsigargin (1 µM). Bath application of thapsigargin (1 µM) elicited a transient stimulation of mIPSC frequency
that returned to control levels 5 min after onset of superfusion. This phenomenon probably reflected the thapsigargin-induced release of
Ca2+ from reticular stores. We then
applied ATP (100 µM) in the steady presence of
thapsigargin (1 µM) at 2 min intervals for periods up to
30 min (15 applications). A typical result is illustrated in Figure
6A. In the presence of
thapsigargin, ATP still elicited an increase in mIPSC frequency in all
cells tested (n = 4). To try to quantify the effect of
thapsigargin on the ATP effect, we compared the control ATP
response to the third or fourth response to ATP in the presence of
thapsigargin. Under these conditions, the response to ATP in the
presence of thapsigargin represented 62 ± 29% (n = 4, range 26-94%) of the control response to ATP. By comparing the
increase in mIPSC frequency triggered by ATP in the absence and in the
presence of thapsigargin (t test), we found that the
apparent inhibition of the ATP response observed in the presence of
thapsigargin was not significant at a 0.05 confidence interval.
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Figure 6.
ATP-evoked increase in mIPSC frequency was
strictly dependent on extracellular Ca2+.
A, Under control conditions, ATP (100 µM)
induced an increase in the frequency of mIPSCs. During bath perfusion
of thapsigargin (1 µM), ATP (100 µM) was
applied at 2 min intervals. The third (t = 6 min)
and fourth (t = 8 min) applications in thapsigargin
are represented. Note that thapsigargin did not block the ATP response.
B, ATP (100 µM) was applied in
extracellular solutions containing different concentrations of
Na+ and Ca2+ ions.
Na+ was replaced by NMDG. The numbers
on top of each trace indicate (in millimolar) the concentrations of
Na+, NMDG, and Ca2+ for each
experimental condition. ATP was still able to increase
mIPSC frequency in the absence of external Na+
(middle trace), but this effect was no longer observed
when the external Ca2+ concentration was lowered
from 2.5 to 0.3 mM (right trace). HP, 60
mV.
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Role of extracellular Ca2+
The stimulatory effect of ATP (100 µM) on mIPSC
frequency was completely and reversibly blocked when the external
Ca2+ concentration was lowered to 0.3 mM. This was observed in all cells tested
(n = 5) and indicated a complete dependence of the presynaptic effect on Ca2+ influx. We then
tried to assess the eventual contribution of a direct
Ca2+ influx through presynaptic P2X
receptors. We used an experimental protocol similar to the one that we
had developed to characterize the Ca2+
permeability of neuronal nicotinic receptors under physiological conditions, i.e., in intact cells (Poisbeau et al., 1994). After having
applied ATP (100 µM) in standard
Na+- and
Ca2+-containing extracellular medium, we
superfused the preparation with a Na+-free
NMDG-containing extracellular solution to prevent
Na+-dependent membrane depolarization
associated with the activation of presynaptic P2X receptors, which are
nonselective cation channels. For applications lasting <20 sec, P2X
receptors are essentially impermeable to NMDG (Khakh et al., 1999a;
Virginio et al., 1999) and ATP-induced
Ca2+ influx can only occur through
activated P2X receptors and not through voltage-dependent
Ca2+ channels (Poisbeau et al., 1994).
Under such experimental conditions, ATP (100 µM) still caused an increase in mIPSC frequency
(Fig. 6B) in all cells tested (n = 4). This response represented 25 ± 10% of the ATP response
recorded under control conditions. When the extracellular
Ca2+ concentration was reduced to 0.3 mM in the absence of external Na+, the stimulatory effect of ATP on
mIPSC frequency was no longer observed (n = 4) (Fig.
6B). Returning to a medium containing 2.5 mM Ca2+ resulted in
a the recovery of the ATP effect. Similar observations were made in all
cells tested (n = 3).
Taken together, these results indicated that the presynaptic effect of
ATP depended essentially on Ca2+ influx, a
substantial fraction of which (~25%) was probably the result
of influx through presynaptic P2X receptors.
Effect of ATP on GABAergic eIPSCs
Electrical stimulation of a presynaptic neuron that was visually
identified as establishing an apparent physical contact with the neuron
from which we recorded was performed as described previously (Jo et
al., 1998a; Jo and Schlichter, 1999). These experiments were performed
in the absence of TTX but in the presence of strychnine, CNQX, and APV.
The equilibrium for Cl ions was set at
90 mV and that for cations at 0 mV to detect the synaptic corelease
of ATP and GABA (see Materials and Methods). Under these conditions the
eIPSCs could be recorded in isolation at an HP of 0 mV, and the P2X
receptor-mediated EPSCs could be recorded at an HP of 90 mV.
The effect of ATP (100 µM) was tested in a total of 11 cells. In six cases, ATP and GABA were coreleased synaptically from the
presynaptic neuron, whereas in the five other cases GABA was released
alone. Table 1 summarizes the results
that we obtained. ATP potentiated GABAergic eIPSCs in 64% (7 of 11) of
the neurons tested. This potentiating effect was detected in neurons
that released only GABA (two of seven, 29%) but was preferentially observed in neurons coreleasing GABA and ATP (five of seven neurons, 71%). Interestingly, the neurons in which ATP had no effect on eIPSCs
belonged mainly to the category of neurons that released only GABA
(three of four, 75%).
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Table 1.
Effect of ATP (100 µM) on electrically evoked
GABAergic IPSCs from neurons coreleasing ATP and GABA or releasing
only GABA
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Figure 7 illustrates a typical effect of
ATP (100 µM) on GABAergic eIPSCs evoked by paired-pulse
stimulation of the presynaptic neuron (see Materials and Methods). ATP
increased on average the amplitude of the first eIPSC by 25 ± 9%
(n = 7) and reduced the paired-pulse inhibition or
facilitation ratio by 54 ± 26% (n = 7). These
effects were reversible and indicated that ATP facilitated GABAergic
transmission by acting presynaptically. The monoexponential decay time
constants of the first and second eIPSCs were 89 ± 16 msec and
101 ± 30 msec under control conditions and 92 ± 14 msec and
101 ± 26 msec in the presence of ATP (n = 7).
There was no significant difference between the decay time constants of the first and second eIPSCs under either condition (control or in the
presence of ATP) (t test, p > 0.05). ATP
did not modify the monoexponential decay time constants of the first
and second eIPSCs with respect to control (t test,
p > 0.05).
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Figure 7.
Effect of ATP on GABAergic eIPSCs. A paired-pulse
stimulation protocol consisting of two identical electrical pulses (0.1 msec duration) separated by 400 msec was applied at a frequency of 0.1 Hz. Arrows indicate the stimulation artifacts
corresponding to the electrical stimuli. In the presence of ATP (100 µM, bold trace), the amplitude of both
eIPSCs was increased (potentiation of the first eIPSC, 18%;
potentiation of the second eIPSC, 29%), and the paired-pulse ratio was
decreased (by 50%), indicating a presynaptic mechanism of action. The
effect of ATP was completely reversible (data not shown). The control
and wash traces are averages of 20 individual consecutive traces and
that in ATP is an average of 10 traces. HP, 0 mV.
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DISCUSSION |
Our study demonstrates the presence of functional receptors for
ATP (P2 receptors) on a subpopulation of cultured laminae I-III DH
neurons. The novel finding was that ATP facilitated GABAergic synaptic
transmission by a presynaptic mechanism involving
Ca2+-permeable ionotropic P2X receptors,
the pharmacological profile of which appeared to be dominated by the
P2X2 subunit.
Existence of functional postsynaptic and presynaptic
ATP receptors
ATP (10-100 µM) induced an inward current in a
subset (38%) of cultured laminae I-III DH neurons. This proportion of
neurons was comparable to that displaying P2X receptor-mediated
excitatory postsynaptic currents in the same preparation (Jo and
Schlichter, 1999), and our results are in line with those of earlier
studies demonstrating the presence of functional ATP receptors on DH
neurons (Jahr and Jessell, 1983; Salter et al., 1993; Li and Perl,
1995).
A novel finding was that ATP increased the frequency of
GABAA receptor-mediated mIPSCs and the amplitude
of eIPSCs. ATP did not modify the amplitude of mIPSCs or the kinetic
properties of mIPSCs or eIPSCs, indicating a purely presynaptic effect
on receptors located close to the presynaptic terminal. This effect was
similar to the previously described P2X receptor-mediated facilitation of glutamate release from terminals of primary afferent neurons in the
DH (Li and Perl, 1995; Gu and MacDermott, 1997; Li et al., 1998) and in
the brain stem (Khakh and Henderson, 1998) or of noradrenaline release
from sympathetic neurons (Boehm, 1999), but it constitutes the first
demonstration of a modulatory effect of ATP on inhibitory synaptic
transmission between neurons of the CNS. Moreover, the effect of ATP
was observed in a subset (~22%) of GABAergic neurons and in a small
proportion (9%) of glycinergic neurons but was never detected in the
case of glutamatergic neurons, suggesting a specific effect of ATP on
inhibitory versus excitatory transmission between DH neurons.
The presynaptic effect of ATP was completely dependent on
Ca2+ influx and was not significantly
affected by thapsigargin, indicating a minor contribution of
intracellular Ca2+ stores. This situation
is therefore clearly distinct from that in cultured DH astrocytes in
which the cellular response to ATP is entirely dependent on the release
of Ca2+ from thapsigargin-sensitive
intracellular stores after activation of metabotropic P2Y/P2U receptors
(Salter and Hicks, 1994, 1995; Ho et al., 1995).
Pharmacological properties of ATP effects
Both presynaptic and postsynaptic effects of ATP were sensitive to
low concentrations of suramin and PPADS, indicating the involvement of
ionotropic P2X-type receptors (Ralevic and Burnstock, 1998). Neither
UTP, an agonist at many P2Y (except P2Y1) receptors, nor ADP--S, an
agonist at P2Y1 receptors (North and Barnard, 1997; Ralevic and
Burnstock, 1998), reproduced the effects of ATP, indicating that
metabotropic P2Y purinoceptors were probably not involved. ADP mimicked
the presynaptic effect of ATP in a subset of neurons but was usually
less potent than ATP when tested at the same concentration, consistent
with the activation of P2X receptors by ADP (Ralevic and Burnstock,
1998).
Subunit composition of P2X receptors in DH neurons
In situ hybridization and immunocytochemical
experiments have demonstrated that P2X2, P2X4, and P2X6 subunits are
expressed in laminae I-II of the DH (Collo et al., 1996; Vulchanova et
al., 1997; Le et al., 1998b; Kanjhan et al., 1999). Interestingly, P2X2
is expressed only in lamina II, and lamina III expresses none of the
known (P2X1-7) subunits (Collo et al., 1996). P2X4 (Le et al., 1998b)
and P2X2 (Kanjhan et al., 1999) have also been localized to presynaptic
terminals and could therefore underlie the facilitation of synaptic
GABA release observed in our study.
Most P2X subunits can form functional homomeric or heteromeric
receptors or both, but interestingly only a limited number of
combinations are likely to occur in our system. P2X2 and P2X4 but not
P2X6 subunits can form functional homomeric receptors (Collo et al.,
1996; Le et al., 1998a; Khakh et al., 1999b; Torres et al., 1999).
However, P2X6 can heteromerize with both P2X2 and P2X4, whereas P2X2
and P2X4 do not assemble (Collo et al., 1996; Le et al., 1998a; Khakh
et al., 1999b; Torres et al., 1999). These considerations suggest that
the combinations that might occur in the superficial DH include
homomeric P2X2 or P2X4 receptors or both as well as heteromeric
P2X2/P2X6, P2X4/P2X6 and speculatively P2X2/P2X4/P2X6 receptors.
Homomeric P2X2 receptors are sensitive to low concentrations of the
antagonists suramin and PPADS and are insensitive to the agonist
-me-ATP. By contrast, P2X4 homomers are insensitive to suramin
and PPADS at concentrations <100 µM and show low
sensitivity to -me-ATP (Collo et al., 1996; North and Barnard,
1997; Le et al., 1998a; Ralevic and Burnstock, 1998; Khakh et al.,
1999b). Interestingly, heteromeric P2X4/P2X6 receptors give rise to a novel phenotype that is sensitive to suramin, PPADS, and -me-ATP and is potentiated by reactive blue (Le et al., 1998a), which otherwise
is an antagonist at P2X receptors (Ralevic and Burnstock, 1998). In our
preparation, the postsynaptic and presynaptic effects of ATP were
antagonized by suramin, PPADS, and reactive blue, and -me-ATP
(100 µM) was never able to reproduce the effects of ATP.
This pharmacological profile was similar to that of homomeric P2X2
receptors and indicated that neither homomeric P2X4 nor heteromeric P2X4/P2X6 receptors were involved. However, the presence of P2X4 receptors on presynaptic boutons and on cell bodies and dendrites in
laminae I-II was clearly demonstrated in situ (Le et al.,
1998b). Although we cannot completely rule out the possibility that the expression profile of P2X subunits is slightly different from that
in situ, two major possibilities can be envisaged: (1) ATP receptors are P2X2 homomers, and the P2X4 subunits localized by electron microscopy were not assembled into functionally detectable receptors (Garcia-Lecea et al., 1999; Khakh et al., 1999b), or (2) the
receptors comprised both P2X2 and P2X4 subunits, their pharmacological
profile being governed by P2X2. P2X2 and P2X4 subunits do not
coassemble, but P2X6 subunits can heteromerize with either P2X2 or P2X4
(Torres et al., 1999). It is therefore tempting to speculate that the
functional P2X receptors of DH neurons, in particular those controlling
GABA release, are heteromers composed of three distinct subunits (P2X2,
P2X4, and P2X6), P2X6 playing the role of a linker subunit between P2X2
and P2X4 and possibly contributing to limiting the formation of P2X4
homomers that otherwise form easily and preferentially (Le et al.,
1998a; Torres et al., 1999). However, a direct confirmation of this
hypothesis will have to await pharmacological tools that allow a clear
discrimination between different native P2X subunit combinations and
co-precipitation experiments designed to determine the assembly of
three different types of subunits.
Origin of ATP and physiological significance
ATP is present at high concentration in synaptic vesicles
containing classical neurotransmitters and is therefore likely to be
released synaptically with these transmitters. However, the detection
of ATP release relies on the presence of functional postsynaptic P2X or
P2Y receptors and on the fact that a sufficient amount of ATP is
released to reach the postsynaptic membrane without being degraded by
extracellular or secreted ectonucleotidases (Zimmermann, 1994; Todorov
et al., 1997). Thus, corelease of ATP with classical transmitters could
be a common phenomenon but might be detected only in a small number of
cases (Jo and Schlichter, 1999). Our electrical stimulation experiments
showed that presynaptic P2X receptors are preferentially, but not
exclusively, localized on terminals of DH neurons that corelease ATP
and GABA, suggesting that they might act as facilitatory autoreceptors
(Boehm, 1999). We have preliminary evidence that the amplitude of
GABAergic eIPSCs is inhibited by ~10% during the application of
PPADS (5 µM) to neurons coreleasing ATP and GABA (our
unpublished observation). Although this effect is similar to that
reported for sympathetic neurons (Boehm, 1999), this finding must be
interpreted with caution, and the clear demonstration of functional P2X
autoreceptors will have to await the development of selective
antagonists that do not interact with GABAA receptors.
It is known that ATP is released from primary afferent terminals in the
DH (for review, see Salter et al., 1993), from intrinsic DH neurons
(Sawynok et al., 1993; Jo and Schlichter, 1999) (which might be
different from those coreleasing ATP and GABA), and from astrocytes
(Queiroz et al., 1997), and ATP appears to be the extracellular messenger that allows the propagation of
Ca2+ waves between astrocytes (Guthrie et
al., 1999). In a physiological context, one could speculate that the
initial release of ATP from neurons could elicit
Ca2+ waves in perisynaptic glial cells, as
has already been demonstrated at the neuromuscular junction
(Robitaille, 1995), which could then propagate locally via the
astrocyte network and regulate presynaptically or postsynaptically the
transmission at nearby synapses via either ATP receptors or receptors
to another neuroactive substance released by glial cells. Although this
type of modulation has to be clearly established, it certainly would
represent an interesting form of local and activity-dependent
modulation of neurotransmission involving neuroglial interactions.
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FOOTNOTES |
Received Sept. 24, 1999; revised Dec. 23, 1999; accepted Jan. 4, 2000.
This work was supported by a grant from the Institut UPSA de la
Douleur. We acknowledge additional support from Université Louis
Pasteur (Strasbourg), Centre National de la Recherche Scientifique (CNRS, France), and Institut Universitaire de France. Many thanks to
Dr. Yves de Koninck (Mc Gill University, Montreal, Canada) for
providing his synaptic current analysis program and to Dr. Jean-Luc
Rodeau (UPR9009, CNRS, Strasbourg) for his Kolmogorov-Smirnov analysis
software. We thank Catherine Moreau and Madeleine Roth for excellent
technical assistance.
Correspondence should addressed to R. Schlichter, Unité Mixte de
Recherche 7519-Centre National de la Recherche Scientifique, Université Louis Pasteur, 21 rue Descartes, 67084 Strasbourg Cedex, France. E-mail:
schlichter{at}neurochem.u-strasbg.fr.
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TOP
ABSTRACT
INTRODUCTION
