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. 2006 Apr 13;573(Pt 3):753–763. doi: 10.1113/jphysiol.2006.108613

Direct excitation of deep dorsal horn neurones in the rat spinal cord by the activation of postsynaptic P2X receptors

Hiroaki Shiokawa 1, Terumasa Nakatsuka 1,2, Hidemasa Furue 1, Makoto Tsuda 3, Toshihiko Katafuchi 1, Kazuhide Inoue 3, Megumu Yoshimura 1
PMCID: PMC1779754  PMID: 16613873

Abstract

ATP mediates somatosensory transmission in the spinal cord through the activation of P2X receptors. Nonetheless, the functional significance of postsynaptic P2X receptors in spinal deep dorsal horn neurones is still not yet well understood. Using the whole-cell patch-clamp technique, we investigated whether the activation of postsynaptic P2X receptors can modulate the synaptic transmission in lamina V neurones of postnatal day (P) 9–12 spinal cord slices. At a holding potential of −70 mV, ATPγS (100 μm), a nonhydrolysable ATP analogue, generated an inward current, which was resistant to tetrodotoxin (1 μm) in 61% of the lamina V neurones. The ATPγS-induced inward current was accompanied by a significant increase in the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in the majority of lamina V neurones. The ATPγS-induced inward current was not reproduced by P2Y receptor agonists, UTP (100 μm), UDP (100 μm), and 2-methylthio ADP (100 μm), and it was also not affected by the addition of guanosine-5′-O-(2-thiodiphosphate) (GDPβS) into the pipette solution, thus suggesting that ionotropic P2X receptors were activated by ATPγS instead of metabotropic P2Y receptors. On the other hand, α,β-methylene ATP (100 μm) did not change any membrane current, but instead increased the mEPSC frequency in the majority of lamina V neurones. The ATPγS-induced inward current was suppressed by pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) (10 μm), but not by trinitrophenyl-ATP (TNP-ATP) (1 μm). Furthermore, we found that ATPγS (100 μm) produced a clear inward current which was observed in all lamina V neurones over P16 spinal cord slices, in contrast to P9–12. These results indicate that distinct subtypes of P2X receptors were functionally expressed at the post- and presynaptic sites in lamina V neurones, both of which may contribute to the hyperexcitability of lamina V in a different manner. In addition, the data relating to the developmental increase in the functional P2X receptors suggest that purinergic signalling may thus be more common in somatosensory transmission with maturation.

Extracellular ATP plays a crucial role in nociceptive transmission in the central and peripheral nervous systems (Burnstock & Wood, 1996; Hamilton & McMahon, 2000; Chizh & Illes, 2001; Kennedy et al. 2003; Liu & Salter, 2005). ATP receptors are divided into two classes, ionotropic P2X receptors (Khakh, 2001; North, 2002) and G-protein-coupled metabotropic P2Y receptors (von Kügelgen & Wetter, 2000). To date, seven P2X receptor subunits (P2X1 to P2X7) have been cloned (North & Surprenant, 2000). Each P2X subunit has two transmembrane domains with a cysteine-rich extracellular loop, which contains an ATP-binding site. A functional P2X receptor is composed of three or more P2X subunits, forming a pore structure that is permeable to cations including Ca2+. Assembled from seven P2X subunits, at least 11 subtypes of functional P2X receptors can thus be formed in heterologous expression systems. These 11 P2X receptors are homomeric P2X1 to P2X7 receptors, heteromeric P2X1/5, P2X2/3, P2X2/6 and P2X4/6 (Khakh et al. 2001; North, 2002).

The spinal dorsal horn (DH) is the first site in the central nervous system where somatosensory information is processed. Deep DH (DDH) neurones transmit a variety of sensory inputs, including nociceptive and non-nociceptive inputs, while superficial DH (SDH) neurones receive nociceptive specific sensory inputs (Willis & Coggeshall, 1991). Six of seven P2X receptor subunits (P2X1 to P2X6) are expressed in the DH (Collo et al. 1996; Vulchanova et al. 1997; Guo et al. 1999). Multiple subtypes of P2X receptors were located at the central terminals of primary afferents that innervate onto DH neurones (Vulchanova et al. 1997; Guo et al. 1999). The activation of these distinct P2X receptors enhances glutamate release in different manners (Nakatsuka & Gu, 2001; Nakatsuka et al. 2003; Chen & Gu, 2005). The modulation of glutamate release by presynaptic P2X receptors is mainly transient in lamina II neurones (Nakatsuka et al. 2003). In contrast, P2X receptor-mediated modulation of glutamate release is long-lasting in lamina I and lamina V neurones (Nakatsuka & Gu, 2001; Nakatsuka et al. 2003; Chen & Gu, 2005). Moreover, the activation of presynaptic P2X receptors modulates not only excitatory, but also the inhibitory synaptic transmission in SDH neurones (Li & Perl, 1995; Li et al. 1998; Hugel & Schlichter, 2000; Rhee et al. 2000). The activation of certain types of presynaptic P2X receptors also increases the GABA and glycine release onto SDH neurones (Hugel & Schlichter, 2000; Rhee et al. 2000).

Although these functional roles of presynaptic P2X receptors have been well established, little is known about the postsynaptic P2X receptors in the DH. Jahr & Jessell (1983) first reported the action of ATP on cultured DH neurones. In spinal cord slice preparations, the bath application of ATP also induces inward currents in lamina II neurones of spinal cord slices (Li & Perl, 1995). However, it is not clear whether the ATP-evoked depolarization or inward current in these studies are mediated by postsynaptic P2X receptors in DH neurones or secondary responses. In acutely dissociated neurones from the SDH, ATP induces inward currents (Bardoni et al. 1997; Rhee et al. 2000) and evokes Ca2+ transients (Bardoni et al. 1997). In addition, excitatory postsynaptic currents (EPSCs) cannot be completely blocked by glutamate receptor antagonists, but they can be blocked by the addition of P2X receptor antagonists in a small population of lamina II neurones (Bardoni et al. 1997). These results suggest that postsynaptic P2X receptors in a subpopulation of SDH neurones can mediate somatosensory transmission in the spinal cord. However, the possible roles of postsynaptic P2X receptors in DDH neurones have never been documented up to now. DDH neurones, especially lamina V neurones, generate long-lasting afterdischarges in response to nociceptive inputs (Woolf & King, 1987). As a result, the development of hyperactivity in DDH neurones is involved in a variety of pathological pain sensations (Willis & Coggeshall, 1991; Mao et al. 1992). Therefore, the aim of this study was to evaluate the effects of postsynaptic P2X receptors on synaptic transmission in DDH neurones of spinal cord slices.

Methods

All the experimental procedures involving the use of animals were approved of by the Ethics Committee on Animal Experiments, Kyushu University, and were in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines.

Spinal cord slice preparation

The methods used to obtain rat spinal cord slice preparations have been previously described (Nakatsuka et al. 1999). In brief, Sprague-Dawley rats at a postnatal age 9–19 days were deeply anaesthetized with urethane (1.2 g kg−1, intraperitoneal), and then a lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in preoxygenated Krebs solution at 1–3°C. Immediately after the removal of the spinal cord, the rats were given an overdose of urethane and then were killed by exsanguination. The pia-arachnoid membrane was removed after cutting all the ventral and dorsal roots near the root entry zone. The spinal cord was mounted on a vibratome and then a 500-μm-thick transverse slice was cut. The slice was placed in the recording chamber, which had a volume of 0.5 ml, and placed on the stage of an upright IR-DIC microscope (Axio Skop 2, Carl Zeiss). Next, the slice was superfused at a rate of 5 ml min−1 with Krebs solution saturated with 95% O2 and 5% CO2 at room temperature. The Krebs solution contained (mm) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3 and 11 glucose.

Patch-clamp recordings from DDH neurones

The lamina regions were identified with a 5× objective lens, and individual neurones were identified with a 40× objective lens under IR-DIC microscope. The microscope was coupled with a CCD camera (XC-EI30, SONY) and a video monitor screen. Whole-cell patch-clamp recordings were made from lamina V neurones with patch-pipette electrodes having a resistance of 4–8 MΩ (Nakatsuka & Gu, 2001). The composition of the patch-pipette solution was as follows (mM): 135 potassium gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 Hepes, 5 ATP-Mg, pH 7.2. Guanosine-5′-O-(2-thiodiphosphate) (GDPβS) was added at a concentration of 2 mm to the patch-pipette solution when necessary. The signals were acquired with an amplifier (EPC-9, HEKA). The data were digitized with an A/D converter (MacLab, ADInstruments), stored on a personal computer using a data acquisition program (Chart 3.6.9, ADInstruments), and then were analysed using a software program (AxoGraph 4.6, Axon Instruments). The lamina V neurones were viable for up to 12 h in slices perfused with preoxygenated Krebs solution. However, all the recordings described here were obtained within 8 h. The firing patterns of the lamina V neurones were determined in current clamp by passing 1 s long depolarizing pulses through the recording electrode from a holding potential of −60 mV. Whole-cell patch-clamp recordings were stable for up to 3 h. A holding membrane potential of −70 mV was used unless otherwise mentioned.

Cell identification

The location and morphological features of the recorded cells were further confirmed in some instances by an intrasomatic injection of neurobiotin (0.2% in recording electrode solution). After terminating the electrophysiological recordings, the slices were fixed overnight with 4% paraformaldehyde in 0.1 m phosphate buffer (PB, pH 7.4) at 4°C and rinsed in 0.1 m PB. Visualization of neurobiotin-labelled cells was performed by diaminobenzidine (DAB)-based histochemistry. Free-floating sections were incubated in Vectastain (Elite kit; Vector Laboratories) according to the manufacturer's protocol. The peroxidase activity was revealed with DAB in the presence of hydrogen peroxide, and sections were mounted on gelatinized slides. The sections were viewed and photographed with a microscope (Eclipse E600, Nikon).

Application of drugs

Drugs were dissolved in Krebs solution and applied by perfusion via a three-way stopcock without any change in the perfusion rate. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord slice was approximately 20 s. The drugs used in this study were adenosine 5′-O-(3-thiotriphosphate) (ATPγS), α,β-methylene ATP (αβmeATP), uridine 5′-triphosphate (UTP), uridine 5′-diphosphate (UDP), 2 methyltio ADP (2meSADP), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS), trinitrophenyl-ATP (TNP-ATP), GDPβS, and baclofen from Sigma (St Louis, MO, USA); tetrodotoxin (TTX) from Wako (Osaka, Japan); 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) and d(−)-2-amino-5-phosphonovaleric acid (APV) were purchased from Tocris Cookson (Bristol, UK).

Statistical analysis

All numerical data were expressed as the mean ± standard error (s.e.m.). Statistical significance was determined as P < 0.05 using Student's paired t test to compare the amplitude of the inward currents and as P < 0.05 using non-parametric Kolomogorov–Smirnov's test to compare the frequency of the miniature excitatory postsynaptic currents (mEPSCs). In the electrophysiological data, n refers to the number of neurones studied.

Results

Dual effect of ATPγS on synaptic transmission in DDH neurones

Whole-cell patch-clamp recordings were performed from lamina V neurones of P9–12 spinal cord slices. Stable recording was obtained from a single lamina V neurone for up to 4 h. In the current-clamp mode, all neurones were classified into tonic firing, showing regular firing throughout the current pulse (Fig. 1B). The majority of lamina V neurones possessed the morphological features of a rounded soma with dendrites branching off dorsally and ventrally (Fig. 1A). In the voltage-clamp mode, perfusion with ATPγS (100 μm) for 1 min induced an inward current in 67 out of 109 lamina V neurones at a holding potential of −70 mV (Fig. 1C). The average amplitude of the peak currents induced by ATPγS (100 μm) was 11.0 ± 0.8 pA (n = 67). In the presence of CNQX (20 μm) and APV (50 μm), ATPγS (100 μm) also produced an inward current (Fig. 1C). In the same lamina V neurones examined, the average amplitude of the peak currents induced by ATPγS in the presence of CNQX and APV was 14.4 ± 8.3 pA, and was not significantly different from that in the absence of CNQX and APV (15.6 ± 9.3 pA, n = 4). In 55 out of 67 neurones, the ATPγS-induced inward current was accompanied by a significant increase in the frequency of glutamatergic sEPSCs (Fig. 2A). The average increase in the sEPSC frequency was 200 ± 14% of control (n = 55, Fig. 2B). The bath application of ATPγS (100 μm) did not change any membrane current in the remaining 42 neurones tested, but significantly increased the frequency of glutamatergic sEPSCs in 29 neurones (Fig. 2C). The average increase in the sEPSC frequency was 184 ± 11% of control (n = 29, Fig. 2D). When ATPγS (100 μm) was applied repeatedly at 10 min intervals, it produced similar inward currents with almost the same amplitude (n = 4, Fig. 3A). Moreover, these ATPγS-induced currents were resistant to TTX (1 μm, Fig. 3B). In the same lamina V neurones, the average amplitude of the ATPγS-induced currents in the presence of TTX was 104 ± 4% of that in the absence of TTX (n = 5). When examined in the concentration range of 10–300 μm, the ATPγS-induced inward currents were enhanced in amplitude with increasing concentrations (Fig. 4A and B). We further examined the changes in the membrane conductance of the ATPγS-induced currents in the presence of TTX (1 μm) (Fig. 4C). Voltage steps (duration: >50 ms) from a holding potential of −70 mV to voltages ranging from −40–40 mV in steps of 10 mV were given to lamina V neurones in the absence or presence of ATPγS. Figure 4D demonstrates the relationships between the step voltage and the steady current at the end of its pulse in the absence (s) and presence (•) of ATPγS (100 μm). The net ATPγS-induced current (○) estimated from a difference between the two currents exhibited a reversal potential of −1.3 ± 4.7 mV (n = 4), which was compatible with the activation of a non-selective cation conductance.

Figure 1. ATPγS excites lamina V neurones in spinal cord slices.

Figure 1

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A, a recorded lamina V neurone identified with an intracellular injection with neurobiotin. Scale bar is 100 μm. B, all lamina V neurones examined displayed tonic firing in response to depolarizing current injection of 143.6 pA (upper trace), 93.6 pA (middle trace), or 43.6 pA (lower trace) in current-clamp mode. C, in voltage-clamp mode, ATPγS (100 μm) produced a clear inward current at a holding potential of −70 mV (upper trace). In the presence of CNQX (20 μm) and APV (50 μm), ATPγS (100 μm) still induced an inward current without a significant decrease in amplitude in the same lamina V neurone (lower trace).

Figure 2. Post- or presynaptic effect of ATPγS in lamina V neurones.

Figure 2

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A, bath application of ATPγS (100 μm) for 1 min induced a clear inward current associated with a marked increase in the sEPSC frequency in a P9–12 lamina V neurone. The ATPγS-induced inward current was observed in approximately 60% of lamina V neurones examined. B, a summary of relative sEPSC frequency before (control) and during application of ATPγS in lamina V neurones which exhibited the ATPγS-induced inward current (*P < 0.05). C, ATPγS (100 μm) did not change any membrane current, but largely increased the sEPSC frequency in a P9–P12 lamina V neurone. D, a summary of the relative sEPSC frequency before (control) and during the application of ATPγS in lamina V neurones which did not exhibit any ATPγS-induced inward current (*P < 0.05). Error bars s.e.m.

Figure 3. ATPγS repeatedly induces an inward current in lamina V neurones.

Figure 3

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A, when ATPγS was repeatedly applied at 10 min intervals, it produced a similar inward current (upper trace, the first application of ATPγS; lower trace, the second application of ATPγS). B, in the presence of TTX (1 μm), ATPγS induced an inward current without any decrease in the amplitude (upper trace, in the absence of TTX; lower trace, in the presence of TTX).

Figure 4. Dose and voltage dependency of the ATPγS-induced inward current.

Figure 4

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A, the ATPγS-induced inward currents showed an enhanced amplitude with increasing concentrations. B, normalized amplitude of the ATPγS-induced inward currents was plotted against the ATPγS concentration. The vertical bar indicates s.e.m. (n = 3–6). C, to examine a change in membrane conductance of the ATPγS-induced currents, a voltage step (duration, >50 ms) from −60 mV to + 40 mV in steps of 10 mV was given to lamina V neurones before and during application of ATPγS (100 μm) in the presence of TTX (1 μm). D, the amplitude of membrane currents in response to voltage pulses from −60 mV to +40 mV was plotted against voltages in the absence (▴) and presence (•) of ATPγS (100 μm). The current–voltage relationship for net ATPγS current was estimated based on the difference between the current responses in the absence and presence of ATPγS (○).

Effects of P2Y receptor agonists

To determine whether the ATPγS-induced inward current is mediated by metabotropic P2Y receptors, the effect of P2Y receptor agonists on membrane currents was observed in the lamina V neurones. The bath application of UTP (100 μm), an agonist for P2Y2 and P2Y4 receptors, for 1 min affected neither the membrane currents nor the glutamatergic excitatory synaptic transmission in lamina V neurones where ATPγS induced an inward currents (n = 4, Fig. 5A). UDP (100 μm), an agonist for P2Y6 receptors, and 2meSADP (100 μm), an agonist for P2Y1, P2Y12 and P2Y13, also changed neither membrane currents nor glutamatergic excitatory synaptic transmission in lamina V neurones (n = 4, Fig. 5A).

Figure 5. The ATPγS-induced inward current was not mediated by P2Y receptors.

Figure 5

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A, ATPγS (100 μm, upper trace) produced an inward current, while P2Y agonists, 2meSATP (100 μm, middle trace) or UTP (100 μm, lower trace) did not cause any inward current in the same lamina V neurones. B, the baclofen- and ATPγS-induced currents were recorded with the pipette solution containing GDPβS. Both baclofen and ATPγS affected the holding currents just after establishing whole-cell configuration in the same lamina V neurones (upper traces). When baclofen or ATPγS was again applied 1 h after establishing whole-cell configuration, the peak amplitude of the baclofen-induced outward current was clearly inhibited, while the ATPγS-induced inward current did not change (lower traces). C, a summary of the relative amplitude of the baclofen- and ATPγS-induced currents 1 h after establishing whole-cell configuration by the addition of GDPβS into the pipette solution. Under the same conditions as for B, the baclofen-induced outward current was significantly suppressed when baclofen was applied 1 h later (*P < 0.05). Error bars s.e.m.

To examine the involvement of intracellular G-proteins in the ATPγS-induced inward current, GDPβS (2 mm), a nonhydrolysable analogue of GDP that competitively inhibits G-proteins, was added to the pipette solution. When ATPγS (100 μm) was applied just after establishing the whole-cell configuration with pipettes containing potassium gluconate and GDPβS, an inward current was observed (n = 4). When ATPγS was again applied 1 h later, it produced similar inward currents with almost the same amplitude (96.5 ± 13.8% of the control, P > 0.05, Fig. 5B and C). Under the same conditions, the baclofen-induced outward currents were significantly suppressed by the addition of GDPβS into the pipette solution in four lamina V neurones examined (29.4 ± 14.2% of the control, Fig. 5C). These results indicated the absence of a relationship with the activation of intracellular G-proteins. As a result, the ATPγS-induced inward current was mediated by ionotropic P2X receptors, rather than metabotropic P2Y receptors.

Effects of P2X receptor agonist and antagonists

To clarify which subtype of P2X receptors is involved in the ATPγS-induced inward current, the effect of P2X receptor agonist and antagonists was examined. The bath application of αβmeATP (100 μm), an agonist for P2X1, P2X3, P2X2/3, P2X1/5 and P2X4/6 receptors, did not induce any inward current in any of 28 lamina V neurones tested (Fig. 6A and C), although glutamatergic mEPSC frequency significantly increased in 17 out of 28 lamina V neurones (Fig. 6A and D). In addition, the effects of the P2X receptors antagonists, PPADS and TNP-ATP on the ATPγS-induced inward currents were examined. In the presence of PPADS (10 μm), the average amplitude of the ATPγS-induced inward current was −1.3 ± 0.2 pA, and it significantly decreased to 15 ± 4% of that in the absence of PPADS in the same lamina V neurones examined (n = 7, Fig. 6B and C). On the other hand, the average amplitude of the ATPγS-induced inward currents was −11.7 ± 1.6 pA in the presence of TNP-ATP (1 μm), which was not substantially different from that in the absence of TNP-ATP in the same neurones recorded (−11.0 ± 1.7 pA, n = 7, Fig. 6B and C). We further examined which subtype of P2X receptors is involved in the ATPγS-mediated increase in sEPSC frequency in lamina V neurones. The bath application of ATPγS (100 μm) significantly increased the frequency of glutamatergic EPSCs (203 ± 38% of the control, n = 7, Fig. 6D). The ATPγS-induced increase in sEPSC frequency was completely blocked by 10 μm PPADS (96 ± 10% of the control, n = 7), but it was not significantly affected by 1 μm TNP-ATP (182 ± 27% of the control, n = 7, Fig. 6D).

Figure 6. Effect of P2X agonist and antagonists.

Figure 6

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A, ATPγS (100 μm) induced an inward current (upper trace), while αβmeATP (100 μm), a P2X receptor agonist, did not cause any inward current in the same lamina V neurones (lower trace). B, TNP-ATP (1 μm), a P2X receptor antagonist, did not affect the ATPγS-induced inward current (middle trace). On the other hand, in the presence of PPADS (10 μm), the ATPγS-induced inward current was completely abolished in the same neurons (lower trace). C, a summary of the averaged amplitude of the inward currents by αβmeATP (100 μm), ATPγS (100 μm), ATPγS (100 μm) in the presence of TNP-ATP (1 μm), and ATPγS (100 μm) in the presence of PPADS (10 μm). The ATPγS-induced inward currents in the presence of PPADS were significantly smaller than those in the absence of PPADS (*P < 0.05). D, a summary of the relative sEPSC frequency before (control) and during the application of αβmeATP (100 μm), ATPγS (100 μm), ATPγS (100 μm) in the presence of TNP-ATP (1 μm), and ATPγS (100 μm) in the presence of PPADS (10 μm). Both αβmeATP and ATPγS significantly increased sEPSC frequency (*P < 0.05). The ATPγS-induced increase in sEPSC frequency was not significantly inhibited by TNP-ATP, but was markedly suppressed by PPADS (*P < 0.05). Error bars s.e.m.

Developmental changes in P2X receptors in DDH neurones

To examine whether or not a developmental change in the P2X receptors existed in the lamina V neurones, whole-cell patch-clamp recordings were performed from lamina V neurones of P16–19 spinal cord slices. Interestingly, the bath application of ATPγS (100 μm) produced a clear inward current in all 11 lamina V neurones recorded at a holding potential of −70 mV (Fig. 7B and C). The average amplitude of the peak currents induced by ATPγS (100 μm) was −22.6 ± 4.2 pA (n = 11), which was significantly larger than that of P9–12 (Fig. 7D). In 10 out of these 11 neurones, the ATPγS-induced inward current was accompanied by a significant increase in the frequency of glutamatergic sEPSCs. The average increase in the sEPSC frequency by ATPγS (100 μm) was 318 ± 45.7% of the control (n = 11). On the other hand, αβmeATP (100 μm) did not affect the membrane current, but significantly increased glutamatergic sEPSC frequency in all seven lamina V neurones in which ATPγS produced an inward current (data not shown). These results suggest that a larger population of lamina V neurones elicited not only postsynaptic but also presynaptic P2X receptor-mediated actions in the later developmental days.

Figure 7. Developmental change of postsynaptic P2X receptors.

Figure 7

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A, ATPγS (100 μm) did not produce any membrane current in a P9–12 lamina V neurone. B, ATPγS (100 μm) induced a clear inward current in a P16–19 lamina V neurone. In contrast to P9–12 lamina V neurones, the ATPγS-induced inward current was observed in all P16–19 lamina V neurones examined. C, a summary of the incidence of the ATPγS-induced inward currents in P9–12 and P16–19 lamina V neurones. The population of P16–19 lamina V neurones which exhibited the inward current was significantly larger than that of P9–12 lamina V neurones (*P < 0.05). D, a summary of the averaged amplitude of the ATPγS-induced inward currents in P9–12 and P16–19 lamina V neurones at these stages. The average amplitude of the ATPγS-induced inward currents in P16–19 was significantly larger than that in P9–12 (*P < 0.05). Error bars s.e.m.

Discussion

In this study, we show that the distinct subtypes of P2X receptors are functionally expressed in both the post- and presynaptic sites of DDH neurones. Activation of the postsynaptic P2X receptor directly depolarizes the majority of lamina V neurones in spinal cord slices, in addition to our previous finding of presynaptic P2X receptor-mediated enhancement of glutamate release onto lamina V neurones (Nakatsuka & Gu, 2001; Nakatsuka et al. 2002, 2003). Furthermore, both the P2X receptor-mediated post- and presynaptic responses in the lamina V neurones were significantly greater in P16–19 than in P9–12, thus indicating that there might be a progressive expression of P2X receptors during maturation.

Expression of P2X receptors in DDH neurones

It has been demonstrated that all P2X mRNAs except P2X3 are distributed in the DH (Collo et al. 1996). P2X2, P2X4, and P2X6 mRNAs were strongly expressed in the SDH. Consistently, several electrophysiological studies have also described the direct action of ATP or its agonists on postsynaptic P2X receptors in SDH neurones. Jahr & Jessell (1983) first demonstrated that pressure application of ATP produced a rapid and marked depolarization in 27% of cultured SDH neurones. The ATP-induced inward current was reproduced by ATPγS, but not by P2Y receptor agonists in 38% of cultured SDH neurones (Hugel & Schlichter, 2000). Furthermore, in a small population (<5%) of acutely dissociated SDH neurones from P7–12 rats, intracellular Ca2+ concentration was significantly elevated by ATP (100 μm), but not by α,βmeATP (Bardoni et al. 1997). ATP also generated an inward current in 24% of the mechanically dissociated lamina II neurones from P10–P14 rats (Rhee et al. 2000). In hamster spinal cord slices (P21–28), high dose of ATP or ATPγS produced an inward current in approximately half of lamina II neurones (Li & Perl, 1995). However, there has been no report concerning postsynaptic P2X receptor-mediated actions in DDH neurones. In the present study, the bath application of ATPγS induced an inward current in the majority of lamina V neurones. The finding of no effect of TTX on the ATPγS-induced inward current suggests that ATPγS acts directly on the lamina V neurones, but not through an activation of interneurones. To eliminate the possibility of the involvement of P2Y receptors in the ATPγS-induced inward current, the effects of P2Y receptor agonists on synaptic transmission were examined in lamina V neurones. UTP, UDP, and 2meSADP, did not mimic the ATPγS-induced inward current. In addition, the ATPγS-induced inward current was not affected by the addition of GDPβS into the pipette solution. These findings suggested that ATPγS-induced inward current in lamina V neurones is mediated by ionotropic P2X, but not by metabotropic P2Y receptors. Consistent with the previous observations of functional P2X receptors in heterologous expression systems (Khakh et al. 2001; North, 2002), the reversal potential of the ATPγS-induced current in the present study was close to 0 mV, a value that was compatible with the activation of a non-selective cation conductance. As well as lamina V neurones, the ATP-induced inward current has been shown to be postsynaptically activated by ionotropic P2X, but not by metabotropic P2Y receptors in a subpopulation of SDH neurones (Bardoni et al. 1997; Hugel & Schlichter, 2000; Rhee et al. 2000).

ATPγS also significantly increased glutamatergic mEPSC frequency in 77% of lamina V neurones. This effect was similar to our previous findings of presynaptic P2X receptor-mediated enhancement of glutamate release onto lamina V neurones (Nakatsuka & Gu, 2001; Nakatsuka et al. 2002; Nakatsuka et al. 2003). Compared with ATPγS (77%), αβmeATP increased glutamatergic mEPSC frequency in a smaller population of lamina V neurones (61%). This result may be explained by the different sensitivities between the P2X receptor agonists, ATPγS and αβmeATP. However, we cannot conclusively rule out the possibility that ATPγS might activate P2X receptors in a subset of glutamatergic interneurones, because spinal DH neurones are directly excited by ATPγS, but not by αβmeATP (Bardoni et al. 1997; Nakatsuka & Gu, 2001). There is no reported evidence regarding this interesting issue. As a result, further investigations will be required to address whether functional P2X receptors are expressed in spinal glutamatergic interneurones.

Pharmachological property of postsynaptic P2X receptors

The ATPγS-induced inward current was accompanied by a significant increase in the frequency of glutamatergic mEPSCs in the majority of lamina V neurones. On the other hand, αβmeATP significantly increased glutamatergic mEPSC frequency, but it did not produce any inward currents in lamina V neurones. These results suggested that a subtype of postsynaptic P2X receptors is different from that of presynaptic P2X receptors. Due to a lack of good selective agonists and antagonists for each subtype of P2X receptors, it is hard to pharmacologically distinguish post- and presynaptic P2X receptors in lamina V neurones. Seven P2X receptor subunits have been identified and cloned (North & Surprenant, 2000). To date, six homomeric (P2X1–5, P2X7) and four heteromeric (P2X1/5, P2X2/3, P2X2/6, P2X4/6) P2X receptors have been characterized in heterologous expression systems (Khakh et al. 2001). ATPγS is a non-hydrolysable ATP analogue and a common agonist for all P2X receptors. On the other hand, αβmeATP selectively activates P2X1, P2X3, P2X1/5, P2X2/6 and P2X4/6 receptors (Khakh et al. 2001). PPADS (10 μm) inhibits almost all P2X receptors except P2X4 and P2X6, although it is unknown whether the P2X2/6 receptor is sensitive to PPADS (Khakh et al. 2001). TNP-ATP (1 μm) is an antagonist for P2X1, P2X2, P2X3, P2X2/3 and P2X1/5, but it remains unknown whether P2X5, P2X6, P2X2/6, and P2X4/6 receptors are sensitive to TNP-ATP (Virginio et al. 1998; Surprenant et al. 2000; Khakh et al. 2001). In the present study, the ATPγS-induced inward current in DDH neurones was not reproduced by αβmeATP (100 μm), and was blocked by PPADS (10 μm) but not by TNP-ATP (1 μm). The pharmacological profile of postsynaptic P2X receptors in DDH neurones may be consistent with the involvement of P2X5 and/or P2X2/6 receptors. Consistent with these electrophysiological findings, in situ hybridization studies have shown that P2X2, P2X5, and P2X6 mRNAs are expressed in the DDH (Collo et al. 1996). However, the involvement of P2X2 or P2X4 receptors could not be excluded in the present study, because P2X2 and P2X4 receptors are relatively insensitive to TNP-ATP (Virginio et al. 1998). So far, few tools have been developed to discriminate P2X receptors on naïve neurones.

Developmental changes of P2X receptors

The circuitry in the central nervous system might be altered by somatosensory inputs in early life (Anand, 2000; Peters et al. 2005). In addition, recent studies into the development of excitatory and inhibitory synaptic transmission have provided that nociceptive circuits in the spinal DH are organized and strengthened during the first postnatal weeks (Fitzgerald, 2005). It has been demonstrated that the presynaptic P2X receptor subtype in SDH changes during postnatal development (Jang et al. 2001). Although αβmeATP did not elicit any presynaptic or postsynaptic effects in mechanically dissociated P10–13 lamina II neurones, αβmeATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals innervated onto P16–18 lamina II neurones. However, it remains unclear whether the expression of post- or presynaptic P2X receptors in DDH neurones can be altered during postnatal development. In the present study, we showed that ATPγS induced an inward current in 61% of P9–12 lamina V neurones and all P16–19 lamina V neurones. Interestingly, the average amplitude of the peak currents induced by ATPγS in P16–19 lamina V neurones was significantly larger than that in P9–12 lamina V neurones. In addition, ATPγS increased the mEPSC frequency in 77% of P9–12 lamina V neurones and 91% of P16–19 lamina V neurones. αβmeATP also increased the mEPSC frequency in 61% of P9–12 lamina V neurones and all P16–19 lamina V neurones. These results suggested that post- and presynaptic P2X receptors are therefore expressed in larger populations of lamina V neurones in later development. As a result, purinergic signalling in the DDH may become more common and important with the postnatal development.

Functional implications

DDH neurones participate in the processing of somatosensory information, including nociceptive inputs, and relay the information to supraspinal structures (Willis & Coggeshall, 1991). DDH neurones may generate prolonged afterdischarges in response to the nociceptive information (Woolf & King, 1987; De Koninck & Henry, 1991), and the hyperactivity of DDH neurones is believed to be associated with the development of pathological pain sensations, including neuropathic pain or inflammatory pain. The present study provides evidence that extracellular ATP could elicit two different actions in facilitating neuronal excitability through distinct post- and presynaptic P2X receptors in the DDH. Although the original source of endogenous ATP is unknown at present, ATP could be released in the central nervous system as a consequence of neuronal hyperactivity (Khakh & Henderson, 1998; Khakh et al. 2003; Koizumi et al. 2003). As a result, the activation of post- and/or presynaptic P2X receptors in DDH neurones may therefore contribute to a variety of pain sensations.

Acknowledgments

We thank Ms. H. Mizuguchi-Takase for the histological support. This work was supported by The Japanese Health Sciences Foundation (No. KH21006) to T.N., and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 17700370) to T.N. and (Nos 16650089 and 17200027) to M.Y.

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