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NEUROPHARMACOLOGY

Increased C-Fiber Nociceptive Input Potentiates Inhibitory Glycinergic Transmission in the Spinal Dorsal Horn

Department of Anesthesiology and Pain Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas (H.-Y.Z., H.-M.Z., S.-R.C., H.-L.P.); and Program in Neuroscience, University of Texas Graduate School of Biomedical Sciences, Houston, Texas (H.-L.P.)

Received for publication October 23, 2007
Accepted December 11, 2007.

	Abstract

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Glycine is an important inhibitory neurotransmitter in the spinal cord, but it also acts as a coagonist at the glycine site of N-methyl-D-aspartate (NMDA) receptors to potentiate nociceptive transmission. However, little is known about how increased nociceptive inflow alters synaptic glycine release in the spinal dorsal horn and its functional significance. In this study, we performed whole-cell recordings in rat lamina II neurons to record glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs). The transient receptor potential vanilloid receptor 1 agonist capsaicin caused a prolonged increase in the frequency of sIPSCs in 17 of 25 (68%) neurons tested. The potentiating effect of capsaicin on sIPSCs was blocked by ionotropic glutamate receptor antagonists or tetrodotoxin in most lamina II neurons examined. In contrast, the P2X agonist β-methylene-ATP increased sIPSCs in only two of 16 (12.5%) neurons. The glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid either increased or reduced the basal frequency of sIPSCs but did not significantly alter the potentiating effect of capsaicin on sIPSCs. Furthermore, the groups II and III metabotropic glutamate receptor antagonists had no significant effect on the capsaicin-induced increase in the sIPSC frequency. Although capsaicin reduced the amplitude of evoked excitatory postsynaptic currents at high stimulation currents, it did not change the ratio of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/NMDA currents. This study provides the important new information that increased nociceptive inflow augments synaptic glycine release to spinal dorsal horn neurons through endogenous glutamate release. Potentiation of inhibitory glycinergic tone by stimulation of nociceptive primary afferents may function as a negative feedback mechanism to attenuate nociceptive transmission at the spinal level.

TRPV1 receptors expressed in primary sensory neurons play an important role in acute thermal nociception and inflammatory pain (Caterina et al., 1997, 2000). Mice lacking TRPV1 receptors have impaired ability to detect noxious heat (Caterina et al., 2000), and removal of TRPV1-expressing primary afferent neurons diminishes thermal nociception in adult rats (Pan et al., 2003; Chen and Pan, 2006). We have shown that stimulation of nociceptive primary afferents can suppress GABAergic synaptic transmission in the spinal dorsal horn through the activation of groups II and III metabotropic glutamate receptors (mGluRs) (Zhou et al., 2007). Glycinergic interneurons are largely restricted to the spinal cord and brainstem (Rampon et al., 1996; Todd et al., 1996), and glycine-like immunoreactive axons, dendrites, and cell bodies are present in the spinal superficial dorsal horn (Todd, 1990; Todd et al., 1996). In addition to its role in activating postsynaptic glycine receptors, glycine is a coagonist for the glycine binding site of NMDA receptors (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). In this regard, increased glycine levels may enhance nociceptive transmission by enhancing NMDA receptor activity (Vaccarino et al., 1993; Lutfy and Weber, 1996; Ahmadi et al., 2003). P2X receptors are also expressed in a subpopulation of nociceptive dorsal root ganglion neurons and their central terminals in the superficial dorsal horn (Gu and MacDermott, 1997; Tsuda et al., 2000). However, little is known about how stimulation of TRPV1- and P2X-expressing nociceptive afferents influences glycinergic synaptic transmission in the spinal cord.

The aim of this study was to determine how stimulation of TRPV1- and P2X-expressing primary afferents alters synaptic glycine release in the spinal dorsal horn. We also investigated the circuitry involved and the potential functional significance of altered glycinergic input to dorsal horn neurons after stimulation of TRPV1-expressing primary afferents. The data from this study demonstrate that brief stimulation of nociceptive primary afferents induces a sustained increase in synaptic glycine release to most lamina II neurons, which probably contributes to the dynamic modulation of nociceptive inflow at the spinal level.

	Materials and Methods

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Spinal Cord Slice Preparation. Male Sprague-Dawley rats (3–4 weeks old; Harlan, Indianapolis, IN) were used in this study. The surgical preparations and experimental protocols were approved by the Animal Care and Use Committee of the University of Texas M.D. Anderson Cancer Center. Under 2 to 3% isoflurane anesthesia, the lumbar segment of the spinal cord was removed through laminectomy. The spinal tissue was immediately placed in ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O₂ and 5% CO₂. The sucrose aCSF contained 234 mM sucrose, 3.6 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 12.0 mM glucose, and 25.0 mM NaHCO₃. The tissue was then placed in a shallow groove formed in a gelatin block and glued onto the stage of a Vibratome (Technical Products International, St. Louis, MO). Transverse spinal cord slices (400 µm) were cut in the ice-cold sucrose aCSF and preincubated in Krebs' solution oxygenated with 95% O₂ and 5% CO₂ at 34°C for at least 1 h before they were transferred to the recording chamber. The Krebs' solution contained 117.0 mM NaCl, 3.6 mM KCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂, 1.2 mM NaH₂PO₄, 11.0 mM glucose, and 25.0 mM NaHCO₃.

Electrophysiological Recordings. Recordings of postsynaptic currents were performed using the whole-cell voltage-clamp method, as we described previously (Li et al., 2002; Pan and Pan, 2004; Zhang et al., 2005). The slice was placed in a glass-bottomed chamber (Warner Instruments, Hamden, CT) and fixed with parallel nylon threads supported by a U-shaped stainless steel weight. The slice was continuously perfused with Krebs' solution at 5.0 ml/min at 34°C maintained by an inline solution heater and a temperature controller (TC-324; Warner Instruments). The lamina II is identified as a distinct translucent band across the superficial dorsal horn under a microscope with transmitted illumination. The neurons in the lamina II in the spinal slice were identified under a fixed-stage microscope (BX50WI; Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. The electrode for the whole-cell recordings was pulled from borosilicate glass capillaries with a puller (P-97; Sutter Instruments, Novato, CA). The impedance of the pipette was 3 to 5 M when filled with internal solution containing 110 mM Cs₂SO₄, 5 mM TEA, 2.0 mM MgCl₂, 0.5 mM CaCl₂, 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM ATP-Mg, mM 0.5 Na-GTP, 1 mM guanosine 5'-O-(2-thiodiphosphate), and 10 mM lidocaine N-ethyl bromide, adjusted to pH 7.2 to 7.4 with 1 M CsOH (290–300 mOsm). Lidocaine N-ethyl bromide was added into the internal solution to suppress the action potential generation from the recorded cell.

Recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) began approximately 6 min after whole-cell access was established and the current reached a steady state. The input resistance was monitored, and the recording was abandoned if it changed more than 15%. Signals were recorded using an amplifier (MultiClamp 700B; Axon Instruments, Union City, CA) at a holding potential of 0 mV, filtered at 1 to 2 kHz, digitized at 10 kHz, and stored into a computer with pCLAMP 9.2 (Axon Instruments). All glycinergic sIPSCs and mIPSCs were recorded in the presence of 10 µM bicuculline, a GABA_A receptor antagonist. Although GABA_C receptor mRNA and protein are expressed on the motoneurons in the spinal cord, they are not present in the superficial spinal dorsal horn (Rozzo et al., 2002). Furthermore, many studies have documented that IPSCs recorded in the spinal dorsal horn are only mediated by GABA_A and glycine receptors (Gerber et al., 2000; Kerchner et al., 2001; Zhang et al., 2005; Wang et al., 2006). Tetrodotoxin (1 µM) was added into the perfusion solution to record the mIPSCs. To determine whether increased glycine release by capsaicin can enhance NMDA receptor activity, we used electrical stimulation (0.2 Hz) of the dorsal root entry zone (Li et al., 2002) to evoke NMDA and AMPA excitatory postsynaptic currents (EPSCs). The impedance of the pipette was 5 to 10 M when filled with internal solution containing 135.0 mM potassium gluconate, 5.0 mM TEA, 2.0 mM MgCl₂, 0.5 mM CaCl₂, 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM ATP-Mg, 0.5 mM Na-GTP, and 10 mM lidocaine N-ethyl bromide, adjusted to pH 7.2 to 7.4 with 1 M KOH (290–300 mOsm). The AMPA-EPSCs were recorded at a holding potential of –60 mV in the presence of 10 µM bicuculline and 1 µM strychnine. The NMDA-EPSCs were recorded at +40 mV in the presence of 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 µM bicuculline, and 1 µM strychnine (Panatier et al., 2006).

Capsaicin, β-methylene-ATP, guanosine 5'-O-(2-thiodiphosphate), CNQX, and MK801 were obtained from Sigma-Aldrich (St. Louis, MO). Bicuculline, strychnine, (RS)--cyclopropyl-4-phosphonophenylglycine (CPPG), LY341495, and L-trans-pyrrolidine-2,4-dicarboxylic acid (L-PDC) were purchased from Tocris Cookson Inc. (Ellisville, MO). TTX and lidocaine N-ethyl bromide were obtained from Alomone Labs (Jerusalem, Israel). Drugs were dissolved in Krebs' solution and perfused into the slice chamber by using syringe pumps.

Data Analysis. Data are presented as means ± S.E.M. The glycinergic sIPSCs and mIPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft, Decatur, GA). Measurements of the amplitude and frequency of sIPSCs and mIPSCs were performed over a period for at least 1 min during the control, drug application, and recovery. The sIPSCs and mIPSCs were detected by the fast rise time of the signal over an amplitude threshold (typically 6–8 pA) above the background noise. We manually excluded the event when the noise was erroneously identified as the sIPSCs by the software program. The cumulative probability of the amplitude and the interevent interval of sIPSCs were compared using the Kolmogorov-Smirnov test. This test was used to determine whether the drug effect on sIPSCs and mIPSCs was significantly different. The background noise level was constant throughout the recording of the same neuron. We used the same threshold level to analyze the cumulative plot data before and after drug application in a given cell. The amplitude of evoked AMPA-EPSCs and NMDA-EPSCs was analyzed off-line with Clampfit 9.2 (Axon Instruments). The effect of capsaicin was determined by two-tailed Student's t test or one-way analysis of variance. The influence of L-PDC and groups II and III mGluR antagonists on the potentiating effect (percentage increase) of capsaicin on the frequency of sIPSCs was analyzed using the Mann-Whitney test. P < 0.05 was considered statistically significant.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Effect of capsaicin on glycinergic sIPSCs in spinal lamina II neurons. A, original traces showing sIPSCs during control, application of 2 µM capsaicin, and washout in an increase-type neuron. Note that 1 µM strychnine abolished the sIPSCs. B, cumulative plot analysis of sIPSCs of the same neuron showing the distribution of the interevent interval and amplitude of sIPSCs during control, application of 2 µM capsaicin, and washout. C, summary data showing different effects of capsaicin on the frequency and amplitude of sIPSCs in a total of 25 lamina II neurons. Capsaicin increased the sIPSC frequency in 17 neurons but had no effect in the other eight neurons. D, summary data showing different effects of capsaicin on the frequency and amplitude of mIPSCs in a total of 22 lamina II neurons. Capsaicin increased the mIPSC frequency in five neurons but had no effect in the other 17 neurons. *, P < 0.05 compared with the respective control.

	Results

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Effect of Capsaicin on Glycinergic mIPSCs in the Spinal Dorsal Horn. To determine whether increased glycine release is due to the excitation of glycinergic and glutamatergic interneurons by nociceptive primary afferents (presynaptic receptors versus excitability), we recorded the glycinergic mIPSCs of lamina II neurons. In 17 of 22 (77.3%) lamina II neurons, capsaicin did not significantly alter the frequency and amplitude of glycinergic mIPSCs in the presence of 1 µM TTX (Fig. 1D). In the remaining five (22.7%) cells, capsaicin still significantly increased the frequency of glycinergic mIPSCs (Fig. 1D). These data suggest that stimulation of TRPV1-expressing afferents increases synaptic glycine release primarily through the increased excitability of glutamatergic and/or glycinergic interneurons in the spinal dorsal horn.

View larger version (49K): [in this window] [in a new window]   Fig. 2. Effect of capsaicin on glycinergic sIPSCs of spinal lamina II neurons in the presence of CNQX and MK801. A, raw traces showing sIPSCs during control, application of 20 µM CNQX and 20 µM MK801 alone, and 2 µM capsaicin plus CNQX and MK801 in one neuron. B, cumulative plot analysis of sIPSCs of the same neuron showing the distribution of the interevent interval and amplitude of sIPSCs during control and application of 2 µM capsaicin in the presence of CNQX and MK801. C, summary data showing different effects of capsaicin on the frequency and amplitude of sIPSCs in a total of 21 lamina II neurons in the presence of 20 µM CNQX and 20 µM MK801. When CNQX and MK801 were present, capsaicin increased the sIPSC frequency only in three neurons but had no effect in the other 18 neurons. *, P < 0.05 compared with the control. Caps, capsaicin.

Effect of β-Methylene-ATP on Glycinergic sIPSCs in the Spinal Dorsal Horn. Because P2X receptors can stimulate a subpopulation of nociceptive primary afferent neurons and their central terminals in the spinal dorsal horn (Gu and MacDermott, 1997; Tsuda et al., 2000), we next examined how stimulation of P2X-expressing primary afferents alters synaptic glycine release to lamina II neurons. Bath application of 100 µM β-methylene-ATP, a P2X receptor agonist (Gu and MacDermott, 1997; Rhee et al., 2000), for 3 min increased the frequency of glycinergic sIPSCs in only two of 16 (12.5%) neurons tested (Fig. 3). The latency of the potentiating effect of β-methylene-ATP was 1 to 2 min. The effect of β-methylene-ATP lasted for 5.83 min in one neuron and 7.55 min in another neuron. However, in the majority of (14 of 16, 87.5%) neurons examined, 100 µM β-methylene-ATP had no significant effect on glycinergic sIPSCs (Fig. 3). These results suggest that most of P2X-expressing primary afferents do not synapse directly with glycinergic interneurons in the lamina II. The following protocols were focused on capsaicin-induced glycine release in the spinal dorsal horn.

View larger version (37K): [in this window] [in a new window]   Fig. 3. Effect of β-methylene-ATP on glycinergic sIPSCs in lamina II neurons. A, frequency histogram showing the time course and duration of the effect of 100 µM β-methylene-ATP on glycinergic sIPSCs in an increase-type neuron. B, original traces showing sIPSCs recorded during control and application of β-methylene-ATP in the same neuron in A. C, summary data showing lack of the effect of 100 µM β-methylene-ATP on the frequency and amplitude of sIPSCs in 14 of 16 lamina II neurons.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Effect of capsaicin on glycinergic sIPSCs of spinal lamina II neurons in the presence of L-PDC. A, representative traces showing sIPSCs during control, application of 10 µM L-PDC alone, and 2 µM capsaicin plus 10 µM L-PDC in one neuron. B, cumulative plot analysis of sIPSCs of the same neuron showing the distribution of the interevent interval and amplitude of sIPSCs during control and application of 2 µM capsaicin plus 10 µM L-PDC. C, summary data showing the effect of 10 µM L-PDC alone and 10 µM L-PDC plus 2 µM capsaicin on the frequency of sIPSCs in a total of 25 lamina II neurons. L-PDC (10 µM) alone decreased sIPSC frequency in 16 neurons, increased sIPSC frequency in four neurons, and had no effect in the remaining five neurons. In the presence of L-PDC, capsaicin increased the sIPSC frequency in 16 neurons but had no effect in the other nine neurons. D, summary data showing the effect of 1 mM L-PDC alone and 1 mM L-PDC plus 2 µM capsaicin on the frequency of sIPSCs in a total of eight lamina II neurons. L-PDC (1 mM) alone decreased sIPSC frequency in four neurons, increased sIPSC frequency in three neurons, and had no effect in the remaining one neuron. In the presence of L-PDC, capsaicin increased the sIPSC frequency in five neurons but had no effect in the other three neurons. E, summary data showing lack of inhibitory effect of 10 µM L-PDC on sIPSCs in the presence of CPPG and LY341495. *, P < 0.05 compared with the respective control. #, P < 0.05 compared with the value obtained with L-PDC alone. Caps, capsaicin.

We also determined the effect of a higher concentration of L-PDC in eight separate lamina II neurons. Bath application of 1 mM L-PDC significantly decreased the glycinergic sIPSC frequency in four of eight (50%) neurons but significantly increased the frequency of sIPSCs in three of eight (37.5%) neurons tested (Fig. 4D). In the remaining one (12.5%) neuron, 1 mM L-PDC had no evident effect on sIPSCs. In five of the above eight (62.5%) neurons, 2 µM capsaicin still significantly increased the frequency of glycinergic sIPSCs in the presence of 1 mM L-PDC (Fig. 4D). The percentage increase (236.8 ± 88.2%) in the sIPSC frequency by capsaicin was not significantly different from that obtained in the absence of L-PDC. In the other three neurons, 2 µM capsaicin had no significant effect on glycinergic sIPSCs (Fig. 4D).

L-PDC increases synaptic glutamate levels by inhibition of its uptake, and this leads to activation of inhibitory group II and III mGluRs; as a consequence, synaptic glutamate release is reduced (Bird et al., 2001). Thus, we determined whether these mGluRs play a role in the inhibitory effect of L-PDC on glycinergic sIPSCs. Bath application of 200 µM CPPG and 100 nM LY341495, which block groups II and III mGluRs (Jane et al., 1996; Chung et al., 1997), respectively, had no significant effect on the basal frequency of glycinergic sIPSCs in another eight lamina II neurons. Subsequent application of 10 µM L-PDC failed to reduce glycine release in the presence of 100 nM LY341495 and 200 µM CPPG in all neurons tested (Fig. 4E). These findings suggest that an increase in synaptic glutamate levels and spillover of glutamate can activate presynaptic groups II and III mGluRs to reduce the basal release of glycine. However, increased synaptic glutamate level has little effect on glycine release triggered by stimulation of TRPV1-expressing primary afferents.

Role of Presynaptic Glutamate Receptors in the Control of Glycine Release in the Spinal Dorsal Horn. To examine the role of presynaptic glutamate receptors in the control of synaptic glycine release, we determined the effect of exogenous glutamate on glycinergic mIPSCs in the presence of 1 µM TTX. In seven of the 14 (50%) lamina II neurons examined, bath application of 1 mM glutamate for 3 min significantly decreased the frequency of mIPSCs (Fig. 5). In contrast, 1 mM glutamate increased significantly the frequency of glycinergic mIPSCs in three of 14 (21.4%) neurons. In the remaining four (28.6%) neurons, 1 mM glutamate had no significant effect on mIPSCs (Fig. 5C). These results suggest that different presynaptic ionotropic and metabotropic glutamate receptors may have distinct roles in the control of synaptic glycine release to dorsal horn neurons.

View larger version (35K): [in this window] [in a new window]   Fig. 5. Effect of exogenous glutamate on glycinergic mIPSCs in spinal lamina II neurons. A, raw traces showing mIPSCs during control and application of 1 mM glutamate in a decrease-type neuron. Cumulative plot analysis of mIPSCs of the same neuron showing the distribution of the interevent interval and amplitude of mIPSCs during control and application of 1 mM glutamate. B, representative records showing sIPSCs during control and application of 1 mM glutamate in an increase-type neuron. Cumulative plot analysis of mIPSCs of the same neuron showing the distribution of the interevent interval and amplitude of mIPSCs during control and application of 1 mM glutamate. C, summary data showing the different effects of 1 mM glutamate on the frequency and amplitude of mIPSCs in a total of 14 lamina II neurons. Glutamate decreased sIPSC frequency in seven neurons, increased sIPSC frequency in three neurons, and had no effect in the remaining four neurons. *, P < 0.05 compared with the control.

Role of Groups II and III mGluRs in the Potentiating Effect of Capsaicin on Glycine Release. Activation of groups II and III mGluRs suppresses glycine release in the spinal dorsal horn (Gerber et al., 2000). Capsaicin evokes a large increase in endogenous glutamate release (Pan and Pan, 2004), which could activate presynaptic groups II and III mGluRs in the spinal cord to limit further increase in glycine release by capsaicin. To assess this possibility, we examined the role of groups II and III mGluRs in capsaicin-induced glycine release in the spinal dorsal horn. In the presence of 200 µM CPPG and 100 nM LY341495, 2 µM capsaicin significantly increased the frequency, but not the amplitude, of glycinergic sIPSCs in 16 of 21 (76.2%) neurons studied (Fig. 6). However, the potentiating effect of capsaicin on glycinergic sIPSCs in the presence of CPPG and LY341495 was not significantly different from that without CPPG and LY341495 (206.5 ± 38.4% versus 229.9 ± 47.5% increase, P > 0.05). Thus, it seems that groups II or III mGluRs do not play a major role in the control of synaptic glycine release evoked by stimulation of TRPV1-expressing primary afferents in the spinal dorsal horn.

View larger version (46K): [in this window] [in a new window]   Fig. 6. Effect of capsaicin on glycinergic sIPSCs of spinal lamina II neurons in the presence of CPPG and LY341495. A, original traces showing sIPSCs during control, application of 200 µM CPPG and 100 nM LY341495 alone, and 2 µM capsaicin plus CPPG and LY341495 in one neuron. B, cumulative plot analysis of sIPSCs of the same neuron showing the distribution of the interevent interval and amplitude during control and application of 2 µM capsaicin in the presence of CPPG and LY341495. C, summary data showing different effects of capsaicin on the frequency and amplitude of sIPSCs in a total of 21 lamina II neurons in the presence of 200 µM CPPG and 100 nM LY341495. When CPPG and LY341495 were present, capsaicin increased the sIPSC frequency in 16 neurons but had no effect in the other five neurons. *, P < 0.05 compared with the control. Caps, capsaicin.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effect of capsaicin on the amplitude of evoked NMDA and AMPA receptor-mediated currents in lamina II neurons. A, original traces showing evoked AMPA-EPSCs and NMDA-EPSCs during control and application of 2 µM capsaicin at low-intensity stimulation in one neuron. B, summary data showing the effect of capsaicin on the amplitude of evoked NMDA and AMPA receptor-mediated currents and their ratio at low-intensity stimulation (n = 8 neurons). C, original traces showing evoked AMPA-EPSCs and NMDA-EPSCs during control and application of 2 µM capsaicin at high-intensity stimulation in one neuron. D, summary data showing the effect of capsaicin on the amplitude of evoked NMDA and AMPA receptor-mediated currents and their ratio at high-intensity stimulation (n = 10 neurons). *, P < 0.05 compared with the control.

	Discussion

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In this study, we determined the influence of stimulating TRPV1- and P2X-expressing nociceptive primary afferents on synaptic glycine release to the spinal dorsal horn neurons. We found that stimulation of nociceptive afferents with capsaicin caused a sustained increase in the frequency of glycinergic sIPSCs in most lamina II neurons. In contrast, activation of P2X-expressing primary afferents with β-methylene-ATP increased the frequency of glycinergic sIPSCs in only two of 16 lamina II neurons. In addition, the iGluR antagonists or TTX largely blocked the potentiating effect of capsaicin on glycinergic sIPSCs. Although inhibition of glutamate reuptake with the glutamate transporter inhibitor L-PDC reduced glycinergic sIPSCs in many lamina II neurons through activation of groups II and III mGluRs, L-PDC did not significantly alter the capsaicin effect on glycinergic sIPSCs. Groups II and III mGluR antagonists did not alter the potentiating effect of capsaicin on glycinergic sIPSCs in the spinal cord. Furthermore, capsaicin failed to significantly change the ratio of AMPA/NMDA currents evoked by stimulation of primary afferents in lamina II neurons. Therefore, these findings suggest that the inhibitory glycinergic tone in the spinal dorsal horn is increased through endogenous glutamate release after stimulation of nociceptive primary afferents. Increased synaptic glycine release probably serves as an inhibitory input to modulate nociceptive transmission at the spinal level.

The spinal lamina II is an important integration site for the relay and processing of nociceptive information (Cervero and Iggo, 1980; Yoshimura and Nishi, 1995; Lu and Perl, 2003; Pan and Pan, 2004). Because the spinal cord slices were used to measure synaptic glycine release in real time, it is not possible to apply "physiological stimuli" in this in vitro preparation. Thus, we chose to stimulate TRPV1 and P2X receptors to elicit nociceptive input to dorsal horn neurons in the spinal cord slice. The capsaicin receptors (TRPV1 channels) are localized largely to the central terminals of primary afferent neurons in the superficial dorsal horn (Guo et al., 2001; Pan et al., 2003; Chen and Pan, 2006). In contrast to our recent study showing that capsaicin suppresses GABAergic sIPSCs in 50% of lamina II neurons (Zhou et al., 2007), we found in our present study that brief application of capsaicin produced no inhibitory effect on glycinergic sIPSCs but instead caused a sustained potentiation of glycinergic sIPSCs in approximately 70% of lamina II neurons examined. These data strongly suggest that stimulation of TRPV1-expressing primary afferents can rapidly (within minutes) enhance the inhibitory glycinergic tone in the spinal cord. It has been suggested that glycine and GABA may be coreleased from the same synaptic terminals in the spinal cord (Jonas et al., 1998). Nevertheless, some important technical differences exist between this and the previous studies. The study by Jonas et al. (1998) used synaptically coupled pairs of interneurons and putative motor neurons in the deep spinal laminae, although in our present study, the recording was performed on lamina II neurons in the spinal cord slices. It is important to note that synaptic GABA and glycine release to lamina II neurons is differentially regulated after stimulation of muscarinic acetylcholine receptors (Zhang et al., 2005; Wang et al., 2006). The present study and our previous work (Zhou et al., 2007) provide further evidence that a different and more complex mechanism is responsible for differential regulation of synaptic release of these two inhibitory neurotransmitters in the spinal dorsal horn when nociceptive input is increased.

Immunocytochemical and electrophysiological studies have documented that TRPV1-expressing primary afferent terminals are glutamatergic (Hwang et al., 2004; Pan and Pan, 2004). A longer onset latency of the capsaicin effect on glycinergic sIPSCs would suggest that the effect of capsaicin on glycinergic transmission is probably an indirect effect secondary to increased glutamatergic input. As shown in this study, the iGluR antagonists abolished most of the stimulating effect of capsaicin on glycinergic sIPSCs. This finding suggests that the capsaicin-induced potentiation of glycine release is critically dependent upon endogenous glutamate release and iGluR activation. It has been reported that capsaicin has no effect on the glycinergic IPSCs in lamina II neurons (Yang et al., 1998). The possible reason for the lack of capsaicin effect on synaptic glycine release in that study may be the short duration (30 s) of capsaicin application. In our study, we found that the potentiating effect of capsaicin on glycinergic sIPSCs was largely blocked by TTX. Thus, increased glycine release by capsaicin occurs largely through the increased excitability of glutamatergic and/or glycinergic interneurons in the spinal dorsal horn. However, the effect of capsaicin on glycine release was still observed in the presence of iGluR antagonists or TTX in a few neurons. In this subpopulation of lamina II neurons, the glycinergic terminals may receive direct input from primary afferents. Alternatively, a neurotransmitter other than glutamate, such as substance P (Vergnano et al., 2004) or ATP (Rhee et al., 2000; Jang et al., 2001), may be involved in the potentiating effect of capsaicin on synaptic glycine release in a small population of lamina II neurons. In fact, when β-methylene-ATP was used to activate ionotropic P2X (not metabotropic P2Y) receptors in the spinal cord slice preparation, we did find that it increased the frequency of glycinergic sIPSCs in about 10% of lamina II neurons studied. It should be noted that all P2X subtypes, except the P2X7, are expressed on the primary sensory neurons (Collo et al., 1996; Vulchanova et al., 1997). The P2X2, P2X4, and P2X6 subunits are also expressed in the superficial laminae of the spinal dorsal horn (Collo et al., 1996; Vulchanova et al., 1997). Because β-methylene-ATP has little effect on P2X2 receptors, the potential role of the P2X2 receptor-expressing afferent terminals in the regulation of spinal glycine release was not specifically determined in our study.

Groups II and III mGluRs are involved in the regulation of glutamatergic and GABAergic/glycinergic transmission in the spinal dorsal horn (Gerber et al., 2000; Zhou et al., 2007). Increased nociceptive inflow increases glutamate release (Pan and Pan, 2004), which, in turn, activates both groups II and III mGluRs expressed on GABAergic interneurons to reduce GABAergic transmission in the spinal dorsal horn (Zhou et al., 2007). In the present study, we observed that blocking groups II and III mGluRs had no significant effect on capsaicin-induced synaptic glycine release. Because glutamate is closely involved in synaptic glycine release, an increase in synaptic glutamate level would be expected to potentiate synaptic glycine release by capsaicin. To test this hypothesis, we used the glutamate transporter inhibitor L-PDC to elevate the endogenous glutamate levels (Bird et al., 2001). Interestingly, we found that at low and high concentrations, L-PDC alone either increased or decreased the basal frequency of glycinergic sIPSCs in lamina II neurons. Because the inhibitory effect of L-PDC was completely blocked by groups II and III mGluR antagonists, it seems that activation of these mGluRs by endogenous glutamate can reduce basal glycine release in the spinal cord. Thus, a properly maintained glutamate level in the synaptic cleft is critical for the potentiation of glycinergic transmission by capsaicin. Similar to the effect of L-PDC, we found that exogenously applied glutamate either increased or decreased the basal frequency of glycinergic mIPSCs. Activation of presynaptic AMPA receptors (Engelman et al., 2006) and kainate receptors (Kerchner et al., 2001) can increase glycine release to lamina II neurons. In addition, stimulation of presynaptic group I mGluRs increases synaptic glutamate release to lamina II neurons (Park et al., 2004) and may play a role in the potentiating effect of glutamate on glycinergic mIPSCs. On the other hand, activation of presynaptic groups II and III mGluRs inhibits glycine release to lamina II neurons (Gerber et al., 2000). These results suggest that different ionotropic and metabotropic glutamate receptors expressed on the presynaptic terminals of glycinergic neurons may be involved in the complex regulation of glycine release (Fig. 8). Because the potentiating effect of capsaicin on synaptic glycine release was not significantly altered by L-PDC, it seems that groups II and III mGluRs may not play a major role in the control of glycine release triggered by stimulation of nociceptive primary afferents.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Schematic drawing illustrating the interaction between glutamatergic and glycinergic synapses in the spinal dorsal horn in response to increased nociceptive input. Stimulation of TRPV1-expressing primary afferents evokes glutamate release onto glycinergic interneurons from primary afferent terminals (and possibly glutamatergic interneurons). Subsequently, glutamate activates iGluRs located on the glycinergic interneurons. As a result, excitation of glycinergic interneurons increases glycine release to a postsynaptic lamina II neuron. In a subpopulation of lamina II neurons, primary afferents can synapse directly with the terminal of glycinergic neurons to either increase (via AMPA, kainate receptors, or group I mGluRs) or decrease (via groups II/III mGluRs) glycine release. Note that stimulation of TRPV1-expressing primary afferents also can release other neurotransmitters such as substance P and ATP to glycinergic interneurons and their terminals (data not shown).

In summary, this study provides new evidence for the dynamic interactions between glutamatergic and glycinergic synapses in the spinal dorsal horn after stimulation of nociceptive primary afferents. Stimulation of TRPV1-nociceptive afferents (and to a lesser extent, P2X-expressing primary afferents) largely leads to increased glycinergic input to lamina II neurons. As illustrated in Fig. 8, the endogenous glutamate and iGluRs located on glycinergic interneurons are principally responsible for increased glycinergic input to dorsal horn neurons after the stimulation of TRPV1-expressing primary afferents. Increased glutamatergic input is directly linked to the augmentation of inhibitory glycinergic tone in the spinal dorsal horn. We found no evidence that synaptic glycine release by stimulation of nociceptive primary afferents can increase NMDA receptor activity. Therefore, increased glycinergic input by stimulation of TRPV1-expressing primary afferents probably functions as a negative feedback mechanism to attenuate nociceptive transmission at the spinal level. After acute stimulation of nociceptive afferents, the initial pain often subsides. For example, s.c. injection of formalin induces a characteristic biphasic pain response in animals (Vaccarino and Melzack, 1992). Our study provides new insights into the mechanism and circuitry involved in the dynamic modulation of nociceptive inflow at the spinal level.

	Footnotes

 
This study was supported by National Institutes of Health Grants GM64830 and NS45602.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.133470.

ABBREVIATIONS: iGluR, ionotropic glutamate receptor; TRPV, transient receptor potential vanilloid receptor; mGluR, metabotropic glutamate receptor; NMDA, N-methyl-D-aspartate; aCSF, artificial cerebrospinal fluid; sIPSC, spontaneous inhibitory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; EPSC, excitatory postsynaptic current; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; MK801, (5R,10S)-(–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cylcohepten-5,10-imine maleate; CPPG, (RS)--cyclopropyl-4-phosphonophenylglycine; LY341495, (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid; L-PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; TTX, tetrodotoxin.

Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology and Pain Medicine, Unit 110, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: huilinpan{at}mdanderson.org

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