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1Department of Anesthesiology and Pain Medicine, The University of Texas M.D. Anderson Cancer Center; and 2Program in Neuroscience, The University of Texas Graduate School of Biomedical Sciences, Houston, Texas
Submitted 8 September 2006; accepted in final form 14 November 2006
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ABSTRACT |
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INTRODUCTION |
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; Torebjork et al. 1992; Woolf et al. 1988). The release of inflammatory mediators from injured tissues and inflammatory cells, including prostaglandins (Martin et al. 1987; Trebino et al. 2003) and bradykinin (Liang et al. 2001; Rueff and Dray 1993), can stimulate and sensitize nociceptors (peripheral sensitization). Furthermore, increased release of glutamate from primary afferents and activation of postsynaptic ionotropic glutamate receptors (iGluRs) in the spinal cord can enhance the excitability of dorsal horn neurons (central sensitization) (Dougherty et al. 1992; Leem et al. 1996). However, the cellular mechanisms for the hypersensitivity of dorsal horn neurons after tissue and nerve injury are not fully known.
The lamina II of the spinal cord is a critical site for the relay and processing of dynamic modulation of sensory information. Yet the detailed circuitry and synaptic control of lamina II neurons involved in the integration and regulation of nociceptive input are largely unclear. Both excitatory and inhibitory interneurons in the lamina II can modulate the input of primary afferents through presynaptic and postsynaptic mechanisms (Lu and Perl 2003; Pan and Pan 2004; Yoshimura and Nishi 1995). Glutamate is an important excitatory neurotransmitter and iGluRs are essential for nociceptive transmission and sensitization of dorsal horn neurons in the spinal cord (Dougherty et al. 1992; Leem et al. 1996; Lu and Perl 2003; Pan and Pan 2004; Yoshimura and Nishi 1995). By contrast, 
-aminobutyric acid (GABA) is the dominant inhibitory neurotransmitter in the spinal cord and spinal nociceptive transmission and dorsal horn neurons in different laminae are under tonic inhibitory control mediated largely by GABA (Light and Kavookjian 1988; Pan and Pan 2004; Yoshimura and Nishi 1995). In this regard, blockade of GABAA receptors results in hypersensitivity of dorsal horn neurons and allodynia (Sivilotti and Woolf 1994; Sorkin et al. 1998). Reduced GABAergic input to lamina II neurons is considered an important contributor to chronic neuropathic pain (Moore et al. 2002). The primary afferent terminals and the glutamatergic and GABAergic interneurons are closely intermingled in the superficial dorsal horn. Although stimulation of primary afferents potentiates glutamatergic transmission in second-order and higher-order neurons in the spinal dorsal horn, little is known whether, and how, increased nociceptive input affects local inhibitory GABAergic input to dorsal horn neurons.
The aim of this study was to determine whether, and how, stimulation of nociceptive primary afferents rapidly modulates GABAergic synaptic transmission in the spinal cord. We found that glutamate spillover after primary afferent stimulation produces a heterosynaptic action to affect GABAergic transmission. Reduction of GABAergic tone can occur rapidly (within minutes) after stimulation of primary afferents, which could facilitate transmission of nociceptive information in the spinal cord. Through group II and group III metabotropic glutamate receptors (mGluRs) on GABAergic interneurons, modulation of spinal GABAergic tone is directly linked to increased glutamatergic input from nociceptive primary afferents. This new information is important for our understanding of the integrative mechanisms underlying the regulation of nociceptive inflow by spinal dorsal horn neurons.
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METHODS |
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Male Sprague–Dawley rats (3–4 wk old; Harlan, Indianapolis, IN) were used in this study. All 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. Some rats were treated with intrathecal pertussis toxin (PTX) to inactivate inhibitory Gi/o proteins (Chen and Pan 2004; Zhang et al. 2005). Intrathecal catheters were inserted in rats anesthetized using 2–3% isoflurane. The catheters (polyethylene-10 tubing) were inserted through an incision in the cisternal membrane and advanced 4.5 cm caudal so that the tip of each catheter was positioned at the lumbar spinal level. Rats were injected intrathecally with 1 µg of PTX 5–7 days before the final electrophysiology experiments.
Spinal cord slice preparation
Under isoflurane anesthesia, the lumbar segment of the spinal cord was removed through laminectomy at the L2–L5 level. The segment of the spinal cord was immediately placed in an ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O2-5% CO2. The sucrose aCSF contained (in mM) 234 sucrose, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 12.0 glucose, and 25.0 NaHCO3. The tissue was then placed in a shallow groove formed in a gelatin block and glued onto the stage of a vibratome (Technical Product International, St. Louis, MO). Transverse spinal cord slices (400 µm) were cut in the ice-cold sucrose aCSF and incubated in Krebs' solution oxygenated with 95% O2-5% CO2 at 34°C for 
1 h before they were transferred to the recording chamber. The Krebs' solution contained (in mM) 117.0 NaCl, 3.6 KCl, 1.2 MgCl2, 2.5 CaCl2, 1.2 NaH2PO4, 11.0 glucose, and 25.0 NaHCO3.
Electrophysiological recordings
Recordings of postsynaptic currents were performed using the whole cell voltage-clamp method, as previously described (Li et al. 2002; Pan and Pan 2004; Zhang et al. 2005). The slice was placed in a glass-bottom chamber (Warner Instrument, Hamden, CT) and fixed with parallel nylon threads supported by a U-shape 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 Instrument). The lamina II was 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 cord slice were visualized under a fixed-stage microscope (BX50WI; Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. Lamina II neurons with a smooth surface were selected for recordings from the translucent region of the spinal dorsal horn (typically 50–150 µm from the dorsal surface the spinal dorsal horn). Neurons in both the outer and inner zones of lamina II were selected for recording, as previously described in detail (Pan and Pan 2004). 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 3–5 M
when filled with internal solution containing (in mM) 110 Cs2SO4, 5 TEA, 2.0 MgCl2, 0.5 CaCl2, 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, and 10 lidocaine N-ethyl bromide, adjusted to pH 7.2–7.4 with 1 M CsOH (290–320 mOsm). Lidocaine N-ethyl bromide was added to the internal solution to suppress the action potential generation from the recorded cell.
Recordings of spontaneous inhibitory postsynaptic currents (sIPSCs) at the holding potential of 0 mV began about 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 >15%. Signals were recorded using an amplifier (MultiClamp 700B; Axon Instruments, Union City, CA), filtered at 1–2 kHz, digitized at 10 kHz, and stored in a computer with pCLAMP 9.2 (Axon Instruments). All GABAergic sIPSCs and the current elicited by puff application of GABA were recorded in the presence of 0.5 µM strychnine, a glycine receptor antagonist. The internal solution for recording of the GABAA current elicited by puff application of GABA contained (in mM) 130 KCl, 1.0 MgCl2, 1.0 CaCl2, 5.0 HEPES, 5.0 ATP-Mg, 0.5 Na-GTP, 1 guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S), and 10 lidocaine N-ethyl bromide, adjusted to pH 7.2 to 7.4 with 1 M KOH (290–300 mOsm).
Capsaicin, GDP--S, strychnine, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), Dizocilpine (MK-801), and GABA were obtained from Sigma–Aldrich (St. Louis, MO). (R,S)-Cyclopropyl-4-phosphonophenylglycine (CPPG) and (2S,1'S,2'S)-2-(2-carboxycyclopropyl)-2-(9H-xanthen-9-yl)glycine (LY341495) were obtained from Tocris Cookson (Ellisville, MO). PTX was purchased from List Biological Laboratories (Campbell, CA). QX314 was 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 ± SE. The sIPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft, Decatur, GA). Measurements of the amplitude and frequency of sIPSCs were performed for 1 min during control, drug application, and recovery. The peak effect of capsaicin on sIPSCs was analyzed over a period of 1.5–2 min after capsaicin application and used for the comparison of the capsaicin effect with the baseline value. The sIPSCs were detected by the fast rise time of the signal over an amplitude threshold above the background noise signal (typically 6–10 pA). We manually excluded the event when the noise was erroneously identified as the sIPSCs by the software program. The background noise level was typically constant throughout the recording of a single neuron. The cumulative probability of the amplitude and interevent interval of sIPSCs before and after drug application was compared using the Kolmogorov–Smirnov test, which estimates the probability if the two cumulative distributions are statistically different. The effects of capsaicin and bradykinin on the amplitude and frequency of sIPSCs were determined by paired two-tailed Student's t-test or one-way ANOVA. The percentage of increase- and decrease-type neurons affected by capsaicin in different protocols was compared using Fisher's exact test. P < 0.05 was considered to be statistically significant.
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RESULTS |
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To test whether GABAergic synaptic transmission in the spinal cord is altered by increased nociceptive input from primary afferents, we tested the effect of capsaicin, a known agonist for TRPV1 channels, on GABAergic sIPSCs of lamina II neurons. TRPV1 is located predominantly on primary afferent neurons and terminals in the superficial laminae of the spinal cord (Chen and Pan 2006; Guo et al. 2001; Pan et al. 2003). We used capsaicin to stimulate primary afferents (Pan and Pan 2004; Yang et al. 1998). Bath application of 2 µM capsaicin for 3 min significantly decreased both the frequency and amplitude of GABAergic sIPSCs in 13 of 26 (50%) neurons recorded (Fig. 1). These 13 neurons consisted of nine inner and four outer lamina II neurons. The cumulative probability analysis of sIPSCs revealed that the distribution pattern of the interevent interval but not the amplitude of sIPSCs was shifted toward the right in response to capsaicin (Fig. 1A). The mean sIPSC frequency and amplitude were reduced 53.0 ± 5.2% (n = 13) and 16.4 ± 3.7% (n = 13), respectively, by capsaicin. In contrast, in nine of the 26 neurons (34.6%), capsaicin significantly increased the frequency but not the amplitude of sIPSCs (Fig. 1, B and C). The latency of both the inhibitory and stimulatory effects of capsaicin was about 1.5 min and these effects lasted for 6 min after capsaicin application was discontinued (Fig. 1). In the remaining four (15.4%) neurons, capsaicin had no significant effect on the frequency and amplitude of sIPSCs (Fig. 1). We did not observe the biphasic effect of capsaicin on sIPSCs. Bath application of 10 µM bicuculline, a GABAA-receptor blocker, abolished sIPSCs of all lamina II neurons tested (Fig. 1). These results suggest that activation of nociceptive primary afferents inhibits GABAergic input to a population of dorsal horn neurons.
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To determine whether changes in the postsynaptic GABAA receptors account for the inhibitory effect of capsaicin on sIPSCs, we recorded the GABAA current elicited by puff application of GABA in lamina II neurons before and after bath application of 2 µM capsaicin for 3–10 min. In all 10 neurons tested, the current elicited by puff application of 1 mM GABA was not significantly altered by capsaicin. The mean amplitude of the current elicited by GABA was 1,169.0 ± 289.7 and 1,142.2 ± 323.5 pA (P > 0.05) before and after capsaicin, respectively (Fig. 2). These data suggest that stimulation of nociceptive primary afferents acutely modulates GABAergic transmission through a presynaptic mechanism.
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To determine whether the Gi/o-protein–coupled receptors are involved in the inhibitory effect of capsaicin on synaptic GABA release, a group of rats was pretreated with intrathecal PTX to inactivate inhibitory Gi/o proteins (Chen and Pan 2004; Zhang et al. 2005). In none of the lamina II neurons from PTX-treated rats did capsaicin cause an inhibition of sIPSCs. In most (23 of 25, 92%) neurons tested, 2 µM capsaicin significantly increased the frequency but not the amplitude of GABAergic sIPSCs and shifted the distribution pattern of the interevent interval of sIPSCs to the left (Fig. 3). In the remaining two (8%) neurons, capsaicin had no evident effect on the frequency and amplitude of GABAergic sIPSCs. These data suggest that the Gi/o-coupled receptors on GABAergic interneurons are involved in the inhibitory effect of capsaicin on GABAergic transmission in the spinal cord.
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Stimulation of primary afferents evokes glutamate release, which activates ionotropic glutamate receptors (iGluRs) on the postsynaptic dorsal horn neurons in the spinal cord (Pan and Pan 2004; Wang et al. 2005; Yoshimura and Nishi 1995). We next examined whether endogenous glutamate release plays a role in the effect of capsaicin on GABAergic transmission in the spinal cord. In PTX-treated rats, in the presence of both 20 µM CNQX [a non–N-methyl-D-aspartate (NMDA) receptor antagonist] and 20 µM MK-801 (an NMDA receptor antagonist), bath application of 2 µM capsaicin failed to inhibit GABAergic sIPSCs in all cells recorded (n = 21, Fig. 4A). Capsaicin did not significantly alter the frequency of sIPSCs in 17 of 21 (81%) cells in the presence of CNQX and MK-801. In the remaining four cells (19%, Fig. 4A), capsaicin still significantly increased the frequency of sIPSCs. These data suggest that the potentiating effect of capsaicin on synaptic GABA release in the spinal cord of PTX-treated rats is primarily caused by increased glutamate release and iGluRs.
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Effect of capsaicin on GABAergic mIPSCs of lamina II neurons
To determine whether the dorsal horn interneurons are involved in the stimulating effect of capsaicin on synaptic GABA release, we tested the effect of capsaicin on GABAergic mIPSCs in 21 separate lamina II neurons. In most neurons (17/21, 81%) tested, bath application of 2 µM capsaicin did not significantly alter the frequency and the amplitude of mIPSCs in the presence of 1 µM of TTX (Fig. 4C). In the remaining four (19%) neurons, 2 µM capsaicin significantly increased the frequency but not the amplitude of mIPSCs (Fig. 4C). Thus increased nociceptive inflow can inhibit synaptic GABA release to most dorsal horn neurons through interneurons.
Role of group II and group III metabotropic glutamate receptors in the inhibitory effect of capsaicin on GABA release
Group II and group III metabotropic glutamate receptors (mGluRs) are Gi/o-protein–coupled receptors (Prezeau et al. 1992; Tanabe et al. 1992). It was previously shown that activation of presynaptic group II and group III mGluRs depresses GABA release in spinal dorsal horn neurons (Gerber et al. 2000). The results described in the previous section suggest that inhibition of synaptic GABA release by capsaicin is mediated by endogenous glutamate release. Also, because the inhibitory effect of capsaicin on synaptic GABA release was abolished by PTX treatment, we hypothesized that the group II and group III mGluRs play a role in the attenuation of synaptic GABA release during stimulation of primary afferents with capsaicin. In the presence of 200 µM CPPG and 100 nM LY341495, which block group II and group III mGluRs, respectively (Conn and Pin 1997; Gerber et al. 2000; Schoepp et al. 1999), 2 µM capsaicin had no inhibitory effect on sIPSCs. In nine of 17 (52.9%) neurons studied, capsaicin significantly increased the frequency of sIPSCs (Fig. 5). These nine neurons consisted of six inner and three outer lamina II neurons. In the remaining eight neurons, capsaicin had no significant effect on the frequency and amplitude of sIPSCs (Fig. 5B).
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Role of group II and group III mGluRs in the inhibitory effect of bradykinin on GABA release
Because capsaicin can cause rapid desensitization of TRPV1 (Szallasi and Blumberg 1999), we were unable to examine the capsaicin effect before and after application of group II and group III mGluRs antagonists in the same dorsal horn neuron. Bradykinin is a nonapeptide, which can be used repeatedly to stimulate glutamate release from nociceptive primary afferents in the spinal cord (Jeftinija 1994; Wang et al. 2005). We chose bradykinin to stimulate nociceptive primary afferent terminals not restricted to TRPV1-expressing nerves in the spinal cord. Bath application of 10 µM bradykinin for 3 min significantly decreased the frequency of GABAergic sIPSCs in 18 of 49 (36.7%) neurons studied (Fig. 7). These 18 neurons consisted of 15 inner and three outer lamina II neurons. In separate (14 of 49, 28.6%) neurons, bradykinin significantly increased the frequency of sIPSCs (Fig. 7). The bradykinin effect persisted for 6 min during washout of bradykinin. In the remaining 17 (34.7%) neurons, bradykinin had no significant effect on the frequency or amplitude of sIPSCs (Fig. 7). Repeated application of 10 µM bradykinin had a reproducible effect on sIPSCs (data not shown).
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DISCUSSION |
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The lamina II is critical for the relay of nociceptive information in the spinal cord (Cervero and Iggo 1980; Lu and Perl 2003). In this study, we used capsaicin and bradykinin to stimulate nociceptive primary afferents in the spinal cord slice preparation. The capsaicin receptors (TRPV1 channels) were localized to the central terminals of primary afferent neurons in the superficial dorsal horn (Chen and Pan 2006; Guo et al. 2001; Pan et al. 2003). Previous immunocytochemical and electrophysiological studies documented that TRPVi-expressing primary afferent terminals are glutamatergic (Chen and Pan 2006; Hwang et al. 2004; Pan et al. 2003). Furthermore, bradykinin receptors (kinin B2 receptors) are also expressed on primary sensory neurons (Prado et al. 2002; Wang et al. 2005) and bradykinin can elicit glutamate release from primary afferent terminals (Jeftinija 1994; Wang et al. 2005). We found that although capsaicin and bradykinin increased synaptic GABA release in a population of lamina II neurons, they also induced a long-lasting inhibition of GABAergic input to other lamina II neurons. Because we measured GABAergic sIPSCs [not miniature (m)IPSCs], the decrease in the sIPSC amplitude by capsaicin or bradykinin can be explained by the reduced presynaptic GABA release. This presynaptic action is further supported by our finding that the postsynaptic GABAA current was not altered by capsaicin. Notably, capsaicin was reported to have no effect on the GABAergic and glycinergic inhibitory synaptic transmission (Yang et al. 1998). The lack of capsaicin effect on synaptic GABA release in the previous studies might have been attributable to the short duration (30 s) of capsaicin application. We found in the present study that the effect of capsaicin on GABAergic sIPSCs took about 2 min, reflecting a disynaptic or multisynaptic effect. Also, a longer onset latency of capsaicin effect on sIPSCs compared with that on excitatory postsynaptic currents (<30 s) (Pan and Pan 2004; Yang et al. 1998) suggests that the effect of capsaicin on GABAergic transmission is probably an indirect effect secondary to increased glutamatergic input. The use of iGluR antagonists to isolate IPSCs in the previous study also may account for the failure to observe the inhibitory effect of capsaicin (Pan and Pan 2004; Yang et al. 1998). As shown in this study, the iGluR antagonists abolished the inhibitory effect of capsaicin on GABAergic input, suggesting that this capsaicin effect is critically dependent on endogenous glutamate and activation of iGluRs. This finding suggests that stimulation of primary afferents can rapidly (within minutes) reduce GABAergic tone in the spinal cord, which possibly can increase glutamatergic synaptic efficacy in the dorsal horn. Thus "disinhibition" of lamina II neurons could contribute to the sensitization of dorsal horn neurons, leading to long-lasting pain hypersensitivity after tissue and nerve injury.
We found that in rats treated with PTX to inactivate Gi/o proteins, capsaicin failed to inhibit synaptic GABA release in all lamina II neurons tested. Therefore the receptors coupled to Gi/o proteins are probably involved in the inhibitory effect of capsaicin. Glutamate released from primary afferents and interneurons acts through two broad classes of glutamate receptors: ionotropic (
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, NMDA, and kainate) receptors and G-protein–coupled metabotropic receptors (mGluRs). Eight mGluRs have been cloned and are classified into three groups. Group I receptors (mGluRs 1 and 5) couple to phospholipase C by Gq/11 proteins (Houamed et al. 1991; Masu et al. 1991) and increase neuronal firing and synaptic transmission. Stimulation of group II (mGluRs 2 and 3) and group III (mGluRs 4, 6, 7, and 8) receptors coupled to Gi/o proteins (Prezeau et al. 1992; Tanabe et al. 1992, 1993), on the other hand, generally reduces neuronal excitability and synaptic transmission (Conn and Pin 1997; Macek et al. 1996; Schoepp et al. 1999). Both group II and group III mGluR agonists can reduce chronic neuropathic pain (Chen and Pan 2005; Fisher et al. 2002). Bath application of exogenous group II and group III mGluR agonists reduces both glutamatergic and GABAergic transmission in the rat spinal cord (Gerber et al. 2000). We hypothesized that when glutamate release is enhanced from primary afferents and glutamatergic interneurons after high-intensity stimulation, glutamate can overspill at the synapse to activate presynaptic inhibitory mGluRs. This use-dependent activation of mGluRs may influence the strength of synaptic transmission in the spinal dorsal horn (Scanziani et al. 1997). We found that the inhibitory effect of both capsaicin and bradykinin on the frequency of GABAergic sIPSCs in lamina II neurons was completely blocked by specific group II and group III mGluRs antagonists LY341495 and CPPG. Because either antagonist alone only partially reduced the inhibitory effect of capsaicin and bradykinin on GABAergic sIPSCs, both group II and group III mGluRs must be involved. Because bradykinin also decreased the frequency of GABAergic sIPSCs in a population of lamina II neurons, those non-TRPV1–expressing afferent terminals can also inhibit spinal GABAergic tone. Collectively, our results suggest that increased nociceptive input leads to endogenous glutamate release from glutamatergic interneurons in the dorsal horn, which activates both group II and group III mGluRs expressed on GABAergic interneurons (to function as heteroreceptors) to reduce GABAergic transmission. Capsaicin-induced reduction in GABAergic tone and increase in glutamatergic input can act in concert to facilitate nociceptive transmission in the spinal cord.
The mGluR2/3 is present at the afferent terminals in the spinal superficial dorsal horn (Jia et al. 1999; Tang and Sim 1999). Furthermore, two subtypes of group III mGluRs—mGluR4 and mGluR7—are located in the rat spinal dorsal horn (Azkue et al. 2001; Ohishi et al. 1995). Immunocytochemical labeling studies suggest that group II mGluRs appear to be in the inner zone of lamina II, but group III mGluRs are mainly in the lamina I and the outer zone of lamina II (Jia et al. 1999; Ohishi et al. 1995; Tang and Sim 1999). In our study, group II mGluRs that mediated the inhibitory effect of capsaicin and bradykinin on sIPSCs appeared to occur mostly in the outer lamina II neurons. Also, the group III mGluRs that mediated the decrease in sIPSCs by capsaicin and bradykinin were predominantly in the inner lamina II neurons. However, the histological evidence does not necessarily conflict with the results of our electrophysiologcal study. It is important to note that the GABAergic interneurons expressing group II and group III mGluRs may be located at a distance from the recorded lamina II neurons. Although group II and group III mGluRs may be present on glutamatergic, GABAergic, or glycinergic neurons in the neuroanatomical studies, we focused on only mGluRs on GABAergic neurons in the present study. Because iGluR antagonists abolished the inhibitory effect of capsaicin, the source of glutamate that stimulates group II and group III mGluRs on GABAergic interneurons is probably released from glutamatergic interneurons rather than from primary afferents. If glutamate released directly from primary afferents was involved in activation of group II and group III mGluRs on GABAergic interneurons, the inhibitory effect of capsaicin on sIPSCs would have been observed in the presence of iGluR antagonists. Furthermore, the finding that capsaicin had no significant effect on GABAergic mIPSCs in most lamina II neurons is also consistent with the involvement of glutamatergic interneurons in the spinal dorsal horn.
Although we focused primarily on the mechanism responsible for the depression of GABAergic transmission by increased nociceptive inflow, we observed that primary afferent stimulation produced a stimulatory effect on synaptic GABA release to a subpopulation of lamina II neurons. This effect was especially prominent in PTX-treated rats. However, in the presence of CNQX and MK-801, capsaicin increased GABA release in only a few lamina II neurons recorded from PTX-treated rats. These data suggest that iGluRs expressed on GABAergic interneurons are also involved in the stimulatory effect of capsaicin on synaptic GABA release. Consistent with these data, it was previously shown that electrical stimulation of primary afferents elicits GABAergic IPSCs primarily through non-NMDA receptors (Yoshimura and Nishi 1995). Nevertheless, we observed that the stimulatory effect of capsaicin was not blocked by iGluR antagonists in a small group of lamina II neurons. Thus neurotransmitters other than glutamate, such as substance P (Vergnano et al. 2004) or ATP (Hugel and Schlichter 2000), may be responsible for this action. Alternatively, group I mGluRs on GABAergic interneurons (Dang et al. 2002; Jia et al. 1999; Tao et al. 2000) may mediate part of this capsaicin effect. Thus glutamate released from the primary afferents may increase GABAergic input to some lamina II neurons, which could modulate nociception during acute stimulation of primary afferents. It is uncertain how increased synaptic GABA release influences nociceptive transmission in the spinal dorsal horn. Precisely which phenotype of recorded postsynaptic neurons receiving GABAergic input will ultimately dictate their contribution to modulation of nociceptive information. This heterosynaptic interaction may be important for dynamic integration of sensory signals at the spinal levels, through the lamina II along subsequent projection pathways. An important observation is that both the increase-type and the decrease-type lamina II neurons in response to primary afferent stimulation do not overlap. Had this been the case, group II and group III mGluR antagonists would have reversed the bradykinin effect, although we never observed such a response. The dual effect of capsaicin and bradykinin on GABAergic input to two separate populations of lamina II neurons suggests a rather complex mechanism of integrating nociceptive inflow at the spinal level. The major limitation of this study is that the phenotype of the recorded postsynaptic neurons was not characterized. The spinal lamina II has excitatory and inhibitory (both GABAergic and glycinergic) interneurons. Characterizing the phenotype of the postsynaptic neuron would be important in explaining the differential effect of capsaicin on synaptic GABA release.
In summary, the findings from our study provide new evidence for the interaction between glutamatergic and GABAergic synapses in the spinal dorsal horn after stimulation of the nociceptive primary afferents. Distinct populations of dorsal horn interneurons are involved in the integration of nociceptive information from primary afferents. As illustrated in Fig. 9, the group II and group III mGluRs located on the somatodendritic and/or terminal sites of GABAergic interneurons are critically involved in the inhibition of GABAergic input to dorsal horn neurons after stimulation of the primary afferents. Glutamate released from primary afferents can heterosynaptically reduce GABAergic input to dorsal horn neurons by group II and group III mGluRs. Therefore increased glutamatergic input is directly linked to the reduction of GABAergic tone in the spinal dorsal horn and the reduction of GABAergic transmission appears to be an important "downstream" effect of increased glutamatergic input from primary afferents. This information is important for our understanding of the synaptic interaction in the integration of nociceptive information at the spinal level and extends our understanding of the microcircuitry of the spinal cord dorsal horn involved in nociceptive signaling.
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GRANTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: H.-L. Pan, Department of Anesthesiology and Pain Medicine, Unit 409, The University of Texas M. D. Anderson Cancer Center, 1400 Holcombe Blvd., Houston, TX 77030 (E-mail: huilinpan{at}mdanderson.org)
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