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Department of Anesthesiology and Pain Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Texas
Submitted 5 June 2006; accepted in final form 17 October 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Villiger and Faull 1985; Yamamura et al. 1983). Intrathecal administration of mAChR agonists or acetylcholinesterase inhibitors produces a potent analgesic effect in many different species including rats, mice, and humans (Chen and Pan 2003; Duttaroy et al. 2002; Ellis et al. 1999; Hood et al. 1997; Iwamoto and Marion 1993; Naguib and Yaksh 1994). Furthermore, the analgesia produced by mAChR agonists or acetylcholinesterase inhibitors is blocked by the mAChR antagonist atropine (Naguib and Yaksh 1994, 1997). Five mAChR subtypes (M1–M5) have been identified, all of which are G-protein–coupled receptors (Bonner 1989; Caulfield and Birdsall 1998). Whereas M1, M3, and M5 subtypes are selectively linked to Gq/11 proteins and activate phospholipase C, M2 and M4 subtypes are preferentially coupled to the pertussis toxin (PTX)–sensitive Gi/o proteins and inhibit adenylyl cyclase (Caulfield and Birdsall 1998; Felder 1995; Wess 1996). Previous studies documented that M2, M3, and M4 (but not the M1) subtypes are present in the spinal cord dorsal horn (Chen et al. 2005; Duttaroy et al. 2002; Hoglund and Baghdoyan 1997; Wei et al. 1994; Yung and Lo 1997). Although the role of the M2 and M4 receptor subtypes in the mAChR agonist-induced analgesia was previously established (Duttaroy et al. 2002; Ellis et al. 1999; Gomeza et al. 1999), the mechanisms by which each mAChR subtype contributes to the muscarinic analgesia in the spinal cord are not well understood.
The spinal lamina II neurons receive glutamatergic excitatory and GABAergic/glycinergic inhibitory inputs. Our previous studies showed that M2, M3, and M4 subtypes all contribute to increased GABAergic tone in the rat spinal cord (Zhang et al. 2005). Also, the M3 subtype is mainly responsible for potentiation of glycinergic input to spinal dorsal horn neurons in rats (Wang et al. 2006). Glutamate released from primary afferents is a major excitatory neurotransmitter that conveys nociceptive information to the superficial dorsal horn neurons (Pan et al. 2002; Yoshimura and Jessell 1990). It was previously shown that stimulation of mAChRs inhibits glutamate release from primary afferents in the rat spinal cord (Li et al. 2002), although the mAChR subtypes that contribute to this action are still unclear. In this study, we determined the role of mAChR subtypes in the control of glutamatergic inputs from primary afferents and interneurons to spinal lamina II neurons.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Intrathecal treatment with PTX
To determine the involvement of M2/M4 subtypes in the inhibitory effect of oxotremorine-M on synaptic glutamate release, a group of rats was pretreated with intrathecal PTX to inactivate inhibitory Gi/o proteins (Chen and Pan 2004; Wang et al. 2006). Intrathecal catheters were inserted in rats anesthetized using 2% 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 with intrathecal 1 µg of PTX 5–7 days before the final electrophysiology experiments.
Spinal cord slice preparations
Rats were anesthetized with 2% isoflurane in O2 and the lumbar segment of the spinal cord was rapidly removed through a limited laminectomy. The rats then were killed by inhalation of 5% isoflurane. The segment of the lumbar 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): sucrose, 234; KCl, 3.6; MgCl2, 1.2; CaCl2, 2.5; NaH2PO4, 1.2; glucose, 12.0; and NaHCO3, 25.0. 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 then preincubated 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): NaCl, 117.0; KCl, 3.6; MgCl2, 1.2; CaCl2, 2.5; NaH2PO4, 1.2; glucose, 11.0; and NaHCO3, 25.0. Recordings of postsynaptic currents were performed using the whole cell voltage-clamp method, as we described previously (Li et al. 2002; Zhang et al. 2005). The neurons located in 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 P-97 puller (Sutter Instrument, Novato, CA). The impedance of the pipette was 3–5 M
when filled with internal solution containing (in mM): K-gluconate, 135; KCl, 5; MgCl2, 2.0; CaCl2, 0.5; HEPES, 5.0; EGTA, 5.0; ATP-Mg, 5.0; Na-GTP, 0.5; and guanosine 5'-O-(2-thiodiphosphate) (GDP-
-S), 1; QX-314, 10; adjusted to pH 7.2–7.4 with 1 M KOH (290–320 mOsm). GDP--S was added to the internal solution to block the possible postsynaptic effect mediated by mAChR agonists through G proteins (Li et al. 2002; Zhang et al. 2005). QX-314 was added to the internal solution to suppress the action potential generation from the recorded cell. The slice was placed in a glass-bottom 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).
Electrophysiological recordings
The evoked excitatory postsynaptic currents (eEPSCs) were induced by electrical stimulation (0.3 ms, 0.2–0.5 mA, and 0.2 Hz) of the dorsal root or dorsal root entry zone. The eEPSCs or spontaneous EPSCs (sEPSCs) were recorded in the presence of 2 µM strychnine (a glycine receptor antagonist) and 10 µM bicuculline [
-aminobutyric acid type A (GABAA) receptor antagonist]. Because activation of mAChRs increases spinal GABA release (Li et al. 2002; Zhang et al. 2005), which in turn activates presynaptic GABAB receptors to depress presynaptic glutamate release (Li et al. 2002), the GABAB receptor antagonist CGP55845 (2 µM) was also bath applied in all the protocols. We started to record eEPSCs or sEPSCs after about 5 min after whole cell access was established and the EPSCs reached a steady state. To record the miniature EPSCs (mEPSCs), 1 µM tetrodotoxin (TTX) was added to the perfusion solution. The input resistance was monitored and the recording was abandoned if it changed >15% (Li et al. 2002; Pan et al. 2002). Signals were recorded using an amplifier (MultiClamp700A, Axon Instruments, Foster City, CA) at a holding potential of –70 mV, filtered at 1–2 kHz, digitized at 10 kHz, and stored in a Pentium computer with pCLAMP 9.0 (Axon Instruments).
Oxotremorine-M, himbacine, AFDX-116, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), GDP--S, strychnine, CNQX, and bicuculline were obtained from Sigma–Aldrich (St. Louis, MO). PTX was purchased from List Biological Laboratories (San Jose, CA). QX-314 and TTX were obtained from Alomone Labs (Jerusalem, Israel). CGP55845 was obtained from Tocris Cookson (Ellisville, MO). Muscarinic toxin-3 (MT-3) was purchased from Peptide Institute (Osaka, Japan). Drugs were dissolved in Krebs solution and perfused into the slice chamber using syringe pumps.
Data analysis
Data are presented as means ± SE. Peak amplitudes of eEPSCs were analyzed using Clampfit (Axon Instruments). Measurements of the peak amplitude of eEPSCs were performed by averaging of 10 consecutive eEPSCs at 0.2 Hz during control, drug application, and recovery. The effects of oxotremorine-M on the peak amplitude of eEPSCs were determined by paired two-tailed Student's t-test or one-way ANOVA. The sEPSCs and mEPSCs were analyzed off-line with a peak detection program (MiniAnalysis, Synaptosoft, Leonia, NJ). Neurons were considered to be responsive to oxotremorine-M if the peak amplitude of eEPSCs or the frequency of sEPSCs and mEPSCs was altered >20% (Wang et al. 2006; Zhang et al. 2006). P < 0.05 was considered to be statistically significant.
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RESULTS |
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To determine the effect of oxotremorine-M on glutamate release from primary afferents to lamina II neurons, the effects of oxotremorine-M on identified monosynaptic and polysynaptic eEPSCs were studied. The eEPSCs were considered to be monosynaptic if: 1) the latency was constant after electrical stimulation (0.2 Hz) and 2) there was no conduction failure or increased latency for monosynaptic eEPSCs when stimulation frequency was increased to 20 Hz (Li et al. 2002), although the amplitude of eEPSCs was appreciably attenuated at 20 Hz (Fig. 1A). In contrast, the latency of polysynaptic eEPSCs was variable and conduction failure occurred at the higher stimulation frequency (20 Hz, Fig. 1B). Because the recordings were made in the outer zone of lamina II, we observed that 60–70% of the eEPSCs were monosynaptic.
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To investigate the contribution of M2/M4 subtypes to the effect of the mAChR agonist on synaptic glutamate release to lamina II neurons, rats were pretreated with intrathecal 1 µg PTX to inactivate inhibitory Gi/o proteins 5–7 days before final electrophysiology experiments. Oxotremorine-M (1–10 µM) failed to alter the peak amplitude of monosynaptic eEPSCs in all 12 cells tested (Fig. 3A). Oxotremorine-M significantly inhibited the peak amplitude of polysynaptic eEPSCs from 228.9 ± 28.6 to 129.5 ± 19.8 pA in seven of 16 cells (44%, Fig. 3B), but it did not significantly alter the peak amplitude of polysynaptic eEPSCs in another nine cells (56%, Fig. 3C).
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; Yigit et al. 2003; Zhang et al. 2005, 2006), was used. The inhibitory effect of 5 µM oxotremorine-M on polysynaptic eEPSCs was blocked by 50 nM 4-DAMP (n = 7, Fig. 3B). These results suggest that M2/M4 subtypes are probably responsible for the muscarinic inhibition of synaptic glutamate release from the primary afferents and a subpopulation of interneurons in the spinal cord. The M3 subtype present on a subpopulation of interneurons in the spinal cord also contributes to inhibition of glutamatergic synaptic transmission. Role of M2 and M4 subtypes in the effect of oxotremorine-M on monosynaptic eEPSCs
To delineate the role of M2/M4 subtypes in the inhibitory effect of oxotremorine-M on glutamate release from primary afferents to lamina II neurons, himbacine, a relatively selective antagonist for M2/M4 subtypes (Doller et al. 1999; Dorje et al. 1991; Miller et al. 1992; Zhang et al. 2005, 2006), was perfused 3 min before reapplication of oxotremorine-M. In our previous studies using spinal cord slices from rats and muscarinic receptor knockout mice (Wang et al. 2006; Zhang et al. 2005, 2006), we determined that 1–2 µM himbacine is the minimal concentration that blocks the M2 and M4 subtypes in the spinal cord. In 12 cells tested, initial application of 5 µM oxotremorine-M significantly decreased the peak amplitude of monosynaptic eEPSCs from 206.4 ± 34.9 to 140.7 ± 28.4 pA (31.8% inhibition, Fig. 4A). After washout of the initial effect of oxotremorine-M for 10 min, subsequent administration of 5 µM oxotremorine-M failed to inhibit the peak amplitude of eEPSCs in all 12 monosynaptic eEPSCs in the presence of 2 µM himbacine (Fig. 4A). These data suggest that M2/M4 subtypes are involved in the inhibitory effect of the mAChR agonist on glutamate release from the primary afferents, which is consistent with the results obtained from PTX-treated rats.
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; Douglas et al. 2001; Wang et al. 2006), was used in another 11 lamina II neurons. As shown in Fig. 4B, the peak amplitude of monosynaptic eEPSCs was inhibited by 5 µM oxotremorine-M from 230.3 ± 23.4 to 161.2 ± 20.4 pA (30.0%) in these 11 cells. Application of 10 µM AFDX-116 abolished the inhibitory effect of oxotremorine-M on the peak amplitude of monosynaptic eEPSCs in all 11 cells tested (Fig. 4B).
Because AFDX-116 at the concentration used in this study may also act on the M4 subtype (Caulfield and Birdsall 1998), we also assessed the potential role of the M4 subtype in the inhibitory effect of oxotremorine-M on monosynaptic eEPSCs. MT-3, a specific M4 subtype antagonist, was studied in another nine cells. MT-3 has a >500-fold selectivity for the M4 over M2, M3, and M5 subtypes (Jolkkonen et al. 1994; Liang et al. 1996). As shown in Fig. 4C, bath application of 100 nM MT-3 failed to alter the inhibitory effect of 5 µM oxotremorine-M on the amplitude of monosynaptic eEPSCs. Collectively, the above data suggest that the M2, but not M4, subtype is likely responsible for muscarinic inhibition of glutamate release from the primary afferents to spinal dorsal horn neurons.
Role of the M3 and M2/M4 subtypes in the effect of oxotremorine-M on polysynaptic eEPSCs
To examine the role of the mAChR subtypes in the effect of oxotremorine on polysynaptic eEPSCs, 14 additional lamina II neurons were studied. Bath application of 5 µM oxotremorine-M significantly decreased the peak amplitude of polysynaptic eEPSCs from 201.8 ± 19.0 to 92.4 ± 14.2 pA (54.2% inhibition) in all 14 cells tested. In six of 14 (43%) neurons tested, 2 µM of himbacine significantly attenuated the effect of oxotremorine-M on polysynaptic eEPSCs (Fig. 5A). However, oxotremorine-M still significantly inhibited the peak amplitude of polysynaptic eEPSCs in the presence of 2 µM of himbacine in these cells (from 252.3 ± 16.3 to 194.4 ± 15.4 pA, 22.9% inhibition, Fig. 5A). This remaining effect of oxotremorine-M was abolished by subsequent application of 50 nM 4-DAMP (Fig. 5A). In another eight (57%) cells, the effect of oxotremorine-M on polysynaptic eEPSCs was completely blocked by 2 µM himbacine (from 208.7 ± 40.4 to 196.7 ± 35.3 pA, Fig. 5B). These data provide further evidence that both the M3 and the M2/M4 subtypes, possibly located on separate populations of glutamatergic interneurons, are involved in the inhibitory effect of oxotremorine-M on glutamatergic synaptic transmission in the spinal cord.
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The effect of oxotremorine-M on the frequency of sEPSCs was determined in 22 lamina II neurons. In 15 of 22 neurons, 5 µM oxotremorine-M had no significant effect on the frequency of sEPSCs (Fig. 6A). In the rest of seven (31.8%) cells, 5 µM oxotremorine-M significantly decreased the frequency of sEPSCs from 3.4 ± 0.6 to 2.1 ± 0.4 Hz (Fig. 6B). In four of these seven cells tested, subsequent application of 50 nM 4-DAMP blocked the inhibitory effect of 5 µM oxotremorine-M on the frequency of sEPSCs (from 2.6 ± 0.8 to 2.5 ± 0.8 Hz). However, oxotremorine-M had no significant effect on mEPSCs in all nine cells recorded (from 9.8 ± 1.8 to 9.2 ± 1.8 Hz). These results suggest that activation of the M3 subtype inhibits synaptic glutamate release from a subpopulation of glutamatergic interneurons in the spinal dorsal horn.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Although stimulation of spinal mAChRs produces potent analgesia, the functional roles of these M2, M3, and M4 subtypes in regulation of spinal synaptic transmission and nociception remain to be established. The M2 subtype is the most predominant subtype in the spinal cord (Duttaroy et al. 2002; Hoglund and Baghdoyan 1997; Yung and Lo 1997). The M2 and M3 subtypes are particularly concentrated in the superficial laminae of the spinal cord (Hoglund and Baghdoyan 1997; Li et al. 2002; Yung and Lo 1997). Also, there is a small but functionally significant M4 subtype in the spinal cord (Chen et al. 2005). The excitability of spinal dorsal horn neurons is tonically controlled by glutamatergic excitatory and GABAergic/glycinergic inhibitory inputs. Our previous studies showed that M2, M3, and M4 subtypes all contribute to increased GABAergic tone in the rat spinal cord (Zhang et al. 2005). Furthermore, the M3 subtype is responsible for potentiation of glycinergic input to dorsal horn neurons in rats (Wang et al. 2006). Glutamate released from primary afferents is a major excitatory neurotransmitter that conveys nociceptive information to the superficial dorsal horn neurons (Yoshimura and Jessell 1990). To determine the role of the M2/M4 subtypes in oxotremorine-M–reduced synaptic glutamate release, we examined whether inactivation of Gi/o proteins with intrathecal pretreatment of PTX alters the effect of oxotremorine-M on eEPSCs. In rats pretreated with PTX, oxotremorine-M failed to decrease the monosynaptic eEPSCs in all neurons tested. The relatively selective M2/M4 antagonist himbacine also abolished the inhibitory effect of oxotremorine-M on monosynaptic eEPSCs, further suggesting the role of the M2/M4 subtypes in the regulation of glutamate release from primary afferents. To determine the relative contribution of M2 and M4 subtypes to the inhibitory effect on eEPSCs, the relatively selective M2 antagonist AFDX-116 (Coelho et al. 2000; Douglas et al. 2001; Wang et al. 2006) and the specific M4 antagonist MT-3 (Jolkkonen et al. 1994; Liang et al. 1996) were used in this study. We found that AFDX-116 abolished the effect of oxotremorine-M on monosynaptic eEPSCs in all cells tested. By contrast, MT-3 failed to attenuate the effect of oxotremorine-M on monosynaptic eEPSCs. Therefore these findings strongly suggest that the M2, but not M4, subtype is likely involved in regulation of glutamate release from the primary afferent terminals in the spinal cord.
In the present study, we unexpectedly found that the inhibitory effect of oxotremorine-M on polysynaptic eEPSCs was much greater than that on monosynaptic eEPSCs. These results suggest that mAChRs are located not only on primary afferent terminals but also on glutamatergic interneurons. Because the effect of oxotremorine-M on polysynaptic eEPSCs was not abolished by PTX and himbacine in some neurons, mAChR subtypes that are not coupled to PTX-sensitive Gi/o proteins (i.e., non-M2/M4) may be present on a subpopulation of glutamatergic interneurons. The radioligand binding study suggests that the M3 subtype is present in the rat spinal cord (Hoglund and Baghdoyan 1997). We previously showed that the M3 subtype is involved in the potentiation of both GABA (Zhang et al. 2005) and glycine release (Wang et al. 2006) from interneurons in the rat spinal cord. In the current study, we found that the remaining inhibitory effect of oxotremorine-M on polysynaptic eEPSCs in PTX-treated rats was blocked by a relatively selective M3 antagonist 4-DAMP. Thus the M3 subtype is also likely involved in the inhibition of synaptic glutamate release from some glutamatergic interneurons in the spinal cord. In support of this conclusion, the effect of oxotremorine-M on polysynaptic eEPSCs in the presence of himbacine in some neurons was abolished by 4-DAMP. Importantly, although oxotremorine-M had no effect on mEPSCs in all cells tested, it decreased the frequency of sEPSCs in 31.8% lamina II neurons, an effect that was blocked by 4-DAMP. Collectively, these results suggest that not only the M3 subtype but also the M2 and M4 subtypes play a role in regulation of synaptic glutamate release from the interneurons in the spinal cord. Because oxotremorine-M had no significant effect on mEPSCs in all neurons tested, these electrophysiological data suggest that the M3 and M2/M4 subtypes are probably located on the somatodendritic site of the interneurons. It should be noted that although the M2 and M4 subtypes are essential for the analgesic effect of mAChR agonists in mice (Duttaroy et al. 2002; Gomeza et al. 1999), the functional role of the M3 subtype in the spinal analgesic effect of mAChRs agonists remains to be defined.
The M2 subtype is coupled to the Gi/o protein, which inhibits adenylate cyclase activity (Caulfield 1993; Fields and Casey 1997; Wess 1996). Thus activation of the M2 subtype is expected to depress the synaptic glutamate release in the spinal cord. However, the M3 subtype is mainly coupled to the Gq/11 protein to activate phospholipase C (Caulfield 1993; Fields and Casey 1997; Wess 1996). Its inhibitory effect on synaptic glutamate release in the spinal cord is not anticipated. The signaling mechanism underlying the inhibitory effect of the M3 subtype on synaptic glutamate release in the spinal cord is not well understood. It was previously shown that stimulation of the M3 subtype reduces excitatory synaptic transmission in dopaminergic neurons of the rat mesencephalon (Grillner et al. 1999). Also, activation of the M3 subtype inhibits both excitatory and inhibitory transmission in rat subthalamic neurons in vitro (Shen and Johnson 2000). Activation of the M3 subtype produces a K+ current (IKM3), a Gq-protein–coupled, delayed rectifier-like K+ current (Shi et al. 1999, 2004). Opening of these potassium channels can cause membrane hyperpolarization and shortening of action potential duration (Shi et al. 2003). This could be a possible mechanism responsible for, at least in part, the inhibitory effect of the M3 subtype on glutamatergic interneurons in the spinal cord.
It is worth noting that many antagonists used in this study have limited selectivity for mAChR subtypes. In our previous study using the rat spinal cord, we extensively tested and determined the minimal concentrations of the antagonists (himbacine, 4-DAMP, and AFDX-116) by using various combinations of the drugs and PTX treatment (Wang et al. 2006; Zhang et al. 2005). Furthermore, using muscarinic receptor knockout (KO) mice, we clearly demonstrated that activation of M2/M4 subtypes and the M3 subtype produces an opposite effect on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the spinal dorsal horn (Zhang et al. 2006). In the mouse spinal cord, these distinct functions of M2/M4 and M3 subtypes constitute the basis for determining the selectivity of various muscarinic subtype antagonists (Zhang et al. 2006). For instance, in M3 KO and M1/M3 double-KO mice, 2 µM himbacine is the minimal concentration that completely blocks the effect of 3–5 µM oxotremorine-M on GABA release. More important, in wild-type mice, 2 µM himbacine completely reverses the effect of oxotremorine-M on synaptic GABA release. This converging evidence strongly suggests that 2 µM himbacine blocks only M2/M4 subtypes without affecting the M3 subtype in the spinal cord. In the present study, the data obtained from PTX-treated rats are consistent with those findings with himbacine, suggesting that the concentration of himbacine used is selective for M2 and M4 subtypes. 4-DAMP cannot exclusively determine the M3 subtype because it has almost the same PA2 values at cloned M1, M3, and M5 subtypes (Watson et al. 1999). However, both radioligand binding and immunocytochemistry experiments demonstrated that only M2, M3, and M4 subtypes are present in the spinal cord dorsal horn (Chen et al. 2005; Duttaroy et al. 2002; Gomeza et al. 1999; Hoglund and Baghdoyan 1997). Thus the confounding effect of 4-DAMP on M1 and M5 subtypes is not a critical concern to this particular study using the spinal cord slice. Using M2/M4 double-KO mice, we determined that 25–50 nM 4-DAMP is the minimal concentration that abolishes the effect of 3–5 µM oxotremorine-M on GABA release (Zhang et al. 2006). To minimize the possibility that 4-DAMP may interfere with the M2 and M4 subtypes in the spinal cord, 4-DAMP was the last antagonist perfused to the spinal slices in our protocol. Although AFDX-116 has a higher affinity for M2 than for M4 subtypes (Caulfield and Birdsall 1998), this antagonist at the concentration used may also act on the M4 subtype in the spinal cord. Therefore we used a more specific M4 subtype antagonist, MT-3 (Jolkkonen et al. 1994; Liang et al. 1996), to delineate the role of M2 and M4 subtypes in the effect of oxotremorine-M on monosynaptic eEPSCs. We found that 100 nM MT-3 failed to alter the inhibitory effect of oxotremorine-M on monosynaptic eEPSCs, although it significantly attenuates the effect of oxotremorine-M on GABA release (Zhang et al. 2005). These data provide strong evidence that the M2, but not M4, subtype is present on the primary afferent terminals.
In summary, we found in this study that the M2 subtype controls the synaptic glutamate release from the primary afferents in the spinal cord (Fig. 7). Furthermore, our data suggest that the M3 and M2/M4 subtypes are involved in regulation of glutamate release from a subpopulation of interneurons in the rat spinal cord (Fig. 7). Therefore the present and other recent studies (Wang et al. 2006; Zhang et al. 2005) demonstrate that different mAChRs subtypes are involved in regulation of the excitatory and inhibitory synaptic transmission in the spinal cord. By potentiation of the inhibitory tone and suppression of glutamatergic inputs, mAChR agonists produce a profound antinociceptive effect in the spinal cord. This new information is important for our understanding of the muscarinic regulation of nociceptive transmission in the spinal cord and the cellular mechanism underlying the analgesic action produced by spinally administered mAChR agonists.
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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, The University of Texas M.D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030 (E-mail: huilinpan{at}mdanderson.org)
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REFERENCES |
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