Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

The spinal cholinergic system and muscarinic acetylcholine receptors (mAChRs) 2 are important for the control of noci-ceptive transmission. For example, neurons and nerve terminals expressing choline acetyltransferase and acetylcholinesterase (enzymes for acetylcholine synthesis and degradation, respectively) are located in the spinal dorsal horn (1,2). The superficial laminae contain the highest density of mAChRs in the spinal dorsal horn (3)(4)(5). Stimulation of mAChRs attenuates the responses of dorsal horn neurons to noxious stimuli (6), whereas blocking spinal mAChRs with atropine causes a large increase in pain sensitivity (7). Furthermore, spinally administered mAChR agonists or acetylcholinesterase inhibitors produce potent analgesia in both animals and humans (8 -11). Because agonists and antagonists that are highly selective for all mAChR subtypes are still lacking at this time, it is difficult to rely on pharmacological approaches alone to define which individual mAChR subtypes are involved in the regulation of synaptic and nociceptive transmission at the spinal level.
Molecular cloning studies have revealed the existence of five molecularly distinct mAChR subtypes (M 1 -M 5 ) (12). The odd-numbered subtypes (M 1 , M 3 , and M 5 ) couple efficiently through the G q/11 class of G proteins to activate phospholipase C, which leads to inositol triphosphate-mediated calcium release from the endoplasmic reticulum and diacylglycerol-mediated activation of protein kinase C. The evennumbered mAChRs (M 2 and M 4 ) inhibit adenylyl cyclase activity through activation of the G i/o class of G proteins (12,13). In the spinal dorsal horn, M 2 is the major mAChR subtype, and the M 3 and M 4 subtypes represent only a fraction of the total mAChRs at the spinal level (11, 14 -16). Using mAChR subtype knock-out (KO) mice and an siRNA approach, we have shown that both the M 2 and M 4 subtypes mediate the analgesic effect of mAChR agonists in both rats and mice (11,14,17). In addition, using mAChR subtype-KO mice, we have demonstrated that the M 2 , M 3 , and M 4 subtypes are differentially involved in the control of GABAergic and glycinergic inhibitory synaptic transmission in the spinal dorsal horn (18,19). Glutamate is the predominant excitatory neurotransmitter involved in nociceptive transmission in the spinal dorsal horn. However, it remains unclear how individ-ual mAChR subtypes contribute to the regulation of glutamatergic input to dorsal horn neurons.
Therefore, in this study, we recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type and mAChR subtype-KO mice to define the role of the individual mAChR subtypes in the regulation of synaptic glutamate release to the superficial dorsal horn neurons. In addition to providing unequivocal evidence showing differential regulation of glutamatergic input by the presynaptic M 2 , M 3 , and M 4 subtypes, we found, unexpectedly, that M 5 is the predominant subtype that potentiates glutamatergic transmission in the spinal dorsal horn. Our study also revealed a reciprocal interaction between the M 2 /M 4 and M 3 /M 5 subtypes in the control of glutamatergic synaptic transmission in the spinal cord. Furthermore, we found that group II/III metabotropic glutamate receptors (mGluRs) are involved in the M 3 /M 5 -mediated feedback loop that regulates glutamatergic input in the spinal cord. This new information is important for our understanding of the complex interactions among mAChR subtypes in the dynamic control of glutamatergic transmission in the spinal cord. Clearly, our findings are crucial for the development of novel mAChR subtype-selective analgesic drugs endowed with increased efficacy and reduced side effects.

EXPERIMENTAL PROCEDURES
Animals-All WT and mAChR subtype single and double KO mice (6 -9 weeks old) used in this study were obtained from the National Institute of Diabetes and Digestive and Kidney Diseases (National Institutes of Health, Bethesda, MD). The genetic background of the M 2 -KO, M 4 -KO, and M 2 /M 4 double-KO mice was C57/BL6 and that of the M 3 -KO, M 5 -KO, and M 1 /M 3 double-KO mice was 129SvEv/CF1. The genetic background of the M 2 -KO, M 4 -KO, and M 2 /M4 double-KO mice was C57/BL6. In studies using these mutant mice, C57/BL6 WT mice (Taconic) were used as controls. The M 3 -KO, M 5 -KO, and M 1 /M 3 double-KO mice were maintained on a mixed 129SvEv (50%)/CF1 (50%) background. In studies using these mutant mice, WT mice with the same mixed genetic background served as controls. The generation and breeding of the M 2 -KO, M 3 -KO, M 4 -KO, M 5 -KO, M 1 /M 3 double-KO, and M 2 /M 4 double-KO mice have been described previously (11, 17, 20 -23). The mouse genotyping was carried out by Southern blotting and polymerase chain reaction analysis of mouse-tail DNA. The experimental protocols and procedures were approved by the Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center and conformed to the National Institutes of Health guidelines for the ethical use of animals.
Spinal Cord Slice Preparation-Mice were anesthetized with 2% isoflurane, and the lumbar segment of the spinal cord was rapidly removed through laminectomy. The mice were then killed by inhalation of 5% isoflurane. The spinal cord segment was immediately placed in an ice-cold sucrose artificial cerebrospinal fluid (aCSF) presaturated with 95% O 2 and 5% CO 2 . The sucrose aCSF contained (in mM) 206 sucrose, 2.8 KCl, 1.0 MgCl 2 , 1.0 CaCl 2 , 1.2 NaH 2 PO 4 , 25.0 glucose, and 26.0 NaHCO 3 . The tissue was then placed in a shallow groove formed in a gelatin block and glued onto the stage of a vibratome. Transverse spinal cord slices (350 m) were cut in the ice-cold sucrose aCSF and preincubated in Krebs' solution oxygenated with 95% O 2 and 5% CO 2 at 34°C for at least 1 h before they were transferred to the recording chamber. The Krebs' solution contained (in mM) 117.0 NaCl, 3.6 KCl, 1.2 MgCl 2 , 2.5 CaCl 2 , 1.2 NaH 2 PO 4 , 11.0 glucose, and 25.0 NaHCO 3 . Each slice was placed in a glass-bottomed chamber and fixed with parallel nylon threads supported by a stainless steel weight. The slice was continuously perfused with Krebs' solution at 5.0 ml/min at 34°C, which was maintained by an inline solution heater and a temperature controller.
Electrophysiological Recordings-Recordings of postsynaptic currents were performed in lamina II neurons using the whole-cell voltage clamp method, as we described previously (18,19,24). The neurons located in the lamina II were identified under a fixed-stage microscope (BX51WI; Olympus, Tokyo, Japan). The electrode was pulled from borosilicate glass capillaries. Patch electrodes with a resistance of 5-10 M⍀ were filled with an internal solution containing (in mM) 135.0 potassium gluconate, 5.0 KCl, 2.0 MgCl 2 , 0.5 CaCl 2 , 5.0 HEPES, 5.0 EGTA, 5.0 ATP-Mg, 0.5 Na-GTP, 1.0 GDP-␤-S, and 10.0 QX314, adjusted to pH 7.2-7.4 with 1 M KOH (290 -300 mOsm). sEPSCs were recorded at a holding potential of Ϫ60 mV. To record the miniature EPSCs (mEPSCs), 0.5 M tetrodotoxin (TTX) was added to the perfusion solution. The input resistance was continuously monitored, and the recording was abandoned if it changed more than 15%. Signals were processed with an amplifier (MultiClamp 700A; Axon Instruments, Union City, CA), filtered at 1-2 kHz, digitized at 10 kHz, and stored in a computer with pCLAMP 9.0 (Axon Instruments).
Data Analysis-Data are presented as means Ϯ S.E. The amplitudes and frequencies of sEPSCs and mEPSCs were analyzed off-line using a peak detection program (MiniAnalysis; Synaptosoft, Decatur, GA). The detection of events was accomplished by setting a threshold above the noise level. The sEPSCs and mEPSCs were detected by the fast rise time of the signal over an amplitude threshold (typically 6 -10 pA) above the background noise. We manually excluded the event when the noise was erroneously identified as a sEPSC by the software program. The cumulative probability of the amplitude and inter-event interval of the sEPSCs and mEPSCs was compared using the Kolmogorov-Smirnov test, which estimates the probability that two distributions are similar. This test was used first to determine whether the effect of oxotremorine-M on the sEPSCs and mEPSCs was significantly different in individual neurons. The effect of oxotremorine-M on the frequency and amplitude of sEPSCs and mEPSCs was determined by one-way ANOVA using Dunnet's or Tukey's post hoc test. p Ͻ 0.05 was considered to be statistically significant.

Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in WT
Mice-To determine the role of mAChRs in the control of synaptic glutamate release to lamina II neurons, we first examined the effect of oxotremorine-M, a specific agonist that stimulates all mAChR subtypes, on glutamatergic sEPSCs in WT mice. The effect of oxotremorine-M on glutamatergic sEPSCs was similar among the two groups of WT mice (C57/BL6 or 129SvEv/CF1 genetic background). Specifically, there were no significant differences in the proportion of neurons in which oxotremorine-M increased (70.5% versus 63.6%) or decreased (20.5% versus 22.7%) the frequency of sEPSCs between C57/BL6-WT mice and 129SvEv/CF1-WT mice. Therefore, the data obtained from the two groups of WT mice were pooled. To examine the concentration-dependent effect of oxotremorine-M, the drug was perfused in a cumulative fashion (1, 3, 5, and 10 M; each concentration applied for 3 min) onto the slice chamber. Oxotremorine-M significantly increased the frequency, but not the amplitude, of the sEPSCs in 21 neurons in a concentration-dependent manner (Fig. 1, A-C). The cumulative probability analysis of glutamatergic sEPSCs revealed that the distribution pattern of the inter-event interval of sEPSCs was shifted toward the left in response to oxotremorine-M (Fig.  1B). In another 13 neurons, oxotremorine-M decreased the frequency, but not the amplitude, of the sEPSCs in a concentration-dependent fashion (Fig. 1D).
In a total of 66 neurons randomly recorded from WT mice, bath application of 3 M oxotremorine-M for 3 min significantly increased the frequency of sEPSCs in 45 (68.2%) neurons ( We next used himbacine, an M 2 /M 4 subtype-preferring antagonist (18,19,(25)(26)(27), to determine the potential role of the M 2 /M 4 subtypes in the inhibitory effect of oxotremorine-M on sEPSCs in WT mice. The effective concentration of himbacine has been determined in our previous studies (18,19,25). Oxotremorine-M (3 M) initially increased the frequency of sEPSCs in 14 neurons. In these 14 neurons, subsequent application of 2 M himbacine significantly potentiated the stimulatory effect of oxotremorine-M on the frequency of sEPSCs (Fig. 2, A and B). Interestingly, in another 9 neurons in which initial application of oxotremorine-M significantly inhibited the sEPSCs, the drug significantly increased the fre-quency of sEPSCs in the presence of 2 M himbacine (Fig.  2C). These data suggest that the M 2 /M 4 subtypes mediate the inhibitory effect of the mAChR agonist on synaptic glutamate release to spinal dorsal horn neurons.
To determine whether the M 3 subtype is involved in the stimulatory effect of oxotremorine-M on glutamate release in the spinal cord of WT mice, we tested the effect of J104129, an M 3 subtype-preferring antagonist (28). In the preliminary experiments, we confirmed that 50 nM J104129 did not alter the inhibitory or excitatory effects of 3 M oxotremorine-M on sEPSCs in M 3 -KO mice. In 6 of 21 neurons in which oxotremorine-M caused a small increase in the frequency of sEPSCs, 50 nM J104129 completely blocked the excitatory effect of oxotremorine-M on the sEPSCs (Fig. 2D). However, in the remaining 15 neurons in which oxotremorine-M produced a large increase in the frequency of sEPSCs, J104129 did not significantly alter its excitatory effect (Fig. 2E). These results suggest that although the M 3 subtype contributes to the increased glutamate release induced by mAChR activation in a subpopulation of neurons, a non-M 3 subtype seems to be involved in the potentiation of glutamatergic input in the majority of dorsal horn neurons.
We next examined the possible subcellular location (i.e. presynaptic terminals versus somatodendritic sites) of the mAChR subtypes in the spinal dorsal horn. If the mAChR subtypes are present on the somatodendritic site of the glutamatergic neurons, oxotremorine-M would be expected to have little effect on the mEPSCs (i.e. EPSCs recorded in the presence of 0.5 M TTX). Bath application of 3 M oxotremorine-M either increased or decreased the frequency of the mEPSCs in a similar manner as the sEPSCs, i.e. in neurons where oxotremorine-M increased the sEPSCs, the mEPSCs were also increased and vice versa (Fig. 3). These data suggest that the mAChRs that modulate glutamatergic transmission in the spinal dorsal horn are primarily present on presynaptic terminals. In 17 neurons in which oxotremorine-M initially increased frequency of sEPSCs, subsequent application of 2 M himbacine further increased the stimulatory effect of 3 M oxotremorine-M on the frequency of sEPSCs (Fig. 4, B and C). In another 7 neurons from M 1 /M 3 double-KO mice, himbacine converted the initial inhibitory effect of oxotremorine-M to an excitatory effect (Fig. 4D). In an additional 11 neurons, 3 M oxotremorine-M produced a similar stimulatory effect on the frequency of sEPSCs and mEPSCs (Fig. 4E). Because the oxotremorine-M-induced increases in the fre- Fisher's exact test). In addition, the magnitude of the increase in the sEPSC frequency induced by 3 M oxotremorine-M was significantly smaller in the neurons from the M 5 -KO mice than in those from all other groups of mice (Fig. 5A). Notably, the potentiating effect of oxotremorine-M was readily washed out within 5 min after cessation of the bath application.

Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in M 1 /M 3 Double-KO mice-In
In 10 neurons from M 5 -KO mice, bath application of 2 M himbacine significantly potentiated the initial stimulatory effect of oxotremorine-M on the sEPSCs (Fig. 5, B and C). In another 7 neurons, himbacine blocked the inhibitory effects of oxotremorine-M on the sEPSCs (Fig. 5D). In 9 additional neurons in which 3 M oxotremorine-M initially increased the frequency of sEPSCs, 50 nM J104129 converted the stimulatory effect of oxotremorine-M on the sEPSCs to an inhibitory effect (Fig. 6, A and B). Furthermore, 3 M oxotremo-   DECEMBER 24, 2010 • VOLUME 285 • NUMBER 52 rine-M significantly inhibited the frequency of both sEPSCs and mEPSCs in 8 neurons tested (Fig. 6C). These results strongly suggest that stimulation of mAChRs potentiates synaptic glutamate release primarily through the M 5 subtype in the spinal dorsal horn.

Muscarinic Control of Glutamatergic Transmission
Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in M 3 -KO Mice-In 30 of 60 (50.0%) lamina II neurons from M 3 -KO mice, 3 M oxotremorine-M significantly increased the frequency of sEPSCs (Fig. 7A). Oxotremorine-M increased the sEPSCs frequency in significantly fewer neurons from M 3 -KO mice than from WT mice (50.0% versus 68.2%; p Ͻ 0.05, Fisher's exact test). Oxotremorine-M significantly reduced the frequency of sEPSCs in 17 of 60 (28.3%) neurons and had no effect on the frequency of sEPSCs in the remaining 13 (21.7%) neurons (Fig. 7A). In 11 neurons in which oxotremorine-M increased the frequency of sEPSCs, 2 M himbacine further increased the frequency of sEPSCs (Fig. 7B). In another 11 neurons from M 3 -KO mice in which oxotremorine-M had an inhibitory effect on the sEPSCs, himbacine abolished the inhibitory effect (Fig. 7C).   In 6 neurons from M 3 -KO mice, himbacine only partially reduced the inhibitory effect of oxotremorine-M on the sEPSCs. We speculated that the mAChR agonist may stimulate M 5 in the M 3 -KO mice to cause excessive glutamate release, which could activate the group II/III mGluRs (29) to subsequently inhibit synaptic glutamate release in this subpopulation of neurons. We therefore further tested the effect of oxotremorine-M in the presence of 100 nM LY341495 and 200 M CPPG, which are selective antagonists for group II and III mGluRs, respectively (29 -32). In these 6 neurons, LY341495 and CPPG completely blocked the inhibitory effect of oxotremorine-M (Fig. 7D).  (Fig. 8, B and C). However, in another 19 neurons, J104129 did not significantly alter the oxotremorine-M-induced increases in the sEPSC frequency (Fig. 8D). In 11 additional neurons, oxotremorine-M increased the frequencies of the both sEPSC and mEPSCs in a similar manner (Fig. 8E).

Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in M 2 /M 4 Double-KO Mice-To
We noticed that even in the M 2 /M 4 double-KO mice, 3 M oxotremorine-M still increased the sEPSC frequency in a   DECEMBER 24, 2010 • VOLUME 285 • NUMBER 52 small population (7.1%) of lamina II neurons. Because stimulation of the M 3 /M 5 subtypes in M 2 /M 4 double-KO mice could result in a large increase in synaptic glutamate release, it is possible that the overflow of glutamate can access and stimulate presynaptic group II and III mGluRs to subsequently reduce the glutamatergic input to some lamina II neurons. In 4 neurons in which the frequency of sEPSCs was initially inhibited by 3 M oxotremorine-M, bath application of 100 nM LY341495 and 200 M CPPG completely blocked the inhibitory effect of oxotremorine-M (Fig. 8F). Bath application of 2 M himbacine did not significantly alter the stimulating effect of 3 M oxotremorine-M on sEPSCs in 24 neurons tested (Fig. 9B). However, in another 11 neurons, himbacine converted the initial inhibitory effect of oxotremorine-M to an excitatory effect (Fig. 9C). In addition, 3 M oxotremorine-M either increased or decreased the frequency of sEPSC and mEPSCs in neurons from M 2 -KO mice in a similar manner (Fig. 9, D and E).

Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in M 2 -KO
Effects of Oxotremorine-M on sEPSCs and mEPSCs of Lamina II Neurons in M 4 -KO Mice-In M 4 -KO mice, 3 M oxotremorine-M significantly increased the frequency of sEPSCs in 63 of 84 (75.0%) neurons (Fig. 10A). In 13 of 84 (15.5%) neurons, oxotremorine-M significantly inhibited the frequency of sEPSCs (Fig. 10A). The percentage of neurons in which oxotremorine-M inhibited sEPSCs was not significantly different between M 4 -KO mice and WT mice (15.5% versus 21.2%; p Ͼ 0.05, Fisher's exact test).
In 21 neurons from the M 4 -KO mice, 2 M himbacine did not significantly alter the stimulatory effect of oxotremorine-M on the frequency of sEPSCs (Fig. 10B). In another 8 neurons, himbacine converted the inhibitory effect of oxotremorine-M on sEPSCs to an excitatory effect (Fig. 10C). In an additional 16 neurons, 3 M oxotremorine-M significantly increased the frequency of both the sEPSCs and mEPSCs (Fig.  10D). Collectively, the electrophysiological data obtained from M 2 /M 4 double-KO and M 2 and M 4 single-KO mice suggest that the M 2 and M 4 subtypes are mainly present on the presynaptic terminals to inhibit synaptic glutamate release in the spinal dorsal horn.

DISCUSSION
In the present study, we used genetic and electrophysiological approaches to determine the functional activity of individual mAChR subtypes in the control of spinal glutamatergic transmission. We found that the mAChR agonist oxotremorine-M significantly increased the frequency of the glutamatergic sEPSCs in the majority of lamina II neurons but re- Both the M 2 and M 4 subtypes in the spinal cord are critically involved in the inhibition of nociceptive transmission by stimulation of mAChRs in mice (11,17) and in rats (14). Previous studies have shown that the M 2 subtype, representing the majority (ϳ90%) of spinal cord mAChRs, is particularly expressed in the superficial dorsal horn (11,14,16,24,33). In addition, the M 4 subtype is expressed at low levels in the spinal cord (11,14,34). We found that the percentage of neurons in which oxotremorine-M inhibited the sEPSC frequency was greatly reduced in M 2  In addition, the inhibitory effect of oxotremorine-M on the frequency of sEPSCs was not significantly attenuated by TTX in M 2 -KO, M 4 -KO, and WT mice, suggesting that these two inhibitory mAChR subtypes are primarily located on the presynaptic terminals. Furthermore, we found that the percentage of neurons in which oxotremorine-M inhibited sEPSCs was not significantly reduced in M 2 and M 4 single-KO mice. This is likely due to the fact that the M 2 and M 4 both play a significant role in the attenuation of glutamatergic transmission by the mAChR agonist. This notion is further supported by our finding that himbacine significantly attenuated the inhibitory effect of oxotremorine-M on sEPSCs in the M 2 and M 4 single-KO mice.
In this study, we found that in neurons from WT mice, oxotremorine-M significantly increased the frequency of glutamatergic sEPSCs in the majority (68.2%) of neurons, while it inhibited the sEPSCs in 26.7% of neurons. These data are distinctly different from what we found in the rat spinal cord, where oxotremorine-M only inhibited the sEPSC frequency in lamina II neurons (35). These distinct effects likely reflect a species difference between rats and mice. For example, we have shown that oxotremorine-M increases synaptic GABA release in the rat spinal cord but primarily reduces GABA release in the mouse spinal cord (18,25). Because the excitatory effect of oxotremorine-M persisted in the presence of the voltage-gated Na ϩ channel blocker TTX (i.e. mEPSCs) in WT and M 2 /M 4 double-KO mice, our data suggest that the mAChR subtypes that stimulate synaptic glutamate release are primarily present at the presynaptic terminals in the mouse spinal cord.
Previous studies have shown that the M 2 , M 3 , and M 4 subtypes are all involved in the control of spinal synaptic transmission (18,19,25,35,36). Although the M 5 subtype has been shown to be present in the mouse spinal cord (37,38), the important function of M 5 in the spinal cord has not been recognized previously. We found that oxotremorine-M still increased the frequency of sEPSCs in about 50% neurons from the M 3 -KO and M 1 /M 3 double-KO mice. In addition, the M 3preferring antagonist J104219 did not abolish the excitatory effect of oxotremorine-M on sEPSCs in WT mice. Thus, we hypothesized that the M 5 subtype may be critically involved in potentiating synaptic glutamate release in the spinal cord. Consistent with this hypothesis, we observed that both the baseline frequency of sEPSCs and the magnitude of the increases in the sEPSC frequency induced by oxotremorine-M were significantly less in the neurons from M 5 -KO mice than in those from all other groups of mice. Furthermore, we found that the percentage of neurons in which oxotremorine-M increased the sEPSC frequency (26.3%) was markedly smaller in the M 5 -KO mice than in the WT mice. Our findings provide the first evidence showing that the M 5 subtype plays a major role in the increased synaptic glutamate release induced by mAChR activation in the spinal dorsal horn. Nevertheless, the potential role of M 5 in the control of spinal nociceptive transmission remains to be defined in more detail.
Unlike the M 2 and M 4 subtypes, knockdown of M 3 in the spinal cord with specific siRNA does not significantly affect the analgesic effect of mAChR agonists in rats (14). Because of the dominance of M 5 in the potentiation of synaptic glutamate release, the functional activity of M 3 is probably masked by the presence of M 5 in the spinal cord. In M 5 -KO mice, we found that blocking the M 3 subtype with J104219 completely blocked the remaining stimulatory effect of oxotremorine-M on the sEPSC frequency. We also found that the percentage of neurons in which oxotremorine-M increased the frequency of sEPSCs was significantly less in the neurons from the M 3 -KO and M 1 /M 3 double-KO mice than in those from the WT mice. Furthermore, in both WT and M 2 /M 4 double-KO mice, J104219 significantly attenuated the stimulatory effect of oxotremorine-M on sEPSCs in a subpopulation of lamina II neurons. Therefore, these data suggest that the potentiation of synaptic glutamate release induced by mAChR activation is mediated by both the M 3 and M 5 subtypes in the spinal cord. We obtained no evidence for the role of M 1 subtype in the control of glutamatergic synaptic transmission in the spinal dorsal horn. This conclusion is based on (1) the effect of oxotremorine-M on sEPSCs in M 3 single-KO mice was nearly identical to that in M 1 /M 3 double-KO mice and (2) the stimulatory effect of oxotremorine-M on sEPSCs in M 5 KO mice was completely blocked by the M 3 -preferring antagonist J104219.
Interestingly, we found that oxotremorine-M still inhibited the sEPSCs in a subpopulation of neurons in M 2 /M 4 double-KO mice. In these neurons, blocking group II/III mGluRs with LY341495 and CPPG completely abolished the inhibitory effect of oxotremorine-M. These data clearly suggest that the increased glutamate release owing to stimulation of the M 3 and M 5 subtypes can access and activate presynaptic group II and III mGluRs (39) to subsequently reduce glutamatergic input in these neurons. Thus, group II/III mGluRs are indirectly involved in the feedback regulation of glutamatergic input by the M 3 and M 5 subtypes in the spinal cord.
Another interesting finding of our study is the complex function and dynamic interactions between four mAChR subtypes in the control of synaptic glutamatergic transmission in the spinal cord. For example, we found that himbacine further The glutamatergic synaptic terminals expressing these four mAChR subtypes are likely intermingled in the superficial spinal dorsal horn. Therefore, the M 2 /M 4 and M 3 /M 5 subtypes, located either on the same or separate presynaptic glutamatergic terminals, can greatly influence the amount of synaptic glutamate release to a given dorsal horn neuron by activation of mAChRs in the spinal cord. Because M 2 and M 4 are coupled to G i/o proteins, stimulation of these two subtypes can inhibit synaptic glutamate release through inhibition of voltage-gated calcium channels (40 -42). On the other hand, M 3 and M 5 are coupled to G q/11 proteins, and activation of these two subtypes could increase presynaptic glutamate release through stimulation of phospholipase Cinositol triphosphate, which increase the intracellular calcium level (43,44). It would be interesting to determine whether M 3 /M 5 stimulation "antagonizes" the muscarinic analgesic effect at the spinal level by comparing the effects of mAChR agonists on nociception in WT, M 3 -KO, and M 5 -KO mice.
In summary, our study using mAChR subtype-KO mice provides unequivocal evidence that the M 5 , and to a lesser extent, M 3 subtypes contribute to the potentiation of glutamatergic input to spinal dorsal horn neurons in mice. Also, we demonstrated that the M 2 and M 4 subtypes mediate the inhibition of synaptic glutamate release induced by mAChR activation in the spinal dorsal horn. Furthermore, the reciprocal interactions between the inhibitory M 2 and M 4 subtypes and the stimulatory M 3 and M 5 subtypes are involved in the dynamic regulation of glutamatergic synaptic transmission in the spinal cord. Finally, we found that the group II/III mGluRs are involved in the M 3 /M 5 -mediated feedback loop that regulates glutamatergic input in the spinal cord. The diverse functions and interactions among different mAChR subtypes are important for our understanding of the complex actions produced by spinally administered mAChR agonists or acetylcholinesterase inhibitors. Our findings are critical in guiding the development of mAChR subtype-selective analgesic drugs endowed with increased efficacy and reduced side effects.