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Opposing Functions of Spinal M₂, M₃, and M₄ Receptor Subtypes in Regulation of GABAergic Inputs to Dorsal Horn Neurons Revealed by Muscarinic Receptor Knockout Mice

Department of Anesthesiology, Pennsylvania State University College of Medicine, the Milton S. Hershey Medical Center, Hershey, Pennsylvania (H.-M.Z., S.-R.C., H.-L.P.); Division of Neuronal Network, Institute of Medical Science, University of Tokyo, Tokyo, Japan (M.M.); and Laboratory of Bioorganic Chemistry, National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland (D.G., J.W.)

Received August 14, 2005; accepted December 19, 2005

	Abstract

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Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 µM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M₂ and M₄ subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M₃ KO and M₁/M₃ double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M₂/M₄ double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M₃ subtype-preferring antagonist. In M₂ or M₄ single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M₃ subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M₂ and M₄ subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M₂ and M₄ subtypes in the spinal cord of rats.

The spinal lamina II neurons are tonically controlled by glutamatergic excitatory and GABAergic/glycinergic inhibitory inputs. Glutamate released from primary afferent nerves is a major excitatory neurotransmitter that conveys nociceptive information to the superficial lamina neurons (Yoshimura and Jessell, 1990). Our previous study in rats has shown that stimulation of mAChRs inhibits glutamate release and inhibition of glutamate release are indirectly mediated by increased GABA release and activation of presynaptic GABA_B receptors (Li et al., 2002). In addition, muscarinic agonists probably produce analgesia through the inhibition of deep dorsal horn projection neurons of rats through GABA_B receptors in rats (Chen and Pan, 2004). Thus, stimulation of spinal GABA release and GABA_B receptors is considered an important mechanism by which muscarinic agonists exert their analgesic effects (Li et al., 2002; Chen and Pan, 2004).

Using a proper combination of mAChR subtype-preferring antagonists and the muscarinic toxin, we recently found that the M₂, M₃, and M₄ receptor subtypes all contribute to the potentiation of GABAergic tone in the spinal cord of rats (Zhang et al., 2005). However, the currently available "selective" muscarinic antagonists are endowed with only a relatively small degree of subtype specificity (Wess, 1996; Caulfield and Birdsall, 1998). Therefore, it is essential to further substantiate the role of mAChR subtypes in the regulation of GABAergic inputs to spinal dorsal horn neurons using other approaches, such as mAChR subtype knockout (KO) mice (Wess, 2004). In the present study, we determined the role of individual mAChR subtypes in the regulation of synaptic GABA release to spinal lamina II neurons using mAChR KO mice. This study revealed an unexpected species difference in the specific role of individual mAChR subtypes in regulating GABAergic inputs to spinal dorsal horn neurons.

	Materials and Methods

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Animals. All wild-type and mAChR subtype single- and double-KO mice were obtained from University of Tokyo (from Minoru Matsui) and the National Institute of Diabetes and Digestive and Kidney Diseases (from Jürgen Wess). The generation and breeding of M₂ KO, M₄ KO, M₃ KO, M₁/M₃ double-KO, and M₂/M₄ double-KO mice have been described elsewhere (Gomeza et al., 1999; Duttaroy et al., 2002; Matsui et al., 2002; Fukudome et al., 2004). All of the KO mice from Minoru Matsui originated from 129/SvJ ES cells and were backcrossed onto the C57BL/6JJcl background (CLEA Japan, Inc., Tokyo, Japan) for more than seven generations. Some of the M₃ KO and M₂/M₄ double-KO mice were obtained from Jürgen Wess. The M₃ KO mice of Jürgen Wess were backcrossed for 10 generations onto the C57BL/6NTac background. The M₂/M₄ double-KO mice of Jürgen Wess were maintained on a 129J1/CF1 mixed genetic background (Duttaroy et al., 2002). Age-(5-7 weeks old) and sex-matched descendants derived from the wild-type mice resulting from intercrossing of these mutants were used in this study. Because the effects of oxotremorine-M on sIPSCs and mIPSCs were identical in the two strains of wild-type, M₃ KO, and M₂/M₄ double KO mice, the electrophysiology data from M₃ KO and M₂/M₄ double KO mice were pooled. Mouse genotyping was carried out by Southern blotting and polymerase chain reaction analysis of mouse-tail DNA, as described previously (Duttaroy et al., 2002; Fukudome et al., 2004). The experimental protocols and procedures were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine and conformed to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Spinal Cord Slice Preparations. Mice were anesthetized with 2% halothane in O₂, and the lumbar segment of the spinal cord was rapidly removed through a limited laminectomy. Mice were then killed by inhalation of 5% halothane. The segment of the lumbar spinal cord was immediately placed in an 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. Transverse spinal cord slices (350 µm) were cut in the ice-cold sucrose aCSF and then 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.

The lamina II has a distinct translucent appearance and can be identified easily under the microscope. In this study, neurons in the outer zone of lamina II were selected for recording (Li et al., 2002; Pan et al., 2002). The neurons located in the dorsal one third of lamina II in the spinal slice were used for recording under a fixed-stage microscope (BX51WI; Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. The electrode for the whole-cell recordings was pulled from borosilicate glass capillaries using a puller (P-97; Sutter Instrument, Novato, CA). The impedance of the pipette was 4 to 7 M when filled with internal solution containing 110 mM Cs₂SO₄, 5 mM KCl, 2.0 mM MgCl₂, 0.5 mM CaCl₂, 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM ATP-magnesium, 0.5 mM sodium-GTP, 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDP--S), and 10 mM QX314, adjusted to pH 7.2 to 7.4 with 1 M CsOH (290-320 mOsm). GDP--S was added to the internal solution to block the possible postsynaptic effect mediated by muscarinic agonists through G proteins (Li et al., 2002; Zhang et al., 2005). QX314, a sodium-channel blocker, was added to the internal solution to suppress the action potential generation from the recorded cell. 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).

Electrophysiological Recordings. Recordings of postsynaptic currents were performed using the whole-cell voltage-clamp method, as described previously (Li et al., 2002; Zhang et al., 2005). Recordings of postsynaptic currents began 5 min later 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 by more than 15%. Signals were recorded using an amplifier (MultiClamp 700A; Axon Instruments, Foster City, CA) at a holding potential of 0 mV, filtered at 1 to 2 kHz, digitized at 10 kHz, and stored in a Pentium computer with pCLAMP 9.0 (Axon Instruments). All spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in the presence of 2 µM strychnine, a glycine receptor antagonist, and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione, a specific glutamate non-N-methyl-D-aspartate antagonist. To record the miniature inhibitory postsynaptic currents (mIPSCs), 1 µM tetrodotoxin was added in the perfusion solution.

Oxotremorine-M, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), GDP--S, atropine, himbacine, strychnine, 6-cyano-7-nitroquinoxaline-2,3-dione, and bicuculline were obtained from Sigma (St. Louis, MO). Tetrodotoxin and QX314 were obtained from Alomone Labs (Jerusalem, Israel). Drugs were dissolved in Krebs solution and perfused into the tissue chamber using syringe pumps.

Data Analysis. Data are presented as means ± S.E.M. The sIPSCs and mIPSCs were analyzed offline with a peak detection program (MiniAnalysis; Synaptosoft, Decatur, GA). Measurements of the amplitude and frequency of sIPSCs and mIPSCs were performed over a period of at least 1 min during control, drug application, and recovery. For each analysis, at least 300 events were included. The sIPSCs and mIPSCs were detected by the fast rise time of the signal over an amplitude threshold above the background noise. Neurons were considered to be responsive to oxotremorine-M if the frequency or amplitude of sIPSCs and mIPSCs was altered >20%. The cumulative probability of the amplitude and interevent interval of sIPSCs and mIPSCs was compared using the Komogorov-Smirnov test, which estimates the probability that two cumulative distributions are similar. The effects of oxotremorine-M on the frequency and amplitude of sIPSCs and mIPSCs were determined by paired two-tailed Student's t test or repeated-measures analysis of variance. P < 0.05 was considered to be statistically significant.

	Results

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All lamina II neurons tested exhibited spontaneous IPSCs at a holding potential of 0 mV. There were no significant differences in the input resistance and the baseline frequency and amplitude of sIPSCs in different types of mAChR KO and wild-type mice (Table 1).

Effect of Oxotremorine-M on sIPSCs and mIPSCs in Wild-Type Mice. To determine the concentration-dependent effect of oxotremorine-M on sIPSCs in spinal cord lamina II neurons of wild-type mice, 1, 3, 5, and 10 µM oxotremorine-M, a nonselective agonist for all mAChR subtypes, was perfused to the tissue chamber. Each concentration was applied for a duration of 3 min. In a total of 15 lamina II neurons, 1 µM oxotremorine-M had no significant effect on the frequency and amplitude of sIPSCs. Oxotremorine-M from 3 to 10 µM significantly decreased the frequency of sIPSCs in 12 (80%) of 15 neurons in a concentration-dependent manner (Fig. 1, A-C). Oxotremorine-M decreased the amplitude of sIPSCs in 7 (58.3%) of the above 12 cells (from 15.76 ± 3.78 to 10.11 ± 1.37 pA at the concentration of 5 µM, P < 0.05) but did not significantly alter the IPSC amplitude in the remaining 5 cells (from 12.46 ± 1.12 to 11.44 ± 0.96 pA at the concentration of 5 µM). Oxotremorine-M had no significant effect on sIPSCs in the remaining three (20%) neurons. The sIPSCs were completely eliminated by 10 µM bicuculline, a specific GABA_A receptor antagonist (Fig. 1A).

View larger version (28K): [in this window] [in a new window]   Fig. 1. Effect of oxotremorine-M on sIPSCs and mIPSCs of spinal lamina II neurons in wild-type mice. A, original tracings of sIPSCs during control, application of 3 and 5 µM oxotremorine-M (Oxo), and washout in one lamina II cell. Note that the sIPSCs were abolished by 20 µM bicuculline. B, cumulative plot of sIPSCs of the same neuron in A showing the distribution of the interevent interval and amplitude during control, application of 5 µM oxotremorine-M, and washout. C, summary data showing that the effect of oxotremorine-M on the sIPSCs in 12 cells in wild-type mice. D, summary data showing the effect of oxotremorine-M on the frequency of mIPSCs in wild-type mice (n = 14). E, group data showing the effect of 3 µM oxotremorine-M on sIPSCs before and during the application of 2 µM himbacine (Him) in 11 lamina II neurons. Data are presented as means ± S.E.M. *, P < 0.05 compared with the control.

In 11 additional lamina II neurons, we tested the effect of the M₂ and M₄ subtype-preferring antagonist himbacine (Dorje et al., 1991; Miller et al., 1992; Doller et al., 1999) on oxotremorine-M-induced decreases in the frequency of sIPSCs in wild-type mice. In all 11 cells tested, 3 µM oxotremorine-M initially decreased the frequency of sIPSCs (Fig. 1E). Oxotremorine-M induced a large increase in the frequency of sIPSCs in the presence of 2 µM himbacine (Fig. 1E).

Effect of Oxotremorine-M on sIPSCs and mIPSCs in M₁/M₃ Double-KO and M₃ Single-KO Mice. To assess the role of the M₂ and M₄ mAChR subtypes in the inhibitory effect of oxotremorine-M on synaptic GABA release, we tested the effect of oxotremorine-M on sIPSCs in M₁/M₃ double-KO and M₃ single-KO mice. In M₁/M₃ double-KO mice, 3 to 10 µM oxotremorine-M significantly decreased the frequency of sIPSCs in a concentration-dependent manner in all 12 cells tested (Fig. 2A). Oxotremorine-M decreased the amplitude of sIPSCs in 6 of 12 cells (from 23.99 ± 4.45 to 16.32 ± 3.88 pA at the concentration of 5 µM, P < 0.05) but did not significantly alter the IPSC amplitude in the remaining 6 cells (from 16.68 ± 3.69 to 16.05 ± 4.19 pA at the concentration of 5 µM, P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 2. Effect of oxotremorine-M on sIPSCs in M1/M3 double-KO and M3 single-KO mice. A, summary data showing the dose-response effect of oxotremorine-M on the frequency of sIPSCs in M1/M3 double-KO mice. B, group data showing the effect of 3 µM oxotremorine-M on sIPSCs before and during application of 2 µM himbacine (Him) in six lamina II neurons from M1/M3 double-KO mice. C, summary data showing the dose-response effect of oxotremorine-M on the frequency of sIPSCs in M3 single-KO mice. Data are presented as means ± S.E.M. *, P < 0.05 compared with respective controls.

Previous pharmacological studies have suggested the presence of the M₁ subtype in the spinal dorsal horn (Iwamoto and Marion, 1993; Naguib and Yaksh, 1997). Thus, both M₃ KO and M₁/M₃ double-KO mice were used to determine whether the M₁ subtype plays a role in the effect of oxotremorine-M on synaptic GABA release in the spinal cord. In M₃ single-KO mice, oxotremorine-M also decreased the frequency of sIPSCs in a concentration-dependent manner in all 15 cells tested (Fig. 2C). Oxotremorine-M decreased the amplitude of sIPSCs in 6 of 15 cells (from 22.68 ± 3.40 to 14.91 ± 2.52 pA at the concentration of 5 µM, P < 0.05) but had no effect on another 9 cells (from 10.98 ± 1.65 to 9.82 ± 1.58 pA at the concentration of 5 µM, P > 0.05). Application of 5 µM oxotremorine-M significantly decreased the frequency (from 1.15 ± 0.27 to 0.51 ± 0.11 Hz) but not the amplitude (11.82 ± 0.53 verses 11.28 ± 0.45 pA) of mIPSCs in 11 of 13 cells. Oxotremorine-M had no significant effect on the frequency (from 1.22 to 1.20 Hz in one cell and from 1.26 to 1.31 Hz in another cell) of mIPSCs in the remaining two cells in M₃ single-KO mice.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Effect of oxotremorine-M on sIPSCs in M2/M4 double-KO mice. A, raw tracings of sIPSCs during control, application of 1, 3, and 5 µM Oxo, and washout in one cell. Note that the sIPSCs were abolished by 20 µM bicuculline. B, cumulative probability plot of sIPSCs of the same neuron in A showing the distribution of the interevent interval and amplitude of sIPSCs during control, application of 5 µM oxotremorine-M, and washout. C, summary data showing the dose-response effect of oxotremorine-M on sIPSCs in 15 cells. D, effect of 3 µM oxotremorine-M on the frequency of sIPSCs in six cells before and after application of 25 nM 4-DAMP. Data are presented as means ± S.E.M. *, P < 0.05 compared with control.

To determine whether the M₃ subtype mediates the stimulating effect of oxotremorine-M on synaptic GABA release in M₂/M₄ double-KO mice, we further tested the effect of 3 µM oxotremorine-M on sIPSCs in the presence of 25 nM 4-DAMP, an M₃ subtype-preferring muscarinic antagonist (Ehlert, 1996; Yigit et al., 2003; Zhang et al., 2005). The effect of 3 µM oxotremorine-M on sIPSCs was completely blocked by subsequent application of 25 nM 4-DAMP in all six cells examined (Fig. 3D).

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effect of Oxo on mIPSCs in M2/M4 double-KO mice. A, representative tracings of mIPSCs during control, application of 3 µM Oxo, and washout in one cell. B, cumulative probability plot of mIPSCs of the same neuron in A showing the distribution of the interevent interval and amplitude of mIPSCs during control, application of 3 µM oxotremorine-M, and washout. C, effect of 3 µM oxotremorine-M on the frequency of mIPSCs in 13 cells from M2/M4 double-KO mice. D, Effect of 3 µM oxotremorine-M on the frequency of mIPSCs before and after application of 25 nM 4-DAMP. These eight neurons were part of 13 cells shown in C. Data are presented as means ± S.E.M. *, P < 0.05 compared with the control.

Effect of Oxotremorine-M on sIPSCs in M₂ Single-KO and M₄ Single-KO Mice. To further delineate the role of the M₂ and M₄ receptor subtypes in the inhibitory action of oxotremorine-M on sIPSCs, the effect of oxotremorine-M on sIPSCs was tested in M₂ single-KO and M₄ single-KO mice, respectively. In M₂ single-KO mice, 3 to 10 µM oxotremorine-M significantly increased the frequency of sIPSCs in a concentration-dependent manner in 16 (66.6%) of 24 neurons (Fig. 5, A-C). In contrast, oxotremorine-M had no significant effect on the frequency of sIPSCs in 4 (16.6%) of 24 neurons and significantly decreased the frequency of sIPSCs in 4 (16.6%) of 24 neurons. Oxotremorine-M increased the amplitude in 4 of the 24 cells (Fig. 5C) but had no significant effect on the amplitude of sIPSCs in 18 cells and even decreased the IPSC amplitude of 2 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effect of oxotremorine-M on the frequency of sIPSCs in M2 KO mice. A, original tracings of sIPSCs during control, application of 3 and 5 µM Oxo, and washout in one lamina II neuron. B, cumulative probability plot of sIPSCs of the same neuron in A showing the distributions of the interevent interval and amplitude during control, application of 5 µM oxotremorine-M, and washout. C, group data showing differential effects of 5 µM oxotremorine-M on the frequency of sIPSCs in 24 cells. Data are presented as means ± S.E.M. *, P < 0.05 compared with control.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Effect of oxotremorine-M on the frequency of sIPSCs in M4 KO mice. A, representative tracings of sIPSCs during control, application of 5 µM Oxo, and washout in one cell. B, group data showing differential effects of oxotremorine-M on the frequency of sIPSCs increased in 17 cells. Data are presented as means ± S.E.M. *, P < 0.05 compared with respective controls.

	Discussion

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In this study, we used mAChR KO mice to define the role of individual mAChR subtypes in regulation of GABAergic inputs to spinal dorsal horn neurons. We found, unexpectedly, that oxotremorine-M decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. Further studies showed that in M₁/M₃ double-KO and M₃ single-KO mice, oxotremorine-M significantly decreased the frequency of sIPSCs and mIPSCs. It is noteworthy that oxotremorine-M significantly increased the frequency of sIPSCs and mIPSCs in M₂/M₄ double-KO mice. These results indicate that the M₂ and M₄ subtypes are responsible for the muscarinic inhibition of spinal GABA release in mice. On the other hand, stimulation of the M₃ subtype is predicted to potentiate GABAergic tone in the spinal cord dorsal horn of mice. Therefore, this study provides new information that the activation of the M₂ and M₄ subtypes inhibits GABAergic inputs to spinal dorsal horn neurons in mice, which is opposite to the functional role of M₂ and M₄ mAChRs in the control of spinal GABA release in rats (Zhang et al., 2005). Nevertheless, stimulation of the M₃ subtype potentiates synaptic GABA release in the spinal dorsal horn in both mice and rats.

The mAChRs in the dorsal horn of the spinal cord are important for regulation of nociception. In this regard, intrathecal administration of muscarinic agonists or acetylcholinesterase inhibitors produces a potent analgesic effect in many different species, including rats, mice, and humans (Iwamoto and Marion, 1993; Naguib and Yaksh, 1994; Hood et al., 1997; Ellis et al., 1999; Duttaroy et al., 2002; Chen and Pan, 2004). Three mAChR subtypes, M₂, M₃, and M₄ receptors, are present in the spinal cord dorsal horn, and the M₂ and M₃ subtypes are particularly concentrated in the superficial laminae of the spinal cord (Hoglund and Baghdoyan, 1997; Yung and Lo, 1997; Li et al., 2002). Because of a lack of highly specific agents for the individual mAChR subtypes, the receptor subtypes that mediate the analgesic effect of muscarinic agonists were not established until recently. The role of the M₂ and M₄ receptor subtypes in mediating the analgesic effect of mAChR agonists has been documented using M₂ KO and M₂/M₄ double-KO mice (Gomeza et al., 1999; Duttaroy et al., 2002). The frequency of sIPSCs and mIPSCs is the most important measure of synaptic GABA release in this study. The change in the sIPSC amplitude by oxotremorine-M may reflect various pools of GABA-containing vesicles released from GABAergic terminals. We found in this study that oxotremorine-M inhibited sIPSCs and mIPSCs of dorsal horn neurons in wild-type mice, which is in contrast to the potentiating effect of oxotremorine-M on GABAergic IPSCs of dorsal horn neurons in rats (Zhang et al., 2005). In M₃ single-KO and M₁/M₃ double-KO mice, oxotremorine-M consistently decreased the frequency of sIPSCs. Oxotremorine-M also significantly decreased the frequency, but not the amplitude, of mIPSCs in M₃ KO mice, suggesting that the M₂ and M₄ subtypes are present on the presynaptic terminals of GABAergic interneurons in the mouse spinal cord. The role of M₂ and M₄ subtypes in the inhibition of synaptic GABA release in the mouse spinal dorsal horn is further supported by our finding that the M₂ and M₄ subtype-preferring antagonist himbacine reversed the inhibitory effect of oxotremorine-M on sIPSCs in wild-type mice and abolished the inhibitory effect of oxotremorine-M on sIPSCs in M₁/M₃ double-KO mice. Because oxotremorine-M consistently decreased the frequency of GABAergic sIPSCs and mIPSCs of dorsal horn neurons in wild-type mice, M₂/M₄ subtypes play a critical role in muscarinic inhibition of synaptic GABA release in the spinal dorsal horn of mice.

Contrary to the inhibitory effect of oxotremorine-M on IPSCs in wild-type and M₃ KO mice, oxotremorine-M consistently increased the frequency of GABAergic sIPSCs in M₂/M₄ double-KO mice. Furthermore, the M₃ subtype-preferring muscarinic antagonist 4-DAMP completely blocked the effect of oxotremorine-M on sIPSCs in M₂/M₄ double-KO mice. These data strongly suggest that activation of the M₃ subtype potentiates synaptic GABA release in the spinal dorsal horn of mice. In wild-type mice, the role of the M₃ subtype in muscarinic modulation of GABA release seems to be overwhelmed by the dominant action of the M₂/M₄ subtypes in the spinal dorsal horn of mice. This may explain why the potential role of the M₃ subtype in muscarinic potentiation of spinal GABA release is only revealed by the use of M₂/M₄ double-KO mice and himbacine. Because oxotremorine-M significantly increased the frequency but not the amplitude of mIPSCs in M₂/M₄ double-KO mice, the M₃ subtype is also probably located on the presynaptic terminals of GABAergic interneurons in the mouse spinal cord. The presence of the M₃ receptor subtype in the rat spinal cord has been suggested in a radioligand binding study (Hoglund and Baghdoyan, 1997). We have shown that the M₃ subtype plays a role in the potentiation of spinal GABA release by oxotremorine-M in rats (Zhang et al., 2005). However, the physiological function of the M₃ subtype in the spinal cord remains largely unknown, and its functional role in the spinal analgesic effect of muscarinic receptor agonists needs to be further defined in M₃ KO mice. Although previous pharmacological studies have suggested the presence of the M₁ subtype in the spinal dorsal horn (Iwamoto and Marion, 1993; Naguib and Yaksh, 1997), we found no difference in the inhibitory effect of oxotremorine-M on sIPSCs between the M₃ single-KO and M₁/M₃ double-KO mice. Hence, these data suggest that the M₁ subtype is not involved in the muscarinic regulation of synaptic GABA release to spinal dorsal horn neurons.

The variable effect of oxotremorine-M on sIPSCs in the M₂ and M₄ single-KO mice most probably occurs because the recorded neurons receive different afferent inputs with distinct presynaptic mAChR subtypes. It is unlikely that this variability can be explained by the presence of different types of lamina II neurons because the effect of oxotremorine-M on sIPSCs is consistent in M₁/M₃ double-KO, M₃ single-KO, and M₂/M₄ double-KO mice. This notion is further supported by our finding that after the blockade of the M₂ and M₄ subtypes with himbacine, oxotremorine-M consistently increased (but not decreased) the frequency of sIPSCs in wild-type mice. Because of the opposing functions of the M₃ and M₂/M₄ subtypes in regulating spinal GABA release in mice (increase versus decrease in GABA release, respectively), the net effect of oxotremorine-M on synaptic GABA release depends on the distribution and the relative density of the M₃ and M₂/M₄ subtypes on the nerve terminals that synapse with the recorded neuron. We observed that oxotremorine-M increased the frequency of sIPSCs in most (66.6%) lamina II neurons but decreased the IPSC frequency in some (16.6%) cells in M₂ single-KO mice. These results suggest that there may be more M₃ than M₄ mAChRs on the presynaptic terminals of GABAergic neurons in the mouse spinal dorsal horn. On the other hand, oxotremorine-M can stimulate both M₂ (to decrease GABA release) and M₃ (to increase GABA release) subtypes in M₄ single-KO mice. We found that oxotremorine-M decreased the frequency of sIPSCs in 58.8% cells, whereas it increased the IPSC frequency in 23.5% of the dorsal horn neurons tested in M₄ single-KO mice. These data suggest that the M₂ subtype plays a greater role than the M₃ subtype in muscarinic regulation of spinal GABA release. Therefore, these mAChR subtypes contribute to a different extent (i.e., M₂ > M₃ > M₄) to the muscarinic modulation of spinal GABA release in mice. These electrophysiological data are consistent with the receptor binding and behavioral studies showing that M₂ is the predominant mAChR subtype in the mouse spinal cord (Gomeza et al., 1999; Duttaroy et al., 2002; Chen et al., 2005; Oki et al., 2005).

View larger version (24K): [in this window] [in a new window]   Fig. 7. Diagram illustrating two possible mechanisms of inhibition of dorsal horn projection neurons as a result of decreased synaptic GABA release to lamina II interneurons by activation of M2 and M4 mAChR subtypes in the mouse spinal cord. See Discussion for details.

View larger version (32K): [in this window] [in a new window]   Fig. 8. Schematic drawing highlighting the major differences in the subcellular location and physiological function of M2, M3, and M4 mAChR subtypes in control of synaptic GABA release to lamina II neurons in the spinal cord dorsal horn of mice (A) and rats (B). Note that M2, M3, and M4 mAChR subtypes may be located on the same or different GABAergic neurons and terminals. AC, adenylyl cyclase; -, inhibition; +, potentiation.

	Footnotes

 
This study was supported by grants GM64830 and NS45602 from the National Institutes of Health (to H.-L.P.) and grant-in-aid for Scientific Research on Priority Areas 16067101 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M.M.).

ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; sIPSC, spontaneous inhibitory postsynaptic current; mIPSC, miniature inhibitory postsynaptic current; GDP--S, guanosine 5'-O-(2-thiodiphosphate); 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; QX314, 2-((2,6-dimethylphenyl)amino)-N,N,N-triethyl-2-oxoethanaminium; aCSF, artificial cerebrospinal fluid; KO, knockout; Oxo, oxotremorine-M.

Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology and Pain Medicine, University of Texas M.D. Anderson Cancer Center, 1400 Holcombe Blvd., Unit 409, Houston, TX 77030.

	References

Top Abstract Materials and Methods Results Discussion References

 
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