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NEUROPHARMACOLOGY
Department of Anesthesiology and Pain Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas (H.-M.Z., H.-Y.Z., S.-R.C., H.-L.P.); and Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland (D.G., J.W.)
Received June 26, 2007; accepted September 17, 2007.
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Abstract |
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). Intrathecal administration of mAChR agonists or acetylcholinesterase inhibitors produces a potent analgesic effect in many 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, 2003). Molecular cloning studies have revealed five molecularly distinct mAChRs (Caulfield, 1993; Wess, 1996). The odd-numbered subtypes (M1, M3, and M5) are selectively linked to Gq/11 proteins, and the even-numbered subtypes (M2 and M4) are preferentially coupled to the pertussis toxin (PTX)-sensitive Gi/o proteins (Felder, 1995; Wess, 1996; Caulfield and Birdsall, 1998). The highest density of spinal mAChRs is located in the superficial laminae in both rats and humans (Yamamura et al., 1983; Scatton et al., 1984; Villiger and Faull, 1985; Höglund and Baghdoyan, 1997; Li et al., 2002). Previous studies have documented that M2, M3, and M4 mAChR subtypes are present in the spinal dorsal horn and that the M2 and M3 subtypes are particularly concentrated in the superficial laminae (Höglund and Baghdoyan, 1997; Duttaroy et al., 2002; Chen et al., 2005). Although the role of the M2 and M4 subtypes in mAChR agonist-induced analgesia has been established (Ellis et al., 1999; Gomeza et al., 1999a; Duttaroy et al., 2002; Li et al., 2002), the mechanisms by which each mAChR subtype contributes to the regulation of nociceptive transmission in the spinal cord are not clear.
The spinal lamina II neurons receive nociceptive inflow from primary afferent neurons and synaptic input from glutamatergic excitatory and GABAergic/glycinergic interneurons in rats and mice (Yoshimura and Nishi, 1995; Pan and Pan, 2004; Zhang et al., 2005, 2006; Wang et al., 2006). Glycine is an important inhibitory neurotransmitter in the spinal cord, and blockade of glycine receptors in the spinal cord leads to hypersensitivity of dorsal horn neurons and allodynia (Yaksh, 1989; Cronin et al., 2004). Glycine-like immunoreactive axons, dendrites, and cell bodies are present in the spinal superficial dorsal horn (Todd, 1990; Todd et al., 1996). In the rat spinal cord, the M3 subtype is mainly responsible for muscarinic potentiation of synaptic glycine release (Wang et al., 2006). However, because highly selective agonists and antagonists for specific mAChR subtypes are not available, the definitive function of the individual mAChR subtypes in the regulation of glycinergic input to spinal dorsal horn neurons have yet to be established. In the present study, we used mAChR subtype-knockout (KO) mice to determine the role of the M2, M3, and M4 subtypes in the control of glycinergic input to spinal lamina II neurons. This study provides unambiguous evidence about the specific function of these three mAChR subtypes in the control of glycinergic synaptic transmission in the spinal dorsal horn.
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Materials and Methods |
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,b; Yamada et al., 2001; Duttaroy et al., 2002; Fukudome et al., 2004). Mouse genotyping was carried out by Southern blotting and polymerase chain reaction analysis of mouse-tail DNA, as described previously (Gomeza et al., 1999a,b; Yamada et al., 2001; Duttaroy et al., 2002). The experimental protocols and procedures were approved by the Animal Care and Use Committee of the University of Texas MD Anderson Cancer Center and conformed to the Institute of Laboratory Animal Resources (1996).
Intrathecal PTX Treatment. Mice were anesthetized with 2% isoflurane in O2 via a nose cone. The lumbar region was shaved and prepared with Betadine solution, and the intervertebral spaces were widened by placing the animal on a Plexiglas tube. The animals were then injected at the L5-L6 level by using a 30-gauge needle connected to a Hamilton syringe filled with 1 µg of PTX dissolved in 4 µlof water. Correct subarachnoid positioning of the tip of the needle was verified by a tail-flick reflex (Hylden and Wilcox, 1980). Animals were allowed to recover for 3 days in their home cage before the final electrophysiological experiments.
Spinal Cord Slice Preparation. Mice were anesthetized with 2% isoflurane in O2, 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% O2 and 5% CO2. The sucrose aCSF contained 234 mM sucrose, 3.6 mM KCl, 1.2 mM MgCl2, 2.5 mM CaCl2, 1.2 mM NaH2PO4, 12.0 mM glucose, and 25.0 mM NaHCO3. The tissue was then placed in a shallow groove formed in a gelatin block and glued onto the stage of a Vibratome (Technical Products International, Inc., St. Louis, MO). Transverse spinal cord slices (350 µm) were cut in the ice-cold sucrose aCSF and preincubated in Krebs' solution oxygenated with 95% O2 and 5% CO2 at 34°C for at least 1 h before they were transferred to the recording chamber. The Krebs' solution contained 117.0 mM NaCl, 3.6 mM KCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 1.2 mM NaH2PO4, 11.0 mM glucose, and 25.0 mM NaHCO3. Each 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 we described previously (Li et al., 2002; Zhang et al., 2005). The lamina II has a distinct translucent appearance and can easily be distinguished under the microscope. The neurons located in the lamina II in the spinal slice were identified under a fixed stage microscope (BX51WI; Olympus, Tokyo, Japan) with differential interference contrast/infrared illumination. The electrode for the whole-cell recordings was triple pulled from borosilicate glass capillaries with a puller (P-97; Sutter Instrument Company, Novato, CA). The impedance of the pipette was 4 to 7 M
when filled with internal solution containing 110.0 mM Cs2SO4, 5.0 mM KCl, 2.0 mM MgCl2, 0.5 mM CaCl2, 5.0 mM HEPES, 5.0 mM EGTA, 5.0 mM ATP-Mg, 0.5 mM Na-GTP, 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 mAChR 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.
Recordings of postsynaptic currents began approximately 5 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 the resistance changed more than 15%. Signals were recorded using an amplifier (MultiClamp700A; 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 10 µM bicuculline, a GABAA receptor antagonist, and 20 µM 6-cyano-7-nitroquinoxaline-2,3-dione, a specific glutamate non-N-methyl-D-aspartic acid (NMDA) antagonist. To record the miniature inhibitory postsynaptic currents (mIPSCs), 1 µM tetrodotoxin was added in the perfusion solution. The conditions for measuring sIPSCs and mIP-SCs were the same except that tetrodotoxin was used during the recording of mIPSCs.
Oxotremorine-M (oxo), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), himbacine, GDP--S, strychnine, and 6-cyano-7-nitroquinoxaline-2,3-dione were obtained from Sigma-Aldrich (St. Louis, MO). Tetrodotoxin and QX314 were obtained from Alomone Labs (Jerusalem, Israel). AFDX-116, CGP55845, and bicuculline were purchased from Tocris Cookson Inc. (Ellisville, MO). Muscarinic toxin 3 (MT-3) was purchased from Peptide Institute (Osaka, Japan). Drugs were dissolved in Krebs' solution and perfused into the tissue chamber with the use of syringe pumps.
Data Analysis. Data are presented as means ± S.E.M. The sIPSCs and mIPSCs were analyzed off-line with a peak detection program (MiniAnalysis; Synaptosoft, Decatur, GA). Measurements of the amplitude and frequency of sIPSCs and mIPSCs were performed for at least 2 min during control, drug application, and washout recovery. The sIPSCs and mIPSCs were detected by the fast rise time of the signal over an amplitude threshold above the background noise (Zhang et al., 2005, 2006). The amplitude detection threshold was typically 6 to 8 pA. We manually excluded an event when the noise was erroneously identified as sIPSCs or mIPSCs by the computer program. The background noise level was typically constant throughout the recording in a given neuron. The cumulative probabilities of the amplitude and interevent interval of sIPSCs and mIPSCs were compared using the Komogorov-Smirnov test. The effects of oxotremorine-M on the frequency and amplitude of sIPSCs and mIPSCs were determined using paired two-tailed Student's t test or one-way analysis of variance. The Komogorov-Smirnov test was used to determine whether the drug effect on sIPSCs and mIPSCs was significantly different. P < 0.05 was considered statistically significant.
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In another nine lamina II neurons in which 5 µM oxotremorine-M significantly decreased the frequency of sIP-SCs (from 0.86 ± 0.19 to 0.57 ± 0.17 Hz), the effect of this agent on mIPSCs was tested. In the presence of 1 µM tetrodotoxin, oxotremorine-M significantly decreased the frequency of mIPSCs (from 0.84 ± 0.16 to 0.60 ± 0.14 Hz; Fig. 1D). These results suggest that stimulation of mAChRs primarily reduces synaptic glycine release and that the mAChRs involved in this activity are present on the presynaptic terminals of glycinergic interneurons in the mouse spinal dorsal horn.
Effect of Oxotremorine-M on Glycinergic sIPSCs in M3-KO Mice. To assess the role of the M2 and M4 mAChR subtypes in the inhibitory effect of oxotremorine-M on synaptic glycine release, we tested the effect of oxotremorine-M on glycinergic sIPSCs in M3-KO mice. Oxotremorine-M (1–10 µM) decreased the frequency of sIPSCs in a concentration-dependent manner, and this effect was observed in all 12 neurons tested (Fig. 2). These data strongly suggest that activation of the M2 and M4 subtypes decreases inhibitory glycinergic input to spinal dorsal horn neurons.
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; Douglas et al., 2001; Wang et al., 2006). In 12 additional neurons from M3-KO mice, the effect of oxotremorine-M was completely blocked by 2 µM himbacine (Fig. 3C), an M2/M4-preferring antagonist (Dörje et al., 1991; Miller et al., 1992; Doller et al., 1999; Zhang et al., 2005). These data provide further evidence that the M2 and M4 subtypes contribute to the inhibition of spinal glycine release by stimulation of mAChRs in mice.
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). Thus, the increased GABA release could spillover and activate presynaptic GABAB receptors on glycinergic neurons to inhibit glycine release. To test this hypothesis, we used the GABAB receptor antagonist CGP55845 (Li et al., 2002; Wang et al., 2006). In the presence of 1 µM CGP55845, oxotremorine-M significantly increased the frequency of sIPSCs in a concentration-dependent manner in all 12 neurons studied (Fig. 4D). These findings indicate that activation of the M3 subtype contributes to muscarinic potentiation of spinal glycine release and that spinal glycine release by the M3 subtype is influenced by concurrent M3 receptor-mediated stimulation of GABAergic interneurons, which evokes spinal GABA release and activate presynaptic GABAB receptors on glycinergic interneurons to limit the further release of glycine.
It has been documented that M2, M3, and M4 mAChR subtypes are present in the spinal dorsal horn (Höglund and Baghdoyan, 1997; Duttaroy et al., 2002; Chen et al., 2005). To confirm the role of the M3 subtype in the potentiating effect of oxotremorine-M on synaptic glycine release in M2/M4 double-KO mice, we further tested the effect of 5 µM oxotremorine-M on glycinergic sIPSCs in the presence of 50 nM 4-DAMP, an M3 subtype-preferring mAChR antagonist (Cembala et al., 1998; Moriya et al., 1999; Zhang et al., 2005). The potentiating effect of 5 µM oxotremorine-M on sIPSCs was completely blocked by subsequent application of 50 nM 4-DAMP in all seven neurons examined (Fig. 5A).
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Effect of Oxotremorine-M on sIPSCs in PTX-Treated WT Mice. To further define the role of the M3 subtype in the potentiating effect of oxotremorine-M on sIPSCs, a group of WT mice were treated intrathecally with 1 µg of PTX to inactivate Gi/o proteins that are coupled to M2/M4 subtypes (Zhang et al., 2005). In all 14 neurons from PTX-treated mice, oxotremorine-M consistently increased the frequency of sIP-SCs in a dose-dependent manner (Fig. 6). These data are similar to those obtained from M2/M4 double-KO mice in the presence of the GABAB receptor antagonist CGP55845 and are consistent with the notion that the M2/M4 subtypes and GABAB receptors are coupled to PTX-sensitive Gi/o proteins.
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Effect of Oxotremorine-M on sIPSCs in M2-KO Mice. To delineate the relative role of the M3 and M4 subtypes in the action of oxotremorine-M on glycinergic sIPSCs, the effect of oxotremorine-M on sIPSCs was tested in M2-KO mice. In this protocol, 1 µM CGP55845 was used to eliminate the influence of GABAB receptors on the effect of oxotremorine-M on spinal glycine release. In 19 of 32 (59.4%) neurons, 3 to 10 µM oxotremorine-M dose-dependently increased the glycinergic sIPSCs (Fig. 7A). Oxotremorine-M had no significant effect on the frequency of glycinergic sIPSCs in the remaining 13 neurons (40.6%).
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; Ellis et al., 1999), oxotremorine-M significantly increased the frequency of glycinergic sIPSCs in all neurons tested (Fig. 7B). These findings suggest that in the absence of the M2 subtype, the M3 subtype plays a major role in the control of spinal glycine release. Furthermore, the effect of the M3 subtype on glycinergic input was suppressed by the M4 subtype in a population of dorsal horn neurons. Effect of Oxotremorine-M on sIPSCs in M4-KO Mice. To assess the relative role of the M2 and M3 subtypes, we examined the effect of oxotremorine-M in M4-KO mice. CGP55845 (1 µM) was also used to remove the influence of GABAB receptors on oxotremorine-M-induced glycine release. In 13 of 33 (39.4%) neurons from M4 single-KO mice, oxotremorine-M dose-dependently increased the frequency of sIPSCs (Fig. 8). In contrast, oxotremorine-M decreased the frequency of sIPSCs in another 13 (39.4%) neurons, and it had no effect on the frequency of sIPSCs in the remaining 7 (21.2%) neurons. Together with the above-mentioned results, these data suggest that in the absence of the M4 subtype, the M2 and M3 subtypes have a similar important role in the regulation of spinal glycine release.
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Discussion |
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Stimulation of mAChRs in the spinal cord inhibits nociceptive transmission. In this regard, application of mAChR agonists to the spinal cord suppresses the response of dorsal horn neurons to nociceptive stimuli (Chen and Pan, 2004). Three mAChR subtypes (M2, M3, and M4) are present in the spinal cord dorsal horn (Höglund and Baghdoyan, 1997; Duttaroy et al., 2002; Chen et al., 2005). The mAChR agonist-induced antinociception is abolished in M2/M4 double-KO mice (Duttaroy et al., 2002). We found that oxotremorine-M significantly inhibited the frequency but not the amplitude of mIPSCs and sIPSCs in WT mice; this observation suggested that the inhibitory mAChRs are probably located on the presynaptic terminals of glycinergic interneurons. We also determined the functional role of the M2/M4 subtypes in the control of synaptic glycine release in M3 single-KO mice: compared with its effect in WT mice, oxotremorine-M consistently decreased the frequency of sIPSCs in all tested neurons in M3-KO mice. The inhibitory action of the M2/M4 subtypes is further supported by our finding that the M2/M4 subtype-preferring antagonist himbacine abolished the inhibitory effect of oxotremorine-M in M3 KO mice. Because the M2 subtype-preferring antagonist AFDX-116 only partially blocked the inhibitory effect of oxotremorine-M, both the M2 and M4 subtypes seems to contribute to the inhibition of synaptic glycine release in the spinal cord by mAChRs.
Despite the findings from our current study, the physiological function of the M3 subtype in the control of spinal nociceptive transmission remains largely unknown. We found that in M2/M4 double-KO mice, the overall effect of oxotremorine-M was to increase glycinergic sIPSCs. Our observation that the M3 subtype-preferring mAChR antagonist 4-DAMP (Cembala et al., 1998; Moriya et al., 1999; Zhang et al., 2005) abolished the potentiating effect of oxotremorine-M on sIP-SCs in M2/M4 double-KO mice provides further support for our conclusion that the M3 subtype contributes to the potentiation of spinal glycine release. In M2/M4 double-KO mice, oxotremorine-M significantly increased the frequency of mIPSCs, but significantly less than it increased the frequency of sIPSCs. Thus, the M3 subtype is likely located on the presynaptic terminals as well as on somatodendritic sites of glycinergic interneurons in the mouse spinal cord. We were surprised that the potentiating effect of oxotremorine-M on the frequency of sIPSCs was attenuated at higher concentrations. Because oxotremorine-M evokes GABA release through the M3 subtype to dorsal horn neurons in M2/M4 double-KO mice (Zhang et al., 2006), we hypothesized that the increased GABA release could activate GABAB receptors to limit synaptic glycine release in the spinal cord of M2/M4 double-KO mice. We found that when the GABAB receptor antagonist CGP55845 was used, oxotremorine-M at higher concentrations produced a consistent increase in glycinergic sIPSCs in M2/M4 double-KO mice. Thus, GABAB receptors present on glycinergic interneurons function as important heteroreceptors in the dynamic feedback control of glycine release following concurrent activation of the M3 subtype on GABAergic interneurons in the spinal dorsal horn. This conclusion is supported by our finding that in PTX-treated WT mice, oxotremorine-M dose-dependently increased the frequency of sIPSCs and mimicked the effect of oxotremorine-M in M2/M4 double-KO mice in the presence of GABAB receptor antagonist. These findings are consistent with the notion that the M2 and M4 subtypes and the GABAB receptors are all coupled to PTX-sensitive Gi/o proteins. By using M2/M4 double-KO mice, we have discovered a close interaction between GABAergic and glycinergic interneurons through GABAB receptors in the spinal dorsal horn.
Because of the opposing functions of the M3 subtype and the M2 and M4 subtypes in regulating spinal glycine release (increased versus decreased glycine release, respectively), oxotremorine-M may increase or decrease synaptic glycine release to spinal dorsal horn neurons, depending on the distribution and the relative density of these three subtypes on the glycinergic interneurons that make synaptic contact with the postsynaptic neuron. This notion is clearly supported by our finding that blocking the M4 subtype with MT-3 toxin revealed a potentiating effect of the M3 subtype in M2-KO mice. Because the major effect of oxotremorine-M on glycinergic sIPSCs of dorsal horn neurons is inhibitory in WT mice, the role of the M3 subtype in the modulation of glycine release seems to be masked by the dominant action of the M2 and M4 subtypes in the spinal dorsal horn. Thus, the M2 and M4 subtypes play a more important role than the M3 subtype in mAChR regulation of synaptic glycine release in the spinal dorsal horn of mice. In M2-KO mice, oxotremorine-M (by activating M3 and M4 subtypes) increased the frequency of sIPSCs in approximately 60% of the neurons. Because this agent did not inhibit the glycinergic sIPSCs in these mice, the function of the M4 subtype on glycinergic interneurons is likely masked by the presence of the M3 subtype. We also assessed the relative roles of the M2 (to decrease glycine release) and the M3 (to increase glycine release) subtypes in M4-KO mice. We found that oxotremorine-M decreased the frequency of sIPSCs in approximately 40% of the neurons, increased the frequency of sIPSCs in approximately 40% of neurons, and had no effect on sIPSCs in approximately 20% neurons in M4-KO mice. These data suggest that the M2 and M3 subtypes are equally important in the regulation of spinal glycine release. Thus, our findings provide important evidence that the three mAChR subtypes in the spinal cord contribute to a different extent (i.e., M2 = M3 > M4) to the muscarinic modulation of glycinergic input to dorsal horn neurons.
Results from our previous studies using relatively selective mAChR antagonists suggested that the activation of mAChRs potentiates spinal glycine release in rats (Wang et al., 2006). In the present study, however, stimulation of mAChRs primarily inhibited glycine release in the mouse spinal cord. Hence, there exist important species differences regarding the function and subcellular distribution of mAChR subtypes in the regulation of spinal glycine release. For example, activation of the M2 subtype seems to increase spinal glycine release in rats (Wang et al., 2006), whereas in the mouse spinal cord the function of the M2 subtype is to inhibit glycine release. Despite this difference, activation of the M2 and M4 subtypes in the spinal cord has a potent antinociceptive action in both species (Ellis et al., 1999; Gomeza et al., 1999a; Duttaroy et al., 2002; Li et al., 2002). It is not yet clear how the reduction of glycine release by spinal M2 and M4 subtypes contributes to the analgesic effect of mAChR agonists in mice. Because the recorded lamina II neurons are probably interneurons, it is possible that reduced glycinergic input may lead to disinhibition of inhibitory neurons in spinal dorsal horn to reduce nociceptive transmission (Zhang et al., 2006). Furthermore, glycine is a coagonist for the glycine binding site of NMDA receptors. Decreased synaptic glycine release may reduce nociceptive transmission by decreasing NMDA receptor activity in the superficial dorsal horn.
In summary, our study using subtype selective KO mice provides unequivocal evidence that activation of the M2 and M4 mAChR subtypes inhibits glycine release and that activation of the M3 subtype potentiates glycine release in the spinal dorsal horn of mice (Fig. 9). Another interesting finding is that the GABAB receptor is involved in the regulation of spinal glycine release through activation of the M3 subtype expressed on GABAergic interneurons (Fig. 9). These findings provide new insight into the distinct functions of the M2, M3, and M4 subtypes in the regulation of glycinergic input to spinal dorsal horn neurons. This information has broad implications in the study of spinal dorsal horn circuitry involved in the regulation of nociceptive transmission because the activity of glycinergic interneurons in the spinal cord might be modified through the use of different mAChR subtype-KO mice.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: mAChR, muscarinic acetylcholine receptor; PTX, pertussis toxin; KO, knockout; WT, wild-type; aCSF, artificial cerebrospinal fluid; GDP--S, guanosine 5-O-(2-thiodiphosphate); sIPSC, spontaneous inhibitory postsynaptic current; NMDA, N-methyl-D-aspartate; mIPSC, miniature inhibitory postsynaptic current; oxo, oxotremorine-M; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; MT-3, muscarinic toxin 3; QX314, lidocaine N-ethyl chloride; AFDX-116, 11-([12-[diethylamino)methyl]-1-piperidinyl]acetyl)-5,11-dihydro-6-pydido[2,3-b][1,4]-benzodiazepin-6-one; CGP55845, (3-N[[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl)-phosphinic acid.
Address correspondence to: Dr. Hui-Lin Pan, Department of Anesthesiology and Pain Medicine, Unit 110, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030-4009. E-mail: huilinpan{at}mdanderson.org
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