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The Journal of Neuroscience, July 16, 2003, 23(15):6200-6208
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Muscarine-Induced Increase in Frequency of Spontaneous EPSCs in Purkinje Cells in the Vestibulo-Cerebellum of the Rat
Yukihiro Takayasu,1,2,3 Masae Iino,1,3 Nobuhiko Furuya,2 andSeiji Ozawa1,3 Departments of 1Physiology and 2Otolaryngology, Gunma University School of Medicine, Maebashi, Gunma, 371-8511, Japan, and 3Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 322-0012, Japan
Abstract
Top
Abstract
Introduction
Cholinergic projections are relatively sparse in the cerebellum compared with other parts of the brain. However, some mossy fibers in the vestibulo-cerebellum are known to be cholinergic. To clarify the functional roles of cholinergic mossy fibers in the vestibulo-cerebellum, we investigated the effects of acetylcholine (ACh) on the membrane electrical properties of both granule cells and Purkinje cells in slices of the cerebellar vermis of the rat using whole-cell patch-clamp techniques. The bath application of ACh induced a marked increase in the frequency of spontaneous EPSCs (sEPSCs) in Purkinje cells specifically in the vestibulo-cerebellum. This effect of ACh was mimicked by muscarine but not by nicotine. It was abolished by application of either tetrodotoxin or the antagonist of AMPA receptors, indicating that the ACh-induced enhancement of sEPSCs occurred indirectly via the activation of neurons sending glutamatergic projections to Purkinje cells. In15% of granule cells tested in the vestibulo-cerebellum, muscarine elicited membrane depolarization accompanied by a decrease in membrane conductance and increased the neuronal excitability. The muscarine-induced depolarization of granule cells in the vestibulo-cerebellum was attributable to the inhibition of standing-outward K+ currents (IKSO) most likely via the activation of muscarinic M3 receptors. Taken together, these results indicate that ACh increases the firing frequency of granule cells by inhibiting IKSO, which in turn increases the frequency of sEPSCs in Purkinje cells in the rat vestibulo-cerebellum.
Key words: acetylcholine; vestibulo-cerebellum; Purkinje cell; granule cell; sEPSCs; muscarinic receptor; potassium current
Introduction
Cholinergic projections are relatively sparse in the cerebellum compared with other parts of the brain, such as the striatum, hippocampus, and basal forebrain (Schäfer et al., 1998). However, previous studies using ligand-binding autoradiography (Spencer et al., 1986
The vestibular system has a close relationship with the cerebellum. The labyrinth sends primary afferents ipsilaterally to the nodulus and uvula in the cerebellum. Neurons of the superior, medial, lateral, and descending vestibular nuclei send their axons bilaterally to the nodulus, uvula, lingual, flocculus, and ventral paraflocculus. These cerebellar regions are referred to as the vestibulo-cerebellum (Altman and Bayer, 1997
To clarify the functional roles of cholinergic mossy fibers in the vestibulo-cerebellum, we investigated the effects of ACh on the membrane electrical properties of both Purkinje cells and granule cells in the cerebellum using whole-cell patch-clamp techniques. We found that ACh directly induced depolarization of granule cells through their muscarinic receptors, resulting in a marked increase in the frequency of spontaneous EPSCs (sEPSCs) in Purkinje cells specifically in the vestibulo-cerebellum. We also found that the ACh-induced depolarization of granule cells was attributable to the inhibition of standing-outward K+ currents (IKSO) most likely via the activation of muscarinic M3 receptors.
Materials and Methods
Slice preparation. Experiments were performed in slices of the cerebellar vermis of postnatal 19- to 22-d-old Wistar rats. Procedures for preparation and maintenance of slices were similar to those described previously (Iino et al., 2001Whole-cell patch-clamp recordings. Slices were visualized using a 60x water-immersion objective illuminated with near-infrared light. The image was collected with a CCD camera (C2741; Hamamastu Photonics, Hamamastu, Japan) with contrast enhancement and displayed on a video monitor. Whole-cell patch-clamp recordings (Edwards et al., 1989
for recording of Purkinje cells and 6–8 M
Data analysis. sEPSCs and miniature EPSCs (mEPSCs) were counted and analyzed using the MiniAnalysis program (Synaptosoft). Spontaneous events at the holding potential of -70 mV were initially detected automatically using our tentative criteria, which were set to an amplitude threshold of 15 pA and an area threshold of 60 fC for sEPSCs and 10 pA and 7 fC for mEPSCs and then visually accepted or rejected on the basis of the rise and decay times. A charge transfer of sEPSC was calculated by summing up the areas of detected events every 10 sec. Origin (Microcal Software, Northampton, MA) was also used for data analysis. Data are given as mean ± SEM. For statistical analysis, the Wilcoxon test, Kolmogorov–Smirnov test, or Mann–Whitney U test was performed.
Solutions. The control external solution contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. The external solution with high K + concentration contained (in mM): 102.5 NaCl, 25 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4 at 32°C when bubbled with 95% O2 and 5% CO2. In all experiments, 100 µM picrotoxin (Wako Chemicals, Osaka, Japan) was continuously added to the external solution to block GABAA receptor-mediated currents. For voltage-clamp recordings of granule cells, 0.3 µM tetrodotoxin (TTX) was also added to block voltage-dependent Na + currents. The internal solution used for conventional whole-cell recordings of Purkinje cells contained (in mM): 150 Cs-gluconate, 8 NaCl, 2 Mg-ATP, 10 HEPES, 0.1 spermine, and 5 N-ethyl bromide quaternary salt (QX-314) (Sigma, St. Louis, MO). The pH was adjusted to 7.2 with gluconic acid. QX-314 was added to the internal solution to avoid Na + current-mediated escape from the voltage clamp. The internal solution for perforated patch recording of granule cells contained (in mM): 125 K-gluconate, 5 MgCl2, 10 HEPES, and 0.2 EGTA. The pH was adjusted to 7.2 with gluconic acid. As a permeabilizing agent, 240 µg/ml amphotericin B (Sigma) was added to the internal solution.
Drugs. Drugs used in this study were ACh, (+)-muscarine chloride, (-)-nicotine, mecamylamine, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine tetrahydrochloride, (+)-himbacine, p-fluoro-hexahydrosiladifenidol hydrochloride (p-F-HHSiD) (Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (Tocris Cookson, Bristol, UK), and TTX (Wako Chemicals). Drugs were dissolved in distilled water or DMSO at concentrations of 1–10 mM and stored. The final concentrations of the drugs in Results were obtained by diluting the stock solutions with the recording external solution. The final DMSO concentration was lower than 0.1%. All drugs were bath-applied via gravity feed at a rate of 4–5 ml/min, resulting in solution exchange within 30 sec.
Results
Effects of ACh on spontaneous EPSCs in Purkinje cells in the vestibulo-cerebellum
sEPSCs were recorded in Purkinje cells voltage-clamped at–70 mV in the cerebellar vermal lobule X (nodules) in the vestibulo-cerebellum. Bath application of ACh (100 µM) markedly increased the frequency of sEPSCs (Fig. 1A). These sEPSCs were completely and reversibly abolished by the bath application of 10 µM CNQX (Fig. 1B). Furthermore, these inward currents reversed at
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Figure 1. Effects of ACh on sEPSCs in Purkinje cells in lobule X. A, Increase in the frequency of sEPSCs in Purkinje cells induced by ACh (100 µM, 90 sec). B, C, Suppression of effects of ACh by either CNQX (10 µM) or TTX (1 µM). D, Effects of mecamylamine (10 µM)and atropine (1 µM)on ACh-induced enhancement of sEPSCs. E, Effects of nicotine (30 µM) and muscarine (30 µM). The membrane potential was held at–70 mV.
To determine whether nicotinic or muscarinic receptors contributed to the ACh-induced event, we examined the effects of additional applications of mecamylamine (10 µM), a nicotine receptor antagonist, and atropine (1 µM). The ACh-induced event was unaffected by mecamylamine but inhibited by atropine (Fig. 1D). In addition, the effect of ACh was mimicked by bath application of 30 µM muscarine but not by 30 µM nicotine (Fig. 1E). These results indicated that the effect of ACh was attributable to the indirect action on muscarinic receptors of presynaptic neurons. For the rest of this study, we used muscarine as the cholinergic receptor agonist.
We examined the effects of muscarine on the frequency, amplitude, and charge transfer of sEPSCs in Purkinje cells in the vestibulo-cerebellum. Figure 2, A and B, shows typical current traces recorded in a Purkinje cell and the time courses of changes in the frequency (Fig. 2B, a), amplitude (Fig. 2B, b), and charge transfer (Fig. 2B, c) of sEPSCs every 10 sec before and during bath application of muscarine. Muscarine (10 µM) increased the frequency, mean amplitude, and charge transfer of sEPSCs in 10 sec to 18.4-, 2.3-, and 46.1-fold of the control, respectively (n = 8; p < 0.01 in all three parameters) (Fig. 2C). In contrast, the bath application of 10 µM muscarine caused no change in both frequency and amplitude of TTX-insensitive mEPSCs (Fig. 2D). Because muscarine invariably increased both frequency and amplitude of the sEPSC, we estimated the degree of the muscarine-induced effects using sEPSC charge transfer in this study.
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Figure 2. Effects of muscarine on the frequency, amplitude, and charge transfer of sEPSCs. A, Typical traces of sEPSCs 10 sec before and during the bath application of muscarine (10 µM). B, Time course of effects of muscarine on the frequency, mean amplitude, and charge transfer of sEPSCs in the experiment shown in A. a, Time course of change in sEPSC frequency. The number of events every 10 sec was summed and plotted. b, Time course of change in mean amplitude of sEPSCs. Each plot represents the mean ± SEM of sEPSC amplitudes every 10 sec. c, Time course of charge transfer of sEPSCs. The area of events every 10 sec was summed and plotted as the charge transfer of sEPSC in 10 sec. Horizontal bars indicate the period during muscarine application (90 sec). C, Means ± SEM of sEPSC frequency (a), amplitude (b), and charge transfer (c) before and during application of muscarine (n = 8). All columns were normalized to the respective control (dotted lines; *p < 0.01, Wilcoxon test). Cont, Control; Mus, muscarine. D, a, Typical traces of mEPSCs recorded in the presence of TTX (1 µM) from the same neuron shown in A before and during application of muscarine (10 µM). b, Cumulative probabilities of interval and amplitude of mEPSCs before (n = 135) and during (n = 137) application of muscarine (p = 0.517, interval; p = 0.625, amplitude; Kolmogorov–Smirnov test).
Dose dependence of muscarine effect
The muscarine-induced increase in sEPSC charge transfer was reproducible even after repeated applications of muscarine. Figure 3, A and B, shows typical current traces and time courses of sEPSC charge transfer every 10 sec during repeated applications of various concentrations of muscarine to a Purkinje cell. To obtain a dose–response relationship for the muscarine effect on sEPSCs, we bath-applied various concentrations of muscarine from 1 nM to 30 µM and measured the increase in sEPSC charge transfer every 10 sec. In Figure 3C, we plotted the peak value of the increase in charge transfer in 10 sec at various concentrations of muscarine normalized to that at 1 µM muscarine against muscarine concentration. The response to muscarine was negligible at <1 nM and almost saturated at 30 µM. The maximum charge transfer in 10 sec was 205.0 ± 111 pC (n = 5), and the best nonlinear least squares fit to these data predicted a half-maximal response at 0.289 µM and a Hill coefficient of 0.739.
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Figure 3. Dose dependence of effects of muscarine. A, Typical traces of sEPSCs before and during application of different concentrations of muscarine (10 and 100 nM, 1 and 10 µM). B, Time course of the increase in sEPSC charge transfer induced by the successive applications of different concentrations of muscarine. The sEPSC charge transfer every 10 sec was plotted against time. The current traces in A and the plots in B were obtained from the same cell. C, Dose–response relationship of the effect of muscarine. The peak value of the increase in sEPSC charge transfer induced by each muscarine concentration as shown in B relative to that induced by 1 µM muscarine is plotted against the concentration of muscarine. Each circle and bar represent the mean ± SEM of the relative peak value obtained from 5–13 cells. The line through the circles is the best nonlinear least squares fit to the equation S = Smax /[1 + (EC50/C)n], where S is the relative peak value; Smax is the maximal peak value; and C is the concentration of muscarine. The best fit yields an EC50 of 0.289 µM and a Hill coefficient of 0.739.
Difference in muscarine effects among Purkinje cells in different cerebellar lobules
The presence of muscarinic receptors in the cerebellum has been demonstrated by previous studies (Neustadt et al., 1988Figure 4A shows histograms giving the distribution of sEPSC charge transfer in 10 sec in the control solution and in the presence of 1 µM muscarine in 89 Purkinje cells in lobules IX and X and 23 cells in lobules IV–VI. In lobules IX and X, the sEPSC charge transfer was <10 pC/10 sec, and its mean value was 3.8 ± 0.3 pC/10 sec in the control solution (n = 89). In the presence of 1 µM muscarine, it increased markedly, and the peak value was widely distributed between 14.9 and 392.5 pC/10 sec, with a mean of 141.7 ± 9.6 pC/10 sec (n = 89; p < 0.0001) (Fig. 4A, a). In lobules IV–VI, the corresponding value in control solution was slightly smaller than that in lobules IX and X (1.1 ± 0.5 pC/10 sec; n = 23; p = 0.027). The application of 1 µM muscarine caused no significant increase in this value in 20 cells (p = 0.191), whereas it caused a substantial increase in the remaining three cells, in which the peak sEPSC charge transfer in 10 sec ranged between 44.4 and 92.6 pC/10 sec (Fig. 4A, b). As a whole, therefore, the sEPSC charge transfer in the presence of 1 µM muscarine was much greater in lobules IX and X than that in lobules IV–VI (141.7 ± 9.6 pC/10 sec; n = 89; vs 10.8 ± 2.8 pC/10 sec; n = 23, respectively; p < 0.0001) (Fig. 4 B). These results indicated that the muscarine-induced increase in sEPSC charge transfer in Purkinje cells occurred much more prominently in the vestibulo-cerebellum.
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Figure 4. Difference in the effects of muscarine on sEPSC enhancement among Purkinje cells in the different lobules. The sEPSC charge transfer every 10 sec before and during the application of 1 µM muscarine was measured in Purkinje cells in the vestibulo-cerebellar lobules (IX, X) and other lobules (IV–VI). A, a, Distributions of sEPSC charge transfers in 10 sec in the control condition (the average of the value for 1 min; top histogram, open bars) and during the application of 1 µM muscarine (bottom histogram, filled bars) in 89 Purkinje cells in the vestibulo-cerebellar lobules (IX, X). The peak value was taken for the sEPSC charge transfer in 10 sec during the application of muscarine. A, b, Distribution of sEPSC charge transfers in 10 sec in the control condition (open bars) and during the application of 1 µM muscarine (filled bars) in 23 Purkinje cells in lobules IV–VI. B, Means ± SEM of sEPSC charge transfers in the presence of 1 µM muscarine in Purkinje cells in lobules IV–VI (n = 23; left column), and IX and X (n = 89; right column). **p < 0.0001, Mann–Whitney U test. C, Recording sites of Purkinje cells on the schematic view of parasagittal slices of the vermis.
Suppression of the effect of muscarine by muscarinic M2 and M3 receptor antagonists
It has been reported that the majority of muscarinic receptors in the cerebellum are of the M2 type, and very few M3-type receptors are detected (Tice et al., 1996To obtain concentration–response relationships of the suppressive effects of the antagonists, 1 µM muscarine was repeatedly bath-applied first in the absence and then in the presence of various concentrations of the antagonists in the same neuron. Figure 5, A, a, and B, a, exemplifies typical traces of sEPSCs in the solution containing 1 µM muscarine without and with various concentrations of the nonselective antagonist atropine or the M3-selective antagonist 4-DAMP. We calculated the relative increase in charge transfer, that is, the peak increase in charge transfer in 10 sec in the presence of various concentrations of the antagonists divided by that obtained without them. In the graphs, we plotted these relative values against the concentrations of the antagonists (Fig. 5A, b, B, b). The best nonlinear least squares fit to the concentration–response curves predicted IC50 values of 3.34 and 2.50 nM for atropine and 4-DAMP, respectively. The IC50 values for the other antagonists were 2.79 µM (methoctramine), 423 nM (himbacine), and 55.8 nM (p-F-HHSiD).
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Figure 5. Dose–response relationship of the effect of muscarinic receptor antagonists. A, a, Typical traces of 1 µM muscarine-induced sEPSCs in the control condition and in the presence of two different concentrations of atropine. b, Dose–response relationship of the effect of atropine. The relative peak value of the increase in sEPSC charge transfer induced by 1 µM muscarine in the presence of each concentration of atropine, with that induced by 1 µM muscarine alone as a reference, is plotted against the concentration of atropine. Plots were fitted to equation S/Scont = 1/[1 + (C/IC50)n], where S is the peak value of the increase in sEPSC charge transfer in the presence of different concentrations of antagonist; Scont is that in the absence of antagonist; C is the concentration of antagonist; and IC50 is the concentration that inhibits the muscarine-induced increase in sEPSC charge transfer by half. The best fit for the effect of atropine yields an IC50 of 3.34 nM and a Hill coefficient of 1.39. B, a, Typical traces of 1 µM muscarine-induced sEPSCs in the control condition and in the presence of 4-DAMP (M3 antagonist). b, Dose–response relationship of the effect of 4-DAMP. The best fit yields an IC50 of 2.50 nM and a Hill coefficient of 1.56. Each circle and bar in A, b, and B, b, represent the mean ± SEM of the relative value of the increase in sEPSC charge transfer obtained from 5–13 cells.
The receptor binding studies for these antagonists of muscarinic receptors revealed that Ki values of methoctramine and himbacine for muscarinic M2 receptors were
Effects of muscarine on excitability of cerebellar granule cells
The results described above strongly suggested that the muscarine-induced enhancement of sEPSCs in Purkinje cells was attributable to the increase in the excitability of granule cells that send excitatory signals to Purkinje cells via parallel fibers. Therefore, we decided to examine whether muscarine affects the excitability of granule cells in lobule X using a perforated patch-clamp technique.The granule cells in the control condition were held at approximately -70 mV with current injection, and depolarizing current pulses were injected at 0.1 Hz to assess the membrane conductance. Bath application of 5 µM muscarine for 90 sec caused membrane depolarization that lasted for several minutes in 13 of 84 cells tested (15.5%) (Fig. 6A). The peak amplitude of this depolarization was 8.3 ± 3.0 mV (n = 13). Action potentials were occasionally elicited by the injection of depolarizing current pulses during the muscarine-induced depolarization (Fig. 6A, b). We also estimated the muscarine-induced changes in the membrane conductance by passing hyperpolarizing current pulses in granule cells that were sensitive to muscarine. For this experiment, the membrane potential was held at -60 mV before and during application of 5 µM muscarine by adjusting the intensity of injection current by hand. Muscarine at 5 µM reduced the membrane conductance to 74 ± 7% of the control (n = 4) (Fig. 6B). This suggested that the muscarine-induced depolarization was attributable to a decrease in the K+ conductance.
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Figure 6. Effects of muscarine on membrane potential and conductance of cerebellar granule cells. A, Membrane depolarization induced by 1 µM muscarine. The membrane conductance was estimated by recording potential change evoked by 10 pA depolarizing current pulses for 250 msec at 0.1 Hz. a–c, Voltage changes induced by the depolarizing pulses before, during, and after application of muscarine, respectively. Action potentials were occasionally elicited during muscarine application. Holding potential was maintained at approximately–70 mV by injecting DC current before muscarine application. B, Muscarine-induced decrease in membrane conductance estimated by injecting 12 pA hyperpolarizing current pulses. The DC membrane potential level was maintained at–60 mV by adjusting the holding current. a–c, Voltage traces before, during, and after application of 1 µM muscarine, respectively.
In contrast, no muscarine-induced depolarization and reduction of membrane conductance were observed in all granule cells tested in lobules IV–VI (n = 39). These results obtained in granule cells were consistent with those in Purkinje cells in the lobular localization of muscarinic effects.
Inhibition of leak K+ current by muscarine in granule cells
Previous studies in a variety of preparations (Madison et al., 1987
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Figure 7. Inhibition of IKSO by muscarine in granule cells in the vestibulo-cerebellum. Granule cells were voltage-clamped in the presence of 1 µM TTX, and 5 µM muscarine was bath-applied. A zero current level (0 pA) was set at the resting potential level at approximately–60 mV. A, a, IKSO detected as a noninactivating current at–30 mV. It was instantaneously reduced in amplitude when the membrane potential was stepped down to–80 mV for 800 msec at 0.1 Hz. The current was partly inhibited by muscarine. b, Time course of inhibition of IKSO by muscarine at–30 mV. The horizontal bar indicates the application of muscarine for 90 sec. B, C, I–V relationships of muscarine-induced changes in IKSO in the normal solution containing 2.5 mM K + (B) and high (25 mM)-K + solution (C). a, Current responses to ramp voltages. Cells were held at–30 mV and then hyperpolarized to–100 mV by ramping the membrane potential at a rate of 70 mV/sec for 1 sec in the presence and absence of 5 µM muscarine. b, I–V curves between–30 and–100 mV. The I–V relationships of muscarine-sensitive current were obtained by subtracting the I–V curve in control solution from that in solution containing muscarine.
To obtain the current-voltage relationship and the reversal potential of muscarine-sensitive current, we used the ramp voltage protocol in which the membrane potential was ramped from the holding potential of–30 to–100 mV at a rate of–0.07 mV/msec. Figure 7B shows typical current traces obtained with this protocol in the presence and absence of 5 µM muscarine. The muscarine-sensitive current was obtained by subtracting the current in the presence of muscarine from that in its absence. In the control external solution containing 2.5 mM K+, in which EK was calculated to be–102 mV, the muscarine-sensitive current was outwardly rectifying and not measurable at potentials more negative than–90 mV (n = 6). In high-K+ solution containing 25 mM K+ prepared by isomolar replacement of NaCl with KCl, the muscarine-sensitive current reversed at–44.5 ± 2.6 mV(n = 4) (Fig. 7C). This reversal potential was close to EK calculated to be–42.0 mV, indicating that the muscarine-sensitive current detected here was a selective K+ current. Thus, the muscarine-sensitive outward current recorded here was identical to IKSO reported previously as noninactivating K+ current (Watkins and Mathie, 1996
Inhibition of the leak K+ current by muscarine is abolished by muscarinic M3 receptor antagonist
We suggested that muscarine-induced enhancement of sEPSCs in Purkinje cells was attributable to the activation of muscarinic M3 receptors (Fig. 5). If this is the case, it would be expected that the leak K+ current is inhibited by activation of muscarinic M3 receptors. Therefore, we examined the effects of methoctramine (M2 antagonist) and 4-DAMP (M3 antagonist) on muscarine sensitivity of leak K+ current in granule cells (Fig. 8). In the presence of 1 µM methoctramine, the amplitude of muscarine-sensitive current at–30 mV was 35.0 ± 5.9 pA (n = 4), which was similar to 35.8 ± 7.2 pA (n = 4) in the control condition. In contrast, muscarine produced no detectable inhibition of leak K+ current in the presence of 30 nM 4-DAMP (n = 4). This strongly suggested that the activation of muscarinic M3 receptors was responsible for the inhibition of leak K+ current by muscarine in granule cells as well as the muscarine-induced enhancement of sEPSCs in Purkinje cells.
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Figure 8. Effects of subtype-selective antagonists of muscarinic receptors on muscarine-induced inhibition of IKSO in granule cells. A, Muscarine-induced inhibition of IKSO in the control condition. The outward current at–30 mV was inhibited by 1 µM muscarine. B, No effect of 1 µM methoctramine (M2 antagonist) on muscarine-sensitive current. C, Abolition of muscarine-sensitive current by 30 nM 4-DAMP (M3 antagonist). Cont, Control; Mus, muscarine.
Discussion
Mechanism underlying ACh-induced increase in the frequency of sEPSCs in Purkinje cells in the vestibulo-cerebellum
In this study, we showed that the bath application of ACh markedly increased the frequency of sEPSCs in Purkinje cells specifically in the vestibulo-cerebellum. This effect of ACh was mediated exclusively by the activation of muscarinic receptors, because it was mimicked by muscarine but not by nicotine. The finding that the effect of ACh was abolished by adding TTX to the bathing medium indicated that ACh or muscarine did not act directly on Purkinje cells but increased the excitability of presynaptic excitatory neurons, thereby augmenting the frequency of sEPSCs in Purkinje cells indirectly. It is most likely that these presynaptic neurons are granule cells. In fact, muscarine caused membrane depolarization accompanied by a decrease in membrane conductance by suppressing the leak K+ current even in the presence of TTX in a population of granule cells in the vestibulo-cerebellum. The above notion is compatible with the previous anatomical and pharmacological findings that muscarinic receptors in the cerebellum are concentrated in the granule cells in the vestibulo-cerebellum (Neustadt et al., 1988An unexpected finding in this study was that the inhibition by muscarine of the leak K+ conductance was detected in only
The muscarine-induced increase in the frequency of sEPSCs in Purkinje cells was accompanied by an increase in the mean amplitude of sEPSCs (Fig. 2B). There was no change in the rise and decay time constants of sEPSCs during this event. Moreover, muscarine did not alter the amplitude of mEPSCs in the presence of TTX. Parallel fiber synapses have a low probability of release (Dittman et al., 2000
Effect of muscarine on the leak K+ current in cerebellar granule cells
Neurons in the CNS possess multiple types of K+ channels that may be subject to modulation by ACh via muscarinic receptors (Storm, 1990With respect to the cholinergic inputs to cerebellar granule cells, the vestibulo-cerebellum including the uvula and nodulus contains dense cholinergic mossy fiber projections originating from neurons in the vestibular nucleus (Ojima et al., 1989
Inhibition of effects of muscarine by M3 antagonist
Molecular cloning studies have revealed the existence of five distinct subtypes of muscarinic receptors (m1–m5), which are widely expressed in the CNS (Bonner et al., 1987In this study, we attempted to determine which of the M2 or M3 receptors underlies the effects of muscarine on the occurrence of sEPSCs in Purkinje cells using the five antagonists of muscarinic receptors. Atropine, a subtype-nonselective antagonist, has Ki values of 0.5–1.2 nM for M2 receptors and 0.2–1.2 nM for M3 receptors (Hulme et al., 1990
It has been reported that m5 receptors are expressed in the cerebellum (Wei et al., 1994
Footnotes
Received Feb. 3, 2003; revised May. 6, 2003; accepted May. 13, 2003.We thank Drs. Keisuke Tsuzuki, Yasuhiko Saito, and Wataru Kakegawa for helpful suggestions and discussions on this manuscript.
Correspondence should be addressed to Dr. Seiji Ozawa, Department of Physiology, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail: ozawas{at}med.gunma-u.ac.jp.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236200-09$15.00/0
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