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The Journal of Neuroscience, December 15, 2001, 21(24):9701-9712

Roles of Glutamate Receptor 2 Subunit (GluR2) and Metabotropic Glutamate Receptor Subtype 1 (mGluR1) in Climbing Fiber Synapse Elimination during Postnatal Cerebellar Development

Kouichi Hashimoto¹, Ryoichi Ichikawa², Hajime Takechi³, Yoshiro Inoue⁴, Atsu Aiba⁵, Kenji Sakimura⁶, Masayoshi Mishina⁷, Tsutomu Hashikawa⁸, Arthur Konnerth⁹, Masahiko Watanabe⁴, and Masanobu Kano¹

¹ Department of Physiology, Kanazawa University School of Medicine, Takara-machi, Kanazawa 920-8640, Japan, ² Department of Anatomy, Sapporo Medical University, Sapporo 060-8556, Japan, ³ Department of Gerontology, Kyoto University Faculty of Medicine, Kyoto 606-8507, Japan, ⁴ Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan, ⁵ Division of Cell Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan, ⁶ Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan, ⁷ Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, University of Tokyo, and Cure Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Tokyo 113-0033, Japan, ⁸ Laboratory for Neural Architecture, Brain Science Institute, RIKEN, Wako-shi, Saitama 351-0198, Japan, and ⁹ Institut für Physiologie, Ludwig-Maximilians-Universität München, 80802 München, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor 2 subunit (GluR2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluR2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluR2 mutant PCs were associated with voltage-dependent Ca²⁺ signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca²⁺ signals involved both proximal and distal dendrites. Morphologically, CFs of GluR2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluR2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluR2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.

Key words: climbing fiber; parallel fiber; cerebellum; Purkinje cell; synapse; glutamate receptor; postnatal development; mutant mouse

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the adult cerebellum, each Purkinje cell (PC) is innervated by a single climbing fiber (CF) that originates in the inferior olive and forms strong excitatory synapses onto proximal PC dendrites (Palay and Chan-Palay, 1974; Ito, 1984; Strata and Rossi, 1998). This one-to-one relationship is preceded by a developmental stage of redundant innervation of PCs by multiple CFs (Crépel, 1982). Elimination of supernumerary CFs proceeds in parallel with synaptogenesis onto PCs from parallel fibers (PFs), bifurcated axons of granule cells and the other excitatory input to PCs (Ito, 1984). The importance of PF-PC synapses in CF synapse elimination has been substantiated by the fact that multiple CF innervation persists in animals with impaired PF-PC synaptogenesis, such as x-irradiated rats (Woodward et al., 1974; Crépel and Delhaye-Bouchaud, 1979; Bravin et al., 1995; Sugihara et al., 2000) and agranular cerebellar mutants (weaver, reeler, and staggerer mice) (Crépel and Mariani, 1976; Mariani et al., 1977; Crépel et al., 1980; Mariani and Changeux, 1980).

Glutamate or its related amino acids is considered to be released from CF and PF terminals and to activate glutamate receptors (GluRs) on PCs. The GluR 2 subunit (GluR2) is highly enriched in PCs (Araki et al., 1993; Lomeli et al., 1993) that are localized at the junctional site of PF-PC synapses but not at the site of CF-PC synapses (Landsend et al., 1997). In PCs of GluR2-deficient mice, the number of PF-PC synapses is reduced to nearly one-half that of wild-type mice, leaving approximately one-third of PC dendritic spines free of innervation (Kurihara et al., 1997). GluR2-deficient mice display persistent multiple CF innervation, impaired long-term depression (LTD), and motor discoordination (Hirano et al., 1995; Kashiwabuchi et al., 1995). The metabotropic GluR subtype 1 (mGluR1) is also abundant in PCs that are localized at the perijunctional site of both the PF-PC and CF-PC (Baude et al., 1993; Nusser et al., 1994). Inactivation of the mGluR1 gene results in persistent multiple CF innervation, impaired LTD, and motor discoordination, without apparent defects in PF-PC synaptogenesis (Aiba et al., 1994; Conquet et al., 1994; Kano et al., 1997; Lévénès et al., 1997; Ichise et al., 2000).

To differentiate the roles of these two GluR molecules in CF synapse formation and elimination, we comparatively examined the GluR2 and mGluR1 mutant mice by recording CF-mediated EPSCs (CF-EPSCs), imaging CF-induced Ca²⁺ signals in PCs, and labeling CFs anterogradely. We present evidence that surplus CFs form ectopic synapses onto distal dendrites of GluR2 mutant PCs, whereas CFs innervate proximal dendrites of mGluR1 mutant PCs. These findings suggest that GluR2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendritic domain, whereas the mGluR1-signaling pathway is involved in eliminating excess CF synapses at the proximal dendrites.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. The GluR2 mutant mice (Kashiwabuchi et al., 1995) and the mGluR1 mutant mice (Aiba et al., 1994) were produced as described. The GluR2 mutant mice and their littermates were of C57BL/6 × TT2 genetic background (Yagi et al., 1993). The mGluR1 mutant mice and their littermates were of the 129/Sv × C57BL/6 genetic background. Mice were kept in the same room at the animal facility with a 12 hr light/dark cycle. Because we have not found any electrophysiological or morphological difference so far between the wild-type (+/+) mice of C57BL/6 × TT2 genetic background and those of 129/Sv × C57BL/6 genetic background, nonmutant (+/+) littermates including both genotypes were used in electrophysiological analyses as wild-type controls.

Electrophysiology. Sagittal cerebellar slices of 200-250 µm thickness were prepared from wild-type, GluR2 mutant, or mGluR1 mutant mice as described previously (Edwards et al., 1989; Llano et al., 1991; Kano and Konnerth, 1992; Aiba et al., 1994). Whole-cell recordings were made from visually identified PCs using a 40× water-immersion objective attached to either an Olympus (BH-2 or BX50WI, Olympus Optical, Tokyo, Japan) or a Zeiss (Axioskop) upright microscope (Edwards et al., 1989; Llano et al., 1991) at 32°C. The resistance of patch pipettes was 3-6 M when filled with an intracellular solution composed of (in mM): 60 CsCl, 30 Cs D-gluconate, 20 TEA-Cl, 20 BAPTA, 4 MgCl₂, 4 ATP, and 30 HEPES; pH 7.3, adjusted with CsOH. The composition of standard bathing solution was (in mM): 125 NaCl, 2.5 KCl, 2 CaCl₂, 1 MgSO₄, 1.25 NaH₂PO₄, 26 NaHCO₃, and 20 glucose, bubbled continuously with a mixture of 95% O₂ and 5% CO₂. Bicuculline (10 µM) was always present in the saline to block spontaneous IPSCs (Konnerth et al., 1990; Kano et al., 1992). Ionic currents were recorded with either an Axopatch-1D (Axon Instruments, Foster City, CA) or an EPC-9 (HEKA, Lambrecht, Germany) patch-clamp amplifier. The pipette access resistance was compensated as described by Llano et al. (1991). Stimulation and on-line data acquisition were performed using the PULSE software on a Macintosh computer (HEKA). Signals were filtered at 3 kHz and digitized at 20 kHz. The decay phase of EPSCs was fitted with the PULSE-FIT software (HEKA). A glass pipette with a 5- to 10-µm-diameter tip that was filled with standard saline was used for stimulating CFs. To search for multiple CFs innervating the recorded PC, the stimulation pipette was moved systematically in the granule cell layer, and the stimulus intensity was gradually increased at each stimulation site (pulse width 0.1 msec; strength 0-100 V).

Ca²⁺ imaging.PCs were loaded for at least 20 min with a Ca²⁺ indicator (Oregon Green 488 BAPTA-1, Molecular Probes, Eugene, OR; 100 µM) through the patch pipette at room temperature. A high-speed confocal laser-scanning microscope (Noran Odyssey or Oz) attached to an upright microscope (Zeiss Axioskop or Olympus BX50WI) was used to acquire fluorescence images at 30 or 60 Hz in parallel to the whole-cell recordings (Eilers et al., 1995a; Takechi et al., 1998). Full-frame images were recorded on an optical disk (TQ2026F, Panasonic) and analyzed off-line with the Image-1 software (Universal Image). In some experiments, fluorescence images were acquired at 30 Hz by using a cooled CCD camera system (IMAGO, T.I.L.L. Photonics, Gräfelfing, Germany) attached to an upright microscope (Zeiss Axioskop). The Ca²⁺-dependent fluorescence signals from selected regions of interest were background corrected and expressed as increases in fluorescence divided by the prestimulus fluorescence values (F/F₀) using Igor Pro software (Wavemetrics). In PCs with multiple CF innervation, it was often difficult to detect CF-induced Ca²⁺ signals under normal resting potentials with an extracellular Ca²⁺ concentration of 2 mM. This was presumably because the CF-induced EPSPs were too small to evoke spikes or, even if they induced spikes, the associated Ca²⁺ entry was too small to be detected. In these cases, we depolarized the PCs by constant current injection so that CF-EPSPs evoked spikes and increased the extracellular Ca²⁺ concentration to 3 mM.

Anterograde labeling of CFs. Under deep anesthesia with chloral hydrate (350 mg/kg of body weight), a glass pipette (inner tip diameter 10-20 µm) filled with 2-3 µl of a 10% solution of biotinylated dextran amine (10,000 molecular weight; Molecular Probes) in 0.01 M PBS, pH 7.4, was inserted into the right inferior olive by dorsal approach (Rossi et al., 1995). Biotinylated dextran amine was injected iontophoretically by positive current (7 µA for 30 min with a protocol of 700 msec on and 1300 msec off). After 6-8 d of survival, mice were anesthetized with chloral hydrate (350 mg/kg) and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Coronal brainstem sections were examined to check the injection site of biotinylated dextran amine. Mice with the injection site restricted to the caudal part of the medial accessory olive were chosen for the preparation of microslicer cerebellar sections (50 µm in thickness). Parasagittal cerebellar sections were incubated overnight in avidin-peroxidase (Amersham Biosciences, Buckinghamshire, UK) diluted with PBS containing 1% Tween 20 and visualized with diaminobenzidine and cobalt. Low-power photographs were taken with a Normarski interference contrast microscope (Axiophoto, Zeiss), whereas high-power ones were taken with a bright-field microscope (AX-80, Olympus). To quantity the extent of arbors of labeled CFs, the relative vertical height of the most distal tip was measured for at least 30 CFs in each mouse and expressed as the percentage of the molecular layer thickness. In each genotype, the mean value and SEM were obtained from three mice, and the difference between the genotypes was tested by Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A novel slow time course CF-EPSC in GluR2 mutant PCs

In parasagittal cerebellar slices, PCs were recorded in the whole-cell configuration, and CFs were stimulated with a glass pipette placed in the granule cell layer near the recorded PCs (Aiba et al., 1994; Kano et al., 1995, 1997). To search PCs with multiple CF innervation, the stimulation pipette was systematically moved by ~20 µm steps, and the stimulus intensity was gradually increased at each stimulation site (pulse width 0.1 msec; strength 0-100 V) (Kano et al., 1995, 1997, 1998; Offermanns et al., 1997; Watase et al., 1998; Ichise et al., 2000). EPSCs were elicited in an all-or-none fashion in most of wild-type PCs (Fig. 1A), whereas EPSCs with more than one discrete step were recorded in mGluR1 mutant PCs (Fig. 1B) with much higher incidence than in wild-type PCs (Kano et al., 1997; Lévénès et al., 1997; Ichise et al., 2000). These EPSCs displayed clear paired-pulse depression (PPD) to a stimulus pair with varying interpulse intervals (Fig. 2A,B, wild-type) (Konnerth et al., 1990; Aiba et al., 1994; Kano et al., 1995, 1997; Hashimoto and Kano, 1998).

View larger version (29K): [in this window] [in a new window]   Figure 1.   Time course of CF-EPSCs in wild-type, mGluR1 mutant, and GluR2 mutant PCs. A-C, EPSCs elicited by stimulation of CFs in the granule cell layer in a wild-type (A, P28), a mGluR1 mutant (B, P49), and a GluR2 mutant (C, P64) PC. With gradually increasing stimulus intensities, EPSCs of the wild-type were elicited in an all-or-none fashion (A), whereas those of the mGluR1 mutant (B) and the GluR2 mutant (C) occurred at multiple discrete steps, indicating that they were innervated by multiple CFs. Note that the rise time of the largest CF-EPSC of the GluR2 mutant is similar to that of the wild-type or the mGluR1 mutant CF-EPSC, whereas the rise times of the smaller two CF-EPSCs of the GluR2 mutant are significantly slower. One to three traces are superimposed at each threshold intensity. Stimuli were applied at 0.2 Hz. Holding potentials were 10 mV for both the wild-type and GluR2 mutant PCs and 20 mV for the mGluR1 mutant PC to inactivate voltage-dependent conductances. D-F, Summary histograms showing the 10-90% rise time of CF-EPSCs in wild-type (D), mGluR1 mutant (E), and GluR2 mutant (F) PCs. Data were obtained from mice at P23-P70.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Paired-pulse depression of CF-EPSCs in the wild-type and GluR2 mutant PCs. A, Single-sweep examples of paired-pulse depression (interpulse interval of 50 msec) of CF-EPSCs from a wild-type (top panel, P28) and a GluR2 mutant (middle and bottom panels, P62) PC. The fast (middle panel) and slow (bottom panel) CF-EPSCs were elicited separately from the same GluR2 mutant PC. Stimulus pairs were applied at 0.2 Hz. Holding potential was 10 mV for all cells. B, Summary graphs showing paired-pulse depression for the wild-type (), the fast GluR2 mutant (), and the slow GluR2 mutant () CF-EPSCs. The second response (expressed as a percentage of response to the first pulse; mean ± SEM) is plotted as a function of interpulse interval. Stimulus pairs were applied at 0.2 Hz. **p < 0.01; *p < 0.05, compared with the values of the wild-type CF-EPSCs (Student's t test).

In the GluR2 mutant mice, stimulation in the granule cell layer elicited multiple EPSCs in 88 of 122 PCs (Fig. 1C). Each of these multiple EPSCs displayed clear PPD to a similar or greater extent than that of wild-type CF-EPSCs (Fig. 2A,B, GluR2 mutant). These multiple EPSCs in GluR2 mutant PCs were judged to be elicited by stimulating CFs and not by activating mossy fiber-granule cell-PF circuits, because EPSCs elicited by PF stimulation display clear paired-pulse facilitation in GluR2 mutant PCs as in wild-type PCs (Kashiwabuchi et al., 1995; Kurihara et al., 1997). These results indicate that most of the GluR2 mutant PCs were innervated by multiple CFs.

In the multiple CF-EPSCs of the GluR2 PC shown in Figure 1C, the time course was significantly slower for the smaller two EPSCs than for the largest EPSC. The 10-90% rise time of the wild-type CF-EPSCs displayed a normal distribution with a peak around 0.5 msec (0.46 ± 0.11 msec; mean ± SD; n = 105) (Fig. 1D). That of the mGluR1 mutant had a similar distribution (0.57 ± 0.58 msec; mean ± SD; n = 88) (Fig. 1E). In contrast, the rise time of GluR2 mutant CF-EPSCs segregated into two distinct populations (Fig. 1F). The mean value for the fast (<1.0 msec) population was 0.48 msec (±0.15 msec SD; n = 127), which was similar to wild-type, whereas that for the slow (>1.0 msec) population was 1.81 msec (±0.59 msec SD; n = 80). These results suggest that GluR2 mutants have two distinct populations of CF inputs in terms of the EPSC rise time. The CF-EPSCs with fast rise time were similar to those of wild-type and mGluR1 mutant mice, whereas the slower CF-EPSCs appear to be unique to GluR2 mutant PCs. We also measured the decay time constant of CF-EPSCs in the three strains of mice by fitting the CF-EPSC decay with single exponentials (Llano et al., 1991; Aiba et al., 1994; Kano et al., 1995, 1997). The decay time constants of CF-EPSCs in wild-type and GluR2 mutant mice were not significantly different, being 6.73 msec (±2.04 msec; n = 84) for wild-type and 6.48 msec (±2.92 msec; n = 174) for the GluR2 mutant. The decay time constants were 4.75 msec (±2.36 msec; n = 88) for the mGluR1 mutant, which is significantly shorter than wild-type mice as reported previously (Aiba et al., 1994; Kano et al., 1997).

The GluR2 mutant PCs could be classified into four categories in terms of the pattern of CF innervation. These are as follows: (1) PC innervated by a single CFs that generates typical fast CF-EPSCs (Fig. 3A); (2) PC multiple-innervated by more than one CF that generates fast CF-EPSCs (Fig. 3B); (3) PC multiple-innervated by one CF that generates fast CF-EPSCs and one or more CFs that generate slow CF-EPSCs (Fig. 3C); and (4) PC multiple-innervated by more than one CF that generates fast CF-EPSCs and one or more CFs that generate slow CF-EPSCs (Fig. 3D). Frequency distribution of all PCs in terms of number of CF-EPSC steps indicates that 72.1% of PCs in GluR2 mutant mice were innervated by multiple CFs (Fig. 3E). The histogram constructed only from PCs without slow CF-EPSCs indicates that 36.2% of such PCs were innervated by two CFs and 5.2% by three CFs (Fig. 3F). On the other hand, the histogram constructed only from PCs with slow CF-EPSCs shows that most of the PCs were multiple-innervated (Fig. 3G). In particular, 68.7% are innervated by three or more CFs (Fig. 3G). These results indicate that the existence of the slow CF-EPSCs significantly contributes to the high percentage of multiple-innervated PCs in the GluR2 mutant mice.

View larger version (33K): [in this window] [in a new window]   Figure 3.   Four patterns of CF innervation in GluR2 mutant PCs. A-D, Sample records of GluR2 mutant CF-EPSCs representing four distinct CF innervation patterns. A, Single fast CF-EPSC; B, multiple fast CF-EPSCs; C, single fast plus one or more slow CF-EPSCs; D, multiple fast plus one or more slow CF-EPSCs. One to three traces are superimposed at each threshold intensity. Stimuli were applied at 0.2 Hz. Holding potentials were 10 mV for A, C, and D and 20 mV for B. E-G, Summary histograms showing the number of discrete steps of CF-EPSCs for all PCs (E), for PCs without slow EPSCs (F), and for PCs with slow EPSCs (G). Open and filled columns represent the wild-type and GluR2 mutant PCs, respectively. Data were obtained from mice at P24-P64.

Slow CF-EPSCs of GluR2 mutant PCs appear during the second postnatal week

In rodents, PF synaptogenesis onto PC dendrites is known to continue postnatally up to approximately postnatal day (P) 20 (Altman and Bayer, 1997). The structure of the PF-PC synapse is immature around P7, when no significant differences in the morphology and electrophysiology are found between the wild-type and GluR2 mutant mice (Kurihara et al., 1997). Impaired PF synapse formation is obvious in the GluR2 mutant mouse at P14 (Kurihara et al., 1997). In contrast, PF-PC synapse formation appears normal in mGluR1 mutant mice (Kano et al., 1997). We followed the developmental course of CF-EPSCs in the three strains of mice to examine whether the impaired PF synapse formation correlated with the appearance of atypical slow CF-EPSCs in GluR2 mutant mice.

The 10-90% rise time of the wild-type CF-EPSC was longer during P3-P9 than at later developmental stages (Fig. 4A) (1.0 ± 0.4 msec; mean ± SD; n = 43). The rise times of the mGluR1 mutant and GluR2 mutant CF-EPSCs from P3 to P9 had a similar distribution (1.0 ± 0.5 msec, n = 71 for the mGluR1 mutant; 0.9 ± 0.4 msec, n = 64 for the GluR2 mutant) (Fig. 4B,C) to that of wild-type. During P10-P14, the rise times of the wild-type and mGluR1 mutant CF-EPSCs decreased to adult levels (Fig. 4A,B). In contrast, CF-EPSCs with significantly slower rise times appeared during P10-P14 in the GluR2 mutant (Fig. 4C). These data indicate that atypical slow CF-EPSCs become evident during the second postnatal week when a defect in the PF to PC synapse formation occurs in the GluR2 mutant mouse.

View larger version (23K): [in this window] [in a new window]   Figure 4.   Postnatal change of CF-EPSC time course in the three strains of mice. The 10-90% rise times of CF-EPSCs are plotted against postnatal day for wild-type (A), mGluR1 mutant (B), and GluR2 mutant (C) PCs. Note that CF-EPSCs have similar rise times in the three strains of mice during P3-P9, whereas those with significantly slower rise times are obvious after P10 in the GluR2 mutant.

Electrophysiological properties of slow CF-EPSC in GluR2 mutant PCs

To examine whether the CF-EPSC amplitudes of mGluR1 mutant and GluR2 mutant mice differ from those of wild-type mice, the peak amplitudes of CF-EPSCs were measured at a fixed holding potential (10 mV). In cases of multiple-innervated PCs (Fig. 1B,C), the amplitude of a given CF-EPSC was measured by digitally subtracting other CF-EPSCs that were induced by lower stimulus strength. The peak CF-EPSC amplitude was plotted against the 10-90% rise time. CF-EPSCs in wild-type (Fig. 5A) and mGluR1 mutant (Fig. 5B) mice were in a single population with only a few CF-EPSCs with slow rise time and small amplitude. In contrast, two distinct populations of CF-EPSCs, those with fast rise time and relatively large amplitude and those with slow rise time and small amplitude, were present in GluR2 mutant mice (Fig. 5C).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Two distinct populations of CF-EPSCs in the GluR2 mutant mice. The amplitudes of CF-EPSCs (measured at a holding potential of 10 mV) are plotted against the 10-90% rise time for wild-type (A), mGluR1 mutant (B), and GluR2 mutant (C) mice. Note that two distinct populations of CF-EPSCs, those with a fast rise time and relatively large amplitude and those with a slow rise time and small amplitude, were evident in C.

We examined other electrophysiological and pharmacological properties of atypical slow CF-EPSCs of GluR2 mutant PCs. They displayed a linear current-voltage (I-V) relationship (data not shown). In Mg²⁺-free saline containing glycine (10 µM), atypical slow CF-EPSCs were not affected by an NMDA receptor antagonist, DL-2-amino-5-phosphonopentanoate (50 µM) but were totally suppressed by a non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM) (data not shown). These results indicate that atypical CF-EPSCs of GluR2 mutant PCs are mediated exclusively by non-NMDA receptors, which is similar to typical CF-EPSCs of wild-type and those of GluR2 PCs (Kashiwabuchi et al., 1995).

Passive membrane properties of GluR2 mutant PCs

It has been shown morphologically that CF synapses are located on the proximal dendrites of PCs, whereas PF synapses oppose spines at the distal dendrites (Palay and Chan-Palay, 1974; Ito, 1984; Strata and Rossi, 1998). The 10-90% rise times of PF-EPSCs are significantly slower than those of CF-EPSCs in both the rat (Llano et al., 1991) and mouse (Aiba et al., 1994; Kano et al., 1995, 1997), suggesting that the EPSC rise time in PCs reflects the electrotonic length from the soma to the site of the synapse. It is likely, therefore, that the atypical slow CF-EPSCs in GluR2 mutant PCs arise from synapses located electrotonically distant from the soma, presumably on the distal dendrites. Because the EPSC rise time depends on the passive membrane properties, we compared several parameters of wild-type and GluR2 mutant PCs in response to hyperpolarizing voltage steps from 70 to 80 mV. As reported previously in rat PCs (Llano et al., 1991), the decay of the current was biphasic and could be described by the sum of two exponentials in both wild-type and GluR2 mutant PCs (data not shown). The time constants for the fast (1) and slow (2) components were 0.35 ± 0.04 and 13.4 ± 1.9 msec (mean ± SD; n = 9) for the wild-type and 0.33 ± 0.11 and 11.6 ± 2.0 msec (n = 11) for the GluR2 mutant. Both 1 and 2 values were not significantly different between the wild-type and the GluR2 mutant (p > 0.05; Student's t test). We then calculated several parameters representing passive properties of PCs based on the model equivalent circuit of PCs described by Llano et al. (1991) (Table 1). As shown in Table 1, the lumped dendritic capacitance (C2) was smaller (p < 0.01; Student's t test) and lumped resistances were larger (p < 0.05 for R2; p < 0.01 for R3; Student's t test) in the GluR2 mutant than in the wild-type mice. These results suggest that the average total membrane area of the dendritic tree of the GluR2 mutant PCs is smaller than that of wild-type PCs. This is consistent with morphological data that the molecular layer of the GluR2 mutant is thinner than that of the wild-type cerebellum (Kurihara et al., 1997). These results indicate that the slow rise times of a subpopulation of GluR2 mutant CF-EPSCs cannot be explained by the altered passive properties of the PC dendrites.

View this table: [in this window] [in a new window]   Table 1.   Passive properties of PCs

Imaging CF-induced Ca²⁺ transients suggests that slow CF-EPSCs of GluR2 mutant PCs arise from synapses located at distal dendrites

We measured Ca²⁺ signals associated with CF responses by using a high-speed confocal laser-scanning microscope or a cooled CCD camera system. Whole-cell recording from PCs was conducted simultaneously with the Ca²⁺ imaging. The Ca²⁺ signals were measured in small regions at three different levels of PC dendritic arborization: the primary smooth branch, the tertiary smooth branch, and terminal branchlets. In the following figures, the primary and tertiary smooth branches are collectively termed "proximal dendrites," and the terminal branchlets are termed "distal dendrites." In wild-type animals, it is shown morphologically that CFs form extensive synaptic contacts onto proximal dendrites, whereas PFs form en passant synapses onto spines of distal dendrites (Palay and Chan-Palay, 1974; Ito, 1984; Strata and Rossi, 1998).

In the mono-innervated wild-type PC (Fig. 6A), stimulation of the CF induced a typical complex spike (Fig. 6B) that accompanied a clear Ca²⁺ signal over the entire dendritic tree (Fig. 6C,D). The Ca²⁺ rose in both proximal (R1, R2) and distal (R3, R4) dendrites (Fig. 6C,D). In the double-innervated mGluR1 mutant PC (Fig. 7A), stimulation of each CF induced a large EPSP with spike(s) (Fig. 7B,E) and induced a clear Ca²⁺ transient (Fig. 7C,F). Stimulation of one CF (CF1) caused elevation of Ca²⁺ that was confined to the left dendritic tree (Fig. 7C,D), whereas stimulation of the other CF (CF2) caused Ca²⁺ transients in the right dendritic tree (Fig. 7F,G). It should be noted that Ca²⁺ was elevated in both proximal and distal portions of each dendritic tree. In 14 multiple-innervated mGluR1 mutant PCs, CF-induced Ca²⁺ signals always involved proximal dendrites. In 16 mono-innervated PCs from mGluR1 mutant mice, the pattern of CF-induced Ca²⁺ signals was identical to that of the mono-innervated wild-type PCs (data not shown). These results suggest that mGluR1 mutant PCs are innervated by CFs at proximal dendrites as wild-type PCs whether they are mono- or multiple-innervated. The Ca²⁺ signals in the distal dendrite presumably reflected the spread of depolarization and activation of voltage-gated Ca²⁺ channels.

View larger version (65K): [in this window] [in a new window]   Figure 6.   CF-induced Ca2+ transients in a mono-innervated wild-type PC. A, Reconstructed image of a wild-type PC. R1-R4 indicate the dendritic regions from which the Ca2+ signals shown in C are measured. Scale bar, 20 µm. B, Complex spike induced by CF stimulation under the current-clamp recording mode. C, Time course of CF-induced Ca2+ signals measured in the regions indicated in A. D, Pseudocolor image showing the relative increase in Ca2+-dependent fluorescence (F/F0) evoked by CF stimulation. For this and the following figures (Figs. 7-9), the F/F0 values for pseudocolor images were calculated from the prestimulus and poststimulus fluorescent values that were obtained by averaging five consecutive images (132 msec periods) just before and after CF stimulation.

View larger version (43K): [in this window] [in a new window]   Figure 7.   CF-induced Ca2+ transients in a double-innervated mGluR1 mutant PC. A, Reconstructed image of a mGluR1 mutant PC. R1-R5 indicate the dendritic regions from which the Ca2+ signals shown in C and F are measured. Scale bar, 20 µm. B, E, Responses induced by stimulating two different CFs, CF1 (B) and CF2 (E), under the current-clamp recording mode. C, F, Time course of Ca2+ signals measured in the regions indicated in A in response to stimulation of CF1 (C) and CF2 (F). D, G, Pseudocolor images showing the relative increase in Ca2+-dependent fluorescence (F/F0) evoked by CF1 (D) and CF2 (G) stimulation.

In GluR2 mutant PCs, we detected different pattern of CF-induced Ca²⁺ signals from those seen in wild-type or mGluR1 mutant PCs. The GluR2 mutant PC in Figure 8 was innervated by at least three distinct CFs. Stimulation of one CF (CF1) generated typical complex spike (Fig. 8B), whereas stimulation of other two CFs (CF2 and CF3) elicited slow responses (Fig. 8E,H). Stimulation of CF1 induced Ca²⁺ transients that spread over the entire dendritic tree involving both proximal and distal dendrites (Fig. 8C,D, R1-R6). In contrast, stimulation of CF2 induced Ca²⁺ transients that were confined to the distal portions (R4, R5) of the left dendritic branch (Fig. 8F,G). Stimulation of CF3 induced small Ca²⁺ transients that were confined to the most distal dendrites (R6) near the pial surface (Fig. 8I,J). Another GluR2 mutant PC shown in Figure 9 was innervated by at least three distinct CFs that generated a fast response (Fig. 9B, CF1) and slow responses (Fig. 9E,H, CF2 and CF3). Stimulation of CF1 induced Ca²⁺ transients that spread over the middle dendritic tree and spared the small trees in the left and the right branches (Fig. 9C,D). The Ca²⁺ rose in both proximal (R1, R2) and distal (R4, R5) portions of the middle dendritic tree (Fig. 9C,D). Stimulation of CF2 induced Ca²⁺ transients that were confined to the distal portions (R5) of the right dendritic tree and spared its proximal (R1, R2) portions (Fig. 9F,G). Stimulation of CF3 induced Ca²⁺ transients that were confined to the left dendritic branch (Fig. 9I,J). The Ca²⁺ rose slightly in the tertiary dendrite (R3) and clearly in distal dendrites (R6) but showed no change in the primary dendrite (R1). In 24 GluR2 mutant PCs, we found that slow CF responses were always associated with Ca²⁺ transients that were confined to the distal dendrites. In a few slow CF responses, small Ca²⁺ signals could be detected also in the tertiary dendrites (Fig. 9, CF3); however, the Ca²⁺ transients never involved the primary dendrites. This pattern of CF-induced Ca²⁺ transients was observed in no PCs from the wild-type and in only one PC from the mGluR1 mutant.

View larger version (51K): [in this window] [in a new window]   Figure 8.   CF-induced Ca2+ transients in a multiple-innervated GluR2 mutant PC. A, Reconstructed image of a GluR2 mutant PC. R1-R6 indicate the dendritic regions from which the Ca2+ signals shown in C, F, and I are measured. Scale bar, 20 µm. B, E, H, Responses induced by stimulating three different CFs, CF1 (B), CF2 (E), and CF3 (H), under the current-clamp recording mode. C, F, I, Time course of Ca2+ signals measured in the regions indicated in A in response to stimulation of CF1 (C), CF2 (F), or CF3 (I). D, G, J, Pseudocolor images showing the relative increase in Ca2+-dependent fluorescence (F/F0) evoked by CF1 (D), CF2 (G), or CF3 (J) stimulation.

View larger version (42K): [in this window] [in a new window]   Figure 9.   CF-induced Ca2+ transients in another multiple-innervated GluR2 mutant PC. Reconstructed image of the PC (A), responses to CF stimulation (B, E, H), time course of Ca2+ signals (C, F, I), and pseudocolor images (D, G, J) are illustrated in a manner similar to Figure 8. Scale bar, 20 µm.

To quantify the differences in CF innervation patterns, the distance from the soma to the dendrites with detectable Ca²⁺ signals was measured along the main dendrites (Fig. 10). As exemplified in Figure 10A, the actual dendritic branches were traced, and the shortest distance was measured from the origin of the primary dendrite to the most proximal point of the dendritic tree where the Ca²⁺ signal was detected. In CF responses of wild-type and mGluR1 mutant mice, the distance was almost 0 µm (Fig. 10B), indicating that the Ca²⁺ rose in the primary dendrite. In GluR2 mutant mice, the length was almost 0 µm for the fast response, whereas it was ~50 µm on an average for the slow response (Fig. 10B). These results suggest that atypical slow CF responses of GluR2 PCs arise from synapses located on the distal dendrites.

View larger version (40K): [in this window] [in a new window]   Figure 10.   Distance from the soma to the dendrites with detectable Ca2+ signals. A, Pseudocolor image from the GluR2 mutant PC shown in Figure 9 exemplifying how the distance was measured. The actual dendritic branches were traced (thick red line), and the shortest distance was measured from the origin of the primary dendrite to the most proximal point of the dendritic tree where the Ca2+ signal was detected. The distance was 75.8 µm in this case. Scale bar, 20 µm. B, Summary histogram of the distance from the soma for the three strains of mice.

Morphology of CFs from GluR2- and mGluR1-deficient mice

To investigate the CF innervation patterns morphologically, anterogradely labeled CFs were visualized in parasagittal cerebellar sections (Fig. 11). Genotypic differences were noted in the extent of CF arbors within the molecular layer. Labeled CFs of GluR2 mutant mice extended up to more superficial regions of the molecular layer than those of wild-type and mGluR1 mutant mice (Fig. 11A-C). Because the thickness of the molecular layer is slightly reduced in GluR2 mutant mice (Kurihara et al., 1997), we evaluated the extent of CF arbors by measuring the height of the most distal tip of each labeled CF relative to the thickness of the molecular layer. We selected cerebellar sections from each mouse that contained staining of entire CF arbors from the Purkinje cell layer to the top of the molecular layer. Then, we examined at least 30 labeled CFs in the straight portion of the lobule VI. The most distal tip of CFs in GluR2 mutant mice reached 95.0 ± 0.1% (mean ± SEM; n = 3) of the molecular layer thickness. This score was significantly higher than that of the wild-type (83.1 ± 0.5%; n = 3; p < 0.0001) or the mGluR1 mutant (77.8 ± 0.7%; n = 3; p < 0.0001) mouse. Our previous study indicates that PC dendrites extend to the surface of the molecular layer in GluR2 mutant mice as well as wild-type mice (Kurihara et al., 1997). Taken together, these results suggest that CFs of GluR2 mutant mice innervate more distal portions of PC dendrites than those of wild-type and mGluR1 mutant mice.

View larger version (108K): [in this window] [in a new window]   Figure 11.   Anterogradely labeled CFs in the cerebellar lobule VI of the wild-type (A), GluR2 mutant (B), and mGluR1 mutant (C) mice. Dotted lines in A-C indicate the pial surface of the molecular layer. Asterisks indicates PC somata. Scale bars, 10 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Distinct CF innervation patterns in GluR2 mutant and mGluR1 mutant PCs

GluR2 mutant PCs had atypical CF-EPSCs with slow 10-90% rise times that coexisted with typical fast rise time CF-EPSCs, suggesting that slow CF-EPSCs arise from synapses located electrotonically distant from the soma. Imaging of CF-induced Ca²⁺ signals through voltage-gated Ca²⁺ channels revealed that GluR2 mutant PCs had atypical small CF responses with Ca²⁺ signals that were confined to PC distal dendrites. In contrast, CF-induced Ca²⁺ signals involved both proximal and distal dendrites in wild-type PCs. Morphologically, CFs of GluR2 mutant mice extended to the superficial regions of the molecular layer, whereas CFs of wild-type mice extended to the four-fifths of the molecular layer and spared the external one-fifth. On the other hand, the CF innervation pattern of mGluR1 mutant mice resembled that of the wild-type mice in the following respects: (1) CF-EPSCs had fast rise times (1 msec), (2) CF-induced Ca²⁺ signals involved proximal dendrites, and (3) anterogradely labeled CFs extended to the four-fifths of the molecular layer and spared the external one-fifth.

Previous electron microscopic studies in mouse and rat indicate that CFs form extensive synaptic contacts on protuberance of the proximal dendrites of PCs, whereas PFs form en passant synapses on spines of the distal dendrites of PCs (Larramendi and Victor, 1967; Palay and Chan-Palay, 1974; Ito, 1984). Staining of CFs in rats also revealed that CFs innervate PC proximal dendrites (Strata and Rossi, 1998; Sugihara et al., 1999; Kreitzer et al., 2000). The range of CF-induced Ca²⁺ signals, however, does not precisely reflect the location of CF synapses on PC dendritic trees. Previous studies consistently indicate that CF-induced Ca²⁺ transients spread over the entire dendritic tree of PCs involving both proximal and distal dendrites (Ross and Werman, 1987; Kano et al., 1992; Konnerth et al., 1992; Miyakawa et al., 1992; Eilers et al., 1995b). The Ca²⁺ transients in distal dendrites are thought to reflect the spread of depolarization from proximal dendrites. CF-EPSPs are generated at proximal dendrites of PCs. However, because they are quite large in size, they can readily activate voltage-gated Ca²⁺ channels not only at proximal dendrites but also at distal dendrites. In contrast, the Ca²⁺ transients induced by PF stimulation are confined to the distal dendrites, and the range of the Ca²⁺ signals reflects the site of PF synapses (Eilers et al., 1995a; Finch and Augustine, 1998; Takechi et al., 1998; Wang et al., 2000). This is presumably because PF-EPSPs are relatively small in size and can only activate voltage-gated Ca²⁺ channels locally at the site of PF-EPSP generation. The atypical CF responses in GluR2 mutant mice were also relatively small in size and presumed to induce Ca²⁺ signals that reflect the site of their generation. In these respects, the atypical CF responses resemble PF-mediated responses and presumably reflect synaptic activity at PC distal dendrites.

Taken together, the results of the present study suggest that surplus CFs persist in GluR2 mutant mice by forming ectopic synapses onto distal dendrites of PCs, whereas CFs of mGluR1 mutant mice form synapses onto PC proximal dendrites similar to wild-type mice.

Roles of GluR2 in PF and CF synapse formation

Our previous study in the GluR2 mutant indicates that the abnormality of PF synaptogenesis is not found at P7 but is evident at P14 and thereafter (Kurihara et al., 1997). Thus, the appearance of atypical slow CF-EPSCs coincides temporally with the manifestation of impaired PF synaptogenesis. Previous studies indicate that the developmental change in CF from multiple- to mono-innervation of cerebellar PCs depends on the presence of intact granule cells and the formation of PF to PC synapses (Woodward et al., 1974; Crépel and Mariani, 1976; Mariani et al., 1977; Crépel and Delhaye-Bouchaud, 1979; Crépel et al., 1980; Mariani and Changeux, 1980; Mariani et al., 1990; Bravin et al., 1995; Sugihara et al., 2000). Thus, impairment of PF to PC synapse formation in GluR2 mutant mice (Kashiwabuchi et al., 1995; Kurihara et al., 1997) can cause persistent multiple CF innervation. In wild-type mice, massive formation of PF to PC synapses occurs onto dendritic spines of PC distal dendrites during the second and third postnatal weeks. CF synapses may not be maintained at distal dendrites and become confined to the thick proximal dendrites. Then, a competition may follow between the remaining CFs for postsynaptic sites on the proximal dendrites, and finally, a single CF may survive to innervate the PC. In GluR2 mutant mice, CF synapses may remain at distal dendrites because of the impairment of PF to PC synapse formation during the second and third postnatal weeks. However, the competition may occur between CFs for postsynaptic sites on the proximal dendrites, and in the majority of PCs, one CF may win to form extensive synapses on the proximal dendrites. As a consequence, many PCs lacking GluR2 have one typical CF that generated fast CF-EPSCs and additional atypical CFs that generated slow CF-EPSCs.

A previous study indicates that the  receptor immunoreactivity is prominent at CF synapses at P10 and P14 and then becomes weaker at later developmental stages (Zhao et al., 1998). On the other hand,  receptors are abundant on postsynaptic membranes at PF synapses from P10 to adult (Takayama et al., 1995, 1996; Landsend et al., 1997; Zhao et al., 1998). Thus, the developmental stage at which the impaired PF synapse formation becomes obvious in GluR2 mutant mice corresponds well with the period during which the  receptors are transiently expressed at CF synapses. These results suggest that GluR2 may also play active roles in CF synapse formation during P10-P14 in addition to stabilizing PF synapses.

It is reported that the spontaneously occurring ataxic mouse mutation lurcher is a gain-of-function mutation in the GluR2 gene (Zuo et al., 1997). Mutated GluR2 channels behave as depolarizing leak channels independent of glutamate. Consequently, PCs display a very high membrane conductance and are constitutively depolarized and, perhaps, permit continuous Ca²⁺ inflow. This appears to disturb various signal transduction cascades within PCs, including the pathway from mGluR1 to the  isoform of protein kinase C (PKC) that is shown to be required for CF synapse elimination (Kano et al., 1995, 1997, 1998; Offermanns et al., 1997; Hashimoto et al., 2000). This may be a cause of persistent multiple CF innervation in lurcher mutant mice (Rabacchi et al., 1992). We studied the kinetics of CF-EPSCs in lurcher mice, but atypical slow CF-EPSCs abundant in GluR2 mutant mice were not found (K. Hashimoto and M. Kano, unpublished observation).

Roles of mGluR1 in CF synapse elimination

We have reported that multiple CF innervation persists into adulthood in four strains of mutant mice lacking mGluR1 (Kano et al., 1997), G-protein q subunit (Gq) (Offermanns et al., 1997; Hashimoto et al., 2000), phospholipase C 4 (PLC 4) (Kano et al., 1998; Hashimoto et al., 2000), and PKC (Kano et al., 1995) caused by the defects in CF synapse elimination during the third postnatal week. In contrast to GluR2 mutant mice, these mutant mice have normal PF-PC synaptogenesis and normal rise time of the CF-EPSCs. We also found that blockade of NMDA receptors during P15 and P16, but not before or after this period, resulted in a higher incidence of multiple CF innervation and caused a mild but persistent loss of motor coordination (Kakizawa et al., 2000). Neither basic synaptic functions nor cerebellar morphology was affected by this manipulation. During P15 and P16, NMDA receptors were not functional at either PF to PC or CF to PC synapses but were rich in mossy fiber to granule cell synapses (Kakizawa et al., 2000). We also demonstrated that the mGluR1 to PKC cascade must work within PCs to complete CF synapse elimination (Ichise et al., 2000). We introduced the mGluR1 transgene into mGluR1-null mutant mice using a PC-specific promoter, L7. The mGluR1-rescue mice showed normal regression of multiple CF innervation and apparently normal motor coordination (Ichise et al., 2000). These results suggest that the signal transduction cascade from mGluR1 to PKC is activated at PF-PC synapses (Kano et al., 1995, 1997) in a manner dependent on NMDA receptor-mediated activity at mossy fiber to granule cell synapses (Kakizawa et al., 2000).

In GluR2 mutant mice a substantial number of PCs had multiple CF-EPSCs with fast rise-times in addition to atypical slow CF-EPSCs (Fig. 3). It is possible that the impaired PF-PC synapse formation in GluR2 mutant leads to a reduction of PF-induced drive of mGluR1 and causes persistent multiple CF innervations that are similar to those of the mGluR1, Gq, PLC 4, and PKC mutant PCs.

In conclusion, we propose that GluR2 is required for consolidating PF synapses onto PC distal dendrites and restricting CF synapses to the proximal dendritic domain, whereas the mGluR1 to PKC signaling pathway is involved in eliminating excess CF synapses at the proximal dendrites. By the actions of these two glutamate receptors, synaptic connections of PCs mature normally during postnatal cerebellar development.

	FOOTNOTES

Received June 13, 2001; revised Aug. 20, 2001; accepted Sept. 11, 2001.

This work has been partly supported by grants from the Japanese Ministry of Education, Science, Sports, Culture and Technology (M.K.), the Kato Memorial Bioscience Foundation (K.H.), and the Human Frontier Science Program (M.K., A.K.). This work has also been supported by Special Coordination Funds for promoting Science and Technology from the Japanese Government (K.H.) and Research for the Future Program of Japan Society for the Promotion of Science (A.A). We thank Dr. N. Kawai for continuous encouragement throughout the course of this study, K. Matsumoto and Y. Hirano for excellent technical assistance, and T. Hensch for critically reading this manuscript.

Correspondence should be addressed to Masanobu Kano, Department of Physiology, Kanazawa University School of Medicine, Takara-machi, Kanazawa 920-8640, Japan. E-mail: mkano{at}med.kanazawa-u.ac.jp.

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