Influence of parallel fiber–Purkinje cell synapse formation on postnatal development of climbing fiber–Purkinje cell synapses in the cerebellum

Neuroscience 162 (2009) 601– 611

REVIEW INFLUENCE OF PARALLEL FIBER–PURKINJE CELL SYNAPSE FORMATION ON POSTNATAL DEVELOPMENT OF CLIMBING FIBER–PURKINJE CELL SYNAPSES IN THE CEREBELLUM K. HASHIMOTO,a,b,g T. YOSHIDA,c K. SAKIMURA,d M. MISHINA,e M. WATANABEf AND M. KANOa,g*

P7 to around P11, and “late phase” of CF synapse elimination from around P12. Normal PF–PC synapse formation is required for the “late phase” of CF synapse elimination. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113– 0033, Japan

Key words: GluR␦2, Purkinje cell, climbing fiber, parallel fiber, cerebellum, synapse elimination.

b

Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Saitama 332– 0012, Japan c Department of Pharmacology, School of Medicine, Hokkaido University, Sapporo, 060 – 8638, Japan

Contents Functional differentiation of multiple CFs and “early phase” of CF synapse elimination 602 Strengthening of single CFs in individual PCs 602 Functional differentiation of multiple CF inputs is independent of PF–PC synapse formation 603 The “early phase” of CF synapse elimination follows functional differentiation of multiple CF inputs 604 The “Late phase” of CF synapse elimination during the second and third postnatal weeks 605 CF-EPSCs with slow rise times in GluR␦2-KO PCs 605 CF-EPSCs with slow rise times in hypogranular cerebella caused by metylazoxy methanol acetate (MAM) treatment 605 Ca2⫹ transients associated with slow rise time CF-EPSCs indicate locations of their synapses on PC distal dendrites 605 Morphological evidence for CF-EPSC with slow rise time in 606 GluR␦2-KO PCs Discussion and conclusions 607 Acknowledgments 609 References 609

d Department of Cellular Neurobiology, Brain Research Institute, Niigata University, Niigata 951– 8585, Japan e Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113– 0033, Japan f Department of Anatomy, School of Medicine, Hokkaido University, Sapporo, 060 – 8638, Japan g Department of Cellular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita 565– 0871, Japan

Abstract—The climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum provides an ideal model for the study of developmental rearrangements of neural circuits. At birth, each PC is innervated by multiple CFs. These surplus CFs are eliminated during postnatal development, and mono innervation is attained by postnatal day 20 (P20) in mice. Earlier studies on spontaneous mutant mice and animals with “hypogranular” cerebella indicate that regression of surplus CFs requires normal generation of granule cells and their axons, parallel fibers (PFs), and normal formation of PF–PC synapses. Our understanding of how PF–PC synapse formation affects development of CF–PC synapse has been greatly advanced by analyses of mutant mice deficient in glutamate receptor ␦2 subunit (GluR␦2), an orphan receptor expressed selectively in PCs. Deletion of GluR␦2 results in impairment of PF–PC synapse formation, which leads to defects in development of CF–PC synapses. In this article, we review how impaired PF–PC synapse formation affects wiring of CFs to PCs based mostly on our data on GluR␦2 knockout mice. We propose a new scheme that CF–PC synapses are shaped by the three consecutive events, namely functional differentiation of multiple CFs into one strong and a few weak inputs from P3 to P7, “early phase” of CF synapse elimination from

A widely accepted view of functional neural circuit formation during postnatal development is that immature neurons initially make synaptic connections not only to their final targets but also to other neurons. Then, necessary synapses are strengthened, and less important synapses are weakened and finally eliminated morphologically (Purves and Lichtman, 1980; Lichtman and Colman, 2000). Mechanisms underlying such neural circuit rearrangements have been studied intensively in the neuromuscular junction and autonomic ganglia (Purves and Lichtman, 1980; Lichtman and Colman, 2000). However, it is difficult to do detailed analyses in the CNS, because of the heterogeneity and abundance of synaptic inputs to individual neurons and the complexity of synaptic organization. In this respect, the climbing fiber (CF) to Purkinje cell (PC) synapse in the cerebellum has been an excellent model to study the cellular and molecular mechanisms of synapse elimination in the developing CNS. PCs in the adult cerebellum receive

*Corresponding author. Tel: ⫹81-3-5802-3314; fax: ⫹81-3-5802-3315. E-mail address: [email protected] (M. Kano). Abbreviations: AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate; CF, climbing fiber; CF-EPSC, climbing fiber–mediated excitatory postsynaptic current; EPSC, excitatory postsynaptic current; GC, granule cell; GluR␦2, glutamate receptor ␦2 subunit; KO, knockout; LTD, long-term depression; MAM, metylazoxy methanol acetate; mGluR1, type 1 metabotropic glutamate receptor; NBQX, 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide; PC, Purkinje cell; PF, parallel fiber.

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.12.037

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two major excitatory inputs, namely parallel fibers (PFs) and CFs (Palay and Chan-Palay, 1974; Ito, 1984). PFs are bifurcated axons of cerebellar granule cells (GCs) and form synapses on spines of PC’s distal dendrites. Each synaptic input is weak but as many as 100,000 PFs form synaptic contacts on a single PC (Palay and Chan-Palay, 1974; Ito, 1984). In contrast, the majority of PCs in the adult cerebellum are innervated by single CFs (mono innervation) but each CF makes strong synaptic contacts on PC’s proximal dendrites (Palay and Chan-Palay, 1974; Ito, 1984). In early postnatal days, however, all PCs are innervated by multiple CFs (multiple innervation) (Crepel et al., 1976; Crepel, 1982; Lohof et al., 1996). These surplus CFs are eliminated eventually with the progress of postnatal development, and mono innervation is attained by the end of the third postnatal week in mice (Kano et al., 1995, 1997, 1998; Offermanns et al., 1997). Earlier studies on spontaneous mutant mice (Crepel and Mariani, 1976; Mariani et al., 1977; Crepel et al., 1980; Mariani and Changeux, 1980) and animals with experimentally-induced “hypogranular” cerebella (Woodward et al., 1974; Crepel and Delhaye-Bouchaud, 1979; Bravin et al., 1995; Sugihara et al., 2000) have revealed that the presence of intact GCs and normal formation of PF–PC synapses are prerequisite for CF synapse elimination. Moreover, Crepel et al. (1981) showed that elimination of surplus CFs consists of two distinct phases, the early phase up to around P8 and the late phase from around P9 to P17 (Crepel et al., 1981). The early phase occurs normally in animals with “hypogranular” cerebella, whereas the late phase is severely impaired by inhibiting GC production, indicating that the early phase CF synapse elimination proceeds independently of PF–PC synapse formation, whereas the late phase critically dependent on it. However, since manipulation to produce “hypogranular” or “agranular” cerebella such as X-ray irradiation to newborn rats or mice may affect cerebellar development other than GC genesis and PF–PC synapse formation (Lovell and Jones, 1980; Chen and Hillman, 1988; Garcia-Ladona et al., 1991), there remains a possibility that CF synapse elimination might be influenced by such developmental defects. Mice deficient in glutamate receptor ␦2 subunit (GluR␦2) are impaired in PF–PC synaptogenesis, resulting in about half the number of the wild-type mice, whereas their histoarchitecture of the cerebellum is largely normal and the PCs developed well-arborized dendritic trees with numerous spines (Kashiwabuchi et al., 1995; Kurihara et al., 1997). GluR␦2 is classified as a member of ionotropic glutamate receptors, but its ligand is unknown and it does not seem to function as an ion channel (Araki et al., 1993; Lomeli et al., 1993; Mandolesi et al., 2009). GluR␦2 is expressed exclusively in cerebellar PCs particularly in PC’s dendritic spines forming synaptic contacts with PFs (Takayama et al., 1996; Landsend et al., 1997; Zhao et al., 1998). Therefore, primary defects in GluR␦2 knockout (KO) mice should be confined to PCs. GluR␦2-KO mice have been used as a tool to gauge the influence of the defects in PF–PC synapse formation on postnatal development of CF synapses with minimal effects of other developmental defects of the cerebellum (Hashimoto

et al., 2001a; Ichikawa et al., 2002). For more details about GluR␦2, several excellent review articles are available (Yuzaki, 2003, 2004; Hirano, 2006; Mandolesi et al., 2009; Watanabe, 2008). In this review article, we will integrate the current knowledge of postnatal development of CF synapses based mostly on our own data. We first show how CF synapses undergo developmental changes during the first postnatal week before functional PF–PC synapse formation occurs. Then we describe how impaired PF–PC synapse formation affects wiring of CFs to PCs and elimination of redundant CF synapses during the second postnatal week and in adulthood. We propose a new scheme of postnatal development of CF synapses, which consists of three distinct phases: (1) functional differentiation of multiple CFs into one strong and a few weak inputs that occurs from P3 to P7, (2) “early phase” of CF synapse elimination from P7 to around P11, (3) “late phase” of CF synapse elimination from around P12.

FUNCTIONAL DIFFERENTIATION OF MULTIPLE CFs AND “EARLY PHASE” OF CF SYNAPSE ELIMINATION Strengthening of single CFs in individual PCs Multiple CFs initially form synapses around the somata of PCs in newborn mice (Chedotal and Sotelo, 1993). Excitatory postsynaptic currents (EPSCs) elicited by stimulating such multiply-innervating CFs are much smaller than those of mature CFs (Hashimoto and Kano, 2003, 2005; Bosman et al., 2008). Therefore, CF inputs become stronger during postnatal development, while redundant CFs are eliminated during the same period. Mariani and Changeux, (1981) showed that some PCs had two CF-mediated EPSPs whose amplitudes were quite different around P10 to P13, suggesting that only one CF was strengthened relative to the others prior to the completion of synapse elimination. Changes in the relative synaptic strengths of multiple CFs innervating the same PC were systematically investigated during postnatal development in mice (Hashimoto and Kano, 2003, 2005). In this study, whole-cell patch clamp recording was made from the soma of a PC in a cerebellar slice prepared from mice from P2 to P21. CFs were stimulated with a glass pipette positioned in the GC layer and sometimes in the molecular layer, and climbing fiber–mediated excitatory postsynaptic currents (CF-EPSCs) were recorded. The stimulation pipette was moved systematically by 20 ␮m steps and the stimulus strength was increased gradually from 0 V to around 50 V at each stimulation site. The number of CFs innervating each PC was judged by the number of discrete CF-EPSCs, and the strengths of individual CF inputs were estimated from the sizes of CF-EPSCs. This systematic study shows that in PCs from neonatal mice around P4, more than five discrete CF-EPSCs with similar amplitudes are commonly recorded. In contrast, in the second postnatal week, PCs with multiple CF-EPSCs have one large CF-EPSC and a few small CF-EPSCs. These results suggest that synaptic strengths of multiply-innervating CFs are relatively uniform

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in neonatal mice, and one CF is selectively strengthened during postnatal development (Hashimoto and Kano, 2003, 2005). To quantify this developmental change, the disparity among the amplitudes of multiple CF-EPSCs was estimated by calculating a parameter, named disparity ratio (see Fig. 1 legend). This value represents the predominance of the CF eliciting the largest EPSC over the other CFs in each PC. The results show the disparity ratio progressively decreases from P3 to P6 and reaches a plateau level at P7. This result indicates that one CF is selectively strengthened among multiple CFs innervating the same PC during the first postnatal week (Hashimoto and Kano, 2003, 2005). A morphological study by Sugihara (2005) shows that the innervation pattern of CFs over PCs drastically changes during this postnatal period in rats. At P4, CFs have many creeping terminals in the PC layer and their swellings do not aggregate at particular PC somata (creeper type). Then, from P4 to P7, CFs surround several specific PC somata and form aggregated terminals on them (nest type). This anatomical observation is consistent with the electrophysiological finding that one CF is selectively strengthened among multiple CFs innervating the same PC during the first postnatal week (Hashimoto and Kano, 2003, 2005). Functional differentiation of multiple CF inputs is independent of PF–PC synapse formation The aforementioned view was challenged by Scelfo and Strata (2005) who reported that the disparity among multiply-innervating CF-EPSC amplitudes became progressively smaller from P4 to P7, and then increased from P7 to P10 (Scelfo and Strata, 2005). They also showed that regression of CFs did not occur until P7 but it started at around P7 and proceeded until P14 (Scelfo and Strata, 2005). They claimed that CF synapse elimination proceeds during the second postnatal week which is critically dependent on PF–PC synapse formation (Scelfo and Strata, 2005). To address this controversial issue, we estimated the average number of CFs innervating each PC from P5 to P14 in C57BL/6 mice. As shown in Fig. 1A, there was no significant reduction in the average value until P6 (Fig. 1A, C57BL/6). Although one day earlier, these data are almost consistent with the data by Scelfo and Strata (2005). However, they reported that the average number of CFs innervating individual PCs at P4 –P7 was around 3.3 in CD1 mice, which is much smaller than that of our measurements in C57BL/6 mice (more than 6, Fig. 1A, C57BL/6). This may be attributable to the difference in mouse strain (Scelfo and Strata, 2005). Alternately, the difference may result from the electrophysiological methods for searching for multiple CFs. Even with the systematic stimulation protocol as adopted in our studies, all the CFs innervating the PCs under recording may not necessarily be stimulated in slice preparation. Scelfo and Strata (2005) might have missed more CFs than we might have, when searching for multiple CFs. In support of our results, Bosman et al. (2008) have reported recently in rat that the amplitudes of the largest CF-EPSC progressively become larger, while those of the 2nd and 3rd largest CF-EPSCs are rather

Fig. 1. Postnatal development of CF-EPSCs in GluR␦2-KO mice, their control littermates (Control), and C57BL/6 mice. (A) Average number of CFs innervating each PC during postnatal development. Values are mean⫾SEM. Number of cells for each point is 19 – 46 for C57BL/6 mice, 10 –20 for Control, and 8 –27 for GluR␦2-KO mice. The values of GluR␦2-KO mice are significantly larger than Control at P12, P13 and P14 (** P⬍0.01, Mann–Whitney U-test). (B) Average values of disparity ratio for each PC during postnatal development. The amplitudes of individual CF-EPSCs in a given multiply-innervated PC are measured and they are numbered in the order of their amplitudes (A1, A2, . . . Ai, . . . AN, Nⱖ2, N is the number of CFs innervating a given PC. Ai is the EPSC amplitude for the CFi recorded at the same holding potential. AN represents the EPSC amplitude for the largest CF). N⫺1

Ai

兺A i⫽1

N

N ⱖ 2兲 N⫺1共 If all CF-EPSCs in a given PC have the same amplitudes, the disparity ratio will be 1. Conversely, if the differences between AN and other smaller CF-EPSCs are large, the disparity ratio will be small. Data for P9 –P11 and those for P12–P14 are pooled and indicated with triangles. Because EPSCs with slow rise times elicited by ectopic CF synapses are considered to suffer from strong distortions due to dendritic filtering and their amplitudes will be underestimated, such slow EPSCs are not included for the calculation of disparity ratio after P12. Number of cells for each point is 19 –72 for C57BL/6 mice, 10 –20 for Control, and 10 –25 for GluR␦2-KO mice. It should be noted that from P3 to P6 (shaded area in A and B), the average number of CFs per PC is constant or has a tendency to increase, whereas the disparity ratio becomes progressively smaller. Disparity ratio ⫽

constant, during the first postnatal week (Bosman et al., 2008).

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We also examined whether functional differentiation of CF inputs during the first postnatal week is affected in GluR␦2-KO mice. Although GluR␦2 is localized predominantly in dendritic spines facing PFs from P14 (Takayama et al., 1996), it is expressed widely in dendritic shafts and spines of PCs at early postnatal period (Zhao et al., 1998). Kurihara et al. have demonstrated that formation of immature PF–PC synapses at P7 is apparently normal in GluR␦2-KO mice, but maturation and stabilization of mature PF–PC synapses during the second postnatal week are impaired in GluR␦2-KO mice, which leads to reduction of mature PF–PC synapses to about half the number of control mice (Kurihara et al., 1997). When developmental change in disparity ratio was followed in GluR␦2-KO mice, the value of GluR␦2-KO was similar to that of control mice and C57BL/6 mice from P5 to P14 (Fig. 1B). These results support that functional differentiation of multiple CF inputs into one strong CF and the other weak CFs proceeds during the first postnatal week before formation of functional PF–PC synapses, which indicates that this process is independent of PF–PC synapse formation. The results also indicate that GluR␦2 present in PC dendritic shafts and spines during this early developmental stage does not contribute to functional differentiation of multiple CF inputs.

The “early phase” of CF synapse elimination follows functional differentiation of multiple CF inputs From P3 to P6 when functional differentiation of multiple CFs occurred, there was no significant reduction but rather a tendency of increase in the average number of CFs (Fig. 1A, C57BL/6). Then, the value decreased progressively from P6 to P14 (Fig. 1A, C57BL/6). These results indicate that CF synapse elimination does not proceed in parallel with functional differentiation of multiple CFs but starts after the strengthening single CFs in individual PCs. In GluR␦2-KO mice, the average number of CFs innervating each PC was similar to that of control mice from P5 to P11 (Fig. 1A, Control, GluR␦2-KO). However, the value was significantly larger than that of control mice from P12 to P14 (Fig. 1A, Control, GluR␦2-KO), which corresponded to the period when small CF-EPSCs with slow rise times appeared in GluR␦2-KO mice (Hashimoto et al., 2001a) (see Fig. 2). As described in the following, this atypical CF-EPSC is characteristic of GluR␦2-KO mice and reflects ectopic CF innervation of PCs’ distal dendrites which results from decreased number of PF–PC synapses (Hashimoto et al., 2001a; Ichikawa et al., 2002). These results collectively indicate that CF synapse elimination in mice can be classified into two distinct phases, namely the “early phase” from P6 to around P11 and the “late phase”

Fig. 2. Time course of CF-EPSCs in GluR␦2-KO and control mice. (A, B) EPSCs elicited by stimulating CFs in a Control PC (A, P28) and a GluR␦2-KO PC (B, P64). With gradually increasing stimulus intensities, EPSCs of the Control PC were elicited in an all-or-none fashion (A), while those of the GluR␦2-KO (B) occurred at multiple discrete steps, indicating that this PC was innervated by multiple CFs. Note that the rise time of the largest CF-EPSC of the GluR␦2-KO PC is similar to that of the Control PC, whereas the rise times of the smaller two CF-EPSCs of the GluR␦2-KO PC are significantly slower. One to three traces are superimposed at each CF-EPSC step. Stimuli were applied at 0.2 Hz. Holding potentials were ⫺10 mV. (C, D) Summary histograms showing the 10%–90% rise time of CF-EPSCs in Control (C) and GluR␦2-KO (D) PCs. Data were obtained from mice at P23-70. (E, F) Postnatal change in the 10%–90% rise times of CF-EPSCs in Control (E) and GluR␦2-KO (F) PCs. Note that CF-EPSCs have similar rise times in the two strains of mice during P3–P9, whereas those with significantly slower rise times are obvious after P12 in the GluR␦2-KO PCs. Adapted from (Hashimoto et al., 2001a).

K. Hashimoto et al / Neuroscience 162 (2009) 601– 611

from around P12 and thereafter. The analysis of GluR␦2-KO mice indicates that the “late phase” is dependent on PF–PC synapse formation (see below). In the following, we describe how PF–PC synapse formation influences CF synapse elimination during the second and third postnatal weeks. We introduce our data on GluR␦2-KO mice and a mouse model of hypogranular cerebellum and describe how the “late phase” of CF synapse elimination proceeds.

THE “LATE PHASE” OF CF SYNAPSE ELIMINATION DURING THE SECOND AND THIRD POSTNATAL WEEKS CF-EPSCs with slow rise times in GluR␦2-KO PCs Our electrophysiological analysis of CF-EPSCs in GluR␦2-KO mice has revealed an aberrant CF innervation pattern of PCs. In most of PCs of adult control mice, EPSCs are elicited in an all-or-none fashion, indicating that they are innervated by single CFs (Fig. 2A). The 10%–90% rise time of CF-EPSC is fast and displays a normal distribution with a peak around 0.5 ms (Fig. 2C) (Hashimoto et al., 2001a). In contrast, multiple EPSCs are elicited in more than two-thirds of PCs from adult GluR␦2-KO mice (Fig. 2B) (Hashimoto et al., 2001a). Notably, EPSCs with slow rise times (⬎1.0 ms) are frequently elicited. The rise times of GluR␦2-KO CF-EPSCs are segregated into two distinct populations (Fig. 2D). The mean value for the fast (⬍1.0 ms) population is similar to the value of control mice, whereas the slow (⬎1.0 ms) population is unique to GluR␦2-KO mice. The atypical slow EPSCs are not considered to be elicited by stimulating PFs, because they appears with discrete steps around threshold stimulus intensity and exhibits clear paired-pulse depression (Hashimoto et al., 2001a). The 10%–90% rise time of the control CF-EPSC is longer during P3-P9 than at later developmental stages (Fig. 2E). The rise times of the GluR␦2-KO CF-EPSCs from P3 to P9 display a similar distribution to that of control mice (Fig. 2E, F). During P10 –P14, the rise times of the control CF-EPSCs become similar to the adult levels (Fig. 2E). In contrast, CF-EPSCs with significantly slower rise times appear at P12 in the GluR␦2-KO mice (Fig 2F). The developmental stage when atypical slow CF-EPSCs appear well corresponds to the stage when defect in PF to PC synapse formation becomes evident in the GluR␦2-KO mouse (Kurihara et al., 1997). Under somatic voltage clamp recording, EPSCs originating from distal dendrites are strongly attenuated by dendritic filtering (Roth and Hausser, 2001; Lisman et al., 2007). In rodents, CF synapses are located on the proximal dendrites of PCs, whereas PF synapses are on spines at the distal dendrites (Palay and Chan-Palay, 1974; Ito, 1984). 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). These results indicate that the EPSC rise time reflects the electrotonic length from the soma to the site of the synapse, provided kinetics of postsynaptic glu-

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tamate receptors and glutamate release from presynaptic terminals are the same. These lines of evidence collectively suggest the possibility that the atypical slow CFEPSCs in GluR␦2-KO PCs arise from CF synapses located electrotonically distant from the soma, presumably on the distal dendrites. The existence of the slow CF-EPSCs significantly contributes to a high percentage of multiply-innervated PCs in the GluR␦2-KO mice. In addition to these slow CF-EPSCs, surplus CF-EPSCs with fast rise times are also present in GluR␦2-KO PCs (Hashimoto et al., 2001a). Such fast CFEPSCs are considered to be generated by CFs at proximal dendrites. Thus, these electrophysiological data strongly suggest that multiple CF innervation of GluR␦2-KO PCs results from surplus CF inputs forming ectopic synapses on distal dendrites and those forming synapses on proximal dendrites. CF-EPSCs with slow rise times in hypogranular cerebella caused by metylazoxy methanol acetate (MAM) treatment To examine whether atypical CF-EPSCs with slow rise times are also observed in cerebella with impaired PF–PC synaptogenesis that is caused by manipulation other than GluR␦2 deletion, CF-EPSCs were examined in mice with hypogranular cerebella by neonatal injection of MAM (Bejar et al., 1985; de Barry et al., 1987; Garcia-Ladona et al., 1991; Bravin et al., 1995; Takacs et al., 1997). MAM (20 mg/kg) was injected s.c. into mice at P3 to kill the proliferating GCs (Bravin et al., 1995). Similarly to GluR␦2-KO mice, PCs in MAM-treated mice have numerous free spines that are not occupied by PF terminals (de Barry et al., 1987; Takacs et al., 1997). At P22 to P70, parasagittal cerebellar slices were prepared from the MAM-treated mice and the saline-injected control mice, and their CFEPSCs were examined. Stimulation in the GC layer readily induced EPSCs with discrete amplitude steps that displayed paired-pulse depression (Konnerth et al., 1990), indicating that these responses were elicited by stimulating CFs. Notably, CF-EPSCs with slow rise times (⬎1.0 ms) were frequently elicited in the MAM-treated mice (Fig. 3B, D), while such slow CF-EPSCs were very rare in the salinetreated control mice (Figs. 3A, C). As a result, most PCs of the MAM-treated mice have one large CF-EPSC with fast rise time and a few surplus CF-EPSCs with small amplitudes and slow rise times. Since these features are similar to those of GluR␦2-KO mice, these results suggest that a major cause for the appearance of atypical slow rise time CF-EPSCs in GluR␦2-KO mice is impaired PF–PC synaptogenesis. Ca2ⴙ transients associated with slow rise time CF-EPSCs indicate locations of their synapses on PC distal dendrites Location of CF synapse on PC dendritic tree can be estimated by imaging Ca2⫹ transients associated with CF responses. Because a Ca2⫹ signal is confined to the region of the cytoplasm where Ca2⫹ is elevated (Eilers et al., 1995), it is a good indicator of the site of Ca2⫹ entry

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Fig. 3. Time course of CF-EPSCs in MAM-treated and control saline-injected mice. (A, B) EPSCs elicited by stimulating CFs in a Control PC (A, P54) and a MAM-treated PC (B, P34). Note that the rise times of the smaller two CF-EPSCs of the MAM-treated PC are slow. One to three traces are superimposed at each CF-EPSC step. Stimuli were applied at 0.2 Hz. Holding potentials were ⫺10 mV. (C, D) Summary histograms showing the 10%–90% rise time of CF-EPSCs in Control (C) and MAM-treated (D) PCs. Data were obtained from mice at P23-66.

through voltage-gated Ca2⫹ channels activated by CF synaptic input (Hashimoto et al., 2001a). In a mono-innervated wild-type PC, stimulation of the CF induces a typical complex spike that accompanies a clear Ca2⫹ signal over the entire dendritic tree (Hashimoto et al., 2001a). In GluR␦2-KO PCs, a different pattern of CF-induced Ca2⫹ signals from those seen in wild-type can be recorded (Hashimoto et al., 2001a). For example, the GluR␦2-KO PC in Fig. 4 was innervated by at least three distinct CFs. Stimulation of one CF (CF1) generated typical complex spikes (Fig. 4A), whereas stimulation of the other two CFs (CF2 and CF3) elicited slow responses (Fig. 4C, E). Stimulation of CF1 induced Ca2⫹ transients that spread widely over the middle dendritic tree involving both proximal and distal dendrites (Fig. 4B). In contrast, stimulation of CF2 induced Ca2⫹ transients that were confined to the distal portions of the left dendritic branch (Fig. 4D). Stimulation of CF3 induced Ca2⫹ transients that were confined to the distal dendrites of the right dendritic branch (Fig. 4F). In 24 GluR␦2 mutant PCs, slow CF responses were always associated with Ca2⫹ transients that were confined to the distal dendrites (Hashimoto et al., 2001a). This pattern of CF-induced Ca2⫹ transients was not observed in any PCs

from the wild-type mice (Hashimoto et al., 2001a). These Ca2⫹ imaging data strongly suggest that the CF synapses generating slow responses are located on the distal dendrites of GluR␦2-KO PCs. Morphological evidence for CF-EPSC with slow rise time in GluR␦2-KO PCs By taking advantage of grossly normal cytodifferentiation in GluR␦2-KO PCs, we addressed how and where multiple CF innervation is formed when PF synaptogenesis is impaired (Ichikawa et al., 2002). CFs were selectively labeled with anterograde tracer (biotinylated dextran amine). In control mice, the mean relative height of CFs in the molecular layer was 84%. The remainder of the superficial molecular layer was a CF-free zone, composed of distal dendrites innervated only by PFs. In GluR␦2-KO mice, the mean relative height increased to 95% and some CF terminals were distributed very close to the pial surface. By serial electron microscopy, the innervation of PC dendrites by tracer-labeled CFs was followed from the soma to the tips of PC dendrites (Fig. 5). Control PCs had

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Fig. 4. CF-induced Ca2⫹ transients in a multiply innervated GluR␦2-KO PC. (A, C, E), Responses induced by stimulating three different CFs, CF1 (A), CF2 (C) and CF3 (E), under the current clamp recording mode. (B, D, F) Pseudocolor images showing the relative increase in Ca2⫹-dependent fluorescence (⌬F/F0) at the peak of the responses evoked by CF1 (A), CF2 (C) or CF3 (E) stimulation. Adapted from (Hashimoto et al., 2001a).

no free spines in the proximal (defined as being innervated by CFs only), intermediate (by both CFs and PFs), or distal (by PFs only) domains of PC dendrites (Fig. 5A). In PCs of GluR␦2-KO mice, free spines were not found in the proximal domain, but they were present in the intermediate (37.4% of the total spines) and distal (55.7%) domains of PC dendrites (Fig. 5B). In the latter two domains, the numbers of PF–PC synapses were reduced by the same extent as the fractions of free spines. Importantly, CFs in GluR␦2-KO mice had aberrant distal extensions to innervate free spines emerging from the intermediate and distal domains. The spine takeover can be classified into two distinct types, intracellular (or intra-dendritic) and intercellular types. In the intracellular type, CFs innervated only one or a few spines emanating from intermediate and distal dendritic domains of the same PC, which did not cause multiple CF innervation (Fig. 5B). In the intercellular type, when CFs reached the distal end of innervation territory of dendrites, CFs abruptly jumped and innervate free spines on distal dendrites of neighboring PCs (Fig. 5B). Because the proximal dendrites of the neighboring PCs were innervated by other CFs, the intercellular type of free spine takeover inevitably caused multiple CF innervation. Both types of aberrant CF innervations were observed in all three PCs reconstructed from GluR␦2-KO mice, but in none of the three PCs from control mice (Ichikawa et al., 2002). Therefore, multiple CF innervation in GluR␦2-KO mice is caused by aberrant takeover of free spines on the distal and intermediate domains of GluR␦2-deficient PC dendrites. This morphological evidence is consistent with

the data from the electrophysiological and Ca2⫹ imaging experiments as mentioned above (Figs. 2 and 4). These findings collectively indicate that through its primary function of ensuring PF–PC synapse formation, GluR␦2 plays an important role in restricting CF innervation to proximal dendrites by preventing aberrant CF innervations onto distal dendrites. Recently, knock-in mice carrying mutant GluR␦2 lacking the C-terminal region of GluR␦2 called T-site (GluR␦2⌬T mice) have been generated (Uemura et al., 2007). In the GluR␦2⌬T mice, CFs display gradual distal extension in the molecular layer. The distal extension of CFs becomes evident at P21, and causes ectopic CF innervation onto distal dendrites without forming surplus innervation onto proximal dendrites (Uemura et al., 2007). Accordingly, electrophysiological analyses demonstrates that PCs from GluR␦2⌬T mice have CF-EPSCs with slow rise times besides one main CF-EPSC with a fast rise time, but they do not have surplus CF-EPSCs with fast rise times (Uemura et al., 2007). These results suggest that the T-site is essential for regulation of the distal border of CF territory and restriction of CF innervation to proximal dendrites, whereas it is dispensable for PF–PC synapse formation during cerebellar development.

DISCUSSION AND CONCLUSIONS The primary defect of GluR␦2-KO mouse is failure to stabilize PF–PC synapses and resultant reduction in the number of PF–PC synapses, whereas the foliation and laminar

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Fig. 5. Schematic illustrations of synaptic organizations of control (A) and GluR␦2-KO (B) PCs. PCD, Purkinje cell dendrite. Adapted from (Ichikawa et al., 2002).

organization of the cerebellum and cytoarchitecture of PCs and other cell-types are grossly normal (Kurihara et al., 1997; Ichikawa et al., 2002). By analyzing the postnatal development of GluR␦2-KO mice as well as control C57BL/6 mice, three distinct phases of postnatal development of CF synapses are clearly demonstrated (Fig. 1). From P3 to P6, one CF is selectively strengthened among multiple CFs in individual PCs. There is no net reduction of the number of CFs innervating each PC until P6, indicating that regression of CF synapse apparently does not occur in this phase. Development of CF synapses in GluR␦2-KO mice appears normal during the first postnatal week (Fig. 1), indicating that GluR␦2 present in PC dendritic shafts and spines during this early developmental stage (Zhao et al., 1998) does not contribute significantly to functional differentiation of multiple CF inputs. Since PF–PC synapses are formed during the second postnatal week (Altman, 1972), the functional differentiation of CF inputs during the first postnatal week is independent of PF–PC synapse formation. From P7 to around P11, the number of CFs innervating each PC decreases progressively with age in both control and GluR␦2-KO mice (Fig. 1A), which we propose to term the “early phase” of CF synapse elimination. In contrast, the previous analyses of X-ray irradiated hypogranular rats shows that the “early phase” is up to around P8 which is independent of GC genesis and the “late phase” starts at around P9 which is significantly influenced by PF–PC synapse formation (Crepel et al., 1981; Crepel, 1982). The apparent discrepancy between our results in mice (Fig. 1A) and the data by Crepel et al. (1981) in rats might be attributable

to species difference and/or recording techniques. One possibility would be that the “early phase” of mice might persist longer than that in rats, which includes the stage of surplus CF regression from P7 to around P11 (Fig. 1A). Around P12 and thereafter, when massive PF–PC synapse formation occurs (Altman, 1972), atypical CF-EPSCs with slow rise times appear in GluR␦2-KO mice (Fig. 2) (Hashimoto et al., 2001a). We propose to term this stage the “late phase” of CF synapse elimination, which is critically dependent on PF–PC synapse formation. In this period, the average number of CFs per PC of GluR␦2-KO mice becomes significantly divergent from that of control mice (Fig. 1A). The slow CF-EPSCs are also frequent in PCs with hypogranular cerebella induced by neonatal MAM treatment (Fig. 3), indicating that they result from impaired PF–PC synaptogenesis. The slow CF responses elicit Ca2⫹ transients that are confined to PC distal dendrites (Fig. 4) (Hashimoto et al., 2001a), suggesting that the Ca2⫹ transients arise from ectopic CF synapses on PC distal dendrites. Morphological data from anterograde labeling of CFs and serial electron microscopic analysis demonstrate that CFs of GluR␦2-KO mice extend distally to spiny branchlets and form ectopic synapses, where about half of spines are free of PF innervation (Fig. 5) (Ichikawa et al., 2002). Notably, CFs extend collaterals to form ectopic synapses on adjacent spiny branchlets, whose proximal portions are innervated by different CFs (Fig. 5) (Ichikawa et al., 2002). These results strongly suggest that PF–PC synapse formation and the occupancy of spines prevent CFs from invading distal spiny branchlets, and that there is a heterosynaptic competition

K. Hashimoto et al / Neuroscience 162 (2009) 601– 611

between PFs and CFs for postsynaptic spines. By contrast, CF innervation is markedly regressed and PF innervation territory is expanded reciprocally to proximal dendrites in null mutant mice deficient in the Cav2.1, a poreforming component of P/Q-type voltage-dependent Ca2⫹ channel (Miyazaki et al., 2004). Because the P/Q-type is the major voltage-gated Ca2⫹ channel in PCs, Ca2⫹ influx into PCs during CF activity is considered to strengthen CF synapses and expel PF synapses from proximal dendrites (Miyazaki et al., 2004). Thus, GluR␦2 and CaV2.1 have opposite actions on heterosynaptic competition between CFs and PFs. Such heterosynaptic competition becomes important during the second postnatal week in mice when massive PF–PC synaptogenesis occurs (Altman, 1972). However, a similar mechanism is at work also in the adult cerebellum. Blockade of neural activity by infusion of tetrodotoxin or an AMPA receptor blocker, NBQX to adult cerebellum causes proximal regression of CF innervation and a reciprocal expansion of PF innervation (Bravin et al., 1999; Kakizawa et al., 2005; Cesa et al., 2007). GluR␦2 appears in the proximal dendritic domain where PFs form ectopic synapses in TTX-treated adult cerebella (Morando et al., 2001; Cesa et al., 2003). Furthermore, conditional deletion of GluR␦2 in adult cerebellum in mice causes regression of PF–PC synapses and distal extension of CF innervation (Takeuchi et al., 2005). Thus, heterosynaptic competition between CFs and PFs is crucial for maintaining territorial innervation of PCs by the two excitatory inputs both during postnatal development and in adulthood. GluR␦2 plays a pivotal role for this heterosynaptic competition by continuously reinforcing PF–PC synapses. Recent reports by Uemura et al. (2007) and Kakegawa et al. (2008) have revealed the functions of the C-terminal region of GluR␦2 (T-site) which contains PDZ binding domains. In addition to impaired PF–PC synapse formation and resultant defect in CF synapse development, GluR␦2-KO mice have multiple deficits including deficient long-term depression (LTD) at PF–PC synapses, severe ataxia (Kashiwabuchi et al., 1995), and impaired motor learning (Kishimoto et al., 2001a,b; Katoh et al., 2005). Uemura et al. (2007) generated knock-in mice carrying mutant GluR␦2 lacking the T-site (GluR␦2⌬T), while Kakegawa et al. (2008) generated transgenic mice that expressed mutant GluR␦2 on GluR␦2-KO background. The two reports consistently demonstrate that the GluR␦2 Tsite is crucial for LTD induction and motor learning but not for PF–PC synapse formation. Importantly, Uemura et al. (2007) showed that CF innervation territory expanded to distal dendrites in mature GluR␦2⌬T mice, although their PF–PC synapses were formed normally. This observation suggests that the distal extension and ectopic innervation of CF axons in mature cerebellum do not necessarily require a decrease in the number of PF–PC synapses or the appearance of free spines during postnatal development. Signaling through the T-site of GluR␦2 itself may prevent expansion of CF innervation territory and thus may underlie heterosynaptic competition between PFs and CFs in mature cerebellum. Uemura et al. (2007) also reported that developmental regression of surplus CFs at proximal den-

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drites of PCs was normal in GluR␦2⌬T mice. Since the CF synapse elimination at proximal dendrites is clearly impaired in GluR␦2-KO mice (Hashimoto et al., 2001a), it is possible that domains of GluR␦2 other than the T-site may be responsible for this phenomenon. Alternatively, the impaired PF–PC synapse formation itself may be a cause of the defect in GluR␦2-KO mice. Previous studies have shown that neural activity along mossy fiber-GC-PF pathway is crucial for the CF synapse elimination at proximal dendrites by driving type 1 metabotropic glutamate receptors (mGluR1) and downstream cascade in PCs (Kano et al., 1995, 1997, 1998; Offermanns et al., 1997; Hashimoto et al., 2000, 2001b; Ichise et al., 2000; Kakizawa et al., 2000). These strains of mouse retain multiple CF innervation of PCs in adulthood, but the atypical slow CF-EPSCs are rare and PF–PC synapse formation is normal. Their postnatal development of CF innervation until the middle of the second postnatal week is normal but the defect in CF synapse elimination becomes evident at around P12. These results indicate that mGluR1 signaling in PCs is crucial for eliminating surplus CFs from somatodendritic domain of PC during the “late phase” of CF synapse elimination. Therefore, the impaired CF synapse elimination at proximal dendrites in GluR␦2-KO mice may be attributable at least partly to reduced PF-mediated drive of mGluR1. Taken together, these lines of evidence strongly suggest that PF–PC synapse formation plays two distinct roles in CF synapse elimination during the “late phase” of CF synapse elimination. First, PF synapses restrict CF innervation territory to PC’s proximal dendrites by heterosynaptic competition. Second, PFs convey neural activity that drives mGluR1 and downstream cascade in PCs. Acknowledgments—This study was supported by Grants-in Aid for Scientific Research (17,023,001 to M.W. and 17,023,021 and 17,100,004 to M.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

REFERENCES Aiba A, Kano M, Chen C, Stanton ME, Fox GD, Herrup K, Zwingman TA, Tonegawa S (1994) Deficient cerebellar long-term depression and impaired motor learning in mGluR1 mutant mice. Cell 79:377–388. Altman J (1972) Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J Comp Neurol 145:399 – 463. Araki K, Meguro H, Kushiya E, Takayama C, Inoue Y, Mishina M (1993) Selective expression of the glutamate receptor channel ␦2 subunit in cerebellar Purkinje cells. Biochem Biophys Res Commun 197:1267–1276. Bejar A, Roujansky P, de Barry J, Gombos G (1985) Different effect of methylazoxymethanol on mouse cerebellar development depending on the age of injection. Exp Brain Res 57:279 –285. Bosman LW, Takechi H, Hartmann J, Eilers J, Konnerth A (2008) Homosynaptic long-term synaptic potentiation of the “winner” climbing fiber synapse in developing Purkinje cells. J Neurosci 28:798 – 807. Bravin M, Morando L, Vercelli A, Rossi F, Strata P (1999) Control of spine formation by electrical activity in the adult rat cerebellum. Proc Natl Acad Sci U S A 96:1704 –1709.

610

K. Hashimoto et al / Neuroscience 162 (2009) 601– 611

Bravin M, Rossi F, Strata P (1995) Different climbing fibres innervate separate dendritic regions of the same Purkinje cell in hypogranular cerebellum. J Comp Neurol 357:395– 407. Cesa R, Morando L, Strata P (2003) Glutamate receptor␦2 subunit in activity-dependent heterologous synaptic competition. J Neurosci 23:2363–2370. Cesa R, Scelfo B, Strata P (2007) Activity-dependent presynaptic and postsynaptic structural plasticity in the mature cerebellum. J Neurosci 27:4603– 4611. Chedotal A, Sotelo C (1993) The “creeper stage” in cerebellar climbing fiber synaptogenesis precedes the “pericellular nest”— ultrastructural evidence with parvalbumin immunocytochemistry. Brain Res Dev Brain Res 76:207–220. Chen S, Hillman DE (1988) Developmental factors related to abnormal cerebellar foliation induced by methylazoxymethanol acetate. MAM. Brain Res 468:201–212. Crepel F, Delhaye-Bouchaud N, Dupont JL (1981) Fate of the multiple innervation of cerebellar Purkinje cells by climbing fibers in immature control, X-irradiated and hypothyroid rats. Brain Res 227: 59 –71. Crepel F, Delhaye-Bouchaud N, Guastavino JM, Sampaio I (1980) Multiple innervation of cerebellar Purkinje cells by climbing fibres in staggerer mutant mouse. Nature 283:483– 484. Crepel F, Delhaye-Bouchaud N (1979) Distribution of climbing fibres on cerebellar Purkinje cells in X-irradiated rats. An electrophysiological study. J Physiol 290:97–112. Crepel F, Mariani J, Delhaye-Bouchaud N (1976) Evidence for a multiple innervation of Purkinje cells by climbing fibers in the immature rat cerebellum. J Neurobiol 7:567–578. Crepel F, Mariani J (1976) Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the weaver mutant mouse. J Neurobiol 7:579 –582. Crepel F (1982) Regression of functional synapses in the immature mammalian cerebellum. Trends Neurosci 5:266 –269. de Barry J, Gombos G, Klupp T, Hamori J (1987) Alteration of mouse cerebellar circuits following methylazoxymethanol treatment during development: immunohistochemistry of GABAergic elements and electron microscopic study. J Comp Neurol 261:253–265. Eilers J, Augustine GJ, Konnerth A (1995) Subthreshold synaptic. Ca2⫹ signalling in fine dendrites and spines of cerebellar Purkinje neurons. Nature 373:155–158. Garcia-Ladona FJ, de Barry J, Girard C, Gombos G (1991) Ectopic granule cell layer in mouse cerebellum after methyl-azoxy-methanol (MAM) treatment. Exp Brain Res 86:90 –96. Hashimoto K, Ichikawa R, Takechi H, Inoue Y, Aiba A, Sakimura K, Mishina M, Hashikawa T, Konnerth A, Watanabe M, Kano M (2001A) Roles of glutamate receptor ␦ 2 subunit (GluR␦2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development. J Neurosci 21:9701–9712. Hashimoto K, Kano M (2003) Functional differentiation of multiple climbing fiber inputs during synapse elimination in the developing cerebellum. Neuron 38:785–796. Hashimoto K, Kano M (2005) Postnatal development and synapse elimination of climbing fiber to Purkinje cell projection in the cerebellum. Neurosci Res 53:221–228. Hashimoto K, Miyata M, Watanabe M, Kano M (2001B) Roles of phospholipase C␤4 in synapse elimination and plasticity in developing and mature cerebellum. Mol Neurobiol 23:69 – 82. Hashimoto K, Watanabe M, Kurihara H, Offermanns S, Jiang H, Wu Y, Jun K, Shin HS, Inoue Y, Wu D, Simon MI, Kano M (2000) Climbing fiber synapse elimination during postnatal cerebellar development requires signal transduction involving G alpha q and phospholipase C ␤4. Prog Brain Res 124:31– 48. Hirano T (2006) Cerebellar regulation mechanisms learned from studies on GluR␦2. Mol Neurobiol 33:1–16. Ichikawa R, Miyazaki T, Kano M, Hashikawa T, Tatsumi H, Sakimura K, Mishina M, Inoue Y, Watanabe M (2002) Distal extension of

climbing fiber territory and multiple innervation caused by aberrant wiring to adjacent spiny branchlets in cerebellar Purkinje cells lacking glutamate receptor ␦2. J Neurosci 22:8487– 8503. Ichise T, Kano M, Hashimoto K, Yanagihara D, Nakao K, Shigemoto R, Katsuki M, Aiba A (2000) mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination. Science 288:1832–1835. Ito M (1984) The cerebellum and neural control. New York: Raven Publishing. Kakegawa W, Miyazaki T, Emi K, Matsuda K, Kohda K, Motohashi J, Mishina M, Kawahara S, Watanabe M, Yuzaki M (2008) Differential regulation of synaptic plasticity and cerebellar motor learning by the C-terminal PDZ-binding motif of GluR␦2. J Neurosci 28: 1460 –1468. Kakizawa S, Miyazaki T, Yanagihara D, Iino M, Watanabe M, Kano M (2005) Maintenance of presynaptic function by AMPA receptormediated excitatory postsynaptic activity in adult brain. Proc Natl Acad Sci U S A 102:19180 –19185. Kakizawa S, Yamasaki M, Watanabe M, Kano M (2000) Critical period for activity-dependent synapse elimination in developing cerebellum. J Neurosci 20:4954 – 4961. Kano M, Hashimoto K, Chen C, Abeliovich A, Aiba A, Kurihara H, Watanabe M, Inoue Y, Tonegawa S (1995) Impaired synapse elimination during cerebellar development in PKC␥ mutant mice. Cell 83:1223–1231. Kano M, Hashimoto K, Kurihara H, Watanabe M, Inoue Y, Aiba A, Tonegawa S (1997) Persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking mGluR1. Neuron 18: 71–79. Kano M, Hashimoto K, Watanabe M, Kurihara H, Offermanns S, Jiang H, Wu Y, Jun K, Shin HS, Inoue Y, Simon MI, Wu D (1998) Phospholipase C ␤4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum. Proc Natl Acad Sci U S A 95:15724 –15729. Kashiwabuchi N, Ikeda K, Araki K, Hirano T, Shibuki K, Takayama C, Inoue Y, Kutsuwada T, Yagi T, Kang Y, Aizawa S, Mishina M (1995) Impairment of motor coordination, Purkinje cell synapse formation, and cerebellar long-term depression in GluR␦2 mutant mice. Cell 81:245–252. Katoh A, Yoshida T, Himeshima Y, Mishina M, Hirano T (2005) Defective control and adaptation of reflex eye movements in mutant mice deficient in either the glutamate receptor ␦2 subunit or Purkinje cells. Eur J Neurosci 21:1315–1326. Kishimoto Y, Kawahara S, Fujimichi R, Mori H, Mishina M, Kirino Y (2001a) Impairment of eyeblink conditioning in GluR␦2-mutant mice depends on the temporal overlap between conditioned and unconditioned stimuli. Eur J Neurosci 14:1515–1521. Kishimoto Y, Kawahara S, Suzuki M, Mori H, Mishina M, Kirino Y (2001b) Classical eyeblink conditioning in glutamate receptor subunit ␦2 mutant mice is impaired in the delay paradigm but not in the trace paradigm. Eur J Neurosci 13:1249 –1253. Konnerth A, Llano I, Armstrong CM (1990) Synaptic currents in cerebellar Purkinje cells. Proc Natl Acad Sci U S A 87:2662–2665. Kurihara H, Hashimoto K, Kano M, Takayama C, Sakimura K, Mishina M, Inoue Y, Watanabe M (1997) Impaired parallel fiber–Purkinje cell synapse stabilization during cerebellar development of mutant mice lacking the glutamate receptor ␦2 subunit. J Neurosci 17:9613–9623. Landsend AS, Amiry-Moghaddam M, Matsubara A, Bergersen L, Usami S, Wenthold RJ, Ottersen OP (1997) Differential localization of ␦ glutamate receptors in the rat cerebellum: coexpression with AMPA receptors in parallel fiber-spine synapses and absence from climbing fiber-spine synapses. J Neurosci 17:834 – 842. Lichtman JW, Colman H (2000) Synapse elimination and indelible memory. Neuron 25:269 –278. Lisman JE, Raghavachari S, Tsien RW (2007) The sequence of events that underlie quantal transmission at central glutamatergic synapses. Nat Rev Neurosci 8:597– 609.

K. Hashimoto et al / Neuroscience 162 (2009) 601– 611 Llano I, Marty A, Armstrong CM, Konnerth A (1991) Synaptic- and agonist-induced excitatory currents of Purkinje cells in rat cerebellar slices. J Physiol 434:183–213. Lohof AM, Delhaye-Bouchaud N, Mariani J (1996) Synapse elimination in the central nervous system: functional significance and cellular mechanisms. Rev Neurosci 7:85–101. Lomeli H, Sprengel R, Laurie DJ, Kohr G, Herb A, Seeburg PH, Wisden W (1993) The rat ␦-1 and ␦-2 subunits extend the excitatory amino acid receptor family. FEBS Lett 315:318 –322. Lovell KL, Jones MZ (1980) Partial external germinal layer regeneration in the cerebellum following methylazoxymethanol administration: effects on Purkinje cell dendritic spines. J Neuropathol Exp Neurol 39:541–548. Mandolesi G, Cesa R, Autuori E, Strata P (2009) An orphan ionotropic glutamate receptor: the ␦2 subunit. Neuroscience 158:67–77. Mariani J, Changeux JP (1980) Multiple innervation of Purkinje cells by climbing fibers in the cerebellum of the adult staggerer mutant mouse. J Neurobiol 11:41–50. Mariani J, Changeux JP (1981) Ontogenesis of olivocerebellar relationships. I. Studies by intracellular recordings of the multiple innervation of Purkinje cells by climbing fibers in the developing rat cerebellum. J Neurosci 1:696 –702. Mariani J, Crepel F, Mikoshiba K, Changeux JP, Sotelo C (1977) Anatomical, physiological and biochemical studies of the cerebellum from reeler mutant mouse. Philos Trans R Soc Lond B Biol Sci 281:1–28. Miyazaki T, Hashimoto K, Shin HS, Kano M, Watanabe M (2004) P/Q-type Ca2⫹ channel ␣1A regulates synaptic competition on developing cerebellar Purkinje cells. J Neurosci 24:1734 –1743. Morando L, Cesa R, Rasetti R, Harvey R, Strata P (2001) Role of glutamate ␦-2 receptors in activity-dependent competition between heterologous afferent fibers. Proc Natl Acad Sci U S A 98: 9954 –9959. Offermanns S, Hashimoto K, Watanabe M, Sun W, Kurihara H, Thompson RF, Inoue Y, Kano M, Simon MI (1997) Impaired motor coordination and persistent multiple climbing fiber innervation of cerebellar Purkinje cells in mice lacking G␣q. Proc Natl Acad Sci U S A 94:14089 –14094. Palay SL, Chan-Palay V (1974) Cerebellar cortex. New York: Springer-Verlag. Purves D, Lichtman JW (1980) Elimination of synapses in the developing nervous system. Science 210:153–157.

611

Roth A, Hausser M (2001) Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patchclamp recordings. J Physiol 535:445– 472. Scelfo B, Strata P (2005) Correlation between multiple climbing fibre regression and parallel fibre response development in the postnatal mouse cerebellum. Eur J Neurosci 21:971–978. Sugihara I, Bailly Y, Mariani J (2000) Olivocerebellar climbing fibers in the granuloprival cerebellum: morphological study of individual axonal projections in the X-irradiated rat. J Neurosci 20:3745–3760. Sugihara I (2005) Microzonal projection and climbing fiber remodeling in single olivocerebellar axons of newborn rats at postnatal days 4 –7. J Comp Neurol 487:93–106. Takacs J, Gombos G, Gorcs T, Becker T, de Barry J, Hamori J (1997) Distribution of metabotropic glutamate receptor type 1A in Purkinje cell dendritic spines is independent of the presence of presynaptic parallel fibers. J Neurosci Res 50:433– 442. Takayama C, Nakagawa S, Watanabe M, Mishina M, Inoue Y (1996) Developmental changes in expression and distribution of the glutamate receptor channel ␦2 subunit according to the Purkinje cell maturation. Brain Res Dev Brain Res 92:147–155. Takeuchi T, Miyazaki T, Watanabe M, Mori H, Sakimura K, Mishina M (2005) Control of synaptic connection by glutamate receptor ␦2 in the adult cerebellum. J Neurosci 25:2146 –2156. Uemura T, Kakizawa S, Yamasaki M, Sakimura K, Watanabe M, Iino M, Mishina M (2007) Regulation of long-term depression and climbing fiber territory by glutamate receptor ␦2 at parallel fiber synapses through its C-terminal domain in cerebellar Purkinje cells. J Neurosci 27:12096 –12108. Watanabe M (2008) Molecular mechanisms governing competitive synaptic wiring in cerebellar Purkinje cells. Tohoku J Exp Med 214:175–190. Woodward DJ, Hoffer BJ, Altman J (1974) Physiological and pharmacological properties of Purkinje cells in rat cerebellum degranulated by postnatal X-irradiation. J Neurobiol 5:283–304. Yuzaki M (2003) The ␦2 glutamate receptor: 10 years later. Neurosci Res 46:11–22. Yuzaki M (2004) The ␦2 glutamate receptor: a key molecule controlling synaptic plasticity and structure in Purkinje cells. Cerebellum 3:89 –93. Zhao HM, Wenthold RJ, Petralia RS (1998) Glutamate receptor targeting to synaptic populations on Purkinje cells is developmentally regulated. J Neurosci 18:5517–5528.

(Accepted 24 December 2008) (Available online 31 December 2008)