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EMBO reports 6, 1, 90–95 (2005)
doi:10.1038/sj.embor.7400312
Published online: 10 December 2004
Rescue of abnormal phenotypes of the  2 glutamate receptor-null mice by mutant 2 transgenes
Hirokazu Hirai1, 2, 3, Taisuke Miyazaki4, Wataru Kakegawa1, 5, Shinji Matsuda1, 5, Masayoshi Mishina6, Masahiko Watanabe4 & Michisuke Yuzaki1, 5
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1 Deptartment of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, Tennessee 38105, USA
2 Advanced Science Research Center, Kanazawa University, Kanazawa 920-8640, Japan
3 PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan
4 Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan
5 Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan
6 Department of Molecular Neurobiology, School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
To whom correspondence should be addressed
Michisuke Yuzaki Tel: +81 3 5363 3749; Fax: +81 3 3359 0437; E-mail: myuzaki@sc.itc.keio.ac.jp
Received 2 July 2004; Accepted 18 November 2004; Published online 10 December 2004.
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Abstract
The 2 glutamate receptor (GluR2) has a crucial role in cerebellar functions; disruption of GluR 2 alleles in mice (2-/-) impairs synapse formation and long-term depression, which is thought to underlie motor learning in the cerebellum, and consequently leads to motor discoordination. However, it has been unclear whether GluR2 is activated by glutamate analogues. Here we introduced a GluR2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino-acid-binding proteins, into 2-/- mice. Surprisingly, a mutant GluR2 transgene, as well as a wild-type GluR2 transgene, rescued all abnormal phenotypes of 2-/- mice. Therefore, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluR2 functions in 2-/- mice.
EMBO reports 6, 1, 90–95 (2005)
doi:10.1038/sj.embor.7400312
Published online: 10 December 2004
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Introduction
Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by L-glutamate, which activates postsynaptic ionotropic glutamate receptors (iGluRs):  -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors. On the basis of amino-acid sequence, the 2 glutamate receptor (GluR2) family is positioned at equal distances in a phylogenetic tree from these three receptor families (Araki et al, 1993; Lomeli et al, 1993). Although GluR2 is predominantly expressed at parallel fibre (PF)–Purkinje cell synapses in the cerebellum, it does not seem to mediate normal synaptic transmission, which is completely blocked by the AMPA receptor-specific antagonist (Kano & Kato, 1987). Instead, GluR2 has a crucial role in forming PF–Purkinje cell and climbing fibre (CF)–Purkinje cell synapses during development, and impaired synaptogenesis is associated with motor discoordination in 2-/- mice (Kurihara et al, 1997; Morando et al, 2001; Ichikawa et al, 2002). In addition to its role during development, GluR2 signalling has a unique role in modulating existing synapses in adult mice (Hirai et al, 2003).
Despite their importance, the mechanisms by which GluR2 participates in cerebellar functions have been elusive: GluR2 does not form functional glutamate-gated ion channels when they are expressed, either alone or with other iGluRs, in heterologous cells (Araki et al, 1993; Lomeli et al, 1993), and radio-ligand-binding assays failed to detect binding of GluR2 to glutamate analogues (Lomeli et al, 1993). Interestingly, the extracellular amino-terminal region of GluR2 contains a putative ligand-binding motif conserved in all mammalian iGluRs but not in metabotropic glutamate receptors (Fig 1). An arginine residue in this domain is especially conserved from the ancestral bacterial periplasmic amino-acid-binding proteins to mammalian iGluRs; X-ray crystallographic analyses demonstrated its essential interaction with the -carboxyl moieties of amino-acid ligands (Oh et al, 1993; Hsiao et al, 1996; Armstrong et al, 1998; Mayer et al, 2001; Furukawa & Gouaux, 2003). Indeed, a substitution of this arginine residue with lysine completely abolishes the ligand-binding or channel activities of iGluRs (Hirai et al, 1996; Kawamoto et al, 1997; Laube et al, 1997; Jouppila et al, 2002). Therefore, to address the question whether GluR2 is activated by glutamate analogues, we did not rely on in vitro binding or functional assays, but instead we generated mice that express a mutant GluR2 transgene in which lysine replaced the conserved arginine at position 514 (TgR/K) onto a 2-/- background. We hypothesized that the function of GluR2 is abrogated by the mutation if GluR2 is activated by glutamate or related amino acids in a conventional manner similar to that of other iGluRs.
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Figure 1
Amino-acid alignment involving the conserved arginine of iGluRs and bacterial periplasmic binding proteins. The conserved arginine residue of GluR2 aligns with that of the following: bacterial periplasmic binding proteins leucine-arginine-ornithine-binding protein (LAOBP) and glutamine-binding protein (QBP), prokaryotic glutamate receptor (pGluR0), chick and frog kainate-binding proteins (cKBP and fKBP, respectively), AMPA receptor subunits GluR1 and GluR2, kainate receptor subunits GluR5 and GluR6 and NMDA receptor subunits NR1 and NR2B. Letters are shaded according to the percentage of conserved similar amino acids at each position (100%, black shading; 80%, mid-grey; 60%, light grey). The amino-acid position of the first residue of each sequence is indicated. Critical residues in LAOBP, QBP, pGluR0, GluR2 and NR1 that interact directly with ligands are indicated by dots above the sequences (Oh et al, 1993; Hsiao et al, 1996; Armstrong et al, 1998; Mayer et al, 2001; Furukawa & Gouaux, 2003). The arrow indicates the position of the highly conserved arginine residue.
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Results
We used the Purkinje-cell-specific L7 promoter (Tomomura et al, 2001; Fig 2A) to drive the expression of a wild-type GluR2 transgene (Tgwt) and TgR/K. By breeding transgenic mice onto a 2-/- background, we obtained transgenic 'rescue' lines called 2-/-/Tgwt and 2-/-/TgR/K. Proper insertion of transgenes into the genome was confirmed by sequencing the genomic DNA from each transgenic line (supplementary Fig S1 online). We further analysed lines that expressed equivalent levels of construct-encoded proteins (Fig 2B). Immunohistochemical staining showed cerebellum-specific expression of TgR/K (Fig 2C) and Tgwt (data not shown). Although the levels of expression of transgenic GluR2 protein were about 20% of those of endogenous GluR2 protein in wild-type cerebellum (Fig 2B), 2-/-/Tgwt mice showed no ataxic gait and could walk along a straight line (Fig 2D). Surprisingly, the ataxic gait was not observed in 2-/-/TgR/K mice (Fig 2D). Results of the rotarod test confirmed the complete restoration of the motor performance of 2-/-/TgR/K mice (Fig 2E). These results indicated that TgR/K as well as Tgwt could rescue the motor discoordination of 2-/- mice.
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Figure 2
Rescue of the ataxic phenotype of 2-/- mice by GluR2 transgene expression. (A) Structure of the transgene construct. Mouse wild-type or mutant GluR2 cDNA was inserted into the BamHI site of the L7 gene in which the translational start codon was disrupted (Tomomura et al, 2001). E1–E4, exons. (B) Western blot of 2+/+, 2-/-/Tgwt and 2-/-/TgR/K cerebellar cells. Total lysates were blotted, and the blots were incubated with a polyclonal antibody against GluR2. The total amount of protein is indicated above each lane. To quantify the expression level of transgenes (lower graph), band intensities of the GluR2 protein in 10  g of 2-/-/Tgwt or 2-/-/TgR/K cerebellar cell lysates were compared with those in various amounts of wild-type cerebellar cell lysates. (C) Immunohistochemical analysis of parasagittal sections of brains from (i) 2-/-, (ii) 2-/-/TgR/K and (iii) 2+/+ (wild-type) mice. These sections were stained with anti-GluR2 antibody. (D) Footprint patterns. Tottering steps were made by the 2-/- mice, and their feet tended to sweep along as they moved. In contrast, 2-/-/TgR/K and 2-/-/Tgwt mice walked along a straight line as did 2+/+ mice. (E) Results of the rotarod task. Mice were allowed a maximum retention time of 120 s per trial. The number of mice in each group is indicated in the graph. Error bars indicate s.e.m.
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Immature Purkinje cells are innervated by multiple CFs that originate from the inferior olive of the medulla (Crepel et al, 1976). As animals grow, redundant CFs are gradually eliminated, and a relationship of one CF to one Purkinje cell is established by the end of the third postnatal week. Previous studies showed that GluR2 has an essential role in this elimination process (Kashiwabuchi et al, 1995; Hashimoto et al, 2001). To examine whether each Purkinje cell was innervated by a single CF in the 2-/-/TgR/K cerebellum, we recorded the CF-evoked excitatory postsynaptic currents (EPSCs) from postnatal day 21 (P21) to P30 Purkinje cells in slice preparations. Because a single CF has a single threshold for excitation, increasing the stimulus intensity normally elicits CF-evoked EPSCs in an all-or-none manner. Single EPSCs were elicited in about 90% of wild-type P30 Purkinje cells (Fig 3A), whereas only 40% of 2-/- Purkinje cells attained a one-to-one relationship with CFs (Hashimoto et al, 2001; Fig 3B). In contrast, 2-/-/TgR/K mice had almost the same percentage (86%; Fig 3C) of single Purkinje cell–single CF innervation as the wild-type mice, which indicates that TgR/K restored the normal process of CF synapse elimination.
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Figure 3
Restoration of impaired synapse formation of 2-/- Purkinje cells by TgR/K expression. (A–C) Electrophysiological estimation of the number of CFs innervating single Purkinje cells in 2+/+ (A), 2-/- (B) and 2-/-/TgR/K (C) cerebella. EPSCs were elicited by stimulation of CFs in the granule cell layer. The number of EPSCs induced by different stimulus thresholds was counted (inset traces). The number of Purkinje cells tested is indicated. Horizontal scale bars, 20 ms; vertical scale bars, 500 pA. (D–I) Normal distribution of CF terminals in 2-/-/Tgwt (D,E,H) and 2-/-/TgR/K (F,G,I) cerebella. Cerebellar sections were stained with antibodies to VGluT2 (red; a marker of CF terminals) and calbindin (green; a marker of Purkinje cells). The pial surface is indicated by the dotted line. (J,K) Electron micrographs of PF–Purkinje cell synapses in the molecular layer. The asterisks indicate Purkinje cell spines in contact with PF terminals. Scale bars: (D,E) 20 m; (H) 10 m; (J) 500 nm.
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Purkinje cell dendrites have two separate domains (Bravin et al, 1999): proximal regions innervated by CFs and distal regions innervated by PFs. CFs abnormally invade the 'PF domain' of distal dendrites of 2-/- Purkinje cells (Ichikawa et al, 2002). To examine the pattern of CF innervation of Purkinje cells, sections were stained with antibody to vesicular glutamate transporter 2 (VGluT2), which is predominantly expressed in CF terminals. The most distal terminal of CFs penetrated 95.1 0.4% of the molecular layer thickness in 2-/- cerebella (Ichikawa et al, 2002), 811% in 2-/-/Tgwt cerebella (Fig 3D,E) and 802% in 2-/-/TgR/K cerebella (Fig 3F,G; means.e.m. from three mice). In high-power micrographs, CF terminals were mainly associated with shaft dendrites in 2-/-/TgR/K cerebella (Fig 3H,I), whereas they terminate at distal spiny branchlets in 2-/- cerebella (Ichikawa et al, 2002). Similarly, although CFs innervating the distal dendrites of 2-/- Purkinje cells are associated with EPSCs with a slow rise time (Hashimoto et al, 2001), CF-evoked EPSCs in 2-/-/TgR/K Purkinje cells had a fast rise time (0.60.1 ms, n=12) similar to those in wild-type cells (0.60.1 ms, n=13). Therefore, we concluded that the abnormal distal CF innervation of 2-/- Purkinje cells was also rescued by TgR/K.
Previous studies revealed that about 40% of spines on 2-/- Purkinje cells are 'naked' ones that lack presynaptic contact but have postsynaptic density (PSD)-like condensations (Kurihara et al, 1997; Lalouette et al, 2001). Furthermore, the remaining PF–Purkinje cell synapses in 2-/- mice frequently show another specific abnormality—the length of the PSD does not equal that of the opposing presynaptic active zone (Lalouette et al, 2001). However, serial electron microscopic analysis (Fig 3J,K) revealed few free spines in both strains of transgenic mice (0.70.7% in 2-/-/Tgwt cerebella and 0.30.3% in 2-/-/TgR/K cerebella; 300 total spines counted in three representative mice of each line). Similarly, mismatching between the PSD and the active zone at PF synapses was rare (0.30.3% in 2-/-/Tgwt cerebella and 0.30.3% in 2-/-/TgR/K cerebella; 300 spines counted in three representative mice of each line). These results indicate that abnormal PF–Purkinje cell synaptogenesis in 2-/- cerebella was rescued by TgR/K as well as by Tgwt.
Simultaneous activation of PFs and CFs induces long-term depression (LTD) of PF–Purkinje cell transmission (Ito, 1989). CF stimulation can be replaced by direct depolarization of Purkinje cells to rule out any effect due to the CF innervation pattern. This protocol robustly induced LTD of PF-EPSCs in wild-type (Fig 4A,D) but not in 2-/- (Fig 4B,E) Purkinje cells. The amplitudes of PF-EPSCs 40 min after conjunctive stimulation (eight Purkinje cells isolated from seven wild-type mice) were 682% of those before the start of conjunctive stimulation. Similarly, the same protocol induced LTD of PF-EPSCs in 2-/-/TgR/K Purkinje cells (Fig 4C,F). The amplitudes of such EPSCs 40 min after conjunctive stimulation were 715% (seven Purkinje cells isolated from six mice) and were not significantly different from those in wild-type Purkinje cells (P>0.5; as determined by Student's t-test). Cerebellar LTD is believed to occur solely in postsynaptic Purkinje cells, probably by increased endocytosis of postsynaptic iGluRs (Matsuda et al, 2000; Wang & Linden, 2000). Therefore, TgR/K probably has a role similar to that of wild-type GluR2 in postsynaptic signalling pathways involved in synaptic plasticity at PF–Purkinje cell synapses.
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Figure 4
Expression of long-term depression in 2-/-/TgR/K Purkinje cells. Representative EPSC amplitudes over time are shown. (A) Results for eight Purkinje cells isolated from seven wild-type mice. (B) Results for four Purkinje cells isolated from two 2-/- mice. (C) Results for seven Purkinje cells isolated from six 2-/-/TgR/K mice. The means (s.e.m.) for each (wild type (D), 2-/- (E) and 2-/-/TgR/K (F)) are shown. The amplitude of the PF-EPSC was normalized to the baseline value, which was the average of 5-min responses that occurred just before conjunctive stimulation. Inset traces are EPSCs just before (1) and 30 min after (2) the conjunctive stimulation.
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Discussion
Here we demonstrated that TgR/K expression effectively rescued all principal abnormal phenotypes of 2-/- mice: motor discoordination, multiple CF innervation, impaired PF synaptogenesis and abrogated LTD. In contrast, a substitution of the corresponding arginine residue with lysine completely abolishes the ligand-binding or channel activities of other iGluRs (Hirai et al, 1996; Kawamoto et al, 1997; Laube et al, 1997; Jouppila et al, 2002). In addition, the arginine residue is essential for the binding of bacterial periplasmic proteins to amino-acid ligands, including glutamate, aspartate, glutamine, glycine, lysine, serine, arginine, ornithine and histidine (Oh et al, 1993; Hsiao et al, 1996; Armstrong et al, 1998; Mayer et al, 2001; Furukawa & Gouaux, 2003). Therefore, we speculate that GluR2 does not require glutamate-like amino acids to function in Purkinje cells.
However, we do not have direct evidence that TgR/K does not bind any glutamate analogues; it may still bind them in an unconventional way. It is also theoretically possible that, although TgR/K does not bind glutamate analogues, wild-type GluR2 does. For example, if TgR/K makes an abnormal heteromeric channel with other iGluRs, glutamate binding to these subunits may substitute for glutamate binding to wild-type GluR2 and restore the altered phenotypes of 2-/- mice. However, the binding of three or more glutamate molecules to each receptor-channel complex is required for full activation of iGluRs (Rosenmund et al, 1998; supplementary Fig S3 online). Thus, it seems unlikely that a heteromeric channel composed of TgR/K and iGluR subunits could substitute for a channel formed by wild-type GluR2. In any case, GluR2 is unique in that its conserved arginine is not essential for the rescue of the altered phenotypes of 2-/- mice.
Recently, we found that the application of an antibody specific for GluR2's extracellular N-terminal region to Purkinje cells specifically abrogated LTD by disrupting the endocytosis of AMPA receptors and caused transient cerebellar ataxia (Hirai et al, 2003). This finding indicates that GluR2 signalling could be controlled by the binding of a ligand to the extracellular domain. In addition, the role in controlling endocytosis suggests that GluR2 acts as a metabotropic receptor (Yuzaki, 2004). Interestingly, the AMPA receptor GluR2 subunit also activates a metabotropic pathway to induce dendritic spine formation in cultured hippocampal neurons (Passafaro et al, 2003), and this activity is mediated by the extracellular N-terminal domain of GluR2, which is distinct from the conventional glutamate-binding domain. Therefore, further studies using 2-/-/TgR/K mice to explore such mechanisms exploited by GluR2 are warranted.
Methods
Generation of transgenic mice. Mouse 2 complementary DNA was inserted into the BamHI site of pL7 AUG (Fig 2A). The resulting plasmid was digested with KpnI and EcoRI, and the linearized L7-2 construct was injected into fertilized eggs (Tomomura et al, 2001). Seven Tgwt and eight TgR/K founders were bred onto a 2-/- background. Homozygous transgenic lines were established and confirmed by backcrossing with wild-type mice. All procedures relating to the care and treatment of animals were carried out according to NIH guidelines, and the experimental protocol was approved by the Animal Resource Committee of St Jude Children's Research Hospital.
Immunoblotting and microscopic analysis. Whole cerebella of wild-type and transgenic mice were homogenized, and the homogenates were used for immunoblotting as described previously (Hirai et al, 2003). Adult mice were anaesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for light microscopy or with 2% paraformaldehyde plus 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for electron microscopy. Sagittal cryosections (20 m in thickness) were immunostained with anti-GluR2 antibody as described previously (Hirai et al, 2003). To visualize CF terminals under a confocal laser scanning microscope (Fluoview FV1000, Olympus, Tokyo, Japan), microslicer sections (50 m in thickness; VT1000S, Leica, Wien, Austria) were treated overnight with a mixture of calbindin antiserum (dilution, 1:10,000) and anti-VGluT2 antibody (0.5 g/ml) as described previously (Ichikawa et al, 2002). For electron microscopy (H-7100, Hitachi, Tokyo, Japan), serial ultrathin sections (70 nm in thickness; UCT ultramicrotome, Leica) were prepared as described previously (Kurihara et al, 1997). When the edge of the PSD was more than 100 nm from the corresponding active zone in any serial sections, the spine was defined as a free spine.
Electrophysiology. Parasagittal cerebellar slices (200 m) were prepared from wild-type, 2-/- and 2-/-/TgR/K mice. Whole-cell voltage-clamp recordings were made of Purkinje cells identified visually at 24°C as described (Hirai et al, 2003). Patch pipettes were pulled from borosilicate glass capillaries to achieve a resistance of 4–5 M when filled with a solution containing 140 mM cesium methanesulphonate, 2 mM Na2ATP, 0.3 mM Na2GTP, 10 mM HEPES and 0.4 mM EGTA (pH 7.3, 290 mOsm/kg). Square pulses were applied every 10 s using an isolation unit (Axon Instruments, Foster City, CA, USA) for focal stimulation.
After stable PF-EPSCs had been observed for at least 15 min, LTD was induced by conjunctive stimulation that consisted of 30 single PF stimuli together with a 50 ms depolarizing pulse from a holding potential of -70 to +20 mV. A hyperpolarizing pulse (-10 mV, 50 ms) was applied 420 ms before each PF stimulus to monitor the access resistance. If the resistance differed from the original value by more than 20%, the record was discarded. PF-EPSCs were evoked every 10 s by a glass electrode placed in the molecular layer (pulse width, 10 s; strength, 20–100 A) about 100 m away from the pial surface.
Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/v6/n1/extref/7400312-s1.pdf).
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Acknowledgements
We thank T. Torashima for technical assistance and J.C. Jones for editing the manuscript. This work was supported in part by the Uehara Memorial Foundation (H.H. and M.Y.), the National Institutes of Health grant NS36925, Cancer Center Support Core grant CA21765, the American Lebanese Syrian Associated Charities, the Naito Memorial Foundation and a Grant-in-Aid for Scientific Research provided by the Japan Society for the Promotion of Science (M.Y.).
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