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mGluR1 in Cerebellar Purkinje Cells Essential for Long-Term Depression, Synapse Elimination, and Motor Coordination

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Science  09 Jun 2000:
Vol. 288, Issue 5472, pp. 1832-1835
DOI: 10.1126/science.288.5472.1832

Abstract

Targeted deletion of metabotropic glutamate receptor–subtype 1 (mGluR1) gene can cause defects in development and function in the cerebellum. We introduced the mGluR1α transgene into mGluR1-null mutant [mGluR1 (–/–)] mice with a Purkinje cell (PC)–specific promoter. mGluR1-rescue mice showed normal cerebellar long-term depression and regression of multiple climbing fiber innervation, events significantly impaired in mGluR1 (–/–) mice. The impaired motor coordination was rescued by this transgene, in a dose-dependent manner. We propose that mGluR1 in PCs is a key molecule for normal synapse formation, synaptic plasticity, and motor control in the cerebellum.

mGluRs are G protein–coupled glutamate receptors and are implicated in modulation of synaptic transmission and plasticity (1). mGluR1 (−/−) mice have characteristic cerebellar symptoms such as ataxic gait, intention tremor, and motor discoordination (2–4). The blockade of mGluR1 by antiserum to mGluR1 results in ataxia, suggesting that mGluR1 is required for motor coordination (5). In mGluR1 (−/−) mice, the anatomy of the cerebellum, the morphology of PCs, and the synaptogenesis onto PCs from parallel fibers (PFs) are normal. However, developmental transition from multiple to mono-innervation of PCs by climbing fibers (CFs) (6), the other excitatory input to PCs (7), is impaired during the third postnatal week (8). Long-term depression (LTD) at PF-PC synapses is clearly deficient in mGluR1 (−/−) mice (3,4). Thus, mGluR1 is thought to be essential for CF synapse elimination and LTD induction, and its disruption may contribute to motor deficits of mGluR1 (−/−) mice. However, mGluR1 is expressed in various cell types in the central nervous system (CNS) other than PCs. Hence it is not clear to what extent mGluR1 in PCs contributes to these phenotypes.

We introduced a transgene (L7-mGluR1) that expressed mGluR1α under the control of the PC-specific L7 promoter (Fig. 1, A and B) into the mGluR1 (−/−) mice. One line of transgenic mice homozygous mutant for endogenous mGluR1 allele showed the cerebellum-restricted expression of the transgene (Fig. 1C) (9). (We refer to these mice as mGluR1-rescue mice.) The amount of mGluR1α protein in mGluR1-rescue cerebella was about 80-fold less than that in wild-type cerebella (Fig. 1C). mGluR1α immunoreactivity was abundant in the cerebellum, olfactory bulb, and thalamus in wild-type mice, whereas it was restricted to the cerebellum in mGluR1-rescue mice (Fig. 1E) (10). High-magnification micrographs revealed that mGluR1α was selectively localized in dendrites of PCs in mGluR1-rescue mice. mGluR1-rescue mice showed no ataxic gait or tremor and could walk along a straight line as do wild-type littermates (Fig. 1D). However, because mGluR1-rescue mice got more excited than wild-type mice when their tails were grasped, the experimenter easily recognized mGluR1-rescue mice without knowing their genotype. This hyperexcitability was similar to that of mGluR1 (−/−) mice.

Figure 1

Generation of L7-mGluR1α transgenic mice and expression of L7-mGluR1α transgene. (A) (a) Schematic structure of the transgene construct with the probe for Southern blots. Rat mGluR1α cDNA was inserted into the L7 promoter vector. Open boxes represent exons of the L7 gene (E1 to E4). (b and c) Schematic structure of the wild-type (b) and mutant (c) mGluR1 alleles. Closed and hatched boxes show coding and noncoding regions of the mGluR1 exon, respectively. Numbers signify lengths in base pairs, and the letters indicate restriction sites. D, Dra I; P, Pvu II. (B) Southern blot analysis of tail DNA. Genomic DNA was isolated from a litter obtained by breeding mGluR1 (+/−) mice with mGluR1 (+/−) (Tg/+) mice. Endogenous mGluR1 gene and transgene are indicated by the presence of a 0.8-kb and a 2.6-kb Dra I–Pvu II fragment, respectively. (C) Total proteins extracted from wild-type, mGluR1-rescue (Tg/+), and mGluR1-rescue (Tg/Tg) cerebral cortices and cerebella were blotted and probed with polyclonal antibodies to mGluR1. Lanes 1, 2, 3, 7, and 8 contained 40 μg of proteins; lanes 4, 5, and 6 contained 4, 2, and 1 μg of proteins, respectively. (D) Footprint patterns: mGluR1 (−/−) mice walked with a wide-base rolling motion from side to side. Their steps appeared to be shorter, and their feet tended to sweep along. In contrast, mGluR1-rescue mice could walk a straight line, as did their wild-type littermates. (E) Parasagittal sections stained with antibody to mGluR1α, from wild-type (a and b), mGluR1 (−/−) (c and d), and mGluR1-rescue mice (e and f). M, molecular layer; P, Purkinje cell layer; G, granule cell layer.

We asked whether CF synapse elimination could be restored by introducing the mGluR1α transgene (11). When a CF was electrically stimulated, a clearly discernible excitatory postsynaptic current (EPSC) was elicited in an all-or-none fashion in the majority of wild-type and mGluR1-rescue PCs (Fig. 2, A and E, upper traces) and in about 40% of mGluR1 (−/−) PCs (Fig. 2C, upper trace). In the remaining PCs, more than one discrete CF-EPSC was elicited at different stimulus sites or at one stimulus site with different stimulus thresholds (Fig. 2, A, C, and E, lower traces). The number of CFs innervating the recorded PC was estimated based on the number of discrete CF-EPSC steps elicited in that PC (8, 12) (Fig. 2, B, D, and F). As reported in (8), mGluR1 (−/−) mice had a significantly higher percentage of PCs with multiple CF innervation than the wild-type mice (P < 0.01, χ2test, Fig. 2, B and D). In contrast, mGluR1-rescue mice had almost the same percentage of PCs with multiple CF innervation as the wild-type mice (P > 0.05, χ2 test, Fig. 2, B and F).

Figure 2

Normal regression of multiple CF innervation in PCs of mGluR1-rescue mice. (A) Sample records of CF-EPSCs from wild-type PCs. One to three traces each were superimposed at threshold intensities. (B) Summary graph showing frequency distribution of PCs in terms of the number of discrete steps of CF-EPSCs from wild-type mice. (C and D) Data from mGluR1 (−/−) PCs. (E and F) Data from mGluR1-rescue PCs. Numbers of tested PCs: n = 77 (from five mice) for (A) and (B), n = 132 (from nine mice) for (C) and (D), and n = 144 (from nine mice) for (E) and (F).

We next determined if LTD at PF-PC synapses would be restored in mGluR1-rescue mice (13). In PCs of wild-type mice, conjunctive PF and CF stimulation (CJS) at 1 Hz for 5 min (300 stimuli) resulted in LTD of the initial slope of PF-mediated excitatory postsynaptic potentials (PF-EPSPs) (Fig. 3A), whereas the same protocol did not induce LTD in mGluR1 (−/−) mice (Fig. 3B). In contrast, LTD was induced normally in mGluR1-rescue mice (Fig. 3C).

Figure 3

Normal LTD in PCs of mGluR1-rescue mice. (A) In PCs of wild-type mice, conjunctive PF and CF stimulation (CJS) at 1 Hz for 5 min (300 stimuli) resulted in LTD of the PF-EPSP initial slopes (n = 7 from five mice). The data points represent mean ± SEM. Inset shows superimposed PF-EPSP traces recorded before conjunctive stimulation and 30 min after. (B) Data obtained from PCs of mGluR1 (−/−) mice (n = 7 from four mice). (C) Data from PCs of mGluR1-rescue mice (n = 10 from seven mice).

The cerebellum is implicated in neural mechanisms for interlimb coordination during locomotion (3, 14). We analyzed the temporal relation between the footfall of one limb and that of the other limb during treadmill locomotion (Fig. 4A) (15). In wild-type mice, phase intervals between the locomotor cycles of two limbs were sharply distributed around 180°, indicating regular alternations of step cycles between two limbs. However, in mGluR1 (−/−) mice there was wide dispersion of phase intervals, suggesting a severe impairment of interlimb coordination. In mGluR1-rescue mice, phase intervals between two limbs were distributed around 180° with a narrow dispersion, indicating that their interlimb coordination was normal. When the belt velocity of the treadmill was increased, the step cycle duration progressively decreased in wild-type and mGluR1-rescue mice (Fig. 4B). However, mGluR1 (−/−) mice did not adapt to change in the belt velocity, and step-cycle durations were different between the fore- and hindlimbs.

Figure 4

Interlimb coordination, locomotor activity; and rotating rod test. (A) Phase relationships between step cycles of the left and right forelimbs (left) and hindlimbs (right). Data for 90 steps from three mice that were walking on the treadmill at the speed of 14 cm/s. A phase interval of 0° or 360° and that of 180° corresponds to the in-phase and out-phase step cycles, respectively. Variances for both limbs of mGluR1 (−/−) mice were significantly larger than those of two other mouse strains (one-way analysis of variance). (B) The step cycle duration of forelimbs (filled columns) and hindlimbs (hatched columns) against different belt velocities. Data (mean ± SD) for 90 steps obtained from three mice. (C) Spontaneous locomotor activities in the open field. Horizontal activities in a novel environment for wild-type (n = 7), mGluR1 (−/−) (n = 7), and mGluR1-rescue mice (n = 7) were measured every 30 min over a 2-hour period in daytime with a behavioral tracing analyzer (Muromachi Kikai, Tokyo). Error bars represent SEM (in the first session: wild-type, 9602 ± 635.6 cm; mGluR1-rescue, 9914 ± 892.5 cm; P > 0.5, t test,n = 7). (D) Rotating rod task. Data represent average of five consecutive trials (maximum retention time, 120 s; rotation speed, 8 rpm). Number of mice examined:n = 10 for wild-type, mGluR1 (−/−), and mGluR1-rescue (Tg/Tg) mice and n = 8 for mGluR1-rescue (Tg/+) mice. Error bars represent SEM. *P < 0.001, t test wild-type versus mGluR1-rescue (Tg/+) mice; †P > 0.9 and ‡P > 0.1, t test wild-type versus mGluR1-rescue (Tg/Tg) mice.

mGluR1 (−/−) mice showed a reduction in total walking distance in the open field (4). In contrast, we observed no significant differences between wild-type and mGluR1-rescue mice (Fig. 4C) (16). Restoration of locomotor activity in mGluR1-rescue mice suggests that the lack of mGluR1 in brain regions other than the cerebellum did not alter the motivational state for locomotion.

To further examine motor coordination, we used the rotating rod task (17). Wild-type mice quickly learned how to keep themselves on the rod, whereas mGluR1 (−/−) mice fell off immediately once the rod began to turn (Fig. 4D) (3). During any given trial, the average retention time of mGluR1-rescue mice was shorter than that of the wild-type mice (Fig. 4D). However, when we examined the mGluR1-rescue (Tg/Tg) mice, which are homozygous for the L7-mGluR1α transgene and display gene dose-dependent increases in mGluR1α expression (Fig. 1C), they managed to stay on the rod for over 100 s by the fourth trial. There was no significant difference between retention time of wild-type and mGluR1-rescue (Tg/Tg) mice (Fig. 4D). Thus, the level of mGluR1 in PCs appears to be the determining factor for performance of motor coordination on the rotating rod task.

The gene targeting technique is a pertinent and powerful tool to examine the function of a gene in vivo. However, if the gene is expressed in various brain regions or during the course of development, and if there is no regional or temporal restriction in deletion of the gene, it is difficult to attribute the observed abnormality to the lack of the gene product in a specific brain region. Here, we returned the missing mGluR1α only into PC with a PC-specific promoter. The impaired cerebellar CF synapse elimination, deficient LTD, and motor discoordination observed in mGluR1 (−/−) mice were all restored. Our results indicate that mGluR1 in PCs is essential for these three events and suggest that mGluR1 in PC is a key molecule needed for normal development and function of the cerebellum. A rescue experiment with tissue-specific promoter is a most productive approach to specify the brain region or cell type responsible for the phenotype observed in conventional knockout mice.

  • * To whom correspondence should be addressed: E-mail: aiba{at}ims.u-tokyo.ac.jp

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