Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor

Abstract

The application of the glutamate analog L-2-amino-4-phosphonobutyric acid (L-AP4) to neurons produces a suppression of synaptic transmission. Although L-AP4 is a selective ligand at a subset of metabotropic glutamate receptors (mGluRs), the precise physiological role of the L-AP4-activated mGluRs remains primarily unknown. To provide a better understanding of the function of L-AP4 receptors, we have generated and studied knockout (KO) mice lacking the mGluR4 subtype of mGluR that displays high affinity for L-AP4. The mGluR4 mutant mice displayed normal spontaneous motor activity and were unimpaired on the bar cross test, indicating that disruption of the mGluR4 gene did not cause gross motor abnormalities, impairments of novelty-induced exploratory behaviors, or alterations in fine motor coordination. However, the mutant mice were deficient on the rotating rod motor-learning test, suggesting that mGluR4 KO mice may have an impaired ability to learn complex motor tasks. Patch-clamp and extracellular field recordings from Purkinje cells in cerebellar slices demonstrated that L-AP4 had no effect on synaptic responses in the mutant mice, whereas in the wild-type mice 100 microM L-AP4 produced a 23% depression of synaptic responses with an EC50 of 2.5 microM. An analysis of presynaptic short-term synaptic plasticity at the parallel fiber-->Purkinje cell synapse demonstrated that paired-pulse facilitation and post-tetanic potentiation were impaired in the mutant mice. In contrast, long-term depression (LTD) was not impaired. These results indicate that an important function of mGluR4 is to provide a presynaptic mechanism for maintaining synaptic efficacy during repetitive activation. The data also suggest that the presence of mGluR4 at the parallel fiber-->Purkinje cell synapse is required for maintaining normal motor function.

Fig. 3.

Behavioral analysis of wild-type +/+, heterozygous +/−, and homozygous −/− mice in the rotating rod motor-learning task. A, Motor-learning performance (rotation speed, rpm, “learnt to” criterion) on the rotating rod. The homozygous −/− mutant mice were significantly (p < 0.05) impaired, as compared with the +/+ and +/− mice from the fourteenth session onward. B, Falling latency (in seconds) of naive, untrained mice on the rotating rod at 30 rpm. No significant differences (p > 0.05, ANOVA) were seen among the +/+, +/−, and −/− mice. Values represent the mean ± SEM.

Fig. 1.

Targeted disruption of the mouse mGluR4 gene.a, Schematic representation of mGluR4 gene targeting.Hatched boxes denote the two exons contained within the cloned 8.2 kb fragment used to construct the targeting vector. The position of the 5′ probe used in Southern blot analyses is shown by anopen rectangle. The positions of the primers used in the PCR analyses are indicated by filled triangles.EV, EcoRV; H,HindIII; N, NotI;S, Sau3A; X,XbaI restriction sites. b, Southern blot analysis of the mGluR4 locus in the wild-type (+/+), heterozygous (+/−), and homozygous (−/−) KO mice. Genomic DNA was digested withEcoRV and XbaI, separated by electrophoresis, transferred to nitrocellulose, and probed with a³²P-labeled 5′ probe. Arrows indicate the 1.5 and 3.3 kb fragments of the wild-type and mutant alleles, respectively. c, Triple primer PCR differentiating allele combinations of mouse DNA. The wild-type and mutant alleles correspond to the 1068 and 1170 bp bands, respectively. The positions of 3 bands of a 1 kb ladder (Life Sciences, Hialeah, FL) are indicated in the right lane. d, Immunoblot analysis of mouse cerebellar samples using an mGluR4 polyclonal antibody. A prominent 190 kDa immunoreactive form of mGluR4 that is seen in the wild-type and heterozygous mouse cerebellum is absent in the −/− mutant mice.

Fig. 2.

Immunoblot analyses of various glutamate receptors in the cerebella of wild-type and mGluR4 KO mice. All electrophoresis samples contained 25–30 μg of total protein. Except for mGluR4, no differences between the mGluR4 KO mice and wild-type control littermates were observed. Similar results were obtained in samples from two additional wild-type and mutant mice.

Fig. 4.

Effects of l-AP4 on synaptic responses at the parallel fiber→Purkinje cell synapse in +/+ and −/− mice. Representative patch-clamp recordings from Purkinje cells showing the effects of 100 μml-AP4 on wild-type (A) and −/− mutant (B) mice.C, l-AP4 (100 μm) produced a 22.6% depression of the parallel fiber-evoked EPSC in the wild-type mice (n = 10) but no statistically significant effect in the −/− mutant mice (n = 8).D, Dose-dependent effects of l-AP4 on extracellularly recorded field EPSPs from wild-type mice (filled circles; n = 10); the EC₅₀ of l-AP4 was 2.5 μm. No significant effects of l-AP4 were observed in the −/− mice (open circles; n = 10).

Fig. 5.

Analysis of short- and long-term synaptic plasticity in cerebellar slices from wild-type (+/+) and mutant (−/−) mice. A, Paired-pulse facilitation of parallel fiber-evoked EPSCs in cerebellar Purkinje cells; at each interstimulus interval, the data from the two animals groups displayed a statistically significant difference (p < 0.05, ANOVA). B, Comparison of LTD in wild-type and knockout mice. The degree of depression in the two animal groups was not statistically significant, either when data were compared by trend analysis (repeated-measures ANOVA, p > 0.05) or when the data 30 min after the conditioning stimulus (CS) were examined (ANOVA, p > 0.05). Synaptic currents illustrated in A andB are averaged responses (n = 6) to parallel fiber stimulation in Purkinje cells of +/+ mice.

Fig. 6.

Post-tetanic potentiation (PTP) in Purkinje cells induced by repetitive stimulation of parallel fibers. A, B, Responses of Purkinje cells in wild-type (A; +/+) and mutant (B; −/−) mice to a stimulus protocol composed of a brief stimulus train (7 stimuli of 100 μsec duration at 40 Hz), followed by a single test stimulus delivered 300 msec after the burst. To assess the extent of PTP, we compared the amplitude of the synaptic current evoked by the test pulse with that of the first EPSC within the conditioning stimulus train. Synaptic currents in A andB are the averages of six responses to parallel fiber stimulation. C, Shown is the mean amplitude of responses within the conditioning burst and the test stimulus in both groups of mice. Asterisks in C indicate that a significant difference (ANOVA, p < 0.05) between cells from wild-type and −/− mutant mice was observed to the second stimulus of the conditioning train and to the PTP test pulse.D, Scatterplot of the relationship between the absolute magnitude of the synaptic current (amplitude of the first EPSC in a stimulus train) versus the amount of PTP or post-tetanic depression (PTD) observed to the test stimulus (open circles, +/+; closed circles, −/−).Dashed lines represent the best fit linear regression line for the two groups.