mGluR1 in cerebellar Purkinje cells is essential for the formation but not expression of associative eyeblink memory

Abstract

Classical eyeblink conditioning is a representative associative motor learning that requires both the cerebellar cortex and the deep cerebellar nucleus (DCN). Metabotropic glutamate receptor subtype 1 (mGluR1) is richly expressed in Purkinje cells (PCs) of the cerebellar cortex. Global mGluR1 knock-out (KO) mice show a significantly lower percentage of conditioned response (CR%) than wild-type mice in eyeblink conditioning, and the impaired CR% is restored by the introduction of mGluR1 in PCs. However, the specific roles of mGluR1 in major memory processes, including formation, storage and expression have not yet been defined. We thus examined the role of mGluR1 in these processes of eyeblink conditioning, using mGluR1 conditional KO (cKO) mice harboring a selective and reversible expression of mGluR1 in PCs. We have found that eyeblink memory is not latently formed in the absence of mGluR1 in adult mouse PCs. However, once acquired, eyeblink memory is expressed even after the depletion of mGluR1 in PCs. We thus conclude that mGluR1 in PCs is indispensable for the formation of eyeblink memory, while it is not required for the expression of CR.

Figure 1

Climbing fiber innervation of PCs is normal after mGluR1 depletion in adult mGluR1 cKO mice. (A) mGluR1 in mGluR1 cKO PCs is reversibly inactivated by Dox administration. (B) Simplified schematic diagram of the circuits essential for eyeblink conditioning. Most interneurons have been omitted from this diagram. (C) Immunoblotting of cerebellar proteins from Dox-treated and untreated cKO mice with anti-mGluR1 and anti-β-actin antibodies. All lanes contain 10 µg of protein. The lane at the right end contains proteins from a global mGluR1 KO mouse. (D) Immunostaining of cerebellar sagittal slices from Dox-treated and untreated cKO mice with anti-mGluR1 (red) and calbindin (green) antibodies. Scale bar: 1 mm. (E) Summary histograms showing the number of discrete steps for climbing fiber-mediated EPSCs (CF-EPSCs) recorded from mGluR1 cKO PCs before (left, 45 cells), 8 weeks after [middle, 56 cells for Dox (−) and 55 cells for Dox (+)], and 20 weeks after [right, 30 cells for Dox (−) and 57 cells for Dox (+)] Dox treatment. No significant difference was found between Dox-treated and untreated mice after 8 weeks (p = 0.110, Mann-Whitney U-test) or 20 weeks (p = 0. 839). Insets show representative traces of CF-EPSCs recorded from PCs.

Figure 2

Normal distribution of parallel fiber and climbing fiber terminals 37 weeks after the initiation of Dox administration. (A) Immunofluorescence of cerebellar parasagittal sections from untreated and Dox-treated cKO mice 37 weeks after the beginning of Dox administration. Sections were stained with antibodies against mGluR1, calbindin, VGluT1 and VGluT2. In Dox-treated cKO mice, immunoreactivity for mGluR1 was abolished, whereas that for calbindin, VGluT1, or VGluT2 was comparable to untreated mGluR1 cKO mice. Scale bar: 0.5 mm. (B) Triple immunofluorescence for calbindin (blue), VGluT1 (green) and VGluT2 (red). There was no apparent difference between untreated and Dox-treated mGluR1 cKO mice in the distribution of parallel fiber or climbing fiber terminals. Scale bars: 20 µm.

Figure 3

Associative motor memory is not formed latently without mGluR1 in PCs of adult mice. (A) Scheme of an animal implanted with electrodes for recording the electromyographic activity. (B) Typical raw EMG recordings of mGluR1 cKO mice were described under the CS-US representations. Upper EMG is an example of raw EMG before conditioning. Lower EMG is an example of raw EMG confirmed as CR after conditioning. (C) Development of CR% in mGluR1 ON/ON mice (n = 10), mGluR1 OFF/ON mice (n = 10), and mGluR1 OFF/OFF mice (n = 10). Top panel indicates experimental design for the timing and period of eyeblink conditioning in the three mouse gropes. Gray boxes indicate the timing of DOX administration. Mouse age is indicated above the schedules. In mGluR1 ON/ON mice, mGluR1 was continuously expressed. In mGluR1 OFF/ON mice, mGluR1 was not expressed in the first conditioning (days 1–7) but was restored in the second conditioning (days 57–61). In mGluR1 OFF/OFF mice, mGluR1 was not expressed during the entire experimental period. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (ON/ON vs. OFF/ON). ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 (OFF/ON vs. OFF/OFF). (D) Averaged EMG amplitudes on days 7, 57, and 61. All EMG amplitudes obtained in one session (100 trials) were summed, representing the overall response pattern. (E) The intraday CR% for three groups of mGluR1 cKO mice on days 7 and 57. On day 57 the CR% for mGluR1 OFF/ON mice was as low as that for mGluR1 OFF/OFF mice in all blocks. (F) Expression of mGluR1 in mGluR1 OFF/ON mice. Coronal sections from mGluR1 cKO mice at different time points after Dox withdrawal were stained with antibody against mGluR1. (G) Immunoblotting of cerebellar proteins from mGluR1 cKO mice (OFF/ON and ON/ON) with anti-mGluR1 and anti-β-actin antibodies. Proteins from a wild-type mouse (WT), a transgenic mouse harboring L7-mGluR1a transgene (rescue) and a global mGluR1 KO mouse (KO) were also applied. The blots cropped from different parts of a same gel were separately shown with a white space. All lanes contain 10 µg of protein. Scale bar: 1 mm. Data are represented as mean ± SEM.

Figure 4

Acquired motor memory is retrieved without mGluR1 in PCs. (A) Development of CR% in mGluR1 ON/ON mice (n = 10), mGluR1 ON/OFF mice (n = 10), and mGluR1 OFF/OFF mice (n = 10). Top panel indicates experimental design for the timing and period of eyeblink conditioning in the three mouse gropes. Gray boxes indicate the timing and period of DOX administration. Mouse age is indicated above the schedules. In mGluR1 ON/OFF mice, mGluR1 was expressed without Dox in the first conditioning (days 1–7), but the expression was blocked with Dox in the second conditioning (days 57–61). The CR% for mGluR1 ON/OFF mice was as high as those for mGluR1 ON/ON mice on day 57, even though no mGluR1 protein remained in PCs, demonstrating that mGluR1 in PCs is dispensable for the expression of once acquired eyeblink memory. Controls were mGluR1 ON/ON and mGluR1 OFF/OFF mice, and the data for these mice are identical to those shown in Fig. 3. ^*p < 0.05 (ON/ON vs. ON/OFF). ^††p < 0.01, ^†††p < 0.001 (ON/OFF vs. OFF/OFF). (B) Averaged EMG amplitudes on days 7, 57, and 61. All EMG amplitudes obtained in one session (100 trials) were summed, representing the overall response pattern. (C) The intraday CR% for mGluR1 ON/OFF mice on days 7 and 57. On day 57, the CR% for mGluR1 ON/OFF mice was as high as that for mGluR1 ON/ON mice in all blocks. Data are presented as in Fig. 3. (D) Expression of mGluR1 in mGluR1 ON/OFF mice. Coronal sections from mGluR1 cKO mice at different time points after the start of Dox administration were stained with antibody against mGluR1. (E) Immunoblotting of cerebellar proteins from mGluR1 cKO mice (ON/ON and ON/FF) with anti-mGluR1 and anti-β-actin antibodies. Proteins from a wild-type mouse (WT), a transgenic mouse harboring L7-mGluR1a transgene (rescue) and a global mGluR1 KO mouse (KO) are identical to those shown in Fig. 3G. The blots cropped from different parts of a same gel were separately shown with a white space. All lanes contain 10 µg of protein. Scale bar: 1 mm. Data are represented as mean ± SEM.