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Keywords:

  • long-term synaptic plasticity;
  • motor learning;
  • rota-rod

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

Previous studies have implicated the role of Purkinje cells in motor learning and the underlying mechanisms have also been identified in great detail during the last decades. Here we report that cyclin-dependent kinase 5 (Cdk5)/p35 in Purkinje cell also contributes to synaptic plasticity. We previously showed that p35−/− (p35 KO) mice exhibited a subtle abnormality in brain structure and impaired spatial learning and memory. Further behavioral analysis showed that p35 KO mice had a motor coordination defect, suggesting that p35, one of the activators of Cdk5, together with Cdk5 may play an important role in cerebellar motor learning. Therefore, we created Purkinje cell-specific conditional Cdk5/p35 knockout (L7-p35 cKO) mice, analyzed the cerebellar histology and Purkinje cell morphology of these mice, evaluated their performance with balance beam and rota-rod test, and performed electrophysiological recordings to assess long-term synaptic plasticity. Our analyses showed that Purkinje cell-specific deletion of Cdk5/p35 resulted in no changes in Purkinje cell morphology but severely impaired motor coordination. Furthermore, disrupted cerebellar long-term synaptic plasticity was observed at the parallel fiber-Purkinje cell synapse in L7-p35 cKO mice. These results indicate that Cdk5/p35 is required for motor learning and involved in long-term synaptic plasticity.

Purkinje cells play an important role in motor learning, the underlying mechanisms of which have been studied during the last two decades. We report here that the ablation of Cdk5/p35 in Purkinje cells impairs motor coordination, along with deficits in the cerebellar synaptic plasticity, which gives new insights into the mechanism of synaptic plasticity in Purkinje cells.

Abbreviations list
ACSF

artificial cerebrospinal fluid

Cdk

cyclin-dependent kinase

CF

climbing fiber

HRP

horseradish peroxidase

IgG

immunoglobulin G

KO

knockout

LTD

long-term depression

LTP

long-term potentiation

NMDAR

N-methyl-d-aspartate-type glutamate receptors

OCT

optimal cutting temperature

PBS

phosphate buffered saline

PFA

paraformaldehyde

PF

parallel fiber

VDCC

voltage-dependent calcium channel

WT

wild-type

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase identified by its structural homology to the classic members of the Cdk family of cell cycle regulating proteins. Cdk5 is active in post-mitotic neurons. Monomeric Cdk5 has no enzymatic activity and requires association with one of its regulatory, neuron-specific subunits, either p35 or p39, in order to function (Tsai et al. 1994; Tang et al. 1995; Takahashi et al. 2005). The Cdk5/p35 complex plays pivotal roles in neurons, including neuronal migration and differentiation, axonal elongation, intracellular trafficking and transport (Ohshima et al. 1996, 2001; Chae et al. 1997; Wu et al. 2000; Grant et al. 2001; Tanaka et al. 2001; Smith et al. 2003), and is also involved in differentiation of oligodendrocytes (Miyamoto et al. 2007; He et al. 2011). Increasing evidence suggests that Cdk5/p35 is also involved in synaptic plasticity in mature neurons (Hawasli and Bibb 2007; Lai and Ip 2009; Su and Tsai 2011). Induction of hippocampal synaptic plasticity and hippocampal-dependent learning are affected in Cdk5 and p35 transgenic and knockout mice (Plattner et al. 2008).

The cerebellum is essential for particular forms of motor learning by modulating motor commands and integrating various forms of sensory information to refine and fine-tune complex voluntary movements and reflexes. In the cerebellar cortex information conveyed by parallel fibers (PFs) and climbing fibers (CFs) converges to Purkinje cells. Long-term forms of synaptic plasticity, namely long-term depression (LTD) and long-term potentiation (LTP) at the PF -Purkinje cell synapse, have been proposed as cellular mechanisms that underlie the forms of motor learning in the cerebellum (Raymond et al. 1996; Lisberger 1998; Jörntell and Hansel 2006; Andreescu et al. 2007; Schonewille et al. 2010; Gao et al. 2012).

In this study, we employed mouse lines lacking Cdk5/p35 specifically in Purkinje cells and the results showed that the ablation of Cdk5/p35 in Purkinje cells impairs motor coordination, along with deficits in cerebellar synaptic plasticity. Our study here gives new insights into the mechanism of synaptic plasticity in Purkinje cells.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Mutant mice

Animal care was performed in accordance with guidelines outlined in the institutional Animal Care and Use Committee of Waseda University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Waseda University. Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. Mice were fed ad libitum with standard laboratory chow and water in standard animal cages under a 12 h light/dark cycle.

p35−/− mice

p35−/− (p35KO) mice were generated as described (Ohshima et al. 2001) and maintained in 129Sv × C57BL/6J (B6) background. Three-month-old p35KO male mice and littermate wild-type male mice were used for behavioral analysis.

Generation of p35-flox mice

We amplified three genomic fragments of p35 gene, a 5′-side 5.2 kb, a 1.2 kb coding region and a 3′-side 5 kb fragment by PCR from a B6 strain-derived BAC clone (RPC123.C, Clone ID 276J12, Life Technologies, Carlsbad, CA, USA). Correct amplification of these fragments were verified by DNA sequencing, and subcloned into Neo-MC/lox#5 and DT-MC#3 vectors to make targeting construct. Purified DNA of resulting construct was linearized by SfiI digestion for electroporation to ES cells using Gene Pulser (Bio-Rad, Hercules, CA, USA) (250 V, 960 μF, = ∞). We used C57BL/6N ES cell line RENKA (Mishina and Sakimura 2007) and cultured as described previously (Fukaya et al. 2006). G-418 selection (150 μg/mL) was started 36–48 h after electroporation and continued for 1 week. Recombinant clones were identified by Southern blot hybridization analysis using a 3′-flanking probe. Selected clones were subjected to Southern blot analysis using a neo probe and a 5′-flanking probe. Presence of 5′-loxP sequence was further confirmed by PCR using primers (p35 5′ loxF: 5′-GAAAACAAGATGGTTCTCAGC-3′; p35 5′ loxR: 5′-TCTGTCCAAGGTTCT-3′). Recombinant ES cells were injected into eight-cell stage embryos of ICR mice. The embryos were cultured to blastocysts and transferred to pseudopregnant ICR uteri. Resulting chimeric mice were mated to FLP transgenic mice of the B6 strain (Takeuchi et al. 2002), and male offspring were further crossed with B6 mice.

L7-cre mediated p35 conditional KO (L7-p35 cKO) mice

L7-cre mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Briefly, a targeting vector was designed to place a cre recombinase gene under the control of L7 promoter enhancer (from the L7-deltaAUG vector; all upstream ATG's mutated). Resulting vector was co-injected with green fluorescent protein (GFP) expression cassette into pronucleus (Lewis et al. 2004). Transgenic mice were generated on a Black Swiss background and were backcrossed to B6 at least 6 generations before mating with p35−/− mice to obtain p35+/−; L7-cre mice. p35+/−; L7-cre mice were then mated with p35flox/flox (p35flox) mice to obtain p35flox/−; L7-cre mice. For behavioral and electrophysiological studies, we used p35flox/flox; L7-cre mice as L7-p35 cKO mice.

Immunohistochemistry

Mice were given inhalation anesthesia using diethylether and were then perfused transcardially with 4% paraformaldehyde in phosphate buffered saline (PBS). Then, brain samples were fixed in 4% paraformaldehyde in PBS overnight. After dehydration in 20% sucrose, the samples were embedded in optimal cutting temperature compound. Cryosections were cut at 14 μm thickness and stored at −20°C until being used for immunostaining. For immunostaining, the cryosections were incubated with the primary antibodies at 4°C overnight. Then, the secondary AlexaFluor-conjugated antibodies were applied (Molecular Probe, Eugene, OR, USA). After washing with PBS, the sections were mounted with Vectashield (Vector Labs, Burlingame, CA, USA). Mouse monoclonal anti-GFP and rabbit polyclonal anti-p35/p25 antibodies were obtained from MBL (1 : 100; Nagoya, Japan) and Cell Signaling (1 : 100, #2680; Danvers, MA, USA), respectively. Mouse monoclonal anti-calbindin and NeuroTraceTM530/615 (red fluorescent Nissl stain) were obtained from Sigma (1 : 2000; St Louis, MO, USA) and Molecular Probe, respectively.

Analysis of Purkinje cell morphology by introducing Dye with Gene Gun

Introduction of DiO (3, 3′-dioctadecyloxacarbocyanine perchlorate) was conducted according to a previously described method using a Gene Gun (Kumazawa et al. 2013) into cerebellar slices from L7-p35 cKO and control (p35flox) mice. Morphometric analysis was conducted using Image J software (Schneider et al. 2012). Dendritic density, maximum length of a primary dendrite, numbers of branch points per area and dendritic area, which is defined as the area surrounded by straight lines connecting the ends of all terminal dendritic tips of a single Purkinje cell, were calculated as described (Kumazawa et al. 2013).

Western blotting analysis

Cerebella of the mice were collected from L7-p35 cKO mice and their littermate p35-flox mice as control at postnatal day 14. Western blotting analysis was conducted as previously described (Ohshima et al. 2007). Primary antibodies used in the present study were as follows; polyclonal antibody against p35 (C-19, 1 : 1000, Santa Cruz Biotechnology Inc., Dallas, TX, USA), tubulin (1 : 1000; Sigma, Tokyo, Japan). Statistical analysis was conducted by the Student's t test and Mean ± SEMis shown in the graph. < 0.05 was considered to be statistically significant.

Behavioral analyses

Rota-rod test

Three-month-old male p35 KO and wildtype control mice (n = 11 for each genotype) were used in rota-rod test. For test of p35KO mice, we used a rotarod apparatus (KN-75; Natsume Seisakujo Co. Ltd., Tokyo, Japan). 10–11-week-old L7-p35cKO (p35flox/flox; L7-cre) and wildtype control mice (n = 8, ♂ = 3, ♀ = 5 for each) were used. Motor learning was assessed with another Rota-Rod Treadmill (ENV-577/577M: Standalone model, Med. Associates Inc., St. Albans, VT, USA) consisting of a plastic rod (shaft diameter: 3.2 cm; lane width: 5.7 cm; fall height: 16.5 cm and lane divider diameter: 24.8 cm). Rota-rod test was conducted according to a protocol as described (Ohmori et al. 1999) with some modifications. Briefly, mice were placed on the rod to adapt to the environment one day before the test. In the test, each mouse was placed on the rod rotating at 16, 24 or 32 rpm. In case of p35KO mice, each mouse was placed on the rod rotating at 5 or 10 rpm. The latency to fall from the rod was recorded with a maximum score as 120 s at the first day (day 1) to the fourth day (day 4). Mice were allowed to rest for at least 5 min between each trial. Statistical analysis was conducted by Two-way repeated measures anova and Mean ± SEM is shown in the graphs. < 0.05 was considered to be statistically significant.

Balance beam test

Motor coordination was evaluated by balance beam test (Carter et al. 2003). 10-11-week-old L7-p35cKO (p35flox/flox; L7-cre) and wildtype control (n = 8, ♂ = 3, ♀ = 5 for each) mice were used. The beam consisted of a 100 cm-long horizontal steel bar (1.1 or 2.8 cm in diameter) and placed 50 cm above the floor. After habituation trials, mice were placed at the starting point of the bar. Numbers of slip off of hind limb from the beam before reaching an enclosed goal box were counted as described (Carter et al. 2003; Yoshihara et al. 2009).

Traction test

Grip strength of mutant and littermate control mice was measured by using a traction apparatus (FU-1; Muromachi Kikai Co. Ltd., Tokyo, Japan). Mouse was lifted by the tail and allowed to grasp the bar with the forepaws, or fore and hind paws. The experimenter slowly pulled the mouse back by the tail, and the maximum tension in the cable was recorded as described (Ogura et al. 2001). Statistical analysis was conducted by the Student's t test and Mean ± SEM is shown in the graph. < 0.05 was considered to be statistically significant.

Footprint analyses

Eight to twelve month-old L7-p35 cKO (p35flox/flox; L7-cre) mice and the control (p35flox) mice (n = 3 for each) were used for foot printing test. Footprints were recorded in a 30-cm-long, 15-cm-wide runway with 10 cm high walls. Hind feet of the mice were coated bilaterally with black paint. A fresh white sheet of paper was placed on the floor of the runway for each run. Before recordings, a few conditioning trials were performed. The footprint patterns were analyzed for two classical step parameters as described (Alves et al. 2011). (i) Stride length was measured as the average distance of forward movement between each stride. (ii) Hind base width was measured as the average distance between left and right hind footprints. Statistical analysis was conducted by two-tailed paired Student's t test and Mean ± SEMis shown. < 0.05 was considered to be statistically significant.

Electrophysiological study

Electrophysiological experiments were carried out using a set of L7-p35 cKO (p35flox/flox; L7-cre) and p35flox mice or a set of L7-p35 cKO and wild type (C57BL/6J) as indicated. Parasagittal cerebellar slices of 250 μm thickness were prepared according to standard procedures using a vibratome-type tissue slicer (Pro7, Dosaka EM, Kyoto, Japan). The cutting solution for slice preparation contained (in mM): 120 choline Cl, 3 KCl, 8 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose and kept at 0°C during cutting. An artificial CSF for incubation and recording contained (in mM): 124 NaCl, 2.5 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 20 glucose, and bubbled continuously with mixture of 95% O2 and 5% CO2. 100 μM picrotoxin was added to the superfusing artificial CSF during recording. Slices were kept at 24–26°C after cutting and at 31°C during recording except for the LTP experiment. Experiments were performed using whole-cell patch recording under direct visualization using an upright microscope (BX50WI; Olympus, Tokyo, Japan) and an objective lens (40× water immersion, N.A. 0.80; Olympus). Borosilicate pipettes (3–6 MΩ) were used, which were filled with a solution containing (in mM) 65 K-gluconate, 65 Cs-methanesulphonate, 10 KCl, 1 MgCl2, 4 Na2-ATP, 1 Na-GTP, 5 sucrose, 0.4 EGTA, 20 K-HEPES (pH 7.3, 295 mOsm) except for the LTP experiment set. Recordings were made with an AxoClamp 2A amplifier (Axon Instruments, Foster City, CA, USA) in the voltage-clamp mode. Electrophysiological data were recorded at a 10 kHz sampling rate with a Mac computer running custom built software TI WorkBench written by T. I. For stimulation of PFs or CFs, monopolar square pulses (200 μs) were applied through a glass pipette filled with the superfusing saline. For stimulation of PFs, stimulation electrodes were placed on the molecular layer, 100–200 μm from the Purkinje cell somata. For stimulation of CF, stimulation electrodes were placed in the granule cell layer. For the record of paired-pulse facilitation (PPF), pairs of PF stimuli of interval ranging 10–1000 ms were applied at 0.5 Hz. Five sets of series of intervals were averaged. Ten- to 12-week-old L7-p35 cKO (p35flox/flox; L7-cre) and p35flox mice were used. For the record of paired-pulse depression of CF-Purkinje cell synapse, Purkinje cells were voltage-clamped at −10 mV, and pairs of CF stimuli interval ranging 10–3000 ms were applied at 0.1 Hz. Two sets of series of intervals were averaged. Multiple-innervation of CFs onto single Purkinje cells was tested by selecting stimulation points covering enough wide area in the granule cell layer with a range of stimulation intensity and threshold levels of CF-excitatory post-synaptic currents (EPSC) were measured. For these tests of CF-Purkinje cell synapse, 12-month-old L7-p35 cKO (p35flox/flox; L7-cre) and p35-flox mice were used. For test of multiple-innervation of CFs onto single Purkinje cells, 5–12-month-old mice were used. For LTD study, peak amplitudes of the EPSC at the PF-Purkinje cell synapse (PF-EPSC) were monitored every 10 s. Membrane potential was held at −70 mV. Experiments in which the PF-EPSC amplitude was not stable during the 10 min period before LTD induction were discarded. Instability was determined if the average in any 2 min period during the 10 min period exceeded ± 5% range of the average of the 10 min. LTD was induced by pairing depolarization of Purkinje cells (from −70 to 0 mV for 200 ms) with a single PF stimulation for 240 times at 1 Hz. Ten to 12 week-old L7-p35 cKO (p35flox/flox; L7-cre) and p35flox mice were used. LTD experiments were carried out blind to the mouse genotype. For the LTP study, the same experimental procedures were used as the LTD study except for the following conditions. Three- to six-month-old L7-p35 cKO (p35flox/flox; L7-cre) and control wild type mice were used. Pipette solution for the LTP experiments contained (in mM) 130 K-gluconate, 10 KCl, 10 K-HEPES, 1 MgCl2, 4 Na2-ATP, 1 Na-GTP, 16 sucrose, 5 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (pH 7.2, 295 mOsm) [33], and the superfusing solution was kept at 24–26°C throughout the recording period. LTP was induced by 300 stimuli at 1 Hz to PFs in current-clamp mode.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Impaired motor coordination in p35 KO mice

Our previous study had shown that p35 KO mice exhibited subtle abnormalities in the laminar structure of the cerebellar cortex, in which the alignment of Purkinje cells being disrupted in some areas and a significant number of granule cells were found within the molecular cell layer (Ohshima et al. 2001). Since the Purkinje cells are considered to participate in motor coordination, we performed a rota-rod test at two different speeds (5 and 10 rpm) to test whether the p35 KO mice had a defect in motor coordination. The ability of p35 KO mice to remain on the rotating rod was significantly reduced when compared to their littermate controls at the first day to the fourth day at both speeds (Fig. 1a). The results show an impaired ability to maintain balance on the rotating rod in the p35 KO mice and reveal a pronounced deficiency in motor coordination. Then we conducted a traction test to exclude the possibility that the impaired rota-rod performance was caused by reduction of the grip strength of p35 KO mice. As shown in Fig. 1b, there was no significant difference between the performances of the two genotypes. These results indicated that the poor rota-rod performance of p35 KO mouse is not caused by the muscle weakness.

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Figure 1. Behavioral tests of p35 KO mice. (a) Rota-rod test for motor coordination. The rota-rod test was performed to assess the motor coordination of the p35−/− (p35 KO) mice. The mice were trained 3 days and the retention time was recorded (maximum was 120 s) at two different speeds (5 and 10 rpm). Comparing with the wild-type (WT) mice, the retention time of the p35 KO mice was significantly reduced at both speeds (< 0.05, n = 11). (b) Traction test for muscular strength. Muscular strength of forepaws (left) or fore and hind paws (right) was evaluated. Maximum grasp force was recorded. The results showed that there was no significant difference between p35 KO mice and their littermate wild-type (WT) mice (= 0.90 for forepaws; = 0.92 for fore and hind paws, n = 7).

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Generation of Purkinje cell-specific p35 conditional knockout mouse

To exclude the possibility that structural abnormality caused the impaired performance on the rota-rod test, we generated L7-p35 cKO mice by crossing p35-flox mice (Fig. 2a and b) and L7-cre mice. In L7-cre mice, the cre recombinase gene is under the control of the L7 promoter/enhancer, restricting the expression of cre recombinase to Purkinje cells (Oberdick et al. 1990). In L7-p35cKO mice, p35 was thus ablated specifically in Purkinje cells. Western blotting analyses were performed to confirm the excision of the p35 gene from Purkinje cells (Fig. 2c). The protein level of p35 was significantly reduced in the cerebellar homogenates of L7-p35cKO mice as compared with control mice (Fig. 2c).

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Figure 2. Generation of L7-p35 cKO mice. (a) Schematic diagram of the wild-type (WT) p35 allele, the p35 gene-targeting vector, and the predicted p35 gene-targeted allele. Cre-lox and Flp-frt systems were applied in the construct. The neomycin resistant (neor) gene flanked with 2 frt motifs (in the same orientation) was located downstream of the p35 coding region. Two loxp motifs were inserted, the first was upstream of the p35 coding region, and the second was downstream of the second frt motif, which was inserted in the p35 allele. The neor gene was used as a positive selection marker, and DT-A (the diphtheria toxin A gene) was used for negative selection against random insertion. (b) Southern blot analysis of p35-flox ES clone D5, its derivative D5′, and DNA from a B6 mouse (B6). Recombinant clones such as D5 were identified by Southern blot using a 3′-flanking probe. The selected clone D5 was expanded and named the D5′ clone. DNA from D5′ ES cells and B6 mouse was analyzed using the Neo probe and a 5′-flanking probe. (c) Protein level of p35 was examined using cerebellar homogenates from L7-p35 cKO and the littermate control mice at postnatal day 14. Blots were incubated with anti-p35 antibody and reblotted with anti-β-tubulin antibody. In L7-p35 cKO mice, distinct reduction in p35/tubulin ratio was observed compared with the control mice. *p < 0.05.

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Histology of L7-p35 cKO mice

Nissl staining of cerebellar sections showed that L7-p35 cKO mice did not exhibit any gross histological alterations in cerebellum (data not shown). Immunohistochemisty performed with anti-p35 antibody showed that the signals in the Purkinje cell layer and the molecular layer were reduced in the L7-p35 cKO mice as compared to the control mice (Fig. 3a and b), confirming that p35 was successfully eliminated in the Purkinje cells in the L7-p35 cKO mice. GFP immunostaining demonstrated normal alignment of Purkinje cells in the Purkinje cell layer (Fig. 3a). No alteration in the density of Purkinje cells were confirmed by immunostaining with anti-calbindin antibody (data not shown). Cdk5/p35 is implicated in axonal elongation (Paglini et al. 1998). Purkinje cells extend their axons to neurons in deep cerebellar nuclei. We found axonal terminals of Purkinje cells around soma of these cells in L7-p35 cKO mice are comparable to those in controls (Figure S1). This result indicates no detectable deficits in axonal elongation of p35-null Purkinje cells. In addition, we examined the dendritic structure of Purkinje cells by biolistic dye delivery with Gene-gun (Fig. 3c). With this method we recently reported sparse dendritic trees of Purkinje cells in p35 KO mice (Kumazawa et al. 2013). We conducted the morphometric analysis of dendrite, including dendrite density, maximum length of primary dendrite, dendrite area of Purkinje cells, and number of branching points/area as described (Kumazawa et al. 2013). We found no difference in any of these parameters between L7-p35 cKO and control p35-flox mice (Fig. 3d).

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Figure 3. Histology of Purkinje cell-specific conditional KO mice. (a) Immunohistological study with the cryosections from L7-p35 cKO and the littermate control mice. Anti-GFP and anti-p35/25 antibodies were used together with DAPI. In L7-p35 cKO mice. (b) Fluorescence signals of p35 in the molecular layer and in the Purkinje cell layer in L7-p35 cKO mice were weaker than those in control mice. (c) Representative images of single Purkinje cells from the control and L7-p35 cKO mice stained by biolistic delivery of DiO into acute cerebellar slices with a Gene-gun. (d) Morphometric analysis of dendrite was conducted, which included dendrite density, maximum length of primary dendrites, dendritic area of Purkinje cell and branching point number/area of dendrites analyses. No difference was found in all these parameters between L7-p35 cKO and control mice. Scale bars, 50 μm. *p < 0.05.

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Impaired motor coordination in L7-p35 cKO mice

To analyze alterations in behavior concomitant with the loss of p35 in Purkinje cells, we first performed footprint analysis to observe the gait. There were no significant differences in stride length and hind base width between L7-p35cKO and control (p35flox) mice; Stride length: control, 5.90 ± 1.31 cm, L7-p35cKO, 5.40 ± 1.16 cm, = 0.117; hind-base width: control, 2.58 ± 0.56 cm, L7-p35cKO, 2.47 ± 0.44 cm, = 0.532, (n = 3). We then performed a balance beam test and a rota-rod test with L7-p35cKO mice at three different speeds (16, 24, and 32 rpm). In the balance beam test, we measured the number of paw slips that occur before reaching an enclosed goal box. L7-p35cKO mice have increased number of slips from balance beams of the both diameters (Fig. 4a). In the rota-rod test, L7-p35cKO mice could not keep on the rota-rod as well as the control mice did at three different speeds (Fig. 4b). These results suggest a motor coordination deficiency, impaired ability to maintain balance or posture, but no gait disturbances, in the L7-p35cKO mice even in the absence of histological abnormalities. These experiments apparently show that the Cdk5 activity in the Purkinje cell is required for proper motor coordination.

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Figure 4. Impaired motor coordination in Purkinje cell-specific transgenic mouse lines. (a) Balance beam test of the L7-p35 cKO mice and control mice (n = 7). Increased numbers of paw slips were observed in L7-p35 cKO mice at both diameters (p < 0.01 in 11 mm diameter, p < 0.05 in 28 mm diameter). (b) Rota-rod test of the L7-p35 cKO and control mice (n = 7). The retention time was recorded (120 s at the maximun) at three different speeds (16, 24 and 32 rpm). Comparing with control mice, retention time of the L7-p35 cKO mice was significantly shorter at the three different speeds (p < 0.01 at 16 rpm, p < 0.01 at 24 rpm, p < 0.001 at 32 rpm). *p < 0.05; **p < 0.01.

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Impaired long-term synaptic plasticity in L7-p35 cKO mice

Since L7 p35 cKO mice exhibited a significant reduction in the motor coordination ability, we assumed that Cdk5/p35 may be involved in the synaptic plasticity in the cerebellar cortex. Purkinje cells receive excitatory inputs from PFs, axons of granule cells, as well as from CFs. We examined whether the loss of p35 in Purkinje cells alters properties in the PF-Purkinje cell synapse. There was no apparent difference between L7-p35 cKO and control (p35flox) mice in the relationship between PF stimulus intensity and EPSC amplitudes (I-O relationship, Fig. 5a). PPF (a short-term form of synaptic plasticity) at this synapse was not altered in the L7-p35 cKO mice (n = 5 for control (p35flox) and n = 7 for L7-p35 cKO, Fig. 5b). We also observed no changes in paired-pulse depression at the CF-Purkinje cell synapse (n = 8 for control (p35flox) and n = 6 for L7-p35 cKO, Fig. 5c) and no multiple CF innervation in either genotype (Figure S2, n = 8 for control (p35flox) and n = 6 for L7-p35 cKO). These observations suggest that there are no detectable deficits in the presynaptic properties of PF- and CF-Purkinje cell synapses by the loss of p35 activity in the postsynaptic Purkinje cell. We next tested whether long-term forms of synaptic plasticity are altered by the loss of p35 activity. Control (p35flox) Purkinje cells showed depressed PF-EPSC amplitudes after the LTD inducing stimuli, i.e., conjunctive depolarization and PF stimuli for 240 times at 1 Hz, while those from L7-p35 cKO mice did not show apparent LTD (n = 10 and 11, respectively, p < 0.05, Two-way repeated measures anova, Fig. 6a). We also tested an LTP-induction protocol (300 PF stimuli at 1 Hz). Though L7-p35 cKO Purkinje cells showed potentiated PF-EPSCs after the induction period, the amplitude of potentiation was significantly smaller than that in control Purkinje cells (n = 10 and n = 12, respectively, p < 0.05, Two-way repeated measures anova, Fig. 6b). These results indicate that p35 is involved in the mechanisms for long-term synaptic plasticity at the PF-Purkinje cell synapse.

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Figure 5. Normal basal synaptic functions in Purkinje cell of L7-p35 cKO mice. (a) Input-output relation of the parallel fiber (PF)-Purkinje cell synapse recorded from L7-p35 cKO and control (wild type) mice. Top traces show typical ESPC time courses evoked by single stimuli (0, 2, 4, 6, 8 and 10 μA, respectively, left: control, right: p35cKO) after averaging five consecutive traces. Bottom graph shows averaged results from L7-p35 cKO (n = 6) and control (n = 5) Purkinje cells. No difference was found in the I-O relationship between two groups. Scale bars: 20 ms and 200 pA. (b) Paired-pulse facilitation (PPF) at the PF-Purkinje cell synapse was observed in Purkinje cells from control (p35flox, n = 5) and L7-p35 cKO (n = 7) mice as well. Upper traces show excitatory post-synaptic currents (EPSCs) averaged from five consecutive records in a control (left) and a p35cKO (right) Purkinje cell, respectively. Scale bars: 20 ms and 200 pA. (c) Paired-pulse depression (PPD) at the climbing fiber (CF)-Purkinje cell synapse was observed in Purkinje cells from control (p35flox, n = 8) and L7-p35 cKO (n = 6) mice as well. Upper traces show EPSCs averaged from five consecutive records in a control (left) and a p35cKO (right) Purkinje cell, respectively. Scale bars: 20 ms and 500 pA.

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Figure 6. Impaired long-term synaptic plasticity in Purkinje cells of L7-p35 cKO mice. (a) Purkinje cells from control mice (p35flox) showed depressed parallel fiber (PF)-evoked excitatory post synaptic currents (EPSCs) after conjunctive stimuli (n = 10), i.e., long-term depression (LTD), while those from L7-p35 cKO mice (p35cKO) did not show apparent LTD (< 0.05, n = 11. Two-way repeated measures anova). Upper traces show EPSCs averaged from five consecutive records before and 30 min after the LTD induction in a control (left) and a p35cKO (right) Purkinje cell, respectively. Scale bars: 10 msec and100 pA. (b) Purkinje cells both from control (wild type) and L7-p35 cKO mice showed long-lasting potentiated PF-EPSCs (long-term potentiation; LTP) after 300 PF-stimuli at 1 Hz. However the amplitude of potentiation in L7-p35 cKO Purkinje cells was significantly smaller than that of the control (n = 10 and 12, respectively, p < 0.05, Two-way repeated measures anova). Upper traces show EPSCs averaged from five consecutive records before and 30 min after the LTP induction in a control (left) and a p35cKO (right) Purkinje cell, respectively. Scale bars: 10 ms and 100 pA.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

The involvement of the cerebellum in motor learning has long been recongnized, but its exact role remains unclear (Welsh et al. 1995; Raymond et al. 1996; Mauk 1997). Synaptic plasticity in the Purkinje cell was postulated to represent the underlying cellular changes that lead to motor learning (Ito 1989; Linden 1994). Purkinje cells exhibit two patterns of firing activity; these are complex spikes elicited by CFs from olivary neurons, and simple spikes evoked by summation of PF inputs from cerebellar granule cells. Long-term synaptic plasticity at the PF-Purkinje cell synapse in particular remains a widely accepted vertebrate model for the cellular mechanism that underlies synaptic changes during motor learning and memories in the cerebellum (Ito 1989; Linden 1994; Mauk 1997; Lisberger 1998; Coesmans et al. 2004; Belmeguenai and Hansel 2005; Jörntell and Hansel 2006; Schonewille et al. 2010; Gao et al. 2012).

The Cdk5/p35 complex modulates cortical lamination and morphology during development through phosphorylation and interacts with many substrates in the mature nervous system (Dhavan and Tsai 2001). Subsequently, lines of evidence indicated that Cdk5/p35 is involved in both functional and structural aspects of plasticity (Hawasli and Bibb 2007; Lai and Ip 2009; Su and Tsai 2011), i.e., the function of Cdk5/p35 appears critical in modulating synaptic plasticity and motor learning both presynaptically and postsynaptically. At the presynapse Cdk5 functions as a key player for the modulation of neurotransmitter release. Cdk5 modulates dephosphins, dynamin I, amphiphysin I, and PIPKIγ as well as glycogen synthase kinase 3 to affect synaptic vesicle retrieval and priming (Barnett and Bibb 2011). At the postsynapse Cdk5 interacts with molecules controlling cytoskeletal architecture and receptor density, including NR2B, PSD-95, Ca2+/calmodulin-dependent protein kinase (CaMKII), tyrosine phosphatase STEP and DARPP-32 (Barnett and Bibb 2011; Lamont and Weber 2012). These Cdk5/p35 functions have been studied in excitatory synapses onto hippocampal neurons using p35KO (Ohshima et al. 2005) and Cdk5 conditional KO mice (Hawasli et al. 2007, 2009; Guan et al. 2011).

In the present study, we evaluated the effects of Cdk5 and p35 on motor coordination and cerebellar synaptic plasticity. p35 KO mice demonstrated significant impairment in the rota-rod test. The normal result of the traction test indicates that the defective motor coordination in p35 KO mice is not a result of muscular weakness (Fig. 1). Since juvenile mice lacking p35 were reported to display hyperactivity due to a low dopamine turnover (Drerup et al. 2010; Krapacher et al. 2010), hyperactivity could account for the poor rota-rod performance. However, adult p35KO mice we used for the rota-rod test showed normal locomotive activity (Ohshima et al. 2005). To exclude a possibility of contribution of the subtle abnormality in the brain structure to the defective motor coordination, we confined the deletion of p35 to the Purkinje cells. L7-p35 cKO mice, in which p35 was ablated specifically in Purkinje cells, exhibited impaired motor coordination (Fig. 4), similarly to the p35KO mice (Fig. 1), but without detectable histological changes. For p35KO mice, a rota-rod test was conducted as a part of the behavioral test battery in 129SV × B6 hybrid background (Ohshima et al. 2005) in a different apparatus model. In addition to its strain difference, p35KO mice may have severe motor coordination defect because p35 was ablated in other parts of brain including basal ganglia. These experiments were followed by electrophysiological analyses. LTD and LTP at the PF-Purkinje cell synapse were impaired in the L7-p35 cKO mice (Fig. 6). The LTD and LTP observed in this study were elicited by protocols which induce postsynaptically expressed LTD and LTP, respectively (Gao et al. 2012). It has been proposed that postsynaptic LTP at the PF-Purkinje cell synapse contributes to the motor learning (Schonewille et al. 2010, 2011). The defects in LTP and motor coordination observed in this study agree with this proposal. We did not find any functional presynaptic alterations in the L7-p35 cKO Purkinje cells, i.e. synaptic strength and PPF at the PF-Purkinje cell synapse and paired-pulse depression and mono-innervation at the CF-Purkinje cell synapse (Fig. 5). Because we deleted the p35 gene specifically in Purkinje cells, these observations do not necessarily mean that p35 does not play roles in the presynaptic sites, i.e. PF and CF terminals.

Since the elimination of the p35 gene is confined to the Purkinje cell in L7-p35 cKO mice, this mouse model is a valuable tool for studying the function of Cdk5/p35 in the postsynaptic compartment. A prominent component of this key structure is the scaffold protein PSD-95, which structurally links N-methyl-d-aspartate-type glutamate receptors (NMDAR) to the cytoskeleton and signaling molecules. It is suggested that Cdk5-dependent phosphorylation of PSD-95 dynamically regulates the clustering of PSD-95/ NMDAR at synapses, thus modulating the molecular architecture of the postsynaptic terminal (Morabito et al. 2004). CaMKII is another constituent of the postsynaptic density involved in the synaptic remodeling and plasticity in the PF-Purkinje cell synapse (Hansel et al. 2006; van Woerden et al. 2009) as well as in wide range of synapses (Lisman et al. 2012). Cdk5 and its cofactors interact with α subunit of CaMKII within the postsynaptic density in a calcium-dependent manner and may regulate the activity of this kinase (Dhavan et al. 2002; Hosokawa et al. 2002).

Studies have shown that changes in intracellular calcium levels through calcium channel activities regulate induction of LTD (Hirano 1990; Sakurai 1990; Crepel and Jaillard 1991) and LTP (Coesmans et al. 2004; Ly et al. 2013) at the PF-Purkinje cell synapse. Among the voltage-dependent calcium channels (VDCCs) the P/Q-type calcium channels, which are the major VDCC subtype found in Purkinje cell, play a key role in the function of the motor circuitry as evidenced by disrupted motor phenotypes observed in P/Q channel mutants (Dove et al. 1998; Jun et al. 1999; Katoh et al. 2007; Hashimoto et al. 2011). Cdk5 phosphorylates P/Q-type VDCCs at the intracellular loop that connects domains II and III between amino acid residues 724 and 981 (Tomizawa et al. 2002). The L-type VDCC (L-VDCC), another type of VDCCs expressed in Purkinje cells, is also phosphorylated by Cdk5 (Wei et al. 2005). The decreased activity of Cdk5 at the postsynaptic side in the L7-Cre p35 cKO mice could affect the reduced LTD and LTP amplitudes through regulation of the P/Q- and/or L-VDCCs.

Inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is an intracellular calcium channel that mediates calcium release from intracellular stores, namely endoplasmic reticulum. Through a number of specific methods to inhibit IP3R1 function, it was found that IP3R1 is necessary for induction of LTD (Inoue et al. 1998) and motor learning (Ogura et al. 2001). The fact that IP3R1 can be phosphorylated by Cdc2 and Cdk5 (Malathi et al. 2003; T. Ohshima and K. Mikoshiba, unpublished data), together with the deficit in LTD induction in the L7-Cre p35 cKO Purkinje cells (Fig. 6a), raises a possibility that the regulation of IP3R1 by Cdk5 through phosphorylation plays a role in LTD at the PF-Purkinje cell synapse.

Calcium influx elicited by the activation of NMDAR plays a critical role in synaptic plasticity. Although Purkinje cells are thought to express relatively low amount of functional NMDAR (Audinat et al. 1990; Kakegawa et al. 2003), the functional role of NMDAR has been implicated at the postsynapse of the CF-Purkinje cell synapse (Renzi et al. 2007; Piochon et al. 2010). And the possibility of participation of NMDAR at the postsynapse of PF-Purkinje cell synapse in induction of presynaptically expressed LTD is remained to be elucidated (Qiu and Knöpfel 2009). It is reported that Cdk5 associates with and phosphorylates NR2A subunits at Ser-1232 (Li et al. 2001). This raises a possibility that the regulation of NMDA receptors through phosphorylation by Cdk5 is involved in the synaptic plasticity in the Purkinje cell.

The present study suggests that Cdk5/p35 is critical for synaptic plasticity and for motor learning in the cerebellum. Further studies are required to determine the precise mechanism involved in the effects of the lack of Cdk5/p35-mediated phosphorylation on synaptic plasticity that mediates higher brain functions.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

This work was supported by Grants-in-Aid from The Ministry of Education, Culture, Sports, Science and Technology (MEXT). The authors have no conflicts of interest to declare.

All experiments were conducted in compliance with the ARRIVE guidelines.

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  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12756-sup-0001-FigS1-S2.pdfapplication/PDF160K

Figure S1. Comparative axonal terminals of Purkinje cells in soma of neurons in deep cerebellar nuclei in L7-p35 cKO mice.

Figure S2. Single CF innervation in Purkinje cells of L7-p35 cKO mice.

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