Enhancement of Both Long-Term Depression Induction and Optokinetic Response Adaptation in Mice Lacking Delphilin

Generation of Delphilin mutant mice

We isolated a mouse genomic clone carrying exon 2 and 3 of the Delphilin gene by screening a bacterial artificial chromosome library prepared from the C57BL/6 strain (Incyte Genomics, St. Louis, MO). The 34-bp loxP and 16-bp linker sequences were inserted into the AvrII site 93-bp upstream of exon 2, and the 1.9-kb DNA fragment carrying the 34-bp loxP sequence and Pgk-1 promoter-driven neo gene flanked by two frt sites into the SphI site 423-bp downstream of exon 3. Targeting vector pTVDEL1 contained exon 2 and 3 of the Delphilin gene flanked by loxP sequences, the 6.7-kb upstream and 2.3-kb downstream genomic sequences and 4.3-kb pMC1DTpA [23]. Homologous recombination in C57BL/6 embryonic stem cells and chimeric mouse production were carried out as described previously [19]. A chimeric mouse with the floxed Delphilin gene was mated to TLCN-Cre mice [24], [25], which were backcrossed 5 times to the C57BL/6 strain, to yield Del^+/− mice. The cre gene was bred out and heterozygous Delphilin mutant mice were crossed with each other. Resulting homozygous mutant mice (Del ^−/−) and wild-type littermates (Del ^+/+) were used as mutant and control mice, respectively. The wild-type and mutant mice of 9 to 10 weeks old were used for subsequent analyses unless otherwise specified. The genotypes of mice were determined by polymerase chain reaction using primers 5′-GCTGGGAATGCAAGTCTGTT-3′ (DelP1), 5′-TGCGACACCACCTCGTCGAA-3′ (DelP2), and 5′-CTGACTAGGGGAGGAGTAGA-3′ (NeoR). Mice were fed ad libitum with standard laboratory chow and water in standard animal cages under a 12-h light: 12-h dark cycle. All animal procedures were approved by the Animal Care and the Use Committee of Graduate School of Medicine, the University of Tokyo (Approval # 1721T062), the Local Committee for Handling Experimental Animals in the Graduate School of Science, Kyoto University (Approval # H1804-12 and H1804-13), and the Animal Care and Use Committee of Hokkaido University (Approval # 06012).

Western blot analysis

Whole homogenates were prepared from cerebella of mice at postnatal day 42 (P42) as described [26]. Western blot analysis was carried out as described [19]. Primary antibodies were guinea pig anti-Delphilin [22], rabbit anti-GluR2/3 (Upstate, Charlottesville, VA), rabbit anti-GluRδ2 [8], rabbit anti-postsynaptic density (PSD)-93 [27], rabbit anti-PTPMEG [28], rabbit anti-Synapsin I (Merck, Darmstadt, Germany) and rabbit anti-neuron specific enolase (NSE) [29]. Expression levels in the mutant mice were estimated as percentages of those in the wild-type mice using NSE as an internal standard.

Histological analyses

Histological and electron microscopic analyses were carried out as described [19], [30]. Immunoperoxidase staining was carried out using guinea pig anti-Delphilin antibody. Double immunofluorescence was carried out using rabbit anti-calbindin [31], guinea pig anti-vesicular glutamate transporter 1 (VGluT1), guinea pig anti-vesicular glutamate transporter 2 (VGluT2), and rabbit anti-vesicular γ-amino butyric acid transporter (VGAT) [32] antibodies. To count PF-PC synapses on electron micrographs, 20 electron micrographs were taken randomly for each mouse from the molecular layer in the lobule IV/V at an original magnification of ×4,000 with an H-7100 electron microscope (Hitachi High-Technologies, Tokyo, Japan). Post-embedding immunogold analysis was carried out as described [22], [30] using rabbit anti-GluRδ2 antibody or the mixture of rabbit anti-GluR1, GluR2 and GluR3 antibodies [33].

Electrophysiological analyses

Parasagittal cerebellar slices (250-µm thickness) were prepared from mice at P14-P18 unless otherwise stated. Whole-cell voltage-clamp recordings were performed on PCs in the II-VIII lobules of vermal region. A PC was whole-cell voltage-clamped with a patch pipette (2–3 MΩ) filled with the internal solution consisting of (in mM) 150 CsCl, 0.5 EGTA, 9 sucrose, 10 HEPES, 2 Mg-ATP (Sigma-Aldrich, St. Louis, MO) and 0.2 Na-GTP (Sigma-Aldrich), titrated to pH 7.3 with CsOH unless otherwise stated. The slices were continuously perfused with the oxygenated Krebs' solution containing (in mM) 124 NaCl, 1.8 KCl, 1.24 KH₂PO₄, 1.3 MgCl₂, 2.5 CaCl₂, 26 NaHCO₃ and 10 glucose with 95% O₂ and 5% CO₂ at 22–24°C. Bicuculline (20 µM, Sigma-Aldrich) was added to suppress spontaneous inhibitory postsynaptic currents. Ionic currents were recorded with an EPC-9 or an EPC-10 amplifier (HEKA Elektronik, Lambrecht, Germany), and the signal was filtered at 1.5 or 2.9 kHz and digitized at 10 kHz. The membrane potential was held at −80 mV after compensation of the liquid junction potential unless otherwise stated.

To record miniature excitatory postsynaptic currents (mEPSCs), 1 µM tetrodotoxin (Wako Pure Chemical, Osaka, Japan) was applied to prevent action potential (AP) generation. The mean amplitude of mEPSC in a particular neuron was calculated from more than 300 mEPSCs, and the mean±SEM from 20 neurons are presented. The 10–90% rise time and the half-height width were measured in 10–11 mEPSCs in a PC and averaged, and the mean±SEM among 10 PCs was calculated. The metabotropic glutamate receptor type 1 (mGluR1)-mediated slow synaptic response was induced by repetitive stimulation of PFs (50 Hz, 1–20 times) in the molecular layer in the presence of 10 µM α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (Tocris Cookson, Bristol, UK) in addition to bicuculline. The CF response was induced by applying electrical stimulation (200 µs) to the granular layer near the soma of PC prepared from P22-P24 mice voltage clamped at −20 mV. In order to estimate the number of CF innervations, the intensity of stimulation was gradually increased from 0 V to 50 V by 3–5 V, and the number of amplitude steps in EPSCs was counted. It is known that most PCs are innervated by single CF at P22-P24. The Ca²⁺ current through voltage-gated Ca²⁺ channels was recorded by applying 20 ms depolarizing voltage pulses to a PC prepared from a P5 mouse in the presence of 10 mM tetraethylammonium chloride (Sigma-Aldrich), 1 mM 4-aminopyridine (Sigma-Aldrich) and 1 µM tetrodotoxin in addition to bicuculline and NBQX. Immature PCs were used in the Ca²⁺ current measurement to obtain a better voltage- and space-clamp condition. The series resistance compensation was optimized for Ca²⁺ current recording. The resting potential and AP were also recorded under the current-clamp condition with the K-gluconate internal solution in which CsCl and CsOH were replaced with K-gluconate and KOH, respectively. The series resistance compensation was optimized. PC firing frequency was measured under the cell-attached or current-clamp condition.

To monitor LTD, test pulses (1–10 V, 200 µs) were applied to PFs in the molecular layer at 0.05 Hz, except for the period of conjunctive stimulation. The intensity of stimulus was adjusted to evoke PF-EPSC whose initial amplitude was 100–200 pA. After stable recording for at least 7.5 min, the conditioning stimulation was applied to induce LTD. The conditioning stimulation was 200 ms depolarization of a PC to −20 mV coupled with the paired PF stimuli applied at 15 and 65 ms after the onset of depolarization. This conjunctive stimulation was repeated once, twice, 5 times, 10 times or 20 times at 1 Hz. In some experiments, 10 mM EGTA was added to the internal solution. Series resistance (10–30 MΩ) and input resistance were monitored every 2.5 min by applying a +10 mV, 80 ms voltage pulse to −70 mV. The data were discarded if the series resistance changed by more than 20% or the input resistance became <100 MΩ.

OKR recordings

Eye movement recordings were performed by the video method as described [34], [35]. The sampling frequency of the image was 30 Hz. To induce OKR the screen with vertical black and white stripes (14°) that surrounds a mouse was rotated sinusoidally in light. The traces of eye velocity calculated from eye positions, and the stimulus (screen or turntable rotation) velocity were fitted with the respective sine curves by a least square method for at least successive 10 cycles except for the recording at 0.1 Hz (5 cycles or more). The gain of OKR was defined as the amplitude of fitted sine curve of eye velocity divided by that of stimulus. The negative value in phase indicates the lead of eye movement relative to the stimuli, and the positive value indicates the lag. Dynamic properties of OKR were measured twice and averaged values were used for the data analysis. To induce the adaptive change in OKR, the surrounding screen was rotated sinusoidally at 0.2 Hz, ±7.2° for 60 min each day. To prevent extinction of the learned response, the animals were kept in the dark between sessions. During the training paradigms, we made noises by clapping hands every 5 min in order to keep a mouse in an aroused state.

Motor coordination test

Naive male mice were housed individually and were handled for ∼1 min a day for 7–10 days before behavioral tests. An animal was placed in the midpoint of a thin rod (TR-3002; O'Hara, Tokyo, Japan), and given six trials with 30-min inter-trial intervals. For a rotarod test, mice were habituated to an apparatus (RRSW-3002, O'Hara) by placing them on the rod rotating at 2.5 rpm (3×2 min sessions). An animal was placed on the rod rotating at 25 rpm, and given three trials with 45- to 60-min inter-trial intervals for 4 consecutive days.

Statistical analyses

All behavioral experiments were performed in a blind fashion. Data were expressed as mean±SEM. Statistical analysis was performed using Student's t test, Mann–Whitney U test, Fisher's exact probability test or ANOVA with repeated measures as appropriate. Correlation analysis was done using Pearson's coefficient of comparison. Statistical significance was set at p<0.05.

Cerebellar structure of mutant mice lacking Delphilin

To examine the functional role of Delphilin in the cerebellum, we generated mutant mice lacking Delphilin (Fig. 1A). The Delphilin mutant mice grew and mated normally. Western blot analysis confirmed the absence of Delphilin of 135 kDa in the mutant mice (Fig. 1B). Strong immunohistochemical signals of Delphilin in the cerebellar molecular layer as well as faint signals in the thalamus attenuated in the mutant mice (Fig. 1C).

The cerebellum of the mutant mice exhibited normal foliation and laminated cortical structures (Fig. 1D). Double immunostaining for calbindin and VGluT1 revealed that PCs extended well-arborized dendrites studded with numerous spines (Fig. 1E,F), which were tightly associated with PF terminals (Fig. 1I,J). Immunostaining for VGluT2 and VGAT showed that the innervation patterns of CF and inhibitory terminals in the cerebellar molecular layer were comparable between the wild-type and mutant mice (Fig. 1G,H). In both genotypes, PC spines forming asymmetrical synapses were distributed in large numbers in the neuropil of the molecular layer (Fig. 1I,J). The cytoarchitecture and synaptic differentiation in the flocculus and paraflocculus were also indistinguishable between the wild-type and mutant mice (Fig. S1). The numbers of PF-PC synapses per 100 µm² of the neuropil area were comparable between the wild-type (20.9±1.0, mean±SEM, n = 4) and mutant mice (20.7±0.5, n = 6; Mann–Whitney U test, p>0.05). Thus, Delphilin ablation exerted little effect on cerebellar histology, PC cytology and PC synapse formation.

Expression and localization of GluRδ2

Immunoblot analyses of the whole cerebellar homogenates showed that the amounts of GluRδ2 as well as PSD-93, PTPMEG and Synapsin I were comparable between the wild-type and mutant mice (n = 3 for each; Student's t test, p>0.2 in all cases), while those of anti-GluR2/3 antibody-immunoreactive AMPA receptor proteins were slightly increased in the mutant mice (p = 0.005, Fig. 2A). Both Delphilin and GluRδ2 are selectively localized at PF-PC synapses and interact with each other [13], [22]. We thus examined the effect of Delphilin ablation on the synaptic localization of GluRδ2 by the postembedding immunogold technique. In both genotypes, immunogold labeling of GluRδ2 was concentrated at PF-PC synapses (Fig. 2B). GluRδ2-particles were hardly found at CF-PC and interneuron (IN)-PC synapses (Fig. 2C). No significant differences were detected in labeling density between the wild-type and mutant mice in each type of PC synapses (Mann–Whitney U test, p>0.4 in all cases). In the perpendicular synaptic localization, gold particles for GluRδ2 peaked at 0–8 nm bin just postsynaptic from the midpoint of the postsynaptic membrane in both mice (Fig. 2D). In the tangential synaptic localization, gold particles were deposited uniformly along the postsynaptic membrane, except for the marginal 20% (80–100% bin) that showed a slight reduction (Fig. 2E). These results suggest that Delphilin ablation exerted little effect on the synaptic localization of GluRδ2. The synaptic distribution of AMPA receptors was also examined by the postembedding immunogold technique. Gold particles representing AMPA receptors were detected on the postsynaptic membrane of PF-PC synapses in both genotypes (Fig. 2F). When quantified, the number of gold particles of AMPA receptors per profile of PF-PC synapses in the mutant mice (6.4±0.3, n = 300 from 3 mice) was significantly larger than that in the wild-type mice (4.6±0.3, n = 300 from 3 mice; Mann–Whitney U test, p<0.001).

Facilitation of LTD induction

There were no significant differences between the wild-type and mutant PCs in basal electrical properties including input resistance, resting potential, AP firing frequency, amplitude, threshold and half-height width (Table 1). The amplitude of mEPSCs was slightly larger in the mutant mice; however, the frequency and time course of mEPSCs were not significantly different between the genotypes (Table 1).

In cerebellar slices of both genotypes, 10 paired-stimulations of PFs in conjunction with PC depolarization induced robust LTD (Fig. 3A). The amplitude of LTD measured 30 min after the induction in the mutant mice (52.5±13.7%, n = 7) was comparable to that in the wild-type mice (56.0±5.0%, n = 7; Student's t test, p = 0.82). The 5 conjunctions induced weak LTD in the wild-type mice (87.5±13.9%, n = 6). In contrast, robust LTD was induced in the mutant mice (47.8±14.9%, n = 6; p = 0.02; Fig. 3B). Further, 2 conjunctions failed to induce LTD in the wild-type mice (97.3±12.1%, n = 7). However, this weak conditioning successfully induced robust LTD in the mutant mice (55.0±5.4%, n = 10; p = 0.01; Fig. 3C). The amplitudes of LTD induced by the 2-conjunction stimulation in the mutant mice were comparable to those induced by the 10-conjunction stimulation in the wild-type or mutant mice (p = 0.90 and 0.87, respectively; Fig. 3E). Just one conjunction induced weak LTD in the mutant mice (78.9±8.3%, n = 6) but not in the wild-type mice (101.5±18.8%, n = 5; p = 0.28; Fig. 3D). These results do not necessarily mean that LTD can be induced by a single or a few conjunctions of CF and PF activities in vivo, because in the present experiments Cs⁺ was introduced into a PC that should have enhanced the Ca²⁺ influx. However, they suggest that LTD is induced relatively easily in the mutant mice in vivo. The time courses of LTD development (decrease in PF-EPSC amplitude) were similar irrespective of the number of conditioning conjunctions in both the wild-type and mutant mice. Together, these results suggest that Delphilin ablation facilitated LTD induction at PF-PC synapses with little effect on the saturation level of the amplitude.

Ca²⁺ influx through voltage-gated Ca²⁺ channels, mGluR1 activation and AMPA receptor activation are required to induce LTD [36], [37]. The amplitudes of Ca²⁺ influx through voltage-gated Ca²⁺ channels for the two genotypes were similar (Table 1). A repetitive stimulation of PFs induces a slow inward current mediated by mGluR1 [38]. No significant difference was detected between the wild-type and mutant mice in terms of the amplitude and time course of the mGluR1-mediated slow synaptic response (Fig. 4A, Table 1). Cytosolic Ca²⁺ is necessary to induce LTD at PF-PC synapses [37]. When 10 mM EGTA was introduced into a PC, LTD was strongly suppressed in the wild-type mice (95.1±9.4%, n = 5) but only weakly in the mutant mice (63.1±7.2%, n = 5; Student's t test, p = 0.03; Fig. 4B). These results suggest that LTD was induced with less intracellular Ca²⁺ in the mutant mice.

CF responses were recorded from P22-P24 mice, and the number of amplitude steps of EPSCs in response to the stimulation whose intensity was gradually increased was determined. CF responses with multiple amplitudes were limited in both genotypes (wild-type, 2 of 22; mutant, 2 of 17; Fig. 5A). This indicates that multiple innervations of CFs were rare in the mutant mice as in the wild-type mice (Fisher's exact probability test, p = 0.59). The amplitudes and time course of CF-EPSCs were also similar in the two genotypes (Student's t test, p>0.6 in all cases; Fig. 5B,C).

Enhancement of OKR adaptation

To test the learning ability of Delphilin mutant mice, we examined OKR, the eye movement that follows the movements of large visual fields, and its adaptation. The cerebellar flocculus is involved in OKR adaptation, while vestibular nuclei play a central role in OKR [6]. OKR was elicited by sinusoidally oscillating a vertically striped screen surrounding an animal at 0.1–1.6 Hz, ±1.8–14.4° in light. The gain, which is the relative amplitude of eye movement against screen movement and the phase, which is the delay of eye movement from screen movement, were analyzed. The OKR gains of the wild-type and mutant mice decreased as the frequency of screen oscillation was increased at a fixed peak amplitude of 1.8° and as the angular amplitude of screen oscillation was increased at a fixed frequency of 0.4 Hz (Fig. 6A). Under these conditions, the mutant mice showed a slightly larger OKR gain than the wild-type mice (ANOVA with repeated measures, genotype effect, F_(1,25) = 6.0, p = 0.02). The OKR phase lags were less than 20° except at a frequency of 1.6 Hz (Fig. 6B). There were no significant differences in OKR phase lag between the two genotypes (genotype effect, F_(1,25) = 2.2, p = 0.15).

Continuous oscillation of the screen at 0.2 Hz, ±7.2° for 60 min induced an increase in OKR gains in both the wild-type and mutant mice (Fig. 6C,D). The adaptive increase in OKR gain in the mutant mice was significantly larger than that in the wild-type mice (genotype effect, F_(1,23) = 10.2, p = 0.004). The difference in basal OKR gain might have affected the learning process. However, there was no significant correlation between the basal OKR gain and the adaptive increase in OKR gain in each genotype (Pearson: wild-type, r = −0.30, p = 0.42; mutant, r = 0.46, p = 0.09). The adaptive decreases of OKR phase lag were similar for two genotypes (ANOVA with repeated measures, genotype effect, F_(1,23) = 0.35, p = 0.56). When the training was conducted for 5 consecutive days, the OKR gains of both the wild-type and mutant mice increased significantly with the number of training sessions (session effect, F_(9,207) = 82.1, p<0.001; Fig. 6E). On days 1 to 4, the mutant mice showed significantly larger gains than the wild-type mice (genotype effect, p = 0.008-0.04). However, the OKR gains of the wild-type mice became comparable to those of the mutant mice on day 5 (genotype effect, F_(1,23) = 0.28, p = 0.60). The OKR phase lag decreased with the number of training sessions in both genotypes (session effect, F_(9,207) = 25.8, p<0.001; Fig. 6F). There were no significant differences in phase lag between the two genotypes throughout the 5 training days (genotype effect, p = 0.08–0.64). Thus, Delphilin ablation augmented the adaptive increase in OKR gain but not the adaptive decrease in OKR phase lag.

Finally, the motor coordination of the mutant mice was examined. In the thin rod test, the retention times on a thin stationary plexiglass rod for the wild-type and mutant mice were indistinguishable (ANOVA with repeated measures, genotype effect, F_(1,29) = 0.002, p = 0.96; Fig. 7A). No significant differences in the retention time were observed on the rotating rod at 25 rpm between the two genotypes (genotype effect, F_(1,40) = 1.1, p = 0.29; Fig. 7B).