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. 2013 Jun 3;591(Pt 16):3919–3933. doi: 10.1113/jphysiol.2012.244947

The du2J mouse model of ataxia and absence epilepsy has deficient cannabinoid CB1 receptor-mediated signalling

Xiaowei Wang 1, Benjamin J Whalley 1, Gary J Stephens 1
PMCID: PMC3764637  PMID: 23732642

Abstract

Cerebellar ataxias are a group of progressive, debilitating diseases often associated with abnormal Purkinje cell (PC) firing and/or degeneration. Many animal models of cerebellar ataxia display abnormalities in Ca2+ channel function. The ‘ducky’du2J mouse model of ataxia and absence epilepsy represents a clean knock-out of the auxiliary Ca2+ channel subunit α2δ-2, and has been associated with deficient Ca2+ channel function in the cerebellar cortex. Here, we investigate effects of du2J mutation on PC layer (PCL) and granule cell layer (GCL) neuronal spiking activity and, also, inhibitory neurotransmission at interneurone–Purkinje cell (IN-PC) synapses. Increased neuronal firing irregularity was seen in the PCL and, to a less marked extent, in the GCL in du2J/du2J, but not +/du2J, mice; these data suggest that the ataxic phenotype is associated with lack of precision of PC firing, that may also impinge on GC activity and requires expression of two du2J alleles to manifest fully. The du2J mutation had no clear effect on spontaneous inhibitory postsynaptic current (sIPSC) frequency at IN-PC synapses, but was associated with increased sIPSC amplitudes. du2J mutation ablated cannabinoid CB1 receptor (CB1R)-mediated modulation of spontaneous neuronal spike firing and CB1R-mediated presynaptic inhibition of synaptic transmission at IN-PC synapses in both +/du2J and du2J/du2J mutants, effects that occurred in the absence of changes in CB1R expression. These results demonstrate that the du2J ataxia model is associated with deficient CB1R signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity and the ataxic phenotype.

Key points

  • Cerebellar ataxias are progressive debilitating diseases with no known treatment and are associated with defective motor function and, in particular, abnormalities to Purkinje cells.

  • Mutant mice with deficits in Ca2+ channel auxiliary α2δ-2 subunits are used as models of cerebellar ataxia.

  • Our data in the du2J mouse model shows an association between the ataxic phenotype exhibited by homozygous du2J/du2J mice and increased irregularity of Purkinje cell firing.

  • We show that both heterozygous +/du2J and homozygous du2J/du2J mice completely lack the strong presynaptic modulation of neuronal firing by cannabinoid CB1 receptors which is exhibited by litter-matched control mice.

  • These results show that the du2J ataxia model is associated with deficits in CB1 receptor signalling in the cerebellar cortex, putatively linked with compromised Ca2+ channel activity due to reduced α2δ-2 subunit expression. Knowledge of such deficits may help design therapeutic agents to combat ataxias.

Introduction

Cerebellar ataxias comprise a group of progressive diseases associated with motor incoordination and are typically associated with dysfunction and/or degeneration of PCs, which represent the sole efferent output of the cerebellar cortex. A number of mutant mouse models exhibit specific ataxias with diverse behavioural phenotypes at different developmental stages (Green, 1981; Grüsser-Cornehls & Baurle, 2001), including the du2J mutation that exhibits behavioural traits consistent with cerebellar ataxia and absence epilepsy. du2J mice have mutations in the Cacna2d2 gene which encodes the α2δ-2 auxiliary Ca2+ channel subunit (Donato et al. 2006), one of four α2δ subunit isoforms (α2δ-1–4) that exert auxiliary effects on Ca2+ channel biophysical properties and physiological function (Gao et al. 2000; Hobom et al. 2000; Klugbauer et al. 2003; Bauer et al. 2010; Dolphin, 2012; Hoppa et al. 2012). du2J mice are part of a group of mutant mouse strains together with either spontaneous (Cacna2d2entla and Cacna2d2du alleles) or targeted (Cacna2d2tm1NCIF) α2δ-2 disruptions, all of which typically exhibit smaller than normal size, comparable ataxia phenotypes, absence seizures and paroxysmal dyskinesia (Barclay et al. 2001; Brodbeck et al. 2002; Inanov et al. 2004; Brill et al. 2004; Donato et al. 2006; Walter et al. 2006). The Cacna2d2entla allele predicts a full-length protein with an inserted region in the α2 moiety of α2δ-2 and is associated with reduced PC Ca2+ currents (Brill et al. 2004). The Cacna2d2du allele disrupts Cacna2d2 in intron 3, yielding a truncated α2δ-2 protein and resulting in reduced native and recombinant CaV2.1 Ca2+ channel expression (Barclay et al. 2001; Brodbeck et al. 2002). The Cacna2d2du2J allele used here has a 2 bp deletion in exon 9 of Cacna2d2 resulting in complete ablation of α2δ-2 expression and reduced PC Ca2+ currents (Donato et al. 2006). In du mutant mice, a reduction in Ca2+ influx, leading to compromised Ca2+-dependent K+ channel (SK) activity and irregular pacemaking, was proposed to underlie the ataxic phenotype (Walter et al. 2006); similarly, the du2J mutation exhibits increased PC firing irregularity, although this could not be normalised using SK blockers (Donato et al. 2006).

Here, we extend previous studies to examine the effect of du2J mutation on basal neuronal network activity and synaptic transmission and, further, on G protein-coupled receptor (GPCR)-mediated presynaptic inhibition of synaptic transmission in the cerebellum. In particular, CB1 GPCRs are strongly expressed in the cerebellar cortex, where they modulate GABA transmission at IN-PC synapses to modulate PC total output (Ma et al. 2008; Wang et al. 2011). We demonstrate that the du2J phenotype exhibits deficient CB1R signalling at the neuronal network level that reflects, at least in part, ablation of CB1R modulation of inhibitory neurotransmission at IN-PC synapses, but which does not result from reduced CB1R expression. These results suggest that α2δ-2 deficits in du2J mutants affect GPCR-mediated modulation of inhibitory transmission in the cerebellar cortex, with consequential effects upon PC spike firing activity; such deficits may be associated with ataxic phenotypes and, potentially, contribute to disease.

Methods

Ethical approval

All work was subject to Local Ethical Research Panel approval and was conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986; every effort was made to minimise pain and discomfort experienced by animals.

Electrophysiology

Preparation of acute cerebellar slices

Breeding pairs of +/du2J mice (C57Bl/6 background) were originally supplied by Professor Annette Dolphin (University College London, UK) from which progeny were bred in-house at the University of Reading and whose genetic classification was determined by the Sequencing & Genotyping Facility, University College London from ear-notch tissue samples. Acute cerebellar brain slices were prepared from 3- to 5-week-old male mice as previously described (Ma et al. 2008). Briefly, animals were killed by a Schedule 1 method followed by immediate decapitation. The brain was then rapidly removed and submerged in cold, sucrose-based aCSF solution (sucrose 218 mm, KCl 3 mm, NaHCO3 26 mm, NaH2PO4 2.5 mm, MgSO4 2 mm, CaCl2 2 mm and d-glucose 10 mm) and 300 μm thick parasagittal cerebellar slices were prepared using a Vibroslice 725M (Campden Instruments Ltd, UK) or a Vibratome (R. & L. Slaughter, Upminster, UK). Slices were maintained under carboxygenated (95% O2–5% CO2), standard aCSF (NaCl 124 mm, KCl 3 mm, NaHCO3 26 mm, NaH2PO4 2.5 mm, MgSO4 2 mm, CaCl2 2 mm and d-glucose 10 mm) at 37°C for <1 h before being returned to room temperature (22–24°C). Recordings were made at 22–24°C, 2–8 h following slice preparation.

Multi-electrode array (MEA) recording

Spontaneous unit and multi-unit spikes were recorded from acute cerebellar slices with respect to a reference ground electrode using substrate integrated MEAs (Multi Channel Systems, Reutlingen, Germany) that consisted of 59 recording electrodes (30 μm diameter; 200 μm spacing) arranged in an 8 × 8 matrix minus corner electrodes, as previously described (Ma et al. 2008). Briefly, slices were adhered to the MEA surface and imaged via a Mikro-Okular camera (Bresser, Germany); once placed, the slice was submerged in carboxygenated standard aCSF, maintained at 24°C and perfused at a rate of ∼2 ml min−1 and allowed to equilibrate for at least 15 min prior to recordings. Signals were amplified (1100× gain) and high-pass filtered (10 Hz) by a 60-channel amplifier (MEA60 System, MultiChannel Systems) and each channel simultaneously sampled at 10 kHz. Continuous recordings from each channel were made using MC_Rack software (MultiChannel Systems) where control spontaneous neuronal activity was first recorded for ≥10 min. In all experiments, each drug was bath-applied for ≥25 min to achieve steady-state effects before 300 s duration continuous recordings were taken. Spike events within continuous recordings were identified using MC_Rack by threshold detection at 4.5× the standard deviation of the mean of a signal-free recording. All analyses included all detected spike events that occurred during the 300 s recording period. Individual spike timings were defined by the time at which the peak minimum for each spike occurred. Spike cut outs were taken for the period 1 ms prior to and 2 ms following each spike's peak minimum (Fig. 1Aa). Spike timings were exported to Neuroexplorer4 (Nex Technologies, USA) for analysis of spike firing rates. Mean spike amplitudes were determined from spike cutout data analysed using in-house code for MATLAB 7.1 (MathWorks, Natick, MA, USA). Regularity of firing was estimated using the coefficient of variation (CV) of interspike interval (ISI), where CV = standard deviation/mean and increases in CV reflect increases in firing irregularity. MEAs have previously been shown to be well suited to recording single unit activity from acute, cerebellar slices (Egert et al. 2002); the validity of such recordings was routinely confirmed via per electrode autocorrelograms that reliably revealed troughs at t= 0 s in PCs, indicative of single units. Stated replicates undertaken in MEA experiments represent the mean of electrodes for a given cellular population per slice as our unit measurement. Thus, for each slice, measured parameters (firing frequency, spike amplitude, CV) from a particular cell type were calculated for each electrode before averaging to provide a single value per cell type for a given slice. To avoid sampling bias, ≥6 separate slices were used for each treatment group. These data were normally distributed (P < 0.05, D’Agostino and Pearson omnibus normality test). Given the slice-to-slice variability in activity under control conditions, drug effects were normalised by expression of change versus the starting control for each slice. Comparisons between raw measures obtained from wild-type +/+, +/du2J and du2J/du2J mice were performed using one-way analysis of variance followed by Tukey's HSD test or Kruskal–Wallace with Dunn's post hoc test as appropriate. Comparisons between multiple treatment groups were performed using Friedman's test followed by Dunn's post hoc test. Throughout, all data are expressed as mean ± SEM unless stated and differences considered significant if P < 0.05.

Figure 1. Region-specific comparison of basal spontaneous spike firing properties in du2J mutants.

Figure 1

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Aa, sample traces of continuous MEA recording from a single electrode in PCL in +/+ and du2Jmutants where inset shows overlay plot of 50 spikes (grey) and mean spike shape (black) from +/+. Ab–d, summary bar graphs of spike firing frequency (b), spike amplitude (c) and coefficient of variation of interspike interval (CV of ISI; d). du2J/du2J firing was more irregular compared with +/+ and +/du2J. Ba, sample traces of continuous MEA recording from a single electrode in GCL in +/+ and du2J mutants where inset shows overlay plot of 50 spikes (grey) and mean spike shape (black) from +/+. Bb–d, summary bar graphs of spike frequency (b), spike amplitude (c) and CV of ISI (d). du2J/du2J firing was more irregular compared with +/+ and +/du2J. **P < 0.01; ***P < 0.001; Kruskal–Wallis test followed by Dunn's test.

Patch-clamp recording

Individual cerebellar brain slices were placed in a recording chamber maintained at room temperature and superfused with carboxygenated standard aCSF. PCs were identified morphologically using an IR-DIC upright Olympus BX50WI microscope (Olympus, Tokyo, Japan) with a 60× numerical aperture 0.9, water immersion lens. Whole-cell patch-clamp recordings from PCs were made in voltage-clamp mode with an EPC-9 patch-clamp amplifier (HEKA Electronik, Lambrecht, Germany) using Pulse software (HEKA) on a Macintosh G4 computer (Apple Computer, Cupertino, CA, USA). Electrodes were fabricated from borosilicate glass (GC150-F10, Harvard Apparatus, Kent, UK) and had resistances ∼5–7 M′Ω when filled with an intracellular solution (CsCl 140 mm, MgCl2 1 mm, CaCl2 1 mm, EGTA 10 mm, MgATP 4 mm, NaGTP 0.4 mm and Hepes 10 mm, pH 7.3). Series resistance was measured at 15–20 MΩ with 70–90% compensation. sIPSCs were isolated at IN-PC synapses in the presence of the non-selective ionotropic glutamate receptor antagonist, NBQX (5 μm), at a holding potential of −70 mV (Stephens et al. 2001). Data were sampled at 5 kHz and filtered at one-third of the sampling frequency. Drugs were diluted in aCSF and superfused ≥25 min and at least 150 s recording obtained during the steady-state period was used as raw data for event detection.

Data were initially exported using Pulsefit (HEKA) to AxoGraph 4.0 software for event detection using a sliding template function. Data were normally distributed (P < 0.05, D’Agostino and Pearson omnibus normality test). Comparisons between measures obtained from +/+, +/du2J and du2J/du2J mice were performed using a one-way ANOVA test followed by Tukey's HSD test. Comparison of multiple treatment groups was performed using repeated measurement one-way ANOVA, followed by Tukey's HSD test.

Radioligand binding assays

Membrane preparation

Cerebellar tissue was dissected from +/+, +/du2J or du2J mice (3- to 5-week-old, male) and stored separately at –80°C until use, as previously described (Jones et al. 2010). Tissue was suspended in a membrane buffer containing Tris-HCl 50 mm, MgCl2 5 mm, EDTA 2 mm and 0.5 mg ml−1 fatty acid-free BSA and complete protease inhibitor (pH 7.4, Sigma, UK) and subsequently homogenised using an Ultra-Turrax blender (IKA, UK). Homogenates were centrifuged at 1200g for 10 min and supernatants decanted. Resulting pellets were homogenised and centrifugation repeated. Pooled supernatants were then centrifuged at 39,000g for 30 min in a high-speed centrifuge (Sorvall, UK) and supernatants discarded. Remaining pellets were resuspended in membrane buffer and protein content determined by Lowry assay (Lowry et al. 1951).

Saturation binding assay

An initial saturation binding assay was carried out using increasing concentrations of the tritiated CB1R antagonist, [3H]SR141716A; the CB1R antagonist, AM251, was used as the non-specific competitor (as previously described in Jones et al. 2010). All concentrations tested were performed in triplicate in assay buffer (20 mm Hepes, 1 mm EDTA, 1 mm EGTA, 0.5% w/v fatty acid-free BSA, pH 7.4). All drug stocks and membrane preparations were diluted in assay buffer and stored on ice immediately prior to use. Assay tubes contained a final volume of 1 ml with [3H]SR141716A at final concentrations of (in nm): 0.1, 0.2, 0.5, 1, 2, 5, 10, 20 and a final concentration of 10 μm AM251 to determine non-specific binding. Assays were initiated by addition of 30 μg membrane protein and were incubated for 1.5 h at 25°C for ligands to reach equilibrium and terminated by rapid filtration through Whatman GF/C filters using a Brandell cell harvester. This was followed by four washes with 3 ml ice-cold PBS (0.14 m NaCl, 3 mm KCl, 1.5 mm KH2PO4, 5 mm Na2HPO4; pH 7.4) to remove unbound radioactivity. Filters were soaked in 2 ml scintillation fluid overnight. Radioactivity was quantified by liquid scintillation spectrometry using a Wallac 1414 scintillation counter where radioactivity bound to cerebellar membrane was quantified in dpm before conversion to pmol mg−1.

Analyses of saturation binding assay data were conducted by non-linear regression and fitted to a one-binding site model (Jones et al. 2010) to determine the equilibrium dissociation constant Kd (nm) and maximal number of binding sites Bmax (pmol mg−1) using GraphPad Prism software (version 4.03; GraphPad Software Inc., San Diego, CA, USA). One-way ANOVA was used to compare results obtained from +/+, +/du2J and du2J/du2J mouse tissues, followed by Tukey's HSD test when appropriate.

Pharmacology

NBQX, WIN55,212–2 (WIN55; each made up as 1000× stocks) and AM251 (made up as a 5000× stock) were dissolved in DMSO and stored at −20°C. Drug stock solutions were diluted to final desired bath concentration using carboxygenated standard aCSF immediately before application.

Results

We have investigated the effects of du2J mutation on cerebellar function by comparing +/+ wild-type litter-matched controls with heterozygous +/du2J, which have >50% reduction in α2δ-2 protein (Donato et al. 2006), and du2J/du2J mice, which exhibit complete α2δ-2 ablation, reduced whole-cell PC Ca2+ current, an ataxic phenotype and fail to survive to adulthood (Donato et al. 2006).

du2J mutation affects spontaneous neuronal spike activity in the cerebellum

The cerebellum consists of the PCL, whose principal PC cells represent the sole output of the cerebellar cortex, the GCL and the molecular layer that, together, provide a well-defined architecture for acute brain slice investigations of spatio-temporal network activity using multi-electrode methods (Egert et al. 2002; Ma et al. 2008). Within the PCL, du2J mutation significantly increased spike firing irregularity in du2J/du2J compared with +/+ and +/du2J mice (both P < 0.001; Fig. 1Ad); PCL spike firing frequency (Fig. 1Ab) and spike amplitude (Fig. 1Ac) were unaffected. Within the GCL, du2J/du2J exhibited significantly more irregular firing compared to either +/+ or +/du2J mice (both P < 0.01; Fig. 1Bd), with no genotype-specific difference in firing frequency (Fig. 1Bb) or spike amplitude (Fig. 1Bc). Overall, these initial results reveal changes in spontaneous network firing properties resulting from du2J mutation that most clearly manifest as globally increased PCL firing irregularity in homozygous du2J/du2J mice, and suggest that the major effect of du2J mutation was to reduce cerebellar PC firing precision, potentially with secondary effects on GCL firing, an effect requiring two du2J alleles to manifest fully.

du2j mutation attenuates CB1R modulation of spontaneous neuronal spike activity in the cerebellum

CB1Rs are highly expressed in the cerebellum (Tsou et al. 1997), where they strongly regulate PC network activity and, consequentially, modulate the final output of the cerebellar cortex (Ma et al. 2008). Modulation of CB1R function can cause severe motor incoordination (including ataxia), as associated with cerebellar dysfunction (DeSanty & Dar, 2001; Patel & Hillard, 2001). CB1R modulation has been suggested as a precipitating factor for cerebellar ataxias (Smith & Dar, 2006). Therefore, we next examined CB1R ligand effects upon spontaneous neuronal activity in cerebellar slices from +/+, +/du2J and du2J/du2J mice. We first recapitulated our previous study (performed in TO strain mice, Ma et al. 2008) to confirm that the CB1R agonist, WIN55 (5 μm), significantly increased PCL spike firing frequency (P < 0.05 vs. control), an action fully reversed by subsequent application of CB1R antagonist, AM251 (2 μm), in the continued presence of WIN55 in +/+ (P < 0.01 vs. WIN55 only; Fig. 2Aa and b). In these experiments, neither WIN55 nor AM251 affected PCL spike amplitude (Fig. 2Ac) or spike firing regularity (Fig. 2Ad). We next investigated whether du2J mutation consequentially affected CB1R-mediated modulation of PC firing. Importantly, WIN55 (5 μm) and AM251 (2 μm) failed to affect PCL spike firing frequency, spike amplitude or regularity of firing in +/du2J (Fig. 2Ba–d) or du2J/du2J mice (Fig. 2Ca–d).

Figure 2. Differential effects of CB1R ligands on spontaneous PCL spike activity in du2J mutants.

Figure 2

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Aa–Ca, sample traces of continuous MEA recording from a single electrode in PCL showing effect of WIN55 (5 μm) and subsequent application of AM251 (2 μm; in the continued presence of 5 μm WIN55) on spontaneous spike firing in +/+ (Aa), +/du2J (Ba) and du2J/du2J (Ca). Ab–Cd, summary bar graphs showing that WIN55 significantly increased normalised spike firing frequency and subsequent application of AM251 caused a significant decrease in normalised spike firing frequency in +/+ (Ab). By contrast, WIN55 and subsequent application of AM251 had no significant effect on normalised spike firing frequency in +/du2J (Bb) or du2J/du2J (Cb). CB1R ligands had no effect on spike amplitude in +/+ (Ac), +/du2J (Bc) or du2J/du2J (Cc) or on normalised CV of ISI in +/+ (Ad), +/du2J (Bd) or du2J/du2J (Cd). *P < 0.05; **P < 0.01; Friedman test followed by Dunn's test.

We next examined CB1R ligand effects on GCL spontaneous spike firing in the du2J genotypes. In +/+, WIN55 (5 μm) and subsequent AM251 (2 μm) application in the continued presence of WIN55 had no effect on GCL firing frequency (Fig. 3Aa and b), spike amplitude (Fig. 3Ac) or firing regularity (Fig. 3Ad). Similarly, WIN55 and AM251 did not affect GCL spike firing in +/du2J (Fig. 3Ba–d) or du2J/du2J mice (Fig. 3Ca–d). These results most likely reflect the reported lack of CB1R expression in GC neurones (Tsou et al. 1997; Egertova & Elphick, 2000). Overall, these findings show that that CB1R ligands predictably modulate cerebellar PCL network level activity in +/+, but not +/du2J or du2J/du2J, mice and are without effect on GCL firing, independent of genotype.

Figure 3. Lack of effect of CB1R ligands on spontaneous GCL spike activity in du2J mutants.

Figure 3

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Aa–Ca, sample traces of continuous MEA recording from a single electrode in GCL showing lack of effect of WIN55 (5 μm) and subsequent application of AM251 (2 μm; in the continued presence of 5 μm WIN55) on spontaneous spike firing in +/+ (Aa), +/du2J (Ba) and du2J/du2J (Ca). Ab–Cd, summary bar graphs showing that CB1R ligands had no effect on normalised spike firing frequency in +/+ (Ab), +/du2J (Bb) or du2J/du2J (Cb), or on spike amplitude in +/+ (Ac), +/du2J (Bc) or du2J/du2J (Cc), or on normalised CV of ISI in +/+ (Ad), +/du2J (Bd) or du2J/du2J (Cd); as assessed by Friedman test followed by Dunn's test.

du2J mutation affects CB1R-mediated presynaptic inhibition at inhibitory IN-PC synapses

We have previously shown that PC firing can be affected by CB1R-mediated modulation of presynaptic GABA release at IN-PC synapses (Ma et al. 2008). Given our data showing that CB1R-modulation of spontaneous neuronal firing is absent in du2J mutants, we next investigated whether du2J mutation affected CB1R modulation of inhibitory transmission at IN-PC synapses. Presynaptic Ca2+ channels (predominantly CaV2.1) underlie GABA release at IN-PC synapses (Forti et al. 2000; Stephens et al. 2001; Lonchamp et al. 2009) and the du2J mutation has been shown to impair PC Ca2+ channel function (Donato et al. 2006). Therefore, we recorded sIPSCs to allow us to determine the effects of du2J mutation on action potential-induced, Ca2+-mediated vesicular neurotransmitter release (Stephens et al. 2001) and, also, to investigate potential associations between effects at IN-PC synapses and the action potential-dependent spontaneous PC spike firing measurements described above. No significant differences in sIPSC frequency (Fig. 4A and Ba) or regularity (Fig. 4A and Bc) between +/+, +/du2J and du2J/du2J were observed, although +/du2J and du2J/du2J each exhibited significantly increased sIPSC amplitudes when compared with +/+ mice (+/du2J: P < 0.05; du2J/du2J: P < 0.01; Fig. 4Bb).

Figure 4. Comparison of basal spontaneous inhibitory transmission at IN-PC synapses in du2J mutants.

Figure 4

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A, raw sIPSC traces from representative PCs from +/+, +/du2J and du2J/du2J. Ba–c, summary bar graphs showing that there was no significant differences in mean sIPSC frequency (a) and CV of ISI (c), but that mean sIPSC amplitude (b) was significant increased in +/du2J and du2J/du2J compared to +/+. *P < 0.05; **P < 0.01; one-way analysis of variance followed by Tukey's HSD test.

We next confirmed the predicted CB1R modulation of sIPSC frequency at +/+ IN-PC synapses (Takahashi & Linden, 2000; Szabo et al. 2004). Thus, WIN55 (5 μm) significantly decreased sIPSC frequency (P < 0.05), an effect that was reversed and increased beyond control levels by subsequent AM251 (2 μm) application in +/+ mice (P < 0.01; Fig. 5Aa and b). The latter result is consistent with the presence of endocannabinergic tone or constitutive CB1R activity in this system (Ma et al. 2008; Wang et al. 2011). Consistent with the lack of CB1R-mediated effects on neuronal spiking activity described above, WIN55 and AM251 failed to significantly modulate sIPSC frequency in +/du2J (Fig. 5Ba and b) or du2J/du2J (Fig. 5Ca and b), although both WIN55 and AM251 showed a marginal trend (P= 0.07; repeated measurement one-way ANOVA) to modulate sIPSC frequency in +/du2J (Fig. 5Bb), an effect not seen in du2J/du2J (P= 0.19; Fig. 5Cb). In addition, WIN55 significantly increased sIPSC amplitude in +/+ (P < 0.05; Fig. 5Ac) and +/du2J (P < 0.05; Fig. 5Bc), but not du2J/du2J (P= 0.11; Fig. 5Cb). Subsequent AM251 application was without effect on WIN55-induced increases in sIPSC amplitude in +/+ (Fig. 5Ac), +/du2J (Fig. 5Bc) or du2J/du2J mice (Fig. 5Cc). The inability of AM251 to block WIN55-induced increases in sIPSC amplitude suggests a CB1R-independent action here.

Figure 5. Differential effects of CB1R ligands on inhibitory transmission at IN-PC synapses in du2J mutants.

Figure 5

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Aa–Ca, raw sIPSC traces from representative PCs from +/+ (Aa), +/du2J (Ba) and du2J/du2J (Ca) showing effect of WIN55 (5 μm) and subsequent application of AM251 (2 μm; in the continued presence of 5 μm WIN55). Ab–Cb, summary bar graphs showing that WIN55 significantly reduced and AM251 significantly increased normalised sIPSC frequency in +/+ (Ab), but was without effect in +/du2J (Bb) or du2J/du2J (Cb). Ac–Cc, WIN55 significantly increased normalised sIPSC amplitude in +/+ (Ac) and +/du2J (Bc), but was without effect in du2J/du2J (Cc). Subsequent application of AM251 was without effect in each case. *P < 0.05; **P < 0.01; repeated measurement one-way ANOVA followed by Tukey's HSD test.

Taken together, these results demonstrate an attenuation of CB1R modulation at IN-PC synapses in du2J mutants. Such effects could contribute to the observed deficits in network level neuronal function.

Investigation of CB1 receptor expression in du2J mice using [3H]SR141716A saturation binding assay

The data above demonstrate that du2J mutants exhibit deficits in CB1R-mediated signalling in the cerebellum. Such deficits could occur as a consequence of reported defects in α2δ-2 Ca2+ channel subunit expression (Donato et al. 2006); however, an alternative hypothesis is reduced CB1R expression in the cerebella of du2J mutants. To further investigate the latter hypothesis, CB1R expression was investigated using radioligand saturation binding assays. In +/+, +/du2J and du2J/du2J mice, specific binding of the high-affinity CB1R antagonist [3H]SR141716A to cerebellar membranes was concentration dependent and saturable (Fig. 6). There was no significant difference in Kd between +/+, +/du2J and du2J/du2J (P= 0.47; Table 1) and the Hill coefficient (nH, the gradient of the Hill plot) approximated unity for all genotypes (Table 1), indicating that [3H]SR141716A bound at a single site to cerebellar CB1Rs. Most importantly, cerebellar membranes from +/+, +/du2J and du2J/du2J mice exhibited no significant differences in Bmax (P= 0.3; Table 1), indicating that there was no difference in CB1R expression between genotypes investigated. These data demonstrate that the reported deficit in CB1R signalling in du2J mutants was likely not to be due to reduced CB1R expression and, rather, may reflect defects in α2δ-2 expression, as discussed below.

Figure 6. Saturation binding of [3H]SR141716A to cerebellar membranes in du2J mutants.

Figure 6

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Representative saturation binding curve for [3H]SR141716A in cerebellar membranes from +/+ (A), +/du2J (B) and du2J/du2J (C).

Table 1.

[3H]SR141716A saturation binding data for cerebellar membrane in du2J mutants

Genotype Kd (nm) Bmax (pmol mg−1) nH
+/+ (n= 3) 3.1 ± 0.2 2.15 ± 0.08 0.99 ± 0.01
+/du2J (n= 3) 2.9 ± 0.3 2.34 ± 0.12 0.99 ± 0.02
du2J/du2J (n= 4) 2.4 ± 0.3 2.03 ± 0.21 1.01 ± 0.01
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Kd and Bmax were obtained from the saturation binding curves plotted between specific binding vs. free [3H]SR141716A radioligand concentration. No significant differences in Kd (P= 0.47) or Bmax (P= 0.3) were seen; one-way analysis of variance. Hill slope (nH) was obtained from the Hill plot of the data transformed from saturation binding plot

Discussion

α2δ-2 mouse mutants exhibit ataxia. Here, we use the du2J mutation, a reportedly clean α2δ-2 knockout (Donato et al. 2006) permitting clear interpretation of phenotypic differences. In addition to studying homozygous du2J/du2J mice, we also examine heterozygous +/du2J mice to investigate potential progressive disturbances. We demonstrate that du2J mutants exhibit deficits in cerebellar CB1R-mediated signalling.

Effects of du2J mutation on neuronal spike activity in the cerebellum

PCL and GCL spike firing showed negative polarity (Egert et al. 2002; Ma et al. 2008). PCL spikes on a given electrode arose from single cells as supported by characteristic trough autocorrelograms and single distribution ISI histograms (data not shown). Conversely, GCL spikes produced variable distribution ISI histograms and autocorrelograms that suggested multi-cell signals (data not shown), which can be accounted for by larger cell somata diameters in PCL than GCL (Egert et al. 2002). GCL spike recordings using MEAs show some differences in the literature, ranging from reports of regular activity consistent with the present findings (Egert et al. 2002), to recordings that are ‘usually silent’ and where the sparse spontaneous activity seen was attributed to Golgi cell activity (Mapelli & D’Angelo, 2007). However, care should be taken when making comparisons between reports where experimental conditions vary (e.g. recordings made at 22–24°C here and in Egert et al. (2002) vs. 32°C in Mapelli & D’Angelo (2007), which can have a profound effect upon basic firing properties. The above caveats for GCL notwithstanding, the major effect of the du2J mutation was to increase PCL irregularity without affecting firing frequency or spike amplitude. du2J/du2J, but not +/du2J, exhibited increased PC firing irregularity suggesting progressive dysfunction; this effect could be coupled to differential reduction of α2δ-2 protein expression between +/du2J and du2J/du2J mice (50%vs. 100% respectively, Donato et al. 2006). We confirm that expression of two du2J alleles is required for manifestation of increased PC irregularity and an ataxic phenotype. Although du2J cerebella are smaller than +/+, du2J mutants show no differences in dendritic morphology (Donato et al. 2006), arguing against PC degeneration underlying differences in firing regularity. Both PC firing precision and activity patterns play important roles in cerebellar motor control (Womack & Khodakhah, 2002; De Zeeuw et al. 2011), potentially by time-locking PC spiking activity (Person & Raman, 2011). Such precision is affected by behavioural state and tactile stimulation (Shin et al. 2007). Importantly, many Ca2+ channel mutants, including du and du2J, increase PC firing irregularity (Hoebeek et al. 2005; Donato et al. 2006; Walter et al. 2006; Ovsepian & Friel, 2012; Alviña & Khodakhah, 2010), which is predicted to adversely affect cerebellar function; for example, PC firing irregularity in tottering mutants functionally reduces compensatory eye movement amplitude (Hoebeek et al. 2005). Donato et al. (2006) reported reduced spontaneous PC firing frequency in +/du2J that was further reduced in du2J/du2J, although this was not observed here or in studies using du mutants (Walter et al. 2006). These differences may be developmental, since younger animals were used by Donato et al. (2006) in comparison to those used in our study and by Walter et al. (2006); however, it is clear that the major, consistent effect of the du2J mutation is to increase firing irregularity.

It has been proposed that GCL firing, driven by mossy fibre inputs, manifests as precisely timed spike bursts limited by Golgi cell-mediated feedforward inhibition to form discrete time windows (∼5 ms) for control of distinct motor domains; thus, GC spike firing dysfunction could contribute to ataxic symptoms (e.g. hypermetria; D’Angelo & De Zeeuw, 2009). Here, GCL firing irregularity was increased in du2J/du2J mice, although to a far lesser extent than in PCL. During development, GC survival depends upon connectivity with PCs (Lossi et al. 2002) and PC disturbances adversely affect GC (Goldowitz & Hamre, 1998), with PC-dependent GCL degeneration proposed as a mechanism (Ivanov et al. 2004); this phenomenon is also reported for ataxic lurcher mice (Wetts & Herrup, 1982). Interestingly, α2δ-2 subunits are barely expressed in GCL, and GC Ca2+ currents were normal in du mutants (Barclay et al. 2001; Donato et al. 2006), consistent with GC changes reflecting secondary consequences of PC dysfunction. Overall, although connectivity deficiencies between cerebellar layers in du2J mutants remain unproven, our results provide evidence for a role of α2δ-2 in correct PC–GC signalling and suggest that the impact of α2δ-2 loss on the GCL should not be ignored.

Effects of du2J mutation on inhibitory synaptic transmission in the cerebellum

Whilst effects of du2J mutation on synaptic transmission are unknown, ataxic mouse models exhibit differences in excitatory transmission in some studies (Matsushita et al. 2002; Liu & Friel, 2008), but not others (Zhou et al. 2003); leaner mutants exhibit enhanced inhibitory transmission, proposed to underlie reduced PC firing and increased irregularity (Liu & Friel, 2008). In addition to intrinsic properties, tonic inhibitory inputs also regulate PC output and synchronization (Hausser & Clark, 1997; de Solages et al. 2008). Here, sIPSC frequencies were unaffected between genotypes, suggesting that action potential-mediated, basal GABA release is unaltered by du2J mutation. Interestingly, sIPSC amplitude was significantly increased in +/du2J and du2J/du2J (cf. +/+ littermates). A similar increase has been reported for leaner mutants and attributed to increased presynaptic GABA release (Ovsepian & Friel, 2012); such effects are unlikely here due to the reported lack of change to sIPSC frequency. An alternative hypothesis is that the mutation leads to an increase in postsynaptic GABAA receptor responsiveness. Increased intracellular Ca2+ ([Ca2+]i) can suppress postsynaptic GABAA receptor function, potentially by decreasing GABA affinity for GABAA receptors (Inoue et al. 1986; Martina et al. 1994); therefore, reduced [Ca2+]i, resulting from decreased PC α2δ-2 expression in du2J mutants (Donato et al. 2006), may relieve Ca2+-mediated suppression of GABAA receptor function.

CB1R modulation is abolished in du2J mutants

Whilst we found no changes to basal IN-PC inhibitory transmission, it remains possible that du2J mutation disrupts presynaptic regulatory mechanisms, including GPCR-mediated inhibition (Zhou et al. 2003). Here, no CB1R-mediated modulation was seen in +/du2J and du2J/du2J mice, as demonstrated by an absence of CB1R agonist-mediated increases in PC spike firing and no reduction in inhibitory transmission at IN-PC synapses compared to +/+ mice. These findings suggest that deficits in CB1R presynaptic inhibition of GABA release are associated with this model of ataxia and could contribute to compromised normal regulation of total PC output and, potentially, the aberrant motor phenotype associated with deficient PC function.

Unlike changes to PC firing regularity, which were confined to homozygous du2J/du2J mice, heterozygous +/du2J mice showed CB1R signalling deficits similar to du2J/du2J mutants. However, WIN55 and AM251 showed a statistical trend (P < 0.1) to modulate sIPSC frequency in +/du2J not seen in du2J/du2J mice, offering some support for a progressive deficit in modulation of presynaptic inhibition. Somewhat unexpectedly, WIN55 increased sIPSC amplitude in +/+ and +/du2J mice, and this increase may reflect a postsynaptic phenomenon; in this regard, the lack of AM251-induced reversal of WIN55 effects (cf.Wang et al. 2011) suggests that this WIN55 effect is CB1R independent, consistent with the reported lack of postsynaptic CB1R expression (Tsou et al. 1997; Yamasaki et al. 2006). For example, WIN55 has been shown to inhibit CaV2.1 channels in PCs at concentrations used here (Fisyunov et al. 2006; Lozovaya et al. 2009), and such actions could reduce [Ca2+]i to overcome Ca2+-mediated suppression of GABAA receptor function (Inoue et al. 1986; Martina et al. 1994) in +/+ and +/du2J mice; the lack of effect in du2J/du2J mice may reflect reduced PC Ca2+ current levels in homozygotes (Donato et al. 2006). Overall, whilst expression of two du2J alleles is required for increased PC irregularity and ataxia, our results demonstrate that expression of a single du2J allele compromises CB1R signalling, prior to any measurable change in PC firing regularity and any clear ataxic phenotype. Here, disrupted cannabinergic signalling may represent a useful diagnostic biomarker of early or asymptomatic cerebellar dysfunction.

Consequences of du2J mutation for CB1R signalling

We show, for the first time, that α2δ-2 deficits caused by du2J mutation are associated with aberrant CB1R signalling and suggest links between impaired Ca2+ channel function and consequential impairment of GPCR-mediated presynaptic inhibition. We also show that CB1R expression is unchanged in du2J mutants, suggesting that deficiency occurs downstream of receptor activation. α2δ-2 is the major isoform expressed in PCs (Cole et al. 2005) and reduced α2δ-2 expression in du2J affects Ca2+ current levels (Donato et al. 2006). Moreover, α2δ-2 is predominantly associated with CaV2.1 (Barclay et al. 2001), the major CaVα subunit mediating presynaptic GABA release at IN-PC synapses (Stephens et al. 2001). Importantly, PC-specific conditional CaV2.1 knock-out causes cerebellar ataxia (Todorov et al. 2012). The association of α2δ-2 and CaV2.1 subunits suggests that deficits in either subunit could equally cause motor deficits, as supported by similarities in ataxic phenotypes in α2δ-2 mutants, including du2J and CaV2.1 knockouts. The most parsimonious explanation for our results is that altered α2δ-2 expression in axon terminals of basket and stellate interneurones in du2J mutants leads to deficits in CB1R-mediated signalling. Although the expression of α2δ-2 in interneurone terminals in cerebellum has not been studied specifically, α2δ-2 is highly expressed in the molecular layer and in GABAergic interneurones throughout the CNS, as well as in PCs (Barclay et al. 2001; Cole et al. 2005). Recent studies have shown that α2δ subunits affect release properties of the Ca2+ channel complex at presynaptic terminals by improving spatial coupling between Ca2+ influx and exocytosis (Hoppa et al. 2012; Dolphin, 2012), in addition to protecting against block of exocytosis by intracellular Ca2+ chelators (Hoppa et al. 2012). Such findings are consistent with the hypothesis that proper α2δ-2 expression is required for correct modulation of presynaptic release. Presynaptic CB1R activation limits transmitter release via generation of Gβγ subunits which inhibit Ca2+ channels (Twitchell et al. 1997; Stephens, 2009). Here, reduced α2δ-2 in du2J mutants could alter G protein–Ca2+ channel interaction to limit direct effects upon channel gating and so dysfunctionally affect modulation of GABA release onto PCs.

Functional impact of CB1R deficits in cerebellar ataxia

We propose that CB1R signalling deficits in du2J mutants occur as a consequence of reduced α2δ-2 expression, which impairs Ca2+ channel function and affects normal GPCR presynaptic inhibition in ataxic phenotypes. Under normal conditions, CB1R inhibition of GABA release at IN-PC synapses reduces inhibitory drive onto PCs to increase PC spike firing (Ma et al. 2008). Regulation of PC spike firing and regularity modulates activity of deep cerebellar nuclei to control motor function. CB1R signalling also contributes to presynaptically expressed synaptic plasticity in the cerebellar cortex. Whilst long-term depression of transmitter release is typically associated with the excitatory parallel fibre (PF)–PC pathway, endocannabinnoid-mediated short term plasticity, in the form of depolarization-induced suppression of inhibition, is prominent at IN-PC synapses (Kano et al. 2009). Notably, CB1R immunoreactivity is reportedly five times higher at IN than at PF terminals; in particular, at basket cell terminals at the PC axon initial segment (Kawamura et al. 2006). Therefore, deficits in CB1R signalling may directly influence PC output in ataxic phenotypes, both in terms of spike firing and regularity and also synaptic function; such deficiencies may contribute to disease.

Acknowledgments

We thank Professor Annette Dolphin for supply of the original +/du2J breeding pairs.

Glossary

CB1R

cannabinoid CB1 receptor

CV

coefficient of variation

GC

granule cell

GCL

granule cell layer

GPCR

G protein-coupled receptor

IN-PC

interneurone–Purkinje cell

ISI

inter-spike interval

PC

Purkinje cell

PCL

Purkinje cell layer

WIN55

WIN55,212–2

Additional information

Competing interests

None.

Author contributions

All the authors contributed to the conception and design of the experiments, the collection, analysis and interpretation of data, and drafting the article or revising it critically for important intellectual content. All authors approved the final version of the manuscript.

Funding

This work was supported by an Ataxia UK Postgraduate Fellowship awarded to B.J.W. and G.J.S. that supported X.W. X.W. also received a University of Reading Postgraduate Research Studentship.

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