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. 2011 Sep 12;589(Pt 20):4847–4855. doi: 10.1113/jphysiol.2011.215509

Genetic deletion of monoacylglycerol lipase alters endocannabinoid-mediated retrograde synaptic depression in the cerebellum

Peng Zhong 1, Bin Pan 1, Xiu-ping Gao 2, Jacqueline L Blankman 3, Benjamin F Cravatt 3, Qing-song Liu 1
PMCID: PMC3224879  PMID: 21911610

Non-technical summary

2-Arachidonoylglycerol (2-AG) is an endogenous marijuana-like chemical that regulates synaptic transmission via the stimulation of the type I cannabinoid receptor (CB1). It is inactivated by an enzyme called monoacylglycerol lipase (MAGL). 2-AG inactivation is impaired in MAGL knockout mice. We show that 2-AG accumulation in the brain of MAGL knockout mice alters several forms of 2-AG-mediated synaptic depression in the cerebellum via tonic activation and desensitization of CB1 receptors.

Abstract

Abstract

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is hydrolysed primarily by monoacylglycerol lipase (MAGL). Here, we investigated whether eCB-mediated retrograde synaptic depression in cerebellar slices was altered in MAGL knockout (MAGL−/−) mice. Depolarization-induced suppression of excitation (DSE) and metabotropic glutamate receptor (mGluR1)-mediated synaptic depression are mediated by 2-AG-induced activation of CB1 receptors. We show that genetic deletion of MAGL prolonged DSE at parallel fibre (PF) or climbing fibre (CF) to Purkinje cell (PC) synapses. Likewise, mGluR1-mediated synaptic depression, induced either by high-frequency stimulation of PF or mGluR1 agonist DHPG, was prolonged in MAGL−/− mice. About 15% of 2-AG in the brain is hydrolysed by serine hydrolase α-β-hydrolase domain 6 and 12 (ABHD6 and ABHD12). However, the selective ABHD6 inhibitor WWL123 had no significant effect on cerebellar DSE in MAGL+/+ and −/− mice. The CB1 receptor antagonist SR141716 significantly increased the amplitude of basal excitatory postsynaptic currents (EPSCs) in MAGL−/− mice but not in MAGL+/+ mice. Conversely, the CB1 agonist WIN55212 induced less depression of basal EPSCs in MAGL−/− mice than in MAGL+/+ mice. These results provide genetic evidence that inactivation of 2-AG by MAGL determines the time course of eCB-mediated retrograde synaptic depression and that genetic deletion of MAGL causes tonic activation and consequential desensitization of CB1 receptors.

Introduction

Endocannabinoids (eCBs) are lipid molecules that play a critical role in regulating synaptic transmission (Alger, 2005; Lovinger, 2007). eCBs exhibit several unique properties that distinguish them from traditional transmitters. eCBs are not stored in synaptic vesicles but are instead produced ‘on demand’ (Marsicano et al. 2003). eCBs are released from postsynaptic neurones to activate type I cannabinoid receptors (CB1) on presynaptic terminals, leading to retrograde depression of synaptic transmission. Depolarization- induced suppression of excitation (DSE) and inhibition (DSI) are forms of retrograde synaptic depression mediated by eCB-induced CB1 receptor activation (Kreitzer & Regehr, 2001; Ohno-Shosaku et al. 2001; Wilson & Nicoll, 2001). Furthermore, the stimulation of certain G-protein-coupled receptors such as group I metabotropic glutamate receptors (mGluRs) induces retrograde synaptic depression via the release of eCBs (Maejima et al. 2001; Varma et al. 2001; Brown et al. 2003).

Anandamide and 2-arachidonoylglycerol (2-AG) are two known eCBs that bind and activate the CB1 receptor (Alger, 2005; Lovinger, 2007). Anandamide and 2-AG are hydrolysed primarily by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively (Cravatt et al. 1996; Blankman et al. 2007). DSE/DSI and mGluR1-mediated retrograde synaptic depression were abolished in mice lacking the 2-AG biosynthetic enzyme diacylglycerol lipase-α (Gao et al. 2010; Tanimura et al. 2010). MAGL inhibitors, but not FAAH inhibitors or knockout, increased 2-AG levels in the brain (Cravatt et al. 2001; Long et al. 2009) and augment DSE/DSI (Straiker & Mackie, 2005; Safo et al. 2006; Hashimotodani et al. 2007; Pan et al. 2009). Thus, 2-AG is the eCB ligand that is responsible for DSE/DSI and mGluR1-mediated retrograde synaptic depression.

The use of a pharmacological approach to study the role of MAGL in eCB signalling has its limitations because it relies on the specificity of the agents used. Some of the MAGL inhibitors have multiple targets and unspecific effects (Lio et al. 1996). The recent generation of MAGL-deficient mice (MAGL−/−) provides a powerful tool to investigate the role of MAGL in shaping eCB signalling. MAGL-deficient mice exhibited marked and sustained elevations in brain 2-AG levels, a significant decrease in CB1 receptor binding density and behavioral tolerance to CB1 agonists (Chanda et al. 2010; Schlosburg et al. 2010). In the present study, we examined whether DSE and mGluR1-mediated retrograde synaptic depression in cerebellar Purkinje cells (PCs) were altered in MAGL knockout mice. We demonstrated that genetic deletion of MAGL prolonged DSE and mGluR1-mediated retrograde synaptic depression in cerebellar slices and caused tonic suppression of basal excitatory transmission and CB1 receptor desensitization.

A comprehensive profile of brain serine hydrolases revealed that about 85% of total 2-AG in mouse brain is hydrolysed by MAGL and the resting 15% is hydrolysed by other serine hydrolases including serine hydrolase α-β-hydrolase domain 6 and 12 (ABHD6 and ABHD12) (Blankman et al. 2007). A recent study has shown that ABHD6 contributes significantly to the inactivation of 2-AG in BV-2 cells and primary neurones (Marrs et al. 2010). WWL123 is a recently developed, highly selective and potent inhibitor of ABHD6 (Bachovchin et al. 2010). We found that WWL123 had no significant effects on cerebellar DSE in MAGL+/+ and −/− mice. These results suggest that ABHD6 does not make a significant contribution to 2-AG inactivation during DSE, regardless of whether MAGL is intact or genetically deleted.

Methods

Animals

MAGL+/+, +/− and −/− mice on a mixed 129SvEv/C57BL/6J background were generated by the Texas Institute of Genomic Medicine (Schlosburg et al. 2010). Male and female MAGL+/+ and −/− mice as well as male C57BL/6J mice (Jackson Laboratories; Bar Harbor, ME, USA) were used in this study. Genotyping of MAGL+/+, +/− and −/− mice was carried out by PCR using DNA samples obtained from the tail or ear. The MAGL+/+ and −/− mice used in this study were littermates from second- to fourth-generation intercrosses of 129SvJ-C57BL/6 MAGL+/− mice.

Slice preparation

All animal use was in accordance with protocols approved by the Institution's Animal Care and Use Committee of Medical College of Wisconsin. Mice were anaesthetized by isoflurane inhalation and decapitated. Parasagittal cerebellar slices (250 μm thick) were cut using a vibrating slicer (Leica VT1000s). For recording climbing fibre (CF)-mediated EPSCs, slices were prepared from 10- to 14-day-old mice. For recording parallel fibre (PF)-mediated EPSCs, slices were prepared from 10- to 14-day-old or 20- to 25-day-old mice. Slices were prepared at 4–6°C in a solution containing (in mm): 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, 26 NaHCO3, 25 glucose, 11.6 sodium ascorbate and 3.1 sodium pyruvate. The slices were immediately transferred and stored in artificial cerebrospinal fluid (ACSF) containing (in mm): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3 and 10 glucose.

Electrophysiology

Whole-cell voltage-clamp recordings were made from cerebellar Purkinje cells as described previously (Pan et al. 2009). Excitatory postsynaptic currents (EPSCs) were evoked by stimulating climbing fibres (CFs) or parallel fibres (PFs) with a glass pipette that was filled with 1 m NaCl and placed in the granule cell layer or molecular layer, respectively. CF-EPSCs showed all-or-none responses at near-threshold stimulation and exhibited paired-pulse depression (30–50 ms intervals), whereas PF-EPSCs showed graded responses and exhibited paired-pulse facilitation (Kreitzer & Regehr, 2001). The GABAA receptor blocker picrotoxin (50 μm) was present in the ACSF throughout the experiments. Glass pipettes (3–5 MΩ) were filled with one of the following internal solutions containing (in mm): (1) 130 cesium methanesulfonate, 10 CsCl, 2 QX-314, 10 Hepes, 0.2 EGTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2GTP and 10 Na2-phosphocreatine (pH 7.2 with CsOH) for examining DSE and effects of WIN55,212-2 or SR141716 on EPSCs; (2) 120 cesium methanesulfonate, 10 CsCl, 2 QX-314, 10 Hepes, 10 BAPTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2GTP and 10 Na2-phosphocreatine for examining the effects of DHPG on EPSCs; (3) 140 potassium gluconate, 5 KCl, 10 Hepes, 0.2 EGTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2GTP and 10 Na2-phosphocreatine (pH 7.2, adjusted with KOH) for mGluR1-mediated responses induced by high-frequency stimulation of PF (50 Hz, 1 s).

Immunohistochemistry

MAGL−/− and +/+ mice were anaesthetized by intraperitoneal injection of sodium pentobarbital (50 mg kg−1, Sigma) and transcardially fixed with 4% paraformaldehyde in 0.1 m phosphate buffer supplemented with 4% sucrose. The brains were sectioned at 30–40 μm thickness with a cryostat after postfix and dehydration. Parasagittal cerebellar sections were incubated with anti-calbindin antibody (1:250, Sigma) at 4°C for 24 h. After rinsing in PBS three times, the sections were incubated in the secondary antibody goat anti-rabbit IgG-Texas Red (1:200, Santa Cruz Biotechnology) for 4–5 h at room temperature in the dark. The sections were then rinsed in PBS three times, dehydrated and coverslipped. NeuroTrace green fluorescent Nissl stain (N21480, Invitrogen) was performed according to manufacturer's protocol. For both immunofluorescence and Nissl staining, the sections were analysed by using a Nikon Eclipse TE-2000U confocal microscope.

Chemicals

Unless specified otherwise, all drugs were prepared as concentrated stock solutions and stored at −20 or −80°C before use. Picrotoxin (Sigma) and R,S-3,5-dihydroxyphenylglycine (DHPG, Tocris) were solved in water through sonication. WIN55,212-2 (Tocris), SR141716 (rimonabant, Sanofi Aventis), JZL184 and WWL123, which were synthesized as described previously (Long et al. 2009; Bachovchin et al. 2010), were solved in DMSO. Bovine serum albumin (BSA, 0.2 mg ml−1, Sigma) was included in the ACSF to assist penetration of SR141716 into cerebellar slices. When these drugs were used, control slices were treated in the same concentration of the respective solvent (DMSO and BSA) for similar exposure time. Drug-treated slices were interleaved with control slices from the same animal.

Data analysis and statistics

The decay time constant (τ) and magnitude of DSE were measured as described previously (Pan et al. 2009). The magnitude of mGluR1-mediated depression (%) was calculated as follows: 100 × (1 – (mean amplitude of 2 smallest EPSCs after DHPG or PF stimulation/mean amplitude of 5 baseline EPSCs)). Values of two to three trials were averaged for each neurone. The depression (%) of EPSCs by CB1 agonists/antagonists was calculated as follows: 100 × (mean amplitude of EPSCs at last 5 min of drug application/mean amplitude of baseline EPSCs). EPSCs, evoked at 20 s intervals, were first averaged for every minute. Data are presented as the mean ± SEM. Results were analysed with Student's t test. Results were considered to be significant at P < 0.05.

Results

Morphology of the cerebellum of MAGL+/+ and −/− mice

Nissl staining of parasagittal sections of the cerebellum did not show detectable differences in the size and overall morphology of the cerebellum between MAGL+/+ and −/− mice. MAGL−/− mice exhibited well-developed foliation, comparable to that of MAGL+/+ mice (Fig. 1A). The dendritic structure and morphology of cerebellar Purkinje cells (PCs) in MAGL−/− mice appear indistinguishable from those in MAGL+/+ mice, as shown by immunostaining with antibody against calbindin, a neuronal maker for PCs (Garcia-Segura et al. 1984) (Fig. 1B).

Figure 1. Normal morphology of the cerebellum in MAGL −/− mice.

Figure 1

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A, Nissal staining of parasagittal sections of the cerebellum shows similar gross morphology between MAGL+/+ and −/− mice. Scale bar, 1 mm. N = 3 mice each group. B, the morphology of cerebellar PCs are indistinguishable between MAGL+/+ and −/− mice, as shown by immunofluorescence staining with antibody against calbindin, a neuronal maker for PCs. Note that some dendrites of the PCs were truncated by histological sectioning (40 μm thick) in both groups. Scale bar, 20 μm. N = 3 mice each genotype.

Effects of genetic deletion of MAGL on DSE in PCs

Whole-cell voltage-clamp recordings were made from PCs in acute cerebellar slices prepared from 10- to 14-day-old or 20- to 25-day-old MAGL−/− and MAGL+/+ mice. A brief depolarization (1 s from −60 mV to 0 mV) of Purkinje cells induced transient suppression of EPSCs, or DSE, at parallel fibre (PF)−PC and climbing fibre (CF)−PC synapses. DSE at either PF−PC or CF−PC synapses, regardless of animal age, was significantly prolonged in MAGL−/− mice compared with corresponding DSE in MAGL+/+ mice, as shown by an increase in the mean decay time constant (τ) of DSE (n = 7–10 each group, P < 0.001; Fig. 2A and B). Figure 2A depicts DSE at PF−PC synapses in 20- to 25-day-old MAGL+/+ and −/− mice. The prolongation of the decay of DSE in MAGL−/− mice became more apparent when peaks of DSE in both genotypes were scaled to the same degree. The magnitude of DSE was decreased at PF−PC synapses in 20- to 25-day-old MAGL−/− mice but remained unchanged at CF−PC and PF−PC synapses in 10- to 14-day-old MAGL−/− mice (n = 7–10 each group, P < 0.001; Fig. 2C). Thus, the decay of DSE at both CF−PC and PF−PC synapses was prolonged in MAGL−/− mice in both age groups but the magnitude of DSE was significantly decreased only in more mature MAGL−/− mice (20- to 25-day-old). All following experiments were performed in 20- to 25-day-old MAGL+/+ and −/− mice.

Figure 2. Effects of genetic deletion of MAGL on DSE in the cerebellum.

Figure 2

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A, left, sample traces of PF-EPSCs and averaged DSE (bottom) in cerebellar slices prepared from 20- to 25-day-old MAGL+/+ and −/− mice. Right, the peaks of DSE are scaled to show that the decay of DSE is prolonged in MAGL−/− mice compared with that of MAGL+/+ mice. The continuous lines are single exponential fitting curves of the decay of DSE. B and C, summary of the decay time constant (τ) (B) and magnitude (C) of DSE at CF−PC and PF−C synapses in cerebellar PCs in MAGL+/+ and −/− mice at 10- to 14-day- and 20- to 25-day-old ages (n = 7–10 cells, N = 3–4 mice from each group; **P < 0.01; ***P < 0.001).

JZL184 is a recently developed, highly selective and potent MAGL inhibitor (Long et al. 2009). It takes ∼40 min for JZL184 to produce the maximal inhibition of MAGL (Long et al. 2009; Pan et al. 2009). Bath application of a saturating concentration of JZL184 (1 μm) for 40–120 min (Pan et al. 2009) increased the decay time constant of DSE at PF−PC synapses in cerebellar slices in 20- 25-day-old MAGL+/+ mice (vehicle, 18.8 ± 3.6 s, n = 7; JZL184, 86.9 ± 18.2 s, n = 8; P < 0.01; Fig. 3A) but did not affect DSE in the same-aged MAGL−/− mice (vehicle, 62.5 ± 11.8 s, n = 7; JZL184, 80.9 ± 14.7 s, n = 9; P > 0.05; Fig. 3B). JZL184 did not significantly affect the magnitude of DSE in MAGL+/+ and −/− mice (P > 0.05; Fig. 3A and B).

Figure 3. Effects of MAGL inhibitor JZL184 and ABHD6 inhibitor WWL123 on DSE in the cerebellum.

Figure 3

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A, bath application of JZL184 (1 μm) prolonged DSE of PF-EPSCs in cerebellar PCs in MAGL+/+ mice (n = 7–8/N = 3–4 mice; P < 0.01). B, JZL184 did not significantly affected DSE in MAGL−/− mice (n = 7–9/N = 3–5; P > 0.05). C and D, bath application of WWL123 (10 μm) did not significantly affect DSE of PF-EPSCs in cerebellar Purkinje neurons in MAGL+/+ (n = 7–8/N = 3–4; P > 0.05; C) and −/− mice (n = 6–8/N = 3–3; P > 0.05; D).

About 85% of total 2-AG in mouse brain is hydrolysed by MAGL, while the resting 15% is hydrolysed by ABHD6 and ABHD12 (Blankman et al. 2007). WWL123 is a selective inhibitor of ABHD6 (Bachovchin et al. 2010) but a selective inhibitor of ABHD12 is not available. Bath application of a saturating concentration of WWL123 (10 μm) (Bachovchin et al. 2010) had no significant effect on either the decay time constant or the magnitude of DSE at PF−PC synapses in cerebellar slices prepared from MAGL+/+, MAGL−/− (Fig. 3C and D) or C57BL/6J mice (data not shown), indicating that ABHD6 does not make a significant contribution to the inactivation of 2-AG during DSE, regardless of whether MAGL is intact or genetically ablated.

Effects of genetic deletion of MAGL on mGluR1-mediated synaptic depression

In the cerebellum and hippocampus, the mGluR1 agonist DHPG induced retrograde synaptic depression via the release of 2-AG and subsequent activation of CB1 receptors (Maejima et al. 2001; Varma et al. 2001; Tanimura et al. 2010). We investigated whether genetic deletion of MAGL affected DHPG-induced suppression of EPSCs. We found that bath application of DHPG (50 μm) for 5 min induced rapid depression of PF-EPSCs in both MAGL+/+ and −/− mice, and the magnitude of the depression was not significantly different (MAGL+/+, 39.3 ± 5.9% of baseline, n = 9; MAGL−/−, 31.3 ± 6.4%, n = 8; P < 0.05; Fig. 4A). Following washout of DHPG, this suppression was quickly and completely recovered in MAGL+/+ mice but slowly and incompletely recovered in MAGL−/− mice (Fig. 4A).

Figure 4. Effects of genetic deletion of MAGL on mGluR-mediated retrograde synaptic depression in the cerebellum.

Figure 4

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A, the effects of bath application of the mGluR1 agonist DHPG (50 μm) on PF-EPSCs in PCs in MAGL+/+ and −/− mice (n = 8–9/N = 3–4 mice). B, high-frequency stimulation (50 Hz, 1 s, arrow) of PF induced a transient depression of EPSCs in MAGL+/+ mice. The depression was significantly prolonged in MAGL−/− mice (P < 0.001). The continuous lines are single exponential fitting curves of the decay of the depression (n = 7–9/N = 3–4 mice). C, in the presence of SR141716, the 50 Hz stimulation induced similar post-tetanic potentiation in MAGL+/+ and −/− mice (n = 6–6/N = 2–3; P > 0.05). Veh, vehicle; SR, SR141716.

A brief high-frequency stimulation of PF could also induce eCB-mediated retrograde suppression of CF-EPSCs or PF-EPSCs via synaptic activation of mGluR1 (Maejima et al. 2001; Brown et al. 2003). We found that PF stimulation (50 Hz, 1 s) induced suppression of PF-EPSCs in both MAGL+/+ and −/− mice (Fig. 4B). In slices from MAGL+/+ mice, the EPSCs were depressed to maximal levels immediately after the 50 Hz stimulation and rapidly returned to pre-stimulation baseline levels (τ: 9.7 ± 3.8 s, n = 7). In contrast, the depression of EPSCs in MAGL−/− mice had a slow onset and the EPSCs returned to baseline levels at a much slower rate (τ: 166.7 ± 29.6 s, n = 9; P < 0.001). The peak magnitude of the mGluR1-mediated depression was not significantly different between MAGL+/+ and −/− mice (MAGL+/+, 35.3 ± 7.1%, n = 7; MAGL−/−, 35.8 ± 5.7%, n = 9; P > 0.05, Fig. 4B). In the continuous presence of the CB1 receptor antagonist SR141716 (2 μm), the 50 Hz stimulation induced indistinguishable post-tetanic potentiation of EPSCs in MAGL+/+ and −/− mice (Fig. 4C). Thus, genetic deletion of MAGL significantly prolonged the duration of mGluR1-mediated depression.

Tonic activation and partial desensitization of the CB1 receptor in MAGL−/− mice

The dramatic elevations of 2-AG in MAGL−/− mice could induce tonic activation of CB1 receptors and persistent suppression of synaptic transmission. To test this, we examined the paired-pulse ratio (PPR) of PF-EPSCs, which is used as a measure of the probability of transmitter release. The PPR of PF-EPSCs was measured at a number of inter-pulse intervals (50–250 ms). PF-EPSCs displayed paired-pulse facilitation at most of these intervals in both MAGL+/+ and −/− mice. The PPR of PE-EPSCs at 50–100 ms intervals was significantly increased in MAGL−/− mice compared with that of MAGL+/+ mice (Fig. 5A). The increase in the PPR suggests glutamate release is depressed (Kreitzer & Regehr, 2001). To test whether 2-AG-induced activation of the CB1 receptor is responsible for the change in the PPR, we measured the PPR of PF-EPSCs in the presence of the CB1 receptor antagonist SR141716 (2 μm). There was no significant difference in the PPR between MAGL+/+ and −/− mice in SR141716 (P > 0.05), this was because SR141716 significantly decreased the PPR in MAGL−/− mice (P < 0.05) but did not significantly affect the PPR in MAGL+/+ mice (P > 0.05; Fig. 5A).

Figure 5. Alterations of basal synaptic properties and synaptic transmission in MAGL−/− mice.

Figure 5

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A, The paired-pulse ratio (EPSC2/EPSC1) of PF-EPSCs was significantly increased at 50 ms and 100 ms inter-pulse intervals in MAGL−/− mice compared with that of MAGL+/+ mice (n = 9–10/N = 3–3; *P < 0.05). Representative paired-pulse PF-EPSCs at 50–250 ms intervals are shown on the top. B, genetic deletion of MAGL decreased the slope of input−output curves of PF-EPSCs (n = 10–11/N = 3–3; *P < 0.05; **P < 0.01). Top, representative PF-EPSCs were evoked with a range of stimulus intensities in MAGL+/+ and −/− mice. C, bath application of the CB1 receptor antagonist SR141716 (2 μm) increased the amplitude of PF-EPSCs in cerebellar PCs in MAGL−/− mice but did not significantly affect those in MAGL+/+ mice (n = 7–9/N = 3–6 mice; P < 0.05). D, bath application of the CB1 receptor agonist WIN55212-2 (2 μm) induced significantly less depression of PF-EPSCs in MAGL−/− mice than those in MAGL+/+ mice (n = 6–8/N = 3–4; P < 0.05). Veh, vehicle; SR, SR141716.

To further test whether basal synaptic transmission is altered in MAGL−/− mice, we recorded EPSCs in cerebellar PCs while stimulating PF with incremental intensities. The input−output (I−O) relationship of PF-EPSCs was determined by plotting the amplitude of the EPSCs against the stimulus intensities. MAGL−/− mice exhibited significant decreases in the mean amplitude of PF-EPSCs at several stimulus intensities compared with that of MAGL+/+ mice (P < 0.05 or P < 0.01; Fig. 5B). If the decrease in PF-EPSC amplitude is due to 2-AG-induced activation of CB1 receptors, CB1 receptor blockade should enhance PF-EPSCs. We found that bath application of SR141716 (2 μm) significantly increased basal PF-EPSCs in cerebellar PCs in MAGL−/− mice but had no significant effects on PF-EPSCs in MAGL+/+ mice (MAGL+/+, 110.1 ± 10.8% of baseline, n = 7; MAGL−/−, 150.9 ± 9.4%, n = 9; P < 0.05; Fig. 5C). Thus, disruption of MAGL causes tonic activation of CB1 receptor and persistent suppression of synaptic depression.

To test whether 2-AG-induced tonic activation of the CB1 receptor causes CB1 receptor sensitization, we examined the effect of the CB1 receptor agonist WIN55,212-2 on EPSCs in MAGL+/+ and −/− mice. Bath application of a saturating concentration (2 μm) of WIN55,212-2 produced significantly less depression of PF-EPSCs in cerebellar PCs in MAGL−/− mice than that in MAGL+/+ mice (MAGL+/+, 48.9 ± 4.9%, n = 6; MAGL−/−, 31.0 ± 5.9%, n = 8; P < 0.05; Fig. 5D). Taken together, these data indicate that genetic deletion of MAGL causes tonic activation and partial desensitization of CB1 receptors.

Discussion

The endocannabinoid 2-AG is hydrolysed primarily by MAGL (Blankman et al. 2007). The recent generation of MAGL-deficient mice (Chanda et al. 2010; Schlosburg et al. 2010) provides a powerful tool to examine the role of MAGL in determining the strength and duration of eCB signalling in the brain. The cerebellum expresses the highest density of CB1 receptors in the brain (Tsou et al. 1998). Several forms of eCB/CB1 receptor-mediated retrograde synaptic depression, including DSE and mGluR1-mediated synaptic depression, are first discovered and well-characterized in this brain region (Kreitzer & Regehr, 2001; Maejima et al. 2001; Brown et al. 2003). In the present study, we examined how eCB/CB1 receptor-mediated responses were altered in MAGL−/− mice. We found that the decay of cerebellar DSE was prolonged in MAGL−/− mice. Similarly, DSI in hippocampal CA1 pyramidal neurones was also prolonged in MAGL−/− mice (Pan et al. 2011). These findings are congruent with findings that MAGL inhibitors prolong DSE/DSI in rats or mice (Straiker & Mackie, 2005; Hashimotodani et al. 2007; Pan et al. 2009). Thus, both genetic and pharmacological studies indicate that degradation of 2-AG by MAGL is the rate-limiting step that determines the time course of DSE/DSI.

Biochemical studies showed that MAGL knockout or blockade caused elevations in 2-AG levels and decreases in CB1 receptor binding density in the brain (Chanda et al. 2010; Schlosburg et al. 2010). Consistent with the CB1 receptor desensitization, we found that the magnitude of DSE at PF–PC synapses was decreased in more mature (20- to 25-day-old) MAGL−/− mice and that the CB1 receptor agonist WIN55212-2 induced greater depression of EPSCs in MAGL+/+ mice than that in MAGL−/− mice. In contrast, the magnitude of DSE at CF–PC or PF–PC synapses was not significantly different between 10- to 14-day-old MAGL+/+ and −/− mice. The minimal desensitization of the CB1 receptor at this young age may explain the lack of significant change in the magnitude of DSE in MAGL−/− mice.

The CB1 receptor desensitization appears to be caused by 2-AG-induced persistent activation of CB1 receptors. We found that the MAGL inhibitor JZL184 or MAGL knockout prolonged the decay of DSE in cerebellar Purkinje neurones, suggesting that 2-AG degradation is impaired following disruption of MAGL. Furthermore, the PPR of PF-EPSCs was increased in MAGL−/− mice, while the slope of the I–O curve of PF-EPSCs was decreased in MAGL−/− mice, suggesting that excitatory synaptic transmission is suppressed. Finally, bath application of the CB1 receptor antagonist SR141716 significantly decreased the PPR but increased the amplitude of basal EPSCs in PCs in MAGL−/− mice, but not in MAGL+/+ mice. Taken together, these results provide further evidence that MAGL knockout or blockade cause tonic activation and partial desensitization of the CB1 receptor.

Interestingly, the MAGL inhibitor JZL184 did not significantly change the magnitude of DSE in MAGL+/+ mice (Fig. 3) and C57BL/6J mice (Pan et al. 2009). JZL184 produces time-dependent inhibition of MAGL, and it takes ∼40 min for JZL184 to produce the maximal inhibition of MAGL (Long et al. 2009; Pan et al. 2009). A covalent mechanism of inactivation may underlie the time-dependent inhibition (Long et al. 2009). The gradual accumulation of 2-AG during ‘acute’ application of JZL184 may cause slight CB1 receptor desensitization. Furthermore, the magnitude of DSE should be determined by peak levels of 2-AG that reach the CB1 receptor. It is likely that the amount of 2-AG that reaches presynaptic CB1 receptors immediately after depolarization is controlled primarily by 2-AG production, while the continued occupation of CB1 receptors by 2-AG during the decay phase of DSE is mainly determined by 2-AG degradation.

About 15% of MAGL in the brain is hydrolysed by ABHD6 and ABHD12 (Blankman et al. 2007). A recent study indicates that ABHD6 plays a significant role in clearing 2-AG in the BV-2 microglia cell line and in brain tissues (Marrs et al. 2010). Nevertheless, we found that the selective ABHD6 inhibitor WWL123 (Bachovchin et al. 2010) had no significant effect on DSE in MAGL+/+, −/− mice and C57BL/6J mice. As a positive control, the selective MAGL inhibitor JZL184 prolonged DSE in MAGL+/+ mice, but not in MAGL−/− mice. The lack of effect of WWL123 on DSE may be explained by the relative small contribution of ABHD6 to 2-AG hydrolysis. However, the inability of WWL123 to affect DSE in MAGL−/− mice is somewhat surprising since these data indicate that ABHD6 does not up-regulate in the face of genetic deletion of MAGL.

mGluR1 receptor activation, induced by its agonist DHPG or by high-frequency synaptic stimulation, induced retrograde synaptic depression that is mediated by 2-AG- induced activation of CB1 receptors (Maejima et al. 2001; Varma et al. 2001; Brown et al. 2003; Tanimura et al. 2010). We demonstrated here that the duration of DHPG- and 50 Hz stimulation-induced depression of EPSCs was significantly prolonged in MAGL−/− mice compared with those in MAGL+/+ mice. The impaired 2-AG inactivation can explain why mGluR1-mediated synaptic depression was prolonged in MAGL−/− mice. The slow onset of the 50 Hz stimulation-induced depression of EPSCs suggests that mGluR1-induced 2-AG release gradually reaches peak levels in MAGL−/− mice. As the onset of DSE in the cerebellum (the present study) or DSI in the hippocampus (Pan et al. 2011) was not altered in MAGL−/− mice, we suspect that adaptive changes in mGluR downstream signalling following sustained elevations of 2-AG may explain the slow onset of stimulation-induced synaptic depression. During bath application of DHPG, it takes several minutes for DHPG to reach equilibrium of its final concentration (50 μm) in the recording chamber. The asynchronous activation of mGluRs may have masked the slow onset of mGluR1-mediated depression observed during the 50 Hz stimulation.

We found that the magnitude of DSE in mature PF−PC synapses was decreased in MAGL−/− mice, whereas the magnitude of DHPG-induced depression was not significantly changed between MAGL+/+ and −/− mice. A critical difference between DSE and DHPG-induced depression is their temporal resolution. mGluRs are activated asynchronously during bath application of DHPG, which may have truncated the peak amplitude of this form of mGluR1-mediated depression. During DSE, depolarization causes synchronic production of 2-AG and rapid occupation of presynaptic CB1 receptors. Any CB1 receptor desensitization will be manifest as a decrease in DSE magnitude.

The cerebellum plays a critical role in motor coordination and the fine adjustment of movements (Rinaldo & Hansel, 2010). CB1 receptor agonists cause ataxia and other cerebellum-dependent deficits in motor function (Patel & Hillard, 2001). However, MAGL−/− mice exhibit normal locomotor activity and do not exhibit significant rotarod ataxia, a deficit in motor coordination (Chanda et al. 2010). Although partial CB1 receptor sensitization may counter the heightened 2-AG activity in MAGL−/− mice, the net effect should be tonic activation of CB1 receptors and persistent suppression of synaptic transmission. The normal locomotor activity in MAGL−/− mice suggests that the cerebellar circuits make adaptive changes in response to sustained elevations of 2-AG. CB1 knockout or blockade causes severe impairment in discrete motor learning such as delay eyeblink conditioning but does not affect normal motor coordination (Kishimoto & Kano, 2006). It remains to be determined whether cerebellum-related learning behaviours are altered in MAGL−/− mice.

Acknowledgments

This work was supported by National Institutes of Health (DA017259 to B.F.C. and DA024741 to Q.S.L.) and Extendicare Foundation (Q.S.L.).

Glossary

Abbreviations

2-AG

2-arachidonoylglycerol

ABHD6

α-β-hydrolase domain 6

ABHD12

α-β-hydrolase domain 12

ACSF

artificial cerebrospinal fluid

CB1

type I cannabinoid receptor

CF

climbing fibre

DGLα

diacylglycerol lipase-α

DHPG

(RS)-3,5-dihydroxyphenylglycine

DSE

depolarization-induced suppression of excitation

DSI

depolarization-induced suppression of inhibition

eCB

endocannabinoid

FAAH

fatty acid amide hydrolase

MAGL

monoacylglycerol lipase

mGluR

metabotropic glutamate receptor

PC

Purkinje cell

PF

parallel fibre

QX-314

N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide

Author contributions

P.Z., B.P. and Q.-s.L. designed the experiments, P.Z., B.P. and X.-p.G. performed the experiments, collected and analysed the data. P.Z. and Q.-s.L. wrote the manuscript. J.L.B. and B.F.C. provided breeding pairs of MAGL knockout mice. All authors approved the final version. All experiments were conducted at Medical College of Wisconsin.

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