Coordinated regulation of endocannabinoid-mediated retrograde synaptic suppression in the cerebellum by neuronal and astrocytic monoacylglycerol lipase

Abstract

The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates retrograde synaptic depression including depolarization-induced suppression of excitation (DSE) and inhibition (DSI). 2-AG is degraded primarily by monoacylglycerol lipase (MAGL), which is expressed in neurons and astrocytes. Using knockout mice in which MAGL is deleted globally or selectively in neurons or astrocytes, we investigated the relative contribution of neuronal and astrocytic MAGL to the termination of DSE and DSI in Purkinje cells (PCs) in cerebellar slices. We report that neuronal MAGL plays a predominant role in terminating DSE at climbing fiber (CF) to PC synapses, while both neuronal and astrocytic MAGL significantly contributes to the termination of DSE at parallel fiber (PF) to PC synapses and DSI at putative Stellate cell to PC synapses. Thus, DSE and DSI at different synapses is not uniformly affected by global and cell type-specific knockout of MAGL. Additionally, MAGL global knockout, but not cell type-specific knockout, caused tonic activation and partial desensitization of the CB₁ receptor at PF-PC synapses. This tonic CB₁ activation is mediated by 2-AG since it was blocked by the diacylglycerol lipase inhibitor DO34. Together, these results suggest that both neuronal and astrocytic MAGL contribute to 2-AG clearance and prevent CB₁ receptor over-stimulation in the cerebellum.

Figure 1. Effects of global and cell type-specific knockout of MAGL on CF-DSE in PCs.

(A,B) Sample traces (A) and average time courses of CF-EPSCs (B) in response to a brief depolarization in cerebellar slices prepared from MAGL-TKO, -NKO, and -AKO mice and their wild-type (WT) littermates (n = 7–11 cells from N = 3–5 mice per genotype). The solid lines are single exponential fitting curves of the decay of CF-DSE, which yield the decay time constant (τ) of DSE shown in (C). (C) Compared with that of WT control, τ of CF-DSE was significantly increased in MAGL-TKO and -NKO mice but was not changed in MAGL-AKO mice (**p < 0.01, ***p < 0.001). (D) MAGL-TKO, -NKO and -AKO did not significantly alter the magnitude of CF-DSE (p > 0.05).

Figure 2. Effects of JZL184 on PF-DSE in cell type-specific MAGL knockout mice.

(A,B) Average time courses of PF-DSE in MAGL-NKO, -AKO and WT slices (n = 7–9 cells from N = 3 mice per genotype) in the absence (control) or presence of JZL184. (C) τ of PF-DSE was significantly increased in MAGL-NKO and -AKO slices compared with that in WT slices (*p < 0.05, **p < 0.01). JZL184 significantly prolonged DSE in WT, MAGL-NKO and -AKO slices (***p < 0.001), and there were no differences in the τ of DSE among WT, MAGL-NKO and -AKO slices treated with JZL184 (p > 0.05). (D) The magnitude of PF-DSE was not significantly different in WT, MAGL-NKO and -AKO slices with or without JZL184 (p > 0.05).

Figure 3. Effects of global and cell type-specific knockout of MAGL on DSI at putative SC-PC synapses.

(A,B) Sample traces (A) and average time courses of IPSCs (B) in response to a brief depolarization in cerebellar slices prepared from MAGL-TKO, -NKO, and -AKO mice and their WT littermates (n = 10–12 cells from N = 3–4 mice per genotype). (C) τ of DSI was significantly increased in MAGL-TKO, -NKO and AKO mice compared to the WT control (*p < 0.05, **p < 0.01). (D) MAGL-TKO, -NKO and -AKO did not significantly alter the magnitude of DSI (p > 0.05).

Figure 4. MAGL-TKO, but not -NKO or -AKO, caused tonic activation of the CB₁ receptor.

(A–D) Bath application of the CB₁ receptor antagonist AM251 (2 μM) did not significantly alter PF-EPSCs in cerebellar PCs in MAGL-NKO (A,B; p > 0.05) and MAGL-AKO slices (A,C; p > 0.05) but caused a robust increase in the amplitude of PF-EPSCs in MAGL-TKO slices (A,D; p < 0.001, n = 7–9 cells from N = 3–4 mice per genotype). The AM251-induced increase in PF-EPSCs in MAGL-TKO slices was blocked by the DAGL inhibitor DO34 (D; p < 0.001). For the purpose of comparison, the result obtained from WT slices were superimposed in each panel from (B–D). (E) Summarized results show that bath application of AM251 induced a significant increase in PF-EPSCs in MAGL-TKO slices (***p < 0.001), but no significant change was found in MAGL-NKO and -AKO slices (p > 0.05). The increase in PF-EPSCs in MAGL-TKO slices was blocked by the DAGL inhibitor DO34 (***p < 0.001).

Figure 5. MAGL-TKO, but not -NKO or -AKO, caused partial desensitization of the CB₁ receptor.

(A–D) There was no significant difference in WIN55,212-2-induced PF-EPSC depression between WT and MAGL-NKO (A,B; p > 0.05), or between WT and MAGL-AKO slices (A,C; p > 0.05). However, WIN55212-2 induced significantly less depression of PF-EPSCs in MAGL-TKO slices compared with that of WT (A,D; *p = 0.045). For the purpose of comparison, the result obtained from WT slices were superimposed in each panel from (B–D). (E) Summarized results show that WIN55,212-2 induced a significantly smaller depression of PF-EPSCs in MAGL-TKO slices (*p = 0.045) compared with that of WT, but no a significant difference was found between MAGL-NKO vs. WT; or MAGL-AKO vs. WT (p > 0.05, n = 9–12 cells from N = 3–4 mice per genotype).