Monoacylglycerol lipase inhibition blocks chronic stress-induced depressive-like behaviors via activation of mTOR signaling

Abstract

The endocannabinoid (eCB) system regulates mood, emotion, and stress coping, and dysregulation of the eCB system is critically involved in pathophysiology of depression. The eCB ligand 2-arachidonoylglycerol (2-AG) is inactivated by monoacylglycerol lipase (MAGL). Using chronic unpredictable mild stress (CUS) as a mouse model of depression, we examined how 2-AG signaling in the hippocampus was altered in depressive-like states and how this alteration contributed to depressive-like behavior. We report that CUS led to impairment of depolarization-induced suppression of inhibition (DSI) in mouse hippocampal CA1 pyramidal neurons, and this deficiency in 2-AG-mediated retrograde synaptic depression was rescued by MAGL inhibitor JZL184. CUS induced depressive-like behaviors and decreased mammalian target of rapamycin (mTOR) activation in the hippocampus, and these biochemical and behavioral abnormalities were ameliorated by chronic JZL184 treatments. The effects of JZL184 were mediated by cannabinoid CB1 receptors. Genetic deletion of mTOR with adeno-associated viral (AAV) vector carrying the Cre recombinase in the hippocampus of mTORf/f mice recapitulated depressive-like behaviors induced by CUS and abrogated the antidepressant-like effects of chronic JZL184 treatments. Our results suggest that CUS decreases eCB-mTOR signaling in the hippocampus, leading to depressive-like behaviors, whereas MAGL inhibitor JZL184 produces antidepressant-like effects through enhancement of eCB-mTOR signaling.

Figure 1

Selective MAGL inhibitor JZL184 rescued CUS-induced deficit in DSI in hippocampal CA1 pyramidal neurons. (a) CUS significantly decreased the decay time constant (τ) (n=10–11, p<0.01) and magnitude (p<0.001) of DSI, which was induced by 5-s depolarization from −60 to 0 mV. Sample traces of IPSCs are superimposed on the top. The solid lines are single exponential fitting curves of the decay of DSI. (b) Bath application of the CB₁ receptor agonist WIN55212–2 (5 μM) induced similar depression of IPSCs in CA1 pyramidal neurons in hippocampal slices prepared from control and CUS-exposed mice (n=9–9; p>0.05). (c) Representative western blots (bottom) and summarized data (top) showed that CUS did not significantly alter protein levels of CB₁ receptor (CB₁R) in the hippocampus (p>0.05; n=5 animals each group). Immunoreactivity was normalized to GAPDH and presented as percentage of time-matched control mice. (d) CUS decreased the tissue content of 2-AG in the hippocampus (n=6 mice each group; *p<0.05). (e) Bath application of JZL184 (1 μM) potentiated DSI in control and CUS-exposed mice. DSI in these two groups was not significantly different in the presence of JZL184 (n=7–8, p>0.05).

Figure 2

CUS altered mTOR signaling in the hippocampus. (a) Representative western blots and (b) summarized data showed that CUS significantly decreased p-mTOR (S2448), p-p70S6K (T389), and p-rpS6 (S235/236) in the hippocampus (**p<0.01, ^#p<0.001; n=5 animals each group). Immunoreactivity was normalized to GAPDH and presented as percentage of time-matched control mice.

Figure 3

Chronic JZL184 treatment blocked CUS-induced depressive-like behaviors. (a) Timeline of the CUS exposure, drug treatment, and behavioral tests. (b) Neither CUS nor chronic JZL184 (JZL) treatment affected the total distance traveled (p>0.05) and on time in center (p>0.05) during the first 5-min test session in the OPT. (c) CUS decreased the sucrose preference in the SPT (^#p<0.001; Tukey's post hoc test), JZL184 treatment restored CUS-induced reduction of the sucrose preference (^#p<0.001), and the effects of JZL184 were blocked by the CB₁ antagonist rimonabant (RIM) (**p<0.01). (d) CUS increased the latency to feed in the novel environment in the NSF test (**p<0.01); JZL184 treatment decreased the latency to feed (*p<0.05), which was blocked by rimonabant (**p<0.01). (e) CUS significantly increased the immobility time in the FST (^#p<0.001); JZL184 treatment increased the immobility time in CUS-exposed mice (^#p<0.001) but not in control mice. The effects of JZL184 was blocked by rimonabant (**p<0.01). n=11–12 animals each group. OPT, open field test; SPT, sucrose-preference test; NSF, novelty-suppressed feeding; FST, forced swim test; Novelty, novel environment.

Figure 4

CUS and chronic JZL184 treatments altered mTOR and ERK signaling in the hippocampus. (a) Representative western blots for p-mTOR (S2448), p-P70s6k (T389), p-rpS6 (S235/236), total mTOR, p-ERK1/2 (T202/204), and total ERK1/2 from the same groups of mice shown in Figure 3. (b–e) Summarized data showed that CUS significantly decreased p-mTOR (b), p-p70S6K (c), p-rpS6 (d), and p-ERK1/2 (e) in the hippocampus, and these decreases were reversed by JZL184 treatments. CB₁ receptor antagonist rimonabant blocked the effects of JZL184 treatments. The p values for Tukey's post hoc test results are shown on the top (*p<0.05, **p<0.01, ^#p<0.001; n=6 animals each group). Immunoreactivity was normalized to GAPDH and presented as the percentage of the control group with vehicle treatment.

Figure 5

Subchronic JZL184 treatment did not alter anxiety- and depressive-like behaviors induced by CUS. (a) Timeline for CUS exposure, JZL184 treatment, and behavioral tests. (b) Neither CUS nor acute JZL184 treatment affected the total distance traveled (p>0.05) and the time in center (p>0.05) during the first 5-min test session in the OPT. (c–e) CUS significantly changed the sucrose preference (^#p<0.001) in the SPT (c), the latency to feed in the novel environment (**p<0.01) in the NSF test (d), and the immobility time (^#p<0.001) in the FST (e). However, subchronic JZL184 treatment did not reverse these CUS-induced behavioral alterations. Neither CUS nor JZL184 treatment affected the latency to feed in the home cage in the NSF test (d) (p>0.05, n=8 animals each group).

Figure 6

AAV2-Cre-GFP-mediated deletion of mTOR in the hippocampus. (a) X-gal staining of hippocampus following intra-hippocampus microinjections of AAV2-Cre-GFP. AAV2-mediated Cre recombinase expression was labeled by LacZ (blue) when injected into Rosa26 reporter mice in the hippocampus. Scale bar: 0.5 mm. (b, c) Immunofluorescence staining for Neuronal Nuclei (NeuN; neuronal marker) and AAV2-Cre-GFP (green) in the hippocampus. Representative images (b) and summarized data (c) showed that AAV2-Cre-GFP infected ∼80% of all hippocampal CA1 pyramidal neurons in both control and mTORf/f mice (n=3 animals each group). Scale bar: 50 μm. (d and e) Representative (d) and summarized data (e) of western blots showed that intra-hippocampal microinjection of AAV2-Cre-GFP significantly decreased protein levels of mTOR, p-p70S6K (T389), and p-rpS6 (S235/236) in the hippocampus (^#p<0.001; n=6 animals each group). Immunoreactivity was normalized to GAPDH and presented as the percentage of that of the control mice.

Figure 7

Hippocampus-specific deletion of mTOR produced anxiety- and depressive-like behaviors. (a) Timeline for the AAV mciroinjection and behavioral tests. (b) Hippocampus-specific mTOR deletion did not affect the total distance traveled (p>0.05) and the time in center (p>0.05) in the OPT. (c–e) Hippocampus-specific mTOR deletion significantly decreased the sucrose preference in SPT test (*p<0.05) (c), increased the latency to feed in the novel environment in NSF test (*p<0.05) (d), and the immobility time in FST test (*p<0.05) (e) but did not alter the latency to feed in the home cage in NSF test (p>0.05) (d). n=9–11 animals each group. AAV, AAV2-Cre-GFP or AAV2-GFP.

Figure 8

Chronic JZL184 treatments did not affect anxiety- and depressive-like behaviors induced by mTOR deletion in the hippocampus. (a) Timeline for the AAV microinjection, drug treatment, and behavioral tests. (b–d) Hippocampus-specific deletion of mTOR significantly altered the sucrose preference (^#p<0.001) in the SPT (b), the latency to feed in the novel environment (**p<0.01) in the NSF test (c), and the immobility time (**p<0.01) in the FST (d). However, chronic JZL184 treatments did not reverse these mTOR deletion-induced behavioral alterations (p>0.05). Neither mTOR deletion nor JZL184 treatment affected the latency to feed in the home cage in the NSF test (c) (p>0.05). n=9–10 mice per group. AAV, AAV2-Cre-GFP.