Genetic disruption of 2-arachidonoylglycerol synthesis reveals a key role for endocannabinoid signaling in anxiety modulation

Abstract

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety and depressive behaviors and emotional learning. However, the role of the most-abundant endocannabinoid 2-arachidonoylglycerol (2-AG) in the physiological regulation of affective behaviors is not well understood. Here, we show that genetic deletion of the 2-AG synthetic enzyme diacylglycerol lipase α (DAGLα) in mice reduces brain, but not circulating, 2-AG levels. DAGLα deletion also results in anxiety-like and sex-specific anhedonic phenotypes associated with impaired activity-dependent eCB retrograde signaling at amygdala glutamatergic synapses. Importantly, acute pharmacological normalization of 2-AG levels reverses both phenotypes of DAGLα-deficient mice. These data suggest 2-AG deficiency could contribute to the pathogenesis of affective disorders and that pharmacological normalization of 2-AG signaling could represent an approach for the treatment of mood and anxiety disorders.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Characterization and validation of DAGLα−/− mice

(A) Conventional PCR gel showing the amplified products from primers that anneal to endogenous DNA sites that flank the targeting cassette, which was inserted upstream of exons 8. The resulting product in DAGLα−/− mice demonstrates the expected ~250 bp shift as a result of the successful insertion of the targeting cassette. (B) Representative western blot gel from male “M” and female “F” and DAGLα−/− forebrain, which is summarized in bar graph (C). (D) DAGL activity in forebrain extracts from male and female DAGLα−/− mice. (E–H) Forebrain levels of 2-AG, 1-steroyl-2-arachidonoyl glycerol (SAG), arachidonic acid (AA), and AEA in male and female DAGLα−/− and WT mice. (I–J) 2-AG and AA levels in the prefrontal cortex (PFC), amygdala, and striatum of male and female DAGLα−/− mice. (K–M) Plasma levels of 2-AG, AA, ad AEA in DAGLα−/− mice. * P < 0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by Sidak’s post hoc test. Error bars indicate SEM. See also Figure S1.

Figure 2. DAGLα deletion increases anxiety-like and depressive behavior

(A–B) Behavioral analysis of male (A) and female (B) DAGLα−/− mice in the open-field assay. (C–D) Behavioral analysis of male (C) and female (D) DAGLα−/− mice in the light-dark box test (L/D Box). (E–F) Behavioral analysis of male (E) and female (F) DAGLα−/− mice in the NIH assay. (G–H) Behavioral analysis of male (G) and female (H) DAGLα−/− mice in the sucrose preference test (SPT). (I–J) Behavioral analysis of male (I) and female (J) DAGLα−/− mice in the tail suspension test (TST). * P < 0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by ANOVA, t-test, or K-S test as indicated in the panel. Error bars indicate SEM. See also Figure S2.

Figure 3. DAGLα deletion impairs eCB-modulation of amygdala glutamatergic transmission

(A) Representative example of DSE in the BLA with arrows indicating time of 10 second depolarization from −70 mV to +30 mV. (B) Effects of the CB1 receptor antagonist Rimonabant and DAGL inhibitor THL on BLA DSE. (C) Effects of post-synaptic calcium chelation with BAPTA on BLA DSE. (E–F) Analysis of BLA neuron (E) membrane properties, excitability, and (F) sEPSC amplitude and inter-event interval (IEI) in male DAGLα−/− mice. (G–H) Analysis of BLA neuron (G) membrane properties, excitability and (H) sEPSC amplitude and IEI in female DAGLα−/− mice. ** P<0.01, **** P<0.0001, by ANOVA, Sidak’s post hoc analysis, or t-test as indicated in the panel. Error bars indicate SEM. See also Figure S3.

Figure 4. JZL-184 reverses anxiety and depressive behaviors in DAGLα−/− mice

(A–C) Effects of DAGLα deletion and JZL-184 treatment on percent light distance travelled, light time, and total ambulatory distance in the L-D box. (D) Effects of DAGLα deletion and JZL-184 treatment on sucrose preference in female mice. Note JZL-184 was administered on 2 consecutive days 2h prior to testing, while recovery represents testing under vehicle-treatment conditions 24h after the last drug treatment. (E) Effects of DAGLα deletion and JZL-184 treatment on latency to feed in the NIH assay. (F–H) Effects of DAGLα deletion and JZL-184 treatment on 2-AG levels in the PFC (F), amygdala (G), and striatum (H). * P