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. Author manuscript; available in PMC: 2015 Dec 11.
Published in final edited form as: Cell Rep. 2014 Nov 26;9(5):1644–1653. doi: 10.1016/j.celrep.2014.11.001

Genetic Disruption of 2-Arachidonoylglycerol Synthesis Reveals a Key Role for Endocannabinoid Signaling in Anxiety Modulation

Brian C Shonesy 1,†, Rebecca J Bluett 2,3,†, Teniel S Ramikie 2,3, Rita Báldi 2, Daniel J Hermanson 4, Philip J Kingsley 4, Lawrence J Marnett 4, Danny G Winder 1,2,3,5, Roger J Colbran 1,3,5, Sachin Patel 1,2,3,5,*
PMCID: PMC4268380  NIHMSID: NIHMS640754  PMID: 25466252

SUMMARY

Endocannabinoid (eCB) signaling has been heavily implicated in the modulation of anxiety, 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 a novel approach for the treatment of mood and anxiety disorders.

INTRODUCTION

Endogenous cannabinoid (eCB) signaling is mediated by cannabinoid receptors (type 1; CB1, and type 2; CB2), which are activated by several endogenous ligands including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) (Kano et al., 2009; Piomelli, 2003). Over the past decade eCB signaling has been implicated in the regulation of multiple physiological functions in the nervous system and periphery, including a primary role in the modulation of anxiety and depressive behaviors (Hill and Patel, 2013; Kathuria et al., 2003; Lutz, 2009; Ruehle et al., 2012). Specifically, pharmacological blockade or genetic deletion of CB1 receptors increases anxiety in multiple animal models and in humans under some conditions (Christensen et al., 2007; Gamble-George et al., 2013; Hill and Gorzalka, 2004; Moreira et al., 2009; Patel and Hillard, 2006). Consistent with these data, anxiety in various animal models is decreased by low doses of exogenous cannabinoids (Patel and Hillard, 2006; Rey et al., 2012) and by pharmacological or genetic augmentation of eCB levels (Hermanson et al., 2013; Kathuria et al., 2003; Moreira et al., 2008; Patel and Hillard, 2006; Rossi et al., 2010; Sciolino et al., 2011; Sumislawski et al., 2011). These data strongly suggest a key role for eCB signaling in the physiological regulation of anxiety and depressive behaviors.

2-AG is the most abundant eCB ligand in the brain and is the primary mediator of phasic eCB-mediated retrograde suppression of neurotransmitter release in the nervous system (Gao et al., 2010; Ohno-Shosaku et al., 2012; Tanimura et al., 2010). The synaptic signaling pools of 2-AG are synthesized and degraded primarily by the postsynaptic DAGLα and presynaptic monoacylglycerol lipase (MAGL), respectively, in the adult brain (Dinh et al., 2002). Several recent studies have demonstrated that pharmacological elevation of 2-AG signaling can reduce anxiety-like behaviors in animal models (Busquets-Garcia et al., 2011; Sciolino et al., 2011; Sumislawski et al., 2011; Zhong et al., 2014), while some clinical studies have demonstrated reduced peripheral 2-AG levels in patients with PTSD and women with major depression compared to control subjects (Hill et al., 2013; Hill et al., 2008). Although these data suggest a close association between 2-AG signaling and affective pathology, a causal relationship between endogenous 2-AG signaling and the physiological expression of anxiety and depressive behaviors has not been demonstrated. Here we show Dagla knockout mice exhibit reductions in limbic 2-AG levels associated with anxiety-like behaviors and anhedonia, both of which are normalized by acute pharmacological normalization of 2-AG deficiency. These data provide causal evidence supporting 2-AG signaling deficiency as a key mechanism subserving development of affective pathology and support pharmacological 2-AG augmentation as a viable treatment approach for mood and anxiety disorders.

RESULTS

Biochemical Validation of DAGLα−/− mice

Homozygous Dagla knockout mice (DAGLα−/−) mice were generated as described in Experimental Methods and identified by standard PCR approaches, exhibiting the expected shift in size of amplification product associated with targeting cassette insertion upstream of exon 8 of the Dagla gene (Fig. 1A). Adult male and female DAGLα−/− mice showed a complete lack of brain DAGLα protein expression (p<0.0001 for both sexes) as assessed by western blot analysis and reductions in enzymatic activity (p<0.05 for both sexes) confirming effectiveness of the gene disruption strategy (Fig. 1B–D). Mass spectrometry analysis revealed a significant reduction of forebrain 2-AG levels (male p<0.05, female p<0.0001; Fig. 1E). To provide insight into the specific diacylglycerol (DAG) species that may be DAGLα substrates in vivo, we analyzed levels of arachidonate-containing DAGs. The most abundant arachidonic acid (AA)-containing DAG detected, 1-stearoyl-2-arachidonoylglycerol (SAG), was increased in both male (p<0.0001) and female (p<0.001) DAGLα−/− forebrains (Fig. 1F), indicating its importance as a physiological substrate for DAGLα in the brain, and confirming previous work in cultured neuroblastoma cells (Jung et al., 2007). Several less abundant AA containing DAG species were also detected, some of which also displayed sex-specific changes in DAGLα−/− mice (Fig. S1A–J). In contrast, none of the non-AA-containing DAGs measured were different between genotypes of either sex (data not shown). Consistent with reductions in 2-AG levels, the primary hydrolytic metabolite of 2-AG, AA, was also reduced in DAGLα−/− mice (male p<0.001; female p<0.01; Fig. 1G). There were no changes in levels of the other major eCB, anandamide (Fig. 1H). Brain regional analysis revealed reductions in 2-AG and AA in the prefrontal cortex (PFC), amygdala and striatum of both male and female DAGLα−/− mice (Fig. 1I–J). Levels of the non-eCB monoacylglycerol, 2-oleoylglycerol (2-OG), were also significantly reduced in the PFC, amygdala and striatum of male and female DAGLα−/− mice (Fig. S1K). These data indicate DAGLα−/− mice have the expected alterations in DAGLα protein, enzymatic activity, and 2-AG metabolism with reduced 2-AG levels, increased 2-AG precursor levels, and reduced levels of the 2-AG degradation product AA. Lastly, given the increasing interest in using peripheral eCB levels as biomarkers for neuropsychiatric diseases, we measured plasma 2-AG, AEA, and AA concentrations and found no changes in DAGLα−/− mice relative to WT littermates (Fig. 1K–M). Thus, plasma 2-AG levels are not dependent on DAGLα under basal conditions.

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

Figure 1

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(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.

DAGLα deletion increases anxiety-like behaviors

Having established that DAGLα deletion results in reduced central 2-AG levels, we next evaluated the effects of this 2-AG deficiency on anxiety- and depressive-like behaviors. Separate cohorts of male and female DAGLα−/− mice and WT littermates were sequentially tested for a series of anxiety and depressive-like behaviors (Fig. 2). In the open-field assay, male (p<0.01) and female (p<0.05) DAGLα−/− mice showed reduced vertical exploration without changes in total distance traveled indicating decreased exploratory drive without gross motor abnormality (Fig. 2A–B). Only female mice showed a reduction in percent center distance traveled (p<0.05) suggestive of enhanced anxiety-like behavior (Fig. 2A–B). In the light-dark box (L-D Box) test, both male (p<0.01) and female (p<0.01) DAGLα−/− mice showed reduced exploratory distance traveled, reduced percent light-zone ambulation (males p<0.001 and female p<0.01), and reduced time spent in the light compartment (males p<0.05 and females p<0.01; Fig 2C–D). We next tested mice using a more ethologically-relevant behavioral assay, the novelty-induced hypophagia (NIH) test, which is highly sensitive to eCB modulation (Gamble-George et al., 2013). In this test, both male and female DAGLα−/− mice showed an increase in feeding latency as demonstrated by a difference in the cumulative feeding latency distribution (males p<0.0001 and females p<0.0001 by K-S test), increased mean feeding latency (females p<0.001), and reduced consumption (males p<0.05 and females p<0.05; Fig 2E–F). To exclude the possibility that impaired motor coordination contributes to the observed behavioral effects, we tested DAGLα−/− mice on the accelerating rotarod, but did not find any differences in motor performance between genotypes (Fig. S2A–B)

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

Figure 2

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(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.

DAGLα deletion results in sex-specific depressive-like behavior

We next examined more traditional depressive-like behaviors using the sucrose preference test (SPT) and the tail suspension test (TST). No differences in sucrose preference, sucrose consumption, or water consumption (not shown) were observed in male DAGLα−/− mice relative to WT littermates (Fig. 2G). In contrast, female DAGLα−/− mice showed reduced sucrose preference (p<0.001), reduced sucrose consumption (p<0.01), and increased water consumption (p<0.001; not shown) over the course of the experiment after normalization to body weight (Fig. 2H and see Fig. S2C–D for body weight data). Neither male nor female DAGLα−/− mice showed differences in immobility time during the tail suspension test relative to WT littermates (Fig. 2I–J).

DAGLα deletion impairs amygdala eCB retrograde synaptic signaling

eCB signaling, and specifically 2-AG signaling, is known to inhibit excitatory glutamatergic transmission in the amygdala (Azad et al., 2003; Domenici et al., 2006; Yoshida et al., 2011) and eCB-mediated reductions in anxiety likely require CB1-mediated inhibition of glutamate, but not GABA, release (Haring et al., 2012; Rey et al., 2012; Ruehle et al., 2013). Therefore, we hypothesized that DAGLα−/− mice would have impaired eCB-mediated suppression of basolateral amygdala (BLA) glutamatergic transmission, which could contribute to the observed anxiety-like behavioral phenotype. To test this hypothesis, we used the most well established form of 2-AG-mediated retrograde endocannabinoid signaling expressed widely throughout the CNS, depolarization-induced suppression of excitation (DSE) (Alger, 2002; Tanimura et al., 2010). Whole-cell patch-clamp recordings from BLA pyramidal neurons revealed that postsynaptic depolarization caused a transient suppression of excitatory postsynaptic current (EPSC) amplitude (Yoshida et al., 2011) (Fig. 3A), which was blocked by pre-incubation with the CB1 receptor antagonist Rimonabant (p<0.0001). BLA DSE was attenuated by pharmacological inhibition of DAGL with THL (p<0.01; Fig. 3B) and by postsynaptic calcium chelation with 40 mM BAPTA (p<0.01; Fig. 3C). Importantly, DSE was essentially absent in DAGLα−/− mice relative to wild-type littermate controls (p<0.0001; Fig. 3D). Since 2-AG levels were decreased in the striatum as well, we also confirmed that DSE was absent in this brain region of DAGLα−/− mice (Fig. S3). These data support 2-AG as a key mediator of retrograde 2-AG signaling at excitatory synapses (Gao et al., 2010; Tanimura et al., 2010).

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

Figure 3

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(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.

Lastly, we evaluated potential compensatory consequences of the prolonged impairment of eCB-mediated inhibition of glutamatergic signaling on measures of excitatory synaptic transmission, membrane properties, and excitability of BLA principle neurons of male and female DAGLα−/− mice. Neither male nor female DAGLα−/− mice exhibited changes in membrane properties or cellular excitability (Fig. 3E and G). However, there was a slight reduction of spontaneous EPSC (sEPSC) amplitude specifically in females, while neither sex exhibited significant changes in sEPSC inter-event interval (IEI; Fig. 4F and H). Overall, these measures indicate no major compensatory changes in basal glutamatergic transmission or cell excitability as a result of long-term DAGLα deletion.

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

Figure 4

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(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 < 0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by ANOVA followed by Sidak’s post hoc analysis or K-S test as indicated in the panel. Error bars indicate SEM. See also Figure S4.

2-AG restoration normalizes the behavioral phenotype of DAGLα−/− mice

Thus far, our data clearly support necessity for 2-AG signaling in the physiological regulation of anxiety and depressive behaviors. However, establishing a causal link between 2-AG signaling deficiency and the behavioral phenotypes observed in DAGLα−/− mice also requires experimental support for sufficiency of 2-AG signaling in the regulation of physiological anxiety and depressive behaviors. Therefore, we utilized a pharmacological approach to restore 2-AG levels in DAGLα−/− mice and determined the impact on the behavioral phenotype of DAGLα−/− mice. Administration of the MAGL inhibitor JZL-184 (20 mg/kg) robustly increases brain 2-AG levels in WT mice (Long et al., 2009) (Fig. S4), suggesting this approach could also be useful for restoring deficient 2-AG levels in DAGLα−/− mice.

In the L-D box, vehicle-treated DAGLα−/− mice again exhibited anxiety-like behavior relative to WT vehicle-treated mice (p<0.05 for both measures; Fig. 4A–B), while both percent light distance and light time were significantly higher in JZL-184-treated DAGLα−/− mice relative to vehicle-treated DAGLα−/− mice (p<0.05 and p<0.01 respectively). Ambulatory distance was not affected by any treatment (Fig. 4C). In the SPT, baseline sucrose preference was again significantly lower in female DAGLα−/− mice relative to WT mice confirming our previous data (p<0.001; Fig. 4D). After establishment of a baseline preference, JZL-184 was administered to DAGLα−/− mice while WT mice received vehicle injections. After 1 day of JZL-184 treatment, the sucrose preference of DAGLα−/− mice remained significantly lower than that of vehicle-treated WT mice, but on the second day of drug treatment sucrose preference was not significantly different between vehicle-treated WT mice and DAGLα−/− mice treated with JZL-184 (p>0.05; Fig 4D). Importantly, sucrose preference deficits in DAGLα−/− mice relative to WT reemerged after a 24h drug washout period (p<0.05; Fig 4D, recovery). Interestingly, although we replicated the robust anxiety-like effect in DAGLα−/− mice in the NIH assay, JZL-184 did not reverse the increased feeding latency exhibited by DAGLα−/− mice (Fig. 4E). To confirm this dose of JZL-184 increased 2-AG levels in DAGLα−/− mice we measured 2-AG 2h after 20 mg/kg JZL-184 treatment. Vehicle-treated DAGLα−/− mice showed significant reductions in 2-AG levels in the PFC (p<0.01), amygdala (p<0.0001) and striatum (p<0.0001) (Fig. 4F–H). Importantly, treatment with 20mg/kg JZL-184 significantly increased 2-AG levels in the PFC (p<0.01), amygdala (p<0.05), and striatum (p<0.05) relative to vehicle-treated DAGLα−/− mice indicating this dose was sufficient to at least partially restore 2-AG levels in DAGLα−/− mice. These data support sufficiency of 2-AG signaling in regulating anxiety and depressive behaviors.

DISCUSSION

To investigate the physiological role of endogenous 2-AG signaling in the regulation of anxiety and depressive behaviors we utilized a genetic approach to delete a primary 2-AG synthetic enzyme, DAGLα. We find that DAGLα−/− mice have the expected decreases in 2-AG tissue levels and decreases in AA consistent with other DAGLα−/− mouse lines (Gao et al., 2010; Tanimura et al., 2010; Yoshino et al., 2011). Comparison to previous studies revealed some key differences between our findings and published results. For example, Gao et al. reported ~75% reduction in whole brain 2-AG and AA in DAGLα−/− mice, while our bulk 2-AG reductions appeared much less dramatic. Reasons for this discrepancy are unclear but could be related to the fact we specifically analyzed forebrain, rather than whole brain. In addition, Gao et al. showed an ~50% reduction in 2-AG levels in DAGLβ−/− mice (Gao et al., 2010), suggesting this enzyme also contributes to bulk brain 2-AG measurements and that the residual forebrain 2-AG levels observed in our DAGLα−/− mice may be synthesized by DAGLβ. There may also be significant regional heterogeneity in the effects of DAGLα deletion on 2-AG levels such that forebrain analyses may underestimate highly localized decreases observed in the sub-regional analysis. This suggestion is supported by the dramatic region-specific reductions in 2-AG levels observed in DAGLα−/− mice, which confirms robust 2-AG deficiency in key limbic brain regions regulating anxiety and depressive behaviors including the PFC and amygdala. We also show for the first time that the levels of SAG and several other AA-containing DAGs are elevated in the forebrain of DAGLα−/− mice, consistent with previous findings in cultured cells (Jung et al., 2007), revealing these lipids are the precursors of 2-AG formation in vivo. Lastly, reductions in brain AEA have also been reported in DAGLα−/− mice (Gao et al., 2010; Tanimura et al., 2010). While we did not observe reductions in forebrain AEA levels in our mice, future studies will be aimed at enhancing detection and quantification of brain regional AEA differences to clarify this discrepancy.

Upon confirmation that DAGLα−/− mice represented a validated limbic 2-AG deficiency model, we undertook a comprehensive behavioral analysis of DAGLα−/− mice to test the hypothesis that endogenous 2-AG signaling is a critical regulator of affective behavior. Our data revealed anxiety-like behaviors in both male and female DAGLα−/− mice, while only female mice exhibit anhedonia as demonstrated by a reduction in sucrose preference. Overall ambulation in the open field and motor coordination on the accelerating rotarod were unaffected by DAGLα deletion, indicating that the observed differences in anxiety-like and depressive behaviors were not likely confounded by motor deficits per se. With regard to measures of anxiety, DAGLα−/− mice showed increased anxiety-like behavior in the open-field, L-D-box, and NIH assay, thus supporting a primary role for 2-AG signaling in the regulation of anxiety-like behavior (Patel and Hillard, 2008). In contrast, only female DAGLα−/− mice exhibited anhedonia, while neither male nor female DAGLα−/− mice exhibited despair-like behavior in the TST. The data herein suggest that 2-AG signaling is selectively involved in the regulation of a discrete behavioral dimension of depression, namely anhedonia, while having broader multi-dimensional effects on anxiety-related behavior.

Importantly, we also found that pharmacological elevation of 2-AG levels in DAGLα−/− mice reversed anxiety behavior in the L-D box and anhedonia in the SPT, but was unable to reverse anxiety in the NIH assay. Since the NIH assay is extremely sensitive to CB1 signaling deficiency (Gamble-George et al., 2013), it is possible that more complete 2-AG restoration or longer duration of restoration would be required to see reversal in this test. In support of this, 2 days of JZL-184 treatment was required to produce a reversal of the anhedonic phenotype of DAGLα−/− mice. These data are consistent with recent findings demonstrating that pharmacological elevation of 2-AG reduces stress-induced anxiety (Sciolino et al., 2011; Sumislawski et al., 2011; Zhong et al., 2014). Taken together, our data showing that the increase in anxiety and depressive behavior in DAGLα−/− mice is reversed by normalization of deficient 2-AG levels provides causal evidence to support 2-AG signaling deficiency as the mechanism subserving the behavioral phenotype of DAGLα−/− mice. However, some caveats to this interpretation remain. For example, in addition to 2-AG reductions, we detected profound loss of arachidonic acid throughout the brain, potentially confounding our interpretation of the causal role for 2-AG deficiency in our observed phenotypes. However, inhibition of 2-AG degradation also produces dramatic reductions in AA (Long et al., 2009), but reduces anxiety-like and depressive behaviors (Busquets-Garcia et al., 2011; Sciolino et al., 2011; Sumislawski et al., 2011; Zhong et al., 2014); thus, reductions in AA levels cannot explain the anxiety-like behavior observed in DAGLα−/− mice. It is also possible that reductions in levels of other monoacylglycerols such as 2-OG could contribute to the observed phenotype of DAGLα−/− mice; a hypothesis that will require further testing.

Increased anxiety and depressive behaviors in humans and laboratory animals is highly associated with increased activity in the amygdala (Etkin and Wager, 2007; Levine et al., 2001; Roozendaal et al., 2009). Given that one key physiological function of 2-AG signaling is retrograde synaptic suppression, we evaluated the effects of DAGLα deletion on eCB-mediated short-term synaptic suppression at amygdala glutamatergic synapses. BLA DSE was absent in DAGLα−/− mice suggesting an impaired activity-dependent feedback inhibition of BLA glutamatergic transmission in DAGLα−/− mice. These data are consistent with previous studies showing impaired DSE and DSI in hippocampus and cerebellum of DAGLα−/− mice (Gao et al., 2010; Tanimura et al., 2010). Although the behavioral phenotype of DAGLα−/− mice is likely a consequence of perturbed limbic circuit interactions, rather than a consequence of impaired DSE at a single synapse, these data confirm that 2-AG signaling is impaired at one key synapse heavily implicated in the regulation of affective behavior and emotional learning.

These data could also have implications for cannabis use disorders, which are expected to rise given recent shifts in the prohibition against cannabis use. Specifically, the most common reason cited for continued cannabis use in chronic users is reduction in tension and anxiety (Hyman and Sinha, 2009; Reilly et al., 1998), and stress “coping” motives are heavily reported by cannabis users (Bujarski et al., 2012; Chabrol et al., 2005; Fox et al., 2011; Hyman and Sinha, 2009). These data are consistent with the “tension-reduction hypothesis” of substance use, which posits that negative reinforcement is a primary driver of continued substance use and that inherent negative affect associated with anxiety could drive cannabis use in an attempt to reduce symptom severity (see (Buckner et al., 2007)). Our data provide compelling support for an “endocannabinoid deficiency” state causing anxiety and depressive effects (Russo, 2008), which could drive cannabis use in an attempt to relieve these symptoms. That restoration of 2-AG signaling reverses anxiety and depressive behaviors induced by eCB deficiency supports therapeutic approaches aimed at normalizing endocannabinoid deficiency to treat cannabis use disorders and facilitate abstinence in some individuals (Clapper et al., 2009).

CB1 receptor activity and AEA signaling have been heavily implicated in the regulation of anxiety- and depressive-like behaviors (Bambico et al., 2010; Gobbi et al., 2005; Hill and Patel, 2013; Hill et al., 2010; Kathuria et al., 2003; Lutz, 2009; Moreira et al., 2008; Viveros et al., 2005). However, the physiological function of the most abundant eCB ligand in the CNS, 2-AG, has until now remained relatively elusive. Our data provide causal evidence for 2-AG signaling in the physiological regulation of anxiety and depressive-like behaviors and suggest the novel hypothesis that 2-AG deficiency could contribute to the pathogenesis of some mood and anxiety disorders. These data also suggest pharmacological approaches aimed at normalizing 2-AG deficiency could represent a viable eCB-based therapeutic strategy for the treatment of mood and anxiety disorders.

EXPERIMENTAL PROCEDURES

Generation of DAGLα−/− mice

All studies were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Vanderbilt University Institutional Animal Care and Use Committee. DAGLα−/− mice were generated by disruption of exon 8 and were maintained on a C57Bl6/N background by inter-breeding DAGLα+/− mice. Further details are described in Supplemental Experimental Procedures.

Mass spectrometry

Lipids from whole brain and brain regions were extracted in acetonitrile and methanol, respectively, containing the appropriate deuterated standards and analyzed by LC/MS/MS in selective reaction monitoring mode as described previously (Hermanson et al., 2013) and in detail in Supplemental Experimental Procedures.

Electrophysiology

Electrophysiological recordings and DSE experiments were conducted from BLA principle cells in the presence of 50 μM picrotoxin to block fast GABAergic transmission as previously described (Ramikie et al., 2014) and detailed in the Supplemental Experimental Procedures. Membrane properties of BLA neurons were analyzed as previously described (Sumislawski et al., 2011).

Behavioral Analysis

Adult mice (9–16 weeks) were housed on a 12:12 light-dark cycle with lights on at 06:00. All experiments were conducted during the light phase. Food and water were available ad libitum. DAGLα−/− mice were sequentially tested in the open-field test, light-dark box, novelty-induced hypophagia assay, sucrose preference test, tail suspension test, and accelerating rotarod as described in supplemental methods. Tests were conducted at least 48 hours apart. Not all mice were evaluated in every test and male and female cohorts were tested and therefore analyzed separately.

Statistical Analysis

All data were analyzed using GraphPad Prism version 6. Data that included more than 2 groups or 2 factors were analyzed by One-Way or Two-Way ANOVA respectively, as indicated in the figures, with post hoc Sidak’s multiple comparisons test. Multiplicity corrected p values are noted in figures. For analysis of two groups, an unpaired two-tailed t-test was used. For analysis of cumulative feeding latencies, K-S test was used. P<0.05 was considered significant throughout. Data are presented as mean ± SEM throughout.

Supplementary Material

1

Figure S1. Alterations in DAG and 2-OG levels of DAGLα−/− mice, Related to Figure 1.

(A–J) Forebrain levels of various sn-2-AA-containing DAGs in male (M) and female (F) WT and DAGLα−/− mice. Note SAG levels from Fig. 1 are shown in (A) for comparison to less abundant DAG species. (K) Levels of 2-OG in prefrontal cortex (PFC), amygdala and striatum of male and female DAGLα−/− mice 2-way ANOVA results indicated in each panel * P < 0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by Sidak’s post hoc test. Error bars indicate SEM.

Figure S2. Effects of DAGLα deletion on body weight and motor performance evaluated using the accelerating rotarod, Related to Figure 2.

(A–B) No difference in latency to fall was observed between DAGLα−/− and WT mice during three consecutive training sessions. N=9–10 per group. Significance determined by unpaired two-tailed t-test. (C–D) 14–16 week old Female DAGLα−/− mice showed significantly reduced body weight relative to WT littermates; however, there were no significant differences between males at the same age or either gender at 6–8 weeks. * P < 0.05 by Sidak’s post hoc test. Error bars indicate SEM.

Figure S3. Effects of DAGLα deletion on DSE in dorsolateral striatal medium spiny neurons, Related to Figure 3.

DSE time course and total AUC were significantly reduced in the striatum of DAGLα−/− mice relative to WT littermates. * P < 0.05 by two-tailed t-test. Error bars indicate SEM.

Figure S4. Effects of JZL-184 (20 mg/kg) on 2-AG levels in WT mice, Related to Figure 4.

JZL-184 increases 2-AG levels in the amygdala, striatum, and PFC of WT mice. ** P<0.01, **** P<0.0001 by two-tailed t-test. Error bars indicate SEM.

2

Acknowledgments

Generation of chimeric mutant mice was carried out at the Vanderbilt University Transgenic Core facility, eCB measurements were conducted at the Vanderbilt Mass Spectrometry Research Center Facility, all behavioral testing was conducted at the Vanderbilt Neurobehavioral Core Facility. SR141716 was a provided by the NIMH Drug Supply Program. These studies were supported by NIH Grants T32-MH065215 (BCS), R01-NS078291 (RJC), K08-090412 and R01-100096 (S.P), Hobbs Foundation (DGW, SP, RJC), GM15431 (LJM), Rosztoczy Foundation (RB), DA031572 (D.J.H.).

Footnotes

AUTHOR CONTRIBUTIONS

BCS generated DAGLα−/− mice and performed biochemical and rotarod experiments in laboratory of RJC. RJB bred mice and performed behavioral experiments and mass spectrometry analyses, and RJB, BCS, TSR, and RB performed and analyzed electrophysiological experiments in laboratory of SP. BCS completed striatal electrophysiology in the laboratory of RJC. DJH and PJK performed DAG and plasma lipid analysis in laboratory of LJM. DGW contributed to generation of DAGLα−/− mice. BCS, RJB, and SP wrote the paper with input from all authors. SP designed and oversaw the project and is responsible for integrity of all data presented.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1. Alterations in DAG and 2-OG levels of DAGLα−/− mice, Related to Figure 1.

(A–J) Forebrain levels of various sn-2-AA-containing DAGs in male (M) and female (F) WT and DAGLα−/− mice. Note SAG levels from Fig. 1 are shown in (A) for comparison to less abundant DAG species. (K) Levels of 2-OG in prefrontal cortex (PFC), amygdala and striatum of male and female DAGLα−/− mice 2-way ANOVA results indicated in each panel * P < 0.05, ** P<0.01, *** P<0.001, **** P<0.0001 by Sidak’s post hoc test. Error bars indicate SEM.

Figure S2. Effects of DAGLα deletion on body weight and motor performance evaluated using the accelerating rotarod, Related to Figure 2.

(A–B) No difference in latency to fall was observed between DAGLα−/− and WT mice during three consecutive training sessions. N=9–10 per group. Significance determined by unpaired two-tailed t-test. (C–D) 14–16 week old Female DAGLα−/− mice showed significantly reduced body weight relative to WT littermates; however, there were no significant differences between males at the same age or either gender at 6–8 weeks. * P < 0.05 by Sidak’s post hoc test. Error bars indicate SEM.

Figure S3. Effects of DAGLα deletion on DSE in dorsolateral striatal medium spiny neurons, Related to Figure 3.

DSE time course and total AUC were significantly reduced in the striatum of DAGLα−/− mice relative to WT littermates. * P < 0.05 by two-tailed t-test. Error bars indicate SEM.

Figure S4. Effects of JZL-184 (20 mg/kg) on 2-AG levels in WT mice, Related to Figure 4.

JZL-184 increases 2-AG levels in the amygdala, striatum, and PFC of WT mice. ** P<0.01, **** P<0.0001 by two-tailed t-test. Error bars indicate SEM.

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