Functional Redundancy Between Canonical Endocannabinoid Signaling Systems in the Modulation of Anxiety

Abstract

Background: Increasing the available repertoire of effective treatments for mood and anxiety disorders represents a critical unmet need. Pharmacological augmentation of endogenous cannabinoid (eCB) signaling has been suggested to represent a novel approach to the treatment of anxiety disorders; however, the functional interactions between two canonical eCB pathways mediated via anandamide (N-arachidonylethanolamine [AEA]) and 2-arachidonoylglycerol (2-AG) in the regulation of anxiety are not well understood.

Methods: We utilized pharmacological augmentation and depletion combined with behavioral and electrophysiological approaches to probe the role of 2-AG signaling in the modulation of stress-induced anxiety and the functional redundancy between AEA and 2-AG signaling in the modulation of anxiety-like behaviors in mice.

Results: Selective 2-AG augmentation reduced anxiety in the light/dark box assay and prevented stress-induced increases in anxiety associated with limbic AEA deficiency. In contrast, acute 2-AG depletion increased anxiety-like behaviors, which was normalized by selective pharmacological augmentation of AEA signaling and via direct cannabinoid receptor 1 stimulation with Δ⁹-tetrahydrocannabinol. Electrophysiological studies revealed 2-AG modulation of amygdala glutamatergic transmission as a key synaptic correlate of the anxiolytic effects of 2-AG augmentation.

Conclusions: Although AEA and 2-AG likely subserve distinct physiological roles, a pharmacological and functional redundancy between these canonical eCB signaling pathways exists in the modulation of anxiety-like behaviors. These data support development of eCB-based treatment approaches for mood and anxiety disorders and suggest a potentially wider therapeutic overlap between AEA and 2-AG augmentation approaches than was previously appreciated.

Keywords: 2-Arachidonoylglycerol; Amygdala; Anxiety; JZL184; MAGL inhibition; Stress.

Figure 1. Acute MAGL inhibition elevates brain 2-AG signaling and reduces anxiety-like behavior

(A–F) Brain levels of 2-AG, AA, NA-Gly, 2-OG, AEA and OEA from mice treated with JZL184 at indicated dosages (3–15 mg kg⁻¹ intraperitoneally) after 3 h. (G–H) Brain and plasma levels of JZL184 after systemic exposure. (I–L) Plasma-brain correlations for JZL184, 2-AG and AEA. (M-R) Effects of JZL184 on (M) light time, (N) light distance, (O) total distance traveled by time course, and on (P) % light time, (Q) % light distance and (R) total distance travelled in 10 min. Significant F and P values from one-way and two-way ANOVA noted above bar graphs; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. vehicle by Holm-Sidak post hoc multiple comparisons test in bar graphs. Linear regression (solid line) with 95% confidence intervals (dashed lines) shown in figures. 2-AG, 2-arachidonoyl glycerol; 2-OG, 2-oleoylglycerol; AEA, N-arachidonylethanolamine; OEA, oleoylethanolamine; AA, arachidonic acid and NAGly, N-arachidonoyl glycine. Data are presented as means ± s.e.m.

Figure 2. MAGL inhibition reduces stress-induced anxiety-like behavior

(A–C) The effect of 30-min restraint stress on tissue content of AEA, 2-AG and corticosterone within the amygdala and PFC. (D) The magnitude of corticosterone secretion positively correlated with 2-AG. (E–J) Effects of JZL184 and PF-3845 systemic administration on (E) light time, (F) light distance, (G) total distance traveled by time course, and on (H) % light time, (I) % light distance and (J) total distance travelled in 10 min. Significant F and P values from t-test, one-way and two-way ANOVA noted above bar graphs; *p<0.05, **p<0.01, ***p<0.001****p<0.0001 vs. stress group by Holm-Sidak post hoc multiple comparisons test in bar graphs. Data are presented as means ± s.e.m.

Figure 3. CB1 receptor agonist CP55940 reduces stress-induced anxiety-like behavior

(A–F) Effects of CP55940 (0.001–0.3 mg/kg) 45 min prior to behavioral testing on (A) light time, (B) light distance, (C) total distance traveled by time course, and on (D) % light time, (E) % light distance and (F) total distance travelled in 10 min. Data are presented as means ± s.e.m. Significant F and P values from one-way and two-way ANOVA noted above bar graphs; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs. stress group by Holm-Sidak post hoc multiple comparisons test in bar graphs. Data are presented as means ± s.e.m.

Figure 4. Amygdala glutamatergic transmission predicts anxiolytic efficacy of 2-AG augmentation

(A) Schematic diagram depicts the recording of excitatory glutamatergic transmission in the basolateral amygdala (BLA) from brain slices incubated with JZL184 and vehicle. (B–C) JZL184 reduces spontaneous excitatory postsynaptic current (sEPSC) frequency (B). Representative traces of vehicle and JZL184 sEPSC (C). (D–G) JZL184 increased intrinsic excitability (D), reduced action potential threshold (E) and did not change action potential amplitude (F). Representative traces of vehicle and JZL184 action potentials at maximum current injection (G). (H–K) JZL184 increased membrane input resistance (H) and did not affect membrane time constant (I), after hyperpolarization amplitude (J), or resting membrane potential (K). (L) Schematic diagram depicts the recording of excitatory glutamatergic transmission in the BLA of brain slices from mice systemically treated with JZL184 or vehicle. (M–O) Effects of stress exposure and JZL184 treatment on the sEPSC frequency of pyramidal neurons of BLA. Representative traces of vehicle and JZL184 sEPSC (N). (P–R) Correlation between % light time and sEPSC frequency of BLA pyramidal neurons of vehicle treated non-stressed, stressed and JZL184 treated stressed mice (P), vehicle treated non-stressed and stressed mice (Q) and JZL184 treated stressed mice [<15% LT and >15% LT; (R)]. Significant F and P values from t test and one way ANOVA noted above bar graphs; *p<0.05, **p<0.01, ****p<0.0001 vs. control group by Holm-Sidak post hoc multiple comparisons test in bar graphs. Linear regression (solid line) with 95% confidence intervals (dashed lines) shown in figures. Data are presented as means ± s.e.m.

Figure 5. FAAH inhibition and THC administration attenuate 2-AG deficiency-induced anxiety-like behavior

(A–D) 2-AG, AA, AEA and OEA brain levels from the mice treated with DO34 and DO34+PF-3845. (E–J) Effects of DO34 and DO34+PF-3845 on (E) light time, (F) light distance, (G) total distance traveled by time course, and on (H) % light time, (I) % light distance and (J) total distance travelled in 10 min. (K–P) Effects of DO34 and DO34+THC on (K) light time, (L) light distance, (M) total distance traveled by time course, and on (N) % light time, (O) % light distance and (P) total distance travelled in 10 min. Significant F and P values from one-way and two-way ANOVA noted above bar graphs; *p<0.05, ***p<0.001 vs vehicle and ^#p<0.05, ^##p<0.01 ^###p<0.001 vs DO34 by Holm-Sidak post hoc multiple comparisons test in bar graphs. 2-AG, 2-arachidonoyl glycerol; AA, arachidonic acid; AEA, N-arachidonylethanolamine and OEA, oleoylethanolamine. Data are presented as means ± s.e.m.