The Lateral Habenula Directs Coping Styles Under Conditions of Stress via Recruitment of the Endocannabinoid System

Abstract

Background: The ability to effectively cope with stress is a critical determinant of disease susceptibility. The lateral habenula (LHb) and the endocannabinoid (ECB) system have independently been shown to be involved in the selection of stress coping strategies, yet the role of ECB signaling in the LHb remains unknown.

Methods: Using a battery of complementary techniques in rats, we examined the localization of type-1 cannabinoid receptors (CB₁Rs) and assessed the behavioral and neuroendocrine effects of intra-LHb CB₁R manipulations. We further tested the extent to which the ECB system in the LHb is impacted following chronic unpredictable stress or social defeat stress, and whether manipulation of LHb CB₁Rs can bias coping strategies in rats with a history of chronic stress.

Results: Electron microscopy studies revealed CB₁R expression on presynaptic axon terminals, postsynaptic membranes, mitochondria, and glial processes in the rat LHb. In vivo microdialysis experiments indicated that acute stress increased the amount of 2-arachidonoylglycerol in the LHb, while intra-LHb CB₁R blockade increased basal corticosterone, augmented proactive coping strategies, and reduced anxiety-like behavior. Basal LHb 2-arachidonoylglycerol content was similarly elevated in rats that were subjected to chronic unpredictable stress or social defeat stress and positively correlated with adrenal weight. Finally, intra-LHb CB₁R blockade increased proactive behaviors in response to a novel conspecific, increasing approach behaviors irrespective of stress history and decreasing the latency to be attacked during an agonistic encounter.

Conclusions: Alterations in LHb ECB signaling may be relevant for development of stress-related pathologies in which LHb dysfunction and stress-coping impairments are hallmark symptoms.

Keywords: Anxiety; CB(1) receptor; Endocannabinoid; Lateral habenula; Rat; Stress coping.

Figure 1. Type-1 cannabinoid receptors (CB1R) are present on presynaptic terminals, postsynaptic dendrites, and glial cells in the lateral habenula (LHb)

(A-D) Representative electron microscopy images illustrating the presence of CB1R-positive immunogold labeling in the rat LHb. (E) No CB1R labeling was observed in CB1R-KO LHb. Black arrows indicate synapses, small white arrows indicate CB1R-positive immunoparticles on excitatory and inhibitory presynaptic terminals (ter) impinging on postsynaptic dendrites (den), and large white arrows indicate CB1R-positive immunoparticles on glial cells which are indicated with a black asterisk. Scale bars = 500 nm. (F) Bar graph illustrating the percentage of CB1R-positive immunoparticles on excitatory and inhibitory presynaptic terminals (black - horizontal lines), postsynaptic membranes forming synapses with excitatory or inhibitory terminals (white - vertical lines), and glial cells (gray) in the LHb.

Figure 2. Acute restraint stress mobilizes 2-arachidonoylglycerol (2-AG) in the lateral habenula (LHb)

In vivo microdialysis recordings revealed a significant effect of time on the concentration of (A) 2-AG (F_17,136 =3.50, p<0.001, ηp² =0.30) and (B) anandamide (AEA) (F_17,136 =3.30, p<0.001, ηp² =0.29) in the LHb. Area under the curve (AUC) analysis comparing the 30-min pre-stress epoch to the final 30-min post-stress epoch revealed a significant effect of immobilization stress to increase the concentration of (C) 2-AG (t₃₆=2.79, p=0.01, d=3.0), but not (D) AEA (t₃₆=0.96, p=0.69, d=0.82) in the LHb. n=10, * denotes significance at p<0.05.

Figure 3. Local manipulation of type-1 cannabinoid receptors (CB1R) in the lateral habenula (LHb) alters basal corticosterone (CORT) secretion

Animals received an intra-LHb infusion of the CB1R agonist WIN 55212-2 (WIN), the CB1R antagonist rimonabant (RIM), or vehicle (VEH) 15 min prior to a 30-min restraint stress episode and blood samples were collected to assay circulating CORT prior to stress onset (i.e., time 0) and at 30, 60, and 90 min post-stress onset. Acute stress exposure produced a significant elevation in CORT in both (A) males and (C) females (Time: F_3,267=83.87, p<0.001, ηp²=0.15), with females exhibiting higher corticosterone levels at all time points (Time × Sex interaction: F_3,267=16.98, p<0.001, ηp2 =0.16). Subsequent within-sex analyses revealed that intra-LHb WIN administration significantly reduced CORT concentrations at time 0 in males (t₂₇=2.84, p=0.01, d=1.12) and females (t₃₀=2.94, p=0.01, d=1.04), whereas intra-LHb RIM administration significantly increased CORT in males t₃₃=−2.74, p=0.01, d=0.95) but not females (t₂₉=0.34, p=0.74, d=0.49). Area under the curve (AUC) analyses indicated that intra-LHb WIN administration decreased CORT in (B) males (t₂₇=1.82, p=0.04, d=0.70) but not (D) females (t₃₀=0.33, p=0.37, d=0.12). n= 12-19/group/sex; * denotes significance at p<0.05.

Figure 4. Local manipulation of type-1 cannabinoid receptors (CB1R) in the lateral habenula (LHb) dictates coping strategies in the forced swim test (FST)

Rats received an intra-LHb infusion of the CB1R agonist WIN 55212-2 (WIN), the CB1R antagonist rimonabant (RIM), or vehicle (VEH) 15 min prior to a 5-min forced swim session where time spent immobile, swimming, and struggling were quantified. There was a main effect of treatment on (A) immobility time (F_3,92=32.51, p<0.001, ηp² =0.54), (B) swimming time (F_3,92=6.49, p<0.001, ηp²=0.19), and (C) struggling time (F_3,92=7.91, p<0.001, ηp²=0.22), and a sex × treatment interaction but no consistent effect of sex for immobility (F_2,58=31.03, p =<0.001, ηp² =0.52 and F_1,58=2.44, p=0.12, ηp² =0.04), for struggling (F_2,58=7.32, p = 0.001, ηp² =0.20 and F_1,58=1.13, p=0.31, ηp² =0.02), and swimming (F_2,58=2.18, p =0.12, ηp² =0.07 and F_1,58=5.05, p=0.03, ηp² =0.08). Post-hoc analyses revealed that intra-LHb RIM treatment (A) decreased total immobility time (male: t₂₉=2.08, p=0.05, d=0.84, female: t₂₅=5.01, p<0.001, d=1.93), (B) had no significant effect on swimming time (male: t₂₉=1.44, p=0.16, d=0.56, female: t₂₅=1.43, p=0.18, d=0.41) and (C) significantly increased struggling time (male: t₂₉=2.44, p=0.02, d=0.96; female: t₂₅=−4.07, p<0.001, d=1.56). Conversely, intra-LHb WIN treatment (A) significantly increased total immobility time in both sexes (male: t₂₉=6.04, p<0.001, d=2.07; female: t₂₆=−3.73, p<0.001, d=1.41), and (B) decreased swimming time in males (t₂₉=4.33, p<0.001, d=1.61) but not females (t₂₆=1.40, p=0.19, d=0.74), with (C) no differences in struggling time in either sex (male: t₂₉=0.98, p=0.34, d=0.36, female: t₂₆=0.44, p=0.67, d=0.16). n=12-19/group/sex; * denotes significance at p<0.05.

Figure 5. Local blockade of type-1 cannabinoid receptors (CB1R) in the lateral habenula (LHb) decreases anxiety-like behavior

Rats received an intra-LHb infusion of the CB1R agonist WIN 55212-2 (WIN), the CB1R antagonist rimonabant (RIM), or vehicle (VEH) 15 min prior testing in either the elevated plus maze (EPM) or novelty-suppressed feeding test (NSFT). (A) With respect to the percentage of time spent exploring the open arms of the EPM, there was a main effect of treatment (F_2,86=3.98, p=0.03, ηp²=0.14), a main effect of sex (F_1,86=41.89, p<0.001, ηp²=0.45), but no treatment × sex interaction (F_2,86=0.53, p=0.59, ηp²=0.02). Post-hoc analyses revealed that intra-LHb RIM administration significantly increased the percentage of time spent exploring the open arms in male (t₂₀=−2.26, p=0.04, d=0.96) and female (t₂₄=−2.24, p=0.04, d=1.0) rats compared to VEH. (B) There was no effect of treatment on the total number of open arm entries (F_2,86=2.69, p=0.08, ηp²=0.10), a main effect of sex (F_1,86=19.80, p<0.001, ηp²=0.28), and no significant interaction treatment × sex interaction (F_2,86=0.24, p=0.79, ηp²=0.01). (C) There was a main effect of treatment on the total distance travelled in the open arms of the EPM (F_2,86=4.17, p=0.02, ηp²=0.14), a main effect of sex (F_1,86=33.17, p<0.001, ηp² =0.39), and no significant treatment × sex interaction (F_2,86=0.31, p=0.74, ηp²=0.01). Post-hoc analyses revealed that, in male rats, the total distance travelled in the open arms was significantly increased following intra-LHb RIM (t₂₀=−2.45, p=0.02, d=1.03) or WIN (t₂₂=−2.55, p=0.02, d=1.04) treatment compared to VEH. In female rats, there was no effect of RIM (t₂₅=−1.73, p=0.11, d=0.93) or WIN (t₁₃=−1.89, p=0.08, d=0.99) on distance travelled in the open arms. (D) In the NSFT, there was no effect of treatment (F_2,86=0.15, p=0.86, ηp²=0.01), sex (F_{1, 86}=0.20, p=0.66, ηp²<0.01), or treatment × sex interaction (F_2,86=1.39, p=0.26, ηp²=0.05) on the latency to approach the peanut butter chips. (E) However, with respect to the latency to consume the peanut butter chips, there was a main effect of treatment (F_2,86=0.02, p=0.98, ηp²<0.01), sex (F_1,86=28.35, p<0.001, ηp²=0.26), and a significant treatment × sex interaction (F_3,86=5.06, p=0.003, ηp²=0.16). Post-hoc analyses revealed that intra-LHb RIM administration significantly decreased the latency to consume the peanut butter chips compared to VEH in male (t₂₀=3.25, p=0.004, d=1.39), but not female (t₂₅=−1.46, p=0.16, d=0.58) rats. (F) There was no effect of treatment (F_2,86=0.64, p=0.53, ηp²=0.05), sex (F_2,86=0.79, p=0.38, ηp²=0.02), or treatment × sex interaction (F_2,86=1.80, p=0.18, ηp²=0.07) with respect to the weight of peanut butter chips consumed in the home cage after the test, thus indicating that the effects observed were not due to treatment-induced hyperphagia. n=10-15/group/sex; * denotes significance at p<0.05.

Figure 6. Chronic unpredictable stress (CUS), but not social defeat stress (SDS), increases bursting activity of lateral habenula (LHb) cells in vivo

(A) There was a main effect of CUS to increase the firing rate of LHb neurons compared to non-stressed rats (F_1,439=21.30, p<0.0001, ηp²=0.046). There was also a main effect of sex, with females exhibiting reduced LHb firing rates compared to males (F_1,439=7.65, p=0.006, ηp²=0.017), but no significant interaction (F_1,439=0.39, p=0.53, ηp²=0.001). When SDS-exposed rats were compared to non-stressed male rats, there was no significant difference in LHb firing rate (t₂₂₇=−0.30, p=0.76, d=0.04). (B) There was no effect of CUS on the number of spontaneously active LHb cells encountered per track (F_1,100=0.02, p=0.90, ηp²=0.0002), no main effect of sex (F_1,100=0.72, p=0.40, ηp²=0.007), and no significant interaction (F_1,100=0.43, p=0.51, ηp²=0.004). However, SDS-exposed rats exhibited fewer spontaneously active LHb cells compared to non-stressed male rats (t₆₀=2.104, p=0.04, d=0.51) and non-stressed female rats (t₅₈=2.539, p=0.014, d= 0.11). (C) There was a main effect of CUS on the percentage of spikes occurring in burst (F_1,439=24.51, p<0.0001, ηp²=0.052), a main effect of sex (F_1,439=5.83, p=0.016, ηp²=0.013), and a significant interaction (F_1,439=7.01, p=0.0084, ηp²=0.015). Tukey’s post-hoc tests revealed that male (but not female) rats experienced an increase in the percentage of spikes occurring in burst (male: t₂₂₄=5.43, p<0.05, d=0.72; female: t₂₁₅=1.61, p>0.05, d=0.22). There was no difference between SDS-exposed males and non-stressed males (t₂₂₉=0.97, p=0.33, d=0.13). (D) There was a main effect of CUS to increase the mean number of spikes per burst (F_1,439=20.29, p<0.0001, ηp²=0.043), a main effect of sex (F_1,439=4.49, p=0.035, ηp²=0.010), but no significant interaction (F1,439=0.08, p=0.78, ηp²<0.0001). There was no difference between SDS and non-stressed male rats (t₂₂₉=0.79, p=0.43, d=0.0014). (E) With respect to the mean burst interspike interval (ISI), there was a main effect of CUS (F_1,439=21.10, p<0.0001, ηp²=0.045), a main effect of sex (F_1,439=16.82, p<0.0001, ηp²=0.036), and a significant interaction (F_1,439=6.41, p=0.012, ηp²=0.014). Post-hoc tests revealed that non-stressed female rats had a significantly shorter burst ISI compared to male non-stressed rats (t₂₂₄=4.62, p<0.05, d=0.62). Additionally, CUS-exposed female rats had a significantly longer burst ISI compared to non-stressed females (t₂₁₅=4.99, p<0.05, d=0.68). SDS-exposed male rats did not differ significantly from non-stressed male rats (t₂₂₆=0.45, p=0.65, d=0.0059). (F) There was a significant main effect of CUS to increase the mean burst length, (F_1,439=16.97, p<0.0001, ηp²=0.036), no effect of sex (F_1,439=2.54, p=0.11, ηp²=0.006), and no significant interaction (F_1,439=0.11, p=0.74, ηp²<0.0001). SDS-exposed male rats did not differ significantly from non-stressed male rats (t₂₂₉=1.20, p=0.23, d=0.16). n=5/group/sex; * denotes significance at p<0.05.

Figure 7. Six weeks of chronic unpredictable stress (CUS) exposure increases 2-arachidonoylglycerol (2-AG) content, alters monoacylglycerol lipase (MAGL) hydrolytic activity, and decreases type-1 cannabinoid receptor (CB1R) binding in the lateral habenula (LHb)

(A) There was a main effect of CUS to increase 2-AG content in the LHb (F_1,17=24.85, p<0.001, ηp²=0.59), a main effect of sex (F_1,17=24.84, p<0.001, ηp² =0.59), but no stress × sex interaction (F_1,17=2.0, p=0.18, ηp²=0.11). (B) With respect to anandamide (AEA) content, there was no effect of stress (F_1,17=0.11, p=0.74, ηp²=0.01), however, females had significantly higher AEA levels compared to males (F_1,17=11.75, p=0.003, ηp²=0.41) and there was no stress × sex interaction (F_1,17=0.13, p=0.72, ηp²=0.01). Both (C) 2-AG and (D) AEA were positively correlated with scaled adrenal weights (2-AG: r=0.459, p=0.005; AEA: r=0.707, p<0.001). (E) In males, the hydrolytic activity (V_max) of MAGL in the LHb was significantly increased in CUS-exposed rats compared to non-stressed rats (t₉=−2.71, p=0.02, d=1.56). (F) In females, there was a trend for CUS to increase the V_max of MAGL in the LHb (t₈=−1.94, p=0.09, d=1.56). (G) The K_M of MAGL was also significantly increased in CUS-exposed male rats (t₉=−2.29, p=0.05, d=1.34). (H) In females, there was once again a trend for CUS to increase the MAGL K_M in the LHb (t₈=−2.17, p=0.06, d=1.38). (I and J) In males, the B_max and K_D of CB1Rs in the LHb were both significantly decreased following CUS exposure (B_max: t₉=3.38, p=0.004, d=2.17; K_D: t₉=3.06, p=0.007, d=1.96). (K and L) In females, the B_max and K_D for CB1Rs in the LHb was similarly reduced following CUS exposure, however, this did not reach statistical significance (B_max: t₉=1.84, p=0.099, d=1.16; K_D: t₉=1.36, p=0.21, d=0.85). n= 4-6/group/sex; * denotes significance at p<0.05.

Figure 8. Local manipulation of type-1 cannabinoid receptors (CB1R) in the lateral habenula (LHb) dictates coping strategies in chronically stressed male rats

Male rats were exposed to seven days of social defeat stress (SDS), and LHb tissue was collected 24 hr after the final defeat episode (under stress free conditions). SDS exposure significantly increased (A) 2-arachidonoylglycerol (2-AG) (F_1,45=10.74, p=0.002, ηp² =0.19) and (B) anandamide (AEA) (F_1,45=6.57, p=0.01, ηp² =0.13) content compared to non-stressed rats. Both (C) 2-AG and (D) AEA content were positively correlated with scaled adrenal weights (2-AG: r=0.27, p=0.04; AEA: r=0.25, p=0.05). A separate cohort of rats was implanted with bilateral cannula aimed at the LHb and the CB1R agonist WIN 55212-2 (WIN), the CB1R antagonist rimonabant (RIM), or vehicle (VEH) were administered 15 min prior to the final SDS episode. There was no effect of treatment on (E) the latency to defeat (F_2,26=1.50, p=0.24, ηp²=0.10), however, there was a significant effect of treatment on (F) the latency to be attacked (F_2,26=10.20, p=0.001, ηp²=0.44). Post-hoc analyses confirmed that intra-LHb RIM administration significantly increased the latency to be attacked compared to VEH (t₁₈=−3.11, p=0.01, d=1.42), while intra-LHb WIN administration failed to significantly affect the latency to be attacked (t₁₇=1.10, p=0.29, d=0.50). (G) Additionally, RIM-treated animals engaged in significantly more approach behaviors during the SDS episode compared to VEH-treated animals (t₁₈=−3.65, p=0.002, d=1.63), while WIN-treated animals were not significantly different from VEH (t₁₇=0.36, p=0.72, d=0.17). The same effect was also observed in non-stressed rats interacting with a non-aggressive conspecific (t₁₀=−4.69, p=0.001, d=2.64), whereas WIN-treated rats did not differ significantly from VEH-treated rats with respect to the frequency of approach behaviors (t₁₀=−0.09, p=0.93, d=0.05). (H) Intra-LHb WIN treatment significantly increased the frequency of avoidance behaviors in both SDS-exposed (t₁₇=−3.33, p=0.004, d=1.45) and non-stressed (t₁₀=−3.41, p=0.01, d=1.99) rats. Conversely, intra-LHb RIM treatment did not significantly affect the frequency of avoidance behaviors in SDS-exposed (t₁₈=−1.11, p=0.29, d=0.49) or non-stressed (t₁₀=1.11, p=0.29, d=0.63) rats. For biochemical assays, control n=11 and SDS n=36. For behavioral assays, n=6/group for control experiments and n=9-10/group for SDS experiments. * denotes significance at p<0.05.