Role of endocannabinoid signaling in a septohabenular pathway in the regulation of anxiety- and depressive-like behavior

Abstract

Enhancing endocannabinoid signaling produces anxiolytic- and antidepressant-like effects, but the neural circuits involved remain poorly understood. The medial habenula (MHb) is a phylogenetically-conserved epithalamic structure that is a powerful modulator of anxiety- and depressive-like behavior. Here, we show that a robust endocannabinoid signaling system modulates synaptic transmission between the MHb and its sole identified GABA input, the medial septum and nucleus of the diagonal band (MSDB). With RNAscope in situ hybridization, we demonstrate that key enzymes that synthesize or degrade the endocannabinoids 2-arachidonylglycerol (2-AG) or anandamide are expressed in the MHb and MSDB, and that cannabinoid receptor 1 (CB1) is expressed in the MSDB. Electrophysiological recordings in MHb neurons revealed that endogenously-released 2-AG retrogradely depresses GABA input from the MSDB. This endocannabinoid-mediated depolarization-induced suppression of inhibition (DSI) was limited by monoacylglycerol lipase (MAGL) but not by fatty acid amide hydrolase. Anatomic and optogenetic circuit mapping indicated that MSDB GABA neurons monosynaptically project to cholinergic neurons of the ventral MHb. To test the behavioral significance of this MSDB-MHb endocannabinoid signaling, we induced MSDB-specific knockout of CB1 or MAGL via injection of virally-delivered Cre recombinase into the MSDB of Cnr1^loxP/loxP or Mgll^loxP/loxP mice. Relative to control mice, MSDB-specific knockout of CB1 or MAGL bidirectionally modulated 2-AG signaling in the ventral MHb and led to opposing effects on anxiety- and depressive-like behavior. Thus, depression of synaptic GABA release in the MSDB-ventral MHb pathway may represent a potential mechanism whereby endocannabinoids exert anxiolytic and antidepressant-like effects.

Fig. 1.. Expression and function of the endocannabinoid system in the MHb.

a-c. DAGLα, NAPE-PLD, and FAAH mRNA are expressed in the MHb in both Tac1- and ChAT-expressing neurons of the dorsal and ventral MHb. d. mRNA puncta density of eCB system components. ChAT+ neurons had a significantly higher mRNA puncta density than Tac1+ neurons (two-way ANOVA significant main effect: F_1,42 = 14.803, p < 0.001). DAGLα puncta density was significantly higher than NAPE-PLD (p < 0.001) and FAAH puncta density (p < 0.001). FAAH puncta density was significantly higher than NAPE-PLD puncta density (p < 0.001). e. Depolarization (from −70 to 0 mV, 5 s) induced DSI (n = 15 neurons/N = 5 mice), which was blocked by pretreatment with AM251 (p < 0.001, n = 9 neurons/N = 2 mice, vs. Control) or DO34 (p = 0.017, n = 7 neurons/N = 3 mice, vs. Control). The solid line is a single exponential fitting of the decay of DSI. f,g. Perfusion of WIN55212–2 suppressed IPSC amplitude and increased the PPR (50 ms inter-pulse interval; t₈ = 3.3, p = 0.011, n = 9 neurons/N = 5 mice; paired t-test).

Fig. 2.. The MSDB sends an exclusively GABAergic projection to cholinergic neurons in the ventral MHb.

a. Anterograde tracing strategy: cell bodies and axons express tdTomato and putative synaptic puncta express SypEGFP. b,c. tdTomato and SypEGFP expression in the MSDB (b) and MHb (c) after injection of AAV1-Flex-tdTomato-T2A-SypEGFP in the MSDB (n = 3 mice). d. Optogenetic circuit mapping strategy: ChR2(H134R)-GFP was expressed in the MSDB and whole-cell recordings of tdTomato+ MHb neurons were made in ChAT-tdTomato reporter mice. e. Expression of ChR2-GFP in the MSDB. f. ChR2-GFP+ axon terminals were observed in the ventral MHb, where they clustered around tdTomato+ neurons. g. Blue (473 nm) light activation of ChR2 on MSDB afferents evoked IPSCs in ventral MHb neurons which were blocked by picrotoxin (n = 9 neurons/N = 3 mice) but were unaffected by CNQX (n = 9 neurons/N = 3 mice). h. I-V curve of light-evoked IPSCs shows an average reversal potential (E_rev) of −27.8 mV (n = 15 neurons/N = 4 mice). Inset: representative light-evoked IPSCs at different junction potential-corrected holding potentials (rounded to nearest integer). Abbreviations: 3V = third ventricle; fr = fasciculus retroflexus; LHb = lateral habenula; MHb = medial habenula; PVT = paraventricular thalamus.

Fig. 3.. CB1 receptor mRNA is predominantly expressed in GABA neurons in the MSDB.

a. Top: CB1 and Gad1 mRNA are highly expressed in the MSDB. Bottom: There is a high degree of CB1 and Gad1 co-expression in the MSDB. b. Percentage of cells in the MSDB co-expressing CB1 and/or Gad1, VGluT2, or ChAT, as determined by RNAscope in situ hybridization.

Fig. 4.. 2-AG from ventral MHb neurons suppresses MSDB GABA input and is limited by MAGL.

a. AAV_DJ-DIO-ChR2(H134R)-GFP was injected into the MSDB and ChR2-expressing axon terminals were observed bilaterally in the MHb. b. DSI was induced in MHb neurons when IPSCs were evoked by blue laser stimulation of ChR2-expressing MSDB axon terminals. DSI was blocked by AM251 pretreatment (t₁₈ = 6.09, p < 0.001, vehicle: n = 12 neurons/N = 4 mice, AM251: n = 8 neurons/N = 3 mice). c. AAV1-hSyn-Cre-eGFP was injected into the MSDB to induce schematic for MSDB-specific knockout of CB1 or MAGL. d. DSI was abolished in MSDB-CB1-KO mice (MSDB-WT vs. MSDB-CB1-KO, p = 0.023, MSDB-WT: n = 13 neurons/N = 3 mice, MSDB-CB1-KO: n = 8 neurons/N = 3 mice). DSI was prolonged in MSDB-MAGL-KO mice, indicated by an increased time constant of the decay of DSI (MSDB-WT vs. MSDB-MAGL-KO, t₁₉ = −2.22, p = 0.039, MSDB-WT: n = 10 neurons/N = 3 mice, MSDB-MAGL-KO: n = 11 neurons/N = 2 mice). The solid lines are single exponential fits of the decay of DSI.

Fig. 5.. MSDB-specific CB1 or MAGL knockout has opposing effects on anxiety- and depressive-like behavior.

a. Timeline of AAV injection and behavior tests. b. Open field distance traveled was not significantly altered by MSDB knockout (F_2,34 = 1.524, p = 0.232). Center time in the open field was significantly decreased in MSDB-CB1-KO mice relative to MSDB-WT and MSDB-MAGL-KO mice (F_2,34 = 7.169, p = 0.003; MSDB-CB1-KO vs. MSDB-WT, p = 0.028; MSDB-CB1-KO vs. MSDB-MAGL-KO, p = 0.002). The number of center entries was significantly reduced in MSDB-CB1-KO mice relative to MSDB-WT and MSDB-MAGL-KO mice (F_2,34 = 8.207, p = 0.001; MSDB-CB1-KO vs. MSDB-WT, p = 0.004; MSDB-CB1-KO vs. MSDB-MAGL-KO, p = 0.003). The percent distance traveled in the center was significantly reduced in MSDB-CB1-KO mice relative to MSDB-MAGL-KO mice, but was not significantly altered in MSDB-CB1-KO mice relative to MSDB-WT mice (F_2,34 = 4.571, p = 0.017; MSDB-CB1-KO vs. MSDB-WT, p = 0.056; MSDB-CB1-KO vs. MSDB-MAGL-KO, p = 0.020). MSDB-WT: n = 12; MSDB-CB1-KO: n = 11; MSDB-MAGL-KO: n = 14. c. Marble burying behavior was increased in MSDB-CB1-KO mice (F_2,34 = 8.076, p = 0.001; MSDB-CB1-KO vs. MSDB-WT mice, p = 0.015; MSDB-CB1-KO vs. MSDB-MAGL-KO mice, p = 0.002). d. Novelty-suppressed feeding changes induced by MSDB-specific CB1 or MAGL KO (H = 22.461, p < 0.001; MSDB-CB1-KO vs. MSDB-WT, p = 0.0035; MSDB-CB1-KO vs. MSDB-MAGL-KO, p < 0.001). Home cage feeding behavior was not significantly altered between groups (H = 6.149, p = 0.046; MSDB-WT vs. MSDB-CB1-KO, p = 0.0803; MSDB-WT vs. MSDB-MAGL-KO, p > 0.9999; MSDB-CB1-KO vs. MSDB-MAGL-KO, p = 0.1295). e. Sucrose preference increased in MSDB-MAGL-KO mice (F_2,34 = 6.060, p = 0.006; MSDB-MAGL-KO vs. MSDB-WT mice, p = 0.026; MSDB-MAGL-KO vs. MSDB-CB1-KO mice, p = 0.011). (c-e) MSDB-WT: n = 9; MSDB-CB1-KO: n = 13; MSDB-MAGL-KO: n = 15.