Limiting habenular hyperactivity ameliorates maternal separation-driven depressive-like symptoms

Abstract

Early-life stress, including maternal separation (MS), increases the vulnerability to develop mood disorders later in life, but the underlying mechanisms remain elusive. We report that MS promotes depressive-like symptoms in mice at a mature stage of life. Along with this behavioral phenotype, MS drives reduction of GABA_B-GIRK signaling and the subsequent lateral habenula (LHb) hyperexcitability-an anatomical substrate devoted to aversive encoding. Attenuating LHb hyperactivity using chemogenetic tools and deep-brain stimulation ameliorates MS depressive-like symptoms. This provides insights on mechanisms and strategies to alleviate stress-dependent affective behaviors.

Fig. 1

MS-induced depressive-like symptoms and hyperexcitability. a MS protocol (all schematics are original drawing made by authors). b Bar graph and scatter plot of failures in the shuttle box for control (Ctrl) and MS mice (Ctrl vs. MS; n _mice = 24, unpaired t-test, t₄₅ = 3.36 **p < 0.01). c Same as b for TST immobility (Ctrl vs. MS; n _mice = 6–7; unpaired t-test, t₁₁ = 4.63; ***p < 0.001). d Same as b but for sucrose preference (Ctrl vs. MS; n _mice = 16; unpaired t-test, t₃₀ = 32.12; *p < 0.05). e Top, sample traces from Ctrl and MS mice of current-evoked firing (50 pA step). Bottom, action potentials vs. injected current (0–100 pA, steps of 10 pA) in all experimental groups (Ctrl vs. MS; aCSF, nmice = 6 per group; ncells = 23 per group; two-way ANOVA-RM, interaction, F_(10;440) = 2.74; **p < 0.01). Scale bars=0.2 s and 20 mV

Fig. 2

MS drives GABA_B-GIRK plasticity in LHb. a Sample traces, bar graph and scatter plots depicting CGP54626-sensitive I-Baclofen (I-Baclofen Ctrl vs. MS; n _mice = 5/6; n _cells = 13; unpaired t-test, t₂₄ = 2.67 *p < 0.05). I-Baclofen was measured at steady state. b Territorial distribution of I-Baclofen showing MS-dependent reduction of GABA_B-GIRK signaling throughout the LHb. c Examples of the injection site of red retrobeads infused in the VTA (top) and in the RMTg (bottom). Right, retrogradely labeled LHb neurons projecting to VTA or RMTg. d Sample traces, bar graph and scatter plots depicting CGP54626-sensitive I-Baclofen in LHb^VTA and LHb^RMTg neurons (LHB^VTA: I-Baclofen Ctrl vs. MS; n _mice = 2; n _cells = 9; unpaired t-test, t₁₆ = 2.37 *p < 0.05; LHb^RMTg: I-Baclofen Ctrl vs. MS; n _mice = 2; n _cells = 9 vs. 7; unpaired t-test, t₁₄ = 2.35 *p < 0.05). e Stereotactic infusion of AAV2-hSyn-CoChR-GFP within the EPN, and opsin expression in terminals within the LHb f Opto-GABA_A-IPSCs and opto-GABA_B-IPSC sample traces and summary plot (Ctrl vs. MS; n_mice = 6/3; n _cells = 9 per group; unpaired t-test, t₁₆ = 2.21; *p < 0.05). Values for opto-GABA_A-IPSC and opto-GABA_B-IPSC were taken at the maximal peak as indicated. Scale bars=4 min and 50 pA a, 500 and 125 μm c, 5 min and 20 pA d, 0.5 mm and 200 μm e, 0.5 s and 10 pA

Fig. 3

MS-induced LHb neurons hyperexcitability requires reduced GABA_B-GIRK signaling. a Left, sample traces for recordings in Ctrl and MS mice of a current-evoked firing (for a 50 pA step) in the presence of picrotoxin/NBQX. Graph representing the action potentials vs. injected current in all experimental groups. (picrotoxin/NBQX, n _mice = 4/6; n _cells = 20 per group; two-way ANOVA-RM, interaction F_(10;380) = 3.52; ***p < 0.001) b Same than a but in the presence of picrotoxin/NBQX/CGP54626, n _mice = 5/6; n _cells = 20 per group; two-way ANOVA-RM, interaction F_(10;380) = 0.32). Scale bars=0.2 s and 20 mV

Fig. 4

Chemogenetic and DBS approaches reduce LHb activity and ameliorate MS-dependent depressive-like symptoms. a Schematic and image for Gi-DREADD LHb expression. b CNO effects on failures in the shuttle box (AAV-YFP, Ctrl vs. MS and AAV-Gi-DREADD, Ctrl vs. MS; n _mice = 21/22/23/26; two-way ANOVA RM, interaction, F_(6,176) = 3.29, **p < 0.01). c Bar graph and scatter plot for TST immobility (AAV-YFP and AAV-Gi-DREADD, Ctrl vs. MS: n_mice = 23/25/25/ 25; two-way ANOVA, interaction, F_(1,94) = 4, *p < 0.05). d DBS effect on 1st EPSC and PPR (5 pulses, 20 Hz. Normalized eEPSC post-DBS, n _mice = 2; n_cells = 6; One-way ANOVA RM; F_(1.23,6.13) = 99.65, ***p < 0.001; Normalized EPSCs, n_mice = 2; n _cells = 6; Two-way ANOVA RM; DBS effect, F_(2,50) = 25.1, ***p < 0.001). e Sample I-clamp recordings (5 superimposed-sweeps), and DBS effects on action potentials, resting membrane potential and input resistance (Before vs. 5 min post-DBS vs. 10 min post-DBS, n _mice = 2; n _cells = 6: Action potential: F_(1.29,6.49) = 13.5, **p < 0.01; Vm: F_(1.15,5.77) = 6.3, *p < 0.05; Ri: F_(1.06,5.30) = 8.3, ^* p < 0.05; One-way ANOVA-RM) f DBS-electrode placement in LHb. g DBS-induced (130 Hz, 150 µA) reduction of activity in vivo (Firing before vs. post-DBS, n _mice = 4; n _cells = 14; paired t-test, t₆ = 3.1; ^** p < 0.01) h DBS-driven reduction of failures in the shuttle on MS mice (Sham vs. DBS; n _mice = 8/9; Two-way ANOVA RM, interaction, F_(1,15) = 9.476, ^** p < 0.01). Scale bars=, 100 μm a, 25 ms and 100 pA d, 0.1 s and 20 mV e, 250 μm f, 10 s g