Stress while lacking of control induces ventral hippocampal autophagic flux hyperactivity and a depression-like behavior

Abstract

Background: Stressed animals may perform depression-like behavior insomuch as stress-provoking blood-brain barrier (BBB) disruption, central immune activation, and autophagic flux changes. This study was undertaken to assess whether adult mice having (executive) vs. lacking (yoke) of behavioral control in otherwise equivalent stress magnitude condition, may display differences in their BBB integrity, ventral hippocampal (VH) interleukin-6 (IL-6) and autophagic flux level and VH-related depression-like behavior. To further understand the causative relation of enhanced autophagic flux and stress-primed depression-like behavior, we assessed the effects of bilateral intra-VH 3-methyladenine (3-MA), an autophagic flux inhibitor, infusion in stressed mice.

Methods: Adult mice used had comparable genetic background and housing condition. Executive/yoke pairs of mice received a 10-day (1 h/day) footshock stressor regimen. Throughout the regimen, the ongoing footshock was terminated immediately contingent on the executive mouse', while irrelevant to the respective yoke mouse' voluntary behavior, or lasting for 7 s. Each dyad's cage-mate receiving no such regimen served as no stressor controls.

Results: Yoke mice displayed disrupted BBB integrity (escalated Evans blue extravasation and decreased VH ZO-1, claudin-5 expression), increases in VH autophagic flux (increased LC3II/LC3I and decreased p62) and immobility duration in forced swimming test. Most of these indices remained unaltered in executive mice. Administration of 3-MA did not affect immobility duration in control mice, while prevented the increases in immobility duration in yoke mice.

Conclusions: (1) stress susceptibility may be determined by their differences in stress-coping results; (2) VH autophagic flux increase plays a permissive role in priming the stressed animals susceptible to exhibit depression-like behavior.

Keywords: Autophagy; Blood–brain barrier; Executive; Forced swimming; Hippocampus; Yoke.

Fig. 1

Experimental timelines for the 10-day stressor regimen and biochemical assays and behavioral tasks. Assays and tasks were done approximately 72 h after the conclusion of the stressor regimen. BBB, WB, VH, IL-6, Elisa, and 3-MA are short forms of blood–brain barrier, Western immunoblotting, ventral hippocampus, interleukin-6, enzyme-linked immunosorbent assay, and 3-methyladenine, respectively.

Fig. 2

BBB integrity, junction component protein expressions, IL-6 levels and autophagic flux indices in yoke, executive and control mice. (A)Representative photomicrographs of Evans blue leaking in brain slices (Bregma:-1.58 mm). Dark arrows point to the leaking site. (B)Stressor/Yoke mice receiving the 10-day stressor regimen and lacking of behavioral control exhibited higher Evans blue extravasation as compared to the Stressor/Executive mice receiving same stressors while having behavioral control and the control mice receiving No Stressor. ∗Significantly greater than the other groups (ps < 0.05). (C)Stressor/Yoke mice demonstrated lower ZO-1 level in VH as compared to executive and control mice. ^Significantly lower than the other groups (ps < 0.05). VH stands for ventral hippocampus. No S, S/E and S/Y are short forms of No stressor, Stressor/Executive and Stressor/Yoke, respectively. (D)The stressor regimen was found to induce decreases in VH claudin-5 level and yoke mice had the lowest VH claudin-5 level among three groups. ∗Significantly higher than Stressor/Yoke group (p < 0.05). (E)Regardless of the stressor regimen, three groups of mice had comparable occludin levels in VH. (F)Regardless of the stressor regimen, three groups of mice had comparable VE-cadherin levels in VH. (G)Regardless of the stressor regimen, three groups of mice had comparable IL-6 levels in VH. (H)Long-term stress and lacking of behavioral control was found to induce increases in LC3II/LC3I ratio (autophagic flux) in VH, while did not alter such ratios in Stressor/Executive mice. ∗Significantly higher than the other groups (ps < 0.05). (I)Long-term stress and lacking of behavioral control was found to induce decreases in p62 (autophagosome inclusion) level in VH, while did not alter such levels in Stressor/Executive mice. ^Significantly lower than Stressor/Executive and No Stressor group (ps < 0.05). Group differences are analyzed by Kruskal–Wallis analyses followed by Dunn multiple comparisons if appropriate.

Fig. 3

Long-lasting impact of stress and behavioral control on hippocampus-related functions. (A)Stressor/Yoke mice demonstrated greater immobility durations (in second) as compared to Stressor/Executive and No Stressor control mice in a 6-min forced swimming observation approximately 72 h after the conclusion of the stressor regimen. ∗Significantly greater than the other groups (ps < 0.05). (B)Tail suspension task was insensitive to the stressor regimen and behavioral control. Three groups of mice demonstrated indistinctive immobility time (in second) in a 6-min tail suspension. (C)Regardless of the stressor regimen or behavioral control, three groups of mice had comparable recognition ratios in object location task. (D)Following the first forced swimming test, mice received a forced swimming retest at 6 weeks after their conclusion of the stressor regimen. Yoke mice displayed shorter immobility duration as compared to executive and control mice. ^Significantly lower than the other groups (ps < 0.05). Group differences in all behavioral task results are analyzed by one-way analysis of variances (ANOVAs), followed by Bonferroni's post hoc tests if appropriate.

Fig. 4

Effects of intra-VH 3-MA (50 mM/0.2 μL/side) infusions on VH autophagic flux indices (LC3II/I and p62), related class I and III activity and forced swimming immobility duration in control and yoke mice. (A)Using PI(3)P gauge as a phosphoinositide-3 kinase activity index, yoke mice displayed higher phosphoinositide-3 kinase activity in their ventral hippocampal tissues as compared to the control mice. Bilateral intra-VH 3-MA (50 mM/0.2 μL/side) infusions reliably prevented such enhanced phospohoinositide 3-kinase class III activity. PI(3)P is a short form of phosphatidylinositol 3-phosphate. ∗Significantly higher than the remaining three groups (ps < 0.05). (B)Neither the stressor regimen or 3-MA treatment affected class I downstream target protein phosphorylation in mice’ ventral hippocampal tissues. (C)While intra-VH 3-MA infusions did not affect LC3II/I ratio in No Stressor controls, such treatment prevented the stress and lacking of control-primed LC3II/I increase in Stressor/Yoke mice. ∗Significantly greater than the other groups (ps < 0.05). (D)Intra-VH 3-MA infusions did not affect p62 in No Stressor controls, while such treatment prevented the stress and lacking of control-primed p62 decrease in Stressor/Yoke mice. ^Significantly lower than the other groups (ps < 0.05). (E)While intra-VH 3-MA treatment did not affect recognition ratio in No Stressor control mice, such treatment prevented the stress and lacking of control-primed increases in immobility duration in forced swimming test. ∗Significantly greater than the other groups (ps < 0.05). Group differences are analyzed by Kruskal–Wallis analyses followed by Dunn multiple comparisons if appropriate.