Figure modified from the BRAIN ATLAS [31].

<p>Pictures show 13 different substructures analysed at 3 rostrocaudal levels, within each structure, throughout different brain regions. In grey areas affected in stress-susceptible rats; in dark grey with stripes brain regions affected in both CMS phenotypes; in light grey areas affected in stress resilient rats. In beige, areas not affected by the CMS. DG, dendate gyrus; Cg1, cingulate cortex—area 1; IL, infralimbic cortex; PrL, prelimbic cortex; LO, lateral orbital cortex; VO, ventral orbital cortex; Pir, piriform cortex; PVA, paraventricular thalamic nucleus—anterior part; BLA, basolateral amygdaloid nucleus—anterior part; MHb, medial habenula; LHb, lateral habenula; CA3, field CA3 of hippocampus; VLGMC, ventral lateral geniculate nucleus magnocellular part; * p < 0.05; ** p < 0.01; *** p < 0.001.</p

Neuronal substrates underlying stress resilience and susceptibility in rats

<div><p>Background</p><p>Stress and stressful life events have repeatedly been shown as causally related to depression. The Chronic Mild Stress rat model is a valid model of stress-induced depression. Like humans, rats display great heterogeneity in their response to stress and adversity. Hence some individuals are stress-sensitive and prone to develop depression-like behaviour in response to modest stressors, while others are stress-resilient and remain essentially symptom free.</p><p>Objectives</p><p>Compared to the large body of research, which describes stress-induced maladaptive neurobiological changes, relatively little attention has been devoted to understand resiliency to stress. The aim of the present study was to identify changes in neuronal activity, associated with stress-resilient and stress-susceptible chronic mild stress endophenotypes, by examining <i>c-Fos</i> expression in 13 different brain areas. Changes in <i>c-Fos</i> expression have been reported as associated to stressful conditions.</p><p>Methods</p><p>Stress-induced modulation of neuronal activation patterns in response to the chronic mild stress paradigm was mapped using the immediate early gene expression <i>c-Fos</i> as a marker. Quantification of the <i>c-Fos</i>-like immunoreactivity responses was done by semi-automated profile counting procedures and design-based stereology.</p><p>Results</p><p>Exposure to chronic mild stress significantly altered <i>c-Fos</i> expression in a total of 6 out of 13 investigated areas. Chronic mild stress was found to suppress the <i>c-Fos</i> response within the magnocellular ventral lateral geniculate nucleus of both stress subgroups. In the the lateral and ventral orbital cortices of stress-resilient rats, the <i>c-Fos</i> like immunoreactivity response was also repressed by stress exposure. On the contrary the <i>c-Fos</i> response within the amygdala, medial habenula, and infralimbic cortex was increased selectively for the stress-susceptible rats.</p><p>Conclusions</p><p>The study was initiated to characterize neuronal substrates associated with stress-coping mechanisms. Six areas, all of which represents limbic structures, were found to be sensitive to stress exposure. The effects within these areas associate to the hedonic status of the rats. Hence, these areas might be associated to stress-coping mechanisms underlying the chronic mild stress induced segregation into stress-susceptible and stress-resilient endophenotypes.</p></div

Sucrose consumption following four weeks of the CMS regime.

<p>Four weeks exposure to chronic mild stressors resulted in a significant decrease of sucrose consumption of the anhedonic animals (n = 10) when compared to unchallenged controls (n = 10). The graph shows sucrose consumption of the control (n = 10), resilient (n = 10) and anhedonic-like (n = 10) animals at 4 different time points (week 1; week 2; week 3 and week 4). Significant differences on sucrose consumptions were detected among goups (p<0,0001) at different time points (p = 0,0084). The sucrose intake of anhedonic-like animals was significantly diminished when compared to control and resilient animals. Two-way ANOVA followed by post hoc Tukey´s multiple comparison test. * p<0,05; **p<0,01; ***p<0,001. Data is presented as mean (±SEM) sucrose intake, indexed to baseline values.</p

Results summary.

<p>Results summary.</p

Representative microphotograph of c-Fos expression in different brain areas.

<p>High and low magnification pictures of representative sections to compare c-Fos-ir in eight brain regions. IL, infralimbic cortex; LO, lateral orbital cortex; VO, ventral orbital cortex; BLA, basolateral amygdaloid nucleus—anterior part; MHb, medial habenula; LHb, lateral habenula; CA3, field CA3 of hippocampus; VLGMC, ventral lateral geniculate nucleus; magnocellular part. Each panel shows a high and low magnification picture of the area, and a picture of the corresponding area modified from the rat brain atlas [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179434#pone.0179434.ref031" target="_blank">31</a>]. Scale bar in panel A-H = 50um.</p

Stereological estimates of total c-Fos-ir in the habenular complexes.

<p>The estimates of total c-Fos-ir in the lateral (A) and medial (B) habenular complexes analysed with stereology. In plot A and B number of cells counted in the LHb as the sum of dark and medial c-Fos-ir and the sum of dark, medial and light c-Fos-ir, respectively. In plot C and D number of cells counted in the MHb as the sum of dark and medial c-Fos-ir and the sum of dark, medial and light c-Fos-ir, respectively. One-way ANOVA. LHb, lateral habenula; MHb, medial habenula.</p

Study design showing the experimental outline and time course.

<p>(modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179434#pone.0179434.ref070" target="_blank">70</a>]).</p

MicroRNA Profiling in the Medial and Lateral Habenula of Rats Exposed to the Learned Helplessness Paradigm: Candidate Biomarkers for Susceptibility and Resilience to Inescapable Shock

<div><p>Depression is a highly heterogeneous disorder presumably caused by a combination of several factors ultimately causing the pathological condition. The genetic liability model of depression is likely to be of polygenic heterogeneity. miRNAs can regulate multiple genes simultaneously and therefore are candidates that align with this model. The habenula has been linked to depression in both clinical and animal studies, shifting interest towards this region as a neural substrate in depression. The goal of the present study was to search for alterations in miRNA expression levels in the medial and lateral habenula of rats exposed to the learned helplessness (LH) rat model of depression. Ten miRNAs showed significant alterations associating with their response to the LH paradigm. Of these, six and four miRNAs were significantly regulated in the MHb and LHb, respectively. In the MHb we identified miR-490, miR-291a-3p, MiR-467a, miR-216a, miR-18b, and miR-302a. In the LHb miR-543, miR-367, miR-467c, and miR-760-5p were significantly regulated. A target gene analysis showed that several of the target genes are involved in MAPK signaling, neutrophin signaling, and ErbB signaling, indicating that neurotransmission is affected in the habenula as a consequence of exposure to the LH paradigm.</p></div

Learned helplessness.

<p>A) Deficit pattern, number of failures to terminate shock within the first 20 s of a trial (mean ±SEM). B) Failure pattern, failure to terminate shock results in a failed trial (mean ±SEM). C) Latency, the accumulated latency to press the lever (mean ±SEM).</p

Pathway analysis.

<p>Displaying the most significant KEGG pathways predicted to be targeted by the indicated miRNAs for A) miR-18b-5p, B) miR-291-3p, C) miR-760-5p and D) miR-367-3p. miR-18b-5p and miR-291-3p were identified in the medial habenula. miR-760-5p and miR-367-3p were identified in the lateral habenula. Only non-cancer pathways with FDR < 0.05 are shown. Lists are limited to maximum 10 pathways. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160318#sec002" target="_blank">Materials and Methods</a> section for further detail.</p