Results summary.

<p>Results summary.</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

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

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

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

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

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

Data_Sheet_1_A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity.PDF

<p>Gene editing in human embryonic stem cells (hESCs) has been significantly enhanced by the discovery and development of CRISPR Cas9, a programmable nuclease system that can introduce targeted double-stranded breaks. The system relies on the optimal selection of a sgRNA sequence with low off-targets and high efficiency. We designed an improved monomeric red fluorescent protein reporter, GEmCherry2, for assessing CRISPR Cas9 activity and for optimizing sgRNA. By incorporating an out-of-frame sequence to the N-terminal of the red fluorescent protein mCherry, we created a visual tool for assessing the indel frequency after cutting with CRISPR Cas9. When a sgRNA-Cas9 construct is co-transfected with a corresponding GEmCherry2 construct, single nucleotide indels can move the GEmCherry2 sequence back in-frame and allow quantification and comparison of the efficiency of different sgRNA target sites by measuring red fluorescence. With this GEmCherry2 assay, we compared four target sites in the safe harbor AAVS1 locus and found significant differences in target site activity. We verified the activity using TIDE, which ranked our target sites in a similar order as the GEmCherry2 system. We also identified an AAV short inverted terminal repeat sequence within the Cas9 construct that, upon removal significantly improved transient transfection and expression in hESCs. Moreover, using GEmCherry2, we designed a sgRNA to target SORCS2 in hESCs and successfully introduced indels into the coding sequence of SORCS2.</p

Data_Sheet_1_Long-Term Stress Disrupts the Structural and Functional Integrity of GABAergic Neuronal Networks in the Medial Prefrontal Cortex of Rats.docx

<p>Clinical and experimental data suggest that fronto-cortical GABAergic deficits contribute to the pathophysiology of major depressive disorder (MDD). To further test this hypothesis, we used a well characterized rat model for depression and examined the effect of stress on GABAergic neuron numbers and GABA-mediated synaptic transmission in the medial prefrontal cortex (mPFC) of rats. Adult male Wistar rats were subjected to 9-weeks of chronic mild stress (CMS) and based on their hedonic-anhedonic behavior they were behaviorally phenotyped as being stress-susceptible (anhedonic) or stress-resilient. Post mortem quantitative histopathology was used to examine the effect of stress on parvalbumin (PV)-, calretinin- (CR), calbindin- (CB), cholecystokinin- (CCK), somatostatin-(SST) and neuropeptide Y-positive (NPY+) GABAergic neuron numbers in all cortical subareas of the mPFC (anterior cingulate (Cg1), prelimbic (PrL) and infralimbic (IL) cortexes). In vitro, whole-cell patch-clamp recordings from layer II–III pyramidal neurons of the ventral mPFC was used to examine GABAergic neurotransmission. The cognitive performance of the animals was assessed in a hippocampal-prefrontal-cortical circuit dependent learning task. Stress exposure reduced the number of CCK-, CR- and PV-positive GABAergic neurons in the mPFC, most prominently in the IL cortex. Interestingly, in the stress-resilient animals, we found higher number of neuropeptide Y-positive neurons in the entire mPFC. The electrophysiological analysis revealed reduced frequencies of spontaneous and miniature IPSCs in the anhedonic rats and decreased release probability of perisomatic-targeting GABAergic synapses and alterations in GABA_B receptor mediated signaling. In turn, pyramidal neurons showed higher excitability. Anhedonic rats were also significantly impaired in the object-place paired-associate learning task. These data demonstrate that long-term stress results in functional and structural deficits of prefrontal GABAergic networks. Our findings support the concept that fronto-limbic GABAergic dysfunctions may contribute to emotional and cognitive symptoms of MDD.</p