Stress-induced lipocalin-2 controls dendritic spine formation and neuronal activity in the amygdala.

This is a freely-available open access publication. Please cite the published version which is available via the DOI link in this record.Behavioural adaptation to psychological stress is dependent on neuronal plasticity and dysfunction at this cellular level may underlie the pathogenesis of affective disorders such as depression and post-traumatic stress disorder. Taking advantage of genome-wide microarray assay, we performed detailed studies of stress-affected transcripts in the amygdala - an area which forms part of the innate fear circuit in mammals. Having previously demonstrated the role of lipocalin-2 (Lcn-2) in promoting stress-induced changes in dendritic spine morphology/function and neuronal excitability in the mouse hippocampus, we show here that the Lcn-2 gene is one of the most highly upregulated transcripts detected by microarray analysis in the amygdala after acute restraint-induced psychological stress. This is associated with increased Lcn-2 protein synthesis, which is found on immunohistochemistry to be predominantly localised to neurons. Stress-naïve Lcn-2(-/-) mice show a higher spine density in the basolateral amygdala and a 2-fold higher rate of neuronal firing rate compared to wild-type mice. Unlike their wild-type counterparts, Lcn-2(-/-) mice did not show an increase in dendritic spine density in response to stress but did show a distinct pattern of spine morphology. Thus, amygdala-specific neuronal responses to Lcn-2 may represent a mechanism for behavioural adaptation to psychological stress.Marie Curie Excellence Grant from the European Commission.Medical Research Council Project GrantCOST Action ECMNe

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts

The 42nd Symposium Chromatographic Methods of Investigating Organic Compounds : Book of abstracts. June 4-7, 2019, Szczyrk, Polan

Lipocalin-2 regulates basal and stress-induced changes in dendritic spine density.

<p>Dendritic spine density in DiI-labeled neurons was analyzed in basolateral amygdala of wild-type and Lcn-2−/− mice before and after restraint stress. (<b>A</b>) Stress caused an increase in spine density in the neurons of BLA in wild-type mice reaching density observed in Lcn-2-deficient stress naïve mice. (<b>B</b>) Stress induced also significant decrease in proportion of mushroom spines observed in both wild-type and Lcn-2−/− strains. Those changes were accompanied by increase in other morphological groups of spines (B). Panel C represents the example of DiI stained neurons. *p<0.05; **p<0.01; ***p<0.001. Data are expressed as mean ± SEM.</p

List of amygdalar transcripts affected by acute restraint-stress.

<p>The list of transcripts affected by restraint stress as compared to control (stress-naïve) mice. Genes are organised by statistical significance (lowest to highest p-value). P-values were adjusted according to the Benjamini & Hochberg (^BH) or Bonferroni (^B) method. n = 5 microarrays per group, each microarray chip hybridised with pulled RNA extracted from 3 different animals.</p

General characterisation of restraint stress affected transcripts.

<p>Stress affected transcripts divided according to their protein product function (<b>A</b>) and localisation (<b>B</b>).</p

Microarray analysis of gene expression in the amygdala following acute restraint stress.

<p><b>A</b>. Principal component analysis of microarray data obtained by hybridization of mRNA extracted from amygdalae of wild-type control (n = 5) and stressed (n = 5) mice. The figure represented the first three principal components of microarray analysis data (PC1, PC2 and PC3). The analysis of microarrays revealed the existence of two relative distinctive components of 5 metasets of control (red) and stress-affected (blue) gene expression patterns. <b>B.</b> The volcano plot depicting the range of gene expression fold-change (log₂[fold change]) induced by restraint stress and corresponding p-value (-log10[uncorrected p-value]). Using the threshold cut-off of an absolute 1.3 fold-change and corrected p-value ≤0.05, statistically significant genes are marked in red. <b>C</b>. Hierarchical clustering of normalised signal intensities read from microarray probe sets. <b>D</b>. qRT-PCR verification of example genes identified as upregulated in response to restraint stress. The basal level of expression is marked by the red dashed line.</p

Lipocalin-2 is upregulated by psychological stress in mouse amygdala.

<p>Psychological stress induces lipocalin-2 gene expression N = 5, ** p<0.01 (<b>A</b>) followed by protein synthesis (<b>B</b> and <b>C</b>); R-Lcn-2– recombinant lipocalin-2 N = 3, ** p<0.01. Data are expressed as mean ± SEM. Panel C consist representative Western blot. Triple immunohistochemistry revealed (<b>D</b>) that Lcn-2 (green) is localised mostly within and nearby of neurons (a and e) co-localised with neuronal marker (b and f) NeuN (red) and to lesser extend with astrocyte marker (purple) GFAP (c and g) in the nucleus of basolateral amygdala. The secondary antibody showed no signal resulting from nonspecific binding (h). LA, Lateral Amygdala; BLA, Basolateral Amygdala; CA, Central Amygdala. Quantitative RT-PCR reaction confirmed lack of expression of Lcn-2 gene in Lcn-2^−/− animals (<b>E</b>).</p

Statistical analysis of over-represented Gene Ontology groups of restraint-stress regulated transcripts.

<p>Sixteen Gene Ontology (GO) subcategories were over-represented in the group of 38 stress-affected genes. Only GO categories with 5 or more stress affected genes are shown. The p-values were adjusted according to the Bonferroni’s method. The subcategories were ordered accordingly to p-values.</p