Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Example of alternative splicing analysis of Lcn-2 ( A , B ) and Cdkn1a ( C , D ) genes expressed in response to restraint stress. Analysis of mean signal intensities read from probe sets annotated to 5′, 3′ untranslated regions (UTRs) and exons of Lcn-2 (a’ and a” respectively) and Cdkn1a transcripts (c’ and c’’ respectively) revealed no alternative splicing events in Lcn-2 transcript. Analysis of Cdkn1a transcript revealed reduced level of expression of probe set 5530406 annotated to untranslated exon 2 (Cdkn1a transcript variant 2, NM_001111099) in both control and stressed animals suggesting tissue specific alternative splicing unrelated to stress. Scatter plots of summarised intensities of all probe sets annotated to Lcn-2 ( B ) and Cdkn1a ( D ) transcripts revealed two relatively separate populations of stress related data points and no outliers significantly affecting splice variant analysis. Each point represents the mean of all probe set intensities annotated to the gene of interest from one array. Data presented as mean ±SEM.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Expressing, Variant Assay

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Psychological stress induces lipocalin-2 gene expression N = 5, ** p<0.01 ( A ) followed by protein synthesis ( B and C ); 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 ( D ) 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 ( E ).

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Expressing, Recombinant, Western Blot, Immunohistochemistry, Marker, Binding Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Dendritic spine density in DiI-labeled neurons was analyzed in basolateral amygdala of wild-type and Lcn-2−/− mice before and after restraint stress. ( A ) 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 ) 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.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Labeling, Staining

Journal: PLoS ONE

Article Title: Stress-Induced Lipocalin-2 Controls Dendritic Spine Formation and Neuronal Activity in the Amygdala

doi: 10.1371/journal.pone.0061046

Figure Lengend Snippet: Current-clamp experiments revealed that neuronal firing rate in the basolateral amygdala is higher in Lcn-2−/− mice when compared to Lcn-2+/+ animals. Voltage responses were recorded by current steps from −100 to +600 pA in 50 pA (starting membrane potential −80 mV) from principal neurons of the basal nucleus of the amygdala. Number of action potential spikes was counted as a function of depolarizing current injection ( A ). Disruption of the lipocalin-2 gene significantly increased action potential firing rate in Lcn-2−/− animals (p<0.01 at 150 pA; p<0.05 at 200 pA). Panel B shows a significant increase in the mean input resistance in Lcn-2−/− mice compared to wild-type animals. *p<0.05; Panel C is example of neuronal firing trace. Data are expressed as mean ± SEM.

Article Snippet: After blocking (5% skim milk for 1 h at RT) and washing with TBS-T (3×5 mins) the membranes were probed with goat anti-Lcn-2 antibody (R&D, 1∶500) overnight at 4°C.

Techniques: Injection