Computational protocol: Social threat exposure in juvenile mice promotes cocaine‐seeking by altering blood clotting and brain vasculature

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Protocol publication

[…] Leukocytes RNA samples (from 16 different mice) were hybridized on Agilent mouse gene expression microarrays (G4852A) according to the manufacturer's procedure (Callegari et al. ) using the Low Input Quick‐Amp Labeling Kit and one color design (Agilent Technologies) (cyanine 3‐CTP). Images at 3‐µm resolution were generated by Agilent scanner, and the Feature Extraction software (Agilent Technologies) was used to obtain the microarray raw‐data.Data were analyzed with BRB Array Tools (‐ArrayTools.html). Data were subjected to filtration based on signal intensity, spot quality and presence across the data set. A total of 27978 probes passed the filters and were further analyzed. Statistical analysis of genes differentially expressed S‐S and NS‐S was performed using Random Variance Model (Wright & Simon ) with parametric p‐value threshold set to 0.005. Identified genes were further filtered according to fold change (LOG FC > 0.6 for up‐regulated genes, LOG FC < −0.6 for down‐regulated ones). False Discovery Rate of the whole gene list was calculated as the proportion of expected false positive observations on total significant observations. Gene Ontology (GO) analysis was performed on the above selected genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID,; Huang et al. ). Significance of overrepresentation was adjusted for multiple comparisons to control the false discovery rate (FDR) by means of the Benjamini–Hochberg step‐down procedure or the approximated FDR tools provided in DAVID. Fold enrichment was scalculated as the ratio between the number of genes belonging to the family observed in the gene list and the number of genes expected to be randomly present in the gene list. To avoid redundancy, selected statistically enriched GO Biological Processes (BPs) and Panther pathways were reported.The microarray data obtained have been deposited in NCBI's Gene Expression Omnibus (Edgar et al. ) and are accessible through GEO Series accession number GSE69019 ( [...] Specimens were examined under a confocal laser‐scanning microscope (Zeiss CLSM700). The structures of interest were identified by using a 10× objective and captured by using a 20× objective. For acquisition of capillaries in the different brain structures, a series of z‐sections were acquired at interval ranging from 0.87 to 1.2 µm. Specimens were captured using consistent settings for laser power and detector gain.The densitometric analysis of lectin staining was performed with ImageJ software (http://rsb.; National Institutes of Health) on confocal maximum intensity projection images. After background subtraction Lectin‐associated signal was quantified in the areas of interest by manually outlining individual structures. Mean signal intensity of lectin staining (F) in the different structures was performed in a variable number of sections, in order to cover the whole extension of the areas of interest. Specifically, for each animal, in five sections for NAc, one every 150 µm, and in five sections for striatum and hippocampus, one every 300 µm. The F/A ratio defines mean fluorescence of individual samples (F) normalized to total cellular surface (A). Quantification was done on five sections per mouse per structure (four mice per group—total group samples n = 20).Furthermore morphometric analysis of lectin‐stained blood capillaries in the different structures was performed on confocal maximum intensity projection images and analyzed by using Neurolucida software (Neurolucida 7.5, MicroBright‐Field, Germany). For capillary diameters analysis, a total of 240 capillaries per structure (from four mice per group) were considered, and the minor axes of capillaries were measured. The branch points of microvasculature (Czéh et al. ; Kiuchi et al. ) were numerated and counted with ImageJ software on confocal maximum intensity projection images. Data collecting for densitometric and morphometric analyses were performed by experimenters blind to the group analyzed. [...] All data obtained were checked for homogeneity of variance, with measures failing Levene's test analyzed by non‐parametric Mann–Whitney procedures. All other parameters were subjected to parametric either Student's t‐test or repeated‐measure analysis of variance (ANOVA). ANOVA was followed, in cases of significance (P < 0.05), by post‐hoc comparisons using Duncan's test. The comparisons between the capillary diameter population were performed using the Chi‐square test on categorical measure (capillary diameter). All statistical analyses were carried out with the help of Statistica software Version 12.0 (StatSoft, Tulsa, OK, USA). […]

Pipeline specifications

Software tools ImageJ, Neurolucida, Statistica
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Blood Coagulation Disorders, Substance-Related Disorders
Chemicals Cocaine