Prism statistic program 5

All morphological measurements were averaged across the sections, generating one overall value for each animal. CSF samples were assayed in triplicate and averaged, generating one value per animal. Therefore, for each type of assay the variance around the mean of each group reflects the difference between animals. All data were compared with analysis of variance (ANOVA) followed by Newman-Keuls post hoc pairwise comparison. Data were tested for unequal variance and if present, a non-parametric ANOVA was applied (Kruskal-Wallace). Combined group data were compared with a t-test. Data were additionally compared to determine effect size using Cohen’s d analysis (http://www.uccs.edu/~lbecker/). In addition, regression analysis was applied to gene expression and CSF 5HIAA. Serotonin axon density in the LC was previously determined in the same animals with an antibody to serotonin (Bethea et al., 2014 (link)). Therefore, quantitation of serotonin axon density from the previous study was correlated with 5HIAA. Each variable was represented with the group means. All statistical analyses were conducted using the Prism Statistic Program 5.0 (GraphPad, San Diego, CA, USA). A confidence level of p < 0.05 was considered significant.

Three anatomical levels were examined for KISS1 mRNA at 250 μm intervals through the infundibular ARC; 2 sections were examined for GnRH axon density at 250 μm intervals in lateral areas at the rostral end of the Infundibular Recess (IR); 4 levels were examined for DBH axon density at 250μm intervals through the rostral ARC; 4 levels were examined for hypothalamic serotonin axon density at 250 μm intervals in the rostral periventricular area, and 7 anatomical levels were examined for PAG serotonin axon density at 250 μm intervals in sections containing the dorsal raphe nucleus. The section/level results were averaged, generating one average for each animal. The average of the individual animals in each group was subjected to statistical analysis, so the variance around the treatment group is due to animal-to-animal variation. Most data were compared with 2-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc pairwise comparison. The PAG serotonin axon density in the non-stressed and stressed animals was analyzed separately with a t-test due to performance of IHC assays at different times. All statistical analyses were conducted using the Prism Statistic Program 5.0 (GraphPad, San Diego, CA). A confidence level of p<0.05 was considered significant.

All measurements were averaged across the sections, generating one overall average for each animal. Therefore, the variance around the mean of each group reflects the difference between animals. The data were compared with analysis of variance (ANOVA) followed by Newman-Keuls post hoc pairwise comparison. The data were tested for unequal variance and if present, a non-parametric ANOVA was applied (Kruskal-Wallace). Finally, regression analysis was applied to gene expression, serotonin axon and varicosity staining, yawning, fenfluramine-stimulated serotonin-dependent prolactin release and basal prolactin secretion. All statistical analyses were conducted using the Prism Statistic Program 5.0 (GraphPad, San Diego, CA). A confidence level of p<0.05 was considered significant.