Fig. 6

Confirmation of increased BLA dopaminergic bouton density, but not axon density, in males compared to females. A Representative confocal image of tdTomato-labeled dopaminergic axons (white) in a single section of basolateral amygdala (BLA) from the second cohort of mice. The contour of BLA was drawn according to the DAPI-stained cytoarchitecture and Allen Mouse Brain Atlas. The pixels outside the BLA contour were set to zero intensity (dark) for automated quantification. The blue box indicates the area that will be enlarged for display of labeled boutons in D. B Binarized image of detected axons in A. C No sex difference in dopaminergic axon density was detected (t (8) = 0.59, p = 0.57, n = 5 mice per sex). D Representative confocal image of SypGFP-labeled dopaminergic boutons (white) in a single section of basolateral amygdala (BLA) from the second cohort of mice. This region is enlarged from the blue-boxed area in A for display. E Binarized image of detected boutons in D. F Male mice have significantly higher densities of dopaminergic boutons in BLA than females (t (4.4) = 3.26, *p = 0.027, with Welch’s correction for unequal variance, effect size = 2.05, n = 5 male mice and n = 7 female mice). The estrous stage in females was estimated approximately by vaginal cytology and indicated in the plot by the shape of markers: estrus (circles, n = 3), diestrus (triangles, n = 3) and proestrus (square, n = 1). Axon and bouton density were calculated across 20 matched BLA coronal sections (− 3.0 bregma to − 1.0 bregma) from each mouse and expressed as percentage of the average axon or bouton density in males. Images were acquired at 25× magnification. Scale bars represent 500 microns (A, B) and 250 microns (D, E). Bars represent Mean ± SEM