Figure 1.

Developmental nicotine exposure eliminates sexual dimorphisms in male sexual behavior and SDN-POA area. A) Animals were developmentally exposed to either vehicle (DMSO) or nicotine (2mg/kg/day) throughout the window for POA sexualization (GD18-PND10). At PND0 and PND1, male and female pups were subcutaneously injected with either vehicle (sesame oil) or estradiol to induce masculinization. Animals were gonadectomized at PND40 and fitted with a testosterone replacement capsule. In all animals, anxiety-like behavior and locomotion were assessed at PND50, and male sexual behavior at PND60. Brains were harvested at PND76 to examine POA-SDN area. B) Male sexual behavior parameters measured in PND60 animals in the presence of a sexually receptive female. Vehicle control females displayed fewer mounts than vehicle control males (a-priori t-test, p=0.0036), estradiol-masculinized males (a-priori t-test, p=0.0325), and nicotine-treated females (a-priori t-test, p=0.0227). Number of mounts: (Vehicle) Control n = 9 male, n= 7 female; Estradiol n = 9 male, n= 8 female; Nicotine n = 8 male, n= 7 female. Vehicle control females took longer to mount a paired receptive female than vehicle control males (a-priori t-test, p=0.0214), nicotine-treated males (a-priori t-test, p=0.022), and nicotine-treated females (a-priori t-test, p=0.0249). Mount latency: (Vehicle) Control n = 8 male, n= 7 female; Estradiol n = 8 male, n= 8 female; Nicotine n = 8 male, n= 6 female. C) The ratio of male to female mounts was significantly reduced in nicotine-treated and estradiol-masculinized animals (one-way ANOVA p=0.0022, F=8.751). Mount ratio: (Vehicle) Control n = 7 litters; Estradiol n =7 litters; Nicotine n = 7 litters. D) Parameters of anxiety-like behavior and locomotion measured at PND50 using the elevated plus maze apparatus. Vehicle control females spent more time in the open arm of the elevated plus maze apparatus than nicotine-treated females (a-priori t-test, p=0.0442). Percent time in open arm: (Vehicle) Control n = 6 male, n= 6 female; Estradiol n = 7 male, n= 7 female; Nicotine n = 8 male, n= 8 female. Vehicle control females performed more center crosses than estradiol-masculinized males (a-priori t-test, p=0.0418). Center Crosses: (Vehicle) Control n = 6 male, n= 6 female; Estradiol n = 7 male, n= 7 female; Nicotine n = 8 male, n= 8 female. E) SDN-POA area visualized in 40 μm sections using immunofluorescent staining for calbindin. Scale bar represents 100 μm. Vehicle control female SDN-POA area was less than vehicle control males (a-priori t-test, p=0.0133) and nicotine-treated males (a-priori t-test, p=0.0047). SDN-POA Area (mm²): (Vehicle) Control n = 4 male, n= 4 female; Estradiol n = 3 male, n= 3 female; Nicotine n = 5 male, n= 4 female. B-E) P=0.05 a-priori t-test compared to vehicle-treated females. # P<0.05 a-priori t-test compared to vehicle-treated females. *P< 0.005 Student’s t-test post hoc analysis compared to vehicle-treated females following significant one-way ANOVA. N indicates the number of litters. Error bars represent standard error of the mean.