Determination of estrous stage:

The stage of the estrous cycle was determined using interpretation of vaginal cytology. Unstained vaginal smears were viewed immediately upon collection via pipet lavage. Proestrus was indicated when cells were predominantly nucleated epithelial cells. A predominance of cornified epithelial cells classified estrus. Metestrus was recognized by scattered, nucleated or cornified epithelial cells and leukocytes, and diestrus was classified by classic leukocytes in combination with various larger round epithelial cells. (Freeman 1988; Goldman et al. 2007). Vaginal samples were taken in the evening prior to the day of experiment to facilitate planning the dosing and a second smear was obtained ~ 1 hour before THC inhalation to confirm. The estrous and diestrous stages were selected for this experiment because a previous study reported the largest stage-related differential effect of 5 mg/kg THC in estrus versus diestrus in gonadally intact, cycling females (Craft and Leitl 2008).