Diameter of stainless steel needles used to collected tissue and protein content of regions in 200 µm frozen brain sections.

Consecutive coronal sections (200 µm) of frozen quail brain were cut beginning at the level of the end of the tractus septopallio-mesencephalicus tractus (section 1) to approximately the level of the third nerve. These sections were numbered following their order of collection.aAll tissues were taken as single midline punches aimed at the collapsed third ventricle, except for the tissue from the TnA and the PAG that consisted of individual left and right punches. For mBST, 2 punches collected the left and right anterior portions (section 3), while 3 punches collected the lateral and medial parts of the posterior portion (section 4; see fig. 1 for details).bTheoretical protein contents were estimated as follows. The volume of the tissue micropunched was obtained by multiplying the square of the radius of the circle determined by the needle by Pi and by the thickness of the brain slice (For the 514 µm i.d. needle: 3.14×0.262×200 = 0.04 µl; for the 838 µm i.d. needle: 3.14×0.422×200 = 0.110 µl). The theoretical fresh weight of the tissue was calculated assuming that brain tissue has a density close to this of water (0.100 µl = 100 µg). The theoretical protein content was then obtained assuming a protein content of about 5% (2.1 and 5.5 µg/punch for the 514 and 838 µm i.d. needle respectively).cThese data represent the mean ± S.E.M. of micropunches collected in both experiments across treatment groups (n = 75–80).