Delayed neuronal death is barely detectable in ATP-injected SNpc.
(A) Midbrain sections including the entire SNpc were prepared at 3 h and 7 d after ATP injection, and stained with TH antibody. The contralateral side (contra) was used as the control. The lower panel represents higher magnification of boxed regions in the upper panel. The ATP-induced damage area detected at 3 h did not increase at 7 d (dotted lines in the upper panel), although TH+ neurons that appeared rough and unhealthy (pathological neurons, arrows in the ‘e’) disappeared at 7 d (f). (B) Sections obtained at 3 h were labeled with TH antibody, and the nuclei were visualized with DAPI. Nuclei of TH+ neurons and uncharacterized cells were indicated by arrows and arrowheads, respectively. (C) Electron microscopic analysis was performed in the boxed regions in intact and ATP-injected brain represented in (Aa and b). Mitochondria (M, insets) and cytosol (cyt) were indicated. (D) Using stereology, the total numbers of TH+ neurons were estimated in the contralateral and ATP-injected ipsilateral SNpc at 3 h (n = 5) and 7 d (n = 5), as described in “Materials and Methods”. The number of TH+ neurons in the ipsilateral side was normalized to that in the contralateral side (contra) in each animal. Pathological TH+ neurons detected at 3 h were counted separately. Scale bars, 200 µm (A, upper panel); 50 µm (A, lower panel); 10 µm (B); 1.7 µm (C); 0.8 µm (C, inset).