Figure 1. Death of dopaminergic neurons and microglia in the SNpc induced by ATP.

(A) ATP (100 nmol in 2 µl PBS) or PBS (2 µl) was unilaterally injected into SNpc (*, injection sites), and brains were obtained after 3 h. Brain sections (30 µm thickness) of the midbrain including the entire SN were prepared, every sixth serial section selected and stained with TH (upper panel) and Iba-1 (lower panel) antibodies, and visualized with biotin-conjugated secondary antibodies and enzymatic detection with the avidin/biotin system unless indicated. At 100 nmol, mild neuronal and microglial damage occurred, thus, 100 nmol ATP was employed for in vivo experiments in this study. Photographs of the most damaged sections were obtained. The contralateral side (contra) and PBS-injected rat brain sections were used as control. (B) Serial sections obtained at 3 h were labeled with TH (left panel), Iba-1 (middle panel), and TH/Iba-1 (right panel) antibodies. For visualization of the double-labeling, color reactions using DAB (for TH) and DAB/nickel sulfate (for Iba-1) were applied. Dotted lines indicated damage areas. (C) Brain tissue obtained 3 h post ATP treatment was subjected to electron microscopy, as described in “Materials and Methods”. Nuclei of neuron (N, white arrow), astrocytes (A, white arrowhead), and microglia (M, black arrow) were shown in intact rat brain whereas cellular structures were severely disrupted in ATP-injected brain. Data in this study are representative of at least 5 animals. Scale bars, 200 µm (A); 100 µm (B); 5 µm (C).