Figure 1. Mitochondria-related signals in respiratory neurons.

A, images of Rh123 (top) and NADH fluorescence (bottom) at low magnification obtained 2 s before the inspiratory burst began (Expiration, left) and 2 s after its completion (Inspiration, right). Two inspiratory and two expiratory neurons (marked I and E, respectively) were identified as demonstrating increases and decreases, respectively, in both Rh123 and NADH fluorescence signals caused by inspiratory motor output as recorded from XII nerve rootlets. The original 12-bit images were filtered using a 2 × 2 median space filter with MetaMorph software in order to remove scarce bright spots, which sometimes appeared after staining on the slice surface. B, double staining with MitoTracker Green and Rh123. After obtaining control images of MitoTracker Green, NADH and Rh123, the fluorescence being selectively excited and collected as described in Methods, hypoxia was applied for 3 min and the images were acquired again. The acquisition time was 2 s for each image. The cell was identified as inspiratory by correlating changes in mitochondrial variables with the respiratory output. In the images presented in the top and bottom panels, the background (defined as mean fluorescence level measured outside the slice) was subtracted. Vertical bar indicates relative fluorescence scale ΔF/F (white indicates highest fluorescence changes of 10 %).