Figure 8.

Anatomical distribution of ChR2-transfected cells and fiber optic locations. A, Example of fiber optic location (dotted line, the green color within the fiber optic tract is an auto fluorescence artifact) next to the ChR2-mCherry-transfected neurons (mCherry in red). Choline acetyl-transferase-immunoreactive neurons (represented in blue) located in the ambiguus nucleus (Amb) and medial to the retrotrapezoid nucleus are not transfected. Several mCherry-positive neurons in this transverse section are catecholaminergic (tyrosine hydroxylase, TH, in green). These neurons generally occupy a medial location relative to the noncatecholaminergic, noncholinergic neurons (the RTN-Phox2b neurons). B, Native mCherry fluorescence (red) detected along with Phox2b-immunoreactivity (green nuclei). Note the presence of Phox2b in every transfected neuron, including within the marginal layer of the retrotrapezoid nucleus. C, Example of a fiber optic tract (dotted line) found to end next to the labeled axons of ChR2-mCherry-transfected neurons. D, Caudal-to-rostral series of transverse sections (bregma levels in millimeters as indicated) depicting the location of all the ChR2-mCherry-transfected neurons identified in 12 cases. Computer-generated drawings with each ChR2-mCherry-transfected neuron indicated by either a green square (TH+, TH-immunoreactive), brown diamond (ChAT+, choline acetyl transferase-immunoreactive), or magenta circle (TH-, neither TH-immunoreactive nor ChAT-immunoreactive) from the respective sections indicated for each bregma level from 12 animals were optimally superimposed using landmarks such as the ventral medullary surface, the edge of the trigeminal tract, the compact portion of nucleus ambiguus, the inferior olive, and the facial motor nucleus. E, Location of fiber optic tips plotted on three transverse sections at the indicated bregma level (in mm). Scale bars: (in A) A, C, 200 μm, B, 100 μm; (in D, E), 500 μm.