Figure 4.

Double immunofluorescent staining of 5-HT2a and 2b receptors (green) with the retrograde tracer (red) Cholera toxin B fragment (CtB) in phrenic motor neurons in the cervical spinal cord (~C₄ region). (A) 4× magnification of the C₄ spinal cord area containing CtB-labeled phrenic motor neurons (red) located bilaterally in the ventral horn and labeled with the 5-HT2b receptor (green); scale bar: 1mm. (B) Higher magnification (20×) of the clustered phrenic motor neurons within the dashed square region outlined in (A), and a 20× magnification (C) of phrenic motor neurons labeled with 5-HT2a receptors in a different rat. In (B), note the CtB positive phrenic motor neurons (red) are co-labeled with intense nuclear staining (arrows) as well as in the surrounding neuropil for the 5-HT2b receptor (green); scale bar: 100µm. Under 100× magnification (D), the 5-HT2b receptor also appears to be in the membrane (middle and right panels) of phrenic motor neurons (arrow heads). Note, 100× magnification (E) of CtB positive phrenic motoneurons (red) co-labeled with the 5-HT2a receptor (green), which is located in the cytoplasm (arrow heads) of phrenic motor neurons and the surrounding neuropil; scale bar: 10µm.