Double immunocytochemistry for the detection of Fos protein in retrogradely identified neurons using cholera toxin B...

The focus of this paper was to describe a method combining the neuroanatomical technique of retrograde transport of cholera toxin B subunit (CTB) with the technique of Fos functional labeling. This method allowed us to evaluate whether neurons identified by retrograde tracing were activated following chemical stimulation of another brain area. We have used this method at the light microscopic level to determine whether the stimulation of the rostral ventrolateral medulla activated retrogradely labeled adrenal sympathetic preganglionic neurons in the spinal cord. CTB-containing neurons, Fos immunoreactive neurons and double labeled neurons were observed in spinal autonomic areas. These results suggest that the rostral ventrolateral medulla exerts a descending activation upon identified adrenal preganglionic neurons. The method described in this protocol can be applied for other brain areas in order to establish if a given structure can activate an identified population of neurons linked with a particular target of central or peripheral nervous system.