Retrograde double labeling of neurons: the combined use of horseradish peroxidase and diamidino yellow dihydrochloride (DY·2HCl) compared with true blue and DY·2HCl in rat descending brainstem pathways

Abstract

Three series of double-labeling experiments were carried out in a study of the collateralization of brainstem nuclei which project to the spinal cord in the rat. The fluorescent tracer Diamidino Yellow Dihydrochloride (DY·2HCl) was injected in one half of the spinal gray and white matter at T7-T8 or T13-L1. Subsequently, either True Blue (TB), wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or free HRP were injected ipsilaterally in the gray matter at C5–C8. The distributions of single and double retrogradely laabeled neurons were studied in the following cell groups: red nucleus, interstitial nucleus of Cajal, ventrolateral pontine tegmentum, nuclei coeruleus and subcoeruleus and nuclei raphe magnus and raphe pallidus including the adjoining ventral reticular formation. The numbers of TB- or HRP-labeled neurons present in those nuclei were counted and the percentages of double-labeled neurons were calculated when TB or HRP had been used in combination with DY·2HCl. The results indicate: (1) The HRP-TMB reaction product and the DY·2HCl fluorescence can be visualized simultaneously in retrogradely single- or double-labeled neurons. (2) The distributions of single- and double-labeled neurons in the various brainstem nuclei were entirely comparable when using TB with DY·2HCl or HRP with DY·2HCl. (3) The percentages of double-labeled neurons obtained with HRP and DY·2HCl were consistent over a series of cases, and were comparable to those obtained with TB and DY·2HCl in several structures. However, in the red nucleus slightly lower percentages of double-labeled neurons were obtained using HRP and DY·2HCl as compared with the percentages obtained using TB and DY·2HCl.