Fluorescent retrograde triple labeling of brainstem reticular neurons

The present study was aimed at the anatomical identification in the rat of neurons of the lower brainstem reticular formation which give off axonal branches ascending bilaterally to more rostral structures and descending unilaterally to the spinal cord. Three fluorescent tracers were injected in one and the same animal. Fast Blue was injected in the midbrain tegmentum, in the termination areas and fiber bundles of the ascending reticular efferents; Evans blue was injected in the midbrain tegmentum on the other side; either Nuclear Yellow or Diamidino Yellow was injected in the white and gray matter of the upper cervical cord. All three populations of single-labeled cells, as well as cells double labeled either from the midbrain injections or from the ipsilateral injections in the mesencephalon and spinal cord, were intermingled in the medial reticular formation. Very few cells double labeled from the contralateral mesencephalon and ipsilateral spinal cord were also seen. However, the main finding of the present study was the visualization of triple-labeled cells. The latter were mainly located ipsilaterally to the injections in the spinal cord. The present results indicate that reticular cells give off divergent multiple branches descending to the ipsilateral spinal cord and ascending bilaterally to rostral centers.