Further studies on the use of the fluorescent retrograde tracer True Blue in combination with monoamine histochemistry.

Some basic methodological aspects on the use of the retrograde fluorescent tracer True Blue (TB) was studied in freeze-dried material in the rat CNS. On the basis of fluorescence morphology and intensity at different filter settings it was possible to objectively delineate 3 distinct zones of the TB injection site, and define degrees of retrograde labelling from the different zones. Correlative studies of injection sites in subportions of the spinal cord and retrograde labelling in cell bodies of defined nuclei in the brainstem and cortex showed that effective uptake occurred only from the central area of the injection (zone I), while the uptake from the less intensely fluorescent zone II was variable and could only be documented for systems terminating within this zone. Time-course studies revealed that the size of the injection site and the resulting retrograde labelling is stable up to at least two months after injection and that relatively long survival times are often needed for optimal labelling. For practical purposes a bulk transport velocity of 20 mm/day can be used for estimating the survival time required for reasonable retrograde labelling. The accumulation of TB did not interfere with the visualization of monoamines in the same neurones, and the tracer was never seen to be anterogradely or transcellularly transported.