Retrograde tracing techniques influence reported death rates of adult rat nigrostriatal neurons

Injury often causes loss of neuronal markers and prior retrograde labeling can circumvent this problem of identification. We have previously used a time-consuming protocol for labeling all dopaminergic substantia nigra pars compacta neurons in adult rats by injecting the fluorescent tracer DiI into six sites throughout each neostriatum. Here, 2 weeks after injection of DiI into two central locations, only half of these nigrostriatal neurons were labeled. With six sites, more medial and lateral neurons were labeled, and also more in the midportion along the medial-lateral extent of the pars compacta. Less than 0.5% of the contralateral neurons were labeled. Two injections of Fluorogold also labeled fewer neurons, but their morphology was clearer. Two to 4 weeks after injection of the neurotoxin 6-OHDA into the two neostriatal sites, the total number of surviving neurons appeared greater with six sites of DiI than with two. However, within the middle region of the nigra, survival was lower with the six sites. This suggests that neurons that project outside the two central striatal tracer and 6-OHDA injection regions may be spared initially, but that those in the midportion that project to the central region are more vulnerable with the six-site protocol. Some reports suggest that Fluorogold prelabeling increases neuronal death. Here, survival after 6-OHDA or axotomy was similar with DiI or Fluorogold. These results suggest that because of a complex projection pattern of the nigrostriatal neurons, detailed quantification of neuronal survival should rely on extensive labeling. However, for drug screening purposes, faster labeling with Fluorogold using two sites is more suitable and should provide reliable data.