Use of two anterograde axon tracers to label distinct cortical neuronal populations located in close proximity

In order to investigate: converging projections originating from adjacent p opulations of cortical neurons, injections of two different anterograde tra cers, biotinylated dextranamine (BDA) and Phaseolus vulgaris leucoagglutini n (PHA-L), were made in close proximity. When the two injection sites were separated by around 500 mum and the time between injections was 1-4 h, BDA- labeling of neuronal elements was found not only at the BDA injection site but also at the PHA-L injection site. This false-positive BDA labeling of n eurons at the PI-IA-L injection site was so intense that labeled axons coul d be traced, both into the neighboring cortical gray matter and into white matter. Increasing the separation distance to 1000 mum resulted in much few er falsely positive labeled neurons at the PHA-L injection site. Even more effective was: extending the time interval between the two injections. Thus , if the BDA injection preceded the PHA-L injection by more than 12 h, virt ually no false-positive labeling was associated with the PHA-L injection si te. Those procedures may be applied to other combinations of anterograde tr acers, such as BDA with tetramethylrhodamine-conjugated dextran amine. (C) 2001 Elsevier Science B.V. All rights reserved.