Adult mice were anesthetized with sodium pentobarbital and injected with 10% biotinylated dextran amine (BDA; MW10000, Invitrogen) into the right motor cortex. Six 0.2-μl aliquots were injected (0.1 μl/min) with a stereotaxic apparatus at the following coordinates: (i) A (anteriority) = 1, L (laterality) = 2, D (depth) = 1; (ii) A = 1, L = 1, D = 1; (iii) A = − 0.25, L = 2, D = 1; (iv) A = − 0.25, L = 1, D = 1; (v) A = − 1, L = 2, D = 1; and (vi) A= − 1, L = 1, D = 1. At each injection point, the needle was left in place for 5 min to minimize leakage.
Two weeks later, the mice were deeply anesthetized and perfused with 4% PFA. Brain slices were prepared as described above, incubated with streptavidin-Cy3 (1:1000; Jackson ImmunoResearch) and counterstained with Hoechst 33258 (1:2000; Sigma) to visualize BDA labeling.
Two weeks later, the mice were deeply anesthetized and perfused with 4% PFA. Brain slices were prepared as described above, incubated with streptavidin-Cy3 (1:1000; Jackson ImmunoResearch) and counterstained with Hoechst 33258 (1:2000; Sigma) to visualize BDA labeling.
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