FIGURE 8.

Golgi stain-like labeling of CTNs with a viral vector and intracellular staining of pyramidal neurons in rat areas S1, FL, and HL. When high titers of adenoviral vectors expressing myrGFP-LDLRct (A; Kameda et al., 2008) were injected into the VP with 0.6 M NaCl, many L6 pyramidal neurons were retrogradely infected in the somatosensory motor area (B–B″). After the brown immunoperoxidase staining with anti-GFP antibody and diaminobenzidine (DAB; C), the cell body (D), basal dendrites (E,F), and apical dendrites (G,H) of CTNs were fully visualized. Note that even fine spines were visualized effectively. In 500-μm-thick cortical slices, single spiny neurons were labeled intracellularly (I) and visualized black (J–N) by the peroxidase method with DAB and nickel. In L6, retrogradely labeled (M′) and unlabeled neurons (N′) were indicated by L6⁺ and L6^– pyramidal neurons, respectively. Most L6^– neurons were considered to belong to corticocortical projection neurons, because their apical dendrites were short and the basal dendrites were abundant as reported previously (Zhang and Deschênes, 1997). In contrast, L6⁺ CTNs were taller and more slender than L6^– neurons. (O,P) It was examined whether each axon bouton of the intracortical collaterals was in close apposition to the retrogradely labeled CTN dendritic spines (large arrowheads) or not (double arrowheads). (Q,R) In addition, 77% of those appositions were electron-microscopically confirmed to form asymmetric synaptic contacts with the labeled spines (small arrowheads). The reaction products of retrograde labeling are indicated by small arrows. AT, labeled axon terminals; Den, dendritic profile; Sp, spine. Modified from Figures 1, 2, and 4 of Tanaka et al. (2011b). Scale bar in (B″) applies to (B–B″), that in (H) to (E–H), that in (N) to (J–N), that in (N′) to (M′,N′), that in (P) to (O,P), and that in (R) to (Q,R).