Fig. 3.
NOS expression is up-regulated in layer Iα interneurons after bulbotomy, as shown by histochemical (A and B) and biochemical (C) analyses. (A and B) By NADPHd histochemistry, a large portion of nNOS is localized to axonal branches of layer I interneurons that ramify throughout layer I. Density of histochemically reactive layer I fibers increases progressively 8–20 h after bulbotomy, and differences between sham and bulbotomized animals are significant throughout this time period by ANOVA followed by post hoc testing (B). Overall, P < 0.0001; post hoc: *, P < 0.01 and **, P < 0.001. (C) Biochemical studies of nNOS expression in microdissected samples of layer I from three anteroposterior planes of piriform cortex indicated in Upper Left. Layer-specific dissection was aided by staining of fresh slices with nonalcoholic hematoxylin (Lower Left; dissected area is delineated by solid line; layer II staining is indicated with an arrow). By semiquantitative RT-PCR screening (Top Right; each condition represented by two lanes), standardized nNOS mRNA (nNOS/GAPDH) was found to be increased, compared to sham, at 12–24 h after bulbotomy; the difference at 18 and 24 h was significant at P < 0.005. Real-time RT-PCR confirmed the up-regulation of nNOS mRNA 12 h after lesion (Middle Right). Bars represent standardized (ΔΔCt) changes against GAPDH expression in unlesioned piriform cortex (gray column set as 1) and are significant between bulbotomized and sham animals (by t test, P < 0.05). By Western blotting, nNOS protein is also found increased 12–24 h after lesion (Bottom Right); each condition is represented here by a single lane, sham (S) juxtaposed to bulbotomy (B) for each time point. (Scale bar in A,20 μm.)