Fig. 2. BDNF knock-out (KO) mice demonstrate increased apoptosis and decreased proliferation within the basal cell region of the olfactory epithelium. A, TUNEL staining was performed on the olfactory epithelium from WT and BDNF knock-out animals and quantified across the entire epithelium. ***p < 0.0001 (direct cell counts: WT, 148 ± 9; BDNF null, 220 ± 15 TUNEL-positive cells/section).B, To evaluate the number of cells undergoing apoptosis at different times in the ORN life cycle, the epithelium was divided into thirds, representing the basal third containing neuronal precursors and some immature neurons, the middle third containing immature and mature neurons, and the apical or upper third containing mature neurons and sustentacular cell bodies. This schematic of the olfactory epithelium shows regional definitions that were used for TUNEL cell counting. C, The number of TUNEL-positive cells per region within the olfactory epithelium as defined inB is presented as a percentage of the total number per section. Direct cell counts (WT, 7.3 ± 1.1; BDNF null, 22.6 ± 2.2 TUNEL-positive cells within the lower one-third of the OE per section) demonstrate that BDNF null animals show a threefold increase in the number apoptotic cells found in the basal region over WT animals. Statistics were calculated by ANOVA followed by Student'st tests. n = 10–15 sections per mouse, three mice per genotype. ***p < 0.0001. Nestin and Ki67 immunostaining were performed on the olfactory epithelium from P0.5 WT (D, F) and BDNF knock-out (E, G) mice to confirm the results of TUNEL that suggested a decrease in the population of neuronal precursors and immature neurons in BDNF knock-out animals. Scale bar, 50 mm. Nestin- and Ki67-positive cells are indicated by asterisks. Nestin is a marker for neuroepithelial stem cells. Ki67 is a marker for non-Go cells. Both nestin and Ki67 staining were reduced in BDNF knock-out mice. Nestin and Ki67 staining were performed on 6–10 sections per mouse, three to four mice per genotype. H, The total number of H3-phosphohistone-stained cells per section in P0.5 WT and BDNF knock-out mice was quantified. H3-phosphohistone is a marker for cells undergoing mitosis, and was reduced in BDNF knock-out mice.I, Timed-pregnant mice received BrdU at E17, and embryos were harvested at E18 to examine the number of cells that incorporated BrdU in the olfactory epithelium. BrdU incorporation is a marker of DNA synthesis. The number of BrdU-positive cells per 40× field in 24 hr labeled E18 WT and BDNF knock-out mice was quantified. Cell counts were performed on 6–10 sections per mouse, two to three mice per genotype. Statistics were calculated by ANOVA followed by Student'st tests; *p < 0.05; **p < 0.001.