Figure 1.
Dendritic morphology of mitral cells.
A, B: Lucifer yellow-labeled mitral cell at P3 (A) and P28 (B). Reconstructed cells with whole apical dendrite (insets) and their glomerular tufts are shown. High-magnification view of glomerular tuft at P28 is shown in B'. Many spine-like structures are visible. C: Total dendrite length (squares) and number of branching point (triangles) in a glomerulus were quantified at P3, 5, 7, 10 and 28. Asterisks denote statistically significant differences in total dendrite length and number of branching point between P3 and P10. Unpaired t test was used for statistics (**p<0.0001). Neither total dendrite length nor branching point number are significantly different between P10 and 28 (n.s.). D: Lateral dendrite of mitral cell labeled with Lucifer yellow at P7. Growth cones which have filopodial structures are seen at the tips of dendrites (D', D″). E: Another lateral dendrite labeled at P7. At this age, filopodia (arrowheads) are observed along the dendrite. Scale bars: 20 µm in insets of A and B, D, E; 5 µm in A, B; 2 µm in B', D', D″.
Figure 2.
Expression patterns of TrkB and p75NTR in developing OB.
A: Western blot analysis with P5 OB homogenate using anti-TrkBex antibody. About 25 µg of protein were loaded. B: P4 OB stained with anti-TrkBex antibody. Signals are observed in GL and EPL. C, D: High magnification views of GL (C) and EPL (D) stained with anti-TrkBex antibody (red) at P4. Dot-like TrkB signals (red) are apposed to both apical and lateral dendrites of mitral/tufted cells (green). C': Higher magnified images from the region encircled in C. TrkB signals (C'3; red in C'2) are positioned on YFP-positive processes (C'1; green in C'2) (arrowheads). E: P7 OB stained with anti-p75NTR antibody. Signals are seen in GL and MCL. F: High magnification views of a glomerulus stained with anti-p75NTR antibody (F1; red in F2). No p75NTR signal is seen at dendrites of mitral/tufted cells (green) (open arrowheads). The thy1-YFP-G mice are used to visualize dendrites of mitral/tufted cells in C, D, and F. Scale bars: 100 µm in B, E; 10 µm in C, D, F; 5 µm in C'.
Figure 3.
Properties of OB neurons in culture.
A: OB neurons cultured from P1 GAD67-GFP mice. At 4 DIV, cells were doubly stained with anti-GFP antibody (A1; green in A2) and anti-vGluT1 antibody (A3; red in A2). No GFP-positive cells express vGluT1. B: OB neurons cultured from P1 thy1-YFP-G mice. At 4 DIV, cells were doubly stained with anti-GFP (YFP) antibody (B1; green in B2) and anti-vGluT1 antibody (B3; red in B2). Many YFP-positive cells coexpress vGluT1. YFP-positive but vGluT1-negative cell also exists in culture (arrowhead). Scale bars: 50 µm.
Figure 4.
Increase of YFP-positive cells by TrkB activation.
A: OB neurons cultured from P1 thy1-YFP-G mice were doubly stained with anti-YFP antibody (A1; green in A2) and anti-TrkBex antibody (A3; red in A2) at 2 DIV. All YFP-positive cells coexpress TrkB. B: The p75NTR (red) is expressed by fibroblast-like cells and not by YFP-positive cells (green). Scale bars: 50 µm. C–E: Densities of DAPI (C) and YFP-positive cells (D) are measured after being cultured for 4 days with/without neurotrophins, and percentages of YFP-positive cells among DAPI are calculated and graphed after normalizing to control (E). BDNF, NT3, or NT4 were added to medium with final concentration of 1 or 10 ng/ml. When TrkBFc was applied, it was added to medium at a final concentration of 1 µg/ml with 10 ng/ml of neurotrophins. Application of BDNF or NT4 increases the proportion of YFP-positive cells in culture. Every condition was tested at least three times. Asterisks in E denote statistically significant differences from control condition analyzed with unpaired t test (**p<0.0001).
Figure 5.
Stimulation of neurite development by TrkB activation.
A–C: Representative YFP-positive cells cultured from P1 thy1-YFP-G mice. The cells were cultured for 4 days in control medium (A), with 10 ng/ml of BDNF (B), or with 10 ng/ml BDNF and 1 µg/ml of TrkBFc (C). YFP signals were enhanced with anti-GFP antibody. More short branches and primary neurites are formed in the cells treated with BDNF compared to control. D–G: YFP-positive cells were cultured with/without neurotrophins for 4 days as described in figure 4. Numbers of primary neurites (D) and branching points (E), total neurite length (E), and maximum neurite length (F) of YFP-positive cells with different culture conditions are measured and averaged. At least 76 YFP-positive cells were analyzed for each condition and the experiments were repeated three times. Asterisks denote statistically significant differences from control condition or differences between two conditions linked with a bar. Unpaired t tests were used for statistics (**p<0.0001). Scale bars: 25 µm.
Figure 6.
Effects of TrkB activation on filopodia growth.
A–C: Filopodia of YFP-positive cells cultured from P1 thy1-YFP-G mice. Cells cultured for 4 days in control medium (A), with 1 ng/ml of BDNF (B), or with 1 ng/ml BDNF and 1 µg/ml of TrkBFc (C) are shown. D: Filopodia densities at each indicated condition were measured. The experiments were repeated three times, and in total 78, 41, 42, and 39 cells were analyzed for control, 1 ng/ml BDNF, 10 ng/ml BDNF, and 1 ng/ml BDNF and 1 µg/ml TrkBFc, respectively. Asterisks denote statistically significant differences from control condition or differences between two conditions linked with a bar. Unpaired t tests were used for statistics (**p<0.001, *p<0.05). Scale bars: 20 µm.