Article Figures & Data
Figures
- Figure 1.
Detection and quantification of BDNF in mouse hippocampus with different monoclonal antibodies. A, BDNF-IR using mAb#9 antibody in P21 WT C57Bl6/J hippocampus. CTIP-2 expression highlights granule cells in the dentate gyrus and pyramidal projection neurons in cornu ammonis (CA) 1-3. B, BDNF-IR in hippocampal mossy fiber projections of 8 week male WT C57Bl6/J versus NFL-Cre BDNFfl/ko hippocampus, using mAb#9. C, Specificity of 4 independent BDNF antibodies in CA3 mossy fiber terminals of WT C57Bl6/J (left column) and NFL-Cre BDNFfl/ko (right column). Clone 3C11 failed to detect endogenous BDNF. 3B2 produced high background in BDNF depleted hippocampal sections. The signal-to-noise ratio appeared best with mAb#9 and also with 4C8, which showed intense BDNF-IR. D, Double staining of BDNF and Myc in hippocampus of 8 week male BDNF-myc mice. Myc was costained with two independent polyclonal myc antibodies (Abcam, AB9106; Santa Cruz Biotechnology, SC789). E, BDNF protein levels in different CNS mouse brain areas, as determined by Western blot (top) with the 3C11 antibody and ELISA (bottom) using mAb#9 and mAB#1. P21 male WT C57Bl6/J (white bars) mice were compared with male NFL-Cre BDNFfl/ko mice (black bars) with bdnf gene recombination in most pyramidal neurons. Data are mean ± SEM. Hip, Hippocampus; CTX, anterior cortex; STR, striatum; Cereb, cerebellum (n = 3 for WT Hip, n = 5 for CTX, Cereb, STR, n = 2 for NFL Cre BDNFfl/ko Hip, CTX, STR, Cereb). Raw data are provided in Extended Data Figure 1-1 and Table 2. Image type: A, 2D merged single-plane image; B, D, maximum intensity projection; C, average intensity projection. Scale bars: B, D, 200 µm; C, 100 µm.
- Figure 2.
Alterations in brain BDNF levels during postnatal development. A, Sandwich ELISA analysis of relative BDNF protein levels (% of P20 hippocampus) shows a progressive increase of BDNF levels in all analyzed brain areas during the first 3 postnatal weeks. Anterior and posterior cortex shows the relatively highest increase in BDNF expression. Between P20 and P84, BDNF levels are downregulated in cortical and subcortical areas. B, BDNF-IR in hippocampal CA3 area of P21 (rows 1, 2) and P84 (rows 3, 4) mice. Single BDNF-expressing neurons are detected within the pyramidal cell layer in CA3 as well as in mossy fiber terminals at both ages. Voluntary physical activity in a running wheel leads to an increase in hippocampal BDNF-IR, which is more pronounced in 12-week-old animals compared with 3-week-old animals. Statistical analysis: A, One-way ANOVA, Tukey multiple comparison post-test (anterior CTX: F(3,9) = 36.18, p < 0.0001, ANOVA; posterior CTX: F(3,9) = 49.02, p < 0.0001, ANOVA; striatum: F(3,8) = 34.92, p < 0.0001, ANOVA; hippocampus: F(3,9) = 78.51; p < 0.0001, ANOVA). Data are presented as mean with SEM. n, number of independent animals. Raw data are provided in Extended Data Figure 2-1 and Table 2. Image type: B, maximum intensity projection. Scale bar: Hippocampus (CA3), 50 µm; CA3 (Detail), 25 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 3.
Tracing of corticostriatal projections. A, P21 mouse somatosensory cortex showing BDNF-IR in layers II/III, V, and VI. B, Representative coronal brain sections stained with DAPI. Images represent corticostriatal projections from prefrontal motor cortex (orange, boxed area represents the region depicted in D) to the dorsolateral striatum (green). C, Coronal brain section showing the injection site for fluorescent latex beads in the striatum. D, IHC staining of ipsilateral motor cortex (box in B), which corresponds to the region of highest tracer accumulation within corticostriatal projection neurons. Cux-1 and CTIP-2 label layers II/III and layers V/VI, respectively. Traced neurons were identified in layers II/III (top right) and upper layer V (bottom right). E, BDNF-IR in traced, Cux-1-positive neurons in layer II/III dorsal frontal cortex (top row, white arrow) and CTIP-2-positive neurons in layer V (bottom row, white arrow; see also Extended Data Fig. 3-1C,D; for statistics, see Extended Data Tables 4-1, 5-1). Not all BDNF-positive neurons contained retrograde tracer beads (yellow arrow). Raw data are provided in Table 2. Image type: A–C, 2D merged single-plane images; D, E, maximum intensity projection. Scale bars: D, Overview, 150 µm; Detail, 50 µm; E, Overview, 50 µm; Detail, 10 µm.
- Figure 4.
Physical activity increases BDNF expression in layers II/III of the motor cortex. A, BDNF-IR in layers II/III motor cortex (left column in A) shows same image as depicted in Figure 3E. P21 sedentary mice (column 1 from left) and runners (column 2); P84 sedentary (column 3) and runners (column 4; see also Extended Data Fig. 4-1). Number of retrogradely traced cortical neurons is shown in Extended Data Figure 4-2. B, Density of BDNF-IR-positive cells in layers II/III dorsal frontal cortex (see also Extended Data Figs. 4-1, 4-3A). P84 CTR mice show less BDNF-expressing neurons compared with P21 CTR animals. Physical activity leads to a significant increase in the number of BDNF-IR-positive cells at both ages (see Extended Data Figs. 4-1, 4-4A-C). C, BDNF-IR in traced corticostriatal neurons reveals a significant increase in the number of BDNF-positive neurons after physical activity (see Extended Data Fig. 4-1). D, Intensity of BDNF-IR per cell is decreased in layers II/III of P84 compared with P21 mice. Physical activity leads to a significant increase in BDNF-IR per cell at both ages compared with sedentary controls. E, The number of Cux-1-positive, layer II/III neurons and the number of traced neurons (see Extended Data Fig. 4-2A) are not affected by age or physical activity. Statistical analysis: one-way ANOVA, Tukey multiple comparison post-test (B: F(3,28) = 19.21, p < 0.0001, ANOVA; C: F(3,16) = 16.67, p < 0.0001, ANOVA; D: F(3,31) = 15.86, p < 0.0001, ANOVA; E: F(3,28) = 3.290, p = 0.0351, ANOVA). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 4-5 and Table 2. Image type: A, maximum intensity projection. Scale bars: A, 50 µm, Detail, 15 µm. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 5.
Physical activity leads to minor changes in BDNF expression in layer V motor cortex. A, BDNF-IR in layer V motor cortex. P21 sedentary mice (column 1 from left) and runners (column 2; see also Extended Data Fig. 4-3B); P84 sedentary (column 3) and runners (column 4). B, Density of BDNF-IR-positive cells in layer V dorsal frontal cortex (see also Extended Data Figs. 4-4A,B,D, 5-1). P84 CTR mice show less BDNF-expressing neurons compared with P21 CTR animals. At P21, significant differences were only observed when high numbers of sections were analyzed (n = 45), whereas no significant increase was observed in the P84 group. C, BDNF-IR in traced corticostriatal neurons reveals a significant increase in the number of BDNF-expressing neurons after physical activity only in the P84 group (see Extended Data Fig. 5-1). D, Physical activity leads to a significant increase in the intensity of BDNF-IR per cell at P84, but not at P21, compared with sedentary controls. E, The number of CTIP-2-positive, layer V neurons and the number of traced neurons (see Extended Data Fig. 4-2B) are not affected by age or physical activity. Statistical analysis: one-way ANOVA, Tukey multiple comparison post-test (B: F(3,28) = 11.69, p < 0.0001, ANOVA; C: F(3,16) = 11.01, p = 0.0004, ANOVA; D: F(3,31) = 3.474, p = 0.0277, ANOVA; E: F(3,28) = 0.3743, p = 0.7722, ANOVA). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 5-2 and Table 2. Image type: A, maximum intensity projection. Scale bars: A, 50 µm, Detail, 15 µm. *p < 0.05.
- Figure 6.
BDNF expression in somatosensory cortex layer VI during postnatal development and after physical activity. A, BDNF-IR in layer VI somatosensory cortex compared in sedentary and exercised animals at P21 (column 1, 2 from left in A) and P84 (column 3, 4 in A). B, Relative number of BDNF-expressing cells per area in somatosensory cortex layer VI is not changed by physical exercise in a running wheel at P21 or P84. C, Quantification of BDNF-IR intensity per cell reveals no significant effect of age or physical activity. The number of CTIP-2-positive, layer VI neurons is not affected by age or physical activity (see Extended Data Fig. 4-2C). Statistical analysis: one-way ANOVA, Tukey multiple comparison post-test (B: F(3,32) = 2.531, p = 0.0746, ANOVA; C: F(3,24) = 0.5629, p = 0.6447, ANOVA). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 6-1 and Table 2. Scale bars: A, 50 µm, Detail, 15 µm.
- Figure 7.
BDNF is enriched in glutamatergic corticostriatal presynaptic terminals. A, Confocal (top) and SIM (bottom) microscopic images showing BDNF-IR in the same section in glutamatergic (left) versus dopaminergic terminals (right) in the dorsal striatum. B, BDNF-IR is present in VGluT1-positive terminals (magenta arrows). Single BDNF-IR signals overlap with TH (white arrows). VGluT1- and TH-positive terminals reside in direct regional proximity but do not overlap. C, Quantification of BDNF signals in VGluT1-positive terminals and TH-positive terminals. True colocalization between BDNF/VGluT1 was confirmed by Costes p value (Costes p > 0.95) but not between BDNF/TH (Costes p ≪ 0.95). D, Representative Western blots showing recombinant BDNF (lanes 1, 2) versus endogenous BDNF derived from anterior cortex or striatum of P21 NFL-Cre BDNFfl/ko mice (lane 3), P21 sedentary mice (lanes 4, 5), and P21 runners after 72 h voluntary running-wheel exercise (lanes 6, 7); 30 µg of protein lysate was loaded for each sample. BDNF levels were normalized to cytochrome C. Band intensities were determined from extracts of 9 independent mice and presented in % of P21 sedentary mice. Statistical analysis reveals significant increase in BDNF protein levels in both brain areas after running-wheel exercise. Statistical analysis: unpaired t test (anterior CTX: t = 5,312, p < 0.0001; striatum: t = 2,784, p = 0.0133). E, SIM images showing BDNF-IR in VGluT1-positive terminals in the dorsal striatum in sedentary mice (top row) and after 72 h of voluntary running-wheel exercise (bottom row). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 7-1 and Table 2. Scale bars: A, 2.5 µm; B, 1.5 µm; E, Overview, 2 µm; Detail, 1 µm. *p < 0.05; ****p < 0.0001.
- Figure 8.
Behavioral analysis of NFL-Cre BDNFfl/wt mice. A, B, Open Field test: Quantification of distance traveled (A) or time spent (B) in center (n, number indicated below). C, Y-Maze test: Quantification of spontaneous alternations between WT and NFL-Cre BDNFfl/wt mice during an 8 min run. D, E, Y-Maze test: Quantification of spatial reference memory by analysis of distance traveled (D) and number of arm entries (E) into unknown versus known arm during the first minute after entering the maze (individual tracks depicted in Extended Data Fig. 8-1). F, G, Rotarod test with 8-week-old mice: Mean latency spent on rod comparing BDNF wt with NFL-Cre BDNFfl/wt mice (F). Mean latency spent on rod on individual days comparing BDNF wt with NFL-Cre BDNFfl/wt mice (G). H, I, Rotarod test with 34-week-old mice. Mean latency spent on rod comparing BDNF wt with NFL-Cre BDNFfl/wt mice (H). Mean latency spent on rod on individual days comparing BDNF wt with NFL-Cre BDNFfl/wt mice (I). n, number indicated below. Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers (A–F,H) or mean ± SEM (G,I). Statistical analysis: A, B, unpaired t test (A: t = 0.1225, p = 0.9055, unpaired t test; B: t = 0.5981, p = 0.5663, unpaired t test). C, Mann–Whitney test (Mann–Whitney U = 11.000, p = 0.1454). D, E, Unpaired t test (D: CTR t = 3.559, p = 0.0026; NFL-Cre BDNFfl/wt t = 2.873, p = 0.0207; E: CTR t = 2.718, p = 0.0152; NFL-Cre BDNFfl/wt t = 2.111, p = 0.0678). F, H, Unpaired t test (F: t = 0.2778, p = 0.7863; H: 0.09904, p = 0.9235). G, Two-way ANOVA, Friedman test (non-normal data distribution) revealed no significant improvement in rotarod test comparing day 1 with any of the following days within each group (Friedman statistic CTR: 5.957, p = 0.1137; NFL-Cre BDNFfl/wt 1.696, p = 0.6798). One-way ANOVA, Kruskal–Wallis test revealed no difference between WT and NFL-Cre BDNFfl/wt mice on any of the days tested (Kruskal–Wallis statistic 1.592, p = 0.9790). I, One-way ANOVA, Tukey test (normal data distribution WT F(2.264,9.057) = 0.8161, p = 0.4861, ANOVA) or Friedman test (non-normal distribution NFL-Cre BDNFfl/wt Friedman statistic: 1.938, p = 0.6255) revealed no significant improvement in rotarod test comparing day 1 with any of the following days within each group. One-way ANOVA, Kruskal–Wallis test revealed no difference between WT and NFL-Cre BDNFfl/wt mice on any of the days tested (Kruskal–Wallis statistic 3.119, p = 0.8738). Raw data are provided in Extended Data Figure 8-2 and Table 2. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 9.
Motor learning is impaired in mice with reduced BDNF in cortical pyramidal neurons. A, D, Mean score values of 3-month-old mice from 5 runs at days 1 and 2 on an irregular ladder rung walking task (unpaired t test, A: t = 5.170, p = 0.0003; Mann–Whitney test, D: Mann–Whitney U 5.500, p = 0.0476). B, E, Mean score value of individual runs in the irregular ladder rung walking task on day 1 and 2. Asterisks indicate significant difference in mean score value between the individual run and the first run of the particular day of either CTR or NFL-Cre BDNFfl/wt mice. Dashed lines indicate mean score value of first run (data points indicated as mean ± SEM). Two-way-ANOVA, Bonferroni's multiple comparison test: B: F(4,44) = 1.769, p = 0.1522; ANOVA, E: F(4,40) = 4.515, p = 0.0042; ANOVA. C, F, Learning effect indicated as difference in score value between first and last run on the particular day (unpaired t test, C: t = 3.174, p = 0.0089, F: t = 3.536, p = 0.0054). Data are presented as box and whiskers (Tukey). +, Mean. Vertical line indicates median. Black dots indicate outliers. n, number indicated below. Raw data are provided in Extended Data Figure 9-1 and Table 2. *p < 0.05; **p < 0.01; ***p < 0.001.
Tables
Figure Projection mode Type 405 nm 488 nm 550 nm 647 nm 1A 2D merge 0-255 DAPI CTIP-2 (10-160) BDNF (8 bit) (no change) (34-138) 1B Maximum intensity projection 0-4095 DAPI BDNF (12 bit) (2000-4095) (200-2503) 1C
WT CTRAverage intensity projection 0-4095 DAPI mAb#9 BDNF (12 bit) (400-4095) (700-3003) DAPI mAb#4C8 (200-2002) DAPI mAb#3C11, 3B2 (300-2503) 1C
BDNF KOAverage intensity projection 0-4095 DAPI (300-2503) BDNF (12 bit) (700-3003) 1D Maximum intensity projection 0-4095 DAPI BDNF Myc (AB9106) (12 bit) (250-2503) (200-750) (200-450) Myc (SC789) (150-500) 2B Maximum intensity projection 0-4095 DAPI BDNF (12 bit) (300-4095) (1401-3804) 3A 2D merge 0-255 CTIP-2 BDNF (8 bit) (35-140) (65-170) 3D Maximum intensity projection 0-4095 DAPI Tracer Cux-1 (12 bit) (1802-4095) (2002-4095) (600-2002) 3E Maximum intensity projection 0-4095 CTIP-2 Tracer BDNF Cux-1 (12 bit) (400-2002) (901-4095) (1601-4095) (901-4095) 4A Maximum intensity projection 0-4095 Tracer BDNF Cux-1 P21 CTR (12 bit) (1201-4095) (1301-4095) (600-3003) 4A Maximum intensity projection 0-4095 Tracer BDNF Cux-1 P21 RW (12 bit) (600-3003) (1301-4095) (600-4095) 4A Maximum intensity projection 0-4095 Tracer BDNF Cux-1 P84 CTR (12 bit) (1201-4095) (1301-4095) (600-4095) 4A Maximum intensity projection 0-4095 Tracer BDNF Cux-1 P84 RW (12 bit) (600-3003) (1301-4095) (600-4095) 5A Maximum intensity projection 0-4095 CTIP-2 Tracer BDNF P21 CTR (12 bit) (1201-3504) (600-3003) (1301-4095) 5A Maximum intensity projection 0-4095 CTIP-2 Tracer BDNF P21 RW (12 bit) (399-1700) (600-3003) (600-2302) 5A Maximum intensity projection 0-4095 CTIP-2 Tracer BDNF P84 CTR (12 bit) (350-1301) (600-3003) (800-2302) 5A Maximum intensity projection 0-4095 CTIP-2 Tracer BDNF P84 RW (12 bit) (350-1301) (600-3003) (600-1802) 6A Maximum intensity projection 0-4095 CTIP-2 BDNF P21 CTR (12 bit) (700-4095) (1201-4095) 6A Maximum intensity projection 0-4095 CTIP-2 BDNF P21 RW (12 bit) (700-4095) (1201-4095) 6A Maximum intensity projection 0-4095 CTIP-2 BDNF P84 CTR (12 bit) (700-4095) (1201-4095) 6A Maximum intensity projection 0-4095 CTIP-2 BDNF P84 RW (12 bit) (700-4095) (1201-4095) 7A confocal Maximum intensity projection 0-4095 DAPI TH BDNF VGluT1 (12 bit) (100-2002) (150-2002) (400-1697) (500-4095) 7A Maximum intensity projection 0-65535 TH BDNF VGluT1 SIM (16 bit) (1921-25624) (3843-26457) (2562-33888) 7B Maximum intensity projection 0-65535 TH BDNF VGluT1 SIM (16 bit) (1921-25624) (3843-26457) (2562-33888) 7E Maximum intensity projection 0-65535 BDNF VGluT1 P21 sedentary SIM (16 bit) (1300-7000) (1300-25.000) 7E Maximum intensity projection 0-65535 BDNF VGluT1 P21 running-wheel SIM (16 bit) (600-12000) (2500-33.000) 3-1A, left Maximum intensity projection 0-4095 DAPI BDNF pro-BDNF (12 bit) (600-4095) (900-4095) (500-2500) 3-1A, right Maximum intensity projection 0-4095 DAPI BDNF pro-BDNF (12 bit) (300-4095) (500-4095) (600-3500) 3-1B, left Maximum intensity projection 0-4095 DAPI BDNF pro-BDNF (12 bit) (600-4095) (900-4095) (500-2500) 3-1B, right Maximum intensity projection 0-4095 DAPI BDNF pro-BDNF (12 bit) (300-4095) (500-4095) (600-3500) 3-1C Maximum intensity projection 0-4095 BDNF pro-BDNF (12 bit) (900-4095) (400-3500) 3-1D, P21 sedentary Maximum intensity projection 0-4095 CTIP-2 BDNF pro-BDNF (12 bit) (800-2500) (900-4095) (400-3500) 3-1D P28 Maximum intensity projection 0-4095 CTIP-2 BDNF pro-BDNF NFL-Cre BDNFfl/ko (12 bit) (250-1000) (900-4095) (400-2500) 4-3A Maximum intensity projection 0-4095 BDNF Cux-1 (12 bit) (1000-4095) (350-4095) 4-3B Maximum intensity projection 0-4095 CTIP-2 BDNF (12 bit) (425-2500) (1000-4095) aOverview of the number of independent experiments, animal counts (including gender and age), number of images and cell counts.
Experiment Total no. of experiments Total no. of individualanimals Total no. ofconsidered images BDNF detection in hippocampusand striatum Figure 1A–D Hippocampus (WT) Figures 1A–C, 2B 45 105 272 Hippocampus (BDNF-myc) Figure 1D 11 2 21 Hippocampus (BDNF-ko) Figure 1B,C 9 6 20 Hippocampus monoclonalantibodies Figure 1C 4 8 38 Striatum Figure 7A,B 6 6 32 Striatum statistical analysisFigure 7C 1 1 15 Experiment Total no. ofexperiments Total no. of individualanimals Total no. ofconsidered images Total no. of countedBDNF-positive cells BDNF vs pro-BDNF-IR inhippocampus and cortex Figure 3-1 3 week CTR hippocampus 5 2 20 none 4 week BDNF-ko hippocampus 4 1 10 none 3 week CTR cortex 3 2 17 none 4 week BDNF-ko cortex 2 1 12 none Experiment Total no. ofexperiments Total no. of individualanimals Total no. of tracedanimals Total no. ofconsidered images Total no. of countedBDNF-positive cells BDNF detection in cortical neurons Figures 3–6 Figures 4-2, 4-3 3 week CTR layer II/III 5 10 4 65 1353 3 week RW layer II/III 5 8 5 51 1651 12 week CTR layerII/III 4 8 5 47 503 12 week RW layerII/III 3 6 6 40 1114 3 week CTR layer V 5 10 4 64 1400 3 week RW layer V 5 8 5 46 1541 12 week CTR layer V 4 8 5 42 438 12 week RW layer V 3 6 6 40 813 3 week CTR layer VI 4 10 0 52 2194 3 week RW layer VI 4 9 0 48 1930 12 week CTR layer VI 3 9 0 45 1572 12 week RW layer VI 2 8 0 50 1814 Experiment Total no. ofexperiments Total no. of individualanimals Total no. ofconsidered images Total no. of analyzedBDNF-positive cells BDNF-IR intensity Figures 4, 5D, 6C 3 week CTR layer II/III 7 10 90 559 3 week RW layer II/III 5 8 50 384 12 week CTR layerII/III 4 6 72 369 12 week RW layerII/III 3 6 42 288 3 week CTR layer V 7 10 100 680 3 week RW layer V 4 7 48 396 12 week CTR layer V 3 6 56 334 12 week RW layer V 2 6 36 215 3 week CTR layer VI 4 10 60 585 3 week RW layer VI 3 7 42 421 12 week CTR layer VI 2 7 42 386 12 week RW layer VI 1 4 24 225 Experiment Total no. ofexperiments Total no. of individualanimals Total no. of consideredimages Total no. of countedBDNF-positive cells BDNF detection incortical neurons Figure 4-3A,B 3 week CTR layer II/III 1 1 3 None 3 week RW layer II/III 1 1 4 None 4 week cBDNF ko layer II/III 1 1 2 None 3 week CTR layer V 1 1 3 None 3 week RW layer V 1 1 2 None 4 week cBDNF ko layer V 1 1 3 None Experiment Total no. ofexperiments Total no. of individualanimals Total no. of animalsconsidered for figure BDNF qRT-PCR Figure 4-3C 3 week CTR layer II/III 2 3 2 None 3 week cBDNF ko layer II/III 2 1 1 None 3 week CTR layer V 2 3 2 None 3 week cBDNF ko layer V 2 1 1 None Experiment Hippocampus Striatum Anterior cortex Cerebellum BDNF-ELISA Figure 1E WT no. of individual animals 3 5 5 5 NFL-Cre BDNFfl/ko no. of individualanimals 2 2 2 2 Experiment Hippocampus Striatum Cortex anterior Cortex posterior BDNF-ELISA Figure 2A P10 no. of individual animals 3 2 3 3 P14 no. of individual animals 3 3 3 3 P20 no. of individual animals 3 3 3 3 P84 no. of individual animals 4 4 4 4 BDNF Western blot detectionFigure 7D Total no. ofexperiments Total no. of individualanimals Total no. of consideredanimals in Figure Anterior cortex vs striatum:sedentary, runners, cBDNFKO Figure 7D 11 13 sedentary 9 sedentary 13 runner 9 runners 2 NFL-Cre BDNFfl/ko 2 NFL-Cre BDNFfl/ko Open Field Total no. of mice Figure 8A,B WT 4 females, 1 male NFL-Cre BDNFfl/wt 4 females, 1 male Y-Maze Total no. of mice Figure 8C–E WT 8 females, 2 males NFL-Cre BDNFfl/wt 4 females, 1 male Rotarod 8-week-old mice Total no. of mice Rotarod 34-week-oldmice Total no. of mice Figure 8F,G Figure 8H,I WT 7 males WT 4 females, 1 male NFL-Cre BDNFfl/wt 6 males NFL-Cre BDNFfl/wt 4 females, 1 male Irregular ladder rung Figure 9 Total no. of mice Total no. of mice tested day 1 Total no. of mice tested day 2 Total no. of runsper day WT 7 7 6 5 NFL-Cre BDNFfl/wt 6 6 6 5 ↵bImage preparation was performed using ImageJ software. Changes were made exclusively on brightness and contrast. γ values were not altered in any case.
Figure 1-1
Source data of BDNF ELISA depicted in Figure 1:E List of relative BDNF levels measured in cortex, hippocampus, striatum and cerebellum of either P21 C57Bl6/J WT or NFL-Cre BDNF fl/ko mice, in percent of mean BDNF level in P21 hippocampus. Download Figure 1-1, XLSX file.
Figure 2-1
Source data of BDNF ELISA depicted in Figure 2A: List of relative BDNF levels measured in anterior/posterior cortex, hippocampus and striatum of either P10, 14, 20 or P84 C57Bl6/J WT mice, in percent of mean BDNF level in P20 hippocampus. Download Figure 2-1, XLSX file.
Figure 3-1
Detection of BDNF and pro-BDNF in mouse hippocampus and layer II/III & V motor cortex: A) BDNF and pro-BDNF-IR detected with mAb#9 antibody and a rabbit polyclonal antiserum against the BDNF pro-domain (ANT-006) in P21 WT C57Bl6/J hippocampus. Pro- and mature BDNF are expressed in mossy fiber projections and single CA3 pyramidal neurons. B) BDNF and pro-BDNF-IR in P28 NFL-Cre BDNFfl/ko derived hippocampus shows absence of mature BDNF-IR and weak pro-BDNF-IR background noise. C-D) BDNF and pro-BDNF-IR in layer II/III (C) and layer V (D) motor cortex from P21 sedentary control or P28 NFL-Cre BDNFfl/ko mice. Both isoforms are detectable in cell bodies of cortical projection neurons in WT, while immunoreactivity for both antibodies is drastically reduced in NFL-Cre BDNFfl/ko motor cortex. Raw data are provided in Table 2 - Transparent Reporting. Image type: maximum intensity projection; Scale bar: A, B) 150 µm, C, D) 50 µm overview; 15 µm detail image. Download Figure 3-1, TIF file.
Figure 4-1
Summary of BDNF expression in layer II/III motor cortex at P21 and P84 sedentary and exercising mice: A-B) I and III represent mean values depicted in Figure 4, B, C). A-B) II represents BDNF expression quantification in layer specific neurons. Download Figure 4-1, XLSX file.
Figure 4-2
Variance of traced neurons in motor cortex of different groups of mice: A, B) Quantification of tracer positive neurons per area in layers II/III and V motor cortex reveals no significant difference between the four groups. C) CTIP-2 expression in layer VI of somatosensory cortex is not altered by physical activity or age. Statistical analysis: A-C) One-way ANOVA, Tukey multiple comparison post-test (A: F(3, 16)=0.5457, p = 0.6581, ANOVA; B: F(3, 16)=1.490, p = 0.2552, ANOVA; C: F(3, 32)=0.4924, p = 0.6901, ANOVA). Data are presented as box and whiskers (Tukey), “+” indicates mean, vertical line median, outliers shown as black dots; n number indicates the number of independent animals used for the analysis. Raw data are provided in Figure 4-6 - Source data and Table 2 - Transparent Reporting. Download Figure 4-2, TIF file.
Figure 4-3
Cortical layer-specific alterations in BDNF protein and mRNA levels after physical exercise and conditional BDNF ablation: BDNF-IR in layers II/III (A) and layer V (B) motor cortex. P21 sedentary mice (left column), 72 h voluntary running-wheel exercise (middle column) and NFL-Cre BDNFfl/ko mice (right column). C) Images show a representative toluidine blue-stained coronal brain section of motor cortex, used for LMD of layers II/III and V for qRT-PCR analysis. qRT-PCR for BDNF, normalized to GAPDH revealed a reduction of BDNF mRNA by ∼60% in layer II-III and ∼80% in layer V motor cortex in NFL-Cre BDNFfl/ko mice. Data are presented as levels relative to wild-type controls (CTR) in bar graphs for representative visualization of BDNF mRNA reduction (no statistical test was used, because of low n-number); n number indicated below. Raw data are provided in Figure 4-7 - Source data and Table 2 - Transparent Reporting. Image type: A, B) maximum intensity projection; Scale bar: A, B) 50 µm; 15 µm (detail). Download Figure 4-3, TIF file.
Figure 4-4
Quantification of BDNF expression in cortical neurons by different experimenters and automatic quantification using ImageJ: A, B) Quantification of BDNF-expressing neurons in P21 (A) or P84 (B) cortex using ImageJ versus manual counting by non-blinded investigator (n number indicated below). C-E) Correlation analysis between the non-blinded investigator and 4 blinded experts for BDNF-positive cell counts in layers II/III (C), V (D) motor cortex and layer VI somatosensory cortex (E). 3 random sample images were analyzed for each of the following conditions: P21 CTR, P21 RW, P84 CTR, P84 RW. Statistical analysis: A, B) unpaired t test between corresponding pairs (A: LII/III: t = 2.147, p = 0.0475, investigator: t = 3.630, p = 0.0023, LV: t = 0.8230, p = 0.4226, investigator: t = 2.487, p = 0.0243; LVI: t = 0.3224, p = 0.7511, investigator: t = 0.2480, p = 0.8071; B: LII/III: t = 3.598, p = 0.0037, investigator: t = 7.212, p < 0.0001, LV: t = 2.559, p = 0.0251, investigator: t = 3.892, p = 0.0021; LVI: t = 0.9821, p = 0.3416), P84 somatosensory CTX layer VI of non-blinded investigator - Mann–Whitney test (Mann–Whitney U 34.00, p = 0.8619). C-E) Linear regression and correlation analysis, Pearson's R value for correlation is indicated in each graph. Data are presented as box and whiskers (Tukey), “+” indicates mean, vertical line median, outliers shown as black dots; n number indicates the number of independent animals used for the analysis. Raw data are provided in Figure 4-8 - Source data and Table 2 - Transparent Reporting. Download Figure 4-4, TIF file.
Figure 4-5
Source data of Figure 4B-E – Quantification of BDNF and Cux-1 expression in Layer II/III motor cortex: B) List of the number of BDNF-positive neurons per 0.1mm2 in P21 and P84 sedentary controls and runner mice (72 h voluntary running-wheel exercise). C) List of the number of BDNF-positive, traced corticostriatal neurons in percent of the total number of traced neurons in P21 and P84 sedentary controls and runners. D) List of the intensity of BDNF-IR per cell in P21 and P84 sedentary controls and runners, indicated as fold change in percent of P21 sedentary controls. E) List of the number of Cux-1-positive in P21 and P84 sedentary controls and runners. Download Figure 4-5, XLSX file.
Figure 4-6
Source data of Figure 4-2: List of the number of traced neurons per 0.1mm2 in layer II/III (A) and layer V (B) motor cortex of P21 and P84 sedentary controls and runner mice (72 h voluntary running-wheel exercise). C) List of the number of CTIP-2-positive neurons per 0.1mm2 in layer VI somatosensory cortex of P21 and P84 sedentary controls and runners. Download Figure 4-6, XLSX file.
Figure 4-7
Source data of Figure 4-3C: List of relative BDNF mRNA levels in microdissected layer II/III or layer V motor cortex, derived from P21 sedentary C57Bl6/J WT or NFL-Cre BDNF fl/ko mice in percent of P21 sedentary control (top row). Crossing points obtained from qRT-PCR for BDNF (middle row) or GAPDH (bottom row) are indicated. Download Figure 4-7, XLSX file.
Figure 4-8
Source data of Figure 4-4: List of the number of BDNF-positive neurons per 0.1mm2 in layers II/III and V motor cortex and layer VI somatosensory cortex in either P21 (A) or P84 (B) sedentary controls and runner mice obtained by either automatic analysis using Image J or by non-blinded investigator. List of the number of counted BDNF-positive neurons in layer II/III (C) and V (D) motor cortex and layer VI somatosensory cortex (E) (mean value of n = 3 random samples) between non-blinded investigator and four blinded experts. Download Figure 4-8, XLSX file.
Figure 5-1
Summary of BDNF expression in layer V motor cortex at P21 and P84 sedentary and exercising mice: A-B) I and III represent mean values depicted in Figure 5, B, C). A-B) II represents BDNF expression quantification in layer specific neurons. Download Figure 5-1, XLSX file.
Figure 5-2
Source data of Figure 5B-E – Quantification of BDNF and CTIP-2 expression in Layer V motor cortex: B) List of the number of BDNF-positive neurons per 0.1mm2 in P21 and P84 sedentary controls and runner mice (72 h voluntary running-wheel exercise). C) List of the number of BDNF-positive, traced corticostriatal neurons in percent of the total number of traced neurons in P21 and P84 sedentary controls and runners. D) List of the intensity of BDNF-IR per cell in P21 and P84 sedentary controls and runners, indicated as fold change in percent of P21 sedentary controls. E) List of the number of CTIP-2-positive in P21 and P84 sedentary controls and runners. Download Figure 5-2, XLSX file.
Figure 6-1
Source data of Figure 6B, C – Quantification of BDNF expression in Layer VI somatosensory cortex: B) List of the number of BDNF-positive neurons per 0.1mm2 in P21 and P84 sedentary controls and runner mice (72 h voluntary running-wheel exercise). C) List of the intensity of BDNF-IR per cell in P21 and P84 sedentary controls and runners, indicated as fold change in percent of P21 sedentary controls. Download Figure 6-1, XLSX file.
Figure 7-1
Source data of Figure 7C, D: C) List of the raw data used for quantification of BDNF signals in VGluT1 and TH-positive terminals in the striatum. Indicated is the total number and density of counted VGluT1 or TH-positive synapses per µm2, the number and density of VGluT1 or TH-positive synapses with BDNF-IR and the amount of BDNF/VGluT1 or BDNF/TH double positive synapses in percent of either all VGluT1 or TH-positive synapses. Costes P value was calculated with ImageJ and is also indicated. D) List of band intensities of BDNF and cytochrome C, obtained from western blot photograph films using ImageJ. Indicated are the values of anterior cortex and striatum of P21 sedentary controls and runners. In addition, values measured in control tissue, derived from NFL-Cre BDNF fl/ko mice are listed (not shown in Figure 7). BDNF levels were normalized to cytochrome C and indicated as percent of either P21 sedentary control derived anterior cortex or striatum. Download Figure 7-1, XLSX file.
Figure 8-1
Y-Maze track records during the first minute after opening of the closed arm in the spatial reference memory test run: Documentation of tracks for control mice (A) and NFL-Cre BDNFfl/wt mice (B). Animals were placed in the start-arm (top) and were able to choose between the known arm (blue) and the unknown arm (orange). Download Figure 8-1, TIF file.
Figure 8-2
Source data of Figure 8: A) List of values for distance traveled in center of an open field in percent of total distance comparing C57Bl6/J WT and NFL-Cre BDNF fl/wt mice. B) List of values for time spent in center of an open field in percent of total time. C) List of values for spontaneous alternations in a Y-Maze task with all arms open. D) List of values for distance traveled in known versus unknown arm in percent of total distance traveled within the first minute after entering a Y-Maze in a spatial reference memory task. E) Number of arm entries into known versus unknown arm in percent of total arm entries within the first minute after entering a Y-Maze in a spatial reference memory task. List of values for mean latency on a rotarod in seconds obtained from 8 week (F) or 34 week (H) old C57Bl6/J WT and NFL-Cre BDNF fl/wt mice. List of values for latency on a rotarod showing individual 8 week (G) or 34 week (I) old animals on four consecutive days. Download Figure 8-2, XLSX file.
Figure 9-1
Source data of Figure 9: List of mean score values in an irregular ladder rung walking task on day 1 (A) or day 2 (D) comparing C57Bl6/J WT and NFL-Cre BDNF fl/wt mice. List of individual score values of 5 runs comparing C57Bl6/J WT and NFL-Cre BDNF fl/wt mice on day 1 (B) and day 2 (E). List of learning effect values calculated as difference in score value between first and last run on day 1 (C) or day 2 (F). Download Figure 9-1, XLSX file.
