Article Figures & Data
Figures
- Figure 1.
Specificity of Cre expression. A representative image showing YFP signal in Cre+/− offspring from crossing Pomc-cre+/− and cre-dependent ChR2-YFP reporter lines. Consistent with McHugh et al. (2007), we found dense expression in the dentate gyrus and sparser expression in the arcuate nucleus of the hypothalamus and the habenula. We also noted sparse expression in the optic tract, which McHugh et al. (2007) did not mention, but we can observe it in their figures (McHugh et al., 2007; their Fig. 1A,D). Fluorescence signal in the molecular layer of the dentate gyrus and stratum lucidum of the CA3 area reflects the dendrites and axons of granule cells, respectively. Scale bar: 1 mm.
- Figure 2.
Enriched housing used in this study.
- Figure 3.
Cell proliferation and the survival of new neurons were reduced in the dentate gyrus of adult DG-NR1KO mice. A, Representative images visualizing Ki67+ (green) and DAPI-labeled (red) cells in the dentate gyrus of adult control and DG-NR1KO mice. Scale bar: 75 μm. B, Density of Ki67+ cells in the subgranular zone. C, Representative images showing DCX+ (green) and DAPI-labeled (red) cells in the dentate gyrus of adult control and DG-NR1KO mice. The images were maximum intensity projections of confocal Z stacks and formed by joining two overlapping images containing adjacent areas. Scale bar: 25 μm. D, Density of DCX+ cells. E, Representative images showing BrdU+ (green) and Prox1+ (red) cells in adult control and DG-NR1KO mice on 7 and 28 d after BrdU injections. Scale bar: 75 μm. F, Density of BrdU+ cells in the granule cell layer and subgranular zone. G, Proportion of BrdU+ cells expressing Prox1. H, Density of BrdU+/Prox1+ cells in the granule cell layer. I, Density of BrdU+/Prox1– cells in the granule cell layer. J, Survival rate of BrdU+/Prox1+ cells from 7 to 28 dpi. Density at 28 d after BrdU injection was divided by mean values of density at 7 d; *p < 0.05, **p < 0.01, ***p < 0.005, independent-sample t test, two tailed.
- Figure 4.
Reduced size of the granule cell layer in adult DG-NR1KO mice. A, Representative images showing the granule cell layer visualized by DAPI staining in adult control and DG-NR1KO mice. Scale bar: 150 μm. B, Volume of the granule cell layer (GCL) in adult control and DG-NR1KO mice. Summed volume from three analyzed sections is presented. C, Thickness of the upper and lower blade of granule cell layer in adult control and DG-NR1KO mice. D, Length of the subgranular zone (SGZ) in adult control and DG-NR1KO mice; *p < 0.05, ***p < 0.005, independent-sample t test, two tailed.
- Figure 5.
Neurogenesis was impaired in the dentate gyrus of postnatal, developing DG-NR1KO mice. A, Representative images showing Ki67+ (green) and DAPI-labeled (red) cells in the dentate gyrus of four-week-old control and DG-NR1KO mice. Scale bar: 60 μm. B, Density of Ki67+ cells in the subgranular zone in two-, three-, and four-week-old control and DG-NR1KO mice. C, Representative images showing DCX+ (green) and DAPI-labeled (red) cells in the dentate gyrus of two-, three-, and four-week-old control and DG-NR1KO mice. The images were maximum intensity projections of confocal Z stacks. Scale bar: 25 μm. D, Density of DCX+ cells in the granule cell layer of two-, three-, and four-week-old control and DG-NR1KO mice. E, Representative images showing the granule cell layer visualized by DAPI staining in two-, three-, and four-week-old control and DG-NR1KO mice. Scale bar: 150 μm. F, Volume of the granule cell layer (GCL) in two-, three-, and four-week-old control and DG-NR1KO mice. Summed volume from six analyzed sections is presented; *p < 0.05, **p < 0.01, ***p < 0.005, independent-sample t test, two tailed.
- Figure 6.
Exposure to an enriched environment increases the survival of newborn neurons in DG-NR1KO mice. A, Experimental timeline. B, Representative images showing BrdU+ (green) and Prox1+ (red) cells in the dentate gyrus of adult control and DG-NR1KO mice with or without exposure to the enriched environment. Scale bar: 60 μm. C, Density of BrdU+ cells in the dentate gyrus of adult control and DG-NR1KO mice with or without exposure to the enriched environment. D, Proportion of BrdU+ cells expressing Prox1. E, Density of BrdU+/Prox1+ cells in the granule cell layer of adult control and DG-NR1KO mice with or without exposure to the enriched environment. F, Normalized density of BrdU+/Prox1+ cells in the granule cell layer of adult control and DG-NR1KO mice with or without exposure to the enriched environment. Density of each mouse was divided by a mean value of mice without exposure to the enriched environment in the same genotype; *p < 0.05, ***p < 0.005, Tukey’s HSD test (performed because of significant genotype × housing interaction in two-way ANOVA); ###p < 0.005, the main effect of genotype or housing (without significant interaction).
- Figure 7.
Impairment in novelty-induced Arc gene expression in granule cells of DG-NR1KO mice. A, Representative images showing Arc+ (green) and Prox1+ (red) expressing cells in the dentate gyrus of adult control and DG-NR1KO mice, which stayed in their home cage or were exposed to a novel environment. Scale bar: 75 μm. B, Density of Arc+/Prox1+ cells in the granule cell layer of adult control and DG-NR1KO mice with or without exposure to the novel environment. C, Normalized density of Arc+/Prox1+ cells in the granule cell layer of adult control and DG-NR1KO mice with or without exposure to the novel environment. Density of each mouse was divided by a mean value of mice without exposure to the novel environment in the same genotype. D, Depth categories divided along the thickness of the granule cell layer. The granule cell layer (GCL) was divided into five 20%-thickness segments, with 0% starting at the border to the hilus and 100% at the border to the molecular layer (ML). E, Density distribution of Arc+/Prox1+ cells along depth; *p < 0.05, **p < 0.01, ***p < 0.005, Tukey’s HSD test.
- Figure 8.
The DG-NR1KO mice display higher tendency to explore the center of an open field during a novelty-suppressed feeding test and show higher fecal counts in an open field test. A, A square open field used in a novelty-suppressed feeding test. Division of the open field into the peripheral, inner and center zone. B, Survival graph for latency to consume food for adult control and DG-NR1KO mice. C–F, Behavioral measurements in the novelty-suppressed feeding test for adult control and DG-NR1KO mice. G, A square open field used in an open field test. Division of the square open field into the peripheral and inner zone. H–J, Behavioral measurements in the open field test for adult control and DG-NR1KO mice. K, An elevated plus maze. L–O, Behavioral measurements in the elevated plus maze for adult control and DG-NR1KO mice; *p < 0.05, **p < 0.01, independent-sample t test, two tailed.
- Figure 9.
Graphs showing data from female and male mice separately. Data corresponding to Figures 3 (A), 4 (B), 5 (C), 7 (D), and 8 (E) are shown separately for females and males. Statistical results are summarized in Table 1-Table 14; #p < 0.05, ##p < 0.01, ###p < 0.005, the main effect of genotype or sex (without significant interaction); &p < 0.05, &&p < 0.01, &&&p < 0.005, Tukey’s HSD test (home cage vs NE including both sexes together and performed each genotype separately; performed because of significant genotype × exposure interaction).
- Figure 10.
Graphs showing data from <60- and ≥60-d-old mice separately. Data corresponding to Figures 6 (A), 7 (C), and 8 (D) are shown separately for <60- and ≥60-d-old mice. B, Relationship of the densities of BrdU+ and BrdU+/Prox1+ cells with the age of mice at the time of the first BrdU injection. Statistical results are summarized in Table 15-Table 26; ***p < 0.005, Tukey’s HSD test, performed because of significant genotype × housing interaction in two-way ANOVA; #p < 0.05, ###p < 0.005, the main effect of genotype, &p < 0.05, &&p < 0.01, &&&p < 0.005, Tukey’s HSD test (home cage vs novel environment including both age groups together and performed separately for each genotype; performed because of significant genotype × exposure interaction).
Tables
Sex Genotype Interaction Fig. 3B p 0.932 0.027 0.272 F(1,9) 0.008 6.917 1.371 Fig. 3D p 0.937 6 × 10−6 0.645 F(1,9) 0.007 87.273 0.227 Fig. 3F (7 d) p 0.010 0.002 0.465 F(1,12) 9.215 16.405 0.571 Fig. 3F (28 d) p 0.015 5 × 10−6 0.069 F(1,13) 7.750 55.532 3.920 Fig. 3G (7 d) p 0.314 0.567 0.448 F(1,12) 1.104 0.346 0.615 Fig. 3G (28 d) p 0.861 5.4 × 10−5 0.798 F(1,13) 0.032 34.581 0.091 Fig. 3H (7 d) p 0.130 0.028 0.829 F(1,12) 2.637 6.210 0.049 Fig. 3H (28 d) p 0.022 4 × 10−6 0.064 F(1,13) 6.708 57.234 4.113 Fig. 3I (7 d) p 0.171 0.276 0.432 F(1,12) 2.121 1.301 0.662 Fig. 3I (28 d) p 0.360 0.390 0.744 F(1,13) 0.889 0.790 0.112 Fig. 3J p 0.020 1.2 × 10−5 0.081 F(1,13) 6.98 46.673 3.575 Genotype Female Male Fig. 3B,D Control 3 3 KO 3 4 Fig. 3F–I (7 d) Control 3 4 KO 5 4 Fig. 3F–I (28 d) Control 5 3 KO 5 4 Fig. 3J Control 5 3 KO 5 4 Sex Genotype Interaction Fig. 4B p 0.052 3.7 × 10−4 0.524 F(1,9) 5.014 30.588 0.440 Fig. 4C, upper p 0.093 1.9 × 10−4 0.582 F(1,9) 3.53 36.882 0.326 Fig. 4C, lower p 0.792 0.004 0.928 F(1,9) 0.074 14.933 0.009 Fig. 4D p 0.523 0.030 0.894 F(1,9) 0.441 6.667 0.019 Genotype Female Male Control 3 3 KO 3 4 Sex Genotype Interaction Fig. 5B (week 2) p 0.794 0.595 0.224 F(1,9) 0.073 0.304 1.704 Fig. 5B (week 3) p 0.729 0.533 0.525 F(1,11) 0.126 0.414 0.431 Fig. 5B (week 4) p 0.797 0.632 0.947 F(1,11) 0.069 0.243 0.005 Fig. 5D (week 2) p 0.780 0.673 0.028 F(1,8) 0.084 0.192 7.180 Fig. 5D (week 3) p 0.813 0.014 0.818 F(1,11) 0.058 8.443 0.055 Fig. 5D (week 4) p 0.999 0.010 0.463 F(1,11) 0.000 9.805 0.579 Fig. 5F (week 2) p 0.811 0.306 0.162 F(1,9) 0.060 1.176 2.316 Fig. 5F (week 3) p 0.533 0.014 0.977 F(1,11) 0.415 8.621 0.001 Fig. 5F (week 4) p 0.766 0.023 0.790 F(1,11) 0.093 6.928 0.074 - Table 6
Tukey’s HSD test results (control vs DG-NR1KO, two-tailed, df = 8) after significant interaction for Figure 9C (sex analysis for Fig. 5)
Female Male Fig. 5D (week 2) p = 0.253 p = 0.363 Genotype Female Male Fig. 5B,F (week 2) Control 2 3 KO 4 4 Fig. 5D (week 2) Control 2 3 KO 3 4 Fig. 5B–F (week 3) Control 4 3 KO 4 4 Fig. 5B–F (week 4) Control 3 4 KO 4 4 Sex Genotype Exposure Sex ×
genotypeSex ×
exposureGenotype ×
exposureSex × genotype ×
exposureFig. 7B p 0.136 2.2 × 10−11 2.2 × 10−8 0.870 0.884 0.002 0.468 F(1,27) 2.360 118.582 60.911 0.027 0.022 12.068 0.541 Fig. 7C p 0.157 0.011 2.3 × 10−9 0.581 0.157 0.011 0.581 F(1,27) 2.120 7.548 76.610 0.312 2.120 7.548 0.312 - Table 9
Four-way mixed ANOVA results, between-subject effects, for Figure 9D (sex analysis for Fig. 7)
Sex Genotype Exposure Sex ×
genotypeSex ×
exposureGenotype ×
exposureSex × genotype ×
exposureFig. 7E (between-subjects effects) p 0.730 4.1 × 10−10 3.1 × 10−7 0.885 0.417 0.145 0.145 F(1,27) 0.121 90.569 45.456 0.021 0.680 2.250 2.248 - Table 10
Four-way mixed ANOVA, within-subject effects, for Figure 9D (sex analysis for Fig. 7)
Depth Depth ×
sexDepth ×
genotypeDepth ×
exposureDepth ×
sex ×
genotypeDepth ×
sex ×
exposureDepth ×
genotype ×
exposureDepth ×
sex ×
genotype ×
exposureFig. 7E (within-
subjects
effects)p 6.7 × 10−21 0.138 3.7 × 10−5 5.2 × 10−7 0.634 0.690 0.117 0.266 F(4,108) 41.121 1.781 7.162 10.148 0.641 0.563 1.890 1.323 Genotype Exposure Female Male Control Home cage 4 2 Novel 5 4 KO Home cage 5 5 Novel 5 5 - Table 12
Log rank test result (stratified with sex) for Figure 9E (sex analysis for Fig. 8)
Fig. 8B p = 0.538 χ2(1) = 0.380 Sex Genotype Interaction Fig. 8C p 0.303 0.188 0.183 F(1,39) 1.090 1.799 1.836 Fig. 8D (peripheral) p 0.460 0.034 0.619 F(1,39) 0.557 4.813 0.251 Fig. 8D (inner) p 0.776 0.151 0.397 F(1,39) 0.082 2.141 0.734 Fig. 8D (left) p 0.247 0.020 0.758 F(1,39) 1.381 5.880 0.096 Fig. 8E p 0.926 0.010 0.887 F(1,39) 0.009 7.322 0.021 Fig. 8F p 0.593 0.894 0.930 F(1,39) 0.291 0.018 0.008 Fig. 8H p 0.432 0.194 0.915 F(1,32) 0.633 1.761 0.012 Fig. 8I (peripheral) p 0.380 0.983 0.693 F(1,32) 0.792 4.7 × 10−4 0.158 Fig. 8I (inner) p 0.380 0.983 0.693 F(1,32) 0.792 4.7 × 10−4 0.158 Fig. 8J p 0.731 0.030 0.770 F(1,32) 0.120 5.187 0.087 Fig. 8L p 0.447 0.777 0.410 F(1,22) 0.599 0.082 0.704 Fig. 8M p 0.431 0.755 0.457 F(1,22) 0.644 0.100 0.574 Fig. 8N p 0.288 0.646 0.910 F(1,22) 1.186 0.216 0.013 Fig. 8O p 0.144 0.578 0.081 F(1,22) 2.301 0.319 3.335 Genotype Female Male Fig. 8C–F Control 9 13 KO 8 13 Fig. 8G–J Control 12 7 KO 9 8 Fig. 8L–O Control 7 6 KO 6 7 Age Genotype Housing Age ×
genotypeGenotype ×
housingAge ×
housingAge × genotype ×
housingFig. 6C p 0.0016 4.4 × 10−8 1.5 × 10−5 5.7 × 10−4 0.0015 0.431 0.035 F(1,22) 130.003 66.284 30.354 16.203 13.190 0.643 5.039 Fig. 6D p 0.413 0.0021 0.306 0.865 0.906 0.684 0.939 F(1,22) 0.696 12.100 1.097 0.030 0.014 0.170 0.006 Fig. 6E p 0.0011 1.1 × 10−8 1.3 × 10−5 6.5 × 10−4 7.0 × 10−4 0.322 0.037 F(1,22) 14.025 78.211 31.159 15.720 15.538 1.028 4.920 Fig. 6F p 0.279 0.282 4.0 × 10−4 0.185 0.282 0.279 0.185 F(1,22) 1.234 1.216 17.361 1.871 1.216 1.234 1.871 - Table 16
Tukey’s HSD test results (standard vs enrichment, two-tailed, df = 22) after significant interaction for Figure 10A,B (age analysis for Fig. 6)
Genotype <60 d ≥60 d Fig. 6C Control p = 1.9 × 10−4 p = 0.182 KO p = 1.000 p = 0.575 Fig. 6E Control p = 1.7 × 10−4 p = 0.156 KO p = 1.000 p = 0.737 Housing <60 d ≥60 d Control Standard 53.2 ± 4.1 67.0 ± 5.7 Enrichment 51.0 ± 1.7 66.0 ± 3.8 KO Standard 53.8 ± 4.7 65.5 ± 3.7 Enrichment 51.0 ± 1.7 65.6 ± 3.4 Housing <60 d ≥60 d Control Standard 5 2 Enrichment 3 4 KO Standard 4 4 Enrichment 3 5 - Table 19
Pearson correlation between age and density for Figure 10A,B (age analysis for Fig. 6)
BrdU+ BrdU+/Prox1+ Standard Enriched Standard Enriched Control p = 0.202 p = 0.015 p = 0.250 p = 0.017 r = −0.548 r = −0.851 r = −0.503 r = −0.844 KO p = 0.188 p = 0.199 p = 0.118 p = 0.323 r = −0.518 r = 0.507 r = −0.597 r = 0.403 Age Genotype Exposure Age ×
genotypeGenotype ×
exposureAge ×
exposureAge × genotype ×
exposureFig. 7B p 0.197 1.1 × 10−11 4.0 × 10−8 0.536 0.0017 0.541 0.515 F(1,27) 1.752 125.968 57.115 0.394 12.081 0.383 0.435 Fig. 7C p 0.519 0.0072 1.0 × 10−9 0.367 0.0072 0.519 0.367 F(1,27) 0.428 8.444 82.771 0.841 8.444 0.428 0.841 - Table 21
Four-way mixed ANOVA results, between-subject effects, for Figure 10C (age analysis for Fig. 7)
Age Genotype Exposure Age ×
genotypeAge ×
exposureGenotype ×
exposureAge × genotype ×
exposureFig. 7E (between-
subjects effects)p 0.295 1.9 × 10−10 3.8 × 10−7 0.384 0.259 0.246 0.847 F(1,27) 1.141 97.378 44.365 0.784 1.332 1.406 0.038 - Table 22
Four-way mixed ANOVA results, within-subject effects, for Figure 10C (age analysis for Fig. 7)
Depth Depth ×
ageDepth ×
genotypeDepth ×
exposureDepth ×
age ×
genotypeDepth ×
age ×
exposureDepth ×
genotype ×
exposureDepth × age ×
genotype ×
exposureFig. 7E (within-
subjects effects)p 3.4 × 10−20 0.735 2.0 × 10−4 1.0 × 10−5 0.295 0.697 0.101 0.822 F(4,108) 39.068 0.501 6.039 8.041 1.248 0.553 1.992 0.380 Exposure <60 d ≥60 d Control Home cage 4 2 Novel 5 4 KO Home cage 6 4 Novel 6 4 - Table 24
Log rank test results (stratified with age) for Figure 10D (age analysis for Fig. 8B–F)
Fig. 8B p = 0.660 χ2(1) = 0.194 Age Genotype Interaction Fig. 8C p 0.052 0.582 0.489 F(1,39) 4.022 0.308 0.487 Fig. 8D (peripheral) p 0.862 0.058 0.951 F(1,39) 0.031 3.815 0.004 Fig. 8D (inner) p 0.669 0.186 0.900 F(1,39) 0.185 1.808 0.016 Fig. 8D (left) p 0.750 0.044 0.941 F(1,39) 0.103 4.312 0.005 Fig. 8E p 0.495 0.010 0.526 F(1,39) 0.474 7.23 0.410 Fig. 8F p 0.940 0.770 0.742 F(1,39) 0.006 0.087 0.110 <60 d ≥60 d Control 17 5 KO 16 5
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