Article Figures & Data
Figures
- Figure 1.
Diphtheria toxin-induced ablation of DCX+ cells in the dentate gyrus using lentiviral transduction of DTR. A, Schematic representation of the recombinant lentiviral vector construct. LTR: long terminal repeat; DCX: doublecortin; DT: diphtheria toxin; IRES: internal ribosome entry site; GFP: green fluorescent protein; WPRE: Woodchuck hepatitis virus posttranscription regulatory element. B, Diagram of the ablation technique specific for immature neurons. C–E, To evaluate the distribution and specificity of viral transduction, we injected the viral vector into the dentate gyrus of mice and examined GFP expression 7 d after the injection, which corresponds to the time when DT was injected in behavioral experiments (Figs. 6-8). C, Representative images showing the distribution of virus-transduced cells visualized by GFP fluorescence over the AP axis of the dentate gyrus. GFP expression was localized predominantly along the hilar border of the granule cell layer across the AP axis of the dentate gyrus, where adult-born neurons are known to be located. Scale bar: 200 μm. D, Density of GFP+ cells across the AP axis of the dentate gyrus. GFP+ cells were quantified in every 12 coronal sections covering the AP axis of dentate gyrus. Sections 1 and 6 correspond to ∼1.06–1.34 and 3.52–3.80 mm posterior to the bregma, respectively. E, A representative confocal image of the granule cell layer in a section immunostained against GFP and DCX. Among 460 DCX+ cells examined (from one hemisphere in three sections each of two mice), 40.7% of DCX+ cells expressed GFP, indicating that viral transduction was achieved in a large proportion of immature neurons. Among 455 GFP+ cells examined (from one hemisphere in three sections each of two mice), 43.8% of GFP+ cells were DCX+. We noted that GFP expression level was highly variable between GFP+ cells (low GFP-expressing cells are highlighted by arrowheads). Scale bars: 20 μm. F, G, To further evaluate the distribution and specificity of viral transduction, we analyzed sections from nine control mice used for the behavioral experiment described in Figure 6. These sections were from 17 d after virus injection, during which water maze training, probe trials and re-training were performed. F, Representative confocal images showing colocalization of DCX and GFP in neurons in the granule cell layer. Scale bars: 10 μm. G, Top, Proportion of GFP+ cells expressing DCX. Percentage within total population, high and low GFP-expressing cell populations were plotted separately. Bottom, Proportion of DCX+ cells expressing GFP. For each population, two sections from different AP levels corresponding to sections 2 and 4 in D were analyzed separately. We observed some GFP+DCX– cells which appeared to include non-neuronal and granule cells based on their morphology. Without an active degradation mechanism, GFP protein is highly stable even after the transcription activity of the promoter is shut off (Andersen et al., 1998). Therefore, GFP protein is expected to be maintained in new neurons that expressed DCX at the time of virus injection but thereafter lost the expression of DCX (and presumably DTR) during the survival time. Thus, some of GFP+DCX– cells were likely to be new neurons that had just lost DCX expression. In accordance with this possibility, the proportion expressing DCX was higher in high GFP-expressing cells (>50%) than that of low-expressing cells (<20%). Nonetheless, we cannot completely exclude the possibility that some of GFP+ cells were mature neurons. Our estimation revealed that the proportion of GFP+/DCX– cells among the total granule cell population in the dentate gyrus would be small (0.6%; see Materials and Methods). This is consistent with our observation of no ablation of mature granule cells (nine weeks old), shown in Figure 2. H, Fluorescent images of the virus-injected dentate gyrus after the injection of PBS or DT. Top, The granule cell layer visualized by DAPI staining was intact after DT-induced ablation. Middle and bottom, DT injection led to an obvious reduction in DCX+ and GFP+ cells. Scale bar: 100 μm.
- Figure 2.
DT-induced ablation of new neurons at the early maturational stage. A, Experimental design. To label the different ages of newborn neurons, BrdU was injected into mice at four different time points (8 d, 14 d, 4 weeks, and 9 weeks) before DT administration. For each mouse, one dose of BrdU (100 μg/g body weight) was injected intraperitoneally. B, Representative images showing two- or nine-week-old BrdU+ cells from virus-injected and non-injected control hemispheres after DT injection. Scale bar: 150 μm. C, Normalized densities of BrdU+/NeuN+ cells. The densities in the injected hemispheres were normalized by dividing by the densities in the non-injected hemispheres of the same mice. Each data point represents one mouse (8 d: n = 3 mice, two weeks: n = 2, four weeks: n = 4, nine weeks: n = 4). A value of 1 indicates that the densities of both hemispheres are equal. Note that a significant reduction in immature (8- to 14-d-old) neurons but minimal effects on four- and nine-week-old neurons. The densities were measured from three sections (every 12 40-μm-thick sections, corresponding to sections 2–4 in Fig. 1D) for each mouse. These three sections are distributed over the dorsal region of the dentate gyrus. D, Estimation of the numbers of ablated neurons at different ages, using trapezoidal rule integration. Difference in the number of new neurons between the control and injected hemisphere (gray area) reflects the number of ablated new neurons. E, Cumulative percentage of ablated new neurons at different ages, using trapezoidal rule integration. Percentages of ablated neurons younger than different ages in the total ablated population were plotted; ***p < 0.005.
- Figure 3.
Ablation of DCX+ cells occurs over multiple days while keeping the ability of protein synthesis/activity-dependent gene expression intact in the granule cell layer. A, Experimental design. B, Representative images showing the reduction of GFP+ cells over 7 d after DT injection. Scale bars: 150 μm. C, Normalized densities of DCX+ cells in the virus-injected hemisphere after DT injection. The densities in the injected hemispheres were normalized by dividing by the densities in non-injected hemispheres in the same mice. D, Representative images of c-fos expression in the granule cell layer of virus-injected and non-injected control hemispheres. Scale bars: 100 μm. E, Normalized densities of c-fos+ cells over 7 d after DT injection. The same way of normalization as C was used.
- Figure 4.
Ablation method induces Iba1 expression in the dentate gyrus. A, Experimental design. Mice were stereotaxically injected with PBS (control group) or the viral vector (ablated group) into the dentate gyrus. After 7 d of recovery, DT was injected into both groups. Brain sections were prepared from the mice after an additional 10 d of survival. B, Representative images of Iba1 immunostaining of brain sections from the control and ablated groups. C, Density of Iba1+ cells in the dentate gyrus. An increase was observed after the induction of ablation by a combination of the viral vector and DT injections (p = 0.0081, t(4.566) = −4.478, n = 5 for each group, independent sample t test). D, Experimental design. Mice were stereotaxically injected with the viral vector into the dentate gyrus. After 7 d of recovery, PBS (control group) or DT (ablated group) was injected into both groups. Brain sections were prepared from the mice after an additional 10 d of survival. E, Representative images of Iba1 immunostaining of brain sections from the control and ablated groups. F, Density of Iba1+ cells in the dentate gyrus. An increase was observed after the induction of ablation by a combination of the viral vector and DT injections (p = 3.9 × 10−5, t(8) = −8.132, n = 5 for each group, independent sample t test).
- Figure 5.
Overall integrity of the granule cell layer and mature granule cells is intact after DT-induced ablation. A, NeuN immunostaining showed the integrity of granule cell layer after DT-induced ablation. The lentiviral vector was injected into the dentate gyrus in one hemisphere. Seven days later, DT was injected. After another 7-d survival, brain sections were prepared and immunostained with DCX or NeuN. The images were from virus-injected (Injected) and non-injected (control) hemispheres. Scale bar: 50 μm. B, DAPI immunostaining showing the integrity of granule cell layer of mice in the control and ablated group. Scale bar: 50 μm. C, Cell density in the granule cell layer was not affected by the ablation (p = 0.783, t(23) = −0.279, control: n = 13 mice, ablated: n = 12, independent sample t test). The analysis was performed using the sections from the mice used in the experiments described in Figure 7. D, Golgi-stained granule cells of mice in the control and ablated group. The lentiviral vector was injected into the dentate gyrus in both hemisphere, and PBS or DT was systemically injected 7 d later. After another 7-d survival, the mice were euthanized for the Golgi staining. Scale bar: 50 μm. E, Sholl analysis for Golgi-stained granule cells in the control and ablated group. No significant group difference was detected (group: p = 0.600, F(1,271) = 0.276, radius: p = 1.1 × 10−9, F(19,271) = 4.429, group × radius interaction: p = 0.996, F(18,271) = 0.330, n = 10 cells for each group, two-way ANOVA). F, Total dendritic length of the Golgi-stained granule cells. No significant, group difference was detected (p = 0.907, t(18) = 0.119, n = 10 cells for each group, independent sample t test). G, The density of dendritic spines in the Golgi-stained granule cells. No significant group difference was detected (p = 0.923, t(18) = 0.098, n = 10 cells for each group, independent sample t test). H, Intact olfactory bulb neurogenesis after DT-induced ablation in the dentate gyrus. Fluorescent images of DCX+ cells in the olfactory bulb and subventricular zone after DT-induced ablation in the dentate gyrus. Images from virus-injected and control hemispheres are shown. To confirm that the ablation technique did not affect neurogenesis outside the dentate gyrus, we injected the viral vector into the dentate gyrus in one hemisphere and performed DT injection one week after the virus injection. We examined the densities of DCX+ cells one week after the DT injection and did not observe any obvious difference between the virus-injected hemisphere and non-injected control hemisphere either in the olfactory bulb or in the subventricular zone. This observation was confirmed by quantifying the density of DCX+ cells in the subventricular zone (control hemisphere: 2.97 ± 0.02; ablated hemisphere: 3.22 ± 0.15 in 103 cells/mm2, p = 0.248, t(2) = −1.614, n = 3 mice, paired t test) and the granule cell layer of the olfactory bulb (control hemisphere: 5.35 ± 1.47; ablated hemisphere: 5.68 ± 1.14 in 104 cells/mm3, p = 0.825, t(2) = −0.252, n = 3 mice, paired t test). Scale bars: 300 μm (left) and 15 μm (right); **p < 0.01, ***p < 0.005. Scale bars: 300 μm.
- Figure 6.
Posttraining ablation impaired the persistence of platform search behavior in a water maze task. A, Experimental design. B, Representative images of DAPI staining and DCX+ cells in the dentate gyrus of mice in the ablated and control groups. Scale bar: 150 μm. C, The densities of DCX+ cells. D, The volume of the granule cell layer. E, F, Latency to locate the platform during pretraining (E) and training (F). The two groups improved performance similarly well in both pretraining and training. G, The position of the removed platform and the area near the platform in probe trials. The area near the platform is defined as the circular area within 14 cm of the center of the former platform position. H, Occupancy plots showing average time that mice spent at different positions in the pool. The color bar below shows the color code for occupancy time; warmer colors indicate high occupancy, while cooler colors represent low occupancy. Dotted circles indicate the area near the platform. I, Time spent in the area near the platform position, the number of platform entries and swimming speed in probe tests. A dotted line indicates the chance level in time spent near the platform position. J, Latency to reach the position of the removed platform. K, Percentage of time spent in the area near the platform position and the number of platform entries per 10 s after the first entries into the position of the removed platform. L, Duration per visit to the area near the platform. M, Latency to locate the platform during re-training. The two groups improved performance similarly well; *p < 0.05, **p < 0.01.
- Figure 7.
Posttraining ablation impaired the persistence of tone-induced freezing in a tone trace fear conditioning task. A, Experimental design. B, Timings of a tone (conditional stimulus) and an electrical shock (unconditioned stimulus) during training. C, Percentage time in freezing during the baseline period and after the onset of first tone during training. No significant difference was detected between the groups. D, The densities of DCX+ cells. E, The volume of the granule cell layer. F, The definition of the tone and posttone periods in the tone test. G, Percentage time in freezing for every 10 s during the tone test. Gray areas indicate the timing of tone deliveries. H, I, Percentage time in freezing during baseline, tone, posttone 1, 2, 3, and 4 periods, averaged over five tones (H) or for first tone only (I). J, K, Frequency (J) and duration (K) of freezing episodes during the baseline period and after the onset of first tone in the tone test; *p < 0.05, ***p < 0.005.
- Figure 8.
Posttraining ablation impaired the persistence of context-induced freezing in a contextual fear conditioning task. A, Experimental design. B, The densities of DCX+ cells. C, The volume of the granule cell layer. D, E, Percentage time in freezing during the baseline period and after the shock in training (D) and remainder training (E). No significance different was detected between the groups. F, Percentage time in freezing for every 10 s during the context test. G, Percentage time in freezing in the context test. H, Latency to reach 20-s cumulative freezing in the context test. I, J, Percentage time in freezing before (I) or after (J) reaching cumulative freezing of 20 s. K, The number and duration of freezing episodes; *p < 0.05, ***p < 0.005.
Tables
Pretraining (three-way, group × trial × day, control, n = 18 mice; ablated, n = 18 mice) Latency to platform Speed p df F p df F Day 0.016 3, 102 3.607 6.5 × 10−7 2.434, 82.753 14.743 Day × group 0.858 3, 102 0.254 0.630 2.434, 82.753 0.524 Trial 1.4 × 10−11 1.529, 51.982 52.033 0.006 2, 68 5.494 Trial × group 0.885 1.529, 51.982 0.072 0.612 2, 68 0.495 Day × trial 0.459 4.993, 169.754 0.936 0.007 6, 204 3.023 Day × trial × group 0.713 4.993, 169.754 0.583 0.226 6, 204 1.375 Group 0.870 1, 34 0.027 0.157 1, 34 2.089 Training (two-way, block × group, control, n = 18 mice; ablated, n = 18 mice) Latency to platform Speed p df F p df F Block 2.6 × 10−12 4.313, 146.653 17.434 7.8 × 10−5 5.291, 179.911 5.440 Block × group 0.321 4.313, 146.653 1.184 0.474 5.291, 179.911 0.919 Group 0.566 1, 34 0.336 0.533 1, 34 0.398 Re-training (three-way, group × trial × day, control, n = 18 mice; ablated, n = 18 mice) Latency to platform Speed p df F p df F Day 0.034 2, 68 3.565 0.065 1.682, 57.174 3.020 Day × group 0.815 2, 68 0.205 0.826 1.682, 57.174 0.149 Trial 1.7 × 10−30 5.849, 198.874 38.729 0.0002 7, 238 4.199 Trial × group 0.756 5.849, 198.874 0.562 0.494 7, 238 0.917 Day × trial 0.478 9.888, 336.198 0.960 0.127 12.302, 418.255 1.479 Day × trial × group 0.871 9.888, 336.198 0.524 0.627 12.302, 418.255 0.826 Group 0.920 1, 34 0.010 0.265 1, 34 1.282
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