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In vivo imaging of dendritic pruning in dentate granule cells

Nature Neuroscience volume 19, pages 788–791 (2016)Cite this article

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Abstract

We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs.

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Figure 1: Chronic window implantation enables long-term in vivo imaging of DGCs and reveals time course of overgrowth and pruning of dendritic branches.
Figure 2: Exposure to EE and knockdown of CELSR3 lead to different growth and pruning time course but do not change final branch number.
Figure 3: Pruning of DGC dendrites is homeostatic and results in reduced heterogeneity of branch numbers.

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Acknowledgements

We thank L. Moore, E. Mejia, S. Marshall and J. Arroyo for their technical help. We thank P.-C. Lee for his help with the 4D SPA software, C. O'Donnell for his help with analysis, and M.L. Gage for editorial comments. This work was supported by grants from The James S. McDonnell Foundation (J.T.G., S.T.J., S.T.S. and F.H.G.), CIRM Grant TB1-01175 (C.W.B.), G. Harold & Leila Y. Mathers Charitable Foundation, Annette Merle-Smith, JPB Foundation, NIH Grants MH095741 and MH092758, The Leona M. and Harry B. Helmsley Charitable Trust Grant #2012-PG-MED00 (F.H.G.), Salk Innovation Grant (M.S.) and the Lewis Biophotonics Fellowship (S.T.J.).

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Authors and Affiliations

  1. Laboratory of Genetics, Salk Institute for Biological Sciences, La Jolla, California, USA

    J Tiago Gonçalves, Cooper W Bloyd, Matthew Shtrahman, Stephen T Johnston, Simon T Schafer, Sarah L Parylak, Thanh Tran, Tina Chang & Fred H Gage

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  1. J Tiago Gonçalves
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  2. Cooper W Bloyd
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  3. Matthew Shtrahman
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  4. Stephen T Johnston
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  5. Simon T Schafer
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  6. Sarah L Parylak
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  7. Thanh Tran
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  8. Tina Chang
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  9. Fred H Gage
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Contributions

J.T.G. and F.H.G. conceived the project, designed experiments and wrote the manuscript. J.T.G., C.W.B., S.T.J., S.T.S., S.L.P., T.T. and T.C. carried out experiments. J.T.G., C.W.B., M.S. and S.L.P. analyzed the data. J.T.G., S.T.J. and F.H.G. co-developed the DG window implant technique. F.H.G. supervised the project.

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Correspondence to Fred H Gage.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Chronic “window” implant for long-term in vivo imaging of the DG.

(a) Histology sections of implanted mouse brain injected with RV-GFP (green) and immunostained for Iba1(magenta). (b) Close-up of implanted hemisphere. The border of the DG remains intact and GFP-labeled newborn cells extend their dendrites into the molecular layer, despite an increase in inflammation. (c,d) Implanted DG has normal levels of FOS activation following a 15-min exposure to a novel EE, indicating that its synaptic inputs are intact (Surgery hemisphere, Sham: Home Cage: 144.9 ± 11.7 FOS+ cells/mm2 n = 5 mice, EE: 402.3 ± 71.5 FOS+ cells/mm2 n = 6 mice, p=0.0386, DG Window: Home Cage: 200.3 ± 26.0 FOS+ cells/mm2 n = 5 mice, EE: 491.1 ± 82.2 FOS+ cells/mm2 n = 6 mice, p=0.0175, 2-way ANOVA with Tukey’s multiple comparison test, *** p = 0.0003). Sham surgery consisted of a craniotomy permanently covered with a glass cover slip.

Supplementary Figure 2 Recurrent longitudinal in vivo imaging of identified neurons

Z-projection images of representative neuron at different time points. Scale bar = 100 µm (note different zoom at 15 and 19 dpi).

Supplementary Figure 3 Time course of DGC dendrite growth.

(a) Adult-born DGCs grew fast during the third week post-infection (mean daily length change 15-21 dpi, 90.9 ± 8.5 µm/day, n=5 cells) but growth subsided in the fourth week (22-29 dpi, 46.1 ± 7.7 µm/day, n=30 cells). By the end of the fourth week, total dendrite length reached a near plateau with small increments during the fifth (29-37 dpi, 11.6 ± 1.6 µm/day, n= 33 cells) and sixth weeks (37-43 dpi, 7.5 ± 4.1 µm/day, n=11 cells). (b) The maximum extent of branching occurred at 21 dpi (Median: 21, IQR: 19-22 dpi). (c) Sholl analysis of DGC dendrites at different time points.

Supplementary Figure 4 Structural dynamics of adult-born DGC dendrites around maximum branching.

(a) Number of endings of reconstructed GFP+ neurons, aligned by day of maximum branching, which highlights the extent of overgrowth and pruning. (b) Quantification of branch turnover for time points adjacent to maximum branching. Note the sharp reduction in branch addition after the peak (Added: 4.1 ± 0.5 vs. 1.7 ± 0.5 endings/day, n=16 cells, ** p=0.0013, MWU; Pruned: 2.1 ± 0.4 vs. 3.6 ± 0.8 endings/day, n=16 cells, p=0.2081, MWU). (c) Mean length of added and pruned branches (Added: 27.8 ± 1.9 µm, n=124 branches, Pruned: 24.3 ± 1.5 µm, n=118 branches; 18 cells, p=0.4355, MWU). (d) Length added and pruned as a fraction of total dendrite length at maximum branching (Added: 0.14 ± 0.02, Pruned: 0.13 ± 0.03 µm; 18 cells, p=0.5685, MWU). (e) Frequency distribution of the length of added and pruned branches.

Supplementary Figure 5 Differences between newborn DGC dendrites of EE and RC mice are transient.

(a) Mice injected with RV-GFP were exposed daily to an EE from 7 until 60 dpi and imaged at different time points. (b) EE cage size is large (91x91 cm) and contains running wheels. (c) At 31 dpi both RC and EE groups have a similar number of endings (RC: 31 dpi: 10.2 ± 0.3 endings/cell, n= 33 cells; EE: 10.8 ± 0.4 endings/cell, n=28 cells; p=0.0918, MWU). (d) Branch pruning is higher in EE neurons (RC: 4.5 ± 0.5, EE: 6.0 ± 0.5 pruned endings/cell, p=0.0337, MWU). (e) No differences were found in the fraction of endings preserved across both groups (RC: 0.72 ± 0.02; EE: 0.66 ± 0.02, p=0.0778, MWU). (f) Branch order distribution and (g) maximum branch order of RC and EE dendrites (RC: 5.8 ± 0.14; EE: 6.1 ± 0.16, p=0.0806, MWU). (h) Sholl analysis of DGC dendrites at 17 dpi (maximum of branching of EE group) and 31 dpi. (i) Total number of Sholl intersections was larger in EE group at 17 dpi, but no difference was found at 31 dpi (RC: 17 dpi: 47.5 ± 5.2 n=19 cells, 31 dpi: 123.7 ± 3.3 n= 33 cells; EE: 17 dpi: 76.2 ± 5.2 n=27 cells, 31 dpi: 125.4 ± 4.8 n=28 cells, two-way ANOVA with Sidak correction, 17 dpi: p=0.0002, 31 dpi: p=0.9500). (j) Rate of change of the number of endings before (p=0.9480, p=0.8447, MWU) and (k) after maximum branching (p=0.0794, p=0.3098, MWU). (l) Length of added and pruned endings is similar in RC and EE groups (EE Added:206 branches, p=0.5498, EE Pruned:177 branches, p=0.0641, MWU) (m) Length of added and pruned endings as a fraction of total length at maximum branching (EE Added:18 cells, p=0.2418; EE Pruned: 18 cells, p=0.8635, MWU). (n) Frequency distribution of the lengths of added and pruned endings in time points adjacent to maximum branching.

Supplementary Figure 6 Branch structure is unchanged in dendrites of CELSR3-deficient DGCs.

(a) Reconstructions of CELSR3-deficient dendrites. (b) Maximum number of endings (GFP: 14.7 ± 0.5, n= 33 cells; shCELSR3: 16.46 ± 0.8 endings/cell, n=25 cells, p=0.0728, MWU) and (c) fraction of endings preserved (GFP: 0.72 ± 0.02; shCELSR3: 0.66 ± 0.03, p=0.1017, MWU) were similar in GFP and shCELSR3 groups (d) Branch order distribution and (e) maximum branch order of GFP and shCELSR3 dendrites (GFP: 5.8 ± 0.1; shCELSR3: 6.1 ± 0.2, p=0.1805, MWU). (f) Sholl analysis of DGC dendrites at 19 dpi (maximum branching of shCELSR3 group) and 31 dpi. (g) Total number of Sholl intersections was larger in shCELSR3 group at 19 dpi but smaller at 31 dpi due to shorter dendrites (GFP: 19 dpi: 65.5 ± 5.5 n=24 cells, 31 dpi: 123.7 ± 3.3 n= 33 cells; shCELSR3: 19 dpi: 81.7 ± 5.1 n=23 cells, 31 dpi: 103.2 ± 3.4 n=25 cells, two-way ANOVA with Sidak correction, 17 dpi: p=0.0241, 31 dpi: p=0.0013). (h) Rate of change of the number of endings before (p=0.5829, p=0.8814, MWU) and (i) after maximum branching (p=0.2423, p=0.1498 MWU). (j) Length of added and pruned endings is similar in GFP and shCELSR3 groups (shCELSR3 Added: 126 branches, p=0.3364, shCELSR3 Pruned: 121 branches, p=0.7897, MWU). (k) Length of added and pruned endings as a fraction of total length at maximum branching (shCELSR3 Added: 12 cells, p=0.4459, shCELSR3 Pruned: 12 cells, p=0.4714, MWU). (l) Frequency distribution of the lengths of added and pruned endings in time points adjacent to maximum branching.

Supplementary Figure 7 Reduction in the branching variability of DGC dendrites requires homeostatic pruning.

(a) Number of endings pruned (31 dpi – Maximum) for all 86 cells imaged at 31 dpi (Mean=5.5) (b) Fraction of endings preserved (31 dpi / Maximum) for all 86 cells imaged at 31 dpi (Mean=0.68). (c) Whereas pruning a constant number or a constant fraction of endings both result in a reduction in the mean number of endings, only adaptive or homeostatic pruning results in a reduced variation of the number of endings (d). In this simulation, the linear-regression fit of Figure 3a was used to calculate the final number of endings in the homeostatic pruning model.

Supplementary Figure 8 Dendrite growth and morphology is unaffected by “window” implantation and in vivo imaging.

No differences were found in dendrite length (a) (EE, No surgery (slice): 1538 ± 29 µm n=18 cells, 4 mice; EE, surgery (slice): 1521 ± 40.0 µm n=17 cells, 4 mice; EE (in vivo): 1471 ± 52 µm n=28 cells, 6 mice; p>0.9999, p=0.9975, K-W with Dunn’s multiple comparisons test) and number of endings (b) at 31 dpi between adult-born neurons imaged in slices from EE mice that underwent no surgery, “window” implant surgery, and those imaged in vivo (EE, No surgery (slice): 10.2 ± 0.3 endings n=18 cells, 4 mice; EE, surgery (slice): 10.6 ± 0.3 endings n=17 cells, 4 mice; EE (in vivo): 10.8 ± 0.4 endings n=28 cells, 6 mice; p=0.8158, p=0.3084, K-W with Dunn’s multiple comparison test). EE exposure led to (c) longer dendrites (RC, No surgery (slice): 787 ± 81.5 µm n=14 cells, 3 mice; EE, No surgery (slice): 1052 ± 52.0 µm n=17 cells, 4 mice; RC (in vivo): 594 ± 67 µm n=20 cells, 6 mice; p=0.0537, p=0.2051, K-W with Dunn’s multiple comparisons test) and (d) more endings (RC, No surgery (slice): 11.3 ± 0.6 endings n=14 cells, 3 mice; EE, No surgery (slice): 17.1 ± 0.5 endings n=17 cells, 4 mice; RC (in vivo): 10.7 ± 0.9 endings n=20 cells, 6 mice; ** p=0.0020, p>0.9999, K-W with Dunn’s multiple comparison test), results that are in accordance with the in vivo data. By 31 dpi these differences were no longer present (e; Length, p=0.9999, p=0.1666 K-W with Dunn’s multiple comparison test) (f; Endings; p=0.9479, p=0.9999, K-W with Dunn’s multiple comparison test). (g) Representative reconstructions of neurons imaged from fixed slice preparations and in vivo at 31 dpi.

Supplementary Figure 9 Density of dendritic protrusions is unaffected by implant surgery.

Representative images of protrusions on terminal branches of dendrites in fixed slices from mice without (a) and with a “window” implant (b) at 31 dpi. Scale bar = 30 µm. (c) No difference was found between the density of protrusions in both groups (EE No surgery: 1.42± 0.02 protrusions/µm, n=16 cells, 4 mice, 1999 µm; EE Surgery: 1.41± 0.02 protrusions/µm, n=16 cells, 4 mice, 2117 µm; p=0.8748, MWU).

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Gonçalves, J., Bloyd, C., Shtrahman, M. et al. In vivo imaging of dendritic pruning in dentate granule cells. Nat Neurosci 19, 788–791 (2016). https://doi.org/10.1038/nn.4301

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