Doublecortin-Like Is Implicated in Adult Hippocampal Neurogenesis and in Motivational Aspects to Escape from an Aversive Environment in Male Mice

Specific knock-down of DCLK1 splice variant DCL. A, Overview of the three most important DCLK1 splice variants and their functional components. The shRNA target sequence resides in the 3′-UTR of DCL mRNA which is absent in DCLK-long and DCLK-short. B, Western blot analysis reveals splice variant specific knock-down of DCL in dox-induced transgenic (TG) animals compared with dox-induced WT animals. DCLK-long and DCLK-short expression is not affected. C, Although there is some leakage, this leakage does not affect hippocampal DCL expression during embryonic development. There is no significant difference in DCL expression between non-induced WT and TG littermates at embryonic day (ED)14 and postnatal day (PND)1 and PND3. D, After dox induction, in the hippocampal tissue (Hi) an almost 10-fold higher shRNA expression measured compared with non-induced TG littermates (Student’s t test, n = 4, two-tailed, p < 0.0001) In the OB a nearly 25-fold higher shRNA expression is measured (Student’s t test, n = 4, two-tailed, p < 0.0001). E, In both hippocampus (Hi, Student’s t test, two-tailed, control n = 4, dox n = 5, p < 0.01) and OB (Student’s t test, two-tailed, control n = 4, dox n = 5, p < 0.0001), DCL protein expression is reduced to 25% after dox induction compared with non-induced TG littermates.

Adult neurogenesis measurement using BrdU labeling. A, Twenty-four hours after a single BrdU injection, a significant (two-way ANOVA, F₍₃₎ = 6.079, p = 0.004) with an significant interaction between genotype and diet (p = 0.043). The number of BrdU+ cells is significantly increased compared with WT animals on dox diet and DCL-KD and WT mice on control diets (respectively, p = 0.0056, p = 0.0022, and p = 0.0017; n = 6). Effect of dox on DCL-KD mice: 95% CI: 401.7, 1573; p = 0.0022). B, Examples of hippocampi derived from animals killed 24 h after BrdU injection. Both sections are stained for BrdU and show mainly BrdU+ cells in the SGZ. Tissue is derived from dox-induced transgenic animals (dox) and non-induced transgenic littermates (control) C, BrdU/NeuN double staining revealed a trend (F₍₃₎ = 2.77, p = 0.057, two-way ANOVA) in double-positive cells in hippocampal dentate gyrus of dox-induced transgenic animals (dox, n = 5) compared with non-induced transgenic littermates and both WT control groups (control, n = 4). In the dox-fed group, pairwise comparison using t tests with pooled SD shows a significant difference between DCL-KD and WT animals (p = 0.01). Effect of dox on DCL-KD mice: 95% CI: −323.7, 68.6; p = 0.188. D–F, Confocal laser scanning microscopy images showing co localization of BrdU (green in D) and NeuN (red in E). Only cells in the dentate gyrus who are double-positive (yellow in F) were counted. Scale bar in D–F: 25 μm. Significant differences are indicated with an asterisk. Means are indicated with a black bar.

DCX cell morphology. A, DCX-expressing cells in the hippocampal dentate gyrus of a transgenic animal on a control diet showing a normal DCX morphology with cell nuclei close to the SGZ and dendrites toward the molecular layer (ML). B, Hippocampal dentate gyrus of a dox-induced transgenic littermate showing aberrant morphology of DCX+ cells. Hardly any DCX+ cell has dendrites in the granular cell layer (GCL) or ML. C, D, Close-up of DCX-expressing cells in the hippocampal dentate gyrus of a transgenic animal on a control diet (C). Several DCX+ cells show dendritic outgrow (arrows) toward the ML which are absent after DCL-KD (D). E–G, Number of proliferating type 1, 2, and 3 DCX+ cells in transgenic and WT mice on a control or dox diet. Two-way ANOVA testing shows a significant effect in the type 1 and 3 DCX+ cells (respectively, F₍₃₎ = 3.377, p = 0.04, and F₍₃₎ = 3.473, p = 0.04). Effect of dox on DCL-KD mice: 95% CI type 1 cells: 157.0, 5501.8; p = 0.039; 95% CI type 2 cells: −3553.7, 164.5; p = 0.072; 95% CI type 3 cells: −665.5, −12.8; p = 0.042. Significant differences are indicated with an asterisk. Means are indicated with a black bar. For further details, see main text.

Setup of the CHB experiment. A, Animals were put on a dox diet for at least five weeks before the CHB was started. B, The CHB paradigm started with a FET. Seven days later, the animals followed a training for four consecutive days with two trials a day. At day 5, the animals were exposed to a probe trial in which the escape hole was closed. C, The hole board was equipped with 12 holes. During training, one hole (black), by which animals could reach their home cage, was open. D, Photograph of the CHB setup in the lab.

Spatial parameters measured on the CHB. A, First visit latency. All four groups showed a similar decrease over four training days in latency to target (two-way ANOVA for repeated-measures, F₍₃₎ = 39 521, p < 0.001). B, Probe trial. DCL knock-down had no effect on the parameters “latency to target” (two-way ANOVA F₍₁₎ = 0.744, p = 0.392). C, Errors to target. All four groups showed a similar decrease over four training days in errors to target (two-way ANOVA for repeated-measures, F₍₃₎ = 13.230, p < 0.001). D, Probe trial. DCL-KD had no effect on the parameters “errors to target” (two-way ANOVA, F₍₁₎ = 2222, p = 0.141). For further details, see main text.