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. 2020 Aug 1;10(21):9674–9685. doi: 10.7150/thno.43829

Figure 3.

Figure 3

Selective overexpressing GSK-3β in NSCs induces temporal increase but long-term deficits of AHN. (A) AAV-EF1α-DIO-GSK-3β-2A-EGFP or AAV-EF1α-DIO-EGFP was stereotaxically infused into the DG for selective overexpressing GSK-3β in NSCs. (B) Experimental scheme. Tamoxifen (TAM) was injected for 3 consecutive days following AAVs injection, and BrdU was injected before sacrificing the mice. (C) Representative image showing the GFP expression by AAVs in the DG. Scale bar, 300 μm. (D) GSK-3β-2A-EGFP overexpression upregulated GSK-3β and β-catenin mRNA levels, with downregulated Hes5 but unchanged Gli1 mRNAs in EGFP-positive cells. Unpaired t tests. ** P < 0.01, *** P < 0.001. n = 3 mice/group. (E) Representative images showing AHN at 4- or 8-weeks post AAVs injection and TAM (wpt) administration. Scale bars were 50 μm (white) or 10 μm (red). (F) Overexpressing GSK-3β increased the number of BrdU-labeled cells at 4-wpt, but decreased BrdU-labeled cells at 8-wpt. Two-way ANOVA followed by Tukey's multiple comparisons tests (virus effect: F (1, 16) = 74.86, P < 0.0001; time effect: F (1, 16) = 7.268, P = 0.0159; interaction: F (1, 16) = 49.70, P < 0.0001). * P < 0.05, ** P < 0.01. n = 5 mice/group. (G) Overexpressing GSK-3β increased the number of EGFP-positive newborn cells both at 4- and 8-wpt. Two-way ANOVA followed by Tukey's multiple comparisons tests (time effect: F (1, 22) = 0.2388, P = 0.6299; virus effect: F (1, 22) = 45.15, P < 0.0001; interaction: F (1, 22) = 2.032, P = 0.1681). ** P < 0.01. n = 5 mice/group. (H) Overexpressing GSK-3β increased the dendrite complexity of EGFP-labeled neurons at 4-wpt, especially at the distal part, but reduced the complexity of proximal dendrites at 8-wpt. Two-way ANOVA followed by Tukey's multiple comparisons tests (4-wpt, virus effect: F (1, 108) = 1.053, P = 0.3071; distance effect: F (5, 108) = 57.50, P < 0.0001; interaction: F (5, 108) = 3.099, P = 0.0118. 8-wpt, virus effect: F (1, 114) = 60.80, P < 0.0001; distance effect: F (5, 114) = 8.511, P = 0.0042; interaction: F (5, 114) = 3.085, P = 0.0119). ** P < 0.01. n = 5 mice/group. (I) Overexpressing GSK-3β reduced the dendritic spine density of EGFP-labeled newborn neurons at 8-wpt. Two-way ANOVA followed by Tukey's multiple comparisons tests (virus effect: F (1, 44) = 12.52, P = 0.001; time effect: F (1, 44) = 87.60, P < 0.0001; interaction: F (1, 44) = 35.09, P < 0.0001). *** P < 0.001. n= 8~19 neurons/group. (J) Overexpressing GSK-3β did not change the total dendrite length of EGFP-labeled newborn neurons both at 4- and 8-wpt. Two-way ANOVA followed by Tukey's multiple comparisons tests (virus effect: F (1, 32) = 0.1322, P = 0.7185; time effect: F (1, 32) = 4.145, P = 0.0501; interaction: F (1, 32) = 3.244, P = 0.0811). n= 8-12 neurons/group. (K-L) GSK-3β overexpression in NSCs dominantly decreased the pool of nestin-positive radial NSCs in the DG granular cell layer (GCL) and molecular layer (ML). Scale bars were 50 μm (L). SGZ, subgranular cell zone. Unpaired t tests, * P < 0.05. n= 4 mice/group.