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. Author manuscript; available in PMC: 2016 Nov 30.
Published in final edited form as: Nat Neurosci. 2016 May 30;19(7):905–914. doi: 10.1038/nn.4315

Figure 5. The Ash2l/Mef2c complex mediates dendritic remodeling by nicotine in neural progenitor cells and in vivo.

Figure 5

(a, b) shRNAs targeting Ash2l or Mef2c were transfected into neural progenitor cells and spine density was measured on the apical dendrite in control cells (Saline) or following 4 days of exposure to nicotine in the bath (Nicotine). Nicotine-induced increases in spine density are significantly attenuated by Ash2l shRNA and Mef2c shRNA delivery (F(5,108)=28.45, p=0.000001 with LSD correction for multiple comparisons). Scrambled Saline vs. Scrambled Nicotine: p = 0.000001; Scrambled shRNA: Saline, n = 20, Nicotine, n=13; Ash2l shRNA: Saline, n = 26, Nicotine = 11; Mef2c shRNA: Saline, n = 17, Nic = 11. “n” sizes for neural progenitor studies represent total cells counted across 3 independent cultures. Scale bar = 18 μm. (c – f) shRNAs or overexpression constructs for Ash2l and Mef2c were introduced into the developing cortex at E14 by electroporation, with or without nicotine treatment. (c) Representative images of cortical neurons following nicotine treatment or electroporation of shRNA constructs (supplementary Fig. 6 shows extent of construct spread). (d) Nicotine exposure during the first 21 days of postnatal development increased the complexity of cortical neuron dendrites as measured by Sholl analysis (F(1, 174) = 14.570, p = 0.0002) and this was blocked by electroporation of shRNAs targeting Ash2l into the developing cortex at E14. Gene x treatment interaction: F(1, 174) = 16.633, p = 0.00006893 by two-way ANOVA with repeated measures followed by Tukey’s test for multiple comparisons (Scrambled Saccharin (n = 32) vs. Scrambled Nicotine (n = 57): p = 0.000581, Ash2l Saccharin (n = 22) vs. Ash2l Nicotine (n = 67): p = 0.229149. “n” sizes for in vivo studies represent number of slices from 5 mice. (e) Nicotine-mediated changes in dendritic complexity are abolished by electroporation of Mef2c shRNA into developing cortex. Gene x treatment interaction: F(1,110) = 95.076, p = 0.0000001 by two-way repeated measures ANOVA with repeated measures followed by Tukey’s test for multiple comparisons (Scrambled Saccharin (n = 26) vs. Scrambled Nicotine (n = 31): p = 0.000581, Ash2l Saccharin (n = 27) vs. Ash2l Nicotine (n = 30): p = 0.229149). (f) Overexpression of Ash2l or Mef2c increases dendritic complexity (F(2,71) = 100.450, p = 0.0000001 by one-way ANOVA with repeated measures). OE control (n = 28) vs. OE Ash2l (n = 22): p = 0.0000001; OE control (n = 28) vs. OE Mef2c (n = 24): p = 0.0000001 with Tukey’s test for multiple comparisons. (g) Nicotine-induced increases in spine density are significantly attenuated by electroporation of Ash2l or Mef2c shRNAs into developing cortex (F(5,183) = 10.748, p = 0.0000001 with LSD test for multiple comparisons). Scrambled Saccharin vs. Scrambled Nicotine: p = 0.0000001; Scrambled Saccharin vs. Mef2c Saccharin: p = 0.042553; Scrambled Saccharin vs. Mef2c Nicotine: p = 0.023182; Scrambled Nicotine vs. Mef2c Saccharin: p = 0.000158; and Scrambled Nicotine vs. Mef2c Nicotine: p = 0.000295. Scrambled shRNA: (Sac, n = 21; Nic, n = 36). Ash2l shRNA: (Sac, n = 34; Nic, n = 37). Mef2c shRNA: (Sac, n = 23; Nic, n = 38). (h) Overexpression of Ash2l or Mef2c significantly increases spine density increases (F(2,102) = 19.692, p = 0.00000006 with LSD test for multiple comparisons). OE control (n = 34) vs. OE Ash2l (n = 45): p = 0.000033; OE control (n = 34) vs. OE Mef2c (n = 26): p = 0.019220. Differences measured by one-way ANOVA followed by LSD test for multiple comparisons. Whiskers represent minimum to maximum value of data distribution. Error bars represent s.e.m.