Fig. 4

Lower spine density and dendritic alterations of cortical pyramidal layer V neurons in GL mice. a, b H&E staining of frontal sections of the cerebral cortex (P15). CTRL (a) and GL mice b displayed similar layering during development (E.14.5) and isocortical layering at postnatal stages (P0, P15). c-f Rapid Golgi Cox staining of the isocortex in CTRL and GL mice at P15. Representative images of cortical layering (c), layer V pyramidal cells’ apical dendrite length (d), dendritic branching (e) and spines of apical dendrite branches (f). g Decreased apical dendrite length in GL mice compared to CTRL (p = 0.0286, Mann–Whitney test, n = 4 for CTRL, n = 4 for GL). (h) Less dendritic branching in GL mice compared to CTRL (p = 0.0010, Mann–Whitney test, n = 4 for CTRL, n = 4 for GL). (i) Significant reduction of spines in GL mice (p = 0.0001, Mann–Whitney test, n = 4 for CTRL, n = 4 for GL). (j) Spine classification defined as stubby, mushroom, long thin and filopodia by Imaris and MATLAB R2018b rules. (k) Representative spine classification in DiI-stained apical dendrite branches of CTRL and GL mice. (l) Overall decreased spine density (p = 0.0424, t-test, n = 3 for CTRL, n = 3 for GL) and reduction of filopodia (p = 0.0468, t-test, n = 3 for CTRL, n = 3 for GL) in GL compared to CTRL. Scale bar in a is 50 µm for E.14.5; 50 µm for P0 and 50 µm for P15 for a, b; scale bar in c is 100 µm; scale bar in d is 50 µm; scale bar in e is 20 µm for scale bars in f and k are 5 µm. VZ = ventricular zone, IZ = intermediate zone, CP = cortical plate, MZ = marginal zone, I-VI = cortex layer I-VI. CTRL n = 4, GL n = 4 for g-i; CTRL n = 3, GL n = 3 for l. ns = not significant (P > 0.05). P values are shown in figures as p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001