Figure 5.

Altered ratio of synaptic/extrasynaptic α5 GABA_AR disrupts spine maturation. Neurons were transfected with α5^WT, α5^α4GBD, or α5^α2GBD, then fixed and stained under permeabilized conditions with anti-GFP and anti-MAP-2 antibodies. High magnification confocal z-series through dendritic regions were obtained, and 3D reconstructions were used to analyze spine length, density, and morphology. 3D reconstructions of confocal images from neurons expressing α5^WT, α5^α4GBD, or α5^α2GBD at DIV 14 (A) and DIV 21 (E). (B) At DIV 14, neurons expressing α5^α4GBD exhibited a significantly lower mushroom/filopodia spine ratio compared to either α5^WT or α5^α2GBD (for B–H, *p < 0.05, **p < 0.01; number of spines examined: α5^WT 394, α5^α4GBD 311, α5^α2GBD 358). (C) Neurons expressing α5^α4GBD exhibited significantly fewer mushroom spines per 10 μm compared to either α5^WT or α5^α2GBD. (F) At DIV 21, neurons expressing α5^α4GBD exhibited a significantly lower mushroom/filopodia spine ratio compared to α5^WT (number of spines examined: α5^WT 474, α5^α4GBD 453, α5^α2GBD 455). (G) Neurons expressing α5^α4GBD exhibited significantly fewer mushroom spines per 10 μm compared to α5^WT. There was no significant difference between the number of filopodia spines per 10 μm between constructs at DIV 14 (D) or at DIV 21 (H).