Figure 3.

GBZ-induced increase in spines is not reproduced by bumetanide and is not associated with changes in KCC2 expression. (A–C) Sample traces from which IV curves were generated (A) and resulting sample IV curve (B), and summary plots showing BUME hyperpolarizes E_GABA in organotypic slices (C, Ctrl −34.0 ± 2.0 mV, n = 3, GBZ −60.8 ± 3.8 mV, n = 3, p = 0.02). (D,E) Bumetanide does not increase spine density above control levels or change the effect of GBZ on spine density, (E, Control 0.21 ± 0.01 μm⁻¹, n = 102; GBZ 0.38 ± 0.02 μm⁻¹, n = 47; BUME 0.21 ± 0.02 μm⁻¹, n = 88; BUME+ GBZ 0.40 ± 0.02 μm⁻¹, n = 53; N = 3; two-way ANOVA indicated no significant interaction between GBZ and BUME treatment (p = 0.633). Tukey honestly significant difference (HSD) post-test indicates significant differences between Ctrl and GBZ in the absence of BUME (p < 0.001) and in the presence of BUME (p < 0.001). (F,G) Western blot (F) showing no changes in monomeric (KCC2-M) or oligomeric (KCC2-O) KCC2 expression following GBZ from 3 to 4 DIV (p = 0.52 and 0.77, respectively, one-sample t-test, n = 3) and 3–5 DIV (p = 0.76 and 0.87, respectively, one-sample t-test, n = 3) (G). Scale bar 3 μm. *p < 0.05, ***p < 0.001.