Supplementary Figure 8: Altered NG2-cell development in vivo and in vitro following GABAergic drugs.

(a) Representative confocal images of sagittal sections from NG2DsRed mice at P11 following treatment (P5–11) with daily injections of the GABA_A receptor antagonist bicuculline (1 mg/kg i.p.), the GABA-T inhibitor vigabatrin (100 mg/kg i.p.) or the GAT-1 inhibitor tiagabine (50 mg/kg i.p.) (see Methods). NG2-cells (red) were co-labeled with Olig2 (an oligodendrocyte lineage marker; white), Ki67 (a marker of proliferation; white), CC1 (a marker of mature oligodendrocytes; green) and DAPI (blue). White boxes illustrate the approximate regions in which cells were counted (see Methods and Fig. 6a). Scale bars 100 μm. (b) Representative confocal images of purified mouse NG2-cell cultures prepared from P1–P3 CD1 pups (see Methods). Cultures were treated with saline (left) or 100 μM muscimol (right). Cells were labeled with NG2 (red) and BrdU (white). Scale bars 50 μm. Bar graph (right) shows pooled data from 5 cultures, each prepared from 5 mice. Selected culture wells were treated either with saline (control; n = 5), 100 μM muscimol (n = 5) or 50 μM bicuculline (n = 3). One-way ANOVA (Welch heteroscedastic F test) showed a significant difference among the groups (F_{2, 5.37} = 10.34, P = 0.014), and subsequent pairwise comparisons showed an effect of muscimol on the percentage of NG2-cells that were BrdU⁺ (t_7.54 = 3.68, P = 0.014, n = 5 cultures from 5 mice each) but no effect following bicuculline pretreatment (t_4.74 = −0.78, P = 0.47, n = 5 and 3). *P < 0.05 (Welch two-sample t test with Holm's sequential Bonferroni correction for multiple comparisons).