To further analyze synaptic inputs onto the NKX2.1::GFP+ cells from the day 10-18 protocol, we examined spontaneous postsynaptic currents and localization of inhibitory or excitatory synaptic markers. GFP+ cells cultured on the mouse cortical feeder for 30 days expressed high levels of vesicular GABA transporter (VGAT), present within the presynaptic terminal of GABAergic synapses. The subcellular localization of VGAT within GFP+ cells closely matched expression of the GABAergic postsynaptic marker gephyrin (Figure 5A–E). Whole-cell patch clamp analyses demonstrated that NKX2.1::GFP+ cells receive spontaneous inhibitory postsynaptic currents (sIPSCs; Figure 5F) which are reversibly blocked by the addition of the GABA-A receptor antagonist bicuculline. In addition, NKX2.1::GFP+ cells also receive excitatory inputs, as demonstrated by the presence of vesicular glutamate transporter 1 (VGLUT1) expression adjacent to GFP+ putative dendrites, and co-labeling with the post-synaptic excitatory synapse marker PSD-95 (Figure 5G–5K). Consistent with the presence of glutamatergic synaptic inputs, spontaneous excitatory postsynaptic currents (sEPSCs; Figure 5L) were also readily detected in the NKX2.1::GFP+ neurons. Additional analysis of spontaneous synaptic activity in NKX2.1::GFP+ cells is presented in Figure S4 and Table S2.
