Figure 3.
In Vitro Neuronal Differentiation and Whole-Cell Patch-Clamp Recordings of CB-iNCs
(A) Representative phase contrast (a) and immunofluorescence (b–i) images of CB-iNCs at differentiation day 65. Differentiated CB-iNCs expressed postmitotic mature markers such as axonal specific neurofilament SMI 312 (NF), MAP2, TAU (T46 epitope), and NEUN (RBFOX3) (b–e). (d) High-magnification image of TAU-positive cells showing downregulation of SOX2 nuclear staining, indicated by white arrows. CB-iNCs can differentiate into inhibitory GABA-positive neurons (e) and excitatory vGLUT1-positive neurons (f). Mature neurons derived from CB-iNCs after 65 days of differentiation expressed synaptic proteins, such as SYN and SV2 (g–i). Scale bars are as indicated in the panels.
(B) Current-clamp recordings from a GFP-positive (SYN::GFP) cell showing evoked action potentials by injecting 20 pA current steps (middle panel) and spontaneous action potentials at a −40 mV resting membrane potential (I = 0) (right panel). The scale bar is as indicated in the panel.
(C) Current-clamp recordings of another GFP-positive (SYN::GFP) cell in the same culture showing spontaneous activity that was pharmacologically modulated by glutamate (upper right panel) and glutamate antagonists (DNQX: non-NMDA receptor antagonist, MK801: NMDA receptor antagonist, left and middle bottom panels). Representative traces of spontaneous activity are truncated for clarity. Immunofluorescence confirmed expression of vGLUT1, together with GFP (SYN::GFP) and human specific N-CAM in this cell (right bottom panel). Scale bars are as indicated in the panels.
(D) Spontaneously active cells showed more mature electrophysiological properties, as capacitance was significantly higher (t35 = 1.66, p = 0.05) (left panel) and the resting membrane potential significantly lower (t29 = 2.51, p < 0.01) (middle panel) than those recorded from nonactive cells. Membrane resistance was smaller, although not statistically different (t35 = 1.45, p = 0.07) (right panel). ∗p ≤ 0.05; ∗∗p < 0.01; unpaired t test.
See also Figure S3.