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. 2015 Jul 2;17(1):74–88. doi: 10.1016/j.stem.2015.05.014

Figure 4.

Figure 4

Generation of Synaptically Mature Neurons upon Combined Expression of Downstream Targets

(A) Schematic representation of the experimental procedure.

(B–D) Electrophysiological characterization of induced neurons upon overexpression of the constructs indicated by live fluorescence during recordings. Examples of sustained trains of APs generated when recording in current-clamp mode are shown (in B′, C′, and D′ top panel: stimulation protocol). 50% repetitive firing NeuroD4/GFP cells present first spike latency lower than 70 ms, with 50% higher than 150 ms; an example of frequency adaptation is shown (B′′ and B′′′). In (C′), an example of a repetitive AP generated in NeuroD4/Insm1 transduced cells is shown (four generated the first spike with a latency lower than 70 ms and the remaining two did so with a latency higher than 150 ms) and characterized by spike accommodation (C′′) and spike adaptation (C′′′). (D′′ and D′′′) show examples of repetitive spike discharge in NeuroD4+Prox1-expressing neurons. (B′′′′–D′′′′) A pie chart shows the fraction of cells firing bursting (gray), transient (blue), or sustained (yellow) APs.

(E) Table summarizing the electrophysiological parameters measured (brackets indicate the number of cells analyzed).

(F) Example of NeuroD4-induced neurons at 14 DPT (F′). A depolarizing current pulse (1 s, 85 pA) induced a train of APs (F′′). In (F′′′) the autaptic response (black trace, average of 10) could be blocked by NBQX (5 μM, red trace, average of 10).

(G and G′) Example of NeuroD4-Insm1-induced neurons at 14 DPI. A depolarizing current pulse (1 s, 230 pA) induced a train of APs (G′′). In (G′′′) the autaptic responses (black trace, average of 10) could be blocked by NBQX (10 μM, 10 min red trace, average of 10) and partially reversed following washout for 45 min (blue trace).

(H) Micrograph depicting a neuron induced by co-expression of NeuroD4-containing viral vector (red) and Insm-containing viral vector (green) immunostained at 30 DPI for MAP2 (H′, blue) and vGlut1 (H′′, white). Scale bar, 50 μm.

See also Figure S3.