Functional characterization of human induced serotonergic neurons (iSNs). (a, b) Quantification of iSNs at 6 weeks post conversion shows a significantly higher percentage (~38%) of 5-HT+ (green) and (c) TPH+ over MAP2ab+ (red) neurons in the iSN groups as compared with the control induced neuron (iN) group. Arrowheads indicate 5-HT+/MAP2ab+ double positive neurons. (d) High-magnification confocal analysis of 5-HT+ (green) and TPH+ (red) neurons counterstained with DAPI (blue) revealed the presence of 5-HT+ puncta near the cell soma and sparse singular puncta at the dendrites and the axons (arrowheads). (e) Confocal analysis of serotonin transporter, SERT+ (green), TPH+ (red) neurons counterstained with DAPI (blue) shows punctate SERT staining at the cell soma and cell processes as indicated by arrowheads. (f) An example of a 9-week-old iSN (5-HT, red; DAPI, blue). (g–i) Electrophysiological activity was recorded using a glass pipette filled with Rhodamine (red), which diffuses into the patched neuron and allows for visualization of its morphology (left image). (g) Immunocytochemistry for Biocytin (red) and TPH (green) confirmed the patched neuron to be TPH+, as indicated by the arrowhead. (h) Action potentials and voltage-dependent sodium and potassium currents evoked by depolarizing steps. (i) Traces of spontaneous action potentials exhibited by iSNs. (j, k) Calcium transients of 6-week-old induced neurons (iNs) and iSNs were measured using Fluo-4. (j) Representative images from time-lapse calcium imaging (Fluo-4, green) of iSN, labeled with TPH2::TdTomato lentiviral reporter (red, top panel) and in iN, labeled with Synapsin::DsRed lentiviral reporter (red, bottom panel) with time stamps (minutes:seconds). (k) Three individual traces of calcium transients (red, green and blue) represented as change in fluorescence/minimum fluorescence (Delta F/F min) over time of iSN (top panel) with scale on right-hand side and iN (bottom panel) with scale on left-hand side are shown; gray trace represents background fluorescence. iSNs and iNs were generated from fibroblasts of two individuals, run as triplicates in each experiment. Data are represented as mean±s.e.m., *P-value<0.05. Scale bars: (a) 50 μm, (d, e) 5 μm, (f, g) 10 μm. TPH, tryptophan hydroxylase.