1：Experimental Design and Method Selection:

The study uses the Optopatch platform, which combines optogenetics for stimulation (CheRiff) and recording (QuasAr) of action potentials in neurons. Human iPS cells are differentiated into excitatory neurons using the NGN2 transcriptional programming approach and co-cultured with rodent glial cells. Immunocytochemistry and qRT-PCR are used for characterization. Pharmacological assessments with quinidine and retigabine are conducted to modulate ion channels.

2：Sample Selection and Data Sources:

Two control iPS cell lines (11a and 20b) from neurologically healthy male subjects are used. Neurons are produced and cultured up to DIV25. Data is collected from hundreds to thousands of neurons per experiment.

3：Data is collected from hundreds to thousands of neurons per experiment. List of Experimental Equipment and Materials:

3. List of Experimental Equipment and Materials: Includes custom-built ultra-widefield fluorescence microscope (Firefly), sCMOS camera (Hamamatsu ORCA-Flash 4.0), lasers (Dilas 635 nm), LEDs (Luminus 470 nm), culture media (e.g., mTeSR1, Neurobasal A), antibodies, and compounds (quinidine, retigabine).

4：0), lasers (Dilas 635 nm), LEDs (Luminus 470 nm), culture media (e.g., mTeSR1, Neurobasal A), antibodies, and compounds (quinidine, retigabine). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: iPS cells are cultured and differentiated into neurons. Neurons are transduced with lentiviral Optopatch constructs. Imaging is performed in a buffer with synaptic blockers. Blue light stimuli are applied, and fluorescence changes are recorded at 1 kHz. Data is analyzed using MATLAB for spike detection and parameter extraction.

5：Data Analysis Methods:

Fluorescence traces are segmented using PCA and ICA. Spike properties (width, rise time, afterhyperpolarization, frequency) are computed. Statistical significance is assessed using Kolmogorov-Smirnov tests with permutation tests.