Journal: bioRxiv

Article Title: BrainPhys neuronal medium optimized for imaging and optogenetics in vitro

doi: 10.1101/2020.09.02.276535

Figure Lengend Snippet: A-E, optogenetic testing of human iPSC-derived neurons with whole-cell patch-clamp recordings. A, live image of typical neurons expressing the optogene synapsin:ChETA-YFP and filled with rhodamine from the patch pipette. B, optogenetic responses of patch-clamped human neurons to blue LED stimuli. Whole-cell patch-clamped traces of example neurons stimulated with 0.4 mW 10 × 5 ms flashes of blue light at 10 Hz in BPI and ACSF. Top traces show ChR2-mediated currents recorded in voltage-clamp at −70 mV. Bottom traces show action potential evoked by ChR2-evoked membrane depolarization in current-clamp. Corresponding raster plots highlight consistent light-evoked firing over 10 sweeps. C, quantitative comparison shown in the graphs on the right were performed from a total of 8 neurons recorded in ACSF and BPI and tested with identical light stimulation parameters as shown in the trace-examples. Symbols represent human neurons tested first (triangles) or second (circle) in either medium. D , optogenetic responses from the same patch-clamped neuron under 0.1 mW of blue light while alternating perfusate from BPI to NEUMO and back to BPI for recovery. E, quantitative comparison shown on the graphs on the right were performed from a total of 22 neurons across four coverslips. The perfusate was alternated between BPI and NEUMO. The neurons were stimulated with 10 × 5 ms flashes of blue light at 10 Hz under three different blue LED power intensities (0.1, 0.2, 0.4 mW at 475 nm). F-I, optogenetically evoked and spontaneous firing rates of human iPSC-derived neurons expressing synapsin:ChETA-YFP were recorded in a 48-well multielectrode array (MEAs) plate at 37 °C with 5% CO 2 in either BP, BPI or NEUMO basal media with identical supplements. Spontaneous and light-evoked firing rates were recorded for 10 minutes after feeding. Neurons in this dataset were cultured in standard BP for 82 days before testing different media. 240 electrodes across 15 wells were recorded over 40 days during media testing. F-G, recordings collected from one 48-well MEA plate were split into two groups of wells represented in the graph by ‘circles’ (8 wells) and ‘triangles’ (7 wells); Both groups were maintained in BP from neural maturation until they were changed to BPI on day 0. Days 7-13 were the ‘test’ period in which half the wells (7) were switched to NEUMO. From day 14, both groups were cultured in BP to recover for one week and then were switched back to BPI. H-I, optogenetics responses of human iPSC-derived neurons recorded with MEAs. The wells in NEUMO and BPI were optically stimulated with blue light (Lumos) at increasing intensities while recording on MEA. Stimulation lasted 1.1s and consisted of 10 flashes of light, each lasting 10ms at 10 Hz. The cumulative number of spikes was summed in binning windows ( H ): 27.5 ms bins ; ( I ): 2s bins starting at the onset of the first light stimulus) over three replicates. In ( H) the cumulative number spikes were plotted with quadratic non-linear curves at various LED intensity (% of max power: 3.9mW/mm 2 ). Values are shown as mean ± SEM. Significance determined via two-tailed non-parametric unpaired (Mann Whitney) tests. P-values are annotated as follows: ns for P>0.05.

Article Snippet: The 48-well Lumos MEA plate was maintained at 37°C, 5% CO 2 environment within a Maestro Pro MEA system (Axion Biosystems).

Techniques: Derivative Assay, Patch Clamp, Expressing, Transferring, Cell Culture, Optogenetics, Two Tailed Test, MANN-WHITNEY