Physiologic maturation is both extrinsically and intrinsically regulated in progenitor-derived neurons

Abstract

Voltage sensitive dye imaging. Progenitor cells were cultured in 24-well plates on coverslips for 14 days. Coverslips were moved to the imaging chamber and washed with Tyrode's solution. Red FLIPR dye (FLIPR Membrane Potential Assay Kit; Molecular Devices) was reconstituted to a final concentration of 20 µM and added to the imaging chamber. The cells were incubated for 30 mins at 37 °C. Muscimol (100 µM) and KCl (30 mM) were added to the FLIPR dye solution and applied to the cells using gravity perfusion while the fluorescence responses were imaged by taking images every 500 ms. Fluorescence changes were analyzed using Fiji software and values plotted in Excel.

Intrinsic Morphologic and Physiologic Development of Human Derived Retinal Ganglion Cells In Vitro

“…11,20,21 Human RGCs produce excitatory responses to glutamatergic agonists, and GABA receptors intrinsically switch from excitatory to inhibitory, which is important for synaptic development. 11,14,16,22,23 Finally, hRGCs generate voltagegated Na + (NaV) and K + (Kv)-dependent action potentials in response to direct current stimulation, 17,24 and these responses increase over culture duration. 11,25,26 In this study, we investigated the intrinsic morphologic and physiologic development of hRGCs over an extended period.…”

Directly induced human retinal ganglion cells mimic fetal RGCs and are neuroprotective after transplantation in vivo

“…We compared iRGCs after 6 days of differentiation with primary RGCs from post-natal day 2 (P2), which are developmentally immature. iRGCs and primary RGCs showed Ca 2+ influx when exposed to muscimol ( Figure 2 ), which resembles the effect of KCl-induced depolarization ( Figure 2 ) and is in contrast to the lack of depolarization-induced calcium entry in mature RGCs ( Venugopalan et al., 2020 ). These data show that rapidly induced RGCs predominantly exhibit immature electrophysiologic maturation, as measured by response to GABA A receptor activation.…”

“…Previous studies have shown that GABA A receptor activation is excitatory in immature RGCs ( Venugopalan et al., 2020 ). When stimulated by a selective GABA A agonist such as muscimol ( Wang et al., 2007 ), immature RGCs depolarize and demonstrate rapid calcium entry, visualized by calcium imaging with Fluo 4-AM.…”