New article in Translational Psychiatry (2021): FORTIS: a live-cell assay to monitor AMPA receptors using pH-sensitive fluorescence tags

The real-time live fluorescent monitoring of surface AMPA receptors (AMPARs) could open new opportunities for drug discovery and phenotypic screening concerning neuropsychiatric disorders. We have developed FORTIS, a tool based on pH sensitivity capable of detecting subtle changes in surface AMPARs at a neuronal population level. The expression of SEP-GluA1 or pHuji-GluA1 recombinant AMPAR subunits in mammalian neurons cultured in 96-well plates enables surface AMPARs to be monitored with a microplate reader. Thus, FORTIS can register rapid changes in surface AMPARs induced by drugs or genetic modifications without having to rely on conventional electrophysiology or imaging. By combining FORTIS with pharmacological manipulations, basal surface AMPARs, and plasticity-like changes can be monitored. We expect that employing FORTIS to screen for changes in surface AMPARs will accelerate both neuroscience research and drug discovery.

Left, Heat maps showing the changes in SEP/pHuji-GluA1 as a function of pH and after the addition of ammonium chloride (NH4Cl). Each square represents the relative fluorescence in a single well (culture).
Right, Real-time measurements of SEP/pHuji-GluA1 starting at pH 7.4 (time 0), after which the pH was reduced to 5.5 and then increased back to 7.3 and 7.9 with increasing concentrations of NH4Cl.
Left, Fluorescent images of hippocampal cultures infected with SEP-GluA1, both at baseline and immediately after treatment with glycine. o A heat map of the changes in SEP-GluA1 fluorescence (ΔF/F0, %) where each square represents a single hippocampal culture in a 96-well plate. After a 5-min baseline, cLTP was induced by injecting glycine (as indicated by the arrow). The fluorescence was measured at 485/40–528/20 (Ex−Em) every 5 min for 1 h.
Middle, The color map represents the change in fluorescence 60 min after cLTP induction.
Right, Changes in SEP-GluA1 fluorescence following cLTP induction. Each gray line represents the ΔF/F0 in a single culture (well), while the black line with the green symbol represents the average of ΔF/F0 (normalized to the average baseline of each culture) in the cultures treated with glycine (100 μM). The data are presented as the mean ± SEM.

https://www.nature.com/articles/s41398-021-01457-w