Objective·To optimize iSeroSnFR,a genetically encoded 5-hydroxytryptamine(5-HT)fluorescent sensor based on bacterial periplasmic binding proteins(PBPs),to enhance its performance for both in vivo and in vitro 5-HT detection.Methods·iSeroSnFR1.2 was engineered by replacing the circularly permuted superfolder green fluorescence protein(cpsfGFP)sequence in iSeroSnFR1.0 with that from the acetylcholine sensor iAChSnFR using Gibson assembly.The fluorescence response and kinetic properties of iSeroSnFR1.0 and iSeroSnFR1.2 were compared by overexpressing the sensors in HEK293 cells and puffing with exogenous 5-HT.Additionally,to mimic physiological conditions,cultured mouse cortical neurons infected with Sindbis virus carrying each sensor were electrically stimulated to induce endogenous 5-HT release and further evaluate sensor performance.Results·iSeroSnFR1.2 showed significantly improved performance over iSeroSnFR1.0.In HEK293 cells,it exhibited a 1.5-fold increase in fluorescence response(ΔF/F0)to exogenous 5-HT,along with faster kinetics(rise time:36.3 ms vs 44.9 ms;decay time:1 003.6 ms vs 1 730.4 ms).In cortical neurons,it demonstrated a 2.7-fold increase in response to endogenously released 5-HT,with rise and decay times reduced by 44.0%and 26.7%,respectively.Notably,iSeroSnFR1.2 showed increased basal fluorescence,enabling better imaging in high-background environments.Conclusion·The optimized iSeroSnFR1.2 sensor offers a markedly improved fluorescent response and temporal resolution for 5-HT detection,providing an advanced tool for studying 5-HT dynamics in neuroscience and psychiatric research.

Objective·To optimize iSeroSnFR,a genetically encoded 5-hydroxytryptamine(5-HT)fluorescent sensor based on bacterial periplasmic binding proteins(PBPs),to enhance its performance for both in vivo and in vitro 5-HT detection.Methods·iSeroSnFR1.2 was engineered by replacing the circularly permuted superfolder green fluorescence protein(cpsfGFP)sequence in iSeroSnFR1.0 with that from the acetylcholine sensor iAChSnFR using Gibson assembly.The fluorescence response and kinetic properties of iSeroSnFR1.0 and iSeroSnFR1.2 were compared by overexpressing the sensors in HEK293 cells and puffing with exogenous 5-HT.Additionally,to mimic physiological conditions,cultured mouse cortical neurons infected with Sindbis virus carrying each sensor were electrically stimulated to induce endogenous 5-HT release and further evaluate sensor performance.Results·iSeroSnFR1.2 showed significantly improved performance over iSeroSnFR1.0.In HEK293 cells,it exhibited a 1.5-fold increase in fluorescence response(ΔF/F0)to exogenous 5-HT,along with faster kinetics(rise time:36.3 ms vs 44.9 ms;decay time:1 003.6 ms vs 1 730.4 ms).In cortical neurons,it demonstrated a 2.7-fold increase in response to endogenously released 5-HT,with rise and decay times reduced by 44.0%and 26.7%,respectively.Notably,iSeroSnFR1.2 showed increased basal fluorescence,enabling better imaging in high-background environments.Conclusion·The optimized iSeroSnFR1.2 sensor offers a markedly improved fluorescent response and temporal resolution for 5-HT detection,providing an advanced tool for studying 5-HT dynamics in neuroscience and psychiatric research.