Single-wavelength fluorescent reporters allow visualization of specific neurotransmitters with high spatial and temporal resolution. We report variants of the glutamate sensor iGluSnFR that are functionally brighter; can detect sub-micromolar to millimolar concentrations of glutamate; and have blue, green or yellow emission profiles. These variants allow in vivo imaging where original-iGluSnFR was too dim, reveal glutamate transients at individual spine heads, and permit kilohertz imaging with inexpensive, powerful fiber lasers.
Main TextThe intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) 1 has become an invaluable tool for studying glutamate dynamics in diverse systems, including retina 2,3 , olfactory bulb 4 and visual cortex 5 . iGluSnFR also allows mesoscale "functional connectomic" mapping 6 and mechanistic studies of Huntington's disease 7 , synaptic spillover 8 , cortical spreading depression 9 and exocytotic vesicle fusion 10 . However, iGluSnFR is insufficient for some applications due to poor expression (in some preparations), and kinetics that do not match the time courses of some phenomena. Here we describe variants that are functionally brighter (due to increased membrane expression), have tighter or weaker affinity, and fluoresce blue, green, or yellow.Replacement of circularly permuted eGFP with circularly permuted "superfolder" GFP 11 (SFiGluSnFR) yielded 5-fold higher soluble-protein expression levels in bacteria (0.5 µmol/1L growth vs. 0.1 µmol/1L). Circular dichroism indicates an increase in melting temperature transition (T m ) of ~5˚C (Supp. Fig. S1). The 2-photon cross-section and excitation, emission, and absorption spectra of SF-iGluSnFR are similar to the original (Supp. Fig. S2a-d). Head-tohead comparison of SF-iGluSnFR with original-iGluSnFR in mouse somatosensory cortex shows substantially more robust expression by the former (Supp. Fig. S3a,b). Under typical imaging conditions (<20 mW, 130-nanosecond dwell time per pixel), SF-iGluSnFR is bright enough for repeated imaging, while original-iGluSnFR is too dim (Supp. Fig. S3c,d). While we observed a faster 2-photon in vivo photobleaching rate for SF-iGluSnFR in somatosensory cortex (Supp. Fig. S3e), partially-bleached SF-iGluSnFR was still brighter than iGluSnFR. Thus, SF-iGluSnFR will have superior expression in vivo, where the quantity of deliverable DNA can be limiting.While the affinity of membrane-displayed iGluSnFR (4 µM) is adequate for some in vivo applications, tighter variants are needed for circumstances of limiting glutamate concentrations, e.g. at sparsely-firing synapses. Additionally, measuring glutamate release events with raster scanning requires variants with slower off-rates so that the decay time from glutamate binding is long enough to be sufficiently sampled at the operating frame rate (typically <100 Hz). Replacement of eGFP with superfolder GFP increases the in vitro affinity of soluble SFiGluSnFR over original-iGluSnFR (40 µM vs. 80 µM, Supp. Fig. S4a). To further modulate affinity, w...