# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 5 Neurotransmitter Indicators
[PERSON] et al. used the newly identified PBP Pf622 cloned from _Pseudomonas fluorescens_[139] as a GABA-binding domain. The initial version of the sensor, created by insertion of cpsfGFP into the Pf622 domain, was further optimized by side-directed mutagenesis to increase affinity and sensitivity (Table 3). The iGABASnFR was shown to report GABA release events over large volume of brain tissue in vivo in mouse visual cortex and in zebrafish cerebellum with sub-second kinetics, although with rather low fluorescence changes at just 10%. While iGABASnFR is a solely available fully genetically-encoded sensor for GABA, it will most likely require further optimization to enhance affinity, kinetics, and sensitivity before it can be widely adopted by the neuroscience community. Development of the PBP-based sensor for Gly appeared to be more challenging. The wild-type PBP Atu2422 from _Agrobacteriumium tumefaciens_, selected as a binding domain for glycine sensor development, also displays promiscuous binding for L-serine and GABA [140]. To increase specificity of Atu2422, [PERSON] et al. employed computationally-guided mutagenesis. Introducing steric obstructions into the ligand-binding site of Atu2422 produced a binding domain highly selective for glycine. Fusing the engineered binding domain with CFP-YFP FRET pair, but not with cpEGFP as in iGluSnFR, resulted in the functional glycine sensor, denoted GlyFS, characterized by \(\sim\)28% of fluorescence dynamic range (Table 3). Membrane targeting of GlyFS via a PDGFR peptide fusion, as used for the iGluSnFR and iGABASnFR sensors, reduced dynamic range to 4%, which is 7-fold lower than that in vitro, due to inefficient membrane trafficking of the protein. Instead, extracellular space immobilization was performed through a biotinylation-based technique that anchors proteins to the outer side of the plasma membrane (Figure 3b). Taking advantage of the GlyFS ratiometric response, the authors performed measurements of the absolute concentration of Gly in brain slices revealing dependency of Gly concentration on animal age. The suggested immobilization technique requires biotinylation of cell surface in intact brain tissue with subsequent delivery of the GlyFS sensor and streptavidin [141]. While the biotinylation-based technique was demonstrated to be compatible with tumor imaging in live mice [142], it might not be feasible for neuroimaging in vivo thus limiting GlyFS application to in vitro and ex vivo preparations. The GlyFS engineering is an inspiring example showing that PBPs are flexible templates for generating diversity of binding domains. Nevertheless, GlyFS still requires further development to increase its usefulness and applicability for neuroscience research. The second class of ligand-binding domains utilized for GENI engineering is represented by GPCRs. GPCRs share a highly conservative 3D structure comprising of seven \(\alpha\)-helix transmembrane domains (TM) and constitute the majority of receptors with high specificity for endogenous neurotransmitters. Structural studies indicate that the largest conformation changes upon ligand binding to GPCRs by occurs at the cytosolic end of TM6. The \(\alpha\)-helix of TM6 can undergo more than 6 A shift outwards from the helix bundle center at the same time initiating the 2-3 A shift of TM5 [143; 144]. For this reason, the third intracellular loop, located between TM5 and TM6, is a primary target for insertion of reporting moieties to engineer GENIs. Indeed, first generation of GPCR-based sensorswas constructed by insertion of FRET donors and acceptors into the third intracellular loop and at the shortened C terminal domain [145; 146; 147; 148]. The FRET-based sensors exhibited good specificity and affinity to agonists as well as adequate kinetics, however, their utility for in vivo imaging is limited due to the relatively modest \(\sim\)10%, changes in FRET signal. Very recently, an alternative design of GENIs was suggested to overcome this limitation.