# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 5 Neurotransmitter Indicators
\begin{table}
\begin{tabular}{|l|l|l|l|l|l|l|l|l|} \hline
**Sensor** & **Ligand** & **E/Em** & **A/F (\%)** & **K\({}_{d}\) (uM)** & **On** & **Off** & **In Vivo Imaging** & **Ref** \\  & & **(nm)** & & & **(ms)** & **(ms)** & & \\ \hline iGluSnFR & Glu \({}^{a}\) & 490/510 & 100 & 4.9 & 15 & 92 & Single dendritic & [127] \\  & & & & & & & spines & \\ \hline SF-iGluSnFR-A184S & Glu \({}^{a}\) & 490/510 & 69 & 0.6 & 85 & 450 & Single dendritic & [130] \\  & & & & & & spines & \\ \hline SF-iGluSnFR-S72A & Glu \({}^{a}\) & 490/510 & 250 & 34 & 5 & 11 & Single dendritic & [130] \\  & & & & & & spines & \\ \hline iGlu\({}_{a}\) & Glu \({}^{a}\) & 490/510 & 170 & 53 & 0.7 & 3 & Not tested & [129] \\ \hline iGlu\({}_{a}\) & Glu \({}^{a}\) & 562/588 & \(-\)33 & 18 & ND & ND & Not tested & [131] \\ \hline iGABAenFR & GABA & 485/510 & 10\({}^{b}\) & -9\({}^{b}\) & -25\({}^{c}\) & -60\({}^{c}\) & Aggregated & [132] \\  & & & & & & neuropil imaging & \\ \hline GlyPS & Gly & 434/477 & 28.3 & 28 & ND & ND & Not tested & [133] \\  & & 515/528 & 19.0 & 21 & & & \\  & & (FRET) & & & & & \\ \hline dLight1.1 & DA & 490/516 & 220 \({}^{d}\) & 0.33\({}^{d}\) & ND & ND & Aggregated signal & [134] \\ \hline dLight1.2 & DA & 490/516 & 340 \({}^{d}\) & 0.77\({}^{d}\) & 9.5\({}^{e}\) & 90\({}^{e}\) & Aggregated signal & [134] \\ \hline GRAB\({}_{\rm DA1}\) & DA & 490/510 & 90 & 0.13 & 80\({}^{e}\) & 3100\({}^{e}\) & Aggregated signal & [135] \\ \hline GRAB\({}_{\rm DA1}\) & DA & 490/510 & 90 & 0.01 & 110\({}^{e}\) & 17,150\({}^{e}\) & Aggregated signal & [135] \\ \hline GACh2.0 & ACh & 490/510 & 90 & 2 & 280\({}^{d}\) & 760\({}^{d}\) & Single neurons with [136] \\  & & & & & & 15 trials averaging & \\ \hline GRAB\({}_{\rm NZ1}\) & NE & 490/510 & 230 & 1.9 & 72\({}^{d}\) & 680\({}^{d}\) & Aggregated signal & [137] \\ \hline GRAB\({}_{\rm NZ1}\) & NE & 490/510 & 150 & 0.093 & 36\({}^{d}\) & 1890\({}^{d}\) & Not tested & [137] \\ \hline fTango2 & DA & 488/507 & 900 & ND & \(-\)10\({}^{b}\)-10\({}^{b}\) & NA & Single neurons & [138] \\  & & 554/581 & & & & & \\ \hline \end{tabular} Ex–excitation wavelength; Em–emission wavelength; \(\Delta\)F/F—maximally achievable fluorescence changes between ligand-free and ligand saturated states; K\({}_{d}\)—ligand dissociation constant measured for cultured neurons expressing corresponding sensor, unless otherwise stated; ND—not determined; NA—not applicable. \({}^{a}\) shows similar affinity for Asp; \({}^{b}\) characterized in vitro; \({}^{c}\) estimated from the trace for one action potential in cultured neurons presented in the corresponding publication; \({}^{d}\) measured in HEK cells; \({}^{e}\) measured in acute brain slice.  
\end{table} 
Table 3: Characteristics of selected genetically-encoded fluorescent neurotransmitter reporters. in the R-iGluSnFR glutamate sensor with red emission peaked at \(\sim\)590 nm [131]. Despite subsequent intense optimization by random mutagenesis resulting in an additional 12 amino acid mutations throughout the protein, performance of R-iGluSnFR is far inferior to that of green glutamate sensors, with a dynamic range of only -33% compared to 250% for SF-iGluSnFR-S72A, thus precluding its in vivo application. Among chromatic variants of iGluSnFR, only the sensor with yellow emission, SF-Venus-iGluSnFR, was utilized for in vivo imaging in mouse visual cortex. The major advantage of SF-Venus-iGluSnFR over green fluorescence sensors for glutamate is its high brightness under two-photon excitation at 1,030 nm, compatible with powerful femtosecond fiber lasers, which are gaining popularity in the neuroscience community due to their affordability and usability. Currently, fluorescent sensors for glutamate represent the most diverse group of GENIs, however, the most practical variants are still limited to green fluorescence. The versatility of the PBP-based sensor design was also demonstrated by the development of green fluorescent sensors for the chief inhibitory neurotransmitter GABA, called an intensity-based GABA Sensing Fluorescence Reporter or iGABASnFR [132] (Figure 3a).