# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 5 Neurotransmitter Indicators
Similar design approaches enabled the development a pair of NE sensors with altered ligand affinity based on the \(\alpha\)-adrenergic receptor (\(\alpha\)2 AR) [137]. Two NE sensors, named GRAB\({}_{\text{NE1m}}\) and GRAB\({}_{\text{NE1h}}\), are characterized by micromolar and nanomolar apparent affinity for NE, respectively, and exhibit several fold increase in fluorescence upon NE binding when measured in cultured cells (Table 3). Due to its slightly higher dynamic range, GRAB\({}_{\rm NE1m}\) is the preferred variant for in vivo imaging. For example, GRAB\({}_{\rm NE1m}\) allowed reliable detection of NE release in locus coeruleus and hypothalamus in freely moving mice using fiber photometry. In addition, confocal imaging of GRAB\({}_{\rm NE1m}\)-expressing neurites in the optic tectum of zebrafish revealed a time-locked increase in green fluorescence during looming stimuli. Utilizing the human muscarinic acetylcholine receptor 3 as a sensing moiety enabled development of green fluorescence sensor for ACh, named GACh2.0 [136]. While GACh2.0 exhibited similar dynamic range to that of the GRAB\({}_{\rm DA}\) sensors and adequate affinity in cultured cells (Table 3), its in vivo performance was not sufficient for single-trial imaging of the ACh transients when tested under two-photon microscopy in visual cortex in mice. Nevertheless, in intact brain tissue GACh2.0 showed \(\sim\)5% fluorescence changes in a single trial in medial entorhinal cortex during multiple pulses of electrical stimulation, though single pulse stimulation still could not be detected. The poor in vivo performance of GACh2.0 can be attributed to severe sensor mislocalization when expressed in brain tissue; further improvement will certainly require optimization of membrane trafficking in vivo. Furthermore, GACh2.0 activation exhibited downstream coupling to G\({}_{\rm Q}\)-dependent calcium signaling. While the coupling was about seven-fold smaller compared to wild-type M\({}_{3}\)R, it still was much higher than for other analogous GPCR-based neurotransmitter sensors. Therefore, the Sindbis viral expression system, which permitted a more rapid (-18 h) and robust expression, was used for the GACh2.0 expression in brain slices and in vivo, while other GPCR-based GENIs were shown to be performing well with the widely used adeno associated virus (AAV) gene delivery. Activation of the native downstream-coupled pathways by GPCR-based GENIs is usually an undesired drawback due to potential artifacts. However, it is possible to engineer GPCR-coupled downstream pathways for optical signal activation, for example, by inducing the expression of a reporter gene [151; 152; 153]. One such fully genetically-encoded system is the Tango assay. The Tango system couples a transcription factor to a GPCR receptor via a specific tobacco etch virus (TEV) protease-sensitive cleavage site. The GPCR activation recruits TEV protease fused to \(\beta\)-arrestin, which releases tethered transcription factor (tTA) by cleavage TEV site, initiating expression of a reporter gene [152]. Signal amplification via gene expression enables single-cell resolution detection of neurotransmitter release with nanomolar sensitivity. Recent modification of the Tango system, named DRD2-iTango2, enabled its application for in vivo detection of DA release in the mouse nucleus accumbens during locomotion. DA release was read out by GFP expression activated via dopamine receptor 2. As a main difference to the first generation Tango systems, iTango2 incorporated improved light-inducible dimer [154] (iLID; Figure 3d). This modification prevented access by TEV protease in the dark state thus significantly improving signal-to-noise ratio and reducing background fluorescence, thus overcoming the main caveats of the original Tango system. As a result, iTango2 represents three-component fully genetically-encoded system with highly modular design (Figure 3d), that can be easily adopted to the end user needs.