# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 5 Neurotransmitter Indicators
Incorporation of the conformationally sensitive cpEGFP into the third intracellular loop enabled over an order of magnitude larger fluorescence changes during GPCR activation than for the FRET-based sensors while preserving high specificity and reasonable kinetics of fluorescence response (Figure 3c). Typically, this engineering strategy involves three consecutive steps: (i) optimization of the insertion site within third intracellular loop of GPCR; (ii) optimization of the linkers between cpEGFP and GPCR; (iii) tuning ligand affinity by point mutations within the ligand-binding pocket. Moreover, the bulky cpEGFP moiety introduced into the intracellular loop almost completely demolish coupling with major GPCR downstream pathways, presumably due to the steric hindrance imposed for G protein or arrestin interaction with the GPCR [149; 150]. As a result, this design strategy was successfully exploited to develop a series of GPCR-based sensors suitable for in vivo imaging of dopamine (DA), acetylcholine (ACh), and norepinephrine (NE), also called noradrenaline (NA), in multiple model organisms. In addition, as a proof of concept a class of sensors for other neuromodulators and neuropeptides were engineered using G\({}_{\text{s}}\)-coupled \(\beta\)1 and \(\beta\)2 adrenergic receptors, G\({}_{\text{i}}\)-coupled \(\kappa\) and \(\mu\)-type opioid receptors, and G\({}_{\text{q}}\)-coupled serotonin receptor-2A and melatonin type-2 receptor [134]. Two groups in parallel independently developed green fluorescence DA sensors with similar biochemical characteristics (Table 3). [PERSON] et al. reported a series of DA sensors, called dLight, developed based on the human Dopamine Receptor D1 (DRD1) [134]. Two variants in this series, dLight1.1 and dLight1.2, showed optimal combination of apparent DA affinity and sensitivity and were used for in vivo imaging in behaving mice (Table 3). The higher affinity dLight1.1 sensor enabled visualization of spontaneous and optogenetically evoked dopamine transients in the nucleus accumbens by recording aggregated fluorescence signal from multiple cells using fiber photometry. Under these imaging conditions, dLight1.1 could report both activation and inhibition of dopamine transients resolving individual spikes at up to 5 Hz stimulation frequency. Higher spatial resolution of dopamine imaging was achieved under two-photon microscopy of dLight1.2 expressed in layer 2/3 neurons of the cortex during a visuomotor learning task. Two-photon imaging with micron-resolution revealed a dopamine transient map with functionally heterogeneous dopamine signals in the cortex. In contrast, utilizing Dopamine Receptor D2 (DRD2) as a sensing moiety, [PERSON] et al. developed a pair of alternative dopamine sensors, named genetically-encoded GPCR-activation-based-DA sensors (GRAB\({}_{\text{DA}}\)) [135]. Overall, in comparison to the dLight sensors, two selected variants, GRAB\({}_{\text{DA1m}}\) and GRAB\({}_{\text{DA1m}}\), are characterized by several fold smaller dynamic range and slower kinetics but higher apparent affinity to DA (Table 3). Similar to the dLight sensors, the signal-to-noise ratio and temporal resolution of GRAB\({}_{\text{DA}}\) were sufficient to record the dynamic bi-directional changes during DA activation and inhibition in dorsal striatum and nucleus accumbens in behaving mice using fiber photometry. In particular, imaging GRAB\({}_{\text{DA}}\) in nucleus accumbens revealed a time-locked DA elevation aligned to various sexual behaviors, confirming an important role of DA in behavioral motivation, anticipation, or arousal. In addition, the GRAB\({}_{\text{DA}}\) sensors were shown to readily respond to visually induced DA release in the intact brain of the zebrafish larvae as well as reveal compartmentalized DA dynamics in the mushroom body of fruit flies with single neuron resolution. In summary, two groups of the currently available DA sensors demonstrated comparable performance in vivo and selection of a particular variant have to be determined based on the exact intended application.