# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 5 Neurotransmitter Indicators  
Neurons communicate with one another using chemical messengers called neurotransmitters. Neurotransmitters are released by a pre-synaptic cell via vesicle-mediated exocytosis into the synaptic cleft [111], where they reach concentrations in the high micromolar range [112]. Here, neurotransmitters bind to receptors on the post-synaptic cell, initiating second messenger signaling cascades (via metabotropic receptors) or evoking ion flux (via ionotropic receptors) [113], and are subsequently cleared on the millisecond time scale from the post-synaptic cleft by neurotransmitter transporters [114]. In addition to classical neurotransmitters, including glutamate [115], gamma-aminobutyric  
Figure 2: Molecular design of selected genetically-encoded and hybrid fluorescent voltage indicators. **(a)** eFRET-opsin voltage sensors Ace2N and VARNAM. **(b)** Hybrid eFRET-opsin voltage sensor Voltron. **(c)** Opsin-based voltage sensors Archon1 and paQuasAr3. **(d)** VSD-based voltage sensor ASAP3. acid (GABA) [116], and other biogenic amines [117], neurons also signal using non-classical neurotransmitters, such as peptides [118], endocannabinoids [119], and even gases [120]. Recent advance in protein engineering have yielded a number of fully genetically-encoded fluorescent sensors enabling real time optical detection of a few key neurotransmitters with high specificity and sensitivity within the intact brains of several model organisms. All genetically-encoded neurotransmitter indicators (GENIs) utilize two types of ligand-binding proteins as sensing moieties, either periplasmic-binding proteins (PBP) or G protein-coupled receptors (GPCRs; Figure 3). PBPs are bacterial non-enzymatic receptors with specificity for a wide variety of small molecule ligands, including neurotransmitters. Upon ligand binding, PBPs undergo significant conformational changes [121] sufficient to modulate fluorescence of covalently bound optical probes thus providing versatile scaffolds for fluorescence biosensor engineering [122; 123; 124]. To date, PBPs have been used to develop three different classes of genetically-encoded neurotransmitter indicators (GENIs) specialized for the optical detection of glutamate, GABA, and glycine. The first GENIs, FLIPE and superGluSnFR, capable of reporting transients of the chief excitatory neurotransmitter glutamate in cultured neurons, were engineered by fusing the glutamate PBP Gltl from _Escherichia coli_ (also known as ybe) with a FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) [125; 126]. However, a relatively low signal-to-noise ratio, an inherent drawback for most of FRET-based sensors, significantly limited applications of FLIPE and superGluSnFR in vivo. A breakthrough in imaging glutamate transients in vivo was achieved with the development of a single FP-based sensor, iGluSnFR [127], boasting a positive intensiometric fluorescence response (Table 3). iGluSnFR was constructed by insertion of a circularly permuted (cp) EGFP into the interdomain hinge region of Gltl with further optimization of linkers between cpEGFP and Gltl to improve the fluorescence dynamic range (Figure 3a). For expression in neurons, iGluSnFR is targeted to the extracellular side of the plasma membrane by fusion with a PDGFR peptide segment, similar to the first generation of glutamate sensors [125; 126]. Two-photon microscopy of iGluSnFR allowed single trial recordings of glutamate transients at single dendritic spines as well as dendritic branches in the primary motor cortex of awake behaving mice [127]. In addition, iGluSnFR reliably reported synaptic activity in vivo in zebrafish and the nematode _Caenorhabditis elegans_[127]. However, due to its slow off rate, with a reported decay half-time of 92 ms, iGluSnFR failed to resolve fast transients associated with local glutamate release during external electrical stimulations of neurons higher than 10 Hz [128; 129], highlighting a clear need for a glutamate sensor with improved biophysical characteristics.