# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 2 Calcium Indicators  
Calcium is a crucial mediator of neural activity and activity-dependent synaptic plasticity [10; 11]. While most neurons at rest contain cytoplasmic calcium concentrations of 50-100 nM, electrical activity can swiftly and dramatically increase calcium concentrations [12] for tens of milliseconds to several seconds [13; 14]. Therefore, calcium is an excellent proxy for neuronal activity, and, accordingly, genetically-encoded calcium indicators (GECIs) are the most popular and widely used optical sensors of neuronal activity in neuroscience [8; 11; 15; 16]. Since the development of the first proof of principle GECIs almost two decades ago [17; 18; 19; 20], herculean efforts in protein engineering resulted in several excellent calcium sensors [21; 22] providing researchers a selection of tools for diverse applications, including imaging large neural population dynamics [23; 24], dendritic processing [25], as well as synaptic [26; 27] and presynaptic [28] function. Here, we will discuss the major progress in engineering GECIs from the past few years and briefly describe some of their most distinctive applications. Prior work on the development and application of GECIs has been reviewed in earlier publications [8; 11; 15; 16]. Since 2016, two types of molecular designs have prevailed among improved as well as newly developed GECIs (Figure 1a,b). The GECI families based on the GCaMP-like design are the most numerous and widely used GECIs for in vivo imaging [22] (Table 1). Calcium sensors with green fluorescence exhibit the best performance, especially the GCaMP6 variants [26] and, therefore, are the primary choice for most applications. Intensive use of the GCaMP variants in vivo resulted in a wealth of evidence for their interference with normal calcium dynamics and gene expression in mammalian systems that must be taken into account when interpreting calcium imaging data [29; 30; 31]. Typically, the side effects are due to the interaction of the calcium binding domain, which has a mammalian origin, with endogenous proteins, as well as the buffering of cytoplasmic calcium, artifacts more prominent during prolonged sensor expression at high levels [26; 32]. To overcome these side effects, several different modifications of calcium binding domains have been attempted. For example, [PERSON] et al. engineered GCaMP-X by incorporating an extra apoCaM-binding motif into the GCaMP variants [31]. This modification did not significantly affect overall sensor performance but reduced interactions with L-type calcium channels, thus effectively protecting CaV1-dependent excitation-transcription coupling from sensor-induced perturbations [31]. An alternative strategy to minimize GCaMP-induced side effects involve exploiting calcium binding domains cloned from fungi and yeasts that share conserved amino acid identity with their metazoan counterparts used in GCaMPs [33]. For example, the calmodulin and M13-like peptide from _Aspergillus_ fungi swapped with the calcium-binding domain in GCaMP6s prevented interaction with endogenous proteins at low calcium concentrations in cultured mammalian cells [33]. In addition, fungal GCaMP, or FGCaMP, exhibits ratiometric by excitation fluorescence response to calcium ions, with the highest brightness and dynamic range combination among other ratiometric GECIs such as GEX-GECO [34], Pericam [19], and Y-GECO [35]. Furthermore, FGCaMP can report neuronal activity with single cell resolution in zebrafish larvae under light-sheet microscopy. Another way to improve the performance of GECIs is structure-guided mutagenesis of the GFP-CaM interface and the CaM-M13 peptide interactions [36]. For example, further mutagenesis of the calcium binding domain in the GCaMP6 variants resulted in the next generation green sensors, the JGCaMP7 series, characterized by improved sensitivity to one action potential and higher signal-to-noise ratio due to enhanced brightness [37]. However, the jGCaMP7 variants are still less validated in vivo compared to the extremely popular GCaMP6 indicators.