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Genetically encoded voltage indicators (GEVIs) allow visualisation of fast action potentials in neurons but most are bright at rest and dimmer during an action potential. Here, the authors engineer electrochromic FRET GEVIs with fast, bright and positive-going fluorescence signals for in vivo imaging.

Abstract Background Genetically encoded calcium ion (Ca2+) indicators (GECIs) are indispensable tools for measuring Ca2+ dynamics and neuronal activities in vitro and in vivo. Red fluorescent protein (RFP)-based GECIs have inherent advantages relative to green fluorescent protein-based GECIs due to the longer wavelength light used for excitation. Longer wavelength light is associated with decreased phototoxicity and deeper penetration through tissue. Red GECI can also enable multicolor visualization with blue- or cyan-excitable fluorophores. Results Here we report the development, structure, and validation of a new RFP-based GECI, K-GECO1, based on a circularly permutated RFP derived from the sea anemone Entacmaea quadricolor. We have characterized the performance of K-GECO1 in cultured HeLa cells, dissociated neurons, stem-cell-derived cardiomyocytes, organotypic brain slices, zebrafish spinal cord in vivo, and mouse brain in vivo. Conclusion K-GECO1 is the archetype of a new lineage of GECIs based on the RFP eqFP578 scaffold. It offers high sensitivity and fast kinetics, similar or better than those of current state-of-the-art indicators, with diminished lysosomal accumulation and minimal blue-light photoactivation. Further refinements of the K-GECO1 lineage could lead to further improved variants with overall performance that exceeds that of the most highly optimized red GECIs.

In the online version of the article [1], Figure S1 was mistakenly replaced with Figure 1.

Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging.

Calcium imaging is commonly used to measure the neural activity of large groups of neurons in mice. Genetically encoded calcium indicators (GECIs) can be delivered for this purpose using non-invasive genetic methods. Compared to viral gene transfer, transgenic targeting of GECIs provides stable long-term expression and obviates the need for invasive viral injections. Transgenic mice expressing the green GECI GCaMP6 are already widely used. Here we present the generation and characterization of transgenic mice expressing the sensitive red GECI jRGECO1a, driven by the Thy1 promoter. Four transgenic lines with different expression patterns showed sufficiently high expression for cellular in vivo imaging. We used two-photon microscopy to characterize visual responses of individual neurons in the visual cortex in vivo. The signal-to-noise ratio in transgenic mice was comparable to, or better than, mice transduced with adeno-associated virus. In addition, we show that Thy1-jRGECO1a transgenic mice are useful for transcranial population imaging and functional mapping using widefield fluorescence microscopy. We also demonstrate imaging of visual responses in retinal ganglion cells in vitro. Thy1-jRGECO1a transgenic mice are therefore a useful addition to the toolbox for imaging activity in intact neural networks.

Endoplasmic reticulum calcium homeostasis is critical for cellular functions and is disrupted in diverse pathologies including neurodegeneration and cardiovascular disease. Owing to the high concentration of calcium within the ER, studying this subcellular compartment requires tools that are optimized for these conditions. To develop a single-fluorophore genetically encoded calcium indicator for this organelle, we targeted a low affinity variant of GCaMP3 to the ER lumen (GCaMPer (10.19)). A set of viral vectors was constructed to express GCaMPer in human neuroblastoma cells, rat primary cortical neurons, and human induced pluripotent stem cell-derived cardiomyocytes. We observed dynamic changes in GCaMPer (10.19) fluorescence in response to pharmacologic manipulations of the ER calcium store. Additionally, periodic calcium efflux from the ER was observed during spontaneous beating of cardiomyocytes. GCaMPer (10.19) has utility in imaging ER calcium in living cells and providing insight into luminal calcium dynamics under physiologic and pathologic states.

Genetically-encoded calcium indicators (GECIs) facilitate imaging activity of genetically defined neuronal populations in vivo. The high intracellular GECI concentrations required for in vivo imaging are usually achieved by viral gene transfer using adeno-associated viruses. Transgenic expression of GECIs promises important advantages, including homogeneous, repeatable, and stable expression without the need for invasive virus injections. Here we present the generation and characterization of transgenic mice expressing the GECIs GCaMP6s or GCaMP6f under the Thy1 promoter. We quantified GCaMP6 expression across brain regions and neurons and compared to other transgenic mice and AAV-mediated expression. We tested three mouse lines for imaging in the visual cortex in vivo and compared their performance to mice injected with AAV expressing GCaMP6. Furthermore, we show that GCaMP6 Thy1 transgenic mice are useful for long-term, high-sensitivity imaging in behaving mice.

Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, RCaMPs, engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.

A stringent control of homeostasis is critical for functional maintenance and survival of neurons. In the mammalian retina, the basic motif leucine zipper transcription factor NRL determines rod versus cone photoreceptor cell fate and activates the expression of many rod-specific genes. Here, we report an integrated analysis of NRL-centered gene regulatory network by coupling chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) data from Illumina and ABI platforms with global expression profiling and in vivo knockdown studies. We identified approximately 300 direct NRL target genes. Of these, 22 NRL targets are associated with human retinal dystrophies, whereas 95 mapped to regions of as yet uncloned retinal disease loci. In silico analysis of NRL ChIP-Seq peak sequences revealed an enrichment of distinct sets of transcription factor binding sites. Specifically, we discovered that genes involved in photoreceptor function include binding sites for both NRL and homeodomain protein CRX. Evaluation of 26 ChIP-Seq regions validated their enhancer functions in reporter assays. In vivo knockdown of 16 NRL target genes resulted in death or abnormal morphology of rod photoreceptors, suggesting their importance in maintaining retinal function. We also identified histone demethylase Kdm5b as a novel secondary node in NRL transcriptional hierarchy. Exon array analysis of flow-sorted photoreceptors in which Kdm5b was knocked down by shRNA indicated its role in regulating rod-expressed genes. Our studies identify candidate genes for retinal dystrophies, define cis-regulatory module(s) for photoreceptor-expressed genes and provide a framework for decoding transcriptional regulatory networks that dictate rod homeostasis.