A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

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.     

Thy1-GCaMP6 Transgenic Mice for Neuronal Population Imaging In Vivo

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.     

Measuring calcium dynamics in living cells with genetically encodable calcium indicators

Genetically encoded calcium indicators (GECIs) allow researchers to measure calcium dynamics in specific targeted locations within living cells. Such indicators enable dissection of the spatial and temporal control of calcium signaling processes. Here we review recent progress in the development of GECIs, highlighting which indicators are most appropriate for measuring calcium in specific organelles and localized domains in mammalian tissue culture cells. An overview of recent approaches that have been undertaken to ensure that the GECIs are minimally perturbed by the cellular environment is provided. Additionally, the procedures for introducing GECIs into mammalian cells, conducting calcium imaging experiments, and analyzing data are discussed. Because organelle-targeted indicators often pose an additional challenge, we underscore strategies for calibrating GECIs in these locations.     

Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.     

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.     

Green-to-Red Photoconversion of GCaMP

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs—including GCaMP3, GCaMP5 and GCaMP6—can be converted from green to red following exposure to blue-green light (450–500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.     

Structural insight into enhanced calcium indicator GCaMP3 and GCaMPJ to promote further improvement

Genetically encoded Ca²⁺ indicators (GECI) are important for the measurement of Ca²⁺in vivo. GCaMP2, a widelyused GECI, has recently been iteratively improved. Among the improved variants, GCaMP3 exhibits significantly better fluorescent intensity. In this study, we developed a new GECI called GCaMPJ and determined the crystal structures of GCaMP3 and GCaMPJ. GCaMPJ has a 1.5- fold increase in fluorescence and 1.3-fold increase in calcium affinity over GCaMP3. Upon Ca²⁺ binding, GCaMP3 exhibits both monomeric and dimeric forms. The structural superposition of these two forms reveals the role of Arg-376 in improving monomer performance. However, GCaMPJ seldom forms dimers under conditions similar to GCaMP3. St ructural and mutagenesis studies on Tyr-380 confirmed its importance in blocking the cpEGFP β-barrel holes. Our study proposes an efficient tool for mapping Ca²⁺ signals in intact organs to facilitate the further improvement of GCaMP sensors.     

An improved genetically encoded red fluorescent Ca2+ indicator for detecting optically evoked action potentials

Genetically encoded Ca(2+) indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5-2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca(2+) transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca(2+) transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca(2+) transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.     

Use of aequorin-based indicators for monitoring Ca2+ in acidic organelles

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca²⁺ upon cell activation. Hence, reliable recording of Ca²⁺ dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca²⁺ indicators (GECIs) are valuable tools to monitor Ca²⁺ in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs. By contrast, bioluminescent GECIs have a combination of features (marginal pH sensitivity, low background, no phototoxicity, no photobleaching, high dynamic range and tunable affinity) that render them advantageous to achieve an enhanced signal-to-noise ratio in acidic compartments. This article reviews the use of bioluminescent aequorin-based GECIs targeted to acidic compartments. A need for more measurements in highly acidic compartments is identified.     

Visible light excited ratiometric-GECIs for long-term in-cellulo monitoring of calcium signals

Genetically Encoded Calcium Indicators (GECIs) are powerful molecular tools for monitoring calcium (Ca²⁺) signaling in the cytosol and organellar compartments. However, currently available ratiometric indicators that allow measurements of resting Ca²⁺ levels have limitations in long-term Ca²⁺ imaging. They either are ultraviolet (UV)-excited ones with strong photo-toxicity, or have poor performance. To overcome this hurdle, we developed a set of visible light excited ratiometric-GECIs (VR-GECIs) based on existing mono-colored GECIs. With performance comparable to their corresponding mono-color prototypes, this set of VR-GECIs enables long-term measurements of intra-cellular or intra-organellar Ca²⁺ signals. Using these VR-GECIs together with a newly developed off-line analysis tool, we achieved long-term measurements of Ca²⁺ homeostasis of moving or dividing cells. Our tools may find broad applications in decoding Ca²⁺-modulated physiological or pathological processes.     

Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters

Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and 'spotty' in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.     

Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators

While genetically encoded Ca(2+) indicators (GECIs) allow Ca(2+) imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca(2+) imaging, it had a, diminished Ca(2+) response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca(2+) response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca(2+) signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.     

Establishment of a NanoBiT-Based Cytosolic Ca2+ Sensor by Optimizing Calmodulin-Binding Motif and Protein Expression Levels

Cytosolic Ca²⁺ levels ([Ca²⁺]_c) change dynamically in response to inducers, repressors, and physiological conditions, and aberrant [Ca²⁺]_c concentration regulation is associated with cancer, heart failure, and diabetes. Therefore, [Ca²⁺]_c is considered as a good indicator of physiological and pathological cellular responses, and is a crucial biomarker for drug discovery. A genetically encoded calcium indicator (GECI) was recently developed to measure [Ca²⁺]_c in single cells and animal models. GECI have some advantages over chemically synthesized indicators, although they also have some drawbacks such as poor signal-to-noise ratio (SNR), low positive signal, delayed response, artifactual responses due to protein overexpression, and expensive detection equipment. Here, we developed an indicator based on interactions between Ca²⁺-loaded calmodulin and target proteins, and generated an innovative GECI sensor using split nano-luciferase (Nluc) fragments to detect changes in [Ca²⁺]_c. Stimulation-dependent luciferase activities were optimized by combining large and small subunits of Nluc binary technology (NanoBiT, LgBiT:SmBiT) fusion proteins and regulating the receptor expression levels. We constructed the binary [Ca²⁺]_c sensors using a multicistronic expression system in a single vector linked via the internal ribosome entry site (IRES), and examined the detection efficiencies. Promoter optimization studies indicated that promoter-dependent protein expression levels were crucial to optimize SNR and sensitivity. This novel [Ca²⁺]_c assay has high SNR and sensitivity, is easy to use, suitable for high-throughput assays, and may be useful to detect [Ca²⁺]_c in single cells and animal models.     

New red-fluorescent calcium indicators for optogenetics,photoactivation and multi-color imaging

Most chemical and, with only a few exceptions, all genetically encoded fluorimetric calcium (Ca²⁺) indicators (GECIs) emit green fluorescence. Many of these probes are compatible with red-emitting cell- or organelle markers. But the bulk of available fluorescent-protein constructs and transgenic animals incorporate green or yellow fluorescent protein (GFP and YFP respectively). This is, in part, not only heritage from the tendency to aggregate of early-generation red-emitting FPs, and due to their complicated photochemistry, but also resulting from the compatibility of green-fluorescent probes with standard instrumentation readily available in most laboratories and core imaging facilities. Photochemical constraints like limited water solubility and low quantum yield have contributed to the relative paucity of red-emitting Ca²⁺ probes compared to their green counterparts, too. The increasing use of GFP and GFP-based functional reporters, together with recent developments in optogenetics, photostimulation and super-resolution microscopies, has intensified the quest for red-emitting Ca²⁺ probes. In response to this demand more red-emitting chemical and FP-based Ca²⁺-sensitive indicators have been developed since 2009 than in the thirty years before.     

Optogenetic Measurement of Presynaptic Calcium Transients Using Conditional Genetically Encoded Calcium Indicator Expression in Dopaminergic Neurons

Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.     

GCaMP as an indirect measure of electrical activity in rat trigeminal ganglion neurons

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca²⁺ indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca²⁺ concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1–30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the next generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95 %, and the impact of GCaMP6 expression in TG neurons over the period of study (<10 days) on the regulation of intracellular Ca²⁺, as assessed with fura-2, was minimal. Having confirmed that the field stimulation evoked Ca²⁺ transients were dependent on Ca²⁺ influx secondary to the activation of action potentials and voltage-gated Ca²⁺ channels, we also confirmed that the signal-to-noise ratio for each of the isoforms was excellent, enabling detection of a single spike in>90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca²⁺ transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca²⁺ was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.     

Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals

Imaging the activities of individual neurons with genetically encoded Ca²⁺ indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca²⁺ signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F _max/F _min = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca²⁺ imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca²⁺ responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.     

Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein

Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K(d)s ranging from 470 nm to 29 microm, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.     

Preparation of iron oxide-based calcium sensors for MRI

We describe a protocol for the preparation of a family of calcium indicators for MRI. The indicators consist of superparamagnetic iron oxide nanoparticles functionalized with the calcium-sensing protein, calmodulin, and its target peptides. Calcium-dependent protein-protein interactions drive particle clustering and produce large changes in T2 relaxivity. To prepare these indicators, interacting polypeptides are purified, biotinylated and then conjugated to streptavidin-coated nanoparticles. Conjugates are isolated, mixed to obtain functional sensors and incubated in the presence or absence of calcium to test response properties. Calcium-dependent responses are assayed by dynamic light scattering and MRI. The whole series of procedures can be carried out in approximately 12 h. The iron oxide-based calcium sensors discussed here are suitable for the detection of calcium concentration changes of approximately 1 microM in vitro.     

New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol.