Imaging of mitochondrial Ca2+ dynamics in astrocytes using cell-specific mitochondria-targeted GCaMP5G/6s: Mitochondrial Ca2+ uptake and cytosolic Ca2+ availability via the endoplasmic reticulum store

Mitochondrial Ca²⁺ plays a critical physiological role in cellular energy metabolism and signaling, and its overload contributes to various pathological conditions including neuronal apoptotic death in neurological diseases. Live cell mitochondrial Ca²⁺ imaging is an important approach to understand mitochondrial Ca²⁺ dynamics. Recently developed GCaMP genetically-encoded Ca²⁺ indicators provide unique opportunity for high sensitivity/resolution and cell type-specific mitochondrial Ca²⁺ imaging. In the current study, we implemented cell-specific mitochondrial targeting of GCaMP5G/6s (mito-GCaMP5G/6s) and used two-photon microscopy to image astrocytic and neuronal mitochondrial Ca²⁺ dynamics in culture, revealing Ca²⁺ uptake mechanism by these organelles in response to cell stimulation. Using these mitochondrial Ca²⁺ indicators, our results show that mitochondrial Ca²⁺ uptake in individual mitochondria in cultured astrocytes and neurons can be seen after stimulations by ATP and glutamate, respectively. We further studied the dependence of mitochondrial Ca²⁺ dynamics on cytosolic Ca²⁺ changes following ATP stimulation in cultured astrocytes by simultaneously imaging mitochondrial and cytosolic Ca²⁺ increase using mito-GCaMP5G and a synthetic organic Ca²⁺ indicator, x-Rhod-1, respectively. Combined with molecular intervention in Ca²⁺ signaling pathway, our results demonstrated that the mitochondrial Ca²⁺ uptake is tightly coupled with inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release from the endoplasmic reticulum and the activation of G protein-coupled receptors. The current study provides a novel approach to image mitochondrial Ca²⁺ dynamics as well as Ca²⁺ interplay between the endoplasmic reticulum and mitochondria, which is relevant for neuronal and astrocytic functions in health and disease.     

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.     

用于细胞核钙成像的新型钙指示剂的构建与鉴定

细胞核钙离子是基因转录等细胞核反应过程重要的调控因子.然而,细胞核内钙离子信号的调控机制尚不清楚.缺乏稳定的、敏感的细胞核钙指示剂,是导致其调控机制难以研究的重要原因之一.针对这一问题,设计了能够在细胞核内特异性表达的、具有核定位功能的钙指示剂.以基因编码钙指示剂（GECIs）家族成员GCaMP6为模板,首先融合了对钙离子不敏感的红色荧光蛋白tdTomato来对局部的钙信号进行量化,其次融合了核定位信号（NLS）,使GCaMP6能够特异定位于细胞核中.结果表明,NLS-GCaMP6-tdTomato能够在细胞核中有效发挥作用,并且在钙敏感性与动力学上,也与GCaMP6相当. 这一新型细胞核钙指示剂将为研究细胞核钙离子的功能及其调控机制提供新的方法与途径.     

A comparison of fluorescent Ca2+ indicators for imaging local Ca2+ signals in cultured cells

Localized subcellular changes in Ca²⁺ serve as important cellular signaling elements, regulating processes as diverse as neuronal excitability and gene expression. Studies of cellular Ca²⁺ signaling have been greatly facilitated by the availability of fluorescent Ca²⁺ indicators. The respective merits of different indicators to monitor bulk changes in cellular Ca²⁺ levels have been widely evaluated, but a comprehensive comparison for their use in detecting and analyzing local, subcellular Ca²⁺ signals is lacking. Here, we evaluated several fluorescent Ca²⁺ indicators in the context of local Ca²⁺ signals (puffs) evoked by inositol 1,4,5-trisphosphate (IP₃) in cultured human neuroblastoma SH-SY5Y cells, using high-speed video-microscopy. Altogether, nine synthetic Ca²⁺ dyes (Fluo-4, Fluo-8, Fluo-8 high affinity, Fluo-8 low affinity, Oregon Green BAPTA-1, Cal-520, Rhod-4, Asante Calcium Red, and X-Rhod-1) and three genetically-encoded Ca²⁺-indicators (GCaMP6-slow, -medium and -fast variants) were tested; criteria include the magnitude, kinetics, signal-to-noise ratio and detection efficiency of local Ca²⁺ puffs. Among these, we conclude that Cal-520 is the optimal indicator for detecting and faithfully tracking local events; that Rhod-4 is the red-emitting indicator of choice; and that none of the GCaMP6 variants are well suited for imaging subcellular Ca²⁺ signals.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.     

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.     

Age- and sex-dependent alterations in primary somatosensory cortex neuronal calcium network dynamics during locomotion

Over the past 30 years, the calcium (Ca²⁺) hypothesis of brain aging has provided clear evidence that hippocampal neuronal Ca²⁺ dysregulation is a key biomarker of aging. Age-dependent Ca²⁺-mediated changes in intrinsic excitability, synaptic plasticity, and activity have helped identify some of the mechanisms engaged in memory and cognitive decline based on work done mostly at the single-cell level and in the slice preparation. Recently, our lab identified age- and Ca²⁺-related neuronal network dysregulation in the cortex of the anesthetized animal. Still, investigations in the awake animal are needed to test the generalizability of the Ca²⁺ hypothesis of brain aging. Here, we used in vigilo two-photon imaging in ambulating mice, to image GCaMP8f in the primary somatosensory cortex (S1), during ambulation and at rest. We investigated aging- and sex-related changes in neuronal networks in the C56BL/6J mouse. Following imaging, gait behavior was characterized to test for changes in locomotor stability. During ambulation, in both young adult and aged mice, an increase in network connectivity and synchronicity was noted. An age-dependent increase in synchronicity was seen in ambulating aged males only. Additionally, females displayed increases in the number of active neurons, Ca²⁺ transients, and neuronal activity compared to males, particularly during ambulation. These results suggest S1 Ca²⁺ dynamics and network synchronicity are likely contributors of locomotor stability. We believe this work raises awareness of age- and sex-dependent alterations in S1 neuronal networks, perhaps underlying the increase in falls with age.     

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.     

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.     

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP¹, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons^2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding⁶ (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER⁷ with a calcium imaging technique⁸ using GCaMP3.0^{1, 9}, I have established an experimental system, where we can monitor activity of neurons in the feeding center – the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca²⁺ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.     

Effects of piperine and deoxyschizandrin on synchronized Ca2+ oscillations in cultured hippocampal neuronal cells

It has been reported that piperine (PIP) and deoxyschizandrin (DS) can modulate synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. We investigated the modulation effects of four different combinations of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks. The results showed that all four combinations (PIP:DS 4.9:1.9, 2.45:2.85, 7.35:0.95, and 2.45:0.95 mg/L) inhibit Ca²⁺ oscillation intensity to a similar extent. However, the first three combinations had strong inhibitory effects on the frequency of Ca²⁺ oscillations whereas the last combination (2.45:0.95 mg/L) only slightly enhanced the frequency of Ca²⁺ oscillations. We propose an improved Chay’s model to explain the mechanism of the effects of piperine and deoxyschizandrin on synchronized Ca²⁺ oscillations in cultured hippocampal neuronal cells. We concluded that deoxyschizandrin modulated synchronized Ca²⁺ oscillations in cultured hippocampal neuronal networks bidirectionally and the effect depended on concentration. Deoxyschizandrin reduced voltage-gated sodium channel conductance and ATP-sensitive potassium channel conductance, and affected the rate of exchange of intracellular calcium and the pump activity of Ca²⁺-ATPase in the endoplasmic reticulum (ER). Piperine reduced the activity of calcium release in the ER, and reduced the pump activity of calcium in the cytomembrane or enhanced the pump activity of Ca²⁺-ATPase in the ER.     

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.     

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.     

Structural effects of divalent calcium cations on the α7 nicotinic acetylcholine receptor: A molecular dynamics simulation study

The α7 subtype of neuronal nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that is vital to various neurological functions, including modulation of neurotransmitter release. A relatively high concentration of extracellular Ca²⁺ in the neuronal environment is likely to exert substantial structural and functional influence on nAChRs, which may affect their interactions with agonists and antagonists. In this work, we employed atomistic molecular dynamics (MD) simulations to examine the effects of elevated Ca²⁺ on the structure and dynamics of α7 nAChR embedded in a model phospholipid bilayer. Our results suggest that the presence of Ca²⁺ in the α7 nAChR environment results in closure of loop C-in the extracellular ligand-binding domain, a motion normally associated with agonist binding and receptor activation. Elevated Ca²⁺ also alters the conformation of key regions of the receptor, including the inter-helical loops, pore-lining helices and the “gate” residues, and causes partial channel opening in the absence of an agonist, leading to an attendant reduction in the free energy of Ca²⁺ permeation through the pore as elucidated by umbrella sampling simulations. Overall, the structural and permeability changes in α7 nAChR suggest that elevated Ca²⁺ induces a partially activated receptor state that is distinct from both the resting and the agonist-activated states. These results are consistent with the notion that divalent ions can serve as a potentiator of nAChRs, resulting in a higher rate of receptor activation (and subsequent desensitization) in the presence of agonists, with possible implications for diseases involving calcium dysregulation.     

Calcium Signalling and Alzheimer’s Disease

New insights into how Ca²⁺ regulates learning and memory have begun to provide clues as to how the amyloid-dependent remodelling of neuronal Ca²⁺ signalling pathways can disrupt the mechanisms of learning and memory in Alzheimer’s disease (AD). The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway remodels the neuronal Ca²⁺ signalling pathways responsible for cognition by enhancing the entry of Ca²⁺ and/or the release of internal Ca²⁺ by ryanodine receptors or InsP₃ receptors. The specific proposal is that Ca²⁺ signalling remodelling results in a persistent elevation in the level of Ca²⁺ that constantly erases newly acquired memories by enhancing the mechanism of long-term depression (LTD). Neurons can still form memories through the process of LTP, but this stored information is rapidly removed by the persistent activation of LTD. Further dysregulation in Ca²⁺ signalling will then go on to induce the neurodegeneration that characterizes the later stages of dementia.     

Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties

Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca²⁺-bound form in the dark to a Mg²⁺-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca²⁺. Substitutions affect residues directly involved in Ca²⁺ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca²⁺ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca²⁺ and blocking the enzyme in a constitutively active state at physiological levels of Ca²⁺. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca²⁺-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.     

Calcium Signaling throughout the Toxoplasma gondii Lytic Cycle: A STUDY USING GENETICALLY ENCODED CALCIUM INDICATORS*

Toxoplasma gondii is an obligate intracellular parasite that invades host cells, creating a parasitophorous vacuole where it communicates with the host cell cytosol through the parasitophorous vacuole membrane. The lytic cycle of the parasite starts with its exit from the host cell followed by gliding motility, conoid extrusion, attachment, and invasion of another host cell. Here, we report that Ca²⁺ oscillations occur in the cytosol of the parasite during egress, gliding, and invasion, which are critical steps of the lytic cycle. Extracellular Ca²⁺ enhances each one of these processes. We used tachyzoite clonal lines expressing genetically encoded calcium indicators combined with host cells expressing transiently expressed calcium indicators of different colors, and we measured Ca²⁺ changes in both parasites and host simultaneously during egress. We demonstrated a link between cytosolic Ca²⁺ oscillations in the host and in the parasite. Our approach also allowed us to measure two new features of motile parasites, which were enhanced by Ca²⁺ influx. This is the first study showing, in real time, Ca²⁺ signals preceding egress and their direct link with motility, an essential virulence trait.     

Infrared Excitation Induces Heating and Calcium Microdomain Hyperactivity in Cortical Astrocytes

Unraveling how neural networks process and represent sensory information and how these cellular signals instruct behavioral output is a main goal in neuroscience. Two-photon activation of optogenetic actuators and calcium (Ca²⁺) imaging with genetically encoded indicators allow, respectively, the all-optical stimulation and readout of activity from genetically identified cell populations. However, these techniques locally expose the brain to high near-infrared light doses, raising the concern of light-induced adverse effects on the biology under study. Combining 2P imaging of Ca²⁺ transients in GCaMP6f-expressing cortical astrocytes and unbiased machine-based event detection, we demonstrate the subtle build-up of aberrant microdomain Ca²⁺ transients in the fine astroglial processes that depended on the average rather than peak laser power. Illumination conditions routinely being used in biological 2P microscopy (920-nm excitation, ∼100-fs, and ∼10 mW average power) increased the frequency of microdomain Ca²⁺ events but left their amplitude, area, and duration largely unchanged. Ca²⁺ transients in the otherwise silent soma were secondary to this peripheral hyperactivity that occurred without overt morphological damage. Continuous-wave (nonpulsed) 920-nm illumination at the same average power was as damaging as femtosecond pulses, unraveling the dominance of a heating-mediated damage mechanism. In an astrocyte-specific inositol 3-phosphate receptor type-2 knockout mouse, near-infrared light-induced Ca²⁺ microdomains persisted in the small processes, underpinning their resemblance to physiological inositol 3-phosphate receptor type-2-independent Ca²⁺ signals, whereas somatic hyperactivity was abolished. We conclude that, contrary to what has generally been believed in the field, shorter pulses and lower average power can help to alleviate damage and allow for longer recording windows at 920 nm.     

Characterization of calcium signals in human embryonic stem cells and in their differentiated offspring by a stably integrated calcium indicator protein

Intracellular calcium signaling pathways play a major role in cellular responses such as proliferation, differentiation and apoptosis. Human embryonic stem cells (hESC) provide new possibilities to explore the development and differentiation of various cell types of the human body. Intracellular calcium responses to various ligands and the calcium signaling pathways, however, have not been thoroughly studied in embryonic stem cells and in their differentiated progenies. In our previous work we demonstrated that the use of the fluorescent calcium indicator Fluo-4 with confocal microscopy allows sensitive and reliable measurements of calcium modulation in human embryonic stem cells and stem-cell derived cardiomyocytes. Here we developed a human embryonic stem cell line stably expressing a genetically encoded Ca^2 + indicator (GCaMP2) using a transposon-based gene delivery system. We found that the differentiation properties were fully preserved in the GCaMP2-expressing hESC lines and Ca imaging could be performed without the need of toxic dye-loading of the cells. In undifferentiated hES cells the calcium signals induced by various ligands, ATP, LPA, trypsin or angiotensin II were comparable to those in Fluo-4 loaded cells. In accordance with previous findings, no calcium signal was evoked by thrombin, histamine or GABA. Cardiomyocyte colonies differentiated from hES-GCaMP2 cells could be recognized by spontaneous contractions and Ca^2 + oscillations. GCaMP2-expressing neural cells were identified based on their morphological and immuno-staining properties and Ca signals were characterized on those cells. Characteristics of both the spontaneous and ligand-induced Ca^2 + signals, as well as their pharmacological modification could be successfully examined in these model cells by fluorescence imaging.     

Neuronal calcium signaling via store-operated channels in health and disease

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca²⁺) into cells in response to the depletion of intracellular Ca²⁺ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca²⁺ sensor STIM1 and plasma membrane Ca²⁺ channel Orai1 and serves to sustain Ca²⁺ signaling and refill ER Ca²⁺ stores. In contrast, because of the complexity of Ca²⁺ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target.