Calcium Dye Research Articles

Quantification of cytosolic 'free' calcium in isolated coral cells with confocal microscopy.

Despite its prominent role as an intracellular messenger in all organisms, cytosolic free calcium ([Ca2+]i) has never been quantified in corals or cnidarians in general. Ratiometric calcium dyes and cell imaging have been key methods in successful research on [Ca2+]i in model systems, and could be applied to corals. Here, we developed a procedure to quantify [Ca2+]i in isolated cells from the model coral species Stylophora pistillata using Indo-1 and confocal microscopy. We quantified [Ca2+]i in coral cells with and without intracellular dinoflagellate symbionts, and verified our procedure on cultured mammalian cells. We then used our procedure to measure changes in [Ca2+]i in coral cells exposed to a classic inhibitor of [Ca2+]i regulation, thapsigargin, and also used it to record elevations in [Ca2+]i in coral cells undergoing apoptosis. Our procedure paves the way for future studies into intracellular calcium in corals and other cnidarians.

Single Cell Droplet-Based Efficacy and Transcriptomic Analysis of a Novel Anti-KLRG1 Antibody for Elimination of Autoreactive T Cells.

Progress in developing improvements in the treatment of autoimmune disease has been gradual, due to challenges presented by the nature of these conditions. Namely, the need to suppress a patient's immune response while maintaining the essential activity of the immune system in controlling disease. Targeted treatments to eliminate the autoreactive immune cells driving disease symptoms present a promising new option for major improvements in treatment efficacy and side effect management. Monoclonal antibody therapies can be applied to target autoreactive immune cells if the cells possess unique surface marker expression patterns. Killer cell lectin like receptor G1 (KLRG1) expression on autoreactive T cells presents an optimal target for this type of cell depleting antibody therapy. In this study, we apply a variety of in vitro screening methods to determine the efficacy of a novel anti-KLRG1 antibody at mediating specific natural killer (NK) cell mediated antibody-dependent cellular cytotoxicity (ADCC). The methods include single-cell droplet microfluidic techniques, allowing timelapse imaging and sorting based on cellular interactions. Included in this study is the development of a novel method of sorting cells using a droplet-sorting platform and a fluorescent calcium dye to separate cells based on CD16 recognition of cell-bound antibody. We applied this novel sorting method to visualize transcriptomic variation between NK cells that are or are not activated by binding the anti-KLRG1 antibody using RNA sequencing. The data in this study reveals a reliable and target-specific cytotoxicity of the cell depleting anti-KLRG1 antibody, and supports our droplet-sorting calcium assay as a novel method of sorting cells based on receptor activation.

Modelling chemotherapy-induced cardiotoxicity with live myocardial slices: from bedside to bench and back again

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Netherlands Heart Institute & Dutch Heart Foundation (Dekker Senior Clinical Scientist 2019) Introduction Cancer therapy related cardiac dysfunction (CTRCD) is a common side-effect of many oncological regimens. The prevention, diagnosis and treatment of CTRCD requires a well-orchestrated management by a multidisciplinary team composed by oncologists, cardiologists, radiologists, but less often translational scientists. Purpose In this work, we investigated the translational potential of live myocardial slices derived from a patient presenting with acute biventricular failure, with no history of cardiovascular disease, who had previously received neo-adjuvant chemotherapy containing doxorubicin (240 mg/m2) and trastuzumab, for the treatment of a breast carcinoma. In contrast to anthracycline cardiotoxicity, which is often permanent, cardiac dysfunction caused by trastuzumab is commonly reversible after cessation of treatment. Herein, we provide a novel side-by-side comparison of myocardial function ex vivo and clinical outcomes in the patient. Methods After several days on extracorporeal life support and inotropic therapy, the patient’s cardiac function did not improve. The patient was ultimately implanted with a left- and right-ventricular assist device (LVAD and RVAD, respectively). Live myocardial slices were produced from the apical biopsy obtained during LVAD implantation and cultured in a biomimetic system under physiological mechanical and electrical conditions. Slices were subsequently exposed to doxorubicin, trastuzumab or vehicle (0.1% DMSO) for a continued period of 7 days. Contractile force was recorded continuously, and a custom-made stimulation protocol applied every 24h. To evaluate responsiveness to inotropes, slices were incubated with milrinone before culture termination. Calcium transients and action potentials were determined optically using calcium and voltage-sensitive dyes. Results Continued exposure to either doxorubicin or trastuzumab limited the recovery of contractility in myocardial slices. In contrast, vehicle controls showed a robust recovery and development of stronger contractile forces. Doxorubicin-treated slices were less responsive to stimulation, displayed increased stimulation threshold and were less able to follow pacing at higher frequencies. Optical mapping revealed that exposure to doxorubicin led to the development of abnormal calcium handling. In parallel to the ex vivo findings, RVAD/LVAD-mediated cardiac unloading and the absence of any chemotherapy drug, resulted in a significant recovery of right-ventricular function, enabling RVAD removal within 2 weeks. Conclusions The complex nature of chemotherapy-induced cardiotoxicity imposes numerous challenges in clinical decision-making. In this unique case, patient care could be paralleled by concomitant state-of-the-art ex vivo tissue culture. We observed that myocardial tissue slices could partially recapitulate clinical observations, highlightings the promising synergy between translational science and personalized patient care.

Abstract P1118: Engineering A Patient-specific Heart On A Chip Model Of Hypoplastic Left Heart Syndrome

Hypoplastic Left Heart Syndrome (HLHS) is the most expensive congenital cardiac disease and is characterized by underdevelopment of the left side of the heart. The surgical intervention leads to the developmentally disadvantaged right ventricle performing systemic circulation. As such, a subset of HLHS patients develops progressive heart failure that results in limited transplant-free survival. There are no animal models to study the molecular and functional properties of HLHS cardiomyocytes, limiting our understanding of disease mechanisms and human genotype-phenotype relationships. To address this, we are creating a physiologically and functionally relevant ”heart on a chip” model of HLHS hiPSC-derived cardiomyocytes (CMs). Induced Pluripotent Stem Cells (iPSCs) were derived from cord blood mononuclear cells isolated from the umbilical cord blood from 8 antenatally diagnosed HLHS children at birth. Control (iPSCs) from 3 healthy subjects were derived from peripheral blood mononuclear cells. We then differentiated ventricular CMs from these iPSCs using StemDiff ventricular CM kit. We observed HLHS iPSCs formed ~50% fewer CMs compared to healthy controls. We studied calcium handling by iPSC-CMs with Fluo-4 fluorescent calcium dye in HLHS and Healthy iPSC-CMs. We observed that calcium transients in HLHS-CMs have half the beat rate, 1.4 times longer rise time to rise, 1.5 times higher time constant (Tau), compared to Healthy CMs, suggesting that HLHS CMs have dysfunctional calcium handling compared to healthy CMs. Our results demonstrate that in an in vitro model of iPSC-derived CM, HLHS patient CMs demonstrate reduced formation and defective calcium handling, which may potentially explain the pathogenesis of heart failure in HLHS patients. In ongoing experiments, we are evaluating diastolic and systolic stress generation in these cells. We will also establish the underlying molecular mechanisms of functional defects in HLHS CMs and screen potential drug therapies for single-ventricle patients.

1748-P: Assessing the Presence of Delta-Cell–Mediated Electrical Coupling in the Islet

Somatostatin-producing delta cells play an important role in the regulation of paracrine cross talk in the islet through inhibition of insulin and glucagon secretion by beta and alpha cells, respectively. However, recent studies have suggested the presence of electrical coupling via gap junctions between delta and beta cells. Electrical coupling mediated cross-talk would support the correlation between pulsatile insulin and somatostatin release and reveal a structural source of cell-cell communication within the islet. To test whether beta and delta cells are gap junction coupled, we measured the permeability of cell to cell connections and the relative dynamics of Ca2+. Islets of Sst-Cre;Rosa-LSL-tdTomato mice were used which show delta-cell specific labelling. They were then loaded with the cationic dye rhodamine-123 at 5mM glucose to measure performance of fluorescence recovery after photobleaching to assess gap junction coupling of delta cells and non-delta cells. Calcium dynamics were assessed using calcium dye Fluo-4 upon stimulating the islet from low glucose (2mM or 5 mM) to high glucose(11mM). No significant differences between recovery rate were observed when comparing delta cells and beta cells (p>0.05), which indicates some level of gap junction permeability among delta cells exists. The calcium activity of a population of delta cells was similar to those of neighboring beta cells, indicating a functional connection exists between these cell types. Furthermore, first-responder beta cells showed a preferential proximity to delta cells suggesting an impact of delta cells on functional sub-populations of nearby beta-cells. These results suggest a possible feedback system exists in the islet that is governed by electrical coupling of delta cells, which is important for both the function of beta cells and insulin secretion, and may also impact secretion of other islet hormones. Disclosure C.H.Levitt: None. R.K.Benninger: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (P30DK116073, R01DK102950, R01DK106412)

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

The Role of EAAT4 in Epidermal Differentiation and Calcium Homeostasis During Aging

Alzheimer’s Disease modeling using hiPSC neurons and microglia

AbstractBackgroundNeuroinflammation is an important topic in neurodegenerative disease research. Scientists have established multiple protocols for differentiating microglia, neurons, and astrocytes from human iPSCs, which enable the study of neuroinflammation in vitro. Here we describe the use of Vala Sciences’ imaging platform, the IC200 Kinetic Image CytometerTM (KIC), to develop screening assays for Alzheimer’s Disease (AD) in cultures with hiPSC‐derived microglia and neurons.MethodWe cultured iCell Glutaneurons with microglia in 384‐well dishes. To detect microglia phagocytosis, we cultured microglia with pHrodo‐Red Zymosan beads or β‐amyloid fragments and then acquired timelapse videos over 5‐24 hours. To measure neuronal activity, we loaded cells with Rhod‐4 calcium dye and imaged at 4fps. We also fixed and immunolabeled cells for neurite and synapse analysis. All assays were scanned using the IC200 KIC. We developed algorithms for our CyteSeer image analysis software to automate neurite tracing, synapse detection, analysis of calcium activity, and microglia phagocytosis and morphology for single cells in a co‐culture system.ResultIn microglia, we demonstrated an increase in uptake of pHrodo‐Red labeled Zymosan beads with increasing concentration of Zymosan particles and in response to IL‐4. We also demonstrated differential engulfment of β‐amyloid 1‐40 and β‐amyloid 1‐42 by microglia. While investigating the effects of microglia on neuronal health, we observed increased neuronal survival, neurite outgrowth, and pre‐synapse count in glutamatergic neurons co‐cultured with microglia when compared to glutamatergic neurons cultured alone. In neuron‐microglia co‐cultures exposed to increasing doses of β‐amyloid 1‐42 fragments, we observed neuronal hyperactivity (increased calcium event frequency), which may reflect disease‐associated stress.ConclusionOur data demonstrate that healthy microglia regulate neuronal heath. Our system can serve as a model for testing inflammatory insults related to AD. To further our research with genetic variants with AD susceptibility, we have created an isogeneic panel of ApoE variants using CRISPR‐Cas9, and plan to characterize them in the culture systems developed above to validate a disease model from these lines.

Abstract 11976: Functional Characterization of Calcium Handling Kinetics in Patient-Derived Ipsc-Cm Models of Pkp2-Mediated Arrhythmogenic Right Ventricular Cardiomyopathy

Introduction: Pathogenic variants in PKP2 -encoded plakophilin 2 cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Electrocardiographically, PKP2 -mediated ARVC may mimic RYR2 -mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). We compare the underlying mechanism of arrhythmia in ARVC and CPVT using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Methods: iPSC-CMs were generated from 2 ARVC patients with different PKP2 variants [E149X (PKP2-1) and c.2146-1G>C (PKP2-2)], 1 patient with a CPVT variant [RyR2-R176Q (CPVT1)], and a wild-type (WT) control. Fluo-4 AM calcium dye was used to assess calcium kinetics in iPSC-CMs before and during treatment with 100 nM isoproterenol (ISO) at 8-10 min. Results: Compared to WT, PKP2 and RYR2 iPSC-CMs had a greater amplitude before (WT: 0.331±0.031, PKP2-1: 1.048±0.074, PKP2-2: 1.076±0.095, CPVT1: 0.966±0.061; P<0.0001) and during ISO (WT: 0.277±0.022, PKP2-1: 0.971±0.069, PKP2-2: 0.622±0.041, CPVT1: 0.756±0.051; P<0.0001). The Ca 2+ transient duration (CTD100) was longer in PKP2 and RYR2 iPSC-CMs compared to WT before (WT: 1.01±0.05 s, PKP2-1: 1.84±0.01 s, PKP2-2: 1.76±0.02 s, CPVT1: 1.81±0.02 s; P<0.0001) and during ISO (WT: 1.38±0.05 s, PKP2-1: 1.65±0.03 s, PKP2-2: 1.71±0.03 s, CPVT1: 1.61±0.05 s; P<0.001). Before (WT: 259±13 ms, PKP2-1: 538±23 ms, PKP2-2: 493±28 ms, CPVT1: 586±24 ms; P<0.0001) and during ISO (WT: 311±19 ms, PKP2-1: 406±21 ms, P=0.0172, PKP2-2: 487±28 ms, P<0.0001, CPVT1: 453±21 ms, P=0.0001), peak to 50% decay time was slower in PKP2 and RYR2 iPSC-CMs compared with WT. Upstroke time was longer in PKP2 and RYR2 iPSC-CMs compared to WT before (WT: 205±5 ms, PKP2-1: 539±13 ms, PKP2-2: 429±19 ms, CPVT1: 455±12 ms; P<0.0001) and during ISO (WT: 213±7 ms, PKP2-1: 375±16 ms, PKP2-2: 416±20 ms, CPVT1: 389±10 ms; P<0.0001). Conclusion: Here, iPSC-CMs, derived from patients with either PKP2 -mediated ARVC or RYR2 -mediated CPVT, display similar abnormal calcium handling properties such as greater amplitude, longer CTD100, and slower peak to 50% decay time and upstroke time compared to WT iPSC-CMs before and during ISO treatment. This may explain why patients with PKP2 -mediated ARVC often mimic the treadmill stress test seen in patients with CPVT1.

Crosstalk between kisspeptin and gonadotropin-inhibitory hormone in the silence of puberty: preclinical evidence from a calcium signaling study

Kisspeptin and gonadotropin-inhibitory hormone (GnIH) are among suggested neuroendocrine modulators of reproductive function. Intracellular calcium signaling is a critical component in the regulation of a variety of physiological and pathological processes including neurotransmitter release, and, therefore, can be used as signaling indicator for investigating the involvement of kisspeptin, GnIH, and gonadotropin-releasing hormone (GnRH) release. Hence, this study investigated the effects of kisspeptin and GnIH on calcium signaling using immortalized hypothalamic cells (rHypoE-8) as a model. Kisspeptin neurons were loaded with the ratiometric calcium dye (Fura-2 AM, 1 μmol) and intracellular free calcium ([Ca2+]i) responses were quantified using digital fluorescence imaging system. Kisspeptin-10 (100, 300, and 1000 nM) caused a significant increase in [Ca2+]i in rHypoE-8 cells (n = 58, n = 64, and n = 49, respectively, p < 0.001). The kisspeptin receptor antagonist, P234, inhibited the calcium responses to kisspeptin (p < 0.001, n = 32). GnIH (100 and 1000 nM), alone, did not cause any significant change in the mean basal [Ca2+]i levels in kisspeptin cells, but GnIH attenuated the kisspeptin-evoked [Ca2+]i transients (n = 47, p < 0.001). This novel findings of [Ca2+]i signaling in in vitro setting implicate that kisspeptin and GnIH may exert their effects on hypothalamus–pituitary–gonadal (HPG) axis by modulating kisspeptin neurons. These results also implicate that kisspeptin neurons may have an autocrine regulation.

A chitosan based multimodal “soft” hydrogel for rapid hemostasis of non-compressible hemorrhages and its mode of action

Uncontrolled hemorrhages during accidents or surgery require non-compressible dressings to halt bleeding and avoid damage to soft tissue and organs. Here, we report the synthesis of a sodium tripolyphosphate crosslinked chitosan-based hemostatic hydrogel dressing incorporating hemostasis agonists like aluminum chloride, an aggregation enhancer and silica nanoparticles, a contact activator, as confirmed by FTIR analyses. Rheology studies displayed G” > G’ values confirming typical soft gel-like behavior that was maintained upto shear rate 10/s. Further, composite had excellent water absorption capacity (737 ± 50%) and showed 6-fold enhanced in vitro hemostasis as compared to bare hydrogel. Platelet aggregation was enhanced with composite as compared to SFLLRN peptide (a thrombin mimic) and adenosine diphosphate (calcium release activator) while it was absent with heparin (thrombin inhibitor). Enhanced activation and release of calcium were observed by composite with a specific fluorescent calcium dye in A549 cell model. The composite was biocompatible and in vivo evaluation of hemostatic efficiency in rat liver injury revealed enhanced blood clotting (16 ± 2.1 s) of hydrogel composite as compared to commercial HemoStat Clear gel (55 ± 5 s). Thus, developed soft hydrogel composite has potential for application as a safe, easy-to-apply, rapid, non-compressible hemostatic agent.

Dequalinium chloride is an antagonists of α7 nicotinic acetylcholine receptors

Dequalinium chloride has been used primarily as antiseptic compounds, but recently has been investigated for its effects on specific targets, including muscarinic acetylcholine receptors. Here we investigated dequalinium chloride as an antagonist to α7 nicotinic acetylcholine receptors. The pharmacological properties of dequalinium were established using cell lines stably co-transfected with the calcium-permeable human α7 nicotinic acetylcholine receptors and its chaperone NACHO, calcium dye fluorescent measurements or a calcium-sensitive protein reporter, and patch clamp recording of ionic currents. Using calcium dye fluorescence plate reader measurements, we find dequalinium chloride is an antagonist of α7 nicotinic acetylcholine receptors with an IC50 of 672 nM in response to activation with 500 μM acetylcholine chloride and positive allosteric modulator PNU-120596. However, using a membrane-tethered GCAMP7s calcium reporter allowed detection of α7-mediated calcium flux in the absence of PNU-120596. Using this approach revealed an IC50 of 157 nM for dequalinium on 300 μM acetylcholine-evoked currents. Using patch clamp recordings with 300 μM acetylcholine chloride and 10 μM PNU-120596, we find lower concentrations are sufficient to block ionic currents, with IC50 of 120 nM for dequalinium chloride and 54 nM for the related UCL 1684 compound. In summary, we find that dequalinium chloride and UCL1684, which are generally used to block SK-type potassium channels, are also highly effective antagonists of α7 nicotinic acetylcholine receptors. This finding, in combination with previous studies of muscarinic acetylcholine receptors, clearly establishes dequalinium compounds within the class of general anti-cholinergic antagonists.

Comparing synthetic refocusing to deconvolution for the extraction of neuronal calcium transients from light fields.

.SignificanceLight-field microscopy (LFM) enables fast, light-efficient, volumetric imaging of neuronal activity with calcium indicators. Calcium transients differ in temporal signal-to-noise ratio (tSNR) and spatial confinement when extracted from volumes reconstructed by different algorithms.AimWe evaluated the capabilities and limitations of two light-field reconstruction algorithms for calcium fluorescence imaging.ApproachWe acquired light-field image series from neurons either bulk-labeled or filled intracellularly with the red-emitting calcium dye CaSiR-1 in acute mouse brain slices. We compared the tSNR and spatial confinement of calcium signals extracted from volumes reconstructed with synthetic refocusing and Richardson–Lucy three-dimensional deconvolution with and without total variation regularization.ResultsBoth synthetic refocusing and Richardson–Lucy deconvolution resolved calcium signals from single cells and neuronal dendrites in three dimensions. Increasing deconvolution iteration number improved spatial confinement but reduced tSNR compared with synthetic refocusing. Volumetric light-field imaging did not decrease calcium signal tSNR compared with interleaved, widefield image series acquired in matched planes.ConclusionsLFM enables high-volume rate, volumetric imaging of calcium transients in single cell somata (bulk-labeled) and dendrites (intracellularly loaded). The trade-offs identified for tSNR, spatial confinement, and computational cost indicate which of synthetic refocusing or deconvolution can better realize the scientific requirements of future LFM calcium imaging applications.

Pharmacogenomic Screening of Drug Candidates using Patient-Specific hiPSC-Derived Cardiomyocyte High-Throughput Calcium Imaging.

Calcium imaging is an invaluable technique to detect and characterize calcium flux in cells. The use of calcium dye provides information on the concentration and spatial distribution of calcium. Calcium imaging is a well-established technique to assess the calcium-induced calcium release mechanism in cardiomyocytes. It can also be used to characterize mutations in genes crucial for this mechanism that frequently causes arrhythmia. Here we describe a high-throughput methodology of calcium imaging that records individual calcium transients in more than 10,000 humaninduced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in less than 30min.

Fast synaptic excitatory neurotransmission in the human submucosal plexus.

Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce. We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut. Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7±2.4% of all submucosal neurons (n=6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α1 , α2 or α3 -subunit). Hexamethonium (200µM) reduced nerve-evoked chloride secretion by 34.3±18.6% (n=14 patients), whereas D-APV had no effect. Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered.

Quantitative high-throughput assay to measure MC4R-induced intracellular calcium.

The melanocortin-4 receptor (MC4R), a critical G-protein-coupled receptor (GPCR) regulating energy homeostasis, activates multiple signalling pathways, including mobilisation of intracellular calcium ([Ca2+]i). However, very little is known about the physiological significance of MC4R-induced [Ca2+]i since few studies measure MC4R-induced [Ca2+]i. High-throughput, read-out assays for [Ca2+]i have proven unreliable for overexpressed GPCRs like MC4R, which exhibit low sensitivity mobilising [Ca2+]i. Therefore, we developed, optimised, and validated a robust quantitative high-throughput assay using Fura-2 ratio-metric calcium dye and HEK293 cells stably transfected with MC4R. The quantitation enables direct comparisons between assays and even between different research laboratories. Assay conditions were optimised step-by-step to eliminate interference from stretch-activated receptor increases in [Ca2+]i and to maximise ligand-activated MC4R-induced [Ca2+]i. Calcium imaging was performed using a PheraStar FS multi-well plate reader. Probenecid, included in the buffers to prevent extrusion of Fura-2 dye from cells, was found to interfere with the EGTA-chelation of calcium, required to determine Rmin for quantitation of [Ca2+]i. Therefore, we developed a method to determine Rmin in specific wells without probenecid, which was run in parallel with each assay. The validation of the assay was shown by reproducible α-melanocyte-stimulating hormone (α-MSH) concentration-dependent activation of the stably expressed human MC4R (hMC4R) and mouse MC4R (mMC4R), inducing increases in [Ca2+]i, for three independent experiments. This robust, reproducible, high-throughput assay that quantitatively measures MC4R-induced mobilisation of [Ca2+]i in vitro has potential to advance the development of therapeutic drugs and understanding of MC4R signalling associated with human obesity.

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology.

Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

A Novel Alginate-Based Delivery System for the Prevention and Treatment of Pressure-Overload Induced Heart Failure.

Background: α-CGRP (alpha-calcitonin gene related peptide) is a cardioprotective neuropeptide. Our recent study demonstrated that the administration of native α-CGRP, using osmotic mini-pumps, protected against transverse aortic constriction (TAC) pressure-induced heart failure in mice. However, the short half-life of peptides and the non-applicability of osmotic pumps in humans limits the use of α-CGRP as a therapeutic agent for heart failure (HF). Here, we sought to comprehensively study a novel α-CGRP delivery system using alginate microcapsules to determine its bioavailability in vivo and to test for cardioprotective effects in HF mice. Methods: Native α-CGRP filled alginate microcapsules (200 µm diameter) were prepared using an electrospray method. The prepared alginate-α-CGRP microcapsules were incubated with rat cardiac H9c2 cells, mouse cardiac HL-1 cells, and human umbilical vein endothelial cells (HUVECs), and the cytotoxicity of the alginate-α-CGRP microcapsules was measured by a trypan-blue cell viability assay and a calcium dye fluorescent based assay. The efficacy of the alginate-α-CGRP microcapsules was tested in a TAC-pressure overload mouse model of heart failure. Male C57BL6 mice were divided into four groups: sham, sham-alginate-α-CGRP, TAC-only, and TAC-alginate-α-CGRP, and the TAC procedure was performed in the TAC-only and TAC-alginate-α-CGRP groups of mice to induce pressure-overload heart failure. After 2 or 15 days post-TAC, alginate-α-CGRP microcapsules (containing an α-CGRP dose of 6 mg/kg/mouse) were administered subcutaneously on alternate days, for 28 days, and echocardiography was performed weekly. After 28 days of peptide delivery, the mice were sacrificed and their hearts were collected for histological and biochemical analyses. Results: Our in vitro cell culture assays showed that alginate-α-CGRP microcapsules did not affect the viability of the cell lines tested. The alginate-α-CGRP microcapsules released their peptides for an extended period of time. Our echocardiography, biochemical, and histology data from HF mice demonstrated that the administration of alginate-α-CGRP microcapsules significantly improved all cardiac parameters examined in TAC-mice. When compared to sham mice, TAC significantly decreased cardiac functions (as determined by fraction shortening and ejection fraction) and markedly increased heart and lung weight, left ventricle (LV) cardiac cell size, cardiac apoptosis, and oxidative stress. In contrast, the administration of alginate-α-CGRP microcapsules significantly attenuated the increased heart and lung weight, LV cardiac cell size, apoptosis, and oxidative stress in TAC mice. Conclusion: Our results demonstrate that the encapsulation of α-CGRP in an alginate polymer is an effective strategy to improve peptide bioavailability in plasma and increase the duration of the therapeutic effect of the peptide throughout the treatment period. Furthermore, alginate mediates α-CGRP delivery, either prior to the onset or after the initiation of the symptom progression of pressure-overload, improves cardiac function, and protects hearts against pressure-induced HF.

Взаимодействие механизмов угнетения квантового выделения ацетилхолина при активации ваниллоидных (TRPV1) и пуриновых рецепторов в нервно-мышечном синапсе мыши

The main goal of the study was to analyze the relationship between the signaling pathways of the regulation of acetylcholine (ACh) quantal release in the peripheral synapse triggered by the activation of vanilloid (TRPV1) and purine receptors. In electrophysiological experiments carried out at the neuromuscular synapses of the m. Levator Auris Longus, it was found that the frequency of miniature endplate potentials (mEPPs) and the quantal content of endplate potentials (EPPs) decrease in the presence of TRPV1 agonist capsaicin. This effect was completely reversed by SB366791, a specific competitive antagonist of TRPV1 receptors. ATP, like capsaicin, decreased the frequency of mEPPs and the EPP quantal content. Against the background of the TRPV1 antagonist, the inhibitory effect of ATP on ACh secretion was realized in full. At the same time, against the background of TRPV1 channels activation by capsaicin, the effect of ATP on both spontaneous and evoked ACh release was absent. It was suggested that the mechanisms of action of ATP and capsaicin may be associated with a change in Ca2+ entry into the nerve ending. To test this hypothesis, experiments were carried out to assess the changes in the presynaptic calcium level (Ca2+ transient) using a fluorescent calcium dye upon nerve stimulation. The amplitude of the calcium transient did not change either with the application of ATP or with the addition of capsaicin. Thus, in the neuromuscular synapse of mammals, along with purinergic pathway of ACh secretion regulation, there is also a mechanism of neurosecretion modulation mediated by the activation of TRPV1 channels. The triggering of these mechanisms leads to the suppression of the processes of both spontaneous and evoked release of ACh quanta from the motor nerve endings. It was demonstrated that both pathways of regulation are not accompanied by a change in the Ca2+ transient, but have a common link of regulation of the quantal neurosecretion.

Improved hyperacuity estimation of spike timing from calcium imaging

Two-photon imaging is a major recording technique used in neuroscience. However, it suffers from several limitations, including a low sampling rate, the nonlinearity of calcium responses, the slow dynamics of calcium dyes and a low SNR, all of which severely limit the potential of two-photon imaging to elucidate neuronal dynamics with high temporal resolution. We developed a hyperacuity algorithm (HA_time) based on an approach that combines a generative model and machine learning to improve spike detection and the precision of spike time inference. Bayesian inference was performed to estimate the calcium spike model, assuming constant spike shape and size. A support vector machine using this information and a jittering method maximizing the likelihood of estimated spike times enhanced spike time estimation precision approximately fourfold (range, 2–7; mean, 3.5–4.0; 2SEM, 0.1–0.25) compared to the sampling interval. Benchmark scores of HA_time for biological data from three different brain regions were among the best of the benchmark algorithms. Simulation of broader data conditions indicated that our algorithm performed better than others with high firing rate conditions. Furthermore, HA_time exhibited comparable performance for conditions with and without ground truths. Thus HA_time is a useful tool for spike reconstruction from two-photon imaging.

Three-photon imaging of synthetic dyes in deep layers of the neocortex

Multiphoton microscopy has emerged as the primary imaging tool for studying the structural and functional dynamics of neural circuits in brain tissue, which is highly scattering to light. Recently, three-photon microscopy has enabled high-resolution fluorescence imaging of neurons in deeper brain areas that lie beyond the reach of conventional two-photon microscopy, which is typically limited to ~ 450 µm. Three-photon imaging of neuronal calcium signals, through the genetically-encoded calcium indicator GCaMP6, has been used to successfully record neuronal activity in deeper neocortical layers and parts of the hippocampus in rodents. Bulk-loading cells in deeper cortical layers with synthetic calcium indicators could provide an alternative strategy for labelling that obviates dependence on viral tropism and promoter penetration, particularly in non-rodent species. Here we report a strategy for visualized injection of a calcium dye, Oregon Green BAPTA-1 AM (OGB-1 AM), at 500–600 µm below the surface of the mouse visual cortex in vivo. We demonstrate successful OGB-1 AM loading of cells in cortical layers 5–6 and subsequent three-photon imaging of orientation- and direction- selective visual responses from these cells.