Never before has it been observed that Ca2+ can induce fibrillogenesis and the gelation of native, enzyme-free fibrinogen. Enzyme catalysis was ruled out by the addition of thrombin and factor XIII inhibitors. Even more striking, Ca2+ induces gelation even under physiological conditions, leading again to stable and fibrous hydrogels. Although this latter approach is possibly co-induced by residual factor XIII, the resulting gels are for the first time recognized as promising materials and not discounted as unwanted side effects. The finding that these gels again consist of fibers especially renders a new perspective on the role of factor XIII and fibrinogen’s well-known Ca2+ binding sites. In this study, we aim to provide first insights into this highly feasible material and its characteristics.Fibrin is considered a highly promising biomaterial for manifold medical applications. Although it is a well-established material in this field, the required enzyme thrombin bears some striking downsides such as high costs and health risks. Current research discovers more and more ways to use fibrin’s precursor fibrinogen as a substitute. Fibrinogen’s full potential is, however, only retained when using it as fibrous gel, as it is the case for fibrin. In our previous work, we introduced such a kind of material for the first time. This material, called pseudo-fibrin, shows striking similarities to fibrin regarding its supramolecular structure and is created in a facile salt-induced process, which we further improved in this study. In particular, we shine light on the role of Ca The role of biobased and bioinspired materials in modern research expands continuously and covers increasingly broader areas of our everyday life. Decades ago, the potential of these materials was discovered, for example their use as casein-based paints . More reOne biomaterial of high interest is fibrin, a fibrous protein gel responsible for wound healing. Due to its function as a wound sealant, fibrin inherits a unique combination of properties, being especially non-toxicity and strongly adhesive . These f2 [Fibrinogen itself is a rod-like, 340 kDa large glycoprotein, which consists of two pairs of amino acid chains, labeled α, β, and γ. These chains are connected by disulfide bridges in the central region of the molecule. Additionally, the fibrinopeptides A and B are attached to chains α and β, respectively, which is why fibrinogen is also denoted as (AαBβγ)2 ,12. A fu2 .In general, fibrinogen-based materials can be subdivided into several categories, depending on their gel-like or fibrous nature. Non-gelled and non-fibrous fibrinogen is mainly used in drug delivery ,20,21 buA similar method yields fibrinogen fibers via an extrusion-like process. Again, highly concentrated fibrinogen solutions are used, which were channeled through a long, thin tube .Many actual self-assembly processes to gain enzyme-free fibrinogen fibers require a substrate, i.e., surfaces on which fibrillogenesis takes place. In a recent work, fiber formation was observed upon drying when salts are present. This approach yields fibrous fibrinogen scaffolds ,29. SubsSimilar to this approach, several studies reported a substrate-driven fiber formation, which however does not involve salts. Instead, the successful fibrillogenesis solely requires hydrophobic substrates such as polystyrene. This process is most probably mediated by fibrinogen’s αC region ,32.Enzyme-free fibrillogenesis in solution has been observed for decades but none of the methods lead to actual hydrogels. One early example of salt-induced fibrillogenesis was presented by Gollwitzer et al. in 1983, who dialyzed fibrinogen solutions against buffers with low ionic strength .A more recent method to induce fibrillogenesis in solution is by adding ethanol to a low-concentrated fibrinogen solution ,35. HydrRecently, we introduced a new process to yield a fibrous, gel-like fibrinogen material in solution. This is the first method to gain a material close to fibrin in solution. The material called “pseudo-fibrin” is created by adding a defined amount of kosmotropic anions to a cold, salt-free fibrinogen solution at a slightly acidic-to-neutral pH. Pseudo-fibrin can also be lyophilized to gain highly porous aerogels, therefore filling the gap in search for a convincing fibrin alternative .2+ as it already plays an important role in the blood clotting cascade. It is known that fibrinogen has some binding sites for these ions, and the binding of Ca2+ is especially known for significant material improvements. These improvements include stabilization against acid and heat denaturation [2+ ions even accelerate fibrin formation [To further analyze this fascinating material and its underlying mechanism, we now characterize the impact of Caturation  and stabturation . Ca2+ ioormation ,38.2+, Cu2+, and Hg2+. Interestingly, the authors emphasize that Mg2+ and Ca2+ especially do not induce any kind of gelation under the applied conditions [2+ reported in the literature. We rediscovered a 40-year-old approach to trigger an (apparently) Ca2+-induced gelation of fibrinogen. Rapidly, this phenomenon could be explained by the residuals of the enzyme factor XIII in the purchased fibrinogen. Factor XIII, i.e., its activated form factor XIIIa, is normally responsible for the covalent cross-linking of fibrin [2+ activate factor XIII, since this process usually requires thrombin? This is also the natural pathway; thrombin and Ca2+ activate factor XIII to form factor XIIIa. The researchers found out that an activation of the enzyme is indeed possible solely via Ca2+, but requires concentrations of at least 50 mmol/L [In the 1980s, Steven et al. had already reported a gelation of fibrinogen in the presence of some divalent metal cations, for example Fenditions . There a0 mmol/L ,42. In a2+ was reported by Marx, who found that fibrin protofibrils (not the monomers) form a fibrous clot when exposed to 1–2 mmol/L Ca2+. This process crucially requires fibrin protofibrils which have to be created beforehand via thrombin addition and nearly instant quenching. Only in this case is Ca2+ able to “continue” self-assembly. Zn2+ ions have an even more pronounced effect. This procedure does not involve any factor XIII, solely thrombin (in the beginning) and Ca2+ [2+ and Zn2+, respectively. Consequently, it is not the presence of protofibrils that is crucial for the process; the cleavage of fibrinopeptide A simply inevitably leads to such an outcome [Another interesting finding regarding Caand Ca2+ ,44. Late outcome .2+-related effects show how much of an influence it has in many aspects of blood clotting. Although it inherits such enormous potential, it has never been observed before that Ca2+ itself can trigger fibrillogenesis and gelation at the same time. This effect is most likely linked to the occupancy of binding sites under suited reaction conditions and potentially renders a new perspective on their role and functions. Further, this observation potentially fills the gap between all reported Ca2+-related self-assembly approaches.All these insights into Ca2+ to the protein solution. To get an impression of this material, gels are analyzed mainly via scanning electron microscopy. First insights into the mechanical properties are provided by measuring the rheological behavior.We herein aim to introduce a new approach to create fibrous fibrinogen hydrogels, which emerge by adding Ca2, CaCl2, SrCl2, and BaCl2. The reaction time was expanded to 24 h to not oversee any effects, otherwise all conditions were kept identical to the standard “anion process” . As already known from previous studies, a white precipitate is formed upon salt addition. By stirring, these  aggregate and settle down to the bottom, so this has to be strictly avoided [Our previously introduced anion-driven process to create fibrin-like pseudo-fibrin gels requires kosmotropic anions. We now aim to characterize the impact of divalent cations on this approach, as they play an important role in the natural blood clotting process. To exclude anion effects, first the experiments were performed using divalent metal chlorides such as MgCl avoided .2 as the trigger are remarkably stable especially, whereas the others appeared to be simply precipitated. Scanning electron microscopy of the four samples revealed a fibrous structure in the case of CaCl2, as 2, SrCl2, and BaCl2 were less defined and contained more unstructured fibrinogen. These findings were not described before; on the contrary, Mg2+ and Ca2+ were explicitly mentioned to not induce gelation or fibrillogenesis of fibrinogen [Surprisingly, in all cases a gel-like material was formed after 24 h, which was significantly more stable than the ones gained in all our previous experiments. In comparison to fibrin, these are completely white and nontransparent, whereas fibrin under the same conditions shows only slight turbidity. This observation is already a hint that a new material was created here. The gels with CaClbrinogen .2+-induced gelation increases the potential of pseudo-fibrin even more, since actual gels are now easily accessible with non-toxic salts. It is, however, unclear whether this material is actually the same as our previously described pseudo-fibrin. To take a deeper insight into this issue, further experiments with calcium salts were performed.Ca2 yields a fibrin-like hydrogel with a remarkably fibrous sub-structure. This finding brought up some fundamental questions regarding the role of Ca2+ in this process. The first important step is to investigate whether the process involves unintended enzyme catalysis. It is known that the activation of several blood-clotting factors requires calcium [2+-induced self-assembly was repeated in the presence of the thrombin inhibitor AEBSF and factor XIII inhibitor 2-iodoacetamide. Both experiments still lead to the same result. The latter especially proves that our Ca2+-induced approach is different from previously described ones, which either use protofibrils [Replacing the sodium phosphate buffer of our anion-triggered fibrillogenesis with the same amount of CaCl calcium ,46. To rofibrils  or factoofibrils .2+ ions since neither NaCl nor other earth alkaline chlorides lead to such results. Indeed, a similar experiment performed with Ca(NO3)2 leads to the same fibrous gel, strengthening the hypothesis of a calcium-induced process. Most calcium salts, however, are not soluble to such a high extent, which hinders comparison with various calcium salts. A combination with kosmotropic anions would be interesting to fill the gap between the already introduced “anion process” and the calcium one.Although enzyme catalysis can be ruled out as a trigger, it is not clear at this point what specifically leads to the self-assembly of fibrinogen to fibers. Most probably, this process is solely induced via Ca2 is around 23 mmol/L, which is not exceeded). On the other hand, the gelation becomes rapidly incomplete below a pH of 7.0. Also, the resulting fibers become unstructured until the process is inhibited at a pH of 6.0. SEM images of all further samples are provided in the supporting information  are obtained. A gel is, however, still formed with 1 mmol/L Ca2+. The upper limit is around 30 mmol/L Ca2+ and is therefore increased compared to the anion process. The corresponding microscopy images are presented in As a next step to characterize the calcium process, the optimal CaCl2+ concentrations (0.5–5 mmol/L), the diameter is in the range of 100–150 nm, whereas the sample created with 5 mmol/L Ca2+ shows the lowest standard deviation (104 ± 9 nm). Beginning at 15 mmol/L Ca2+, the obtained fibers appear more coalesced and are thicker, i.e., the diameter is approximately 200 nm. The average diameters and their standard deviation can also be found in the supporting information  and to increase the solubility of fibrinogen. The latter was analyzed in detail by means of time-resolved light scattering in our early works regarding fibrinogen self-assembly . Briefly2+-triggered fibrillogenesis has been reported yet; the combination of the high salt concentration, physiological temperature, and rather low Ca2+ concentrations disrupts the process so much that it was overlooked. Finally, many studies regarding fibrin are conducted in phosphate buffered saline, which is incompatible with such high amounts of Ca2+. All these aspects together are the reason why this phenomenon has not been described before.It is not surprising that also elevated temperatures (37 °C) do not inhibit the process either, but significantly decrease yield and kinetics. The product is however less stable and can hardly be considered as “gel-like”. On the other hand, pseudo-fibrin produced at 5 °C only partially redissolves at 37 °C. Regarding the weakness against high ion concentrations and temperature, it becomes clear again that the process cannot be enzyme-driven, especially since the physiological temperature would rather enhance enzyme activity. Furthermore, this brings up a new explanation as to why no Ca2+; at least, it is very probable that the Ca2+ ions occupy them. In general, the specific and unspecific binding sites must be distinguished. The unspecific ones are electrostatic effects due to the net charge of fibrinogen at alkaline pH. These ones are occupied by Mg2+ as well as Ca2+, which is why they are labeled unspecific. The specific ones, however, are only suited for Ca2+ as they involve chelate complexes [2+- induced gels and Ca2+-induced gels is most likely attributed to the occupancy of these high-affinity binding sites, which renders a fascinating new perspective on their role and function in general. Although the exact mechanism(s) of gelation and fibrillogenesis are not clear yet and require deeper investigation (for example via light scattering and IR measurements), we hypothesize the following contribution: theoretical work regarding the charge distribution of fibrinogen show that the outer domains of the protein (the D domains) are negatively charged under physiological conditions [2+ triggers a unique effect. Most likely, fibrinogen undergoes changes in its secondary structure upon Ca2+ addition, which already has been observed in several previously mentioned studies. These changes potentially favor a more orientated aggregation, in this case to fibers. Other ions such as Mg2+ or Sr2+ presumably do not fit into the molecular structure because the specific binding sites are intended for Ca2+, i.e., cations of this respective size. These aspects however have to be proven in future work.It is highly likely that this process is linked to the well-known binding sites for Caomplexes . As a reomplexes , leadingomplexes . The signditions . Consequ2+-induced fibrillogenesis proved to be a facile and even more convincing way to create a fibrin-like material without enzymes. The reaction conditions are more flexible compared to our anion process and include an extended regime of ion concentrations, reaction temperatures, and pH. Only the reaction time is increased to at least 16 h. Most important, it could be shown that this process is neither enzyme-induced nor related to substrates or drying effects.In summary, the Ca2+-induced pseudo-fibrin is significantly more robust against ion concentrations and temperatures than our previously introduced anion process. However, a limitation until now is the necessity for low temperatures and salt concentrations for optimal yield and kinetics. Ideally, the gels can even be fabricated under physiological conditions. Since the process is in principle possible at 37 °C (but with low yield and disadvantageous kinetics), the first step to counterbalance these effects is a much higher concentration of fibrinogen (and consequently of product). We therefore performed a new series of experiments to finally achieve gelation at 37 °C.The Ca2+ system allows for the use of a buffered system to stabilize the pH at 7.5. For this purpose, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (“HEPES”) was chosen. To not disrupt the process too much initially, experiments were carried out without additional NaCl at 37 °C. Dissolution of 20 g/L fibrinogen— fourfold of the previously possible concentration—was achieved rather quickly (approximately 10 min). The addition of the previously successful 15 mmol/L CaCl2, however, only leads to a slight turbidity, followed by unspecific precipitation. This precipitate settles down to the bottom of the reaction vessel. Even after 24 h of reaction time, no gel or fibers are formed.The overall robustness of the Ca2+ concentration to 25 mmol/L Ca2+ changes the outcome, as a genuine gel formation is observed. Even better results are obtained when using 50 mmol/L Ca2+: After 24 h, a stable gel has emerged. Still, even when using this high Ca2+ concentration, an initial precipitate forms directly upon salt addition. Although the samples do gel, the precipitation is undesirable because less fibrinogen is available, and the overall control is lower. To prevent this, 100 mmol/L NaCl are added, which also brings the system even closer to physiological conditions. As a side effect, the high amount of NaCl allows us to dissolve even more fibrinogen. Indeed, 40 g/L of fibrinogen can be brought into solution within minutes using this approach.However, increasing the Ca2 concentration series with additional NaCl. It can clearly be seen that 15 mmol/L of CaCl2 is again not sufficient to induce gelation. The first stable gels are obtained with 25 mmol/L of CaCl2. A further increase to 50 mmol/L of CaCl2 is accompanied by a more complete gelation, as can already be determined by eye regarding the intense white appearance of the gel. The reference sample without calcium did not undergo any change and remained transparent. The microscopic structure of these gels is also shown in 2+, filigree networks are seen, and using 50 mmol/L of Ca2+ yields high amounts of fibers. These appear more undefined, which is however just a side effect of the high fibrinogen concentration combined with the drying step necessary for electron microscopy. An analogously prepared reference sample of fibrin yielded similar results, i.e., many fibers were incorporated into a rather dense clot , however, the presented experiments provide an effective approach to gel commercial fibrinogen via CaAfter introducing this new approach to obtain calcium-induced hydrogels, the last step is to provide the first insights into their properties as a material.First, the water content, yield, and purity of the pseudo-fibrin gels were determined. This requires careful extraction from the original vessel, which was easily possible without damaging the material. A photograph of the gel is shown in To determine the water content, yield, and purity, the gels were either dried directly after their fabrication or after one additional day of storage in a large volume of fresh buffer. As can be seen in 2+ addition. The results of the amplitude and frequency scan are shown in As a last step to obtain the first insights into the “optimized” pseudo-fibrin gels, rheological measurements were performed. These measurements are a common strategy to monitor and compare various important characteristics of hydrogels such as mechanical stability, the degree of cross-linking, and reversibility of cross-linking . The mos2+ addition makes; instead of a barely measurable solution (G’ is approximately 0.2 Pa), a stable gel is obtained.The main purpose of the amplitude scan is to identify the linear viscoelastic regime of a material. For fibrin, G’ and G’’ are independent of the shear deformation until it reaches values of approximately 5%. Even macroscopically, this effect is very prominent as the water is pressed out of fibrin, leaving behind a dense protein clot. This effect is not observed for pseudo-fibrin, which shows constant storage and loss modulus. Regarding the absolute values, G’ and G’’ of fibrin are approximately six times higher than the ones of pseudo-fibrin. More precisely, G’ of fibrin is 600 Pa, whereas G’ of pseudo-fibrin is only 100 Pa. Before pseudo-fibrin can be considered as an actual alternative to fibrin, this issue has to be addressed in future studies. A potential strategy would be a subsequent covalent cross-linking of the gels. Remarkably, G’ and G’’ of pseudo-fibrin are far beyond the ones of native fibrinogen, demonstrating the difference CaWhen characterizing hydrogels via their rheological properties, the most common measurement is the frequency dependence of G’ and G’’. Again, fibrin’s storage and loss modulus still surpass the ones of pseudo-fibrin, indicating a higher degree of cross-linking. Since G’ and G’’ are nearly constant in both cases, the materials remain stable in the measured frequency range. In contrast, the solution of native fibrinogen is again only barely measurable (G’ = 0.03 Pa). Further, the curves of G’ and G’’ intersect at 0.7 Hz, which is not the case for fibrin and pseudo-fibrin.The presented rheological measurements serve as a first insight into pseudo-fibrin’s material properties. Certainly, there are numerous further possibilities to adequately characterize the material, which go beyond the scope of this first work. We herein aim to introduce a new material and provide first insights into its structure. Deeper insights, including into the mechanism of gelation and the actual potential for  applications, will be the subjects of future studies.2+ has a unique ability to induce an intense fibrillogenesis and gelation on its own, which has not been observed anywhere before. These hydrogels are remarkably stable and, most importantly, resemble the fibrous structure of actual fibrin. Unwanted enzyme catalysis, especially via thrombin and factor XIII, was ruled out as trigger, proving a purely salt-induced process. The calcium-induced process is more resistant towards temperature, ion concentration(s), and alkaline pH especially, than our previously introduced anion-driven process. The hypothesis arose that this “Ca2+ process” is based on the occupancy of the already known binding sites for divalent cations. Evidence for this hypothesis especially includes an inhibition of the process at an acidic pH, where most binding sites vanish.Exchanging the cation in our previously introduced process to gain enzyme-free “pseudo-fibrin” has a huge impact on the outcome. Starting with the addition of earth alkaline chlorides, it quickly became obvious that Ca2+-induced fibrillogenesis is even possible under physiological conditions, resulting again in stable hydrogels. Although residuals of factor XIII in commercially available fibrinogen possibly contribute to this discovery, it still yields fibrous structures, opening up a new perspective on factor XIII cross-linking and the role of Ca2+. The hypothesis arose that this process still interferes with the previously described Ca2+-induced fibrillogenesis. Independent of the mechanism(s), these feasible and easy to produce gels were for the first time considered as actual materials instead of side effects.Ca2+ may even shine new light on the role of fibrinogen’s binding sites for divalent cations and contribute to an even more comprehensive view of the protein.To characterize pseudo-fibrin’s potential as an actual alternative to fibrin, profound further studies are necessary. These studies should especially include a detailed mechanical analysis, cell viability tests, and mechanistic insights such as possible changes in protein folding via IR measurements. In general, the findings open up a large new field of research, not only for material optimization but also for fundamental research regarding fibrinogen itself. The newly discovered influence of CaFibrinogen from bovine plasma (≥95% protein of which ≥95% is clottable) was purchased from Merck. The protein was not further purified before use. Thrombin (50 U/mg solid) was obtained from Fisher scientific. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  was purchased from Carl Roth. 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride  was bought from Carl Roth. 2-iodoacetamide (purity 98%) was obtained from thermo scientific. All experiments were performed in pure water  by VWR. All other chemicals were of at least 98% purity and purchased from the usual suppliers.To prepare the salt stock solutions, the respective amount of salt was first weighed in and then dissolved in water. If necessary, the pH was adjusted to 7.0 using NaOH or HCl. If not stated differently, all salt stock solutions had a concentration of 500 mmol/L.Fibrinogen solutions with a concentration of 5 g/L were prepared by suspending 20 mg of the protein in 4 mL of 5 °C cold pure water. The pH was adjusted to 7.0 using 0.1 mol/L NaOH. The solution was stirred at 300 rpm for 10 min until all fibrinogen was dissolved.2 microscopy cover slide was used to collect some of the material for scanning electron microscopy. These samples were dried overnight at room temperature and ambient pressure.Fibrinogen self-assembly was triggered by adding 120 μL of the respective 500 mmol/L of salt stock solution. Addition of the salt stock solution has to be performed carefully, i.e., fast but without stirring. This results in a salt concentration of 15 mmol/L, which served as reference in all experiments. For experiments using different salt concentrations, the volume of the added salt solution was adjusted accordingly. Finally, the reaction mixture was stored for 24 h at 5 °C if not stated differently. A 12 × 12 mm2 solution. The initial fibrinogen solution was still prepared at pH 7.0.Experiments using different pH were performed analogously; the required amount of NaOH/HCl to reach the desired pH was however added together with the CaCl2 concentrations, the added volume of the stock solution was adjusted accordingly.For experiments using different CaClA summary of these steps is provided below in A 50 mmol/L HEPES buffer stock solution was prepared by weighing in the required amount of solid, dissolving it in pure water and adjusting the pH to 7.5 with NaOH at 37 °C.2 solution was added to reach the desired Ca2+ concentration . If different concentrations were used, the added volume was adjusted accordingly. Reaction mixtures were allowed to react for 24 h at 37 °C. The total reaction volume was in all cases 1 mL. A fibrin reference was prepared analogously but the CaCl2 was replaced by 5 U/mL thrombin.Preparation of pseudo-fibrin follows similar steps as described in 2 microscopy cover slide was used to collect some of the material for scanning electron microscopy. These samples were dried overnight at room temperature and ambient pressure.After the respective reaction time, a 12 × 12 mmThrombin catalysis was ruled out by adding 0.5 mmol/L of AEBSF to the reaction mixture prior to the respective trigger salt. Fresh stock solutions of 2-iodoacetamide in ultrapure water with a concentration of 1 mmol/L were prepared prior to each respective experiment. Factor XIII catalysis was ruled out by adding 0.1 mmol/L 2-iodoacetamide to the reaction mixture prior to the respective trigger salt. Both inhibitors were incubated for 10 min at 37 °C. Reference experiments, to control whether this reaction time is sufficient, lasted overnight at 37 °C but yielded the same results.2, 24 h at 37 °C). Two of them were weighed after 24 h. Then they were dried overnight at room temperature and weighed again. The remaining two gels were stored in 15 mL/20 mmol/L of HEPES buffer with a pH of 7.5 and containing 140 mmol/L of NaCl at 37 °C, for one day after their production. Afterwards, these washed gels were extracted, weighed, dried overnight, and then weighed again. To calculate the exact masses of the gels, the additional masses of all used salts  were subtracted from the result.Four pseudo-fibrin gels were prepared as described above  and an InLens detector  at an acceleration voltage of 2 kV.Fibrinogen hydrogels were prepared as described earlier. A 12 × 12 mm glass slide was used to extract some of the material. Then the sample was dried overnight at room temperature and ambient pressure. Prior to microscopy, all samples were sputtered with a 3 nm thick layer of Au/Pd alloy.Fiber diameters were determined using GIMP. In each image, five individual fibers were chosen. Their diameter (in pixels) was measured and converted into an actual length.To obtain the first insights into the rheological properties, an Anton Paar MCR 302 was used. All samples were measured with a cone/plate setup. First, the linear viscoelastic regime was identified by means of an amplitude scan. Based on these results, a consecutive frequency sweep was performed at 1% shear deformation. G’ and G’’ were measured from 0.01 Hz–15 Hz. For each measurement, a fresh sample was used.2, 24 h at 37 °C). Fibrin references were prepared analogously but without CaCl2. Instead, clotting was induced by adding 5 U/mL of thrombin. In addition, a reference sample containing only fibrinogen was prepared analogously to the pseudo-fibrin samples but without the addition of any trigger salt or enzyme. All samples were prepared twice to have an undamaged one for the measurements.Pseudo-fibrin hydrogels were prepared as described above (20 mg/mL of protein, 20 mmol/L of HEPES with a pH of 7.5, 100 mmol/L of NaCl, 50 mmol/L of CaCl


Using the popular model cell line HEK-293, we quantified TG-induced changes in cytosolic and ER Ca2+ levels using FURA-2 and the FRET-based ER Ca2+ sensor D1ER, respectively. Our analysis predicts an ER Ca2+ leak of 5–6 µM⋅s−1 for the typical basal ER Ca2+ level of 335–407 µM in HEK-293 cells. The resulting cytosolic Ca2+ transients reached peak amplitudes of 0.6–1.0 µM in the absence of external Ca2+ and were amplified by SOCE that amounted to 28–30 nM⋅s−1 in 1 mM external Ca2+. Additionally, cytosolic Ca2+ transients were shaped by a Ca2+ clearance of 10–13 nM⋅s−1. Using puromycin (PURO), which enhances the ER Ca2+ leak, we show that TG-induced cytosolic Ca2+ transients are directly related to ER Ca2+ levels and to the ER Ca2+ leak. A one-compartment model incorporating ER Ca2+ leak and cytosolic Ca2+ clearance accounted satisfactorily for the basic features of TG-induced Ca2+ transients and underpinned the rule that an increase in amplitude associated with shortening of TG-induced cytosolic Ca2+ transients most likely reflects an increase in ER Ca2+ leak.Thapsigargin (TG) inhibits the sarco/endoplasmic reticulum Ca Along wolutions . To gene+ levels . In gene2+ in an ATP-dependent manner   decreases after the exposure of cells to TG , revealed that this organelle stores Cat manner . Now, itin (BiP) . Notablym the ER . This foeceptors . So far,lmodulin . Other ieceptors . Therefols to TG . When TGm the ER , as remonly SOCE . Thus, Tgnalling .2+]cyt combined with TG to deplete ER Ca2+ made feasible the detection of SOCE in numerous cell types, electrophysiological techniques used to measure Ca2+ currents defined the prototypical store-operated channels, i.e., the Ca2+-release-activated Ca2+ (CRAC) channels that are formed by ORAI proteins in the plasma membrane , which is a structural analogue of t-RNA and, as such, a substrate of the peptidyltransferase of the 60 S subunit of ribosomes. By its incorporation in the nascent peptides, PURO causes the release of incomplete peptides and enhances the Ca2+ leak from ER by promoting a Ca2+-permeable state of Sec61 translocons (2+ leak to the generation of cytosolic Ca2+ transients and generated a model for the TG-induced Ca2+ mobilisation that comprises quantitative data on the Ca2+ leak from the ER and the clearance of cytosolic Ca2+ in HEK-293 cells.While the introduction of FURA-2 to image cyt) was imaged with FURA-2 as previously described /(Rmax - R), in which R = F340/F380 and KFURA-2 represents the system specific apparent Ca2+ dissociation constant for FURA-2  were imaged simultaneously using the FRET-based D1ER sensor and FURA-2, respectively /(Rmax - R), in which R = FCitrine/FCFP and KD1ER represents the system specific apparent Ca2+ dissociation constant for D1ER  with a 63× oil objective  and 488 nm, 543 nm excitation light using Zen  software. Z-stack images were acquired every 500 nm throughout the entire cell to determine the cell, ER and nucleus area and the total RFP fluorescence. Subsequently, images were analyzed and processed with ImageJ (version 1.53t). Before analyzing the images, the background was subtracted using the rolling ball plugin with a radius of 50 pixels. To measure the area of the ER, a threshold was set at 20 times the mean background intensity, so that the outlines of the cells were not visible, allowing the ER area to be quantified.For visualization of the plasma membrane and the ER, HEK-293 cells were incubated with Cell Mask Green Plasma Membrane Stain (Thermo Fisher) with a dilution of 1:2000 and 1 µM ER-Tracker Red (BODIPY TR Glibenclamide) (Thermo Fisher), respectively, for 20 min at 37°C in HBSS containing Mg2+ imaging recordings with 3–12 coverslips per experimental setting. Because of technical reasons, we imaged only 10–15 cells per coverslip in experiments with D1ER and FURA-2, while 30–50 cells per coverslip were imaged with FURA-2 alone. The total number of analysed cells in each experimental setting is given in the figure legends. Data is given as mean ± SEM. Statistical significance  was calculated using the non-parametric two sample Kolmogorov-Smirnov test.Single cell data has been obtained in independent Ca2+ homeostasis  increased in HEK-293 cells depending on the external Ca2+ concentration ([Ca2+]ext), but it generally remained below 100 nM  and [Ca2+]ext (2+ transients (pΔ[Ca2+]cyt) increased by approx. 0.5 µM by increasing external Ca2+ from zero to 1 mM and, again by approx. 0.5 µM from 1 to 10 mM external Ca2+. The area under the curve (AUC) also followed the increases of [Ca2+]ext  induced by TG in HEK-293 cells:entry, Jleak and Jclear denote the Ca2+ influx from the external space (SOCE), the Ca2+ efflux from internal stores (ER Ca2+ leak) and the Ca2+ efflux from cytosol due to clearance mechanisms, respectively. A similar approach has been used to model the Ca2+ dynamics in various cell types  corresponds to the Ca2+ concentration at the time point when SOCE was abruptly interrupted. In order to facilitate the interpretation of our data, we also calculated τclear, the time constant for Ca2+ clearance that is defined as the inverse of kclear. clear and τclear are presented in clear as given in 2+]cyt due to the Ca2+ clearance is around 10–13 nM⋅s−1 for mid values of [Ca2+]cyt (0.35–0.45 µM). This Ca2+ clearance rate is much lower than described for other cell types (2+ clearance mediated by SERCA pumps. The parameters given in 2+ clearance via the plasma membrane of HEK-293 cells.Ca+cyt (0) ll types  and may 2+ transients in HEK-293 cells  scattered between 300 and 500 µM with a mean of approx. 370 μM  that is defined as the inverse of kdepl . This changes in the kleak translated into faster ER Ca2+ depletion in PURO-treated cells compared to controls . Thus, PURO likely enhanced the ER Ca2+ leak from ER, which produced a decrease of ER Ca2+ levels already during the exposure and then accelerated the Ca2+ depletion in ER when the Ca2+ leak was unmasked with TG. As a consequence, the effects on cytosolic TG-induced Ca2+ transients were sharply dependent on the PURO concentration. At the low PURO concentration of 500 μM, we observed an increase of the amplitude of the cytosolic Ca2+ transients, when the increase in kdepl was moderate. At the concentration of 1000 µM PURO, the loss of ER Ca2+ dominated and the cytosolic TG-induced Ca2+ transients became smaller than in control cells.As shown in the plot of [Ca[Ca2+]ER , the incntration . When we[Ca2+]ER , the lincontrols . The tim2+ leak from ER have been performed in the absence of external Ca2+ to avoid SOCE  that we calculated by subtracting b[Ca2+]cyt from [Ca2+]cyt (t). Accordingly, the general equation that models TG-induced cytosolic Ca2+ transients in the absence of external Ca2+ is:However, this equation predicts a zero value for blER values required to model the Ca2+ transients seem to mirror the PURO-induced changes in b[Ca2+]ER , reflecting the minor loss of ER Ca2+ during the PURO treatment. The considerable loss of ER Ca2+ in cells treated with 1000 µM PURO .The Bateman formalism assumes a one-compartment model, in which the ER Catics Eqs . As a prtics Eqs  to reconeriments . In thes93 cells . Interesor kdepl . For com[Ca2+]ER . The b of the Eq. leak ⋅ t) in the tail of the Ca2+ transient, as shown in the breakdown of the Bateman equation (2+ transient in the presence of 500 µM PURO (leak became slightly higher than kclear (30.07 e−3 s−1 vs. 28.34 e−3 s−1). Therefore, it can be hardly distinguished whether the term [exp (-kleak ⋅ t) - exp (-kclear ⋅ t)] converge towards exp (-kleak ⋅ t) or exp (-kclear ⋅ t) in the tail of the Ca2+ transients ) ⋅ (exp (-kleak ⋅ t) - exp (-kclear ⋅ t))] increased with the PURO treatment (leak/(kclear - kleak)] on kleak. Since [kleak/(kclear - kleak)] is not determined when kleak = kclear, the absolute value of [kleak/(kclear - kleak)] increases asymptotically around kleak = kclear, as shown in clear = 28.34 e−3 s−1. Accordingly, the term [(kleak/(kclear - kleak)) · (exp (-kleak ⋅ t) - exp (-kclear ⋅ t))] increases monotonically with kleak, although the term [exp (-kleak ⋅ t) - exp (-kclear ⋅ t)] decreases ] determined the amplitude of the cytosolic Ca2+ transients in these cells. Conversely, the mild loss of ER Ca2+ in cells treated with 500 µM PURO was apparently compensated by the increase in kleak and the term [kleak/(kclear - kleak)] dominated over b[Ca2+]lER in setting the amplitude of the cytosolic Ca2+ transients. Thus, the magnitude of ER Ca2+ loss produced by Ca2+ leak enhancers dictates whether b[Ca2+]lER or [kleak/(kclear - kleak)] determines the amplitude of TG-induced cytosolic Ca2+ transients.The peak amplitude of the Ca µM PURO , while treatment . The latecreases . Hence, a2+ leak . When TG2+ leak can amplify or attenuate the TG-induced cytosolic Ca2+ transients. The reason for this switch from amplification to attenuation of cytosolic Ca2+ transients is that the Ca2+ leak induces a loss of ER Ca2+, which in turns reduces the amplitude of the cytosolic Ca2+ transients. To illustrate the general principle of this phenomenon, we modelled TG-induced cytosolic Ca2+ transients using Eq. leak values between 8 e−3 and 35 e−3 s−1 . Under control conditions, for instance, a kleak in the range of 14 e−3 to 16 e−3 s−1 will generate a Ca2+ leak of 5–6 µM⋅s−1 when the ER Ca2+ levels are around 370 µM. An enhancement of kleak to 21 e−3 s−1 will increase the Ca2+ leak to about 8 µM⋅s−1 when the ER Ca2+ levels remain constant. This implies that at least a 33% increase of the Ca2+ leak is required to modify the TG-induced Ca2+ transients in a way that the duration becomes shorter and the amplitude larger, as shown in HEK-D1ER cells exposed to PURO (2+ leak in HEK-D1ER cells resemble the levels of the Ca2+ leak mediated by ryanodine receptors in cardiac muscle (2+ leak of HEK-293 cells appears to be similar to those of professional secretory cells (In our modelling of Caof kdepl . This imnditions . In ordele terms . Followile terms , our anaa2+ leak . In conta2+ leak . Hence,  to PURO . These rc muscle . When cory cells .2+ dynamics of HEK-293 and HEK-D1ER cells align with published data that has been obtained in Ca2+ imaging studies with various cell types. A one-compartment model satisfactorily explained the basic features of TG-induced Ca2+ transients and supported the rule that shortening of the TG-induced Ca2+ transients accompanied by an increased amplitude likely reflect the enhancement of ER Ca2+ leak (In conclusion, our quantitative data on parameters of the Caa2+ leak .


Methods: Subretinal stim was applied on retinal explants (blind rd1 mouse) using multielectrode arrays (MEAs) or single metal electrodes, and the GC activity was recorded using Ca2+ imaging or MEA, respectively. Stim parameters, including voltage, phase polarity, and frequency, were investigated using specific blockers. Results: At lower stim frequencies (<5 Hz), GCs responded synaptically according to the stim pulses . In contrast, higher stim frequencies (≥5 Hz) also activated GCs directly and induced a rapid GC spike response outage , while in Ca2+ imaging at the same frequencies, increased intracellular Ca2+ levels were observed. Conclusions: Our study elucidated the mechanisms involved in stim-dependent GC spike response outage: sustained high-frequency stim-induced spike outage, accompanied by electrogenically clamped intracellular Ca2+ levels at elevated levels. These findings will guide future studies optimizing stim paradigms for electrical implant applications for interfacing neurons.Background: The electrical stimulation (stim) of retinal neurons enables blind patients to experience limited artificial vision. A rapid response outage of the stimulated ganglion cells (GCs) allows for a low visual sensation rate. Hence, to elucidate the underlying mechanism, we investigated different stim parameters and the role of the neuromodulator calcium (Ca Blinding diseases, such as hereditary retinitis pigmentosa, affect millions of people worldwide ,2,3. TheRegarding performance, artificial vision technology still faces limitations in terms of spatial and, in particular, temporal resolution. For most patients, the visual sensation rate was limited to 0.3 s at a 3 Hz stim frequency; higher stim frequencies led to vision fading ,20,21. Trd1 mouse model for retinal degeneration [2+) responses are modulated by the strength of the applied stim. Hence, in the present study, we thoroughly investigated the role of Ca2+ in shaping GC responses. Different stim parameters were investigated, including voltage (0.5–2.5 V), polarity (anodic- or cathodic-first), the distance of the stim electrode to the reference electrode, and frequencies (various frequencies up to 50 Hz for 90 s). We utilized multimodal methods to record GC responses (Ca2+ imaging and multielectrode array (MEA)) and applied specific synaptic and channel blockers to determine the role of Ca2+ in the modulation of GC responses.In our previous studies investigating the outer retina of a neration , we obse2+ responses and spike trains revealed interesting yet undescribed findings. At high stim frequencies (sustained application), while the GC spike responses were absent, the intracellular Ca2+ levels were remarkably elevated. We concluded that high-frequency stim prevented electrogenically cellular Ca2+ extrusion and thereby clamped intracellular Ca2+ at high levels with probable electrophysiological implications for the further generation of GC spikes during ongoing sustained stim.The temporal correlation of GC CaThese findings are interesting for studies optimizing stim paradigms for artificial vision to counter vision fading and, overall, for electrical implants’ neural interfaces.rd1 (C3H Pde6brd1/rd1) mouse model  for retinal degeneration with primary rod degeneration and secondary cone dystrophy [This study used a blind ystrophy . The ani3, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, and 20 glucose . A pH of 7.4 was maintained by perfusing it with carbogen (95% CO2/5% O2). To minimize the number of animals used, the explanted retina was cut in half, and each half was used for recording. For Ca2+ imaging, the GCs were loaded by applying electroporation with fluorescent Ca2+ dye Oregon Green 488 BAPTA-1 . The electroporation method for bulk loading described by Brigman and Euler [rd1 retina. For loading , 20 monophasic pulses of 7 V with a pulse duration of 100 ms were applied at 1 Hz . After the electroporation process, the retina was incubated in ACSF for 1 h to recover before being transferred to the MEA recording chamber. Prior to recording, the retina rested for an additional 30 min on the MEA electrode field with ACSF perfusion (1 mL/min).After enucleating both eyes, the retinas were isolated in artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 26 NaHCOnd Euler  was adap2+ imaging, or the stim was applied using a single metal electrode , and the GC activity was recorded with an MEA  equipped with a 20 X water immersion objective , a polychromator , and a CCD camera . Image stacks of the OGB-1 fluorescence were acquired at 50 Hz  using VisiView software . For each experiment, an image of the electrodes (MEA and single microelectrode) and the retina was taken to correlate with the spatial distance of the stimulating electrode and responsive GCs. GC spike activity was recorded via the MEA system at 20 kHz raw data sampling. Ca2+ imaging and MEA recordings were both synchronized with the stim using a trigger, which was controlled by the recording protocol set within the MC_Rack software  and the digital input–output box . For each experiment (Ca2+ imaging or MEA), 20 s of control was recorded before stim; then, the application of the stim (+trigger detection) and 20 s of the post-stim period were recorded. When applying stim with an MEA, MEA_select  was used to define the stim and reference electrodes  To investigate the role of Ca2+ in GC response outage, we applied a sustained 90 s stim at various frequencies , using the stim parameters (cathodicnear and −1.6/+1.5 V) established in (a). Here, both Ca2+ imaging and MEA were used to record GC activity.The retina was placed in an MEA system , Reutlingen, Germany) equipped with a 60HexaMEA40/10iR-ITO-pr  consisting of 59 individual electrodes with a diameter of 10 µm in a 40 µm distance and an internal reference electrode. The built-in temperature system of the MEA was set to 32 °C. The mean impedance of the MEA electrodes was 1.0 MΩ . Either the subretinal stim was applied with an MEA and the GC activity was recorded via Ca MEA cf. A,B. The ectrodes C. The st2+ traces were extracted using ImageJ software . The Ca2+ δ-amplitudes were obtained by calculating the difference between the Ca2+ signal baseline (averaged from the pre-stim phase) and the stim-evoked Ca2+ amplitude (identified by the stim trigger mark). The signal decay (τ) was calculated to estimate the refractory time of the GC’s Ca2+ response . The Ca2+ data were normalized against the highest occurring Ca2+ value in the respective dataset. Further data analysis and the production of plots were carried out using custom-developed scripts . For statistical analysis of the drug data, one-way ANOVA followed by Dunnett’s multiple-comparison test was applied, and for the comparison of the frequencies (ctr), the Wilcoxon–Mann–Whitney test was utilized to compare them pairwise with the next higher frequency .Regions of interest were set manually on GCs in the proximity of the stim electrodes, and the corresponding Cawww.inkscape.org (accessed on 27 December 2022) was used to compile the presented figures.For spike extraction, a high-pass filter was applied to the raw data . The filtered files were exported to HDF5 file format with the MC DataManager  and were read in MATLAB using the McsMatlabDataTools  software bundle. Custom-developed MATLAB scripts were used for further data processing . A stati2+ channel (VGCC) blocker) obtained from Sigma-Aldrich ; and 30 TTX  obtained from Carl Roth .Pharmaceutical agents were directly injected into the MEA chamber, and the retina was incubated for 30 min before recording. We used (in µM) 100 L-AP4 , 20 CNQX  obtained from TocrisBioScience ; 100 verapamil  with OGB-1 and recorded  the GC Ca2+ responses elicited by stim. On the one hand, this method allowed us to assess the stim-dependent GC activity with high spatial resolution at the cellular level  by modulating three parameters: (i) the phase of the biphasic pulse: cathodic- or anodic-first .The evaluation of GC Camplitude A determi2+ signal’s decay time τ , which indicates the cell’s recovery time. This parameter is crucial when investigating higher stim frequencies regarding the overlap of following stim responses. The τ value of anodic-first responses was naturally smaller than the cathodic-first because the generated Ca2+ amplitudes were also smaller. However, in the same category of cathodic-first stim, the cathodicnear constellation performed better than cathodicremote . Overall, cathodic-first, particularly cathodicnear, performed best in subretinal stim compared with others, generating the largest Ca2+ amplitudes with much faster τ.We extended our investigation to evaluate the Canear) was selected: optimal amplitude with relatively low decay time .Based on the given data, as an optimal stim pulse for further investigation of the frequency domain, a cathodic-first pulse .Investigating the role of Ca2+ signals (2+ influx) correlating temporally with the applied stim pulse and the following recovery (Ca2+ efflux). While at 0.5 Hz, the Ca2+ signals were clearly separated  at 1.5 Hz , the following Ca2+ signal continued to the end of the recovery phase of its previous signal.Lower stim frequencies (0.5 and 1.5 Hz) generated typical Ca2+ response amplitudes became smaller and eventually started to diminish. Hence, for a better evaluation and understanding, we discriminated between the responses’ initial, middle, and recovery phases , and Ca2+ responses increased with the increase in stim frequency . With progressing stim, in the middle phase at 3 and 5 Hz stim, the observed Ca2+ transients correlated with the stim pulses, but the amplitudes were significantly smaller and “resided” on elevated intracellular Ca2+ levels. These data indicate that Ca2+ responses did not have enough time to recover; hence, the subsequent responses overlapped with the previous ones. The resulting elevated Ca2+ level of the middle phase was approximately aligned with the response amplitude of the very first pulses within the initial phase . At comparatively higher frequencies , no Ca2+ peaks were observed to be temporally correlated with applied pulses, but again, the intracellular Ca2+ was elevated. The established Ca2+ plateau was level with the initial response  and was maintained during the stim period. At all frequencies, the elevated intracellular Ca2+ level recovered immediately post stimulus.Upon higher stim frequencies (>1.5 Hz), the Ca2+-induced Ca2+ release (CICR) events were rarely observed . Hence, Ca2+ traces exhibiting CICRs were identified easily and not considered for stim/amplitude evaluation.Additionally, it is worth mentioning that stim-evoked Caobserved B. CICR e2+ transient (intrusion) and its recovery (extrusion). With the increase in stim frequencies, larger Ca2+ response transients were observed, and the GC Ca2+ responses melted, forming a plateau. The intracellular Ca2+ remained clamped at elevated levels during the stim period and recovered immediately after stim.Overall, the stim frequency shaped the cellular Ca2+ imaging recording.To assess whether the subretinally applied high stim frequencies  activateLower stim frequencies  promoted network-mediated responses (dPhr => BC => GC or BC => GC), which were diminished by ~95% in the presence of the glutamate receptor blockers  despite the blockage of the glutamatergic synaptic pathway . In contrast, the antagonization of VGCC and VGNaC silenced the GC responses entirely , suggesting the direct electrical activation of the GCs via VGC.GC CaOverall, higher stim frequencies penetrated the tissue deeper during subretinal stim. Mainly, the GCs were activated synaptically (~88%) and directly (~12%) over the GCs’ VGCCs and VGNaCs.2+ levels, we correlated GC spike recordings . Lower stim frequencies  elicited GC spike responses and Ca2+ signals in accordance with the respective stimulus pulse  or extrusion was not noticed, establishing a steady elevated Ca2+ plateau (To elucidate the full extent of the observed elevated intracellular Ca2+ levels and (2) does not further promote intracellular Ca2+ intrusion but facilitates the inhibition of the extrusion clamping of intercellular Ca2+ at elevated levels  leads to a rapid GC spike response outage that is accompanied by elevated intercellular Cad levels B, and  What is the correlation between GC spikes and Ca During electrophysiological activity (depolarization), Ca2+ enters GCs via the VGNaC ) and the additional source of Ca2+ intrusion to the VGCC [2+ amplitudes were measured, indicating the opening of more and more channels facilitating the large Ca2+ intrusions. At a stim frequency of 20 Hz, most channels seemed to be open, achieving a maximum response; however, the 50 Hz stim did not yield a significantly more extensive response. After the stim-induced depolarization, the observed Ca2+ signals required a relatively prolonged recovery period of 300 ms to become responsive again. Eventually, shortening the resting time  by increased frequencies  led to plateau-like elevated intracellular Ca2+. In contrast, the refractory period of intrinsically closing VGNaCs roughly lies by half the action potential time (~ 1 ms), in which the balance of ions is maintained, and the neurons become responsive again. To this end, it remains unclear whether the VGNaCs are affected directly or indirectly  by the elevated Ca2+ leading to GC response outage, which leads us to our further questions.the VGCC . With in2+ plateau established?(ii) How is the Ca High-frequency stim produced larger Ca2+ transient signals within the first few pulses, leading to a steady plateau. With ongoing stim, if Ca2+ had entered via VGNaC , spikes should have been generated, but in the MEA recordings, no spikes were observed. Alternatively, if Ca2+ had entered via VGCC, we should have seen Ca2+ transients, but this was not the case. Since the Ca2+ signal did not increase during the ongoing stim, it can be ruled out that additional Ca2+ had entered the cell. It is likely that most of the channels had been opened by the first pulses, establishing the level of the Ca2+ plateau.2+ level not decreasing?(iii) Why is the Ca Ca2+, intruded by electrophysiological actions into cells , is regulated by several Ca2+ extrusion mechanisms [2+ plateau results from an equilibrium of rapid intrusion and extrusion, appearing to be steady. However, we rejected this hypothesis because no spikes (no VGNaC-mediated Ca2+ influx) were observed. A successive Ca2+ influx mediated by VGCC was also excluded according to the null Ca2+ influx rate. Hence, we concluded that the Ca2+ extrusion must have been affected electrogenically by the stim, which clamped the initially intruded Ca2+ in the cell for the remaining stim period. Higher stim frequencies affected the retinal tissue in depth, stimulating GCs directly (verified by synaptic blockers). First, the electric field excited the GCs but then inhibited the extrusion mechanism electrogenically as a side effect.chanisms ,34. Henc2+ extrusion candidates that could be electrogenically affected by the electric field [2+ exchanger (NCX)) or channels (voltage-gated and Ca2+-modulated potassium (K+) channels) [2+ buffering caused by cytosolic Ca2+-binding protein [2+ pumps  ,38,39. O protein  stores ( protein ) or ATP-s  is unlis  How does a response outage occur? The aforementioned discussion of the evaluated data established that the elevated Ca2+ is not extruded during continuous ongoing high-frequency stim. The extrusion is obviously extremely slowed down or nearly blocked. Given that high stimulation frequency can lead to greater cell depolarization [2+-dominated positive potential. Consequently, it is conceivable that the elevated Ca2+ levels impact the regeneration of the resting membrane potential and thereby the generation of further action potentials, resulting in a response outage (no spikes in MEA recordings). The immediate drop in the Ca2+ levels after stim (at high and low frequencies) allows for the recovery and regeneration of action potentials. Hence, we concluded that high-frequency stim (≥5 Hz) electrogenically prevented cellular Ca2+ extrusion and clamped the intracellular Ca2+ levels at remarkably high levels, with strong electrophysiological implications for the further generation of GC spikes. This electrogenic blockage might also affect dendrites and synapses throughout the retinal neuronal network [2+ levels—the inhibitory pathway is probably equally stim. In addition, Ca2+, Na+, and K+ play a significant role in the maintenance of electrophysiological resting membrane potential [rization , the up  network . A naturotential ,44,45. T(v) What is the difference in outcome between high-rate stimulations from our study and those of others? Comparing previous findings, Weiland, Walston, and Humayun (2016) [2+ data (2+ transient (intrusion) was observed for the very first stimulation pulses despite the frequency . Within an earlier study, Sekirnjak et al. (2006) [2+ recovery and the regeneration of spike activity.n (2016)  have conn (2016) , they in[2+ data , <500 ms. (2006)  reported2+ imaging recordings led us to the most interesting observation: as long as the Ca2+ levels were elevated within the GCs, no further Ca2+ transients were observed in Ca2+ data, and a GC spike response outage was observed in MEA data.Overall, previous findings do not contradict our findings; however, our approach goes beyond the scope of previous investigations in terms of stimulation duration (90 s), stimulation frequency, sustained application, and the discrimination of different stimulation phases . The use of Ca2+ imaging recordings of GCs in conjunction with different pharmaceuticals unequivocally revealed that the applied stim (voltage and frequency) did not electroporate the retinal cells; the GC activity was based on neurotransmitter transmission or voltage-sensitive channels.The presented Ca2+ imaging allowed us to conclude that not all GCs responded to the same stim and not all applied voltages produced GC responses. These findings might guide efforts investigating the differential activation of GCs, for example, as established by Baden et al. (2016) [2+ imaging [The cellular recording resolution of Ca. (2016)  for ligh imaging ,48,49 fo imaging ,50,51,522+ imaging. Thus, our study enriches research in this field by highlighting the role of ions, particularly Ca2+, in shaping physiological responses.Seeking stimulation paradigms to drive the different retinal channels independently is the ultimate goal of artificial vision research. Therefore, different parameters are applied in variations to obtain new insights. In schemes of subretinal stimulations (outer retina), Jensen and Rizzo 2009)  suggeste  suggest2+ to recover  , Li et a2+ baseline due to successive high-frequency stim could also cause secondary neuronal damage by activating Ca2+-triggered cell degenerative pathways [In addition to the electrophysiological impact, the response outage and sustained elevated Capathways .2+ in GC response outages that are dependent on stim frequency. The newly established protocol, stimulating blind mouse retinas in a subretinal configuration and recording GC responses via Ca2+ imaging and MEA, allowed us to elucidate the Ca2+ dynamics and determine the underlying mechanism.Our study highlights the paramount role of the neuromodulator Ca2+ intrusion. On the other end, the disruption of Ca2+ extrusion was found to be responsible for the GC response outage.The combination of stim parameters  influenced the response strength and stim depth, thereby regulating cellular Ca2+ responses and spike trains revealed that, at high-frequency stim (>3 Hz sustained application), a GC spike response outage occurred, while at the same time, the intracellular Ca2+ level was remarkably elevated. We concluded that high-frequency stim prevented electrogenically cellular Ca2+ extrusion and thereby clamped the intracellular Ca2+ at high levels, in turn contributing to the electrophysiological GC spike outage for the remaining duration of the sustained stim.The temporal correlation of GC CaThese findings are of profound interest for studies optimizing electrical stimulation paradigms for artificial vision to prevent vision fading and, overall, for applications of electrical neural interfacing.


Here, we aimed to investigate ERα and ERβ subtypes in the regulation of E2-modulated colonic epithelial HCO3− and Cl− secretion and epithelial restitution. Through physiological and biochemical studies, in combination of genetic knockdown, we showed that ERα attenuated female colonic Cl− secretion but promoted Ca2+-dependent HCO3− secretion via store-operated calcium entry (SOCE) mechanism in mice. However, ERβ attenuated HCO3− secretion by inhibiting Ca2+via the SOCE and inhibiting cAMP via protein kinases. Moreover, ERα but not ERβ promoted epithelial cell restitution via SOCE/Ca2+ signaling. ERα also enhanced cyclin D1, proliferating cell nuclear antigen, and β-catenin expression in normal human colonic epithelial cells. All ERα-mediated biological effects could be attenuated by its selective antagonist and genetic knockdown. Finally, both ERα and ERβ were expressed in human colonic epithelial cells and mouse colonic tissues. We therefore conclude that E2 modulates complex colonic epithelial HCO3− and Cl− secretion via ER subtype-dependent mechanisms and that ERα is specifically responsible for colonic epithelial regeneration. This study provides novel insights into the molecular mechanisms of how ERα and ERβ subtypes orchestrate functional homeostasis of normal colonic epithelial cells.Although it was described previously for estrogen (E Intestinal epithelium either absorbs electrolytes or secretes anions  , etc.3− and Cl− secretion), which in turn is important for nutrient absorption and mucosal protection  in murine duodenal epithelium (2) may protect human duodenum against the acid-induced injury by mediating duodenal HCO3− secretion likely via ER activation, which explains the lower incidence of duodenal ulcer in women than age-matched men  may cause not only imbalance of pH values and electrolytes in whole body but also local disruption of colonic environments, such as epithelial barrier and microbiome  and ERα selective activator propyl pyrazole triol  stimulated rapid duodenal HCO3− secretion; however, ERβ selective activator diarylpropionitrile  did not (A). Second, we tested if there is any regional heterogeneity for the ER subtype-mediated HCO3− secretion in between the duodenum and distal colon. Like in the duodenum, both E2 and PPT stimulated colonic HCO3− secretion but DPN alone did not (B). Moreover, compared to PPT alone, PPT plus DPN did not further stimulate additional colonic HCO3− secretion (B). Thus, ERα activation stimulated HCO3− secretion from the duodenum and distal colon, which has no regional heterogeneity in GI tract. In the subsequent experiments, distal colonic HCO3− secretion was focused because duodenal HCO3− secretion has been extensively studied - or forskolin -stimulated HCO3− secretion. As shown in A and B, the selective ERα activator PPT (10 nM) pretreatment did not affect CCh (100 μM)- or forskolin (20 μM)-induced colonic HCO3− secretion, indicating that ERα-stimulated HCO3− secretion did not utilize the same signaling pathways as CCh or forskolin. In contrast, although the selective ERβ activator DPN (10 nM) did not alter basal colonic HCO3− secretion, it almost abolished both CCh (100 μM)- and forskolin (20 μM)-induced colonic HCO3− secretion (C and D), suggesting that ERβ activation may alter Ca2+- and cAMP-mediated colonic HCO3- secretion.It is well-known that Caecretion , 20, 21.ectively , we examecretion , C and D3− secretion, we further elucidated the underlying mechanisms. Since protein kinase, such as tyrosine kinase (TK), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) pathways play critical roles in intestinal HCO3− secretion , a commonly used TK inhibitor, inhibited basal HCO3− secretion, suggesting its role in basal HCO3− secretion, consistently with the report by Osamu Furukawa et al (3- secretion (E and F), suggesting that ERβ activation inhibits Ca2+- and cAMP-mediated colonic HCO3− secretion likely via TK pathway. Although genistein may also activate ER , suggesting its role in regulating basal HCO3− secretion, which is consistent with the report by Odes HS et al (3− secretion (B and D). Neither rottlerin nor wortmannin affected DPN inhibition of CCh-induced male colonic HCO3− secretion (A and B). In contrast to CCh-induced colonic HCO3− secretion, both rottlerin and wortmannin reversed DPN inhibition of forskolin-induced colonic HCO3− secretion (C and D). These data suggest that ERβ activation inhibits cAMP-mediated colonic HCO3− secretion via PKC and PI3K pathways, which are not involved in ERβ inhibition of Ca2+-mediated HCO3− secretion.Second, rottlerin (10 μM), a selective PKC inhibitor , attenuaecretion , A and CHS et al . HoweverHS et al  did not ecretion , B and Decretion , A and Becretion , C and D3− secretion, we examined their roles in colonic Cl− secretion that is poorly understood although it is known about E2 inhibition of Cl− secretion in rat distal colonic epithelium with a gender-specific mechanism (2 (1 μM), PPT (500 nM), and DPN (500 nM) at high concentrations affected basal colonic short-circuit current (Isc) in both sexes (A and B). Moreover, none of them affected CCh- and forskolin-induced male colonic Isc (C and D), consistently with the previous reports that E2 did not alter colonic epithelial Cl− secretion in male rats  significantly inhibited CCh- and forskolin-induced Isc of female mouse colon, which is consistent with the previous reports that E2 inhibited colonic epithelial Cl− secretion through KCNQ1 channels in female rats . Taken together, ERα subtype is specifically responsible for the inhibition of female colonic Isc.We further examined the role of ER subtypes in female colonic Clale rats , 15, 28.se colon , G and H2+ signaling triggers intestinal HCO3− secretion  plays a pivotal role in controlling Ca2+ signaling. Initially, we applied single cell Ca2+ imaging to characterize the SOCE in human colonic epithelial cell line (HCoEpiC). Cyclopiazonic acid , an inhibitor of the sarcoendoplasmic reticulum Ca2+ ATPase and a commonly used SOCE activator, significantly induced free cytoplasmic Ca2+ [Ca2+]cyt in Ca2+-free solutions and then a sustained [Ca2+]cyt increase in Ca2+-containing solutions, suggesting that intracellular Ca2+ release causes extracellular Ca2+ influx in HCoEpiC  (A). Pretreatment with three different SOCE blockers, GSK7975A (10 μM) (B–J), verifying SOCE function in HCoEpiC.It is well documented that Caecretion , 20, 29,e, SOCE) A. Pretre (10 μM) , ML-9 (1 (10 μM) , and SKF (10 μM) , markedl (10 μM) , B–J, ve2+ transient produced by CPA in Ca2+-free solutions is presumably Ca2+ release from stores, it was reduced by pretreatment with SOCE blockers . We assume it is because the SOCE is important to refill the intracellular Ca2+ store, and Ca2+ release from stores would be reduced by pretreatment with SOCE blockers. To test this possibility, we added SOCE blockers after the store depletion but before extracellular Ca2+ influx. Indeed, SKF96365 (50 μM) markedly reduced CPA-induced Ca2+ influx (K–M). These data verify not only SOCE function per se but also its important role in refilling intracellular Ca2+ store in HCoEpiC.Although the initial Ca+ influx , K–M. Th2+ signaling. ERα selective activator PPT (10 nM) significantly stimulated [Ca2+]cyt in Ca2+-containing solutions (A and C), but ERβ selective activator DPN (10–500 nM) did not alter basal [Ca2+]cyt (B and C). However, DPN (10 nM) significantly inhibited CPA (5 μM)-induced SOCE (D–G). Together, these data suggest that ERα triggers Ca2+ signaling, but ERβ inhibits Ca2+ signaling likely via the SOCE.After characterizing the SOCE in HCoEpiC, we next examined whether ER stimulates colonic epithelial Caolutions , A and CCa2+]cyt , B and Cced SOCE , D–G. To2+ signaling in HCoEpiC. Like SOCE inducer CPA, PPT (10 nM) significantly stimulated [Ca2+]cyt in Ca2+-free solutions and then a sustained [Ca2+]cyt increase in Ca2+-containing solutions (A). Further studies showed that either MPP dihydrochloride (MPP) at 5 to 10 μM (B–E), a ERα selective inhibitor, or ML-9 (100 μM) (F–H) and SKF96365 (50 μM) (I–K), two different inhibitors of the SOCE, markedly eliminated PPT-induced Ca2+ release and Ca2+ influx, suggesting that ERα activation triggers [Ca2+]cytvia the SOCE. Moreover, SKF96365 (50 μM) markedly reduced PPT-induced Ca2+ entry when it was added after the store depletion but before extracellular Ca2+ entry (L–N). Finally, after shERα was applied to successfully knock down the protein expression of ERα in HCoEpiC (O), PPT-induced [Ca2+]cyt was significantly attenuated by shERα-3 as well (P–S), verifying ERα activation of the SOCE in colonic epithelial cells.We further elucidated the underlying mechanisms of ERα-induced Caolutions A. Furtheto 10 μM , B–E, a (100 μM) , F–H and (50 μM) , I–K, tw2+ entry , L–N. Fi HCoEpiC O, PPT-in as well , P–S, veA), which was attenuated by ERα selective inhibitor MPP  and shERα , but not by ERβ selective inhibitor PHTPP (5 μM) . However, DPN at 1 to 50 nM did not affect proliferation of HCoEpiC . Finally, [Ca2+]cyt chelator BAPTA-AM (1 μM) also inhibited PPT-induced cell proliferation . MPP (1 μM), PHTPP (5 μM), and BAPTA-AM (1 μM) were chosen at these concentrations because they per se did not directly alter HCoEpiC proliferation .Since proliferation and migration of epithelial cells are critical for the restitution of injured intestinal epithelium , 34, we Fig. 8I, the pretreatment with PPT (10 nM) for 48 h enhanced the expression of cyclin D1, PCNA, and β-catenin. However, DPN (10 nM) did not affect their expression . Therefore, ERα promotes Ca2+-dependent proliferation of HCoEpiC.Since cyclin D1, proliferating cell nuclear antigen (PCNA), and β-catenin play crucial roles in enterocyte proliferation , 36, we Fig. 8A and B, PPT at 10 to 50 nM promoted cell migration, but DPN (10–50 nM) did not affect it. Moreover, ERα selective inhibitor MPP (1 μM) and BAPTA-AM (1 μM) and shERα abolished PPT-induced cell migration (C–E). Therefore, ERα also promotes Ca2+-dependent migration of HCoEpiC.Next, we performed cell scratch assays to examine the roles of ER subtypes in HCoEpiC migration. As shown in igration , C–E. ThA and B, both ERα and ERβ mRNA expression were detected in HCoEpiC. Western blots analysis further confirmed ERα and ERβ protein expression in HCoEpiC like in HUVEC (C and D). Moreover, we also performed immunofluorescence to examine the expression and localization of ER subtypes in HCoEpiC. As shown in Figure 10E, ERα proteins were predominately expressed in the cytoplasm and nucleus, but ERβ were mostly expressed in the cytoplasm compared to the nucleus. However, the immunofluorescence staining was not observed without the primary antibodies against ERα and ERβ in the negative control, indicating specific staining on these proteins in HCoEpiC.Since there is no information available in literature on ER subtype expression in HCoEpiC, we examined ER subtype expression in HCoEpiC and human umbilical vein endothelial cells (HUVECs) as a positive control , 38. As in HUVEC , C and DF and G, both proteins were expressed in native colonic epithelia in male and female mice, further supporting both ERα and ERβ expression in mice  ERβ attenuates E2-mediated HCO3− secretion by inhibiting SOCE/Ca2+ signaling and by inhibiting cAMP signaling via activation of TK, PKC, and PI3K, (4) Erα but not Erβ promotes E2-mediated epithelial cell restitution via the SOCE, and (5) ERα enhances the expression of cyclin D1, PCNA, and β-catenin in HCoEpiC.Although sex-based differences in Eescribed , 15, 16,2 stimulated murine duodenal HCO3− secretion  to exert various biological actions through genomic and nongenomic pathways  without triggering excessive Cl− secretion that might induce the unwanted diarrheal side effects.We previously showed that activation of the Cadecrease ; the endignaling , 19. Det2+-dependent cell proliferation and migration, leading to colonic epithelial restitution; a process that may be beneficial to colonic disease like UC. Moreover, we found that activation of ERα but not ERβ increases protein levels of cyclin D1, PCNA, and β-catenin, indicating increased proliferation and migration.While epithelial restitution plays a critical role in the healing process of intestinal mucosa , little 2 modulates colonic epithelial ion transports and epithelial restitution via ER subtype-dependent mechanisms. Since ERα and ERβ subtypes usually play opposite roles in regulating colonic epithelial function, E2 may coordinate different ER subtypes to orchestrate functional homeostasis of colonic epithelial cells. Disruption of ERα- and ERβ-coordinated gut homeostasis may have underpinnings behind sex-based differences in GI disease, especially those involving epithelial injury and repair. While this requires further study, we have provided new insights into the cellular mechanisms of E2-mediated colonic epithelial ion transports and epithelial repair via different ER subtypes.In conclusion, we demonstrate for the first time that E2.Human colonic epithelial cells (HCoEpiC) (Cat #2950) and HUVEC (Cat #8000) were purchased from ScienCell Research Laboratory. Cells were cultured in DMEM-HIGH GLUCOSE or RPMI-1640 (HyClone) supplemented with 10% fetal bovine serum (HyClone) in a 37 °C humidified atmosphere containing 5% COAll animal studies were approved by the Ethics Committee of the Qingdao University Medical College. All animal care and experimental procedures complied with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. Animal studies are reported in compliance with the ARRIVE guidelines . The C572). The experiments were performed under continuous short-circuited conditions , and luminal pH was maintained at 7.0 by the continuous infusion of 5 mm HCl under the automatic control of a pH-stat system . The volume of the titrant infused per unit time was used to quantitate HCO3− secretion. Measurements were recorded at 5-min intervals, and mean values for consecutive 5- or 10-min periods were averaged. The rate of luminal HCO3− secretion is expressed as micromoles per square centimeter per hour. The transepithelial Iscs were measured via an automatic voltage clamp, in which μA was used for the original recordings, but μA·cm-2 was used for summary data. After a 30 min basal period, inhibitor or control vehicle was added for another 30 min, followed by addition of stimulus to both sides of the tissue. Electrophysiological parameters and HCO3− secretion were then recorded for 60 min. The mucosal solution contained the following (mM): 115 NaCl, 25 sodium-D-gluconate, 5.2 potassium-D-gluconate, 1.2 CaCl2, 1.2 MgCl2, and 10 mannitol. The serosal solution contained the following (mM): 115 NaCl, 25 NaHCO3, 2.2 K2HPO4, 1.2 CaCl2, 1.2 MgCl2, 0.8 KH2PO4, 10 glucose, and 0.01 indomethacin. The osmolalities for both solutions were ∼300 mosmol·kg-1 of H2O.Ussing chamber experiments were performed as previously described , 53. C5750 and the data from others’ reports.The maximal volumes of concentrated stock solutions of all compounds added to 3 ml Ussing chamber solutions were less than 30 μl. All added compounds did not alter pH values in chamber solutions, which is consistent with other report . The con2+]cyt imaging experiments were performed as previously described . Fluorescence signals were imaged using an intensified CCD camera (ICCD200) attached to an inverted fluorescence microscope (Leica) and recorded with MetaFluor software . Images were acquired every 3 s. The dual wavelength excitation method for the measurement of fura-2 fluorescence was used. The excitation wavelengths were 340 and 380 nm, and the emitted fluorescence was collected at 510 nm. [Ca2+]cyt was presented as fluorescence ratios (F340/F380) after background subtraction. The PSS contained the following: 140 mM Na+, 5 mM K+, 2 mM Ca2+, 147 mM Cl−, 10 mM Hepes and 10 mM glucose (pH 7.4). The 0 Ca2+ solution (0 Ca2+) contained the following: 140 mM Na+, 5 mM K+, 145 mM Cl−, 0.5 mM EGTA, 10 mM Hepes, and 10 mM glucose (pH 7.4). The osmolality for the solution was ∼300 mosmol.kg−1 of H2O.[Caescribed . BrieflyWestern blotting was performed as previously described , 54. BriQuantitative real-time PCR was performed as previously described , 55. BriERα: 5′- GGGAAGTATGGCTATGGAATCTG-3′ (Forward),ERα: 5′- TGGCTGGACACATATAGTCGTT-3′ (Reverse).ERβ: 5′-AGCACGGCTCCATATACATACC-3′ (Forward),ERβ: 5′- TGGACCACTAAAGGAGAAAGGT-3′ (Reverse).GAPDH: 5′- ACAACTTTGGTATCGTGGAAGG-3′ (Forward),GAPDH: 5′- GCCATCACGCCACAGTTTC-3′ (Reverse).After fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, and blocked with bovine serum albumin, the HCoEpiC were incubated with anti-ERα (1:100)  or anti-ERβ (1:200)  antibody overnight at 4 °C. Then, the cells were incubated with Cy3 labeled anti-rabbit secondary antibody . Finally, nuclei were stained with DAPI for 5 min, and images were captured using confocal microscope.Cell proliferation assay was performed as previously described . BrieflyLentiviruses were purchased from Sangon Biotech Co, Ltd. The sequences for ERα shRNA and NC were as follows: shRNA-1 (5′-TTGTGTGCCTCAAATCTATTA-3′), shRNA-2 (5′-AGGCCAAATTCAGATAATCGA-3′), shRNA-3 (5′- CAGGTCCACCTTCTAGAATGT-3′), shNC (5′- TTCTCCGAACGTGTCACGT -3′). HCoEpiC were infected with lentiviruses according to the protocol of the manufacturer.2, forskolin, genistein, rottlerin, wortmannin, GSK-7975A, ML-9, SKF-96365, CPA, and BAPTA-AM were purchased from MedChemExpress and dissolved in DMSO. CCh and indomethacin were purchased from Sigma-Aldrich. CCh was dissolved in ultrapure water. Indomethacin was dissolved in anhydrous alcohol. All salts were supplied by Sangon Biotech and dissolved in ultrapure water.PPT, DPN, MPP, PHTPP, ESCR_002798, USA) software was used for analysis and graph generation. All results shown are means ± SD. All experiments were repeated at least three times. The number of biological repeats (n) in the figures is the number of individual tissues or cells obtained from at least three mice or three independent experiments. The statistical significance of differences in the means of experimental groups was determined using Student's t test or one-way ANOVA followed by post hoc test for multiple pairwise comparisons. Significant differences (∗p < 0.05) are expressed in the figures and figure legends.GraphPad Prism 7.0 (RRID: The data supporting the findings of this study are available within the article.The authors declare no conflicts of interest with the content of this article.


The microscope is one of the most important tools in neuroscience, which aims to reveal the complex intercellular communications underlying brain function and pathology. Many aspects of astrocytes, including the structures of their fine processes and physiological activities in concert with neurons and blood vessels, were revealed in the course of innovations in modern microscopy. The evolution of modern microscopy is a consequence of breakthroughs in spatiotemporal resolutions and expansions in molecular and physiological targets due to the progress in optics and information technology, as well as the inventions of probes using organic chemistry and molecular biology. This review overviews the modern microscopic approach to astrocytes.Microscopy started as the histological analysis based on intrinsic optical properties of tissues such as the refractive index and light absorption, and is expanding to include the visualization of organelles by chemical staining, localization of molecules by immunostaining, physiological measurements such as Ca Microscopy in life sciences addresses structures in living organisms and distributions of biomolecules, as well as measures and manipulates physiological phenomena. The astrocyte polarity between neurons and blood vessels, and gliotransmission, would not have been revealed without the innovations in modern microscopy. Modern microscopic approaches to astrocytes are challenges of optical visualization and manipulation of intricate astrocytes deep in the brain tissue, and their progress is attributed to breakthroughs in optics, information technology, chemistry, and molecular biology. This review overviews astrocyte research from a microscopic perspective and discusses future prospects.The history of optics can be traced back to ancient times. The word “lens” was derived from a Mesopotamian bean, lentil , and convex lenses made of polished quartz crystal have been found in ancient Egyptian ruins, indicating that lenses were used as magnifiers or igniters in ancient times. The scientific revolution in the 17th century proposed in “The Origin of Modern Science (1949)” by Herbert Butterfield is closely associated with the dramatic advances in optics. The astronomical telescope, which was created during this period, brought about the geocentric view of the universe, and the microscope brought the cell-based view of living creatures. The compound microscope, consisting of objective and ocular lens, was invented by Hans and Zacharias Janssen in 1590, and their observation of plant tissue gave rise to the term “cell” . Early microscopic observations were based on the contrast due to the light absorption of dense organelles or the light scattering by the cell wall or membrane. Eventually, the staining methods using chemicals that accumulate in organelles were developed to delineate the distribution and structure of neurons. The advance of microscopy to the discovery of astrocytes is described in detail in a review by Chvatal et al. . This diIn the 20th century, new microscopic techniques such as electron microscopy and fluorescence microscopy were invented and revealed the common aspects of astrocytes including polarity and domain structure . An astrAstrocytic processes are widespread nanoscale structures which are difficult to identify with light microscopy and were also difficult to be imaged in their entirety by electron microscopy for a long time after their discovery. However, the invention of serial block-face scanning electron microscopy (SBF-SEM) is revealing the full structure of astrocytic processes . SBF-SEM2+ imaging and superimposing Ca2+ elevations with STED images have shown the nodes as starting points of Ca2+ elevations. Since STED microscopy requires long dwell times during scanning for obtaining high resolution images, it is not suitable for imaging rapid changes of PAPs. Thus, the dynamics of PAP structure has been quantitatively analyzed by FRAP, which measures the time course of fluorescence recovery after intense light irradiation for bleaching fluorescent molecules. FRAP quantifies the diffusion process of fluorescent molecules, thus the recovery delays in relation to volume reduction. The regression of PAPs in association with synaptic plasticity has been demonstrated by the delay of FRAP measured from astrocyte processes filled with fluorescent dye following LTP induction and glutamate spillover in brain slice [The function of astrocytes cannot be fully understood without the analysis of structures and motility of unfixed astrocytic processes. However, PAPs are nanoscale structures, which is difficult to visualize by conventional light microscopy, due to diffraction limits. The first imaging of unfixed PAPs has been achieved by stimulated emission depletion (STED) microscopy , a type in slice .The distribution of astrocytes in brain tissue has been studied for understanding how these cells fill the brain tissue as neuroglia and form syncytium, which is defined as a cell cluster connected intracellularly by gap junctions. Dye injection into adjacent astrocytes has shown that these cells occupy an exclusive domain in the brain , but the2+ plays crucial roles in the regulation of diverse cellular functions, and intracellular Ca2+ elevation can be evaluated by electrophysiological measurement of calcium current, aequorin bioluminescence, calcium-dependent gene regulation, and fluorescence Ca2+ indicator. A series of fluorescent Ca2+ indicators such as Fura2 and Fluo4 have been developed by introducing the structure of Ca2+ chelator into fluorophore and the acetoxymethyl modification at their carboxylic acid residues in the chelating site has allowed irreversible cell loading [2+ imaging using these indicators with digital video cameras has revolutionized astrocyte research [2+ elevation in a glioma-derived cell line following serotonin treatment [2+ elevation [2+ elevations associated with neural activities and blood flow; however, their consequences still largely remain to be determined [Ca loading . Ca2+ imresearch . Startinreatment , it has levation . These ftermined .2+ indicators change fluorescence intensity and others change excitation or emission spectrum depending on Ca2+ concentration [2+ by time-lapse imaging using conventional image sensors, but their data do not directly reflect absolute Ca2+ concentration. In contrast, the latter represented by Fura2 and Cameleon can provide data reflecting absolute Ca2+ concentration by ratio imaging, which requires switching the excitation wavelengths or dual imaging after splitting fluorescence. However, ratio imaging is limited for high resolution microscopy, such as confocal laser scanning microscopy (CLSM) and two-photon excitation microscopy (2PM), because the focal position depends on wavelength. Thus, the invention of fluorescence lifetime imaging microscopy (FLIM) drastically changed the quantitative accuracy of Ca2+ imaging [2+ indicators reflect absolute Ca2+ concentration. Thus, FLIM can measure absolute Ca2+ concentration by using existing fluorescent Ca2+ indicators and the results are not affected by the intracellular concentration and the fading of indicators. The fluorescence lifetime is also not affected by sample movement, because it is nanosecond order and measured in each pixel independently. In addition, the efficient calculation of absolute Ca2+ concentration and high-speed imaging at over 100 fps have been achieved by measuring fluorescence in time gates synchronized with pulsed lasers [2+ concentration has been achieved by using two photon lasers for FLIM (2P-FLIM) [2+ concentrations in slice, as well as in vivo preparations [Some fluorescent Cantration . The for imaging . Since fd lasers . Further2P-FLIM) . 2P-FLIMarations  . Early Caesponses  as well levation , Ca2+ osllations , and Ca22+ waves  in cultutivities . Furthermulation . Becausetrocytes . Currente matter   on the cell body and stem process [2+ imaging; however, the Ca2+ elevation in PAPs is also not reported by this methodology, presumably due to insufficient fluorescence [2+ imaging of PAPs has been achieved not by microscope technology, but by membrane-anchored genetically encoded Ca2+ indicator (GECI), which distributes proportionally to the cell membrane. The fluorescence of PAPs was comparable to that of the cell body and stem processes, and unique Ca2+ dynamics of PAPs were characterized in two- photon imaging of in vivo astrocytes expressing membrane-anchored GECI [2+ release from endoplasmic reticulum, but showed microdomain Ca2+ transients, which is a Ca2+ efflux through the mitochondrial permeability transient pore. Future imaging of plasma membrane-bound GECI using 2P-FLIM will determine resting concentration and dynamics of Ca2+ in in vivo PAPs.Since PAPs are a nanoscale structure, they are rarely loaded with sufficient fluorescent Ca process , presumarescence . Ca2+ imred GECI . PAPs la2+ elevations in these regions are associated with psychiatric disorders [2+ imaging of the internal brain structures of freely moving animals. For imaging the internal brain structures, the cerebral cortex is removed for direct access of conventional objective [2+ imaging of large 3D-FOV in the internal brain structure [2+ imaging of the internal brain region of freely behaving mice [Internal brain structures such as the hippocampus, basal ganglia, and medial prefrontal cortex, which cannot be directly visualized through cranial windows, have functional association with behavior. It was shown that the astrocytic Caisorders ,39,40 bybjective , or lesstructure . Furthering mice . Head-moing mice  or with ing mice  have bee2+ imaging was first reported by using a transgenic mouse line expressing high levels of GECI both in neurons and astrocytes and temporal skull clearing with a mixture of paraffin oil and glycerol [2+ elevation following transcranial direct current stimulation to astrocytes by using both wide-field Ca2+ imaging at the tissue level and 2PM Ca2+ imaging at the cellular level. The recent progresses of wide-field Ca2+ imaging include the stable skull transparency by clearing with cyanoacrylate glue allowing long-term analysis and the optical compensation of hemoglobin absorbance [Transcranial wide-field Caglycerol . This pasorbance ,47, whicsorbance ,49.2+ elevations using caged compounds [2+ or caged IP3 is uncaged by brief UV flashes [The optical manipulations of astrocytic Caompounds ,51 or byompounds ,53 are t flashes  or local flashes ,53. Scan flashes ,or local flashes  or compu flashes . The DMD flashes . Further flashes . On the  flashes . Both 3D flashes  and has  flashes . The proAstrocytes are widely known to play crucial roles in regulating the brain environment by exchanging nutrients, waste, ions, neurotransmitters, and water. As a component of the blood–brain barrier, astrocytes are a major pathway for uptaking glucose and other metabolic substrates from the bloodstream to brain parenchyma, and synthesizing lipids from glucose for providing building blocks for neuronal processes and myelin . Activit2+ imaging-based fluorescent adenosine biosensor [2+ [Since glutamate transporters are electrogenic, the astrocytic glutamate uptake in hippocampal slices has been imaged by using voltage sensitive dye and photodiode array . Similariosensor  have suciosensor . In ordensor [2+ ; howevernsor [2+ .2O (water) is distinct from the O-D vibration in D2O (heavy water), the water dynamics have been visualized by using heavy water as a tracer in cell culture [The autofluorescence imaging of NAD(P)H in slices or in vivo by epifluorescence microscopy revealed the real-time influences of neural activities on the astrocytic energy metabolism . Other n culture  and tiss culture . Thus, tModern microscopic approaches discussed in this review are summarized in