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Sensory processing is essential for motor control. Climbing fibers from the inferior olive transmit sensory signals to Purkinje cells, but how the signals are represented in the cerebellar cortex remains elusive. To examine the olivocerebellar organization of the mouse brain, we perform quantitative Ca 2+ imaging to measure complex spikes (CSs) evoked by climbing fiber inputs over the entire dorsal surface of the cerebellum simultaneously. The surface is divided into approximately 200 segments, each composed of ∼100 Purkinje cells that fire CSs synchronously. Our in vivo imaging reveals that, although stimulation of four limb muscles individually elicits similar global CS responses across nearly all segments, the timing and location of a stimulus are derived by Bayesian inference from coordinated activation and inactivation of multiple segments on a single trial basis. We propose that the cerebellum performs segment-based, distributed-population coding that represents the conditional probability of sensory events., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Stimulus-induced changes in the intracellular Ca(2+) concentration control cell fate decision, including apoptosis. However, the precise patterns of the cytosolic Ca(2+) signals that are associated with apoptotic induction remain unknown. We have developed a novel genetically encoded sensor of activated caspase-3 that can be applied in combination with a genetically encoded sensor of the Ca(2+) concentration and have established a dual imaging system that enables the imaging of both cytosolic Ca(2+) signals and caspase-3 activation, which is an indicator of apoptosis, in the same cell. Using this system, we identified differences in the cytosolic Ca(2+) signals of apoptotic and surviving DT40 B lymphocytes after B cell receptor (BCR) stimulation. In surviving cells, BCR stimulation evoked larger initial Ca(2+) spikes followed by a larger sustained elevation of the Ca(2+) concentration than those in apoptotic cells; BCR stimulation also resulted in repetitive transient Ca(2+) spikes, which were mediated by the influx of Ca(2+) from the extracellular space. Our results indicate that the observation of both Ca(2+) signals and cells fate in same cell is crucial to gain an accurate understanding of the function of intracellular Ca(2+) signals in apoptotic induction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Monitoring the pattern of intracellular Ca(2+) signals that control many diverse cellular processes is essential for understanding regulatory mechanisms of cellular functions. Various genetically encoded Ca(2+) indicators (GECIs) are used for monitoring intracellular Ca(2+) changes under several types of microscope systems. However, it has not yet been explored which microscopic system is ideal for long-term imaging of the spatiotemporal patterns of Ca(2+) signals using GECIs. We here compared the Ca(2+) signals reported by a fluorescence resonance energy transfer (FRET)-based ratiometric GECI, yellow cameleon 3.60 (YC3.60), stably expressed in DT40 B lymphocytes, using three different imaging systems. These systems included a wide-field fluorescent microscope, a multipoint scanning confocal system, and a single-point scanning confocal system. The degree of photobleaching and the signal-to-noise ratio of YC3.60 in DT40 cells were highly dependent on the fluorescence excitation method, although the total illumination energy was maintained at a constant level within each of the imaging systems. More strikingly, the Ca(2+) responses evoked by B-cell antigen receptor stimulation in YC3.60-expressing DT40 cells were different among the imaging systems, and markedly affected by the illumination power used. Our results suggest that optimization of the imaging system, including illumination and acquisition conditions, is crucial for accurate visualization of intracellular Ca(2+) signals., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ca(2+) microdomains or locally restricted Ca(2+) increases in the cell have recently been reported to regulate many essential physiological events. Ca(2+) increases through the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)/Ca(2+) release channels contribute to the formation of a class of such Ca(2+) microdomains, which were often observed and referred to as Ca(2+) puffs in their isolated states. In this report, we visualized IP(3)-evoked Ca(2+) microdomains in histamine-stimulated intact HeLa cells using a total internal reflection fluorescence microscope, and quantitatively characterized the spatial profile by fitting recorded images to a two-dimensional Gaussian distribution. Ca(2+) concentration profiles were marginally spatially anisotropic, with the size increasing linearly even after the amplitude began to decline. We found the event centroid drifted with an apparent diffusion coefficient of 4.20 ± 0.50 μm(2)/s, which is significantly larger than those estimated for IP(3)Rs. The sites of maximal Ca(2+) increase, rather than initiation or termination sites, were detected repeatedly at the same location. These results indicate that Ca(2+) microdomains in intact HeLa cell are generated from spatially distributed multiple IP(3)R clusters or Ca(2+) puff sites, rather than a single IP(3)R cluster reported in cells loaded with Ca(2+) buffers.

The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular Ca(2+) release channel, and its opening is controlled by IP(3) and Ca(2+). A single IP(3) binding site and multiple Ca(2+) binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca(2+) is unknown. In this study, we visualized conformational changes in IP(3)R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP(3) and Ca(2+) have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca(2+) dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca(2+) alone from the FRET changes evoked by both ligands together. A mutant IP(3)R containing a single amino acid substitution at K508, which is critical for IP(3) binding, did not exhibit this bell-shaped Ca(2+) dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca(2+) sensor, resulted in ∼10-fold reduction in the Ca(2+) dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP(3)R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca(2+) binding affinity, as the mechanism underlying the IP(3)-dependent regulation of the bell-shaped relationship between the IP(3)R activity and cytosolic Ca(2+).

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.

We report ultrasensitive Ca(2+) indicators, yellow cameleon-Nano (YC-Nano), developed by engineering the Ca(2+)-sensing domain of a genetically encoded Ca(2+) indicator, YC2.60 or YC3.60. Their high Ca(2+) affinities (K(d) = 15-140 nM) and large signal change (1,450%) enabled detection of subtle Ca(2+) transients associated with intercellular signaling dynamics and neuronal activity, even in 100,000-cell networks. These indicators will be useful for studying information processing in living multicellular networks.

ATP enhances Ca(2+) release from inositol (1,4,5)-trisphosphate receptors (InsP(3)R). However, the three isoforms of InsP(3)R are reported to respond to ATP with differing sensitivities. Ca(2+) release through InsP(3)R1 is positively regulated at lower ATP concentrations than InsP(3)R3, and InsP(3)R2 has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP(3)R2 and InsP(3)R3 expressed in isolation on a null background in DT40 InsP(3)R knockout cells. We report that the Ca(2+)-releasing activity as well as the single channel open probability of InsP(3)R2 was enhanced by ATP, but only at submaximal InsP(3) levels. Further, InsP(3)R2 was more sensitive to ATP modulation than InsP(3)R3 under similar experimental conditions. Mutations in the ATPB sites of InsP(3)R2 and InsP(3)R3 were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP(3)R3 had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP(3)R2 eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP(3)R2.

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.

Large Ca2+ signals essential for cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses are known to be induced when PF activation precedes climbing fiber (CF) activation by 50-200 ms, consistent with cerebellar learning theories. However, large Ca2+ signals and/or LTD can also be induced by massive PF stimulation alone or by photolysis of caged Ca2+ or inositol 1,4,5-trisphosphate (IP3). To understand the spike-timing detection mechanisms in cerebellar LTD, we developed a kinetic model of Ca2+ dynamics within a Purkinje dendritic spine. In our kinetic simulation, IP3 was first produced via the metabotropic pathway of PF inputs, and the Ca2+ influx in response to the CF input triggered regenerative Ca2+-induced Ca2+ release from the internal stores via the IP3 receptors activated by the increased IP3. The delay in IP3 increase caused by the PF metabotropic pathway generated the optimal PF-CF interval. The Ca2+ dynamics revealed a threshold for large Ca2+ release that decreased as IP3 increased, and it coherently explained the different forms of LTD. At 2.5 microM IP3, CF activation after PF activation was essential to reach the threshold for the regenerative Ca2+ release. At 10 microM IP3, the same as achieved experimentally by strong IP3 photolysis, the threshold was lower, and thus large Ca2+ release was generated even without CF stimulation. In contrast, the basal 0.1 microM IP3 level resulted in an extremely high Ca2+ threshold for regenerative Ca2+ release. Thus, the results demonstrated that Ca2+ dynamics can detect spike timing under physiological conditions, which supports cerebellar learning theories.

The type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is an intracellular Ca(2+) channel protein that plays crucial roles in generating complex Ca(2+) signalling patterns. IP(3)R1 consists of three domains: a ligand-binding domain, a regulatory domain and a channel domain. In order to investigate the function of these domains in its gating machinery and the physiological significance of specific cleavage by caspase 3 that is observed in cells undergoing apoptosis, we utilized various IP(3)R1 constructs tagged with green fluorescent protein (GFP). Expression of GFP-tagged full-length IP(3)R1 or IP(3)R1 lacking the ligand-binding domain in HeLa and COS-7 cells had little effect on cells' responsiveness to an IP(3)-generating agonist ATP and Ca(2+) leak induced by thapsigargin. On the other hand, in cells expressing the caspase-3-cleaved form (GFP-IP(3)R1-casp) or the channel domain alone (GFP-IP(3)R1-ES), both ATP and thapsigargin failed to induce increase of cytosolic Ca(2+) concentration. Interestingly, store-operated (-like) Ca(2+) entry was normally observed in these cells, irrespective of thapsigargin pre-treatment. These findings indicate that the Ca(2+) stores of cells expressing GFP-IP(3)R1-casp or GFP-IP(3)R1-ES are nearly empty in the resting state and that these proteins continuously leak Ca(2+). We therefore propose that the channel domain of IP(3)R1 tends to remain open and that the large regulatory domain of IP(3)R1 is necessary to keep the channel domain closed. Thus cleavage of IP(3)R1 by caspase 3 may contribute to the increased cytosolic Ca(2+) concentration often observed in cells undergoing apoptosis. Finally, GFP-IP(3)R1-casp or GFP-IP(3)R1-ES can be used as a novel tool to deplete intracellular Ca(2+) stores.

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha-thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha-thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha-thrombin receptors may have a second, alternative mechanism for activating CA2+i release.

To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.

Many cellular responses to extracellular stimuli are mediated by the second messenger inositol 1,4,5-trisphosphate (InsP3). InsP3 releases Ca2+ from intracellular stores by binding to an InsP3 receptor (InSP3R), which is an InsP3-gated Ca2+ release channel. The resultant increase in the cytoplasmic Ca2+ concentration modulates various cellular functions, such as gene expression, metabolism, proliferation, secretion, and neural excitation. In these signaling cascades, InsP3R works as a signal converter from InsP3 to Ca2+. We describe here structural and functional properties and localization of InsP3R, a key molecule in the Ca2+ signaling pathway.

The kinetics of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release of the immunoaffinity-purified IP3 receptor (IP3R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca2+ indicator fluo-3. IP3R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with an anti-IP3R type 1 (IP3R1) antibody. The immunoblotting analysis using monoclonal antibodies against each IP3R type showed that the purified IP3R is almost homogeneous, composed of IP3R1. Ca2+ efflux from the proteoliposomes was monitored as fluorescence changes of 10 microM fluo-3, whose concentration was high enough to buffer released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 30 nM, excluding the possibility of Ca(2+)-mediated regulation of IP3-induced Ca2+ release. We also examined IP3-induced Ca2+ release using 1 microM fluo-3, where the deviations of free Ca2+ concentration were within 300 nM. At both fluo-3 concentrations, IP3-induced Ca2+ release showed similar kinetic properties, i.e. little Ca2+ regulation of Ca2+ release was observed in this system. IP3-induced Ca2+ release of the purified IP3R exhibited positive cooperativity; the Hill coefficient was 1.8 +/- 0.1. The half-maximal initial rate for Ca2+ release occurred at 100 nM IP3. At the submaximal concentrations of IP3, the purified IP3R showed quantal Ca2+ release, indicating that a single type of IP3R is capable of producing the phenomenon of quantal Ca2+ of release. The profiles of the IP3-induced Ca2+ release of the purified IP3R were found to be biexponential with the fast and slow rate constants (k(fast) = 0.3 approximately 0.7 s-1, k(slow) = 0.03 approximately 0.07 s-1), indicating that IP3R has two states to release CA2+. The amount of released Ca2+ by the slow phase was constant over the range of 10-5000 nM IP3 concentrations, whereas that by the fast phase increased in proportion to added IP3. This provides evidence to support the view that the fast phase of Ca2+ release is mediated by the low affinity state and the slow phase by the high affinity state of the IP3R. This also suggests that the fast component of Ca2+ release is responsible for the process of quantal Ca2+ release.

Kinetics of Ca2+ release by adenophostin, a novel agonist of inositol 1,4,5-trisphosphate (IP3) receptor, in the purified and reconstituted IP3 receptor type 1 (IP3R1) was investigated using the fluorescent Ca2+ indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca2+ release from the purified IP3R1 as IP3 did. Adenophostin-induced Ca2+ release by the purified IP3R1 exhibited a high positive cooperativity (nH = 3.9 +/- 0.2, EC50 = 11 nM), whereas the IP3-induced Ca2+ release exhibited a moderate one (nH = 1.8 +/- 0.1, EC50 = 100 nM). Inhibition of [3H]IP3 binding to the purified IP3R1 by adenophostin exhibited a positive cooperativity (nH = 1.9, Ki = 10 nM), whereas IP3 did not (nH = 1.1, Ki = 41 nM).

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger that releases Ca2+ from the intracellular stores. The InsP3 receptor (InsP3-R) was purified and its cDNA was cloned. We have found that InsP3-R is identical to the P400 protein identified as a protein enriched in the cerebellar Purkinje cells. We generated an L fibroblast cell transfectant that produced cDNA derived InsP3-R. The expressed protein displays high affinity and specificity for InsP3. InsP3 induces Ca2+ release from the membrane vesicles of the transfected cells. Incorporation of purified InsP3-R into a lipid bilayer showed InsP3 induced Ca2+ release. These result suggest that InsP3-R is a Ca2+ release channel. Immunogold method using monoclonal antibodies against the receptor showed that it is highly condensed on the smooth surfaced endoplasmic reticulum (ER) and slightly on the outer nuclear membrane and rough ER. Cross linking experiments show that the InsP3-R forms a homotetramer. The approximately 650 N-terminal amino acids are highly conserved between mouse and Drosophila melanogaster, and this region has the critical sequences for InsP3 binding. We found novel subtypes of the InsP3-R resulting from RNA-splicing that are expressed in a tissue-specific and developmentally specific manner and also resulting from different genes. It is believed that there are two Ca2+ release mechanisms, InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR). Eggs are good materials to analyse the machanism of Ca2+ signalling: fertilized hamster eggs exhibit repetitive Ca2+ transients as well as the Ca2+ wave.(ABSTRACT TRUNCATED AT 250 WORDS)