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Photochemically initiated intracellular astrocytic calcium waves in living mice using two-photon uncaging of IP(3).

ABSTRACT: We have developed a caged IP(3) analogue for two-photon photolysis in living animals. This probe is a cell permeable version and was coloaded with a fluorescent Ca(2+) dye into astrocytes in layer 1 of the somatosensory cortex of anesthetized mice. Two-photon irradiation of single cells at 720 nm produced rapid and robust increases in intracellular Ca(2+) concentrations monitored using two-photon microscopy at 950 nm. The photoevoked intracellular Ca(2+) waves were similar in magnitude to intrinsic signals in wild type mice. These waves did not propagate to other cells beyond the targeted astrocyte. In contrast, we observed intercellular astrocytic Ca(2+) waves in two mouse models of familial Alzheimer's disease. These data suggest that Alzheimer's might perturb gliotransmission but not IP(3) signaling per se in mouse models of the disease.

SUBMITTER: Crowe SE

PROVIDER: S-EPMC3368680 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Photochemically initiated intracellular astrocytic calcium waves in living mice using two-photon uncaging of IP(3).

Crowe Sarah E SE Kantevari Srinivas S Ellis-Davies Graham C R GC

ACS chemical neuroscience 20100615 8

We have developed a caged IP(3) analogue for two-photon photolysis in living animals. This probe is a cell permeable version and was coloaded with a fluorescent Ca(2+) dye into astrocytes in layer 1 of the somatosensory cortex of anesthetized mice. Two-photon irradiation of single cells at 720 nm produced rapid and robust increases in intracellular Ca(2+) concentrations monitored using two-photon microscopy at 950 nm. The photoevoked intracellular Ca(2+) waves were similar in magnitude to intrin ...[more]

PMID: 22778846

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Project description:Key pointsIn cardiac myocytes, subcellular local calcium release signals, calcium sparks, are recruited to form each cellular calcium transient and activate the contractile machinery. Abnormal timing of recovery of sparks after their termination may contribute to arrhythmias. We developed a method to interrogate recovery of calcium spark trigger probabilities and their amplitude over time using two-photon photolysis of a new ultra-effective caged calcium compound. The findings confirm the utility of the technique to define an elevated sensitivity of the calcium release mechanism in situ and to follow hastened recovery of spark trigger probabilities in a mouse model of an inherited cardiac arrhythmia, which was used for validation. Analogous methods are likely to be applicable to investigate other microscopic subcellular signalling systems in a variety of cell types.AbstractIn cardiac myocytes Ca2+ -induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca2+ release occurs via subcellular Ca2+ signalling events, Ca2+ sparks. Local recovery of Ca2+ release depends on both SR refilling and restoration of Ca2+ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca2+ compound BIST-2EGTA and laser-scanning confocal Ca2+ imaging to probe refractoriness of local Ca2+ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca2+ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyRR420Q mutation were used for further validation and revealed a higher Ca2+ sensitivity of CICR. This new 2P approach provides composite information of Ca2+ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca2+ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.

Dataset Information

Photochemically initiated intracellular astrocytic calcium waves in living mice using two-photon uncaging of IP(3).

Publications

Photochemically initiated intracellular astrocytic calcium waves in living mice using two-photon uncaging of IP(3).

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