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Optically selective two-photon uncaging of glutamate at 900 nm.

ABSTRACT: We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended ?-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

SUBMITTER: Olson JP

PROVIDER: S-EPMC4079120 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Optically selective two-photon uncaging of glutamate at 900 nm.

Olson Jeremy P JP Kwon Hyung-Bae HB Takasaki Kevin T KT Chiu Chiayu Q CQ Higley Michael J MJ Sabatini Bernardo L BL Ellis-Davies Graham C R GC

Journal of the American Chemical Society 20130411 16

We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength-selective two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moiety at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slice ...[more]

PMID: 23577752

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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.

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Optically selective two-photon uncaging of glutamate at 900 nm.

Publications

Optically selective two-photon uncaging of glutamate at 900 nm.

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