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Wavelength-selective one- and two-photon uncaging of GABA.

ABSTRACT: We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of ?-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological buffer. We found that DEAC450-caged GABA is relatively inactive toward two-photon excitation at 720 nm, so when paired with a nitroaromatic caged glutamate that is efficiently excited at such wavelengths, we could photorelease glutamate and GABA around single spine heads on neurons in brain slices with excellent wavelength selectivity using two- and one-photon photolysis, respectively. Furthermore, we found that DEAC450-caged GABA could be effectively released using two-photon excitation at 900 nm with spatial resolution of about 3 ?m. Taken together, our experiments show that the DEAC450 caging chromophore holds great promise for the development of new caged compounds that will enable wavelength-selective, two-color interrogation of neuronal signaling with excellent subcellular resolution.

SUBMITTER: Amatrudo JM

PROVIDER: S-EPMC3894722 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Wavelength-selective one- and two-photon uncaging of GABA.

Amatrudo Joseph M JM Olson Jeremy P JP Lur G G Chiu Chiayu Q CQ Higley Michael J MJ Ellis-Davies Graham C R GC

ACS chemical neuroscience 20131204 1

We have synthesized photolabile 7-diethylamino coumarin (DEAC) derivatives of γ-aminobutyric acid (GABA). These caged neurotransmitters efficiently release GABA using linear or nonlinear excitation. We used a new DEAC-based caging chromophore that has a vinyl acrylate substituent at the 3-position that shifts the absorption maximum of DEAC to about 450 nm and thus is named "DEAC450". DEAC450-caged GABA is photolyzed with a quantum yield of 0.39 and is highly soluble and stable in physiological b ...[more]

PMID: 24304264

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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

Wavelength-selective one- and two-photon uncaging of GABA.

Publications

Wavelength-selective one- and two-photon uncaging of GABA.

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