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1.
Neuron ; 54(4): 535-45, 2007 May 24.
Artículo en Inglés | MEDLINE | ID: mdl-17521567

RESUMEN

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.


Asunto(s)
Conducta Animal/fisiología , Iluminación/métodos , Neuronas/fisiología , Receptores de Ácido Kaínico/fisiología , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de la radiación , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Conducta Animal/efectos de la radiación , Células Cultivadas , Cisteína/genética , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores , Hipocampo/citología , Larva , Leucina/genética , Mutación , Neuronas/efectos de los fármacos , Neuronas/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Estimulación Física/métodos , Ratas , Receptores de Ácido Kaínico/genética , Transfección/métodos , Pez Cebra , Receptor de Ácido Kaínico GluK2
2.
Proc Natl Acad Sci U S A ; 105(46): 17789-94, 2008 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-19004775

RESUMEN

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.


Asunto(s)
Imagenología Tridimensional/métodos , Microscopía de Contraste de Fase/métodos , Actinas , Animales , Supervivencia Celular , Células Cultivadas , Colorantes Fluorescentes/química , Ratones , Microscopía Fluorescente , Músculos/citología , Células 3T3 NIH , Neuronas/citología , Ratas , Xenopus , Pez Cebra
3.
Nat Chem Biol ; 3(5): 263-7, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17401379

RESUMEN

Hydrogen peroxide (H2O2) is emerging as a newly recognized messenger in cellular signal transduction. However, a substantial challenge in elucidating its diverse roles in complex biological environments is the lack of methods for probing this reactive oxygen metabolite in living systems with molecular specificity. Here we report the synthesis and application of Peroxy Green 1 (PG1) and Peroxy Crimson 1 (PC1), two new fluorescent probes that show high selectivity for H2O2 and are capable of visualizing endogenous H2O2 produced in living cells by growth factor stimulation, including the first direct imaging of peroxide produced for brain cell signaling. The combined features of reactive oxygen species selectivity, sensitivity to signaling levels of H2O2, and live-cell compatibility presage many new opportunities for PG1, PC1 and related synthetic reagents for exploring the physiological roles of H2O2 in living systems with molecular imaging.


Asunto(s)
Peróxido de Hidrógeno/metabolismo , Transducción de Señal , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Estructura Molecular , Ratas
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