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Biologie

Электрофизиологические Запись в Drosophila Эмбрионов

Published: May 21, 2009
doi:
10.3791/1348
, ,
1Department of Biological Sciences,University of Illinois, 2Department of Biological Sciences,Vanderbilt University

Summary

Электрофизиологические записи с<em> Drosophila</em> Эмбрионов позволит анализ развития мышц и нейронов электрическим свойствам, а также характеристика функциональных синаптогенез на глутаматергической нервно-мышечном соединении и центральной холинергической и ГАМК-синапсы.

Abstract

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of <8 hours (at 25°C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Protocol

Часть 1: Оборудование и расходные материалы Электрофизиологические записи из эмбрионов дрозофилы в первую очередь требует знания эмбриональных методы вскрытия, которые описаны в другой Юпитер видео. Электрофизиологические записи из эмбрионов дрозофилы использу…

Discussion

Электрофизиологические записи из эмбрионов дрозофилы требует ручного манипулирования и рассечение. Здоровье подготовки, и, как следствие качество записей, зависит от того, один из которых в состоянии быстро и аккуратно подготовить хрупкой эмбриональных тканей для записи, а зате?…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

КБ поддерживается NIH грант GM54544.

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Tags

Electrophysiological RecordingDrosophila EmbryoGenetic ModelEmbryonic DevelopmentFunctional NeuroscienceDevelopmental ProgramsFunctional Electrical Signaling PropertiesFunctional Chemical SynapsesNeural Circuit FormationLate-stage EmbryogenesisEpidermal CuticleWater-polymerizing Surgical GlueControlled DissectionDorsal Longitudinal IncisionVentral Nerve CordBody Wall MusculatureWhole-cell Patch-clamp TechniquesIndividually-identifiable NeuronsSomatic MusclesIonic CurrentsAction Potentials

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Chen, K., Featherstone, D. E., Broadie, K. Electrophysiological Recording in the Drosophila Embryo. J. Vis. Exp. (27), e1348, doi:10.3791/1348 (2009).
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