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National Institute of Health

6 ARTICLES PUBLISHED IN JoVE

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Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx
Kenneth Paradiso 1, Wei Wu 1, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke, National Institute of Health

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

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Biology

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies
Matthias Garten 1, Sophie Aimon 2, Patricia Bassereau 1, Gilman E. S. Toombes 3
1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

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Medicine

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling
Jerome D. Robin 1, Woody E. Wright 1, Yaqun Zou 2, Stacy A. Cossette 3, Michael W. Lawlor 3, Emanuela Gussoni 4
1Department of Cell Biology, UT Southwestern Medical Center, 2National Institute of Neurological Disorders and Stroke, National Institute of Health, 3Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 4Division of Genetics and Genomics, Boston Children's Hospital

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

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Engineering

Fabrication of High Contrast Gratings for the Spectrum Splitting Dispersive Element in a Concentrated Photovoltaic System
Yuhan Yao 1, He Liu 1, Wei Wu 1
1Department of Electrical Engineering, University of Sothern California

The fabrication of high contrast gratings as the parallel spectrum splitting dispersive element in a concentrated photovoltaic system is demonstrated. Fabrication processes including nanoimprint lithography, TiO2 sputtering and reactive ion etching are described. Reflectance measurement results are used to characterize the optical performance.

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Neuroscience

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons
Seth Villarreal 1, Sung Hoon Lee 1,2, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke, 2College of Pharmacy, Chung-ang University

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

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Neuroscience

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells
Sue Han *1, Xin Wang *1, Nicholas Cordero 1, Ling-Gang Wu 1
1National Institute of Neurological Disorders and Stroke

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

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