Accedi

University of California at San Diego

5 ARTICLES PUBLISHED IN JoVE

Neuroscience

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Sarah H. Shahmoradian¹, Mauricio R. Galiano², Chengbiao Wu³, Shurui Chen⁴, Matthew N. Rasband², William C. Mobley³, Wah Chiu⁴

¹Department of Molecular Physiology and Biophysics, Baylor College of Medicine, ²Department of Neuroscience, Baylor College of Medicine, ³Department of Neuroscience, University of California at San Diego, ⁴National Center for Macromolecular Imaging, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

Chemistry

Conducting Miller-Urey Experiments

Eric T. Parker¹, James H. Cleaves^2,3, Aaron S. Burton⁴, Daniel P. Glavin⁵, Jason P. Dworkin⁵, Manshui Zhou¹, Jeffrey L. Bada⁶, Facundo M. Fernández¹

¹School of Chemistry and Biochemistry, Georgia Institute of Technology, ²Earth-Life Science Institute, Tokyo Institute of Technology, ³Institute for Advanced Study, ⁴Astromaterials Research and Exploration Science Directorate, NASA Johnson Space Center, ⁵Goddard Center for Astrobiology, NASA Goddard Space Flight Center, ⁶Geosciences Research Division, Scripps Institution of Oceanography, University of California at San Diego

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth’s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

Neuroscience

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

Xiaobei Zhao¹, Yue Zhou², April M. Weissmiller¹, Matthew L. Pearn^3,4, William C. Mobley¹, Chengbiao Wu¹

¹Department of Neurosciences, University of California, San Diego, ²School of Biomedical Engineering and Med-X Research Institute, Shanghai Jiao Tong University, ³Department of Anesthesiology, University of California, San Diego, ⁴VA San Diego Healthcare System

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

Developmental Biology

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

Michael S. Yu^*1,2, Sean Spiering^*1, Alexandre R. Colas¹

¹Sanford Burnham Prebys Medical Discovery Institute, ²Department of Bioengineering, University of California at San Diego

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

Neuroscience

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Ekaterina Martianova¹, Sage Aronson^2,3, Christophe D. Proulx¹

¹CERVO Brain Research Center, Department of Psychiatry and Neurosciences, Université Laval, ²Center for Neural Circuits and Behavior, Department of Neuroscience and Section of Neurobiology, Division of Biology, University of California at San Diego, ³Neurophotometrics Ltd.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.