Skip navigation
Please use this identifier to cite or link to this item:
Title: Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons
Authors: Calvo Rodríguez, María
Villalobos, Carlos
Nuñez Llorente, Lucía
Issue Date: 2015
Publisher: Journal of Visualized Experiments (JoVE)
Description: Producción Científica
Citation: J Vis Exp. 2015 Dec 1;(106)
Abstract: Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 h, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.
Keywords: Hippocampal neurons
ISSN: 1940-087X
Peer Review: SI
DOI: 10.3791/53330
Sponsor: Ministerio de Economía y Competitividad (BFU2012-37146)
Junta de Castilla y Leòn (BIO103/VA45/11, VA145U13 and BIO/VA33/13)
Publisher Version:
Language: eng
Rights: info:eu-repo/semantics/embargoedAccess
Appears in Collections:DEP06 - Artículos de revista

Files in This Item:
File Description SizeFormat 
JOVE 2015.pdf551,33 kBAdobe PDFThumbnail

This item is licensed under a Creative Commons License Creative Commons

University of Valladolid
Powered by MIT's. DSpace software, Version 5.5