RT info:eu-repo/semantics/article
T1 Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons
A1 Calvo Rodríguez, María
A1 Villalobos Jorge, Carlos
A1 Núñez Llorente, Lucía
K1 Hippocampal neurons
AB Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanismsresponsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activationof glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis orremodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we haveused 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. Thencultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, culturedneurons 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 mitochondrialCa2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as forexample the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure mayyield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selecteddrugs aimed at preventing neuron cell death in age-related diseases.
PB Journal of Visualized Experiments (JoVE)
SN 1940-087X
YR 2015
FD 2015
LK http://uvadoc.uva.es/handle/10324/21819
UL http://uvadoc.uva.es/handle/10324/21819
LA eng
NO J Vis Exp. 2015 Dec 1;(106)
NO Producción Científica
DS UVaDOC
RD 25-nov-2024