Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

María Calvo-Rodríguez; Carlos Villalobos; Lucía Nuñez

doi:10.3791/53330

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Fluorescence and Bioluminescence Imaging of Subcellular Ca²⁺ in Aged Hippocampal Neurons

7.9K views

⸱

DOI:

10.3791/53330

⸱

December 1st, 2015

María Calvo-Rodríguez¹ , Carlos Villalobos¹ , Lucía Nuñez²

¹Instituto de Biologìa y Genética Molecular, Consejo Superior de Investigaciones Cientìficas, ²Departamento de Bioquìmica y Biologìa Molecular y Fisiologìa, Universidad de Valladolid

Summary

Intracellular Ca²⁺ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca²⁺ overload. We aimed at investigating Ca²⁺ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca²⁺ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

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 hr, 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.

Introduction

Excitotoxicity is one of the most important mechanisms contributing to neuronal damage and cell death in neurological insults such as ischemia, and in some neurodegenerative diseases such as Alzheimer's disease ¹. This type of neurotoxicity is mainly mediated by glutamate acting on Ca²⁺-permeable, ionotropic NMDA receptors (NMDAR) ². Exposure of cultured neurons to glutamate can lead to excitotoxicity ³, which causes neuronal apoptosis ⁴. We and others have previously reported that neuronal vulnerability to NMDA-induced apoptosis may change with development in vitro and aging ^5-8.