This work was supported by Ministerio de Econom&#236;a y competitividad (BFU2012-37146) and Junta de Castilla y Le&#242;n (BIO103/VA45/11, VA145U13 and BIO/VA33/13), Spain. MCR was supported by Junta de Castilla y Le&#242;n (Spain) and the European Social Fund. We thank the late Dr. Philippe Br&#251;let (1947-2013) for the mitochondrial GFP Aequorin plasmid.

Ethics Statement: Procedures involving animal subjects have been handled under protocols approved by the Valladolid University animal housing facility in agreement with the European Convention 123/Council of Europe and Directive 86/609/EEC.
1. Short and Long-term Culture of Rat Hippocampal Neurons
<ol>
	<li>Preparation of poly-D-lysine coated, 12 mm glass coverslips.
	<ol>
		<li>Sterilize 12 mm diameter glass coverslips in ethanol for at least 24 hr. Allow them to dry under sterile conditions.</li>
		<li...

<ol>
	<li>Sattler, R., Tymianski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+glutamate+receptor-mediated+excitotoxic+neuronal+cell+death.">Molecular mechanisms of glutamate receptor-mediated excitotoxic neuronal cell death.</a> Molecular Neurobiology. 24 (1-3), 107-129 (2001).</li><li>MacDermott, A. B., Mayer, M. L., Westbrook, G. L., Smith, S. J., Barker, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-receptor+activation+increases+cytoplasmic+calcium+concentration+in+cultured+spinal+cord+neurones.">NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones.</a> Nature. 321 (6069), 519-522 (1986).</li><li>Choi, D. W., Maulucci-Gedde, M., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+neurotoxicity+in+cortical+cell+culture.">Glutamate neurotoxicity in cortical cell culture.</a> The Journal of Neuroscience. 7 (2), 357-368 (1987).</li><li>Kure, S., Tominaga, T., Yoshimoto, T., Tada, K., Narisawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+triggers+internucleosomal+DNA+cleavage+in+neuronal+cells.">Glutamate triggers internucleosomal DNA cleavage in neuronal cells.</a> Biochemical and Biophysical Research Communications. 179 (1), 39-45 (1991).</li><li>Calvo, M., Sanz-Blasco, S., Caballero, E., Villalobos, C., Nunez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+to+excitotoxicity+in+aged+hippocampal+cultures+and+neuroprotection+by+non-steroidal+anti-inflammatory+drugs:+role+of+mitochondrial+calcium.">Susceptibility to excitotoxicity in aged hippocampal cultures and neuroprotection by non-steroidal anti-inflammatory drugs: role of mitochondrial calcium.</a> Journal of Neurochemistry. 132 (4), 403-417 (2015).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor+subunits+have+differential+roles+in+mediating+excitotoxic+neuronal+death+both+in+vitro+and+in+vivo.">NMDA receptor subunits have differential roles in mediating excitotoxic neuronal death both in vitro and in vivo.</a> The Journal of Neuroscience. 27 (11), 2846-2857 (2007).</li><li>Zhou, M., Baudry, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+NMDA+neurotoxicity+reflect+developmental+changes+in+subunit+composition+of+NMDA+receptors.">Developmental changes in NMDA neurotoxicity reflect developmental changes in subunit composition of NMDA receptors.</a> The Journal of Neuroscience. 26 (11), 2956-2963 (2006).</li><li>Brewer, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vulnerability+of+hippocampal+neurons+with+age+in+culture:+temporal+association+with+increases+in+NMDA+receptor+current,+NR2A+subunit+expression+and+recruitment+of+L-type+calcium+channels.">Increased vulnerability of hippocampal neurons with age in culture: temporal association with increases in NMDA receptor current, NR2A subunit expression and recruitment of L-type calcium channels.</a> Brain Research. 1151, 20-31 (2007).</li><li>Berridge, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling+remodelling+and+disease.">Calcium signalling remodelling and disease.</a> Biochemical Society Transactions. 40 (2), 297-309 (2012).</li><li>Stout, A. K., Raphael, H. M., Kanterewicz, B. I., Klann, E., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate-induced+neuron+death+requires+mitochondrial+calcium+uptake.">Glutamate-induced neuron death requires mitochondrial calcium uptake.</a> Nature Neuroscience. 1 (5), 366-373 (1998).</li><li>Pivovarova, N. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+calcium+overload+in+a+subpopulation+of+mitochondria+triggers+delayed+death+in+hippocampal+neurons.">Excitotoxic calcium overload in a subpopulation of mitochondria triggers delayed death in hippocampal neurons.</a> The Journal of Neuroscience. 24 (24), 5611-5622 (2004).</li><li>Morris, R. G., Garrud, P., Rawlins, J. N., O'Keefe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Place+navigation+impaired+in+rats+with+hippocampal+lesions.">Place navigation impaired in rats with hippocampal lesions.</a> Nature. 297 (5868), 681-683 (1982).</li><li>Geinisman, Y., Detoledo-Morrell, L., Morrell, F., Heller, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+markers+of+age-related+memory+dysfunction:+behavioral,+electrophysiological+and+morphological+perspectives.">Hippocampal markers of age-related memory dysfunction: behavioral, electrophysiological and morphological perspectives.</a> Progress in Neurobiology. 45 (3), 223-252 (1995).</li><li>Sodero, A. O., Weissmann, C., Ledesma, M. D., Dotti, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+stress+from+excitatory+neurotransmission+contributes+to+cholesterol+loss+in+hippocampal+neurons+aging+in+vitro.">Cellular stress from excitatory neurotransmission contributes to cholesterol loss in hippocampal neurons aging in vitro.</a> Neurobiology of Aging. 32 (6), 1043-1053 (2011).</li><li>Brini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfected+aequorin+in+the+measurement+of+cytosolic+Ca2++concentration+([Ca2+]c).+A+critical+evaluation.">Transfected aequorin in the measurement of cytosolic Ca2+ concentration ([Ca2+]c). A critical evaluation.</a> The Journal of Biological Chemistry. 270 (17), 9896-9903 (1995).</li><li>Villalobos, C., Alonso, M. T., Garc&#236;a-Sancho, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+of+calcium+oscillations+inside+intracellular+organelles.">Bioluminescence imaging of calcium oscillations inside intracellular organelles.</a> Methods in Molecular Biology. 574, 203-214 (2009).</li><li>Villalobos, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redistribution+of+Ca2++among+cytosol+and+organella+during+stimulation+of+bovine+chromaffin+cells.">Redistribution of Ca2+ among cytosol and organella during stimulation of bovine chromaffin cells.</a> FASEB Journal. 16, 343-353 (2002).</li><li>Rogers, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+local+Ca2++dynamics+with+genetically+encoded+bioluminescent+reporters.">Visualization of local Ca2+ dynamics with genetically encoded bioluminescent reporters.</a> The European Journal of Neuroscience. 21 (3), 597-610 (2005).</li><li>Barreto-Chang, O. L., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+of+cortical+neurons+using+Fura-2+AM.">Calcium imaging of cortical neurons using Fura-2 AM.</a> Journal of Visualized Experiments. (23), 1067 (2009).</li></ol>

The remodeling of intracellular Ca2+ homeostasis in the aging brain has been related to cognitive loss, increased susceptibility to ischemic damage, excitotoxicity and neurodegeneration. To investigate this hypothesis in vitro, Ca2+ imaging procedures are available. Unfortunately, viable cultures of old hippocampal neurons are not reliable. Recently, it has been observed that long-term cultures of rat hippocampal neurons present many of the typical hallmarks of aging in vivo includ...

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&#39;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.
It is widely accepted that an increase in the cytosolic-free Ca2+ concentration ([Ca2+]cyt) leads to cells activation. However, if this rise is too high and/or sustained enough, it can trigger cell death 9. Moreover, it has been proposed that excitotoxicity requires mitochondrial Ca2+ uptake 10, since treating neurons with a mitochondrial uncoupler protected neurons against glutamate-induced cell death 11. If mitochondria take up too much Ca2+, the opening of the mitochondrial permeability transition pore may occur, leading to release of cytochrome c and other pro-apoptotic factors, and inducing apoptosis. We have recently shown that this mitochondrial Ca2+ uptake is directly related to the age-dependant susceptibility to excitotoxicity, by directly measuring NMDA-induced mitochondrial Ca2+ uptake in single hippocampal neurons 5, a method which is reported in this article. The hippocampus, involved in physiological processes such as learning, memory and other cognitive processes 12, is highly vulnerable to aging and neurodegenerative disorders 13. It has been proposed that, after several weeks in vitro, cultured hippocampal neurons show a number of typical characteristics of aged neurons 14. Accordingly, long-term cultured hippocampal neurons may provide a comprehensive model to investigate Ca2+-mediated mechanisms of enhanced excitotoxicity in aging.
The overall goal of the method presented is, therefore, to investigate substantial changes in intracellular Ca2+ homeostasis or Ca2+ remodeling in the aging brain including the differential Ca2+ responses elicited by NMDA receptor agonists in a long-term cultured hippocampal neurons. The method includes a detailed description of the culture of rat hippocampal neurons and the monitoring of cytosolic and mitochondrial Ca2+ concentrations by fluorescence and bioluminescence imaging in individual neurons, respectively. Fluorescence imaging of cytosolic Ca2+ in cultured neurons is a standard procedure. However, this method is less reliable for subcellular Ca2+ measurements including mitochondrial Ca2+. Reasons for this include lack of proper targeting of synthetic probes and inappropriate affinity for Ca2+ concentrations that may change in mitochondria from the low &#181;M level even to the mM level. The use of Ca2+ probes based on proteins as for instance aequorin, has allowed the targeting to subcellular organelles and the use of derivatives different Ca2+ affinities using different coelenterazines or mutated probes lacking specific Ca2+ binding sites 15. In this way, bioluminescence imaging of cells expressing mitochondria-targeted aequorin may allow the monitoring of mitochondrial Ca2+ concentrations in individual neurons. Yet, this procedure may require the use of photon counting cameras or ultrasensitive CCD cameras for bioluminescence imaging 16-18. This method may yield novel results that should be confirmed in more established brain aging models as, for instance, brain slices from old animals.

<table>
	<tbody>
		<tr>
			<td>Neurobasal Culture Medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS medium</td>
			<td>Gibco</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 medium</td>
			<td>Gibco</td>
			<td>31330-038</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (from bovine pancreas)</td>
			<td>Sigma</td>
			<td>D5025-15KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bobine Serum</td>
			<td>Lonza</td>
			<td>DE14-801E</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15750</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-032</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm glass coverslips</td>
			<td>Labolan</td>
			<td>0111520/20012</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Worthington</td>
			<td>LS003127</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well multidish plaques</td>
			<td>Nunc</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Petry dishes</td>
			<td>JD Catalan s.l.</td>
			<td>2120044T</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipettes</td>
			<td>Fisher Scientific</td>
			<td>431030</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura2-AM</td>
			<td>Life Technologies</td>
			<td>F1201</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine2000</td>
			<td>Invitrogen</td>
			<td>11668-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Coelenterazine n</td>
			<td>Biotium</td>
			<td>BT-10115-2250 uG</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma</td>
			<td>D5628</td>
			<td></td>
		</tr>
		<tr>
			<td>NMDA</td>
			<td>Sigma&#160;</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>50046</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Axiovert S100 TV microscope</td>
			<td>Carl Zeiss Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xcite ilumination system</td>
			<td>EXFO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ORCA ER fluorescence camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VIM photon counting CCD camera</td>
			<td>Hamamatsu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>VC-8 valve controller</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SH-27B heating system&#160;</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aquacosmos Software</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fluorescence and bioluminescence imaging of subcellular ca2+ in aged hippocampal neurons

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.

Here we describe a simple method to assess Ca2+ remodeling and the effects of NMDA on cytosolic and mitochondrial [Ca2+] in aged neurons. Figure 1 shows the schematic of the procedure for isolating and culturing hippocampal neurons from neonatal rats. Before starting, we need to prepare sterile, poly-D-lysine coated, glass coverslips and locate them in a 4-well dish. Then, neonatal rats are killed and the brain removed. After isolating the hippocampus, tissue is carefully dispersed ...

Watch this Scientific Journal Video about Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons at JoVE.com

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

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.

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

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

fluorescence-bioluminescence-imaging-subcellular-ca2-aged-hippocampal

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Neuroscience

8.2K ビュー.  Consejo Superior de Investigaciones Cientìficas. 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. 