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>Distribute the coverslips on a strip of parafilm in a Petri dish. Cover the surface of each coverslip with 200 &#181;l of 1 mg/ml poly-D-lysine. Allow treating O/N.</li>
		<li>The next day wash the coverslips with double distilled sterile water every 15 min for 90 min under sterile conditions.</li>
		<li>Fill a four-well multidish plate with 500 &#181;l of Neurobasal Culture Medium per well. Neurobasal Culture Medium should be supplemented with 10% fetal bovine serum, 2 % B27, 1 &#181;g/ml gentamicin and 2 mM L-glutamine.</li>
		<li>Place the coverslips in the multidish plate. Maintain them in a humidified 37 &#186;C and 5% CO2 incubator until use.</li>
	</ol>
	</li>
	<li>Isolation of hippocampal neurons from neonatal rats.
	<ol>
		<li>Prepare 1.8 ml papain solution (directly purchased) in a vial (20 &#181;l/ml) under sterile conditions: To obtain a final concentration of 20 &#181;l/ml add around 40 &#181;l papain in 1.8 ml Hank&#39;s plus 0.6% BSA (&#181;l should be calculated depending on the supplier conditions). Hank&#39;s 0.6% BSA is prepared by mixing 85 ml of HBSS medium with 15 ml of 4% bovine serum albumin (BSA, prepared solving 4 g of BSA in 100 ml HBSS). Prepare it in a sterile hood.</li>
		<li>Incubate the papain in Hank&#39;s plus 0.6% BSA for 30 min at 37 &#186;C before use. Filter the solution using a 0.22 &#181;m filter prior to use.</li>
		<li>Prepare Ham&#39;s F-12 medium by adding Dulbecco&#39;s Modified Eagle&#39;s Medium powder (13.5 g per L) in 900 ml of double distilled sterile water. Then add 6 g HEPES and 336 mg NaHCO3 and adjust the pH to 7.42 using NaOH 4 N. Add sterile water in order to obtain a 1 L solution and filter it using a 0.20 &#181;m polyethersulfone (PES) bottle top filter. Carry this process in a sterile hood. Finally saturate the solution with CO2 under sterile conditions before use. Store solutions at 4 &#186;C and use them chilled.</li>
		<li>Euthanize newborn (P0) rat pups by decapitation and wash the head in sterile HBS medium. Open the skull with the help of sterile scissors. Extract the brain with a spatula and wash it quickly in Ham&#39;s F-12 medium.</li>
		<li>Make a diagonal cut with the aid of a scalpel in each hemisphere (Figure 1B), and carry it to a Petri dish containing Ham&#39;s F-12 medium.</li>
		<li>Discard meninges carefully with the help of dissection forceps and under a magnifying glass.</li>
		<li>Identify the hippocampus in a characteristic concave location over the cortex using magnifying glass. Then, separate the hippocampus from the cortex by pulling carefully from one border and removing it from its position using eye scissors.</li>
		<li>Transfer the hippocampal tissue to a 4-well plate filled with sterile Hank&#39;s medium lacking Ca2+ and Mg2+ plus 0.6% BSA and wash hippocampal tissue. Without removing the media cut the tissue in small pieces (around 2 x 2 mm) using the same eye scissors.</li>
		<li>Transfer hippocampal pieces to a vial containing 1.8 ml pre-filtered papain solution. Then incubate them at 37 &#186;C with occasional, gentle shaking. 15 min later, add 90 &#181;l of DNase I solution (50 &#181;g/ml final) and incubate for 15 additional min.</li>
		<li>Transfer the tissue to a 10 ml centrifuge tube and wash the fragments with fresh Neurobasal Culture Medium. Obtain cell suspension by passing tissue fragments through a 5 ml plastic pipette.</li>
		<li>Centrifuge cell suspension at 160 x g for 5 min. Remove the supernatant using a plastic sterile Pasteur pipette and suspend pellet carefully with a 1 ml automated pipette in 1 ml of Neurobasal Culture Medium.</li>
		<li>Measure cell density using a Neubauer counting chamber. Put the glass coverslip over the Neubauer chamber. Add 10 &#181;l of cell suspension. Make sure the cell suspension enters the chamber uniformly and making no bubbles. Count the number of cells under the microscope and perform corresponding calculations to obtain a suitable drop of 40-80 &#181;l containing 30 x 103 cells. The total number of cells will depend on the number of animals used.</li>
	</ol>
	</li>
	<li>Short-term and long-term culture of rat hippocampal neurons.
	<ol>
		<li>Over the four-well multidish plate containing 500 &#181;l of Neurobasal Culture Medium prepared before and kept in the incubator, plate around 30 x 103 cells in a drop of about 50 &#181;l to each well of the multidish plate containing one poly-D-lysine-coated, 12 mm diameter glass coverslip.</li>
		<li>Maintain primary hippocampal cells in a humidified 37 &#186;C and 5% CO2 incubator for 2-5 days in vitro (DIV, short-term, young cultures) or &#62;15 DIV (Long-term, aged cultures) before experiments without changing the culture media.</li>
	</ol>
	</li>
</ol>
2. Fluorescence Imaging of Cytosolic Ca 2+ Concentration
<ol>
	<li>Preparation of test solutions for fluorescence imaging.
	<ol>
		<li>Prepare an external HEPES-buffered saline (HBS) solution containing, in mM: NaCl 145, KCl 5, MgCl2 1, CaCl2 1, glucose 10, sodium-HEPES 10 (pH 7.42).</li>
		<li>Prepare an external, Mg2+-free, HEPES-buffered saline (HBS) solution containing, in mM: NaCl 146, KCl 5, CaCl2 1, glucose 10 and HEPES 10 (pH 7.42).</li>
		<li>Solve NMDA in Mg2+-free, HEPES-buffered saline (HBS) at a final concentration of 100 &#181;M and supplement it with 10 &#181;M glycine.</li>
		<li>Prepare a HBS solution containing KCl instead of NaCl by solving (in mM): KCl 145, MgCl2 1, CaCl2 1, glucose 10 and HEPES 10 (pH 7.42).</li>
	</ol>
	</li>
	<li>Loading of hippocampal cells with fluorescent calcium probe fura2/AM.
	<ol>
		<li>Take the cell culture containing coverslips off the incubator and wash them with HBS medium at RT by transferring them to a new four-well multidish plate containing 500 &#181;l of HBS per well.</li>
		<li>Incubate cells with fura2/AM 4 &#181;M (prepared in the same HBS medium) for 60 min at RT (25 &#186;C) in a dark place.</li>
		<li>After 60 min, wash coverslips with fresh HBS medium.</li>
	</ol>
	</li>
	<li>Recording fluorescence images of cytosolic [Ca 2+ ] in cultured cells.
	<ol>
		<li>Turn on the lamp, microscope, perfusion system, fluorescence camera and computer.</li>
		<li>Place the coverslips in a thermostated platform for open 12 mm glass coverslips on the stage of the inverted microscope and select a microscopic field using a 40X objective (oil, NA:1.3). Perfuse cells continuously with pre-warmed (37 &#186;C) HBS medium in absence or presence of test substances. Maintain the flow at a rate of about 5-10 ml/min. 
		Note: Cell perfusion system for living cells is mounted in a thermostated platform for open 12 mm glass coverslips. 8-lines gravity-driven perfusion system equipped with a valve controller is used to perfuse the solutions. A vacuum pump is responsible for removing any excess medium. Solutions are heated using an in-tube heating system.</li>
		<li>Capture a background image with the shutter closed at both excitation wavelengths.</li>
		<li>Epi-illuminate cells alternately at 340 and 380 nm. Record light emitted at 520 nm every 5-10 sec with a fluorescence camera, which is filtered by a fura-2 dichroic mirror.</li>
		<li>When the recording period is finished, store the complete sequence of images emitted at 520 nm in the computer for further analysis.</li>
	</ol>
	</li>
	<li>Analysis of recorded fluorescent images.
	<ol>
		<li>Open the experiment file. Using the aquacosmos software, click on &#39;Ratio&#39; and select the desired ratio range. Calculate the pixel by pixel ratio in the resulting images in order to obtain a sequence of ratio images.</li>
		<li>Subtract background by adjusting the &#39;background elimination&#39;. Press &#39;start calculation&#39;.</li>
		<li>Press &#39;all times sequence&#39; button and erase the ancient regions of interest (ROIs). For quantitative analysis of individual cells, establish new regions of interest or ROIs corresponding to individual neurons. Average all ratio values in each pixel corresponding to each ROI and each image to obtain a recording of ratio fluorescence values for individual ROIs (cells).</li>
		<li>To graph the individual recordings export the ratio fluorescence values corresponding to each ROI to a graphing program.</li>
		<li>Make the corresponding calculations for estimating the size of the rises in ratio fluorescences in response to each stimulation using a suitable data analysis and graphing software.</li>
	</ol>
	</li>
</ol>
3. Bioluminescence Imaging of Mitochondrial Ca 2+ Concentration
<ol>
	<li>Transfection of cultured hippocampal neurons with the mitGAmut plasmid. 
	NOTE: Cells are first transfected with the mitGAmut plasmid containing a mitochondria-targeted sequence and a mutated aequorin lacking a Ca2+ binding site fused to the green fluorescent protein (GFP). This construction detects large rises in mitochondrial Ca2+ uptake and allows the identification of transfected cells by the GFP fluorescence. The plasmid was developed and kindly provided by P. Br&#251;let and co-workers 18 (CNRS, Paris).
	<ol>
		<li>Prepare Neurobasal TF, containing Neurobasal Culture Medium, lacking fetal bovine serum, B27, gentamicin and 2 mM L-glutamine.</li>
		<li>Prepare 50 &#181;l of Neurobasal TF plus 2.5 &#181;l transfection reagent in a vial (Solution A). Prepare 50 &#181;l of Neurobasal TF plus 4 &#181;g mitGAmut plasmid in a vial (Solution B). Add gently solution B over solution A. Allow mixing during 20 min, without shaking.</li>
		<li>Transfer the coverslips containing hippocampal neurons to new multidish plates containing 200 &#181;l of fresh Neurobasal TF.</li>
		<li>Add drop by drop 100 &#181;l of solution A + B over every coverslip. Incubate for 30 min. Remove the medium. Wash once cells with fresh Neurobasal TF. Return the coverslips to the original Neurobasal Culture Medium.</li>
		<li>Culture neurons for an additional 24 hr at 37 &#186;C and 5% CO2 after transfection to allow efficient expression and targeting of the probe.</li>
	</ol>
	</li>
	<li>Preparation of test solutions for bioluminescence imaging.
	<ol>
		<li>Prepare HBS solutions containing NMDA 100 &#181;M or 145 mM KCl as above (Step 2.1). Prepare HBS solution containing 10 mM Ca2+ plus digitonin 100 &#181;M.</li>
	</ol>
	</li>
	<li>Reconstitution of mitochondria-targeted aequorin with coelenterazine n.
	<ol>
		<li>Transfer coverslips containing transfected hippocampal neurons to a four-well plate containing 200 &#181;l of HBS.</li>
		<li>Add 4 &#181;l of coelenterazine n (200 &#181;M) to 200 &#181;l of HBS for a final concentration of 4 &#181;M and mix gently. Incubate for 2 hr at RT (25 &#186;C) and in the dark.</li>
	</ol>
	</li>
	<li>Recording bioluminescence images of mitochondrial [Ca 2+ ].
	<ol>
		<li>Turn on the microscope, the lamp, the perfusion system, bioluminescence camera and the computer.</li>
		<li>Transfer reconstituted coverslips to a thermostated (37 &#186;C) perfusion chamber and perfuse continuously with pre-warmed HBS solution at a rate of 5-10 ml/min.</li>
		<li>Using a 40X objective (oil, NA: 1.3), select a microscopic field containing cells expressing the apoaequorins as shown by fluorescence emission associated with expression of green fluorescent protein (GFP) using the FITC filters. A typical field may contain 1-2 transfected cells. Capture the GFP fluorescence image, using a CCD camera.</li>
		<li>Turn off the microscope light. Remove the dichroic-containing box located in the light pathway. Turn off the excitation light and close the dark-box for complete darkness.</li>
		<li>Perfuse cells at 5-10 ml/min with HBS medium with or without test solutions pre-warmed at 37 &#186;C.</li>
		<li>Capture photonic emission images every 10 sec with a photon-counting camera handled with an image processor.</li>
		<li>At the end of each experiment, permeabilize cells with 0.1 mM digitonin in 10 mM CaCl2 in HBS in order to release all remaining photonic emissions. 
		Note: These photonic emissions must be added up in order to estimate the total photonic emissions, a value required for calibration in Ca2+ concentrations.</li>
		<li>Store bioluminescence images in the computer with the GFP associated fluorescence image captured before the photon counting imaging.</li>
	</ol>
	</li>
	<li>Analysis of bioluminescence images reflecting mitochondrial [Ca 2+ ].
	<ol>
		<li>Quantify photonic emissions using specific software. Photonic emissions can be converted to mitochondrial free Ca2+ concentration ([Ca2+]mit) using the algorithm described by Brini et al. 15.</li>
		<li>Open the experiment and the GFP fluorescence image. Select regions of interest (ROIs) with the help of specific software by drawing the shape of the transfected cells according to the fluorescence images captured at the beginning of the experiment.</li>
		<li>Paste the same ROIs on every image. Total photonic emissions in each ROI are computed with specific software to obtain the luminescence emission value (L) for each cell at each point in time.</li>
		<li>Add up all the photonic emissions in the bioluminescence images, including the ones obtained after digitonin permeabilization, using the specific software, to obtain a bioluminescence image containing all the photonic emissions.</li>
		<li>Export values of photonic emissions for every region of interest to a specific software for calculations. To do this, click on &#39;Graph&#39; in order to compute the bioluminescence values for every ROI at each time. Select &#39;total value&#39; and &#39;use current ROI to all images. Press &#39;calculate&#39;. Save the &#39;txt.file&#39; and export data to a worksheet. For every ROI, [Ca2+]mit is calculated using the following algorithm: 
		 
		[Ca2+](M) = [R + (R &#183; KTR) - 1] / [KR - (R &#183; KR)]; 
		 
		where, 
		R = [L/(Ltotal&#955;)]1/n 
		L is the luminescence emitted at the time of measurement in each region of interest. 
		Ltotal is the total luminescence remaining after addition of the counts present in the tissue at that time at the time of measurement for each region of interest. &#955;, KTR, KR and n are constants whose values depend on combination of aequorin (wild type or mutated) and coelenterazin used (wild type or n type) as well as the recording temperature 16. For the combination used here (mutated AEQ and coelenterzine n), at 37 &#186;C, we used the following values: KR = 8.47 x 107, KTR = 165.6 x 103, n = 1,204 and &#955; = 0,138.</li>
	</ol>
	</li>
	<li>Make the corresponding calculations for estimating the size of the rises in bioluminescence in response to each stimulus using a suitable data analysis and graphing software.</li>
</ol>

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None of the authors have competing interests or conflicting interests.

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 including ROS accumulation, formation of lipofuscin granules, heterochromatic foci, activation of pJNK and p53/p21 pathways, cholesterol loss, and changes the density of Ca2+ channels and NMDA receptors 14. Accordingly, Ca2+ imaging experiments in young and old cultures of rat hippocampal neurons may provide new insights of Ca2+ remodeling in the aging brain. A condition that is critical for culturing hippocampal neurons in the long term is two fold. First, it is important to plate the cells at a very low density as stated above. Second, cells are cultured in supplemented Neurobasal Medium without changing the medium. This approach allows the presence of glia but avoiding their overly growth. The schematic for such culture is shown in Figure 1.
Fluorescence imaging with synthetic Ca2+ probes allows the monitoring of cytosolic [Ca2+] in individual neurons 19. The use of this approach in young and aged neurons allows the monitoring of changes in the responses with age in culture. For instance Figure 2 shows typical bright field and Ca2+ images of the increases in [Ca2+]cyt induced by NMDA in young neurons and neurons from aged cultures. Notice that responses in aged neurons are much larger than in young neurons. However, this procedure is not reliable for [Ca2+] measurements within subcellular organelles as, for instance, mitochondria, where [Ca2+] may vary from below the &#181;M to above the mM level. In those cases, protein based, mitochondria-targeted probes have been developed as, for instance, aequorin. This probe is particularly interesting since its affinity for Ca2+ can be finely tuned to monitor very low or very high [Ca2+] like the ones reached inside mitochondria in resting and stimulated conditions, respectively. Specifically, it is possible to select either the wild type aequorin or a mutated aequorin without Ca2+ binding sites to modify the affinity. Fine tuning is achieved when different coelenterazines (wild type, h, n) are used in combination with different aequorins 17. However, whereas the use of fluorescence for Ca2+ imaging is widespread, monitoring bioluminescence is not straightforward and may require photon counting cameras. Fortunately, aequorin probes, like the one used here, have been improved to co-express GFP 18 making them more stable, able to release more photonic emissions and allowing simple identification of transfected cells by the GFP associated fluorescence. Success in bioluminescence imaging in neurons depends not only on the efficiency of the photon counting camera used but also on the efficiency of the transfection of the mitochondria-targeted probe. In the case of hippocampal neurons, a simple chemical transfection may work provided that a high sensitive photon counting camera is available. Figure 2 shows examples of bright field, GFP fluorescence and bioluminescence images of young and aged in culture hippocampal neurons. Also shown are the increases in mitochondrial [Ca2+] induced by NMDA recorded in transfected hippocampal neurons. Notice that the effect of NMDA is much larger in aged cultured neurons than in young neurons. In other cases, the efficiency of the transfection can be increased by using virus-derived vectors 16,18. Alternatively, the development of transgenic mice harboring protein-based, subcellular Ca2+ probes is an emerging option for future approaches, perhaps even in vivo.

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&#039;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&amp;nbsp;</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&amp;nbsp;</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 using papain. Isolated cells are washed and plated on coated coverslips. Then cells are cultured for 2-5 DIV or &#62;15 DIV to get young or aged cultures, respectively, and used for Ca2+ imaging experiments.
Using the above strategy, it is possible to have young and old neurons from the same specimen to test, for example, whether NMDA induces differential effects on cytosolic [Ca2+] in young cultures than in older cultures. Figure 2 shows that NMDA 100 &#181;M induces larger increases in cytosolic [Ca2+] in older cultures than in young cultures. Likewise, NMDA induces cell death in old neurons but not in young neurons 5. In cases where Ca2+ responses are very high, similar experiments can be carried out with Ca2+ probes with less affinity for Ca2+ to avoid dye saturation like, for instance fura4F 5. In the same manner, it is also possible to learn whether resting levels of cytosolic [Ca2+] are different. Moreover, the combination of this protocol with quantitative immunofluorescence using antibodies specific for NMDAR subunits may allow correlate changes in responsiveness with differences in expression of NMDA receptor subunits provided that specific antibodies are available 5. Measurements of cytosolic [Ca2+] can be used also for assessing other relevant parameters including Ca2+ store content, resting permeability to Ca2+, Ca2+ clearance rates and their possible differences between young and aged neurons.
In a similar fashion, it is also possible to test the effects of such stimuli on mitochondrial [Ca2+]. Figure 3 shows an example of bioluminescence imaging of hippocampal neurons transfected with mitochondria-targeted aequorin. Release of photonic emissions after stimulation is a function of the rise in mitochondrial [Ca2+] achieved. Notice that NMDA-induced rises in mitochondrial [Ca2+] are much larger in aged neurons than in young hippocampal neurons, where NMDA barely increases photonic emissions. These results may contribute to explain why NMDA induces apoptosis only in aged neurons but not in young cultures of hippocampal cells 5. In the same manner, it is possible to test for additional differences in mitochondrial Ca2+ handling between young and aged neurons using protocols specifically designed to test, for instance, Ca2+ exit from mitochondria, mitochondrial Ca2+ uptake in permeabilized neurons and mitochondrial Ca2+ uptake induced by Ca2+ release from intracellular stores. Moreover, this methodology could be used to test for drugs affecting mitochondrial Ca2+ overload that can be of interest for neuroprotection against excitotoxicity.
<img alt="Figure 1" src="/files/ftp_upload/53330/53330fig1.jpg" /> 
Figure 1: Procedure for isolation of primary rat hippocampal neurons. (A) Poly-D-lysine, 12 mm glass coated coverslips are prepared in a Petri dish and finally transferred to a 4-well dish. (B) Dorsal view of a rat brain. The dotted line indicates where the cut should be made. (C) Obtaining a suspension of primary hippocampal neuron cells. After removing the hippocampus, a cell suspension is obtained. Then cells are plated in the 4-well dishes containing the poly-D-lysine coated coverslips. (D) Bright field image showing primary rat hippocampal neurons in culture. Bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53330/53330fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53330/53330fig2.jpg" /> 
Figure 2: NMDA increases cytosolic [Ca2+] in hippocampal neurons. Short- and long term cultured hippocampal neurons were loaded with fura2/AM and subjected to fluorescence imaging. Pictures show representative bright field and pseudocolor images (Ratio F340/F380) of short-term (A) and long-term (B) hippocampal neurons before (basal) and after stimulation with 100 &#181;M NMDA. Warmer colors reflect elevated cytosolic [Ca2+] (pseudocolor scales are shown at the bottom). Traces show representative, single-cell recordings of cytosolic [Ca2+] in response to 100 &#181;M NMDA in short-term (A) and long-term (B) cultured hippocampal neurons. Note that cytosolic [Ca2+] increases are much larger in long-term than in short-term cultured neurons. Bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53330/53330fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53330/53330fig3.jpg" /> 
Figure 3: NMDA induces mitochondrial Ca2+ overload in hippocampal neurons. Cultured hippocampal neurons were transfected with the low-affinity, mitochondria targeted aequorin fused to GFP, incubated with 4 &#181;M coelenterazine and subjected to bioluminescence imaging of mitochondrial [Ca2+]. Pictures show the fluorescence (GFP) and accumulated photonic emissions (aequorin bioluminescence) images of representative short- (A) and long-term (B) cultured hippocampal neurons. Luminescence intensity is coded in pseudocolor (1 to 16 photonic emissions per pixel). Recordings show the release of photonic emissions (expressed as mitochondrial [Ca2+]) in short- (A) and long-term (B) cultured hippocampal neurons. NMDA 100 &#181;M increased mitochondrial [Ca2+] in aged neurons but not in young cultured hippocampal neurons. Bar represents 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53330/53330fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.3K Views.  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. 

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

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These amyloid fibrils can sequester endogenous metastable proteins as well as components of the proteostasis network (PN) and thereby exacerbate protein misfolding in the cell. There are a limited number of tools available to assess the aggregation process of amyloid proteins within an animal. We present a protocol for fluorescence lifetime microscopy (FLIM) that allows monitoring as well as quantification of the amyloid fibrilization in specific cells, such as neurons, in a noninvasive manner and with the progression of aging and upon perturbation of the PN. FLIM is independent of the expression levels of the fluorophore and enables an analysis of the aggregation process without any further staining or bleaching. Fluorophores are quenched when they are in close vicinity of amyloid structures, which results in a decrease of the fluorescence lifetime. The quenching directly correlates with the aggregation of the amyloid protein. FLIM is a versatile technique that can be applied to compare the fibrilization process of different amyloid proteins, environmental stimuli, or genetic backgrounds in vivo in a non-invasive manner.

Characterization of Amyloid Structures in Aging C. Elegans Using Fluorescence Lifetime Imaging

Fluorescence lifetime imaging monitors, quantifies and distinguishes the aggregation tendencies of proteins in living, aging, and stressed C. elegans disease models.

Amyloid fibrils are associated with a number of neurodegenerative diseases such as Huntington&#39;s, Parkinson&#39;s, or Alzheimer&#39;s disease. These ...

Biochemistry

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Behavior

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Developmental Biology

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

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

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