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.
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.
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.
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 µM 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.
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
2. Fluorescence Imaging of Cytosolic Ca 2+ Concentration
3. Bioluminescence Imaging of Mitochondrial Ca 2+ Concentration
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 >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 µM 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.
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 µm. Please click here to view a larger version of this figure.
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 µM 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 µM 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 µm. Please click here to view a larger version of this figure.
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 µM 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 µM increased mitochondrial [Ca2+] in aged neurons but not in young cultured hippocampal neurons. Bar represents 10 µm. Please click here to view a larger version of this figure.
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 µM 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.
The authors have nothing to disclose.
This work was supported by Ministerio de Economìa y competitividad (BFU2012-37146) and Junta de Castilla y Leòn (BIO103/VA45/11, VA145U13 and BIO/VA33/13), Spain. MCR was supported by Junta de Castilla y Leòn (Spain) and the European Social Fund. We thank the late Dr. Philippe Brûlet (1947-2013) for the mitochondrial GFP Aequorin plasmid.
Neurobasal Culture Medium | Gibco | 21103-049 | |
HBSS medium | Gibco | 14170-088 | |
Ham's F-12 medium | Gibco | 31330-038 | |
DNase I (from bovine pancreas) | Sigma | D5025-15KU | |
Fetal Bobine Serum | Lonza | DE14-801E | |
B27 | Gibco | 17504-044 | |
Gentamicin | Gibco | 15750 | |
L-glutamine | Gibco | 25030-032 | |
12 mm glass coverslips | Labolan | 0111520/20012 | |
Papain | Worthington | LS003127 | |
4-well multidish plaques | Nunc | 176740 | |
Petry dishes | JD Catalan s.l. | 2120044T | |
Sterile pipettes | Fisher Scientific | 431030 | |
Fura2-AM | Life Technologies | F1201 | |
Lipofectamine2000 | Invitrogen | 11668-027 | |
Coelenterazine n | Biotium | BT-10115-2250 uG | |
Digitonin | Sigma | D5628 | |
NMDA | Sigma | M3262 | |
Glycine | Sigma | 50046 | |
Zeiss Axiovert S100 TV microscope | Carl Zeiss Inc. | ||
Xcite ilumination system | EXFO | ||
ORCA ER fluorescence camera | Hamamatsu | ||
VIM photon counting CCD camera | Hamamatsu | ||
VC-8 valve controller | Warner Instruments | ||
SH-27B heating system | Warner Instruments | ||
Aquacosmos Software | Hamamatsu Photonics |