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

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.

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.

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Protocol

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

1. Preparation of poly-D-lysine coated, 12 mm glass coverslips.
   1. Sterilize 12 mm diameter glass coverslips in ethanol for at least 24 hr. Allow them to dry under sterile conditions.
   2. Distribute the coverslips on a strip of parafilm in a Petri dish. Cover the surface of each coverslip with 200 µl of 1 mg/ml poly-D-lysine. Allow treating O/N.
   3. The next day wash the coverslips with double distilled sterile water every 15 min for 90 min under sterile conditions.
   4. Fill a four-well multidish plate with 500 µl of Neurobasal Culture Medium per well. Neurobasal Culture Medium should be supplemented with 10% fetal bovine serum, 2 % B27, 1 µg/ml gentamicin and 2 mM L-glutamine.
   5. Place the coverslips in the multidish plate. Maintain them in a humidified 37 ºC and 5% CO₂ incubator until use.
2. Isolation of hippocampal neurons from neonatal rats.
   1. Prepare 1.8 ml papain solution (directly purchased) in a vial (20 µl/ml) under sterile conditions: To obtain a final concentration of 20 µl/ml add around 40 µl papain in 1.8 ml Hank's plus 0.6% BSA (µl should be calculated depending on the supplier conditions). Hank'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.
   2. Incubate the papain in Hank's plus 0.6% BSA for 30 min at 37 ºC before use. Filter the solution using a 0.22 µm filter prior to use.
   3. Prepare Ham's F-12 medium by adding Dulbecco's Modified Eagle'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 µm 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 ºC and use them chilled.
   4. 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's F-12 medium.
   5. Make a diagonal cut with the aid of a scalpel in each hemisphere (Figure 1B), and carry it to a Petri dish containing Ham's F-12 medium.
   6. Discard meninges carefully with the help of dissection forceps and under a magnifying glass.
   7. 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.
   8. Transfer the hippocampal tissue to a 4-well plate filled with sterile Hank'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.
   9. Transfer hippocampal pieces to a vial containing 1.8 ml pre-filtered papain solution. Then incubate them at 37 ºC with occasional, gentle shaking. 15 min later, add 90 µl of DNase I solution (50 µg/ml final) and incubate for 15 additional min.
   10. 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.
   11. 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.
   12. Measure cell density using a Neubauer counting chamber. Put the glass coverslip over the Neubauer chamber. Add 10 µl 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 µl containing 30 x 10³ cells. The total number of cells will depend on the number of animals used.
3. Short-term and long-term culture of rat hippocampal neurons.
   1. Over the four-well multidish plate containing 500 µl of Neurobasal Culture Medium prepared before and kept in the incubator, plate around 30 x 10³ cells in a drop of about 50 µl to each well of the multidish plate containing one poly-D-lysine-coated, 12 mm diameter glass coverslip.
   2. Maintain primary hippocampal cells in a humidified 37 ºC and 5% CO₂ incubator for 2-5 days in vitro (DIV, short-term, young cultures) or >15 DIV (Long-term, aged cultures) before experiments without changing the culture media.

2. Fluorescence Imaging of Cytosolic Ca ²⁺ Concentration

1. Preparation of test solutions for fluorescence imaging.
   1. 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).
   2. 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).
   3. Solve NMDA in Mg²⁺-free, HEPES-buffered saline (HBS) at a final concentration of 100 µM and supplement it with 10 µM glycine.
   4. 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).
2. Loading of hippocampal cells with fluorescent calcium probe fura2/AM.
   1. 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 µl of HBS per well.
   2. Incubate cells with fura2/AM 4 µM (prepared in the same HBS medium) for 60 min at RT (25 ºC) in a dark place.
   3. After 60 min, wash coverslips with fresh HBS medium.
3. Recording fluorescence images of cytosolic [Ca ²⁺ ] in cultured cells.
   1. Turn on the lamp, microscope, perfusion system, fluorescence camera and computer.
   2. 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 ºC) 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.
   3. Capture a background image with the shutter closed at both excitation wavelengths.
   4. 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.
   5. When the recording period is finished, store the complete sequence of images emitted at 520 nm in the computer for further analysis.
4. Analysis of recorded fluorescent images.
   1. Open the experiment file. Using the aquacosmos software, click on 'Ratio' 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.
   2. Subtract background by adjusting the 'background elimination'. Press 'start calculation'.
   3. Press 'all times sequence' 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).
   4. To graph the individual recordings export the ratio fluorescence values corresponding to each ROI to a graphing program.
   5. 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.

3. Bioluminescence Imaging of Mitochondrial Ca ²⁺ Concentration

1. 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. Brûlet and co-workers ¹⁸ (CNRS, Paris).
   1. Prepare Neurobasal TF, containing Neurobasal Culture Medium, lacking fetal bovine serum, B27, gentamicin and 2 mM L-glutamine.
   2. Prepare 50 µl of Neurobasal TF plus 2.5 µl transfection reagent in a vial (Solution A). Prepare 50 µl of Neurobasal TF plus 4 µg mitGAmut plasmid in a vial (Solution B). Add gently solution B over solution A. Allow mixing during 20 min, without shaking.
   3. Transfer the coverslips containing hippocampal neurons to new multidish plates containing 200 µl of fresh Neurobasal TF.
   4. Add drop by drop 100 µl 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.
   5. Culture neurons for an additional 24 hr at 37 ºC and 5% CO₂ after transfection to allow efficient expression and targeting of the probe.
2. Preparation of test solutions for bioluminescence imaging.
   1. Prepare HBS solutions containing NMDA 100 µM or 145 mM KCl as above (Step 2.1). Prepare HBS solution containing 10 mM Ca²⁺ plus digitonin 100 µM.
3. Reconstitution of mitochondria-targeted aequorin with coelenterazine n.
   1. Transfer coverslips containing transfected hippocampal neurons to a four-well plate containing 200 µl of HBS.
   2. Add 4 µl of coelenterazine n (200 µM) to 200 µl of HBS for a final concentration of 4 µM and mix gently. Incubate for 2 hr at RT (25 ºC) and in the dark.
4. Recording bioluminescence images of mitochondrial [Ca ²⁺ ].
   1. Turn on the microscope, the lamp, the perfusion system, bioluminescence camera and the computer.
   2. Transfer reconstituted coverslips to a thermostated (37 ºC) perfusion chamber and perfuse continuously with pre-warmed HBS solution at a rate of 5-10 ml/min.
   3. 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.
   4. 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.
   5. Perfuse cells at 5-10 ml/min with HBS medium with or without test solutions pre-warmed at 37 ºC.
   6. Capture photonic emission images every 10 sec with a photon-counting camera handled with an image processor.
   7. 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.
   8. Store bioluminescence images in the computer with the GFP associated fluorescence image captured before the photon counting imaging.
5. Analysis of bioluminescence images reflecting mitochondrial [Ca ²⁺ ].
   1. 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.
   2. 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.
   3. 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.
   4. 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.
   5. Export values of photonic emissions for every region of interest to a specific software for calculations. To do this, click on 'Graph' in order to compute the bioluminescence values for every ROI at each time. Select 'total value' and 'use current ROI to all images. Press 'calculate'. Save the 'txt.file' and export data to a worksheet. For every ROI, [Ca²⁺]_mit is calculated using the following algorithm:
      
      [Ca²⁺](M) = [R + (R · K_TR) - 1] / [K_R - (R · K_R)];
      
      where,
      R = [L/(L_totalλ)]^1/n
      L is the luminescence emitted at the time of measurement in each region of interest.
      L_total 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. λ, K_TR, K_R 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 ºC, we used the following values: K_R = 8.47 x 10⁷, K_TR = 165.6 x 10³, n = 1,204 and λ = 0,138.
6. 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.

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Results

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 ...

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Discussion

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...

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Materials

Name	Company	Catalog Number	Comments
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