Name: Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes (Video) | JoVE Journal
Uploaded: 2009-04-26T23:27:00.000+00:00
Duration: 12 min 48 s
Description: Learn to measure near plasma membrane and global intracellular calcium dynamics in astrocytes using total internal reflection and epifluorescence microscopy. This method enables simultaneous observation of calcium signaling in astrocyte-neuron co-cultures.

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5 minutes

This work was supported by the Uehara Memorial Foundation of Japan (to ES) as well as the Whitehall Foundation, the National Institute of Neurological Disorders and Stroke and a Stein-Oppenheimer Endowment Award (to BSK).

EXPERIMENTAL PROCEDURES
The experimental procedure consists of two key parts that are described in a step wise manner below.
Part 1: PREPARING HIPPOCAMPAL ASTROCYTE CULTURES
Briefly, mixed hippocampal astrocyte-neuron cultures were prepared using a well established protocol1,2,3. We optimized the procedure to yield healthy cultured astrocytes. All the procedures listed below should be carried out in a sterile environment such as a laminar flow hood.
Preparing coverslips
<ul>
	<li>22 mm coverslips (VWR, 48380-068)</li>
	<li>Poly-D-Lysine (PDL, Sigma P0899), aliquots (1 mg/ml)</li>
	<li>Laminin aliquots (20 μg/ml): add 49 ml of sterile water to 1 mg/ml laminin solution (Sigma L2020), make 1.2 ml aliquots and store at -20°C</li>
</ul>
<ol>
	<li>Dissolve PDL in sterile water (50 μg/ml)</li>
	<li>Autoclave the coverslips, rinse with sterile water and place in PDL (20 ml for 100 coverslips). Leave at room temperature overnight.</li>
	<li>Replace PDL with sterile water (3 extensive washes). Then dry the coverslips and store at 4°C.</li>
	<li>The day before plating the astrocytes, place individual coverslips in a sterile well on a 6-well plate (Multiwell Flat-Bottom Plates with Lids, Sterile from BD Bioscience). Put 400 μl of laminin on each coverslip and incubate overnight, in the incubator to avoid evaporation.</li>
</ol>

Dissection
Dissection media:
<ul>
	<li>500 ml Earle’s Balanced Salt Solution (EBSS) (1X), liquid (Invitrogen, 14155-063)</li>
	<li>5 ml HEPES solution (Sigma, H0887)</li>
</ul>

Hippocampal media:
<ul>
	<li>412.5 ml Minimum Essential Medium (MEM) (1X), liquid Contains Earle's salts, but no L-glutamine or phenol red (Invitrogen, 51200-038)</li>
	<li>10 ml glucose (Sigma, D-(+)-GLUCOSE ANHYDROUS SIGMA ULTRA G7528) 1 M in MEM</li>
	<li>5 ml Penicillin-Streptomycin (Invitrogen, 15140-122)</li>
	<li>5 ml Sodium pyruvate solution (Sigma, S8636)</li>
	<li>12.5 ml HEPES solution 1 M (Sigma, H0887)</li>
	<li>5 ml N-2 Supplement (100X), liquid (Invitrogen, 17502-048)</li>
	<li>50 ml Horse Serum, Heat-Inactivated (Invitrogen, 26050-088)</li>
</ul>


Papain solution:
<ul>
	<li>Add 5 ml of dissection media into Worthington PAPAIN-022 vial (LK003178; final concentration, 20 U/ml) and incubate at 37°C for 60 min.</li>
</ul>

<ol>
	<li>Decapitate rat pups aged P1 (typically 2 pups) following guidelines vetted and approved by national regulations and your local Institutional Animal Care and Use Committee.</li>
	<li>Remove skin and skull and place brain in Petri dish filled with cold dissection media.</li>
	<li>Removed meninges, dissect both hemispheres and dissect hippocampi.</li>
	<li>Chop the hippocampi into ~1X1 mm pieces and digest in papain solution at 37°C for 11-13 min.</li>
	<li>Once the pieces of tissue have settled, remove papain carefully and add 5 ml of hippocampal medium to remove all traces of the enzyme. Repeat this step and resuspend in hippocampal medium (2 ml).</li>
	<li>Triturate the cells ~ 5 times until there are no clumps left, with three flame-polished pipettes of progressively smaller bores (1000 μm, ~500 μm and ~300 μm).</li>
	<li>Once triturated, pass the cells through a cell strainer (pore size, 70 μm, BD Bioscience). Count the cells in 10 μl of suspension. Adjust the volume of hippocampal medium in order to have 400000 cells/ml.</li>
	<li>Aspirate the laminin off the coverslips, and before the covers can dry, plate 200 μl of the cell suspension per coverslip.</li>
	<li>Leave them to attach for 60 min in the incubator, and then add 2 ml of hippocampal medium per well.</li>
	<li>The next day, aspirate old hippocampal medium to remove dead cells and debris and add 2 ml of pre-warmed fresh hippocampal medium.</li>
</ol>

Maintenance
Neurobasal media:
<ul>
	<li>500 ml neurobasal (Invitrogen, 12348-017)</li>
	<li>10 ml B27 serum free supplement (Invitrogen, 17504-044)</li>
	<li>5 ml Penicillin-Streptomycin  (Invitrogen, 15140-122)</li>
	<li>1.25 ml L-glutamine (Invitrogen, 25030-149)</li>
</ul>
Feed the astrocyte-neuron cultures twice a week with neurobasal medium, starting four days after plating. Preincubate the media about 30 min in the incubator in a ventilated flask to equilibrate the temperature and CO2.
Part 2: CALCIUM IMAGING
Hippocampal recording buffer: 110 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 10 mM D-glucose, 10 mM HEPES (All chemicals from Sigma) pH 7.4 (adjusted with NaOH).
Loading calcium indicator dye into astrocytes
<ol>
	<li>Place the culture in a well on a 6-well plate filled with 2 ml hippocampal recording buffer containing 2.5 μM Fluo-4, AM (Invitrogen, F-14217) and 0.05% Pluronic® F-127 20% solution in DMSO (Invitrogen, P-3000MP) and incubate at room temperature for 10-30 min.</li>
	<li>Remove Fluo-4 solution and wash coverslips 3 times with hippocampal recording buffer and incubate at room temperature for 30 min.</li>
	<li>Place the coverslip on the chamber, and clean the other side of coverslip with lens cleaning liquid.</li>
	<li>Put one small drop of immersion oil (Immersion Oil TYPE DF from Cargille) on the objective and put chamber (Open chamber for 25 mm round coverslip from Warner Instruments) on the stage of the microscope (Olympus IX71).</li>
	<li>Look at the cells using transmission light to see how the cells look, and get them into focus. Then illuminate the cells with 488 nm from a monochromator (Polychrome V from TILL Vision) and determine if the fluorescence signal of Fluo-4 is uniform and detectable in astrocytes.</li>
	<li>Use EPI and TIRF illumination to measure calcium in astrocytes.</li>
</ol>

TIRF microscopy
Briefly, we use an Olympus IX71 microscope equipped with an Andor IXON DV887DCS EMCCD camera.  The control of excitation and image acquisition is achieved using TILLVision software.  The beams of 454/488/515 nm Argon (100 mW) and 442 nm solid state (45 mW) lasers are combined and controlled with a TILL Polyline laser combiner, TIRF dual port condenser and acoustoptical tuneable filter and controller (AOTF; all from TILL Photonics) and fed into a Kineflex broad band fiber for entry into the TIRF condenser. We use an Olympus 60X 1.45 NA lens to achieve TIRF. The camera gain is adjusted for each astrocyte to provide the best signal to noise images. The background and principles of TIRF microscopy have been recently reviewed4, 5. Most of the optical components we use were purchased from TILL Photonics, which is now part of Agilent Technologies (http://www.till-photonics.com/). The TIR penetration depth can be calculated from the equations below.
<img alt="Optical resolution equation with numerical aperture NA in microscopy formula diagram." src="/files/ftp_upload/1142/equation.jpg" style="float:left;" data-altupdatedat="1787630408000" data-alt="Equation">

d = penetration depth
n1 = refractive index of glass
n2 = refractive index of cell
a = angle of incidence
NAi = numerical aperture of incidence
 
In order to ensure the laser is aligned optimally for TIRF we find it useful to observe 100 nm fluorescent bead (Invitrogen, F8803). We present still frames and videos of beads with EPI and TIRF microscopy. When in TIRF, one observes a dramatic increase in signal-to-noise and the beads display Brownian diffusion. We find it useful to observe the behavior of 100 nm beads with TIRF microscopy on a regular basis (~once per week) to be sure that optimal TIRF occurs, rather than the compromised oblique illumination that would occur if the critical angle were not equal to α (see Fig 1).
Application of G-protein coupled receptor agonists
Astrocytes express a variety of Gq-coupled receptors6, 7 including metabotropic glutamate receptors and P2Y receptors (agonist, ATP, ADP). Activation of these receptors leads to significant increases in intracellular calcium levels within astrocytes. For instance, one can readily observe intracellular calcium elevations during application of ATP (30 μM) to astrocytes8-10. We use a fast solution switcher from Warner Instruments called the VC-77SP Fast-Step Perfusion System (http://www.warneronline.com/index.cfm). With this method solutions can be applied in less than ~10 ms.
<img alt="Spectroscopy setup with Olympus IX71 microscope and Andor iXon EMCCD; includes AOTF and laser system." src="/files/ftp_upload/1142/fig1a.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 
 
 
 
 
<img alt="Total internal reflection fluorescence (TIRF) microscopy setup with protein analysis diagram." src="/files/ftp_upload/1142/fig1c.jpg" data-altupdatedat="1787630408000">
FIGURE 1. Cartoon and photographs of the imaging set up. A. Shows a photograph of the microscope mounted on an airtable, whereas (B) shows a photograph of the laser assembly, controllers and beam boxes. C. Shows a photograph of the microscope stage with the chamber mounted for imaging.  On the left the fast perfusion device can be seen (along with the stepper motor and theta tubing). On the right the headstage of an Axopatch 200A amplifier is seen.  The cartoon schematizes the light path in the set up and how TIRF is achieved. The laser is focused on the back focal plane of the 60X 1.45 NA objective lens and its position is adjusted off center so that it emerges into the immersion oil at the critical angle α. At this point the beam suffers from total internal reflection and decays with a distance λ (see equation in main text) into the medium of lower refractive index. In this case this is the recording buffer surrounding the astrocytes and the astrocytes themselves. The result is optical excitation (and thus imaging) of molecules within ~100 nm of the plasma membrane. In the cartoon of the cell this is shown as green “excited” membrane receptors, whereas those within the cell or on the top surface of the cell are not excited. A full account of TIRF microscopy has been provided by Steyer and Almers4.
<img alt="EPI vs. TIRF microscopy diagram; contrast comparison, cellular imaging, fluorescence technique." src="/files/ftp_upload/1142/fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2">
FIGURE 2. Images of 100 nm fluorescent beads acquired with EPI and TIRF microscopy. A. Shows EPI images of a field of view with several dozen 100 nm fluorescent beads. The red arrows point to beads that have settled onto the glass coverslip, whereas the blue arrowheads point to beads that are diffusing in water. B. Shows a TIRF image of the same field of view as shown in A. In this view only the adherent beads shown by red arrows are visible. This is because these had settled onto the glass coverlsip and were thus within the ~100 nm evanescent field. The beads shown in A by blue arrows are not within this region and are thus invisible in the TIRF images. The lower plots show 3D rendering of the images. It is clear that a large increase in signal-to-noise occurs for beads within the evanescent field when observed by TIRF microscopy. In fact for these images the signal-to-noise for EPI was 7.1 ± 0.6, whereas for TIRF it was 20 ± 0.7.
<img alt="Fluorescence microscopy comparison, EPI vs. TIRF; ATP response, intensity graph, 10 µm scale bar." src="/files/ftp_upload/1142/fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3">
FIGURE 3. Images of astrocytes loaded with Fluo-4 calcium indicator dye acquired with EPI and TIRF microscopy.  A. EPI images of a field of view with five astrocytes. B. A TIRF image of the same field of view shown in B. Note that the images in A and B are significantly different. This is because with TIRF illumination only the plasma membrane regions in close apposition to the glass coverslip are imaged.  The lower panels show ATP-evoked intracellular calcium transients imaged with EPI and TIRF microscopy.

<ol><li>Richler, E., Chaumont, S., Shigetomi, E., Sagasti, A., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+approach+to+image+activation+of+transmitter-gated+P2X+receptors+in+vitro+and+in+vivo%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">An approach to image activation of transmitter-gated P2X receptors in vitro and in vivo.</a> Nature Methods. 5, 87-93 (2008).</li>
<li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51, 773-786 (2006).</li>
<li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+culture+of+hippocampal+neurons+from+P0+newborn+rats%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Primary culture of hippocampal neurons from P0 newborn rats.</a> J Vis Exp. 19, (2008).</li>
<li>Steyer, J. A., Almers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+real-time+view+of+life+within+100+nm+of+the+plasma+membrane%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">A real-time view of life within 100 nm of the plasma membrane.</a> Nat Rev Mol Cell Biol. 2, 268-275 (2001).</li>
<li>Jaiswal, J. K., Fix, M., Takano, T., Nedergaard, M., Simon, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resolving+vesicle+fusion+from+lysis+to+monitor+calcium-triggered+lysosomal+exocytosis+in+astrocytes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes.</a> Proc Natl Acad Sci U S A. 104, 14151-14156 (2007).</li>
<li>Araque, A., Carmignoto, G., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+signalling+between+astrocytes+and+neurons%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Dynamic signalling between astrocytes and neurons.</a> Annu Rev Physiol. 63, 795-813 (2001).</li>
<li>Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GLIA%3A+listening+and+talking+to+the+synapse%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">GLIA: listening and talking to the synapse.</a> Nat Rev Neurosci. 2, 185-193 (2001).</li>
<li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATP+excites+interneurons+and+astrocytes+to+increase+synaptic+inhibition+in+neuronal+networks%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">ATP excites interneurons and astrocytes to increase synaptic inhibition in neuronal networks.</a> J Neurosci. 24, 8606-8620 (2004).</li>
<li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+forms+of+astrocyte+single+vesicle+exocytosis+imaged+with+total+internal+reflection+fluorescence+microscopy%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Two forms of astrocyte single vesicle exocytosis imaged with total internal reflection fluorescence microscopy.</a> Proc Natl Acad Sci U S A. 104, 4212-4217 (2007).</li>
<li>Bowser, D. N., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicular+ATP+is+the+predominant+cause+of+intercellular+calcium+waves+in+astrocytes%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes.</a> J Gen Physiol. 129, 485-491 (2007).</li>
<li>Agulhon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+is+the+role+of+astrocyte+calcium+in+neurophysiology%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">What is the role of astrocyte calcium in neurophysiology.</a> Neuron. 59, 932-946 (2008).</li>
<li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mystery+and+magic+of+glia%3A+a+perspective+on+their+roles+in+health+and+disease%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60, 430-440 (2008).</li>
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<li>Shigetomi, E., Bowser, D. N., Sofroniew, M. V., Khakh, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+forms+of+astrocyte+calcium+excitability+have+distinct+effects+on+NMDA+receptor-mediated+slow+inward+currents+in+pyramidal+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons.</a> J Neurosci. 28, 6659-6663 (2008).</li>
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It is well-established that astrocytes display intracellular calcium elevations. These occur spontaneously, can be triggered by neuronal activity or by application of agonists to activate receptors on the astrocyte surface11. One important and controversial issue is whether astrocyte intracellular calcium elevations can trigger the release of signaling molecules that activate receptors on neurons11, 12. This is controversial because there has been evidence for and against this view, as highlighted i...

<table><tbody><tr><td>VWR&amp;#174; Micro Cover Slips, Round, No. 1</td><td>Tool</td><td>VWR international</td><td>48380-068</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P0899</td><td></td></tr><tr><td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td><td>Reagent</td><td>Sigma-Aldrich</td><td>L2020</td><td></td></tr><tr><td>Earle&amp;rsquo;s Balanced Salt Solution (EBSS) (1X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>14155-063</td><td></td></tr><tr><td>Minimum Essential Medium (MEM) (1X), liquid Contains Earle&amp;rsquo;s salts, but no L-glutamine or phenol red</td><td>Reagent</td><td>Invitrogen</td><td>51200-038</td><td></td></tr><tr><td>Penicillin-Streptomycin liquid</td><td>Reagent</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>Sodium pyruvate solution</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S8636</td><td></td></tr><tr><td>HEPES solution 1 M</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>N-2 Supplement (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17502-048</td><td></td></tr><tr><td>Horse Serum, Heat-Inactivated</td><td>Reagent</td><td>Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>PAPAIN-022</td><td>Reagent</td><td>Worthington Biochemical</td><td>LK003178</td><td></td></tr><tr><td>Neurobasal&amp;#8482; Medium (1X) Liquid without Phenol Red</td><td>Reagent</td><td>Invitrogen</td><td>12348-017</td><td></td></tr><tr><td>B-27 Serum-Free Supplement (50X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>17504-044</td><td></td></tr><tr><td>L-Glutamine-200 mM (100X), liquid</td><td>Reagent</td><td>Invitrogen</td><td>25030-149</td><td></td></tr><tr><td>Cell Strainers</td><td>Tool</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>BD Falcon Multiwell Flat-Bottom Plates with Lids, Sterile</td><td>Tool</td><td>BD Biosciences</td><td>353046</td><td></td></tr><tr><td>NaCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Reagent</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>21108</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; hexahydrate</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M2670</td><td></td></tr><tr><td>HEPES free acid</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>D-(+)-glucose</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G7528</td><td></td></tr><tr><td>Fluo-4, AM 1 mM solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>F-14217</td><td></td></tr><tr><td>Pluronic&amp;#174; F-127 20% solution in DMSO</td><td>Reagent</td><td>Invitrogen</td><td>P-3000MP</td><td></td></tr><tr><td>Immersion Oil TYPE DF</td><td>Microscope</td><td>Cargill Labs</td><td>16242</td><td></td></tr><tr><td>Open chamber for 25 mm round coverslips, 100 &amp;mu;l volume</td><td>Tool</td><td>Warner Instruments</td><td>64-0362 (RC-21BDW)</td><td></td></tr><tr><td>P-2 platform for Series 20 chambers, non-heater</td><td>Tool</td><td>Warner Instruments</td><td>64-0278 (P-2)</td><td></td></tr><tr><td>FluoSpheres carboxylate-modified microspheres, 0.1 &amp;mu;m, yellow-green fluorescent (505/515) 2% solids</td><td>Reagent</td><td>Invitrogen</td><td>F8803</td><td></td></tr></tbody></table>

measuring near plasma membrane and global intracellular calcium dynamics in astrocytes

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

In the first part of this video, we will demonstrate how to prepare the astrocyte neuron co-culture. To do this, several hippo Campi from P one rats are collected into a tube digested with propane, dissociated by ation, and then plated onto cover slips. The second section will show how to measure intracellular calcium dynamics in astrocytes, which involves loading of flu oh four calcium indicator dye into astrocytes and measuring global calcium dynamics by epi microscopy and measuring near membrane calcium dynamics by turf microscopy.By changing the illumination automatically and quickly, both global calcium and near membrane calcium can be observed almost simultaneously. Hi, I'm Aji omi, a postdoctoral researcher from Budget COS lab in the Department of Physiology at the University of California Los Angeles. Today we'll show you a procedure for calcium imaging of asto sites.We use this procedure in our laboratory study near membrane and global calcium dynamics in as osteocyte. By changing the elimination automatically and quickly, we can resolve global calcium and near membrane calcium. Almost simultaneously, I will show how we apply a TP to cause calcium elevation in astrocyte.I will then finish by summarizing and discussing the barrier with the method for studying astrocyte. So let's get studied. To begin the astrocyte culture, remove the skin and skull from the head of a decapitated rat pup.Place the brain in a Petri dish filled with cold dissection media, dissect both hemispheres and remove the meninges. Next, dissect the hippo campi. Chop the hippo campi into approximately one by one millimeter pieces.Digest in Pappa solution at 37 degrees Celsius for 11 to 13 minutes. Remove the dissected tissue from the incubator. Then once the pieces of tissue have settled, remove them from the propane digestion carefully and add five milliliters of hippocampal medium to wash the chopped pieces of hippo Campi.Repeat this step and then finally, resus suspend in hippocampal medium. Iterate the cells about five times with three flame polished progressively smaller pipettes. Once ated passed the cells through a cell strainer with a whole size of 70 micrometers and add four milliliters of medium to the strainer.Count the cells in 10 microliters of suspension. Adjust the volume of hippocampal medium in order to have 400, 000 cells per milliliter. Now prepare cover slips for seeding the cell suspension by aspirating laminate from the cover slips, which have been exposed to a laminate solution overnight.Before the covers can dry plate 200 microliters of the cell suspension per cover slip. Leave them to attach for 60 minutes in the incubator. After incubation, add two milliliters of hippocampal medium per well.The next day aspirate the old hippocampal medium to remove dead cells and debris and add two milliliters of pre-warned fresh hippocampal. Medium maintenance of astrocyte neuron Co cultures should begin four days after plating and adding one milliliter of fresh neuro basal medium twice a week. Be sure to pre incubate the media about 30 minutes in the incubator in a ventilated flask to equilibriate the temperature and carbon dioxide.Before studying the astrocytes, it is useful to look at nano fluorescent beads to optimize laser angle for turf. To do this, put 200 microliters of water on the Petri dish and add one microliter of 100 nanometer fluorescent beads and mix look at the beads by epi fluorescence microscopy. Next, look at the beads by turf microscopy.For calcium imaging, place the culture in a well on a six well plate filled with two milliliters hippocampal recording buffer containing 2.5 micromolar flu oh 4:00 AM and 0.05%onic F1 27, 20%solution in DMSO and incubate at room temperature for 10 to 30 minutes, remember to cover the plate to avoid bleaching the fluoro. Four, remove the flu oh four solution and wash the cover Slips three times with hippocampal recording buffer. Incubate the washed cover slips at room temperature for 30 minutes.Place the cover slip in the chamber, clean the other side of the cover slip with lens cleaning liquid and add 200 microliters of hippocampal buffer buffer. Put one small drop of immersion oil on the objective and put the chamber on the stage of the microscope. Look at the cells using transmission light to focus and see how the cells look.Next, illuminate the cells with 488 nanometers from a monochrome and determine if the fluorescent signal of flu oh four is uniform and detectable. In astrocytes, use epi and turf illumination to measure calcium in the astrocytes. Here, fluorescent beads are observed by epi illumination.In addition to producing background fluorescence, they also display significant brownian motion as the angle of illumination. With the laser approaches the critical angle, the laser beam is totally reflected from the glass water interface. This total internal reflection generates an electromagnetic field called the evanescent field.This has a depth into the cell of lower refractive index of about 100 nanometers. You can excite floral fours in this thin layer on the specimen surface. Here are fluorescent beads observed by turf illumination when compared with epi.Illumination signal to noise ratio significantly increases when you decrease the angle. The beam of the laser does not reflect at the surface and passes through the cover slip to hit the fluorescent beads directly. You can see a lot of background fluorescence.Now we increase the angle. Again, background noise significantly decreases and you can see fluorescent beads near the surface of the cover slip. These images show flu oh four loaded astrocytes and neurons.Astrocytes are flat cells with fine processes, whereas neurons have round cell bodies and thick and long dendrites. Both astrocytes and neurons look healthy and loaded with flu. Oh four properly.Looking at astrocytes by epi or confocal microscopy is useful to know if astrocytes are healthy and if calcium indicators are loaded into astrocyte properly. In the regions of the astrocyte that are closest to the cover glass regions called the footprint, a TP is observed to cause a calcium increase near the plasma membrane. Hippocampal astrocytes are known to express pure anergic receptors and a TP when applied to the bath can cause a global calcium release.We've just shown you how to prepare hippocampal osteocyte culture and how to measure the calcium level of as osteocyte both globally and near plasma membrane. When doing this procedure, it's important to remember to optimize laser angle for the best way to ensure this on the microscope is to observe the behavior of 100 millimeter flow speeds. The simultaneous recording of global calcium and near membrane calcium level will provide us not only detailed description of calcium signal, but new insight of the mechanism of calcium regulation in astrocyte.It also occurred to us that the approach described here is useful for measuring near membrane and global intracellular calcium level in other excitable unknown excitable field. So that's it. Thank you for watching and good luck with your experiment.

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Research

JoVE Journal

Biology

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

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Here we present a protocol for Ca2+ imaging in neurons and glial cells, which enables the dissection of Ca2+ signals at subcellular resolution. This process is applicable to all cell types that allow the expression of genetically encoded Ca2+ indicators.

Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Here we describe an in vitro live-imaging method to visualize intracellular transport of organelles and trafficking of plasma membrane proteins in murine astrocytes. This protocol also presents an image analysis methodology to determine cargo transport itineraries and kinetics.

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

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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.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

This protocol provides a comprehensive description of a method for the simultaneous recording of calcium signals from neurons and astrocytes across multiple memory-related brain regions and demonstrates its application during a learning and memory behavior paradigm.

A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors

Learn to measure near plasma membrane and global intracellular calcium dynamics in astrocytes using total internal reflection and epifluorescence microscopy. This method enables simultaneous observation of calcium signaling in astrocyte-neuron co-cultures.

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

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