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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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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</ol>

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

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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

Source: Bannai, H., et. al., Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators. J. Vis. Exp. (2019)The video demonstrates fluorescence imaging to observe compartment-specific spontaneous calcium dynamics in astrocytes by sequentially monitoring calcium-sensitive proteins localized on the plasma membrane and endoplasmic reticulum.

Spontaneous Calcium Imaging for Compartment-Specific Dynamics in Astrocytes

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

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.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

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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