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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.
The experimental procedure consists of two key parts that are described in a step wise manner below.
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.
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.
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).
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.
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).
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.
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 in the reviews by the Haydon7,13 and McCarthy11 labs. Based on our recent brain slice imaging and electrophysiology data we argued that a better and precise understanding of astrocyte calcium dynamics is needed before new hypothesis driven experiments can be designed to determine how astrocytes impact neurons14. In this video article we present a simple method to image near plasma membrane and global intracellular calcium changes almost simultaneously in cultured astrocytes. An unavoidable technical requirement of TIRF microscopy is that cultured cells have to be used because they adhere to a glass coverslip within the evanescent field depth3. It is worth noting that astrocytes in culture change their gene expression profiles when compared to those in vivo15, and so this caveat should be considered when implementing this method. With this consideration in mind however the simple method we report does allow one to image and quantify near plasma membrane and global intracellular calcium changes almost simultaneously. The further application of the approach to astrocytes holds the promise of providing accurate data on intracellular calcium changes near the plasma membrane of astrocytes. The availability of such quantitative data will be useful for the complete understanding of astrocyte biology.
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).