Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

Eiji Shigetomi,Baljit S Khakh

doi:10.3791/1142

Abstract

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.

Highlights

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 neurons[14]
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 Total internal reflection fluorescence (TIRF) microscopy is that cultured cells have to be used because they adhere to a glass coverslip within the evanescent field depth[3]

Summary

Part 1: PREPARING HIPPOCAMPAL ASTROCYTE CULTURES

Mixed hippocampal astrocyte-neuron cultures were prepared using a well established protocol[1,2,3]. 22 mm coverslips (VWR, 48380-068) Poly-D-Lysine (PDL, Sigma P0899), aliquots (1 mg/ml) 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. 5. Once the pieces of tissue have settled, remove papain carefully and add 5 ml of hippocampal medium to remove all traces of the enzyme. 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). 9. Leave them to attach for 60 min in the incubator, and add 2 ml of hippocampal medium per well. The day, aspirate old hippocampal medium to remove dead cells and debris and add 2 ml of pre-warmed fresh hippocampal medium. Preincubate the media about 30 min in the incubator in a ventilated flask to equilibrate the temperature and CO2

Part 2: CALCIUM IMAGING

Discussion