Ratiometric Calcium Imaging in Chick Optic Tectum Astrocyte Cultures

Undergraduate #45
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Mercedes K. Howard - Delaware State University
Co-Author(s): Sai Kodali, University of Delaware, Newark, DE Karla Sanchez and Murali Temburni, Delaware State University, Dover, DE

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Establishing functional neuronal networks during brain development requires synchronous oscillatory activity among neurons. However, the mechanisms of synchronization are not fully understood. Current models of neuronal synchronous activity assume that it is a process intrinsic to neurons. Astrocytes participate in neuronal communication by releasing ‘gliotransmitters’ like glutamate, ATP and D-serine. Evidence that glial cells particularly astrocytes modulate synchronous activity in networks of neurons is accumulating. During development signaling between astrocytes and neurons is crucial for the establishment of synchronously oscillating neuronal networks.

Our preliminary results indicate that astrocytes are necessary for synchronous activity of embryonic chick optic tectum neurons in culture. To further dissect the molecular pathways involved we are targeting GPCR pathways (mGluR, P2Y1 and GABAB) within astrocytes that mediate intracellular calcium release. Activation of these G-protein coupled receptors by their respective neurotransmitters mobilizes intracellular calcium release leading to exocytosis of either glutamate or ATP. We have also demonstrated that chick optic tectum astrocytes express mGluR1 receptors. We set out to measure intracellular Ca++ release in astrocytes upon simulation with DHPG, an mGluR agonist using ratiometric imaging. Briefly, astrocytes grown on coverslip bottom dishes were loaded with the ratiometric fluorescent dye Fura-2 AM and imaged on an Olympus IX-71 inverted fluorescent microscope equipped with a DG-4 Plus Ultra High Speed Wavelength Switcher. Fura-2 has two excitation wavelengths: 340nm (when bound to Ca++) and 380nm (when free) and emits at 510nm. To measure Ca++ release, time lapse images are acquired every 200 ms for both 340nm and 380nm excitation before and after addition of agonist. As a control for maximal Ca++ release, ionomycin (6uM) was used. We were able to image and measure Ca++ release with ionomycin, but not with DHPG. We conclude that Fura-2AM is not sensitive enough to detect the low amounts of Ca++ released with mGluR activation, especially as it is released in the astrocytic processes as opposed to cytoplasmic release with ionomycin. To increase sensitivity and image Ca++ release in the astrocytic processes, we will use the genetically encoded calcium sensor GCaMP which are much more sensitive compared to Fura-2. We are now generating astrocyte lines expressing membrane targeted versions of the calcium sensor (lck-GCaMP5 and lck-GCaMP6).

Abbreviations: GPCR (G-protein coupled receptor), mGluR ( metabotropic glutamate receptor), P2Y1 (metabotropic purinergic receptor), GABAB (gamma-amino-butyric acid B receptor), GCaMP (Green fluorescent protein-Calmodulin-M13 fusion protein), DHPG (dihydroxy-phenyl-glycine)

Funder Acknowledgement(s): This study is supported by the NSF HBCU-UP Research Initiation Award (HRD 1401426), a pilot from the NIH-COBRE grant (1P20GM103653-01A1) and NSF HBCU-UP HRD-1533631.

Faculty Advisor: Murali Temburni,