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Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Kashanna T. Robinson - Delaware State University
Co-Author(s): Mercedes Howard, Karla Sanchez, and Murali Temburni, Delaware State University, Dover, DE
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 communicate with each other by intracellular calcium waves. Intracellular Ca++ elevation within astrocytes also leads to release of ‘gliotransmitters’ like glutamate, ATP and Dserine and thus participate in neuronal communication. Evidence that glial cells particularly astrocytes modulate synchronous activity in networks of neurons is accumulating.
Central hypothesis: During development signaling between astrocytes and neurons is crucial for the establishment of synchronously oscillating neuronal networks. Preliminary results from our lab indicate that astrocytes are necessary for synchronous activity of embryonic chick optic tectum neurons in culture. To further dissect the molecular pathways involved in these interactions, 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 demonstrated that chick optic tectum astrocytes express mGluR1 receptors. Our previous attempts to assay intracellular calcium release upon DHPG (di-hydroxy-phenylglycine) stimulation of mGluR receptors using the ratiometric dye fura-2 were not successful. To image Ca++ release within the astrocytic processes upon mGluR stimulation with DHPG we are using the genetically encoded calcium sensor GCaMP. We are generating primary astrocyte cell lines expressing membrane targeted versions of the calcium sensor (lck-GCaMP5 and lckGCaMP6) from chick embryonic optic tectum. Astrocytes expressing the GCaMPs will be stimulated with the mGluR agonist and subjected to live time-lapse video imaging an Olympus Fluoview confocal microscope. In order to assess the role of the astrocytic mGluR pathway in astrocyte-neuron signaling, we will disrupt mGluR signaling in astrocytes by expressing a dominant negative construct. The construct (mGluR DN) will be expressed in astrocytes via a lentiviral vector. Astrocytes expressing the GCaMP calcium sensor and infected with the mGluR DN virus or control virus will be stimulated with DHPG and subjected to live time-lapse imaging. Astrocytes expressing the mGluR DN construct are expected to show reduced intracellular Ca++ release compared to control virus infected astrocytes. These astrocytes will be further tested for their ability to generate synchronous activity by co-culturing them with pure optic tectum neurons on multi-electrode arrays. 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) and a pilot from the NIH-COBRE grant (1P20GM103653-01A1).
Faculty Advisor: Murali Temburni,
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