An optogenetic approach to understand the molecular mechanisms of astrocyte-neuron interactions
Board Location: #118
Discipline: Biological Sciences
Subcategory: physiology and health science
Session: 2
Isabella Gondolfo - Delaware State University
Co-Author(s): Naana Twumasi, Delaware State University, Dover, DE; Venkata Ajay Narendra Talabattula, Delaware State University, Dover, DE; Murali Temburni, Delaware State University, Dover, DE
Neurons must fire together in networks to perform a variety of functions but the mechanisms by which they accomplish this synchronization are not entirely understood. Astrocytes have been shown to modulate oscillatory activity in networks of neurons possibly by releasing gliotransmitters like glutamate and ATP. Our preliminary results indicate that astrocytes are necessary for synchronous activity of neurons in culture. Mixed neuron and astrocyte cultures on multi-electrode arrays (MEAs) show random spiking activity which synchronizes over time whereas astrocyte-free neurons only show random activity without synchronization.
We implicate calcium mobilization within astrocytes via metabotropic g-protein coupled receptor (GPCR) pathways leading to gliotransmitter release as a possible mechanism for neuronal synchronization. To test this hypothesis, we use an optogenetic approach – using a lentiviral vector we express the photopigment melanopsin, a Gaq GPCR with an absorption peak of around 470nm, in chick optic tectum astrocytes. We expect that increase in Ca++ flux in astrocytes upon optogenetic stimulation with 470nm blue light. This powerful tool allows us to combine live calcium imaging with multielectrode array experiments to clearly define the role of astrocytic calcium in the development of network activity of neurons, particularly synchronization.
Funder Acknowledgement(s): This study is supported by NSF Research Initiation Award (HRD 1401026) and NSF IOS Neural Systems Awards (IOS 1755341 and 1755033) and by NINDS grant #R25NS095371 and Louis Stokes STEM Pathways and Research Alliance: Greater Philadelphia Region LSAMP (Philadelphia AMP) #HRD-2008197.
Faculty Advisor: Murali Temburni, mtemburni@desu.edu
Role: I am an undergraduate researcher working under Dr. Murali Temburni and my part of the research consists of making plasmid maxipreps of pUltrahot-Melanopsin-GCaMP6F lentiviral vector plasmid and the helper plasmids pD8.91 and pVSV.G. These plasmids are then used to transfect the producer cell line HEK293T in order to make lentiviral preps. Next, I infect primary astrocyte cultures from chick optic tectum and perform calcium imaging experiments on the LSM500 confocal microscope.