Azin Ebrahim Amini and IBBME UofT

February 5, 2020 @ 12:30 pm – 1:00 pm
Red Seminar Room
Donnelly Building

Event Name: Graduate Seminar Series: Clinical Stream

Graduate Seminar Series for the Institute of Biomaterials and Biomedical Engineering (IBBME). This day is for clinical stream presenters.

Location: Red Seminar Room – Donnelly Building

Presentation Title: Contribution of astrocytic gap junctions and membrane potential to potassium redistribution in the neocortex
Background: Extracellular potassium ion concentration ([K]e) is tightly regulated throughout the brain and has a major impact on brain function1. [K]e is significantly increased and plays a pathogenetic role in stroke, migraine, brain trauma and epilepsy2. Increased [K]e causes neuronal depolarization and hyperexcitability, and further increases cause spreading depolarizations/depression, associated with neuronal silence and death in compromised tissue1. Astrocytes are thought to be the key players in buffering [K]e since they express a high number of potassium channels, are very sensitive to changes in [K]e, and are highly interconnected via gap junctions (GJs)3. GJs are cytoplasmic bridges, which permit the intercellular transfer of molecules. [K]e redistribution in the neocortex is remarkably little studied3. Most studies limit their focus on focal potassium dynamics and few are done in vivo. Herein I study [K]e redistribution mechanisms following using 2 novel techniques, focusing on the roles of astrocytic gap junctions and astrocytic membrane potential.
Hypotheses: A) Astrocytic gap junction permeability strongly modulates [K]e redistribution in the neocortex, and B) Hyperpolarizing glia via optogenetics will depress resting [K]e and enhance [K]e reuptake via astrocytic membrane potassium rectifying channels.
Methods/experimental plan: In this project we are using novel well-developed experimental tools in vivo to elucidate role of astrocytes in potassium redistribution dynamics over the mouse neocortex. Two double-barreled K-sensitive electrodes, each coupled with a local field potential (LFP) electrode, are placed about 3-4 mm apart into young adult CD-1 mouse neocortex. 50mM KCl solution is injected focally beside one of the K-LFP electrodes and [K]e levels and LFP changes are measured; i) with application of GJ blockers or openers, and ii) with optical intervention, activating a hyperpolarizing glial construct introduced by a viral vector.
Preliminary results and future experimental plans: Focally increased [K]e is associated with a transient depolarization which spreads into the neighboring tissue. Topical application of the GJ blockers, carbenoxolone (non-specific) or Gap27 (Cx43 GJ blocker) to the exposed cortex increased the amplitude and duration of the [K]e and LFP responses to the raised [K]e in the proximal neocortical recording sites, whereas in the remote site, the [K]e and LFP responses were depressed and prolonged . Topical application of Trimethylamine (a GJ opener) reduced the amplitudes of [K]e and LFP responses to the raised K both in the peri and remote injection sites. Further delineation of these responses will include more specific GJ and hemichannel/pannexin blockers and openers, using Cx30 +/- Cx43 knockouts, and specific in vivo 2 photon confocal imaging of intra- and extracellular K redistribution using K-specific fluorescent dyes.
Optical stimulation of the in vivo transfected neocortex decreased the response to the raised [K]e by about 35% both in the peri- and remote injection sites. Next steps will involve very focal activation of astrocytes (instead of wide field illumination), testing Kir channel modulation, patch clamping and immunohistochemistry of astrocytes in neocortical brain slices.
In summary, this project is examining the markedly understudied but critical neocortical [K]e spatiotemporal dynamics, to provide a strong physiological basis for understanding the pathophysiological contributions of these mechanism on several relevant brain diseases.
Supervisor Name: Peter Carlen
Year of Study: 4
Program of Study: PhD

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