Abstracts

A Novel Strategy to Image Calcium Signaling Mechanisms of NG2-Glia Following CNS Viral Infection

Abstract number : 3.009
Submission category : 1. Basic Mechanisms / 1A. Epileptogenesis of acquired epilepsies
Year : 2021
Submission ID : 1825974
Source : www.aesnet.org
Presentation date : 12/6/2021 12:00:00 PM
Published date : Nov 22, 2021, 06:51 AM

Authors :
Laura Bell, BS - University of Utah; Glenna Wallis - University of Utah; John Wagner - University of Utah; Karen Wilcox - PI, University of Utah

Rationale: NG2-glia are commonly known as oligodendrocyte progenitor cells. However, accumulating evidence suggests that these cells have many additional functions, leading to their classification as a major glial cell-type in their own right. We recently demonstrated that NG2-glia become reactive, increase proliferation, and participate in scar formation in the Theiler’s Murine Encephalomyelitis Virus (TMEV) mouse model of infection-induced epilepsy. To investigate functional changes that may accompany NG2-glia reactive processes, we generated a triple-transgenic mouse to express a fluorescent reporter (tdTomato) and a genetically encoded calcium indicator (GCaMP6f) under control of an inducible NG2-promoter. This provides a novel approach to investigate damage-associated changes in calcium signaling in NG2-glia.

Methods: Mice homozygous for tamoxifen-inducible cre and floxed-tdTomato were bred to mice homozygous for floxed-GCaMP6f to produce mice that are heterozygous for all three genes. Tamoxifen was administered (20mg/kg i.p. in peanut oil every-other-day for a total of three injections) to induce tdTomato and GCaMP6f expression specifically in NG2-glia. Time-series images were acquired using a 2-Photon microscope in coronal brain slices at least 60µM below the surface. Imaging rates were optimized between 1-5 Hz to monitor both spontaneous calcium transients and those evoked by focal ATP (100µM) and ADP (100µM) application. ACSF application served as control for pressure effects.

Results: Both spontaneous and agonist-induced calcium transients were consistently observed in NG2-glia in the hippocampus of acutely prepared brain slices. While the application of ACSF alone did not induce pressure-sensitive calcium transients in NG2-glia, repetitive ATP and ADP application resulted in positive calcium responses throughout both the soma and fine processes of NG2-glia (Average ATP ΔF/F = 0.21 ± 0.11 and Average ADP ΔF/F = 0.11 ± 0.08, n = 2-3 ROIs per mouse in a total of five mice).

Conclusions: Our approach provides a novel strategy to better understand the roles of NG2-glia in inflammatory states and in epilepsy development. We show that NG2-glia react to purinergic damage signals (ATP and ADP) through transient increases in intracellular calcium. Future work will determine whether calcium signaling is altered following TMEV infection at timepoints known to induce reactive changes in NG2-glia.

Funding: Please list any funding that was received in support of this abstract.: This work was supported by R37 NS065434 (KSW), NSF GRFP (LAB), and the Skaggs Scholars Fellowship (GJW).

Basic Mechanisms