Opto-Chemogenetic Modulation of Astrocytic Buffering for Re-Establishing Neuronal Homeostasis Pubblico

Abstract

Astrocytes play an important role in maintaining a stable extracellular environment for neurons, via their maintenance of extracellular potassium ion concentration ([K⁺]O) and subsequent diffusion through the astrocytic network. During seizure activity, the excessive efflux of K⁺ from neurons causes a series of downstream effects which result in astrocyte alkalization and ultimate uncoupling of astrocyte gap junctions. This biochemical chain reaction in astrocytes leads to deterioration of K⁺ uptake and exacerbation of epileptic seizures. To counter these effects, we proposed indirect molecular neuromodulation through hippocampal astrocytes with the novel inhibitory luminopsin iLMO7, which combines the light-activated inward chloride pump halorhodopsin (NpHR) with a bioluminescent luciferase (NanoLuc). Here we explored the ability of iLMO7 to modulate intracellular chloride concentration ([Cl^-]i) and intracellular pH (pHi) using heterologous expression systems, a human astrocytic cell line and a human embryonic kidney cell line. Both chemical activation of NpHR (via coelenterazine-mediated bioluminescence of NanoLuc) and optical activation (via external light) were analyzed in transfected cells using a plate reader. Two ratiometric fluorescent proteins, SuperClomeleon and pHluorin2, were co-transfected to monitor pertinent changes in [Cl^-]i and pHi, respectively, before and after activation of NpHR. Acidification of the astrocyte interior following activation of NpHR indicates potential for a therapeutic treatment by restoring the seizure- and [K⁺]o-induced alkalization of astrocytes.

Table of Contents

Table of Contents

INTRODUCTION 1

Background 1

Hypothesis and Goals 4

RESULTS 6

Bioluminescence 6

Chloride concentration measurements through SCLM 17

Intracellular pH (pHluorin2) 22

DISCUSSION 24

CONCLUSION 26

MATERIALS AND METHODS 27

Molecular Biology 27

Cell Culture 29

Transfection 29

rAAV Production 29

Mouse Protocol 30

Histology 30

Cell Plate Assays and Exclusion 31

Statistical Analysis 31

SUPPLEMENTAL INFORMATION 32

REFERENCES 35

About this Honors Thesis

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School	Emory College
Department	Neuroscience and Behavioral Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular Biology, Neuroscience
Parola chiave	Optogenetics Epilepsy
Committee Chair / Thesis Advisor	Ken Berglund, Emory University
Committee Members	Anita Devineni, Emory University Keith Easterling, Emory University

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