In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Arianna R. Lark; Toshihiro Kitamoto; Jean-René Martin

doi:10.3791/53705

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Method Article

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

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DOI:

10.3791/53705

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January 8th, 2016

Arianna R. Lark¹^,² , Toshihiro Kitamoto²^,³ , Jean-René Martin¹

¹Equipe: Imagerie Cérébrale Fonctionnelle et Comportements (ICFC), Institut des Neurosciences Paris-Saclay (Nero-PSI), UMR-9197, CNRS/Université Paris Sud, ²Interdisciplinary Program in Neuroscience, Graduate College, University of Iowa, ³Department of Anesthesia, Carver College of Medicine, University of Iowa

Summary

Here we present a novel Ca²⁺-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca²⁺ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

Abstract

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca²⁺-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and — with the addition of its cofactor coelenterazine — emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca²⁺-transients and Ca²⁺-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca²⁺-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

Introduction

The fundamental patterning and function of activity within the brain and its discrete structures have long been an area of intense study within the field of neuroscience. Perhaps some of the earliest successful approaches used to address this issue were direct physiological measurement of the change in electrical activity — however alternate methods which allow live imaging of changes in voltage, pH or calcium concentrations (as proxy for activity) have brought additional capabilities to the study of neuronal activity ¹. Imaging techniques carry a wide array of advantages such as reduced invasiveness and the ability to monitor activity within whole br....