Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

Han-Sol Lee; Jin-Hee Han

doi:10.3791/61659

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Method Article

Successful In vivo Calcium Imaging with a Head-Mount Miniaturized Microscope in the Amygdala of Freely Behaving Mouse

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DOI:

10.3791/61659

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August 26th, 2020

Han-Sol Lee¹^,² , Jin-Hee Han¹^,²

¹Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), ²KAIST Institute for BioCentury (KIB), Korea Advanced Institute of Science and Technology (KAIST)

Summary

In vivo microendoscopic calcium imaging is an invaluable tool that enables real-time monitoring of neuronal activities in freely behaving animals. However, applying this technique to the amygdala has been difficult. This protocol aims to provide a useful guideline for successfully targeting amygdala cells with a miniaturized microscope in mice.

Abstract

In vivo real-time monitoring of neuronal activities in freely moving animals is one of key approaches to link neuronal activity to behavior. For this purpose, an in vivo imaging technique that detects calcium transients in neurons using genetically encoded calcium indicators (GECIs), a miniaturized fluorescence microscope, and a gradient refractive index (GRIN) lens has been developed and successfully applied to many brain structures¹^,²^,³^,⁴^,⁵^,⁶. This imaging technique is particularly powerful because it enables chronic simultaneous imaging of genetically defined cell populations for a long-term period up to several weeks. Although useful, this imaging technique has not been easily applied to brain structures that locate deep within the brain such as amygdala, an essential brain structure for emotional processing and associative fear memory⁷. There are several factors that make it difficult to apply the imaging technique to the amygdala. For instance, motion artifacts usually occur more frequently during the imaging conducted in the deeper brain regions because a head-mount microscope implanted deep in the brain is relatively unstable. Another problem is that the lateral ventricle is positioned close to the implanted GRIN lens and its movement during respiration may cause highly irregular motion artifacts that cannot be easily corrected, which makes it difficult to form a stable imaging view. Furthermore, because cells in the amygdala are usually quiet at a resting or anesthetized state, it is hard to find and focus the target cells expressing GECI in the amygdala during baseplating procedure for later imaging. This protocol provides a helpful guideline for how to efficiently target cells expressing GECI in the amygdala with head-mount miniaturized microscope for successful in vivo calcium imaging in such a deeper brain region. It is noted that this protocol is based on a particular system (e.g., Inscopix) but not restricted to it.

Introduction

Calcium is a ubiquitous second messenger, playing a crucial role in almost every cellular functions⁸. In neurons, action potential firing and synaptic input cause rapid change of intracellular free [Ca²⁺]⁹^,¹⁰. Therefore, tracking calcium transients provides an opportunity to monitor neuronal activity. GECIs are powerful tools that allow for monitoring [Ca²⁺] in defined cell populations and intra-cellular compartments¹¹^,¹². Among many different types of protein based calcium indicator, GCaMP, a Ca²⁺