Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Ruotian Jiang; Martin D. Haustein; Michael V. Sofroniew; Baljit S. Khakh

doi:10.3791/51972

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Method Article

Imaging Intracellular Ca²⁺ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

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DOI:

10.3791/51972

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November 19th, 2014

Ruotian Jiang¹ , Martin D. Haustein¹ , Michael V. Sofroniew² , Baljit S. Khakh¹^,²

¹Department of Physiology, University of California Los Angeles, ²Department of Neurobiology, University of California Los Angeles

Summary

The properties and functions of astrocyte intracellular Ca²⁺ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca²⁺ signals within striatal astrocytes in situ.

Abstract

Astrocytes display spontaneous intracellular Ca²⁺ concentration fluctuations ([Ca²⁺]_i) and in several settings respond to neuronal excitation with enhanced [Ca²⁺]_i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca²⁺]_i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca²⁺]_i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca²⁺]_i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca²⁺]_i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca²⁺]_i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca²⁺]_i signals in the striatal microcircuitry.

Introduction

Astrocytes are ubiquitous and abundant glial cells of the brain. It is well established that astrocytes serve vital support and homeostatic roles including buffering of K⁺ concentration in the extracellular space, uptake of neurotransmitters as well as providing nutrients. However, recent studies show that they also display [Ca²⁺]_i signals, which occur spontaneously and are increased by neuronal activity¹. The existence of astrocyte [Ca²⁺]_i signaling has been increasingly thought to trigger their communication with neurons, and as such has been interpreted as a form of “Ca²⁺ excitabilit....