Identification and three-dimensional reconstruction of oxytocin receptor expressing astrocytes in the rat and mouse brain

Ferdinand Althammer; Eric G Krause; Anette D de Kloet; Justin Smith; Valery Grinevich; Alexandre Charlet; Javier E Stern

doi:10.1016/j.xpro.2022.101160

Identification and three-dimensional reconstruction of oxytocin receptor expressing astrocytes in the rat and mouse brain

STAR Protoc. 2022 Feb 10;3(1):101160. doi: 10.1016/j.xpro.2022.101160. eCollection 2022 Mar 18.

Authors

Ferdinand Althammer¹, Eric G Krause^{2

3

4}, Anette D de Kloet^{5

3

4}, Justin Smith^{2

3

4}, Valery Grinevich^{1

6}, Alexandre Charlet^{7

8}, Javier E Stern¹

Affiliations

¹ Center for Neuroinflammation and Cardiometabolic Diseases, Georgia State University, Atlanta, GA 30302-5030, USA.
² Department of Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, FL 32611, USA.
³ Center of Integrative Cardiovascular and Metabolic Diseases, University of Florida, Gainesville, FL 32611, USA.
⁴ McKnight Brain Institute, University of Florida, Gainesville, FL 32611, USA.
⁵ Department of Physiology and Functional Genomics, College of Medicine, University of Florida, Gainesville, FL 32611, USA.
⁶ Department of Neuropeptide Research for Psychiatry, Central Institute of Mental Health, University of Heidelberg, Mannheim, Germany.
⁷ Centre National de la Recherche Scientifique, University of Strasbourg, Strasbourg, France.
⁸ Institute of Cellular and Integrative Neuroscience, INCI CNRS UPR3212 8, Allée du Général Rouvillois 67000 Strasbourg, France.

Abstract

Here, we present a step-by-step protocol for three-dimensional reconstruction of astrocyte morphology, applied to the central amygdala oxytocin receptor-expressing astrocytes. This includes RNAse-free perfusion, combination of RNAscope and immunohistochemistry, and confocal imaging. This protocol provides detailed information about tissue handling and a comprehensive description of the RNAScope technique to label rat and mouse oxytocin receptor mRNA. We also describe three-dimensional reconstruction that allows the assessment of more than 70 different cellular parameters, powerful for studying astrocyte morphology and astrocyte-astrocyte interactions. For complete details on the use and execution of this protocol, please refer to Wahis et al. (2021) and Althammer et al. (2020).

Keywords: Antibody; In Situ Hybridization; Microscopy; Molecular Biology; Neuroscience.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Astrocytes*
Central Amygdaloid Nucleus*
Imaging, Three-Dimensional / methods
Immunohistochemistry
Mice
Rats
Receptors, Oxytocin / genetics

Substances

Receptors, Oxytocin

Abstract

Publication types

MeSH terms

Substances

Grants and funding