The focus of this application is to develop much needed and easy to use optical and genetic tools to permit the study of astrocyte function in physiological compartments for genetically specified and tractable cell populations. Astrocytes interact with neurons via fine specialised distal extensions called peripheral astrocyte processes (PAPs). However, a bottleneck to progress has been lack of methods to monitor calcium signals in PAPs, which is a crucial hurdle to overcome in order to understand diverse astrocyte functions in different parts of the brain. This application is based on advances made in our laboratory that allow us to directly measure calcium signals in PAPs. To develop such a method we modified a genetically encoded calcium indicator (GECI) called GCaMP2 to carry a membrane tethering domain (Lck), thus generating Lck-GCaMP2. Then we improved this ~3-fold to generate Lck-GCaMP3 and expressed this in vivo with adeno associated viruses (AAV). Our recent unpublished findings show that Lck-GCaMP3 allows for non-invasive imaging with spectacular clarity. This is a very exciting innovative breakthrough that for the first time will alow researchers to directly measure physiologically relevant astrocyte signals and functions. Moreover, with recent structure-based refinements we made Lck-GCaMP5G, which is ~3-fold better than Lck-GCaMP3 and ~10-fold better than Lck-GCaMP2. We are now ready to develop novel in vivo tools so that Lck-GCaMP5G can be used by anyone and thus generalise a precise way to study astrocyte function and diversity.
In Aim 1 we will generate knock-in mice expressing Lck-GCaMP5G at the Rosa26 locus.
In Aim 2 we will generate novel BAC transgenic mice expressing Cre/ERT in genetically specified astrocytes.
In Aim 3 we will exploit our novel mice to image astrocyte calcium signals in thalamocortical slices.

Public Health Relevance

We will generate new tools to study the physiological properties and functional roles of calcium signals in astrocytes from regions of the brain known to be involved in mental illness such as insomnia and schizophrenia. Our data will provide new information to explore the roles of astrocytes in the normal healthy brain and in diseases of the nervous system, including the processes that lead to the development of psychiatric disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH099559-05
Application #
9171382
Study Section
Special Emphasis Panel (ZMH1)
Program Officer
Asanuma, Chiiko
Project Start
2013-01-15
Project End
2017-10-31
Budget Start
2016-11-01
Budget End
2017-10-31
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Khakh, Baljit S; Beaumont, Vahri; Cachope, Roger et al. (2017) Unravelling and Exploiting Astrocyte Dysfunction in Huntington's Disease. Trends Neurosci 40:422-437
Shigetomi, Eiji; Patel, Sandip; Khakh, Baljit S (2016) Probing the Complexities of Astrocyte Calcium Signaling. Trends Cell Biol 26:300-312
Srinivasan, Rahul; Lu, Tsai-Yi; Chai, Hua et al. (2016) New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo. Neuron 92:1181-1195
Anderson, Mark A; Burda, Joshua E; Ren, Yilong et al. (2016) Astrocyte scar formation aids central nervous system axon regeneration. Nature 532:195-200
Srinivasan, Rahul; Huang, Ben S; Venugopal, Sharmila et al. (2015) Ca(2+) signaling in astrocytes from Ip3r2(-/-) mice in brain slices and during startle responses in vivo. Nat Neurosci 18:708-17
Olsen, Michelle L; Khakh, Baljit S; Skatchkov, Serguei N et al. (2015) New Insights on Astrocyte Ion Channels: Critical for Homeostasis and Neuron-Glia Signaling. J Neurosci 35:13827-35
Khakh, Baljit S; McCarthy, Ken D (2015) Astrocyte calcium signaling: from observations to functions and the challenges therein. Cold Spring Harb Perspect Biol 7:a020404
Khakh, Baljit S; Sofroniew, Michael V (2015) Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci 18:942-52
Jiang, Ruotian; Haustein, Martin D; Sofroniew, Michael V et al. (2014) Imaging intracellular Ca²? signals in striatal astrocytes from adult mice using genetically-encoded calcium indicators. J Vis Exp :e51972
Zhang, Shanshan; Anderson, Mark A; Ao, Yan et al. (2014) Tunable diblock copolypeptide hydrogel depots for local delivery of hydrophobic molecules in healthy and injured central nervous system. Biomaterials 35:1989-2000

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