Monitoring astrocyte calcium microdomains with improved membrane targeted GCaMP reporters
- 1 August 2010
- journal article
- research article
- Published by Cambridge University Press (CUP) in Neuron Glia Biology
- Vol. 6 (3), 183-191
- https://doi.org/10.1017/s1740925x10000219
Abstract
Astrocytes are involved in synaptic and cerebrovascular regulation in the brain. These functions are regulated by intracellular calcium signalling that is thought to reflect a form of astrocyte excitability. In a recent study, we reported modification of the genetically encoded calcium indicator (GECI) GCaMP2 with a membrane-tethering domain, Lck, to generate Lck-GCaMP2. This GECI allowed us to detect novel microdomain calcium signals. The microdomains were random and ‘spotty’ in nature. In order to detect such signals more reliably, in the present study we further modified Lck-GCaMP2 to carry three mutations in the GCaMP2 moiety (M153K, T203V within EGFP and N60D in the CaM domain) to generate Lck-GCaMP3. We directly compared Lck-GCaMP2 and Lck-GCaMP3 by assessing their ability to monitor several types of astrocyte calcium signals with a focus on spotty microdomains. Our data show that Lck-GCaMP3 is between two- and four-times better than Lck-GCaMP2 in terms of its basal fluorescence intensity, signal-to-noise and its ability to detect microdomains. The use of Lck-GCaMP3 thus represents a significantly improved way to monitor astrocyte calcium signals, including microdomains, and will facilitate detailed exploration of their molecular mechanisms and physiological roles.Keywords
This publication has 57 references indexed in Scilit:
- A genetically targeted optical sensor to monitor calcium signals in astrocyte processesNature Neuroscience, 2010
- Long-term potentiation depends on release of d-serine from astrocytesNature, 2010
- A genetically encoded reporter of synaptic activity in vivoNature Methods, 2009
- Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicatorsNature Methods, 2009
- Transgenic mice expressing a cameleon fluorescent Ca2+ indicator in astrocytes and Schwann cells allow study of glial cell Ca2+ signals in situ and in vivoJournal of Neuroscience Methods, 2009
- Structural Basis for Calcium Sensing by GCaMP2Structure, 2008
- What Is the Role of Astrocyte Calcium in Neurophysiology?Neuron, 2008
- Reporting neural activity with genetically encoded calcium indicatorsBrain Cell Biology, 2008
- Imaging Large-Scale Neural Activity with Cellular Resolution in Awake, Mobile MiceNeuron, 2007
- Purinergic signalling in neuron–glia interactionsNature Reviews Neuroscience, 2006