In:
European Journal of Neuroscience, Wiley, Vol. 21, No. 3 ( 2005-02), p. 597-610
Abstract:
Measurements of local Ca 2+ signalling at different developmental stages and/or in specific cell types is important for understanding aspects of brain functioning. The use of light excitation in fluorescence imaging can cause phototoxicity, photobleaching and auto‐fluorescence. In contrast, bioluminescence does not require the input of radiative energy and can therefore be measured over long periods, with very high temporal resolution. Aequorin is a genetically encoded Ca 2+ ‐sensitive bioluminescent protein, however, its low quantum yield prevents dynamic measurements of Ca 2+ responses in single cells. To overcome this limitation, we recently reported the bi‐functional Ca 2+ reporter gene, GFP‐aequorin (GA), which was developed specifically to improve the light output and stability of aequorin chimeras [V. Baubet, et al ., (2000) PNAS , 97, 7260–7265]. In the current study, we have genetically targeted GA to different microdomains important in synaptic transmission, including to the mitochondrial matrix, endoplasmic reticulum, synaptic vesicles a nd to the postsynaptic density. We demonstrate that these reporters enable ‘real‐time’ measurements of subcellular Ca 2+ changes in single mammalian neurons using bioluminescence. The high signal‐to‐noise ratio of these reporters is also important in that it affords the visualization of Ca 2+ dynamics in cell–cell communication in neuronal cultures and tissue slices. Further, we demonstrate the utility of this approach in ex‐vivo preparations of mammalian retina, a paradigm in which external light input should be controlled. This represents a novel molecular imaging approach for non‐invasive monitoring of local Ca 2+ dynamics and cellular communication in tissue or whole animal studies.
Type of Medium:
Online Resource
ISSN:
0953-816X
,
1460-9568
DOI:
10.1111/ejn.2005.21.issue-3
DOI:
10.1111/j.1460-9568.2005.03871.x
Language:
English
Publisher:
Wiley
Publication Date:
2005
detail.hit.zdb_id:
2005178-5
SSG:
12
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