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Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Ablondi, Eileen F. and Paudel, Sudip and Sehdev, Morgan and Marken, John P. and Halleran, Andrew D. and Rahman, Atiqur and Kemper, Peter and Saha, Margaret S. (2020) Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis. Journal of Visualized Experiments (156). Art. No. e60726. ISSN 1940-087X. https://resolver.caltech.edu/CaltechAUTHORS:20200221-140937830

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Abstract

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.3791/60726DOIArticle
ORCID:
AuthorORCID
Paudel, Sudip0000-0003-4502-8405
Marken, John P.0000-0001-9696-088X
Halleran, Andrew D.0000-0001-8720-1451
Rahman, Atiqur0000-0003-1805-3971
Saha, Margaret S.0000-0003-0096-2667
Additional Information:© 2020 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Date Published: 2/18/2020. We thank Wendy Herbst and Lindsay Schleifer for their contributions to the development of these protocols. This work was supported by grants from the National Institutes of Health (1R15NS067566-01, 1R15HD077624-01 and 1R15HD096415-01) to MSS. No conflicts of interest declared.
Funders:
Funding AgencyGrant Number
NIH1R15NS067566-01
NIH1R15HD077624-01
NIH1R15HD096415-01
Subject Keywords:Developmental Biology, Issue 156, Ca2+, calcium, calcium imaging, calcium activity, neural development, embryonic development, neurotransmitter, neuroscience
Issue or Number:156
Record Number:CaltechAUTHORS:20200221-140937830
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200221-140937830
Official Citation:Ablondi, E. F., Paudel, S., Sehdev, M., Marken, J. P., Halleran, A. D., Rahman, A., Kemper, P., Saha, M. S. Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis. J. Vis. Exp. (156), e60726, doi:10.3791/60726 (2020)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101467
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Feb 2020 22:16
Last Modified:09 Mar 2020 13:19

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