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

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

Eileen F Ablondi¹, Sudip Paudel², Morgan Sehdev³

¹Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University.

Journal of Visualized Experiments : Jove

|March 10, 2020

Summary

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Researchers developed a new method to link calcium activity patterns to specific cell types in developing neurons. This technique allows for precise analysis of how calcium signals influence neural development at a single-cell level.

Area of Science:

Neuroscience
Developmental Biology
Cell Biology

Background:

Spontaneous intracellular calcium activity is crucial for physiological processes, particularly vertebrate neural development.
Calcium activity patterns are implicated in neural tube closure, synaptogenesis, and neurotransmitter phenotype specification.
Previous population-level studies lacked the precision to correlate calcium activity with molecular phenotypes.

Purpose of the Study:

To establish a high-resolution experimental workflow for correlating calcium activity patterns with molecular phenotypes.
To investigate the role of cell-cell interactions in neuronal determination by assaying single-cell states.
To enable the study of calcium signaling in neural development at a single-cell level.

Main Methods:

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Paired time-lapse calcium imaging of dissociated neuronal explants with fluorescence in situ hybridization.

Single-cell level analysis to correlate specific calcium activity patterns with molecular phenotypes.

Distinguished calcium activity patterns in differentiating neural cells versus neural progenitor cells.

Main Results:

Successfully distinguished and characterized distinct calcium activity patterns associated with differentiating neural cells.
Successfully distinguished and characterized distinct calcium activity patterns associated with neural progenitor cells.
Demonstrated the ability to link specific calcium dynamics to cellular states.

Conclusions:

The developed workflow enables unambiguous correlation of calcium activity patterns with molecular phenotypes on a single-cell level.
This approach is adaptable for investigating correlations between various time-series activity profiles and gene expression.
Provides a powerful tool for dissecting the role of calcium signaling in neural development and other biological processes.