Abstract
Calcium is a ubiquitous signaling molecule that plays a vital role in many physiological processes. Recent work has shown that calcium activity is especially critical in vertebrate neural development. Here, we investigated if calcium activity and neuronal phenotype are correlated only on a population level or on the level of single cells. Using Xenopus primary cell culture in which individual cells can be unambiguously identified and associated with a molecular phenotype, we correlated calcium activity with neuronal phenotype on the single-cell level. This analysis revealed that, at the neural plate stage, a high frequency of low-amplitude spiking activity correlates with an excitatory, glutamatergic phenotype, while high-amplitude spiking activity correlates with an inhibitory, GABAergic phenotype. Surprisingly, we also found that high-frequency, low-amplitude spiking activity correlates with neural progenitor cells and that differentiating cells exhibit higher spike amplitude. Additional methods of analysis suggested that differentiating marker tubb2b-expressing cells exhibit relatively persistent and predictable calcium activity compared to the irregular activity of neural progenitor cells. Our study highlights the value of using a range of thresholds for analyzing calcium activity data and underscores the importance of employing multiple methods to characterize the often irregular, complex patterns of calcium activity during early neural development.
Highlights
Calcium serves as a pervasive and essential signaling molecule in virtually all biological systems [1,2,3]
As baseline information for marker gene expression, we performed a Kruskal–Wallis test to determine whether the percentages of cells expressing either gad1.1 or slc17a7 at the various Fluorescence In situ Hybridization (FISH) levels are similar across the three developmental stages
As baseline information for marker gene expression, we performed a Kruskal–Wallis test to determine whether the percentages of cells expressing either sox2 or tubb2b at the various FISH levels are similar across the three developmental stages
Summary
Calcium serves as a pervasive and essential signaling molecule in virtually all biological systems [1,2,3]. Specific patterns of calcium activity have been implicated in many distinct phases of neural development, ranging from neural induction and neurotransmitter fate determination to neurite extension and neural tube closure [18,27,28,29,30,31]. These processes rely on the ability of calcium-dependent signaling pathways to encode amplitude and frequency-based signals that trigger responses from downstream effectors, which in turn regulate gene expression [7,22,32]. There is compelling evidence demonstrating that deregulation of these processes is associated with an array of human disease syndromes (reviewed in [33])
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