Analysis Of Calcium Activity During Early Neural Development

Cells typically maintain very low levels of cytosolic Ca2+ ([Ca2+]i), but transient changes in [Ca2+]i due to influxes of Ca2+ from the extracellular milieu or intracellular stores form the basis of calcium activity. The occurrence of diverse patterns of activity attributes, such as frequency and amplitude, has been considered a potential mechanism by which this simple ion can transmit signals to downstream effector molecules. These molecules in turn regulate a wide array of biological processes, including many aspects of early neural development. However, it remains unclear exactly which attributes of these calcium activity events (CAEs) are translated by the effectors. Therefore, understanding the correlation between CAE patterns and gene expression during early neural development can provide critical insight into the underlying mechanisms of nervous system development and patterning. To this end, we performed both in vitro and in vivo Ca2+ imaging and image analysis during early neural development in Xenopus. First, we compared calcium activity and presynaptic neural phenotype in vitro. We found that a high frequency of low-amplitude spiking activity correlates with neural progenitors and glutamatergic phenotype. In contrast, high-amplitude spiking correlates with GABAergic phenotype and neuronal cells that are committed to differentiation (tubb2b positive cells). Further analysis using entropy measure and Hurst exponent suggested that both tubb2b and GABAergic neurons display relatively predictable and persistent calcium activity. The results of our in vitro analysis necessitated an in vivo imaging to confirm that these characteristic calcium activity patterns in vitro match those present in vivo. More importantly, because of this dramatic impact of data analysis techniques on the conclusions drawn from a given dataset, we then critically examined in vivo Ca2+ imaging literature that focuses on early neural development. We found that techniques that are used to define what constitutes a calcium activity event vary widely across studies. In light of instances in which studies with seemingly similar experimental designs have reported dramatically different conclusions, we have outlined the potential impact of techniques that were used to determine what constituted the background signal, baseline activity, and a CAE. Finally, for our in vivo analysis, we first identified Ca2+ peak detection techniques (PDTs) that were commonly used in the recent in vivo imaging literature, generated a combinatorial range of PDTs, and then assessed how application of different PDTs impact the interpretation of spatial patterns of CAEs using simulated data. Further, we constructed a composite neural plate (CNP) by integrating images from disparate tissue regions of embryos. By using this CNP, we examined spatial patterns of CAEs using all the PDTs mentioned above. As in our in vitro study, tissue-specific clusters of CAEs can be detected in PDT-dependent manner in the placodal primordium and near the blastopore and chordoneural hinge. More importantly, unlike previous reports, we found spiking is not restricted to specific tissue precursors in the developing neural plate. This study not only resolves some of the discrepancies in the literature but also highlights the value of systematic application of multiple methods of data analysis to provide a complete picture of the often irregular, complex patterns of calcium activity during early neural development