Calcium Activity Dynamics Correlate with Neuronal Phenotype at a Single Cell Level and in a Threshold-Dependent Manner

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.

Glycine Receptor Inhibition Differentially Affect Selected Neuronal Populations of the Developing Embryonic Cortex, as Evidenced by the Analysis of Spontaneous Calcium Oscillations

“…Thus, it is important to have robust methods of analysis that allow us to understand the underling biological mechanisms of calcium changes. Common methods of analysis for calcium oscillation have normally involved the visual or automatic identification of events based on criteria, detection thresholds or kinetic [ 39 ]. These methods work well in a number of settings and might be a precise readout of mature circuit activity.…”

“…Sample entropy calculation was applied to calcium imaging raw data, as shown previously [ 49 ]. Hurst exponent analysis was computed with a rescaled range (R/S), as shown before [ 39 ].…”

“…Importantly, the same initial calcium oscillations are modulated or even driven by the activation of neurotransmitter systems. It has been reported that this type of calcium activity can participate in cell fate determination, cell proliferation [ 29 , 36 ], neurogenesis, cell migration [ 6 , 7 , 16 ], morphological maturation [ 37 ] and neuronal specification [ 28 , 38 , 39 ].…”

“…On the other hand, power spectrum analysis is a calculation of the energy of the signal as a function of the frequencies present in the series (computed by transforming the signals to the frequency domain). This analysis is useful when individual events cannot be easily identified, and thus it can offer a better approach to evaluate calcium activity in developing neurons [ 39 ]. Likewise, principal component analysis (PCA) is a projection method searching for a low-dimensional representation of the data.…”

Xenopus leads the way: Frogs as a pioneering model to understand the human brain

“…Conveniently, early embryonic effects of blastomere injection can be circumvented by electroporation techniques that target the brain directly (Bestman & Cline, 2020). Several imaging tools, including injectable calcium dyes, in vivo lineage and axon tracing techniques, and transgenic animals (e.g., GcAMP6:GFP, brainbow, hsp70‐CRE, and I‐SceI lines), make the imaging of live developing brains simple compared to other vertebrate systems (Ablondi et al, 2020; Hiramoto & Cline, 2009; Hiramoto & Cline, 2020; Horb et al, 2019; Koser et al, 2016; Offner, Daume, Weiss, Hassenklöver, & Manzini, 2020; Paudel et al, 2019; Qian et al, 2020; Tandon et al, 2017; Thompson et al, 2019). The Xenopus oocyte is a long‐favored model for electrophysiology studies of channel and other protein function (Kusano, Miledi, & Stinnakre, 1977; Limon, Reyes‐Ruiz, & Miledi, 2008; Miledi, Dueñas, Martinez‐Torres, Kawas, & Eusebi, 2004; Sigel & Minier, 2005; Ullah, Demuro, Parker, & Pearson, 2015; Vindas‐Smith et al, 2016), and electrophysiology tools have been adapted for use in embryos, tadpoles, and adults (Barkan, Zornik, & Kelley, 2017; Pratt & Khakhalin, 2013).…”