Analysing Intercellular Communication in Astrocytic Networks Using “Astral”

Abstract

Astrocytic networks are critically involved in regulating the activity of neuronal networks. However, a comprehensive and ready-to-use data analysis tool for investigating functional interactions between the astrocytes is missing. We developed the novel software package named “Astral” to analyse intercellular communication in astrocytic networks based on live-cell calcium imaging. Our method for analysing calcium imaging data does not require the assignment of regions of interest. The package contains two applications: the core processing pipeline for detecting and quantifying Ca++ events, and the auxiliary visualization tool for controlling data quality. Our method allows for the network-wide quantification of Ca++ events and the analysis of their intercellular propagation. In a set of proof-of-concept experiments, we examined Ca++ events in flat monolayers of primary astrocytes and confirmed that inter-astrocytic interactions depend on the permeability of gap junctions and connexin hemichannels. The Astral tool is particularly useful for studying astrocyte-neuronal interactions on the network level. We demonstrate that compared with purely astrocytic cultures, spontaneous generation of Ca++ events in astrocytes that were co-cultivated with neurons was significantly increased. Interestingly, the increased astrocytic Ca++ activity after long-term co-cultivation with neurons was driven by the enhanced formation of gap junctions and connexin hemichannels but was not affected by silencing neuronal activity. Our data indicate the necessity for systematic investigation of astrocyte-neuronal interactions at the network level. For this purpose, the Astral software offers a powerful tool for processing and quantifying calcium imaging data.

Advocating For Interdisciplinary Collaborations To Unravel The Astrocyte "Calcium Code"

“…Recently, several image analysis algorithms have been developed to quantify astrocyte calcium signals in 2D+time microscopy images. Among them, we can cite GECIquant [43], CaSCaDe [18], FASP [44], AQuA [45] and, more recently, Begonia [46] and Astral [47]. Most of these methods are ROI-based approaches (ROI: region of interest), meaning that calcium signals are analyzed through fixed spatial boundaries in the image.…”

Photonics tools begin to clarify astrocyte calcium transients

“…but also evaluates intercellular propagation of calcium events between neighbors in astrocytic networks. 125 …”

“…but also evaluates intercellular propagation of calcium events between neighbors in astrocytic networks. 125 Another useful image analysis package that has been tailored to suit astrocyte calcium imaging datasets is known as CHIPs, which is a MATLAB-based, open-source toolbox that offers the ability to combine multiple analysis approaches into a single pipeline. 126 The flexible framework of CHIPs is ideal for examining a dataset in a variety of ways and for developers who are looking to further customize the analysis process.…”

Looking to the stars for answers: Strategies for determining how astrocytes influence neuronal activity

“…To date, a significant amount of pioneering work in this field has been done ex vivo , which has provided a solid understanding of the bidirectional chemical communication between neurons and astrocytes, including the astroglial intracellular signaling pathways leading to astrocytic Ca 2+ dynamics, and the neuroactive chemicals released by astroglia as a result of this intracellular signaling modality [25] , [26] , [46] , [47] , [48] . However, current strategies, such as in vivo two-photon calcium imaging in behaving animals [48] , [49] , [50] , combined with advanced analysis pipelines [51] , [52] , now enable the study of astrocytes as potential computational entities, allowing for a more complete understanding of if and how astroglia shape neuronal activity patterns during complex behaviors, including learning and motivated responses. Importantly, while astrocytic Ca 2+ events are remarkably heterogeneous (discussed in length below) and can occur at spatially restricted subcellular domains within individual cells [53] , these events can also expand across multiple cells in astroglial networks [54] , [55] , which likely reflects their ability to influence neuronal network activity.…”

“…2 ). A more recent software package developed to assess intercellular activity within astrocytic networks was developed by Dzyubenko et al (2021), deemed Astral, and is geared at examining astrocyte-astrocyte communication at the population level [51] . The Astral software package employs pipelines for data processing and signal extraction in 3-D + time data sets and groups adjacent pixels together with their change in intensity to evaluate the existence of a calcium waves, evaluated in light of background noise, based on their standard deviation.…”