**1. Introduction**

For a long time, astrocytes have been thought to play an auxiliary role without generally affecting the higher functions of the central nervous system. However, recent experimental evidence has allowed for a more profound view on the functions of astrocytes and their role in adaptive processes in the CNS. Currently, there is no doubt that astrocytes are not simply passive transmitters of energy substrates and structural supports for neurons but also active participants in a large number of metabolic reactions [1]. Several studies have demonstrated the role of astrocytes in the pathological processes at sites of mechanical trauma and in the development of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease [2–4].

While astrocytes are not electrically excitable cells, they are capable of producing and transmitting Ca2+ signals that can propagate between them, resulting in "calcium waves" [5]. It is known that astrocytes interact with neurons, mutually regulating the functional activity of each other [5]. At the same time, it remains unclear whether the astrocytes are able to respond to external stimuli and develop a consolidated response to stress independently on neurons. Investigating the collective dynamics of astrocytic calcium activity, particularly the coordinated activity, which would answer the long-standing problem of the existence of functional astrocytic networks, is highly anticipated.

In particular, a fundamental question is whether astrocytes are able to coordinate their activity over long distances. In other words, can a large-scale dynamic astrocytic network emerge in the absence of neurons? Another key question is whether a functional astrocytic network can be observed in the normal state or if its activity becomes manifested only after stimulation. Generally, the mechanisms that could possibly affect the strength of connections between cells in response to stress factors, thus reshaping the network, remain unknown. In this respect, elucidating the role of astrocytes in the neuroprotective function in the framework of functional astrocytic network reorganization would provide a novel perspective in understanding the processes of CNS adaptation.

Astrocytic interaction relies on gap junctions [6], which enable direct intercellular communication and transport of small molecules for maintaining homeostasis in the brain, glutamate, ATP, and Ca2+ [7]. Gap junctions are formed by two connexons (or hemichannels), and each of them consists of six connexin proteins. Overall, the brain expresses 11 connexins, among which connexin 43 (Cx43) is mostly attributed to astrocytes [6], affecting communication between them.

Understanding the influence of ischemia on the cellular network activity is also of paramount importance. While the resulting disturbance of neuronal activity has been well studied, much less is known regarding the alterations in functional consolidated astrocytic calcium activity. Arguably, elucidating the latter will pave the way to improving the therapy for ischemic stroke.

It is known that astrocytic gap junctions are normally open, while hemichannels formed by connexin 43 demonstrate a low probability of opening [8]. However, after an ischemic interval, the hemichannels become activated, which leads to the uptake of Na+ and Ca2+ into cells, accompanied by the release of ATP and other metabolites. This can cause changes in the calcium activity, an overload of Ca2+ up to toxic values, and osmotic imbalance up to cell death [9,10]. Additionally, ATP released from hemichannels can provoke neuroinflammation due to microglia and astrocyte activation [11]. Instructively, recent evidence demonstrates that hemichannel blockers, such as connexin 43 blocker GAP19, manifest neuroprotective effects in cerebral ischemia (reperfusion) [8].

Gap19 inhibits the opening of hemichannels without affecting gap junctions in astrocytes [12]. According to observations, selective inhibition of hemichannels Cx43 by Gap19 decreases the severity of a stroke and prevents the death of astrocytes in vivo and in vitro [13–15]. However, the effect of connexins 43 blockade on the consolidation of post-ischemic astrocytic calcium activity is currently unknown.

Primary astrocyte cultures could be considered as a model for studying the role of the consolidated astrocytic response because in such cultures, astrocyte–astrocyte interactions form without neuronal regulation, and the reactions of cells to various biochemical and physiological stimuli in isolation from neural activation can be observed.Accordingly, the study aimed to investigate the networks and related parameters of calcium activity in primary astrocytic cultures in a model of ischemic damage, as well as to estimate the neuroprotective effects of connexin 43 Gap19 blocker. For this purpose, we used the calcium event analysis tool for time-lapse fluorescence image recordings of astrocytic cultures [16] and developed an approach for reconstructing functional astrocytic networks based on inferred dynamic correlations. We demonstrate that model stress conditions and biochemical stimuli may produce a dramatic impact on the structure and statistics of these networks, suggesting the underlying changes in consolidated astrocytic activity and indicating the potential of the method for elucidating the effect of the other imposed conditions.
