Revisiting astrocytic calcium signaling in the brain

Abstract

Astrocytes, characterized by complex spongiform morphology, participate in various physiological processes, and abnormal changes in their calcium (Ca²⁺) signaling are implicated in central nervous system disorders. However, medications targeting the control of Ca²⁺ have fallen short of the anticipated therapeutic outcomes in clinical applications. This underscores the fact that our comprehension of this intricate regulation of calcium ions remains considerably incomplete. In recent years, with the advancement of Ca²⁺ labeling, imaging, and analysis techniques, Ca²⁺ signals have been found to exhibit high specificity at different spatial locations within the intricate structure of astrocytes. This has ushered the study of Ca²⁺ signaling in astrocytes into a new phase, leading to several groundbreaking research achievements. Despite this, the comprehensive understanding of astrocytic Ca²⁺ signaling and their implications remains challenging area for future research.

Deciphering Ca²⁺ signaling in astrocytes is crucial for understanding the functional roles of the glial cells. A growing body of evidence suggests that astrocytic Ca²⁺ signaling contributes to a variety of astrocytic functions, including modulation of cerebral blood flow, synaptic transmission, synaptic plasticity, and gliotransmitter release [1]. In this context, our attention is dedicated to advancing our understanding of astrocytic Ca²⁺ events. Collectively, these advancements promise to yield fresh insights into the mechanisms through which astrocytes regulate the brain function and how changes in astrocytic Ca²⁺ signals influence physiological and pathological processes.

1. Astrocyte morphology

The types of glial cells in the central nervous system (CNS) include microglia, astrocytes, and oligodendrocyte precursors [2], [3], [4]. Astrocytes were first described by Rudolf Virchow in the 19th century. Decades later, researchers used the silver chromate staining technique to visualize the morphology of astrocytes, advancing the view that astrocytes act as a “glue” in the CNS [5]. These abundant and morphologically complex cells in mammalian brain are distributed in a lattice-like pattern [6,7], forming a dense network in which other glial cells, blood vessels, and neurons are embedded [6], [7], [8], [9], [10].

Astrocytes exhibit complex spongiform shapes, with volumes of approximately ∼6.6 × 10⁴ µm and diameters ranging from ∼40 to 60 µm [11], [12], [13], [14]. The fine structure of astrocytes, particularly protoplasmic astrocytes in the gray matter, consists of a soma, several major branches, numerous branchlets, leaflets, and end-feet (Fig. 1) [15]. Branches, the major processes emanating from the soma, have diameters in the micrometer range. Secondary to tertiary processes, branchlets have diameters in the sub-micrometer range. Leaflets, the terminal extensions of branchlets, show close contacts with synapses, while end-feet are distal extensions that contact blood vessels. Perisynaptic astrocyte processes (PAPs), a morphologically distinct leaflets adjacent to a synapse, affect information processing by the nerve circuits and the behaviours which rely on it [16,17]. However, emerging evidence has suggested that the morphology of astrocytes varies under different conditions. For instance, the phenotypes of reactive astrocytes show an increased number of processes and a denser morphology with altered soma volume compared to resting astrocytes [18].

Fig. 1.

Astrocytes present a complex morphological appearance. Schematic representation of the astrocyte structure.

As a heterogeneous population of cells, astrocytes vary in morphological appearance [19]. Generally, protoplasmic astrocytes are one of the major types found in grey matter, whereas fibrous astrocytes are found in white matter [20], [21], [22]. An increasing number of studies have shown that astrocytes exhibit morphological deficits that may contribute to the progression of CNS disease [7,14]. For instance, at the onset of Alzheimer's disease (AD), progressive atrophy of astrocytes with decreased GFAP expression was observed in the hippocampus and cortex [23], [24], [25]. Furthermore, several studies have shown that the morphological changes of astrocytes in the substantia nigra pars compacta exhibited GFAP-astrogliosis in patients with Parkinson's disease (PD). These findings imply that astrogloiosis dysfunction is likely to be fundamental to dissecting the cellular mechanisms underlying CNS disease [26], [27], [28]. In summary, the study of astrocytes with varying morphologies has garnered increased attention due to their important contribution to the CNS.

2. Ca²⁺ signaling in astrocytes

Calcium (Ca²⁺) is a crucial intracellular second messenger for the survival and fate of organisms [29,30]. The Ca²⁺ signaling system comprises numerous types of proteins, including channels, pumps, receptors, exchangers, and sensors, several of which are altered in expression or mutated in various diseases [31]. For example, excessive inositol 1,4,5-trisphosphate (InsP₃)/Ca²⁺ signaling contribute to the excitatory-inhibitory imbalance of neurons in bipolar disorder [32,33]. Notably, researchers have studied Ca²⁺ signaling in various cell types and model organisms to understand its physiological and pathological roles [34]. In the CNS, Ca²⁺ serves as a pivotal component in intracellular signaling, facilitating communication within both glial cells and neurons. Its role is integral to the maintenance of brain homeostasis [35].

It is well known that astrocytes are electrically non-excitable glial cells and Ca²⁺ fluctuations have been detected in astrocytes under physiological and pathological conditions [36], [37], [38]. Spatially, Ca²⁺ signaling potentially propagates to neighbouring cells via astrocytic gap junctions [39,40]. This Ca²⁺ wave propagation has been thoroughly proven in vitro systems. Instead of wave propagation, synchronized Ca²⁺ activities of many astrocytes have been detected in awake and naturally sleeping animals in response to different behavioral stimuli [41]. Within a single astrocyte, Ca²⁺ events in different cellular compartments also have different functional roles, and several studies have provided evidence for the diversity of Ca²⁺ signals [15,42].

Analyzing Ca²⁺ in the astrocytic soma is relatively straightforward due to its distinct identification in the soma region. Ca²⁺ signaling in astrocytes is frequent and short in the distal processes, but relatively rare and large in the soma [43]. Furthermore, Ca²⁺ signaling induced by the hypoosmotic test was detected in most compartments of astrocytes except the soma, indicating that the astrocytic soma does not respond to the hypoosmotic test. Interestingly, under selected experimental conditions, Ca²⁺ signals can still be detected in astrocytes at the soma. Thrane et al. reported that the astrocytic soma responded to osmotic swelling, in terms of Ca²⁺ spikes both in vivo and in vitro [44]. The mouse pups used in this study may explain why the somatic Ca²⁺ response occurred, demonstrating that Ca²⁺ signaling at the soma relied on developmentally regulated autocrine signaling pathways. Age is likely to be an important factor regulating somatic Ca²⁺ signaling in astrocytes. Moreover, Duffy et al. showed that osmotically induced Ca²⁺ responses at the soma occur in the late phase of brain swelling [44]. Furthermore, another study supported the idea that astrocytes exhibited an increase in Ca²⁺, mediated by voltage-dependent influx and released from internal stores in response to ischemia [45].

Notably, most Ca²⁺ activity in astrocytes is spatially restricted to microdomains, occurring in fine processes that form a complex meshwork [46]. These localized Ca²⁺ signals were observed to occur more frequently than somatic Ca²⁺ elevations and were firstly described as microdomains in the processes of Bergmann glia [47]. Researchers demonstrated that these astrocyte Ca²⁺ signals were frequently far removed from the soma, such as in branches, branchlets, and end-feet [9,[48], [49], [50]]. Ca²⁺ released from the endoplasmic reticulum (ER) store in an inositol triphosphate receptor 2 (IP₃R2)-dependent manner is one of the important sources of Ca²⁺ microdomain signals [50]. Microdomain activity, which occurs in the absence of IP3-dependent release from the endoplasmic reticulum, is mediated by Ca²⁺ efflux from the mitochondria during brief openings of the mitochondrial permeability transition pore [9]. The Ca²⁺ influx-dependent events are also detected in the fine processes of adult hippocampal astrocytes [51]. Many studies have focused on understanding the Ca²⁺ events at microdomains [1,52,53]. For instance, a fraction of astrocytic Ca²⁺ signaling is caused by spontaneous and action potential–dependent synaptic activity [54]. Microdomain Ca²⁺ signal has been detected in end-feet, mediated by Ca²⁺ entry via TRPV4 channels and Ca²⁺ release from IP₃R2-dependent stores [55,56]. Finally, another type of Ca²⁺ signaling occurs during startle responses and locomotion, and is driven by the volumetric release of neuromodulators [57,58].

3. Genesis of astrocytic Ca²⁺ events

Astrocytic Ca²⁺ signaling is regulated by multiple mechanisms, although these remain to be fully elucidated [59]. To date, numerous studies have shown that astrocytic Ca²⁺ signaling depended not only on intracellular Ca²⁺resources, but also on extracellular transmembrane Ca²⁺ influxes, as shown in Fig. 2. The sources of Ca²⁺ also show spatio-temporal characteristics. For instance, transients in the soma of astrocytes rely on the coordination between the release of intracellular Ca²⁺ stores and extracellular influxes. However, some of the events in fine processes can be solely generated by Ca²⁺ influxes [51].

Fig. 2.

Different Ca²⁺ channels exist in the cell and organelle membranes. Ca²⁺ channels are expressed on both the plasma membrane and the membrane of organelles, as discussed in this review. GPCRs: G-protein coupled metabotropic receptors, PLC: phospholipase C, IP₃R: inositol triphosphate receptor, MAM: mitochondria-associated ER membranes, MCU: mitochondrial Ca²⁺ uniporter, mPTP: mitochondrial permeability transition pore, VGCC: voltage-gated Ca²⁺ channel, TRP: transient receptor potential channels, P2XR: P2X receptors.

3.1. Intracellular Ca²⁺ source

The regulated release of Ca²⁺ from the internal stores of the ER or mitochondria is one of the major mechanisms contributing to astrocytic Ca²⁺ events. Among them, astrocytic Gq-G protein-coupled metabotropic receptor (GPCR)-linked IP₃R-dependent Ca²⁺ signaling is the most extensively studied molecular mechanism of Ca²⁺ signals, which has been investigated in an IP₃R2 knockout mouse [19,50,[60], [61], [62], [63], [64]]. Nagai et al. reported that mice lacking intracellular IP₃R2s showed downregulation of spontaneous and GPCR-mediated Ca²⁺ signaling [64]. Notably, astrocytic Ca²⁺ events in the soma are reduced by silencing IP₃R2 in the ER. However, both the proportion of fast-onset microdomain Ca²⁺ events evoked by nearby synaptic activity and the Ca²⁺ responses in fine processes to sensory stimulation are unaffected [50,60,65]. Nevertheless, IP₃R2 is unlikely to be the only type of receptor mediating Ca²⁺ release from the ER in astrocytes. Okubo et al. recently re-evaluated the assumption that Ca²⁺ release from the ER was abolished in IP₃R₂-KO astrocytes using a highly sensitive imaging technique and demonstrated that IP₃R₂-independent Ca²⁺ release induced small cytosolic Ca²⁺ elevations but robust Ca²⁺ transients in the mitochondria [66]. Accordingly, in IP₃R2-KO mice, ryanodine and IP₃R1/3 receptors may facilitate IP₃R2-independent Ca²⁺ release [67,68].

Mitochondria are another key player in the astrocytic Ca²⁺ microdomain, acting as a single electrically coupled continuum or as multiple separate organelles to modulate Ca²⁺ signals [69]. Recently, the Bergles laboratory demonstrated that mitochondria were critical for the Ca²⁺ activities in astrocytic microdomains and were used to store Ca²⁺, which can influence signals by buffering receptor-induced Ca²⁺ transients. In this study, scientists have shown that spatially restricted Ca²⁺ events in astrocytic processes, which occurred independently of ER Ca²⁺ release, were caused by Ca²⁺ efflux from mitochondria in response to the transient opening of the mitochondrial permeability transition pore (mPTP) [9]. Moreover, studies indicated that opening of the mPTP maintained the levels of mitochondrial Ca²⁺. Reversible mPTP opening-dependent ROS release is an adaptive housekeeping function of mitochondria to avoid potentially toxic levels of ROS (and Ca²⁺) [70]. Agarwal et al. showed that inhibiting the function of astrocytic mPTP or disrupting the mitochondrial membrane potential markedly reduced Ca²⁺ events in the microdomain, whereas increasing mPTP opening increased Ca²⁺ transients [9]. Collectively, these studies showed that opening of mPTP was the cause of spontaneous Ca²⁺ signals in astrocytes lacking IP₃R-mediated Ca²⁺ release. Mitochondrial Ca²⁺ homeostasis is also achieved by the mitochondrial Ca²⁺ uniporter (MCU), mitochondrial H⁺-Ca²⁺ exchangers, and Na⁺-Ca²⁺ exchangers [71].

Interestingly, functional coupling also exists between mitochondria and ER Ca²⁺ stores in astrocytes. The ER and mitochondria are frequently in close proximity and have been shown to form mitochondria-associated ER membranes (MAMs) that promote the exchange of metabolites and ions [72]. This specialized junction may synergistically promote Ca²⁺ release. In PD, the interface of the ER–mitochondria was disrupted, leading to abnormal ER-to-mitochondria Ca²⁺ transfers in patients with PD and Parkin knockout mice with Parkin mutations [73]. Parkinson's disease protein 7 (Park7/DJ-1) which is another PD-related protein closely related to MAMs has also been implicated in Ca²⁺ events and upregulating DJ-1 prevents these alterations by re-establishing the ER-mitochondria tethering [74,75]. Liu et al. identified DJ-1 as a key component of the IP₃R–Grp75–VDAC1 complex, the loss of which resulted in decrease in mitochondrial Ca²⁺ upon IP₃R stimulation [75]. In AD, exposure of neurons to Aβ triggered the upregulation of MAM-associated proteins and increased ER–mitochondrial contact sites [76]. Moreover, MAM-localized Ca²⁺ signals have been implicated in the pathogenesis of other diseases and warrants further investigation [72].

3.2. Extracellular Ca²⁺ influx

Numerous studies have suggested that Ca²⁺ activity in astrocytes also depended on extracellular Ca²⁺ sources. In particular, astrocytes exhibited Ca²⁺ events close to the membrane [49,77]. Genetic and pharmacological evaluations have shown that extracellular Ca²⁺ influx in astrocytes occurred through transient receptor potential cation channel A1 (TRPA1) and others (e.g., TRPC3, TRPC4, TRPC5, TRPV1, TRPV4 and TRPC1) [55,[77], [78], [79], [80], [81], [82], [83], [84]]. Importantly, the studies conducted by Freeman's group on TPR channels contributing to microdomain Ca²⁺ signals deserve attention. They demonstrated that astrocytic Ca²⁺ events mediated by different TRP channels were indispensable for multiple sensory-driven behaviors as well as the regulation of CNS gas exchange [85,86]. However, despite the dedication of numerous scientists to the investigation of these receptors, the molecular basis of this process has not been systematically elucidated. Ca²⁺ signaling in astrocytes in different brain regions may be mediated by different sources.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that can be divided into three types (NMDA receptor, AMPA receptor, and Kainate receptor). iGluRs are also essential mediators of astrocytic Ca²⁺ influx [87], [88], [89], [90], [91]. Porter et al. demonstrated that hippocampal astrocytes responded to the neuronal release of the neurotransmitter glutamate by increasing in Ca²⁺ through iGluRs [92]. Similarly, NMDA triggered local increases in astrocytic Ca²⁺ in the mouse neocortex via functional NMDA receptors [93].

The Na⁺-Ca²⁺ exchanger, also known as “NCX,” exchanges the Ca²⁺ and Na⁺in the astrocytes. Multitudinous researches have attempted to model the function of NCX in astrocytic Ca²⁺ microdomain events, which revealed a novel form of astrocytic Ca²⁺ signaling [94], [95], [96].

P2X receptors, which are thought to contain seven subunits, are ion channels that open in response to the binding of extracellular ATP in astrocytes from the hippocampus, cortex, spinal cord, cerebellum, brain stem, and retina [19,[97], [98], [99]]. P2×_1/5 and P2×₇ are the major subunits associated with Ca²⁺ signals. Rat cortical astrocytes have been reported to express ligand-gated P2X (i.e., P2×₁-P2×₅ and P2×₇) for all clones except the P2×₆ receptor [88,100]. In addition to the cortex, P2×₇ receptors were found to be expressed in the hippocampus and retina [101,102].

Voltage-Gated Ca²⁺ Channels (VGCC), documented at the molecular level, are classified as Ca_v1.1–1.4 or L-type Ca²⁺ channels, Ca_v2.1–2.3 or P/Q/N/R-type channels, and Ca_v3.1–3.3 or T-type Ca²⁺ channels [19,103]. Previous studies have found Ca_v1.2 and Ca_v1.3 channels in rodent cortical astrocytes, while VGCCs subtypes of α1B (N-type), α1C (L-type), α1D (L-type), α1E (R-type), and α1G (T-type) have been found in neonatal cultured cortical astrocytes [104], [105], [106], [107]. Additionally, the primary pituicytes express immunoreactivity for the Ca_v1.2 L-type, Ca_v2.1 P/Q-type, Cav2.2 N-type, Ca_v2.3 R-type, and Cav3.1 T-type subunits [108].

The homomeric α-7 nicotinic acetylcholine receptors (α7nAChRs) are expressed in astrocytes, and activation of α7nAChRs in hippocampal slices or in culture has been shown to induce intracellular Ca²⁺ transients [109,110]. Other Ca²⁺ channels, such as Ca²⁺ release-activated Ca²⁺ channels and plasmalemmal Ca²⁺-ATPase, also contribute to the astrocyte microdomain Ca²⁺ signals and other astrocytic functions [111]. While astrocytic Ca²⁺ events can originate from either pure intracellular or extracellular Ca²⁺ resources, they may also arise from the collaborative action of different sources [112,113].

4. Function of astrocytic Ca²⁺ signals

Ca²⁺ signaling assumes a fundamental role in diverse astrocytic functions, including the modulation of cerebral blood flow, synaptic transmission, synaptic plasticity, release of gliotransmitters, and neuronal spiking [1]. Additionally, the ability to generate Ca²⁺ events promotes ATP production by enhancing glycogenolysis [114]. Significantly, the complex morphology and wide distribution of astrocytes makes them well placed to exert local control over synapses and provide global support to neural circuits [9,115]. Astrocytes express numerous neurotransmitter receptors, particularly metabotropic receptors, which sense the microenvironment and induce intracellular Ca²⁺ signaling. Therefore, there exists a significant relationship between astrocytes and neurons that is mediated by Ca²⁺ signals. From the perspective of astrocytic Ca²⁺, the interaction between astrocytes and neurons can be divided into three models, interactions at individual synapses, interactions at groups of synapses, and interactions between networks (Fig. 3). Delving deeper into the intricacies of these interactions can contribute to a better understanding of synapse-astrocyte interactions.

Fig. 3.

Three modes of interaction between neurons and astrocytes. (a) Ca²⁺ events in perisynaptic astrocytic leaflets induce the release of signaling molecules that affect neuronal excitability, synaptic plasticity, and transmission. (b) Astrocytic Ca²⁺ signaling involves the territories of multiple synapses, which affect their plasticity/ activity. (c) Ca²⁺ events propagate through the astrocytic network and control information processing throughout the network of neurons.

4.1. Synaptic transmission

It is known that some astrocytic Ca²⁺ signals are intrinsic, while others are driven by ATP or neurotransmitters such as norepinephrine (NA), acetylcholine, and dopamine. Indeed, NA regulates Ca²⁺ events in astrocytes [53,57,58,116]. Paukert et al. showed that the NA released after light stimulation greatly enhanced astrocytic Ca²⁺ signals and shifted the gain of astrocytic networks according to the behavioral state, enabling astrocytes to respond to local changes in activity of neuron [57]. Similarly, Chen et al. characterized a previously undescribed glial type in zebrafish that resembles mammalian astrocytes and demonstrated that zebrafish astrocytes exhibit microdomain Ca²⁺ transients and respond to NA [117]. Another study further confirmed that Ca²⁺ levels in radial astrocytes of zebrafish increased with the number of failed swimming attempts [118]. Recently, Poskanzer's group reported that NA-driven astrocytic Ca²⁺ events acted as a distinct neuromodulatory signal to regulate state of cortex and linking arousal-associated desynchrony to cortical circuit resynchronization [119]. Moreover, Ca²⁺ transients were induced by acetylcholine in hippocampal and cortical astrocytes and modulate synaptic transmission [120,121]. Dopamine, which is produced in dopaminergic neurons, induced Ca²⁺ responses in astrocytes in the nucleus accumbens or hippocampus [122,123]. In the nucleus accumbens, dopamine increased Ca²⁺ in astrocytes, stimulated adenosine/ATP release and inhibited excitatory synaptic transmission by activating presynaptic A1 receptors [122]. In the hippocampus, dopamine triggered changes in the concentration of intracellular astrocytic calcium with enhanced associated effects on local synaptic function [123].

4.2. Synaptic plasticity

Long-term potentiation (LTP) is the most researched form of synaptic plasticity and is thought to be achieved by Ca²⁺ signals in astrocytes and neurons [124], [125], [126], [127], [128]. Interestingly, different Ca²⁺ signaling pathways in astrocytes are involved in different forms of LTP [52]. The classical form of LTP relies on NMDA receptors and Goshen's group reported that astrocytic activation induced de novo NMDA-dependent LTP in CA1 to further confirm this concept [129,130]. D-serine is an endogenous ligand for NMDA receptors and it has been reported that astrocytes regulated activation of LTP through Ca²⁺-dependent release of D-serine [131], [132], [133]. Additionally, Shigetomi et al. indicated that astrocytes contributed to NMDA receptor-dependent LTP, the activation of which caused Ca²⁺ influx, possibly through plasma membrane TRPA1 channels [78]. Navarrete et al. showed that cholinergic activity in vivo evoked by electrical or sensory stimulation increased astrocytic Ca²⁺ events and induced LTP in the hippocampus. Similarly, in hippocampal slices, stimulation of cholinergic pathways evoked astrocytic Ca²⁺ signals and induced LTP at single CA3-CA1 synapses [134]. Recently, Liu et al. demonstrated that through the integration of synaptic inputs, astrocyte inositol IP₃R2-dependent Ca²⁺ signaling was crucial for late-phase LTP, which was mediated by astrocyte- and brain-derived neurotrophic factor [135].

4.3. Other functions of Ca²⁺ signals in astrocytes

Previous research has shown that changes in Ca²⁺ signals of astrocytes altered hemodynamics, increase glucose mobilization and influence cell activity by releasing neuroactive substances (e.g., glutamate, D-serine, and ATP) [136]. Moreover, Ca²⁺ signals in astrocytes stimulate the Na⁺- and K⁺-dependent adenosine triphosphatase, leading to a decrease in the extracellular concentration of K⁺ [36]. Furthermore, researchers have discovered widespread astrocytic Ca²⁺ events in the cortex, showing that 11% of astrocytes exhibited Ca²⁺ signaling closely correlated with the behavior of running [137]. Interestingly, some substances can also affect the function of Ca²⁺ events to mediate responses in astrocytes. Nagai et al. reported that a 122-residue inhibitory peptide of β-adrenergic receptor kinase 1 attenuated astrocyte G_q-GPCR-mediated Ca²⁺ signals and contributed to behavioral adaptation and spatial memory [64].

Notably, astrocytic Ca²⁺ signaling has been proposed to be involved in disease progression [59,138]. In AD mice, Kuchibhotla et al. reported that astrocytic Ca²⁺ events were more frequent after CNS damage and were enhanced in the amyloid deposition regions [139]. P2Y1 receptors, highly expressed by reactive astrocytes surrounding plaques, are reported to mediate astrocyte hyperactivity, and blockade of P2Y1 receptors significantly reduced astrocytic Ca²⁺ activity and normalized astrocytic dysfunction. In addition to P2Y1 receptor-mediated Ca²⁺ signals, Bosson et al. demonstrated that astrocytes contributed to early Aβo toxicity by exhibiting a global and local Ca²⁺ hyperactivity involving TRPA1 channels [140,141]. Additionally, Lee et al. demonstrated that picomolar amounts of Aβ peptides was detected via an astrocytic α7-nAChR-dependent mechanism, which responded with increased Ca²⁺ transients [142]. However, astrocytic Ca²⁺ signals were reduced during the early stages of Aβ deposition, while neurons were hyperactive and specific tactile memory loss occurred. Restoration of deficient Ca²⁺ signals attenuated neuronal hyperactivity and alleviated the clinical phenotypes [143,144]. In an HD mouse model, the amplitude, duration, and frequency of astrocyte Ca²⁺ events significantly reduced, but astrocytes responded vigorously to cortical stimulation with evoked action potential-dependent Ca²⁺ signaling [145].

In short, astrocytic Ca²⁺ events are vital for optimal physiological and pathological function. Nevertheless, the underlying mechanisms and the physiological and pathophysiological significance of Ca²⁺ in astrocytes remain poorly understood. Therefore, the study of Ca²⁺ events in astrocytes remains elusive and is envisioned as the focus of future biological research after the development of more advanced detection methods.

5. Analysis tools of astrocytic Ca²⁺ signaling

Compared to the easy identification of somas, astrocytic Ca²⁺ microdomain signaling is difficult to define. The processes of astrocytes extend thin lamellar sheets that contain minimal cytoplasm, posing a challenge to the detection of Ca²⁺ changes with cytosolic indicators. Due to technical difficulties in accessing the small spatial area of calcium microdomains, the role of astrocytic Ca²⁺ microdomain activity remains poorly understood. As a result, many experiments and analysis methods are constantly being updated, to better dissect the characteristics of Ca²⁺ signaling in astrocytes.

5.1. Indicators for detecting Ca²⁺ signals

Observing changes and timelines in intracellular Ca²⁺ signaling is essential for exploring the physiological and pathological states in the CNS. Ca²⁺ activity is varied in a single astrocyte and in astrocytic networks at different locations and times. Various indicators are used to determine the number, size, and position of detected Ca²⁺ signals, which are powerful tools for visualizing the activity of Ca²⁺ signals. Visualization and quantification of intracellular Ca²⁺ signals can be achieved using chemical Ca²⁺ indicators (e.g., Fura-2, Fluo-4, and Mag-Fura-Red) or genetically encoded Ca²⁺ indicators (GECI) (e.g., GCaMPs, Pericams, and TN-XXL).

5.1.1. Chemical Ca²⁺ indicators

Beginning with the studies of Cornell-Bell et al., chemical Ca²⁺ indicators have been introduced into the study of astrocytic Ca²⁺ signal functions. These indicators have been widely used and the resulting data have significantly advanced the understanding of astrocyte function [42,146]. Importantly, the concentrations of the indicators should not exceed the buffering capacity of the cells [147,148]. Additionally, the selection of the most appropriate chemical Ca²⁺ indicator requires multiple considerations, including Ca²⁺ affinities, spectral properties, and the forms of different indicators [149].

Compared to GECIs, chemical Ca²⁺ indicators offer a notable advantage in that they are readily accessible commercially and can be easily employed without the need for cellular transfection. Moreover, cell-loading protocols have been well-established [150]. Thus, traditional chemical synthesis indicators have been widely used to investigate intracellular Ca²⁺ signaling. Selecting an appropriate chemical Ca²⁺ indicator requires various considerations, including Ca²⁺ affinities and spectral properties. However, these indicators have many limitations and problems, such as leakage, uneven dye loading photobleaching, and cytotoxicity. In particular, the cellular localization of the indicators cannot be specifically targeted to a particular organelle [149]. Therefore, they may be inappropriate indicators for the precise study of Ca²⁺ signaling [149].

5.1.2. Genetically-encoded Ca²⁺ indicators

Imaging of Ca²⁺ with protein-based indicators has been extensively used to follow neural activity in intact nervous systems. In recent years, GECIs have become one of the most comprehensive calcium indicators and have made outstanding contributions to the study of physiological functions and developmental mechanisms of different tissues and organisms [151]. GECIs have been particularly valuable in studying Ca²⁺ signals in branches and branchlets of astrocytes [59,152]. According to the luminescence principle, GECIs can be divided into two categories: single-fluorescent protein-based GECIs and fluorescence resonance energy transfer (FRET)-based GECIs [153]. The most commonly used single-fluorescent protein-based GECIs include GCaMPs, Camgaroos, and Pericams, while FRET-based GECIs include TN-XXL, D3cpV, and Cameleons [151,154].

Notably, GCaMP indicators are the most broadly used GECIs for monitoring Ca²⁺ in astrocytes. GCaMP indicators consist of a circularly permuted enhanced GFP moiety linked to the calcium-binding protein calmodulin (CaM) and the CaM-binding peptide M13pep [155]. The first GCaMP indicator was terribly dim and poorly folded at 37 °C, limiting its effectiveness for imaging. Improvement by grafting of GFP-stabilizing mutations and random mutagenesis led to the generation of GCaMP2, which showed significant improvements in the characteristics of fluorescence and folding [156], [157], [158]. Since then, groups at the Janelia Research Campus have continued to develop and optimize several rapid and sensitive GCaMP-type indicators by using structure-guided mutagenesis and large-scale screening. The main approaches to the introduction of single wavelength GECIs for the detection of Ca²⁺ signals are summarized in Table 1.

Table 1.

Summary of the single wavelength GECIs for detecting astrocytic Ca²⁺signaling.

Name		Wavelength (nm)		Ref.
		Excitation	Emission	PMID
GCaMP	GCaMP3	485	510	23868258
	GCaMP5G	485	510	23868258
	GCaMP5K	485	510	23868258
	GCaMP6f	485	510	23868258
	GCaMP6s	485	510	23868258
	GCaMP6m	485	510	23868258
	jGCaMP7s	485	510	31209382
	jGCaMP7f	485	510	31209382
	jGCaMP7b	485	510	31209382
	jGCaMP7c	485	510	31209382
	jGCaMP8s	485	510	36922596
	jGCaMP8m	485	510	36922596
	jGCaMP8f	485	510	36922596
Lck-GCaMP6f		488	510	27939582
jRCaMP1	jRCaMP1a	575	595	27011354
	jRCaMP1b	570	595	27011354
jRGECO1a		565	590	27011354
XCaMP-B	XCaMP-B	405	446	31080068
	XCaMP-G	488	514	31080068
	XCaMP-Gf	488	514	31080068
	XCaMP-Gf0	488	514	31080068
	XCaMP-Y	488	527	31080068
	XCaMP-R	561	593	31080068

Crucially, given the swift advancement of new techniques, there is a growing need for more precise localization of astrocytic Ca²⁺ signals. Scientists have developed membrane and organelle-targeted indicators that have been used in various studies [159], [160], [161]. In 2013, Khakh and his colleagues compared two types of GECIs, cyto-GCaMP3 and Lck-GCaMP3, and provided detailed descriptions to facilitate the systematic research on astrocytic Ca²⁺ signaling [48]. A few years later, O'Donnell et al. observed mitochondrially centered and extramitochondrial Ca²⁺ signals using Lck-GCaMP6s [162]. Mitochondria, as key organelles regulating Ca²⁺ signals, have been considered in the construction of DNA plasmids encoding GECIs. Chen et al. constructed a mito-GCaMP2 by genetic manipulation and demonstrated that the response of mitochondrial Ca²⁺ signals was diverse in different cell lines [163]. Similarly, Zhang et al. observed mitochondrial Ca²⁺ imaging of cultured astrocytes by transfecting with mito-GCaMP5G/6s in astrocytes [164]. The ER acts as a store that maintains Ca²⁺ homeostasis [165]. Aryal et al. developed ER-GCaMP6f and indicated that ER-GCaMP6f was expressed in vivo and used to measure Ca²⁺ activity in brain slices [160]. With the development of technology and the need for ongoing research, numerous membrane- and organelle-targeted indicators are being developed and applied.

5.2. Software

In order to investigate astrocytic Ca²⁺ signals in depth, various software tools that employ an event-based perspective to accurately quantify Ca²⁺ in fluorescence imaging datasets have been developed. Here, we discussed the most commonly used image analysis toolboxes tailored for astrocytic Ca²⁺ image data.

5.2.1. GECIquant

In 2015, Khakh et al. developed GECIquant, which allows for rapid semi-automated detection of regions of interest (ROIs) for Ca²⁺ signaling [50]. As the first software used to specifically analyze Ca²⁺ in GCaMP expressing astrocytes, GECIquant was able to automatically detect the microdomain and expand wave ROIs based on the provided area criteria. The GECIquant software focuses on the single cell level, with the ability to provide raw fluorescence data from different regions. Moreover, traces can be processed by the software to obtain additional features [166].

5.2.2. Ca²⁺ signal classification and decoding (CaSCaDe)

Subsequently, in 2017, CaSCaDe was developed, which was similar to GECIquant and used a machine learning-based algorithm to identify Ca²⁺ signaling. Significantly, the CaSCaDe software can represent the regions that exhibit dynamic fluorescence changes and provide information on frequency, number, time course, and amplitude [9].

5.2.3. Astrocyte quantitative analysis (AQuA)

Both GECIquant and CaSCaDe rely on ROIs and measure along time. Nevertheless, their shortcoming lies in the defined boundaries of microdomains, which would lead to inaccurate signals. Accordingly, Wang et al. presented a new analytical framework that releases researchers from the limitations of ROI-based tools. The AQuA software accurately quantifies complex Ca²⁺ signaling and neurotransmitter activity in fluorescence imaging datasets [167]. In this study, researchers demonstrated that the AQuA software outperformed other analysis methods on simulated datasets and described event detection using multiple GECIs. Further, it applies not only to fluorescent indicators, but also to others tested here, especially those with complex dynamics.

5.2.4. Cellular and hemodynamic image processing suite (CHIPS)

CHIPS is an open source toolbox developed to analyze the cells and blood vessels, primarily from two-photon microscopy [168]. Significantly, it can integrate a range of algorithms and streamline image analysis pipelines. In detail, CHIPS is best suited to examine a dataset using multiple approaches simultaneously. For instance, it can simultaneously analyze cell volume or vascular diameter [169].

5.2.5. Begonia

Begonia is a MATLAB-based two-photon imaging analysis toolbox developed for astrocytic Ca²⁺ signals [170]. The analysis suite includes an automatic, event-based algorithm with few input parameters that can capture a high level of spatio-temporal complexity of astrocytic Ca²⁺ signals. Furthermore, begonia enables the experimentalist to accurately quantify astrocytic Ca²⁺ signals and examine Ca²⁺ transients in conjunction with other time series data [169].

5.2.6. Deconvolution of Ca²⁺ fluorescent patterns (deCLUTTER)

Recently, Grochowska et al. developed a new pipeline for calcium imaging data analysis called deCLUTTER²⁺_, which can be used to discover spontaneous or cue-dependent patterns of Ca²⁺ transients [171]. In their study, to better integrate the data from the different cell lines, deCLUTTER was used to analyze the variability of Ca²⁺ at different time points, with older astrocytes contributing to the clusters of more responsive cells.

6. Conclusion and perspectives

This review focuses on the recent advances in astrocytic Ca²⁺ signaling, contributing to more comprehensive understanding of Ca²⁺ events in astrocytes. In particular, we describe the main characteristics of astrocytic Ca²⁺ signaling, exploring its genesis and functions in the CNS. Finally, we provide an overview of the analytical tools used to study astrocytic Ca²⁺ signaling.

Despite these advances, research in this field is still in its infancy, leaving several fundamental questions unanswered. Firstly, the heterogeneity of astrocytes may be reflected in various types of Ca²⁺ signaling [172,173]. Supporting this perspective, given the demonstrated astrocytic heterogeneity across different brain regions, Khakh et al. identified distinct astrocytic Ca²⁺ signals in the CA3 region compared to other regions of the hippocampus [54,[174], [175], [176], [177], [178]]. Secondly, in vivo changes in astrocytic Ca²⁺ signals are diverse and complex over both short and long time periods. Therefore, a pressing need exists for enhanced experiments that can comprehensively investigate astrocyte responses across different temporal scales, building on the refined comprehension of Ca²⁺ signals in vivo. When scientists can consistently and accurately observe and measure specific astrocyte Ca²⁺ microdomain signaling with spatial and temporal resolution, the field will be well-positioned to delve into the functions of astrocytes in the CNS. Thirdly, the investigation of the properties and biophysics of astrocyte Ca²⁺ signals in processes deserves further attention. Notably, anesthesia, a critical confounding factor, has been confirmed to affect Ca²⁺ responses within astrocytes. Therefore, maintaining mice in an awake state is imperative when researchers investigate Ca²⁺ signals [172,179,180]. Selection of sensitive critical indicators, in particular GECIs, allows in vivo monitoring of astrocytic Ca²⁺ activity in precise regions. Fourth, the present studies may not systematically reflect the functions of astrocytic Ca²⁺ events throughout the entire brain. More attention should be directed towards understanding the synergistic effect between astrocytic Ca²⁺ events and other components of the neurovascular unit. Takano et al. reported that astrocytic Ca²⁺ signals mediated vasodilation in response to increased neural activity [181]. In essence, the study highlights the necessity for more systematic research on astrocytic Ca²⁺ signals in the future. Consequently, it is crucial to adopt more comprehensive strategies and develop new technologies to enhance our understanding of astrocytic Ca²⁺ signaling in vivo.

Numerous challenges in this field warrant in-depth exploration. Key questions that require addressing include: (1) What is the precise molecular mechanism by which astrocytes control Ca²⁺? (2) Can novel methods be developed to regulate astrocyte Ca²⁺ signaling in specific brain regions? (3) Do altered astrocyte Ca²⁺ signals contribute to specific CNS diseases via different pathways?

In consideration of the aforementioned points, despite clear progress, further in-depth studies are needed to explore the molecular mechanisms of Ca²⁺ signals in astrocytes. Hence, the next stage of research into astrocytic Ca²⁺ signals should persistently emphasize investigations into astrocyte-related physiology, pathology, and animal behaviors. This strategic focus will undoubtedly contribute to a more nuanced understanding of the pivotal roles of astrocytic Ca²⁺ signaling in both normal and pathological conditions, paving the way for significant advancements in the foreseeable future.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Biographies

Ying Bai(BRID: 06882.00.86191) is an associate professor at Southeast University. She obtained her Ph.D. degree from Southeast University in 2018. Her current research interests focus on the regulation of glial cells in neuroinflammation and related central nervous system diseases, such as ischemic stroke and depression.

Honghong Yao(BRID: 09227.00.02257) is a professor at Southeast University. Her research interests focus on major neurological diseases, such as stroke and depression. Concentrating on the shared pathological mechanism underlying these diseases, specifically the neuroinflammatory response, she dedicates her efforts to addressing pivotal scientific challenges related to diagnostic markers and therapeutic targets using the field of non-coding RNA molecular biology. The research findings illuminate the pathological mechanisms of major nervous system diseases from a fresh perspective, introducing innovative concepts and potential pharmaceuticals for both diagnosis and treatment.