Calcium signaling in astrocytes and gliotransmitter release

1Nervous systems throughout the animal kingdom vary in structure and complexity and are made up of neurons, specialized cells that can receive and transmit chemical or electrical signals, and glial cells, historically considered to only provide support functions to neurons. Glial cells were first described by Virchow in the 1850s as “nervenkitt” or nerve glue, implying a homogenous population of support cells holding them together. However, several different types of glia can be differentiated based on their different functions and morphology. Among them, there are microglia, oligodendrocytes, and astrocytes. The term astrocyte was coined by Michael von Lenhossek to describe star-shaped cells observed in histological brain specimens. Subsequently, Camillo Golgi and Ramon y Cajal with the development of novel histological stains illustrated several astrocytes with their elaborated processes. Conventionally, two major classes of astrocytes have been distinguished in histological sections of the central nervous system (CNS) based on their morphology and distribution, the fibrous and protoplasmic astrocytes. The fibrous astrocytes are located mainly in white matter with few straight and long processes. Their processes are long (up to 300 μm), though much less elaborate as compared to protoplasmic astroglia. The protoplasmic astrocytes are mainly found in gray matter and are characterized by their extremely elaborate morphology with many branching processes yielding a “bushy” or “spongiform” appearance. Protoplasmic astrocytes extend their endfeet to blood vessels and enwrap them to form the glial limiting membrane, which is the outermost wall of the blood–brain barrier (BBB). More recently, the emergence of molecular approaches such as RNA-sequencing and proteomic analysis has revealed a much larger degree of astrocytic heterogeneity across various brain regions. Excellent reviews related to this topic can be found elsewhere.

2Astrocytes customarily have been identified using the intermediary filament protein Glial Fibrillary Acid Protein (GFAP) as a histological marker. Other markers such as the enzyme glutamine synthetase or a Ca2+ binding peptide S100 have also been applied. Transcriptome analysis of purified astrocytes identified novel molecular markers for astrocytes such as aldehyde dehydrogenase family 1 member L1 or the transcription factor Sox9.

3Electrophysiologically, astrocytes are characterized by their lack of voltage-gated conductances, displaying a quasi-linear voltage-current relationship. The expression of large amounts of inwardly rectifying potassium channels confers astrocytes with their characteristic low input resistance and membrane potential close to the equilibrium potential for transmembrane potassium. The principal potassium channels are the weakly inwardly rectifying Kir4.1 channels although other potassium channels such as the two-pore domain TWIK-1 and TREK-1 channels are also likely to be expressed in astrocytes. Another major conductance found in astrocytes is the connexin channel such as connexin 43 which provides gap junctional coupling among astrocytes. This gap junctional coupling allows the intercellular passive diffusion of endogenous signaling molecules, such as inositol (1,4,5)-triphosphate (IP3), as well as glucose and its metabolites, glutamate, glutamine, and lactate. Therefore, astrocytes are considered to form a functional network of communicating cells.

4Astrocytes also express various transporter proteins on the plasma membrane for the uptake of neurotransmitters. Transporters are vital for the normal CNS physiology by maintaining neurotransmitter homeostasis and modulating synaptic transmission. It is estimated that astrocytes remove about 80% of the glutamate released, whereas the remaining 20% is taken up by neurons. Astrocytes remove extracellular glutamate by excitatory amino acid transporters (EAAT). Five types of EAATs are present in the human brain; the EAAT1 and EAAT2 are expressed almost exclusively in astrocytes (the rodent analogs are known as glutamate/aspartate transporter, GLAST, and glutamate transporter-1, GLT-1).

5Studies in the past few years have shown that astrocytes are spatially organized to form exquisite tridimensional structures. Reconstruction of protoplasmic astrocyte assemblies in the rat hippocampus showed that astrocyte cell bodies are evenly spaced, and their processes overlap only minimally creating a “tiling” of astrocytes. This may be the case in some other brain regions as well though overlap of astrocyte territories have also been described. Perhaps even more surprising is how a single astrocyte with its large territory and complex morphology can massively interact with a neuronal network. Indeed, a single astrocyte in the rat hippocampus is estimated to occupy a territory of 66,000 μm3 of neuropil and contact over 140,000 synapses.

6As discussed above, the role of astrocytes in promoting neurotransmitter clearance at synapses has long been recognized. A more unconventional role of astrocytes at synapses has emerged in the last three decades. The deployment of calcium imaging techniques in cultured cells and in brain slices provided evidence that when neurons communicate with each other they also signal to astrocytes. In turn, astrocytes respond to this neuronal signaling by releasing various neuroactive substances, mentioned in detailed in the section below, such as ATP, glutamate, D-serine, and GABA. Thus, the astrocytes form the third element at the synapses. Not only the information flows from presynaptic to postsynaptic elements but also streams to astrocytes that, in turn, regulate synaptic communication. This intimate morphological and functional association of astroglial processes in a synapse led to the conceptual term of a “tripartite synapse” (Figure 1).

7The plasticity of neuronal connectivity requires dynamic cooperation between neurons and astrocytes. Astrocytes change their morphology and synaptic coverage to scale synaptic strength and modulate neuronal circuit activity. Although not electrically excitable, astrocytes display complex intracellular Ca2+ pathways as a major component of astrocytic signaling. Interaction between synapses and astrocytic arborization promotes astrocyte Ca2+ events to modulate astrocyte neurotransmitter and K+ uptake, the release of neuroactive molecules, and regulation of local blood flow. Astrocyte Ca2+ events manifest differentially in space and time within single astrocytes and across astrocytic networks. In soma and primary branches, Ca2+ events are primarily initiated by intracellular Ca2+ release from Ca2+ stores in the endoplasmic reticulum (ER) and mitochondria. Moreover, astrocytic Ca2+ transients mainly have also been suggested to occur by Ca2+ entry through the plasma membrane following Na+ increases during neurotransmitter uptake via the sodium/calcium exchanger (NCX) or after activation of other ionotropic Ca2+ permeable receptors and transient receptor potential channels.

8In contrast to neurons, astrocytes contain processes with distinct morphology and complement organelles that generate widely distributed Ca2+ loci that allow them to differentially respond to synaptic activity and integrate multiple synaptic inputs. Astrocytic branches are intermingled with neuronal structures and contain Ca2+ stores that can trigger and amplify Ca2+ events by activation of inositol-1,4,5-triphosphate receptors (IP3Rs). IP3Rs are synergistically modulated by IP3 and Ca2+ levels and further Ca2+-dependent phospholipase C activation, stimulating Ca2+ release from the ER. Ca2+ levels can also reach the threshold for activation of IP3Rs by activation of plasmalemmal G-protein-coupled receptors (GPCRs) and via increased diffusion of Ca2+ from multiple daughter leaflets. Intracellular Ca2+ amplification between clusters of IP3Rs can propagate Ca2+ waves within the astrocyte cell body and further astrocytic branches. Ca2+ event generation in leaflets can be additionally enhanced by ER-independent release mechanisms, involving Ca2+ efflux from mitochondria, in response to the transient opening of permeability transition pores (; Figure 1).

9The analysis of the neurotransmitter-evoked astrocyte calcium dynamics has revealed that astrocytes integrate incoming synaptic information. Indeed, synaptic action of excitatory or inhibitory neurotransmitters evoke non-linear calcium elevations and result in the control of the spatial propagation of the intracellular calcium signal within the astrocyte, which is indicative of synaptic information processing by astrocytes. The control of the spatial extent of the calcium signal may have important functional consequences, as it may regulate the spatial extention of the gliotransmitter release and the consequent synaptic regulation. Moreover, converging Ca2+ signals from multiple daughter leaflets can be finally integrated by parent branches as a readout of local network activity. In some circumstances, propagating Ca2+ waves can spread through astrocytes and the astrocytic network to influence neuronal activity. This pathway has been suggested to guide information processing across neuronal networks. Ca2+ events are terminated by Ca2+ removal through the plasma membrane by Ca2+ ATPase (PCMA) or by uptake to Ca2+ stores by ER calcium ATPase (SERCA). Elongated mitochondria in astrocytic branchlets can also actively uptake intracellular Ca2+ by mitochondria Ca2+ uniporters.

10Astrocytic Ca2+ events can be classified as either spontaneous or neurotransmitter-evoked. Spontaneous events are characterized by intrinsic Ca2+ fluctuations that can occur in the absence of external signals. These spontaneous Ca2+ oscillations persist even if neuronal firing or neuronal and astrocytic vesicular release is blocked. Even though the precise mechanisms mediating the triggering of spontaneous Ca2+ transients are not completely understood, it has been proposed that they can be the result of stochastic Ca2+ fluxes through simultaneous multiple pathways. These mechanisms involve both entering Ca2+ from the extracellular space through Ca2+ permeable receptors, Ca2+ channels, and Na+/Ca2+ exchangers at the plasma membrane or intracellular Ca2+ stores through IP3Rs on the ER and mitochondrial permeability via transition pores. The addition of small spatially determined Ca2+ events stimulates local cytosolic Ca2+ oscillations that can trigger Ca2+-dependent Ca2+ release via activation of IP3Rs, leading to amplification and propagation of Ca2+ events. The magnitude of spontaneous Ca2+ activity can be influenced by the intrinsic activity of Gq GPCRs, which stimulates sufficient levels of IP3 to activate IP3Rs, or by focal points of elevated Ca2+ which acts as a co-agonist of IP3Rs. Ca2+ fluxes can be further strengthened or weakened depending on cellular energy states, changes in membrane potential, surface-to-volume ratio, and ER depletion. In soma and primary branches, intracellular Ca2+ waves will mobilize in a specific spatial path within the cell, depending on the proximity of ER IP3Rs, further distance from IP3Rs will terminate the cascade and buffer Ca2+ to basal levels.

11Astrocytic Ca2+ signals are considered to rely mainly on the IP3R pathway, especially in the soma and primary branches, as genetic deletion of IP3R2, which is known to be enriched in astrocytes, reduces spontaneous Ca2+ oscillations with the complete abolition of Ca2+ signals in astrocytic soma. Residual Ca2+ activity in astrocyte processes, even if reduced, is still persistent in astrocytes of IP3R2–/– mice, suggesting IP3R-independent Ca2+ release mechanisms, especially in processes. Such mechanisms involve low cytosolic Ca2+ elevations in mitochondria and transmembrane Ca2+ fluxes mediated by transient receptor potential ion channels (TRPA1), that contribute to the maintenance of basal Ca2+ levels within astrocytes. Importantly, 80% of the astrocyte Ca2+ activity in vivo takes place in astrocytic ramifications, that account for 75% of astrocytic volume. Spatial restriction of spontaneous Ca2+ events has been reported in ex vivo and in vivo preparations. Such events occur predominantly in distal parts of astrocyte processes and do not propagate to the soma, thereby identifying autonomous functional domains called “microdomains”. High-resolution imaging techniques have allowed a deeper understanding of the distinct properties and mechanisms underlying astrocyte somatic and microdomain Ca2+ activity. While somatic Ca2+ increases can be triggered by intense neuronal firing patterns, astrocytic processes also respond to local levels of synaptic activity, suggesting compartmentalized astrocyte neuronal communication integration. Microdomain Ca2+ oscillations are more frequently observed than somatic ones and occur asynchronously in various processes. Microdomain Ca2+ events have been deferentially categorized based on their distinct properties, however, a rich diversity of Ca2+ signals are present within single astrocytes and are modulated by local brain environments in distinct brain areas. Previous elegant classifications have distinguished microdomain Ca2+ activity in focal and expanded microdomains. Focal microdomains, also later referred to in the field, as localized microdomains in branches and branchlets, depend largely on IP3R-dependent Ca2+ transients and seem to be independent of neuronal firing. A distinct hypothesis has suggested that these events could originate from spontaneous neurotransmitter release at neighboring synapses, potentially contributing to plastic adaptations at the tripartite synapse.

12On the other hand, expanded microdomains present different Ca2+ dynamics, compared to focal events, with larger amplitude, duration, and spatial extent, and are highly sensitive to surrounding neuronal firing. The increased magnitude of these Ca2+ events has been suggested to result from the synchronization of several autonomous microdomains and might represent a more coordinated Ca2+ response that could modulate gliotransmitter release probability.

13Astrocytes sense, react and modify the extracellular transmitter homeostasis by responding in situ to neuronal activity. Ex vivo and in vivo examinations have provided strong evidence showing that neuronal inputs trigger astrocyte Ca2+ events by activation of multiple plasma membrane receptors (;;; Figure 1). Engagement of distinct receptor arrays after neuronal input increases cytosolic IP3 levels and IP3R activation, promoting Ca2+ release from ER Ca2+ stores. Additional Ca2+ entry to the cytosol and further triggering of Ca2+ transients can be observed after neuronal-mediated activation of ionotropic receptors, such as glutamate AMPA and NMDA, purinergic P2X, and nicotinic cholinergic receptors or after uptake of glutamate and GABA via Na+ influx via Na+/Ca2+ exchangers. Evidence collected through the last decades has shown that astrocyte GPCR activation mainly leads to intracellular Ca2+ increases. Such a dynamic seems to oppose canonical responses observed in neuronal activation, as increases in astrocytic intracellular Ca2+ are triggered after activation of excitatory or inhibitory transmitter receptors or other Gq, Gs, or Gi-coupled metabotropic receptors. The consequences of GPCR-mediated increase in astrocytic Ca2+ are not fully characterized, however, exciting evidence has suggested that astrocytes can discriminate and integrate metabotropic signaling upstream of internal Ca2+ oscillations. Different activation efficiencies of GPCRs exert equivalent or do not necessarily induce the release of gliotransmitters, contrary to the effects observed after Ca2+ uncaging or IP3 application.

14Neuronal influence on astrocytic activity can occur at individual synapses but also after diffusion of neuromodulators, such as dopamine, acetylcholine, serotonin, and noradrenaline, that modulate spatiotemporal spontaneous Ca2+ events that trigger new Ca2+ fluctuations. Evidence collected during the last decades has suggested that the modulation of astrocyte intracellular Ca2+-induced by neuromodulators finely tunes K+ homeostasis and gliotransmitter release. By integrating the neuromodulatory effects, astrocytes act as crucial players in behavioral states. Neuromodulator effects have been especially evident in astrocyte Ca2+ network activity, as they influence astrocyte activity thresholds in response to local neuronal activity or depending on the brain’s vigilance state. Astrocyte Ca2+ events in leaflets and branchlets can also be triggered by gliotransmitters or other diffuse signals in the local environment, as well as by changes in partial pressures of CO2 and O2, osmotic pressure, pH, and temperature. Astrocytic Ca2+ activity resulting from the interaction between astrocytic processes and synapses can trigger astrocyte morphological remodeling and gliotransmitter release, which feedback to neuronal network excitability and functioning.

15Astrocyte Ca2+ signals in response to external stimulation present different temporal and spatial properties than neuronal activity. The timescale of astrocytic Ca2+ dynamics is generally much slower, with variable intervals between sensory stimulation and the onset of astrocytic Ca2+ event. Single action potentials that can last within a range of a few milliseconds differentiate from astrocytic Ca2+ events, as they can occur over durations of several hundred milliseconds to a few seconds. The differences in Ca2+ dynamics between neurons and astrocytes have raised the question of whether the astrocytic activity can be directly correlated to real-time information processing in the brain. Astrocyte information processing could potentially bridge information received by thousands of synapses belonging to different circuits and neurons and integrate the information in different spatial-temporal scales. Indeed, recent evidence suggests that astrocytes could encode information by evoking specific time and spatial Ca2+ signal patterns, characterized by the different total area of appearance, number, and duration of Ca2+ events.

16Moreover, during information processing, astrocytes could incorporate not only, neuronal information, but also signals resulting from complex interactions with other non-neuronal cells and non-cellular elements part of the extracellular brain microenvironment. Further investigation is needed to elucidate the emerging complexity of mechanisms and dynamics mediating specific types of astrocytic Ca2+ patterns and astrocyte processing of information.

17Since the coining of the term, “tripartite synapse,” researchers have been studying the extent that astrocytes actively communicate with neurons. One of the active mechanisms of astrocytes that impacts synaptic transmission is gliotransmission. Gliotransmission refers to the capacity of astrocytes to release neuroactive molecules that impact synaptic transmission or neuronal signaling. Many of these signaling molecules include classic transmitters such as glutamate and GABA and amino acids like ATP/adenosine and d-serine. Even though the cellular and molecular mechanisms mediating gliotransmitter release are not completely understood, several studies have revealed both calcium-dependent and -independent release mechanisms (;;; Figure 2).

18Calcium-dependent and -independent mechanisms for glutamate release from astrocytes have been proposed. These include (a) exocytosis from vesicles, (b) anion channel opening, (c) glutamate exchange via cystine-glutamate antiporter, (d) release from hemichannels, or (e) ionotropic purinergic receptors. Vesicular gliotransmitter release of glutamate has been supported by morphological and functional evidence. For example, it has been shown that astrocytes possess some of the proteins involved in exocytosis, including the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE) complex, to control vesicle fusion. SNARE proteins, such as VAMP2 or VAMP3, Syntaxin 1, SNAP23, and synaptotagmin isoforms have been detected in astrocytes. Interestingly, the mechanisms involved in glutamate-mediated exocytosis have been highly debated in the last years. Functionally, expression in astrocytes with the light chain of tetanus toxin that selectively cleaves the vesicle-associated SNARE protein potently inhibits the release of glutamate from astrocytes. However, complementary evidence has questioned the exact mechanisms involved in Ca2+-dependent glutamate exocytosis. Deployment of a variety of experimental approaches revealed that fusion events from astrocytic vesicles following intracellular calcium increase occurs in a much slower time scale in comparison to neurons. While in neurons the fusion occurs in less than 0.5 ms following calcium increase, in astrocytes the exocytotic release takes place over two orders of magnitude slower. The release of glutamate may also occur via the opening of glutamate-permeable, two-pore domain potassium channel TREK-1 or the opening of glutamate-permeable, calcium-activated bestrophin anion channel (Best1). Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Recent evidence has also shown that activation of volume-regulated anion channels (VRAC) can lead to glutamate release. When this channel is activated by cell swelling, astrocytes in the hippocampus release glutamate. Lastly, glutamate may also be released via hemichannels which can be blocked by drugs targeting synaptic vesicle protein 2A.

19GABA is an important neurotransmitter for neuronal inhibition. As neurons, astrocytes can also release GABA via transporters, anion channels, and gap junction channels. In contrast to glutamate, GABA release from astrocytes has been reported to be mediated by distinct mechanisms, as the vesicular release of GABA seems unlikely, due to the lack of GABA-containing vesicles in astrocytes. Atypically, astrocytes synthesize GABA from the polyamine putrescine using monoamine oxidase B. Early examples of GABA release from astrocytes have been found in the olfactory bulb, thalamus, and hippocampus. One of the major functional consequences of astrocyte-derived GABA is the tonic inhibition of various neuronal circuits. Various mechanisms of GABA release from astrocytes have been proposed. Calcium-dependent GABA release from astrocytes potentially involving the GABA transporter GAT has been reported in the dorsal root ganglia. Other mechanisms for GABA release from astrocytes such as Best anion channels and gap Junction hemichannels have also been described. “Sniffer-patch” experiments have shown that the Best-1-mediated release of GABA is dependent on intracellular calcium and is triggered by GPCR activation. Tonic inhibition caused by GABA release via glial Best1 anion channels has been reported in the cerebellum and thalamus. This mechanism has also been demonstrated in reactive astrocytes in the hippocampus. Finally, gap junction hemichannels could be another route by which GABA can be released from astrocytes. GABA release via gap junction hemichannels is involved in the regulation of tonic GABA currents of neurons in cultured hippocampal neurons and acute hippocampal slices.

20ATP is a primary energy source in cells and also acts as an important messenger molecule through action on purinergic receptors. ATP plays an important role in calcium wave propagation in astrocytes. Unlike the previously mentioned gliotransmitters, the mechanism for exocytosis was unclear in situ until recent years. This was due primarily to using indirect assays to measure quantal and non-quantal ATP release. Many studies have examined calcium-dependent and independent mechanisms of ATP release. Evidence collected from mice conditionally expressing the SNARE domain of VAMP2 selectively in astrocytes (dn-SNARE mice), has shown Ca2+-dependent ATP release by astrocytes. In addition, ATP release can be mediated by calcium-dependent lysosome exocytosis. Lysosome exocytosis and ATP release occurred after mechanical stimulation in primary hippocampal astrocyte culture in a calcium-dependent manner. In addition, ATP can also be released via connexin 43 (Cx43) hemichannels and anion channels.

21Astrocytes can produce and store D-serine in vesicles. The enzyme, serine racemase converts L-serine to D-serine. Astrocytes play an important role in the serine shuttle by converting L-serine from glucose which can then supply to neurons. Ca2+ dependent vesicle release of D-serine has been demonstrated to modulate long-term potentiation (LTP). Astrocytic glutamate activates on mGluRs and further activates LTP in cholinergic neurons. Moreover, astrocyte release of D-serine also leads to LTP modulating recognition memory. Glial D-serine is relevant for astrocytes across multiple species including Drosophila. In Drosophila, glial D-serine is required for thirst-directed behavior. Many studies have shown that astrocytes can release D-serine under pathological conditions. For instance, preventing the release of d-serine from glia reduce synaptic damage after traumatic brain injury. Astroglial d-serine can also travel through Cx43 hemichannels. The form of release is particularly important for fear memories during fear conditioning. Blocking Cx43 in the basolateral amygdala impaired fear memory consolidation. In addition, astrocytes can also release D-serine via Best1 channels. This has been shown to alter NMDA tone in the hippocampus.

22The development of tools for visualization and manipulation of cell Ca2+ dynamics together with advances in imaging techniques have enabled the monitoring and modulation of astrocyte Ca2+ signaling in in vitro, ex vivo, and in vivo preparations. Advanced optical imaging techniques, sensitive genetically encoded Ca2+ indicators (GECIs), and optogenetic and pharmacogenetic tools allow the selective measuring and activation of astrocyte Ca2+ signaling pathways to study astrocyte-neuron communication, mechanisms of gliotransmitter release, and role of astrocytes in physiology. In particular, selective astrocyte GPCR activation has been useful to explore the functional role of astrocyte Ca2+ signaling in specific brain areas and astrocyte populations. A variety of experimental approaches are now available to increase astrocyte intracellular Ca2+ levels, such as light-gated glutamate receptor, channelrhodopsin-based effectors, melanopsin, optoXRs, and designer receptor exclusively activated by designer drugs (DREADDs). In particular, Gq-GPCR and Gi-GPCR DREADDs have been widely used in the field, as they offer an opportunity for non-invasive and selective in vivo activation of astrocyte GPCR pathways after selective agonist administration. Even though there is a variety of tools to increase astrocyte Ca2+ signaling, till recently, IP3R2–/– mice and IP3 sponges have been the only available options to achieve astrocyte Ca2+ selective attenuation. Recent studies have provided new tools to lessen intracellular Ca2+ release, such as activation of kappa-opioid receptor coupled to a Gi-GPCR selectively activated by salvinorin B or by Cre-dependent expression of hPMCA2, a human plasma membrane Ca2+ ATPase pump that constitutively extrudes Ca2+ from astrocytes. Decreases in astrocyte intracellular Ca2+ levels have also been observed during neuropathology after astrocyte Gs-GPCR activation.

23The evolving genetically targeted optical and pharmacological tools to modulate astrocytic Ca2+ signals have been of value in several studies in the field, showing that astrocyte function and astrocyte-neuron communication is heavily impacted during pathological conditions. Visualization of astrocyte Ca2+ by GECIs monitoring has shown that astrocytes become hyperactive in many neurological diseases such as traumatic brain injury, amyotrophic lateral sclerosis, epilepsy, and Alzheimer’s disease (AD). In addition, modified gliotransmitter release and synaptic transmission have been associated with the development of astrocyte hyperactivity and reactivity. In conclusion, further studies are essential for a precise understanding of the detailed mechanisms by which astrocyte-neuron communication mediates physiological outputs and how the dysregulation of this reciprocal communication affects the development of neuropathology. Tailoring novel molecular tools that specifically modulate astrocyte Ca2+ signaling pathways combined with advanced Ca2+ imaging techniques in vivo will further shed light on the complexity of astrocyte-neuron bidirectional communication and its impact on physiology.

24JG, AA, PK, and DH drafted manuscript and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.