Astroglia-Derived ATP Modulates CNS Neuronal Circuits

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Review

Astroglia-Derived ATP Modulates CNS Neuronal Circuits Peter Illes,1,2,* Geoffrey Burnstock,3 and Yong Tang2 It is broadly recognized that ATP not only supports energy storage within cells but is also a transmitter/signaling molecule that serves intercellular communication. Whereas the fast (co)transmitter function of ATP in the peripheral nervous system has been convincingly documented, in the central nervous system (CNS) ATP appears to be primarily a slow transmitter/modulator. Data discussed in the present review suggest that the slow modulatory effects of ATP arise as a result of its vesicular/nonvesicular release from astrocytes. ATP acts together with other glial signaling molecules such as cytokines, chemokines, and free radicals to modulate neuronal circuits. Hence, astrocytes are positioned at the crossroads of the neuron–glia–neuron communication pathway.

Highlights In contrast to the peripheral nervous system, the functional significance of ATP in exerting rapid, excitatory (co)transmitter effects in the CNS appears to be relatively limited. ATP may be released from glial cells by exocytotic and nonexocytotic mechanisms.

Purinergic Signaling by ATP in the CNS ATP was defined as a neurotransmitter in the peripheral nervous system in a review article published as early as 1972 [1], then later as a neuroeffector (co)transmitter mediating fast and slow synaptic signals via ligand-gated cationic channels (P2X receptors, P2XRs) and G protein-coupled receptors (P2YRs), respectively [2–5]. P2XRs occur in native cells as homomeric (P2X1, 2, 3, 4, 5, 6, 7) and heteromeric (P2X2/3, P2X1/5) assemblies [6]. P2YRs exist in mammals as the P2Y1, 2, 4, 6, 11, 12, 13, 14 types [7]. ATP was also reported to be a fast neuro-neuronal (co)transmitter in the CNS, although it is surprising that hitherto relatively few areas of the brain were found to utilize fast ATP signaling via P2XRs [6,8–10]. Furthermore, ATP-mediated excitatory postsynaptic currents (EPSCs) caused by stimulation of postsynaptic P2XRs are relatively small, infrequent, only observed in a subpopulation of neurons, and require strong electrical stimulation to evoke them [6]. P2YRs may be localized either presynaptically on nerve terminals or postsynaptically on dendrites of (inter)neurons [11]. Therefore, it can be concluded that P2XRs in some areas of the CNS are activated by neuronally released ATP. However, ATP, exactly like several other neurotransmitters/signaling molecules, may stimulate glial ligandgated and G protein-coupled receptors to release ATP, which then modulates neuronal functions mostly via P2YR activation. The aim of this review is to summarize the available evidence on the participation of astroglia-derived ATP in the modulation of CNS neuronal circuits. Given the breadth of the relevant literature, it is beyond the scope of the current review to cover the role of additional neuroglial cells (e.g., oligodendrocytes, NG2 glia) or of microglia, the resident CNS macrophages, in the context of ATP signaling, although their functional significance is likely comparable to that of astrocytes.

In the CNS, astrocytes may be interposed between neuronal afferents/interneurons and effector neurons to exert slow modulation of neuronal circuits by releasing the gliotransmitters glutamate, GABA, ATP, D-serine, and taurine. Gliotransmitter ATP is released from astrocytes in conjunction with several additional signaling molecules (cytokines, chemokines, free radicals, etc.) to modulate neuronal circuits. Although not discussed in the present review, neuroglial cells other than astrocytes (oligodendrocytes, Mu¨ller cells, Bergmann glia, etc.) as well as microglia also release ATP, and thereby exert effects similar to those of their astrocytic counterparts.

Astrocyte–Neuron Interaction The human CNS consists of neuronal and nonneuronal cells in approximately a 1:1 relationship [12,13]. Glia constitute most of the nonneuronal cell population, but other cell types such as pericytes and endothelial cells are also part of it. The relative proportions of neurons and glia vary by region (e.g., grey vs white matter), developmental stage, and species. On the basis of morphological criteria, in the human neocortex oligodendrocytes account for
50–75% of the total glial population, astrocytes for 20–40%, and microglia for 5–10% [14,15]. Oligodendrocytes generate the myelin sheath of axonal processes, whereas diverse types of astrocyte-like cells (grey matter protoplasmic and white matter fibrous astrocytes, Mu¨ller cells in the retina, Bergmann glial cells in the cerebellum, pituicytes in the neurohypophysis, etc.) ensure the homeostasis of the CNS [15]. Astrocytes modulate synaptic transmission, both directly and indirectly. Indirect effects are exerted by changes in astrocytic functions as a result of modifications in K+ uptake and redistribution, Cl and

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1Rudolf Boehm Institute for Pharmacology and Toxicology, University of Leipzig, 04107 Leipzig, Germany 2Acupuncture and Tuina School, Chengdu University of Traditional Chinese Medicine (TCM), 610075 Chengdu, China 3Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC 3010, Australia

*Correspondence: [email protected]

https://doi.org/10.1016/j.tins.2019.09.006 ª 2019 Elsevier Ltd. All rights reserved.

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water fluxes, Na+/K+, Na+/Ca2+, or Na+/HCO3 exchange, and neurotransmitter uptake, etc. [16,17]. Astrocytes are in a position to modify synaptic transmission because they contact and partially ensheath synapses with their perisynaptic processes (a single human astrocyte can contact up to 2 million synapses), and in addition send end-feet to brain capillaries to regulate blood flow [18,19]. Thereby, astrocytes form a neuron–glia network which signals bidirectionally by means of neurotransmitters/ gliotransmitters released from neurons and glial cells, respectively [20,21] (Figure 1A,B). At this point it should be mentioned that, for the sake of simplicity, we will use the term ’gliotransmitter’ throughout to refer to substances released from glial cells, although most of these substances are in fact neurotransmitters. In other words, the only difference in referring to substances as gliotransmitters rather than neurotransmitters relates to their release from glial cells rather than neurons. Effective neuron–glia interactions rely, in part, on the ability of astrocytes to respond to CNS extracellular substances, and accordingly require that astrocytes express appropriate membrane receptors. With respect to ATP-sensitivity, this means that astrocytes are endowed with both ionotropic P2XRs, allowing influx of Ca2+ from the extracellular space, and metabotropic P2YRs that release Ca2+ from intracellular pools. P2X1/5Rs are stimulated by the exocytotic release of neuronal ATP, whereas P2X7Rs are targets of cell damage-induced outflow of large quantities of ATP [22,23]. In fact, in the CNS, P2X7Rs are present at highest density on microglia; neuroglial cells such as astrocytes and oligodendrocytes also possess this receptor type ([23,24]; but see [25]). For several years it was believed that neurons express P2X7Rs presynaptically, inducing transmitter release via Ca2+ influx through the receptor-channel, but recent evidence and reinterpretation of old data tend to disagree with this hypothesis [26–28]. Astrocytes are integrated into networks where individual astrocytes communicate with each other via gap junctions and probably also P2YRs. Three connexins (Cx26, Cx30, Cx43), of which Cx43 is the most abundant, provide the molecular basis for gap junction channels that connect the cytoplasm of adjacent glial cells [15,29,30]. They allow direct exchange of a variety of small molecules of less than
1 kDa, including ions, energy metabolites, neurotransmitters, and signaling molecules, that coordinate the metabolic and functional activities of connected cells [31,32]. Inositol 1,4,5-trisphosphate (IP3) is a second messenger linked to Gq protein-coupled receptors such as P2Y1R. IP3 diffuses freely through gap junctions, in the same way as Ca2+ itself does, and thereby supports the propagation of Ca2+ waves in astrocytic networks in conjunction with the release of ATP from one astrocyte that then stimulates P2Y1Rs on neighboring astrocytes [29,30]. This latter mechanism certainly functions in cultured astrocytes, although under in vivo conditions astrocyte networks interconnected by P2Y1Rs might operate only in pathological situations [33,34]. In addition to the activation of several P2YRs, an increase [Ca2+]i of astrocytes can also be induced by diverse neurotransmitter molecules, such as glutamate [35,36], noradrenaline [37], and dopamine [38], that stimulate metabotropic glutamate receptors (mGluR3, mGluR5), adrenergic a1 adrenoceptors, and dopamine D1 receptors, respectively. The stimulation of these receptors is thought to release gliotransmitters from astrocytes via a Ca2+-dependent exocytotic mechanism, and may consequently establish neuron–glia crosstalk [39]. The idea of gliotransmission is a fundamental constituent of the ‘tripartite synapse’ hypothesis suggesting that presynaptic neuronal elements, postsynaptic dendritic specializations, and astrocytic processes that contact or even enwrap the synapse, together form a mutually interacting unit [40–42]. More recently, the ‘tripartite synapse’ hypothesis has evolved into the idea of an ‘astroglial cradle’ that summarizes all aspects of synaptic function, and not only those mediated by neurotransmitters [43]. Nonetheless, neurotransmitters released from presynaptic axon terminals may induce the release of gliotransmitters (glutamate, GABA, ATP, D-serine, taurine) which act at the postsynaptic specializations of neurons. Glutamate is taken up by astrocytic processes, metabolized to glutamine, and supplied to presynaptic elements of neurons (‘glutamine shuttle’), which use it for the synthesis of either glutamate or GABA. Astrocytic ATP is extracellularly metabolized to adenosine, which, via adenosine A1R and A2AR activation, causes inhibition or excitation of neuronal activity

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Figure 1. Stimulation of Astroglial Release of ATP by Neurotransmitters and Neuroglial/Microglial ATP: Mechanisms of ATP Release from Astrocytes. (A) ATP and classic neurotransmitters may be released from neurons by Ca2+-dependent exocytosis; a similar release mechanism was debated in relation to astrocytes and microglia. In addition, ATP may also outflow according to its concentration gradient from the neuronal/glial cytoplasm through a leaky cell membrane damaged by any type of cellular injury. Exocytotic glutamate (Glu), GABA, noradrenaline (NA), and dopamine (DA) act at their respective receptors (Rs), whereas ATP activates P2X1/5 heteromeric receptors. Passively outflowing ATP activates P2X7Rs and thereby stimulates the diffusion of cytokines/chemokines through the astroglial cell membrane. In addition, release pathways of ATP from astrocytes are P2X7Rs, connexin hemichannels (e.g., Cnx-43), pannexin channels (e.g., Panx-1), and calcium homeostasis modulator 1 (CALHM1). In neurons, exocytotic release of transmitters takes place from neuronal synaptic vesicles (NSVs); in astrocytes, astrocytic secretory vesicles (ASVs), or astrocytic secretory lysosomes (ASLs) are the sources of exocytotically released ATP. (B) Enzymatic degradation of ATP in astrocytes to adenosine, cytoplasmic resynthesis of ATP, and its subsequent uptake into glial secretory vesicles and secretory lysosomes. ATP is degraded enzymatically by ecto-ATPase to ADP and AMP. AMP is further decomposed by 50 -nucleotidase (50 -NU) to adenosine (ADO). Adenosine is also degraded by adenosine deaminase (ADOD) to inosine (INO) which fails to stimulate any of the purinoceptor types. Adenosine may be converted to AMP by adenosine kinase (ADOK). Extracellular ATP is degraded to adenosine and is then taken up by the adenosine transporter (AT) into the glial cytoplasm; from this adenosine, ATP may be resynthesized and concentrated in secretory vesicles or lysosomes by the vesicular nucleotide transporter (VNUT). Artwork by Ms Lumei Huang.

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(see later). D-serine has been suggested to be a gliotransmitter (but see [44]), acting as a coagonist for NMDA-Rs, and thereby (possibly) fulfilling an indispensable requirement for neuronal NMDA-R stimulation [45]. An important experimental tool to study the role of exocytotic gliotransmission in physiological and pathological processes was provided by the generation of mice in which a dominant negative (dn) domain of vesicular soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) was selectively overexpressed in astrocytes [42,46]. The selectivity of dnSNARE expression was later criticized by reports indicating the expression of the transgene also in neurons [47]. Arguments challenging the ‘gliotransmitter’ hypothesis were that (i) deleting IP3R2, that is responsible for Ca2+ release from the endoplasmic reticulum of astrocytes, had virtually no effect on synaptic transmission; (ii) in contrast to the cell body, Ca2+ stores are absent in small perisynaptic processes of astrocytes; and (iii) the expression of astrocytic mGlu5Rs, which were shown to stimulate the release of gliotransmitters, is undetectable after the third postnatal week in mice [46,48]. On the whole, arguments favoring [49,50] and refuting [51,52] the role of astrocytes in fast neurotransmission are roughly balanced. Nevertheless, it is an indisputable fact that the amount of glutamate released from healthy astrocytes is absolutely minuscule compared with its neuronal release. After having clarified some general aspects of neuro/gliotransmission as means of crosstalk between neurons and astrocytes, we turn our attention to ATP as one of the gliotransmitters that is released by exocytotic and nonexocytotic mechanisms from this latter cell type.

Release Mechanisms of ATP from Astrocytes Exocytotic Release It is a fascinating possibility that ATP is released in a [Ca2+]i-dependent manner exocytotically from synaptic-like vesicles of astrocytes. It was shown at first in cultured cortical and hippocampal astrocytes, by combining epifluorescence and total internal reflection fluorescence (TIRF) microscopy, that individual, quinacrine-loaded, ATP-containing vesicles undergo exocytosis [53,54]. In fact, a low percentage of fluorescently labeled vesicles displayed directional motility and underwent fusion with the plasma membrane after stimulation with glutamate or the Ca2+ ionophore ionomycin, and this event was Ca2+-dependent. Moreover, glutamate stimulation of astrocytes was followed by the incidence of small transient inward currents measured in HEK293 cells transfected with recombinant P2X3Rs (’sniffer cells’ [53]). Subsequently, these findings were confirmed in acutely dissociated cortical astrocytes, and also in astrocytes located in neocortical brain slices, which are thought to more closely reflect the in vivo situation than do cultured astrocytes [55]. This latter approach also showed that exocytosis of ATP from astrocytes can activate postsynaptic P2XRs in adjacent neurons, causing a downregulation of synaptic and extrasynaptic GABAA-Rs in cortical pyramidal cells [56]. Fluorescent styryl pyridinium (FM) dyes are widely used to study endocytosis, vesicle trafficking, and organelle organization in living eukaryotic cells. By means of these dyes it was shown that the ATPstoring vesicles in astrocytes may be secretory lysosomes; incubation of cultured hippocampal astrocytes with FM dyes resulted in the appearance of granule-like fluorescent puncta, which costained with specific markers for lysosomes, and partially for cathepsin D, a secretory lysosomal protease [57]. The FM-labeled lysosomes underwent regulated exocytosis because the application of ATP or glutamate induced a substantial decrease in the fluorescence intensity of the puncta. The existence of an exocytotic machinery was confirmed by the Ca2+-dependence of the release [58,59] and by association of the immunocytochemically identified vesicular nucleotide transporter (VNUT) with astrocytic lysosomes [60]. Importantly, vesicular release from synaptic-like vesicles and lysosomes of astrocytes involves SNARE-dependent merger of the vesicle membrane with the plasmalemma [51,61,62]. From these studies it is clear that the vesicular proteins involved in exocytosis are relatively similar in astrocytes and neurons, but that astrocytic release of ATP is much slower than that of its neuronal counterpart [51].

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Nonexocytotic Release Exocytotic, vesicular/lysosomal release of ATP from astrocytes is not an exclusive exit pathway to modulate neuronal circuits. Whereas the large and negatively charged ATP molecule is not able to diffuse through the intact plasmalemma of astrocytes, disruption of the plasma membrane caused by cellular damage removes this hurdle and allows the accumulation of high ATP concentrations in the extracellular space. Several nonexocytotic release mechanisms also operate in astrocytes by means of channel molecules, including connexin hemichannels (mostly Cx43), pannexin 1 channels (Panx-1 [63]), maxi anion channels [64], volume-regulated anion channels, the Ca2+-dependent Cl channel bestrophin [65], and the calcium homeostasis modulator 1 (CALHM1-3 [66]) [32,67]. As mentioned previously, gap junctions in vertebrates comprise two opposed connexin hemichannels that link the cytosol of adjacent cells, whereas in invertebrates they are built up of innexins of related structure. Homologous to innexins are the vertebrate pannexins; however, these do not form gap junctions and exist only as channels. Long-lasting or repetitive activation of P2X7Rs by high concentrations of ATP was suggested to cause either dilation of the receptor channel itself [68] or the recruitment of an accessory protein, believed to be the Panx-1 channel [69]. Thereby, diffusion of molecules of up to 900 Da (e.g., glutamate, ATP, some fluorescent dyes) becomes possible according to their concentration gradients [70]. Although there is a clear consensus regarding the release of glutamate/ATP via P2X7Rs, both the dilation of P2X7R channels [71,72] and the coupling of this receptor to the Panx-1 channel [73,74] have been negated in recent publications. Both connexins and pannexins are pathways to release ATP from astrocytes, which then plays important roles in secondary inflammatory reactions, for example, following traumatic spinal cord or optic nerve damage [75,76]. However, astroglial Cx43 hemichannels can also modulate basal excitatory neurotransmission in the hippocampus under physiological conditions [77]. Absence of pannexin (Panx1/ mouse) or pharmacological blockade of Panx-1 channels with probenecid increased long-term potentiation (LTP) in hippocampal CA1 pyramidal cells induced by high-frequency stimulation of the incoming Schaffer collateral fibers [78,79]. It was concluded that enhanced LTP is due to the lack of endogenous adenosine because of extracellular ATP/adenosine depletion and the consequent loss of A1R-mediated inhibition of excitatory neurotransmission. Panx1/ mice exhibited distinct behavioral alterations, with enhanced anxiety-like behaviors, and impaired object recognition and spatial learning, concordant with the assumption that LTP is an indicator of a cellular learning process [78]. Further, it was reported that Panx-1-mediated ATP release from the CA3 area drives mGlu5R-dependent neuronal oscillations [80]. Both exocytotic and nonexocytotic release of ATP from astrocytes have been shown to modulate the excitability of neighboring neurons. In a simplified scheme this means that astrocytic networks interconnected by gap junctions, and probably also P2Y1Rs, modulate the spread of excitation in neuronal networks that are interconnected by the release of various neurotransmitters. These considerations are the subject of the following section.

Modulation of Neuronal Circuits by Astrocytic ATP Modulation of the Synaptic Plasticity of Neuronal Networks by Astrocytic ATP/ADP In the following sections we report data obtained by a variety of techniques used to examine the modulation of neuronal circuits by astrocytic ATP/ADP (Box 1). One tool for studying gliotransmission has been dnSNARE transgenic mice in which dnSNARE is selectively overexpressed in astrocytes. In these mice, the exocytotic machinery and thereby the release of gliotransmitters is thought to be selectively impaired [41]. In hippocampal slices of wild-type (WT) mice, stimulation of Schaffer collaterals by theta bursts induces stronger LTP in CA1 pyramidal cells than it does in hippocampal slices prepared from dnSNARE mice. This was suggested to be due to the conversion of ATP released from astrocytes to adenosine that activates neuronal A1Rs to persistently suppress excitatory synaptic transmission. However, it is difficult to envisage how less adenosine in dnSNARE mice would cause weaker LTP instead of the expected stronger LTP, as shown also in analogous experiments in Panx1 knockout

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Box 1. Techniques Used To Examine Modulation of Neuronal Circuits by Astrocytic ATP. The following techniques have been used to selectively modulate astrocytic ATP release and study the modulation of neuronal circuits by astrocytic ATP. (i) Intracellularly applied caged/inactive Ca2+ or IP3 into astrocytes can be uncaged by photoactivation. (ii)

Channel rhodopsin 2 (ChR2) is a light-switched cationic channel which generates a large permeability for monovalent and divalent cations. It can be introduced into astroglia and stimulated by blue light.

(iii)

DREADDs (designer receptors exclusively activated by designer drugs) are G protein-coupled receptors (GPCRs) that are normally absent from the organism. They can be introduced into astroglia and stimulated by specific agonists that are inactive at other GPCRs.

(iv)

Astrocytes in mice devoid of IP3-type 2 receptor (IP3R2) fail to release Ca2+ from their intracellular pools upon activation of metabotropic phospholipase C-coupled GPCRs. Otherwise these mice display no neuronal or neurovascular deficits.

(v)

Intracellular injection of the Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) into an astrocyte and its diffusion into neighboring astrocytes blocks Ca2+-dependent reactions in the network.

(vi)

Fluorocitrate is a selective metabolic poison of astrocytes which does not alter neuronal functions.

(vii) Mice selectively expressing the dominant negative (dn) domain of vesicular soluble N-ethylmaleimidesensitive fusion protein attachment protein receptor (SNARE) in their astrocytes fail to exocytotically release gliotransmitters. (viii) Tetanus or botulinum toxin applied intracellularly to astrocytes blocks the exocytotic release of gliotransmitters.

(KO) mice (see above). We would argue that deficiency of ATP itself is involved in this effect, as reported in the somatosensory cortex of dnSNARE mice, where LTP recorded in layer II/III neurons (initiated by stimulation of neuronal afferents from layer IV/V) was weaker than in WT mice [55,81,82]. In support of a role for glia-derived ATP, LTP was rescued by substitution of endogenous ATP by its exogenous structural analog, ATP-g-S, which is thought to be relatively resistant to enzymatic degradation. In perfect accordance with the assumption that the release of gliotransmitters (in particular ATP) may decline with age, the magnitude of LTP was much lower in neocortices of old rather than young mice [55,83]. Electrophysiological experiments in brain slices show that noradrenaline, through a1-adrenoceptor activation, increases the postsynaptic efficacy of astrocytic vesicular ATP release from magnocellular cells of the rat hypothalamic paraventricular nucleus [84]. It was suggested that astrocytic ATP facilitates the insertion of postsynaptic AMPA receptors (AMPARs) and thus enhances glutamatergic signaling in the paraventricular nucleus [84]. Repetitive electrical stimulation of neuronal afferents, or caged molecule photolysis of glutamate, demonstrated that a metabotropic glutamate receptor-mediated rise of [Ca2+]i in the astrocytic processes surrounding neurons and the subsequent release of the gliotransmitter ATP were responsible for the upscaling of synaptic strength [85]. Comparable effects on the strength of glutamatergic neurotransmission in neocortical pyramidal neurons were reduced by perfusion of individual astrocytes with tetanus toxin, blocking the vesicular release of ATP [37]. Moreover, elevation of cytosolic Ca2+ in neocortical (layer II/III) astrocytes by protease activated receptor 1 (PAR-1) stimulation has been shown to trigger the release of ATP, that directly induced quantal purinergic currents in adjacent pyramidal neurons [55]. In consequence, Ca2+ entry through the neuronal P2XRs led to a phosphorylation-dependent downregulation of GABAA-Rs. By contrast, it was reported that exogenous ATP, or noradrenaline-dependent glial release of endogenous ATP, in both cases via P2XR stimulation, lead to internalization of AMPARs at dendrites and

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synapses of cultured hippocampal neurons or CA1 neurons in acutely prepared hippocampal slices [86]. This was due to phosphorylation of two CaMKII sites located in the tail of GluA1 subunits of AMPARs [87]. These divergent results could be explained by the different responses of hippocampal CA1 neurons on the one hand, and by hippocampal magnocellular neurons and neocortical layer II/III pyramidal neurons on the other. Current responses to NMDA in layer V pyramidal neurons of the rat prefrontal cortex were potentiated by the P2R agonists ATP and UTP [11,88]. This potentiation was suggested to be due to astrocytic P2Y4R activation, releasing glutamate, which facilitated NMDA-R currents on layer V pyramidal neurons via stimulation of type I mGluRs. In brain slices of the dorsomedial hypothalamus (DMH), high-frequency stimulation of GABA inputs onto DMH neurons elicited long-term depression (LTD) [89]. Cholecystokinin (CCK) shifted the plasticity of GABA synapses from LTD to LTP. It appeared that this LTP required the activation of both type-2 CCK receptors and group-5 metabotropic glutamate receptors, resulting in a rise of astrocytic [Ca2+]i and subsequent ATP release. ATP then acted on presynaptic neuronal P2XRs to trigger a prolonged increase in GABA release. Another physiological process that needs to be mentioned in this context is heterosynaptic plasticity, which refers to long-term changes in synaptic pathways which are not specifically stimulated, in addition to those which are specifically stimulated. When two independent commissural pathways (S1, S2) were used to stimulate inputs to CA1 pyramidal cells in the hippocampus, high-frequency stimulation of S1 depressed the EPSPs evoked by stimulation of S2. This so-called heterosynaptic depression observed in WT mice disappeared after superfusion with the selective astrocytic metabolic poison fluoroacetate [90], or when hippocampal slices were prepared from dnSNARE mice [41], indicating that gliotransmission is involved in this process.

Network Effects of Astrocytic ATP/ADP Released by Cell Type-Specific Stimulation Extracellular ATP was shown to stimulate [Ca2+]i in astrocytes and inhibitory GABAergic interneurons in the stratum radiatum area of the hippocampus [91]. This appeared to be due to activation of P2Y1Rs on both astrocytes and interneurons. Later it was reported by the same group of authors that the effect of endogenously released astrocytic ATP in the hippocampal CA1 region appears to be stimulus-dependent [92]. Whereas ATP release from astrocytes induced by PAR-1 stimulation led to the appearance of NMDA-R-mediated slow inward currents in pyramidal neurons, P2Y1R activation was ineffective in this regard. In other words, the ATP-mediated effects of astrocyte stimulation were agonist-dependent in these experiments. This agonist-dependency of glial stimulation was no longer a problem when channelrhodopsin 2 (ChR2) was selectively expressed in astrocytes of the CA1 area. It was found that stimulation of these photosensitive receptors by blue light specifically increased the firing rate of CCK-positive, but not parvalbumin-positive interneurons and decreased the firing rate of CA1 pyramidal neurons (Figure 2A) [93]. The release of ATP from the ChR2-stimulated astrocytes activated P2Y1Rs coupled in an inhibitory manner to two-pore domain potassium channels (K2P) of CCK-positive, GABAergic interneurons. The ensuing increase of the GABAergic tone imposed upon the CA1 pyramidal neurons resulted in decreased excitability of pyramidal neurons. In addition, ATP released from the astrocytes also activated A1Rs on pyramidal neurons that, by opening G protein-coupled inwardly rectifying potassium channels (GIRKs), caused hyperpolarization and a decrease of excitability. Another study showed that photostimulation of ChR2 receptors expressed in hippocampal astrocytes increased action potential firing of CA1 neurons [94]. This finding contrasts with the results reported in [93], discussed above. A likely explanation is that, whereas Tan et al. [93] recorded ionic currents in the presence of a wide-spectrum ionotropic glutamate receptor antagonist, in the study of Shen et al. [94] no such antagonist was present. Glutamate receptor antagonists could of course preclude effects relying on glutamate release from astrocytes and the activation of their receptors on neurons.

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Figure 2. Astroglia–Neuron Interactions in Shaping Hippocampal Functions. (A) Stimulation by blue light of channelrhodopsin 2 (ChR2) introduced into astrocytes in the CA1 area of the hippocampus releases ATP which modulates the function of pyramidal neurons both directly and indirectly. First, ATP decomposes enzymatically to adenosine (ADO) which activates the A1 receptors of CA1 neurons. A1 receptors increase the outflow of potassium via G protein-coupled inversely rectifying potassium (GIRK) channels, leading to decreased excitability of pyramidal neurons. Second, ATP activates P2Y1Rs located on cholecystokinin (CCK)-positive interneurons and inhibits two pore-domain potassium (K2P) channels, thus reducing GABA release from these interneurons. Loss of the GABAergic inhibitory tone results in increased excitability of pyramidal neurons. In consequence, the excitability of CA1 pyramidal neurons can be differentially modulated by astrocyte-derived ATP [93]. (B) ChR2, this time introduced into somatostatin-positive interneurons of the CA1 area of the hippocampus, causes the release of GABA from the terminals of these interneurons. There are two different targets of the synaptically released GABA. First, GABA activates GABABRs at astrocytes and is also taken up by these cells via the high-affinity transporter GAT-3; both effects lead to an increased intracellular Ca2+ concentration. This causes the release of ATP from astrocytes, which enzymatically decomposes to adenosine, stimulating dendritic A1Rs located at CA1 pyramidal neurons. These A1Rs then upregulate dendritic GABAA-R functions. Second, dendritic GABAA-Rs may be also directly stimulated by GABA that is released from the terminals of somatostatin-positive interneurons. In this case both the direct and astrocyte-mediated interneuronal effects result in decreased excitability of CA1 pyramidal neurons [95]. Artwork by Ms Lumei Huang.

The light-induced stimulation of selectively expressed ChR2 in somatostatin-positive interneurons led to subsequent release of GABA onto CA1 pyramidal cells (Figure 2B) [95]. Parallel to the appearance of GABAergic inhibitory postsynaptic currents (IPSCs) in CA1 neurons, astrocytes in this area

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responded with a pronounced increase of their [Ca2+]i, resulting in ATP release [95]. This ATP was then enzymatically converted to adenosine, subsequently activating A1Rs on dendrites of pyramidal neurons and increasing synaptic inhibition of pyramidal neurons by somatostatin interneuron-evoked IPSCs. In amygdala slice preparations, astrocytes depressed excitatory synapses from the basolateral amygdala via A1R activation, and enhanced inhibitory synapses from the lateral subdivision of the central amygdala via A2AR stimulation [96]. Adenosine was generated by enzymatic degradation of the gliotransmitter ATP. In these experiments, endocannabinoids and designer receptors exclusively activated by designer drugs (DREADDs) were used as endogenous and exogenous stimuli to activate astrocytes, respectively. In view of the modulation of neuronal circuits by astrocytic ATP, it is necessary to define which neuronal functions are due to the effects of astrocytic ATP at the whole systems level. The next section gives a short compendium of some of the most pertinent effects.

Systemic Effects of Astrocytic ATP in Health and Disease Sleep It has been shown that adenosine is an endogenous sleep-inducing factor. Pharmacologic inhibition of adenosine A1Rs promote wakefulness, and injecting adenosine into the brain promotes sleep [42,46]. The adenosine involved in sleep regulation appears to be of astrocytic origin, and transgenic dnSNARE mice, in which the SNARE-mediated vesicular release of gliotransmitters is inhibited, show deficits in sleep homeostasis. Although adenosine levels normally rise during wakefulness, they remain unchanged and relatively low in dnSNARE mice throughout a 24 h period [46,97,98]. Multineuronal network-driven fluctuations in membrane potential, known as UP states (during which neurons are depolarized for up to hundreds of milliseconds) and DOWN states, occur in the neocortex [99]. Electrical stimulation of individual astrocytes in a local circuit can trigger UP state synchronizations of neighboring neurons by utilizing purinergic signaling [100]. This may be one of the mechanisms that regulate cycling between wakefulness and sleep.

Synaptic Plasticity and Memory An absence of gliotransmitter release in astrocytes, or blockade of adenosine A1Rs, affected neuronal plasticity and cortical oscillations, and also promoted resilience to the sleep pressure and spatial long-term memory impairments caused by sleep deprivation [101–103]. Astrocytic activation by a designer drug was sufficient to induce NMDA-dependent LTP in hippocampus that persisted after astrocytic activation ceased [104]. Further, optogenetic stimulation of CA1 astrocytes in vivo enhanced memory allocation, in other words it increased neuronal activity in a task-specific way, but only when coupled with learning, and this was not seen in control mice. These findings are particularly interesting in view of the recently demonstrated postsynaptic excitatory interaction between NMDARs and A2ARs on hippocampal CA1 neurons [105]. Eventually, it was shown that Gq DREADD activation in astrocytes of the medial central amygdala caused extinction of learned fear memory in a cued fear-conditioning task [96,103]. Another issue that requires consideration in this context concerns the effects caused by activation of P2X7Rs, given that these receptors in astrocytes (and especially in microglia) are targets of pathologically high extracellular ATP concentrations as well as being exit pathways for nonvesicular release of ATP from astrocytes/microglia. In view of the large number of original publications and excellent review articles available on this subject, we limit ourselves to a short summary of the available data in relation to the following disease states.

Alzheimer’s Disease Alzheimer’s disease (AD) is pathologically defined by the presence of amyloid b (Ab) accumulation in extracellular amyloid plaques, hyperphosphorylated tau protein aggregation in intracellular

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neurofibrillary tangles, and brain atrophy caused by loss of neurons and synapses [106]. Astrocytic hyperactivity, consisting of single-cell transients and calcium waves, was most pronounced in reactive astrogliosis around plaques in a mouse genetic AD model [33,34]. This hyperactivity is reduced after P2Y1R blockade or inhibition of nucleotide release through connexin hemichannels. This finding agrees with the observation that Ab excites astrocytes and in consequence causes oversecretion of glio-signaling molecules, including ATP/ADP, which cause neuronal damage [107,108]. Further, P2Y2Rs were shown to enhance a-secretase-mediated processing of Ab in cortical neurons of the larger amyloid precursor protein (APP) to its soluble, neuroprotective fragment (sAPPa) [109]. By contrast, b- and g-secretases produce the toxic Ab aggregates. Neurodegenerative diseases such as AD, Parkinson’s disease, Huntington’s disease, multiple sclerosis, and amyotrophic lateral sclerosis each possess different pathogenetic mechanisms, but share a superimposed enrichment/migration of microglia/reactive astrocytes in the CNS, with subsequent upregulation of their P2X7Rs. P2X7Rs thereafter release neuroinflammatory cytokines [interleukin-1b, (IL-b), tumor necrosis factor-a, (TNF-a)], reactive oxygen and nitrogen species, as well as proteases that damage neurons [110–112]. Therefore, it is conceivable that P2X7R antagonists may have a beneficial therapeutic effect in these diseases, although definitive evidence can be derived only from controlled clinical studies in humans; the lack of bona fide experimental animal models for most of these diseases is a major limitation in the neurodegeneration field.

Major Depression Major depression can be modeled in mice by applying different stressors which lead to the development of a state called ‘learned helplessness’ that is considered to be a depression-like behavioral response. Learned helplessness can be induced by tail suspension, inescapable foot shock, chronic social defeat, etc. [113]. Recently, depression has been conceptualized as a glia-based synaptic dysfunction because it is associated with decreased density and hypofunction of astrocytes and increased microglial activation in frontohippocampal regions [114,115]. Both lack of IP3R2 and transgenic blockade of vesicular ATP release in the prefrontal cortex and hippocampus of mice blocked the development of depressive-like behavior induced by chronic social defeat [116]. Conventional KO and conditioned astrocytic KO of Calhm2 initiated depression-like behaviors in mice, indicating that this channel is the exit pathway for the release of ATP [117]. In partial disagreement with these findings, the antidepressant drug fluoxetine has been shown to increase ATP exocytosis [118]. In consequence, the authors of this latter study concluded that the astrocytic release of ATP involved in depression operates by vesicular exocytosis rather than by CALHM2 opening. The role of astrocytic and microglial P2X7Rs in depression has been reviewed extensively in the past years (e.g., [119–121]) and we will touch on it only briefly. The general idea is that P2X7R activation and the resulting secretion of proinflammatory cytokines of the IL-1 family induce depression. In consequence, P2X7R antagonists are assumed to be antidepressive agents. The assumption that a decrease in extracellular ATP concentration in the prefrontal cortex and hippocampus could lead to a compensatory upregulation of P2X7Rs is in fact an attractive hypothesis.

Epilepsy High levels of neuronal activity and metabolic stress associated with seizures induces the release of ATP into the extracellular space [122], probably largely from astrocytic sources [123]. In epileptic tissue, the evidence points to enhanced chemical signaling and disrupted linkage between water and potassium balance by reactive astrocytes, which together enhance synchrony in hippocampal microcircuits [124,125]. Using a reporter mouse in which green fluorescent protein is generated in response to transcription of P2rx7, it was shown that the expression of the receptor was selectively increased in the hippocampus of mice that developed epilepsy after intra-amygdala kainic acid-induced status epilepticus (SE) [126,127]; the authors suggested that the increased expression of P2X7Rs took place in neurons rather

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Outstanding Questions Astroglial cells are endowed with several ligand-gated ion channels and G protein-coupled receptors. Are these channels and receptors only phylogenetic remnants or do they perform physiological functions and play a role in pathophysiology? There are relatively few examples of ATP-induced and P2X receptormediated fast neuro-neuronal signaling in the CNS. If available, the excitatory postsynaptic currents are relatively small, are only observed in a subpopulation of neurons, and require strong electrical stimulation to evoke. Alternatively, could astrocytes modulate conventional (e.g., glutamate- or GABA-mediated) rapid excitatory/ inhibitory neurotransmission by their gliotransmitters/signaling molecules including ATP? Whereas ATP is released from neurons by a vesicular/exocytotic process that is dependent on the presence of Ca2+ ions, ATP from astroglia may reach the extracellular space via both exocytotic and nonexocytotic mechanisms. Release pathways for ATP from glial cells not only involve secretory vesicles and secretory lysosomes but also connexin hemichannels, pannexin channels, and the calcium homeostasis modulator 1. The question arises of whether the modulation of particular synaptic circuits depends on specific types of release pathways. Slow modulation of neuronal circuits by ATP may involve several mechanisms such as immediate changes due to the facilitation/ inhibition of neurotransmitter release, alterations of postsynaptic sensitivity to neurotransmitters, or protracted changes due to the upor downregulation of transmitter receptors by interfering with their turnover. It is important to clarify which of these mechanisms operate in individual neuronal circuits to regulate, for example, sleep as well as synaptic plasticity and memory. A possible scenario is that glia– neuron interactions are triggered solely by neuronal inputs that

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than astrocytes. A 5 day treatment of epileptic mice with systemic injections of the blood–brain barrier-permeable, specific P2X7R antagonist JNJ-47965567, beginning on day 11 after SE, significantly reduced the otherwise ensuing spontaneous seizures [126]. Moreover, miRNA targeting of P2X7Rs opposed the contralateral epileptogenic focus in the hippocampus [128]. When the maximal electroshock or pentylenetetrazol seizure threshold tests were used instead of intra-amygdala kainic acid injection, neither brilliant blue G nor JNJ47965567 demonstrated anticonvulsant effects [129]. This finding pointed out that not only the epileptogenic signals but also the endpoint of the measured reactions [electroencephalogram (EEG) seizure activity [126] and manifest muscular convulsions [129]] may be important for the conclusions being drawn. Interestingly, P2Y1Rs were also recognized as targets of a pro- or antiepileptic activity, depending on their time-point of activation [130]. Whereas P2Y1R deficiency or P2Y1R antagonist administration before intra-amygdala kainic acid or intraperitoneal pilocarpine exacerbated epileptoform activity, a P2Y1R agonist administered at this time-point was anticonvulsant. However, when these drugs were administered after the onset of SE, their effects on seizure severity were reversed. Further, TNF-a, that is released during temporal lobe epilepsy, appeared to trigger Ca2+-dependent glutamate release from astrocytes that boosted synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1Rs by astrocyte-derived ATP/ADP [131].

terminate at glial cells, subsequently resulting in glial secretion of various signaling molecules. However, in several neurodegenerative diseases (AD, Parkinson’s disease, Huntington’s disease, multiple sclerosis, and amyotrophic lateral sclerosis), damage by the primary illness leads to ATP release from all types of CNS cells, leading to P2X7R stimulation and aggravation of the original pathological state. It should be clarified whether P2X7Rs receptors are located at microglia/astroglia where they initiate the release of signaling molecules that damage neighboring neurons.

The picture becomes even more complex if we consider that SE is known to transiently increase neurogenesis in the neurogenic niches of the brain, such as in the hippocampal subgranular zone [132]. Under these conditions, neural progenitor cells (NPCs) may migrate to aberrant locations (e.g., the hilus hippocampi) and, after maturation to granule cells, initiate chronic recurrent epileptic fits [133,134]. In this context it is important to note that ATP released during SE onto subgranular zone NPCs activates their P2X7Rs; this led to necrosis/apoptosis which decreased the liability of rodents to develop spontaneous recurrent seizures after a one-time SE [135,136]. In agreement with this hypothesis, intracerebroventricular injection of a P2X7R antagonist before SE enhanced the number of spontaneous seizures.

Concluding Remarks Only relatively few examples are known of ATP-induced and P2XR-mediated fast neuroneuronal signaling in the CNS. However, there are many questions related to neuron-induced but astroglia-mediated modulation of neuronal circuits (see Outstanding Questions). Astroglia are endowed with several ligand-gated ion channels (e.g., P2X) and G protein-coupled receptors (e.g., P2Y) that respond to transmitter/signaling molecule stimulation and in consequence release ATP. The released ATP may interfere with CNS functions via facilitation/inhibition of neurotransmitter release, altering the postsynaptic sensitivity to neurotransmitters, or leading to protracted changes due to up- or downregulation of transmitter receptors by modifying their turnover. Hence, astrocytes mediate slow modulatory effects on neurons via the interposed glial signaling molecule ATP. In various neurodegenerative diseases, damage by the primary illness leads to ATP release from all types of CNS cells, causing P2X7R stimulation and aggravation of the original pathological state. In conclusion, glia-derived ATP operates at the neuron–glia interfaces as an important functional regulator of the brain and spinal cord.

Acknowledgments We are grateful to Ms Lumei Huang for expert drawing of the two figures. Our work was made possible by a generous grant (The Project First-Class Disciplines Development; CZYHW1901) from Chengdu University of TCM to Y.T. and P.I. to establish the International Collaborative Center for Purinergic Signaling, and grants from the Sichuan Provincial Administration of Foreign Affairs to support the stays of P.I. in Chengdu (SZD201731, SZD201846). Financial support from the National Natural Science Foundation of China (81774437, 81173320, 81373735) and the Sichuan Science and Technology Program (2019YFH0108) are also gratefully acknowledged.

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References 1. Burnstock, G. (1972) Purinergic nerves. Pharmacol. Rev. 24, 509–581 2. Kennedy, C. and Burnstock, G. (1985) Evidence for two types of P2-purinoceptor in longitudinal muscle of the rabbit portal vein. Eur. J. Pharmacol. 111, 49–56 3. Abbracchio, M.P. and Burnstock, G. (1994) Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol. Ther. 64, 445–475 4. Burnstock, G. (2009) Autonomic neurotransmission: 60 years since Sir Henry Dale. Annu. Rev. Pharmacol. Toxicol. 49, 1–30 5. Burnstock, G. (2016) Short- and long-term (trophic) purinergic signalling. Philos. Trans. R. Soc. Lond B Biol. Sci. 371 6. Khakh, B.S. and North, R.A. (2012) Neuromodulation by extracellular ATP and P2X receptors in the CNS. Neuron 76, 51–69 7. von Ku¨gelgen, I. and Hoffmann, K. (2016) Pharmacology and structure of P2Y receptors. Neuropharmacology 104, 50–61 8. Edwards, F.A. et al. (1992) ATP receptor-mediated synaptic currents in the central nervous system. Nature 359, 144–147 9. Nieber, K. et al. (1997) Role of ATP in fast excitatory synaptic potentials in locus coeruleus neurones of the rat. Br. J. Pharmacol. 122, 423–430 10. Pankratov, Y. et al. (2007) Quantal release of ATP in mouse cortex. J. Gen. Physiol. 129, 257–265 11. Guzman, S.J. and Gerevich, Z. (2016) P2Y receptors in synaptic transmission and plasticity: therapeutic potential in cognitive dysfunction. Neural. Plast. 2016, 1207393 12. Azevedo, F.A. et al. (2009) Equal numbers of neuronal and nonneuronal cells make the human brain an isometrically scaled-up primate brain. J. Comp. Neurol. 513, 532–541 13. Jha, M.K. et al. (2019) Microglia–astrocyte crosstalk: an intimate molecular conversation. Neuroscientist 25, 227–240 14. Pelvig, D.P. et al. (2008) Neocortical glial cell numbers in human brains. Neurobiol. Aging 29, 1754–1762 15. Verkhratsky, A. and Nedergaard, M. (2018) Physiology of astroglia. Physiol. Rev. 98, 239–389 16. Mederos, S. et al. (2018) Astrocyte–neuron networks: a multilane highway of signaling for homeostatic brain function. Front. Synaptic. Neurosci. 10, 45 17. Verkhratsky, A. et al. (2017) Stratification of astrocytes in healthy and diseased brain. Brain Pathol. 27, 629–644 18. Mishra, A. et al. (2016) Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat. Neurosci. 19, 1619–1627 19. Allen, N.J. and Eroglu, C. (2017) Cell biology of astrocyte–synapse interactions. Neuron 96, 697–708 20. Perea, G. et al. (2014) Neuron–glia networks: integral gear of brain function. Front. Cell Neurosci. 8, 378 21. Durkee, C.A. and Araque, A. (2019) Diversity and specificity of astrocyte–neuron communication. Neuroscience 396, 73–78 22. Palygin, O. et al. (2010) Ionotropic NMDA and P2X1/ 5 receptors mediate synaptically induced Ca2+ signalling in cortical astrocytes. Cell Calcium 48, 225–231 23. Illes, P. et al. (2012) P2X receptors and their roles in astroglia in the central and peripheral nervous system. Neuroscientist 18, 422–438 24. Rivera, A. et al. (2016) A central role for ATP signalling in glial interactions in the CNS. Curr. Drug Targets 17, 1829–1833 25. Jabs, R. et al. (2007) Lack of P2X receptor mediated currents in astrocytes and GluR type glial cells of the hippocampal CA1 region. Glia 55, 1648–1655

12

Trends in Neurosciences, --, Vol. --, No. --

26. Illes, P. et al. (2017) Neuronal P2X7 receptors revisited: do they really exist? J. Neurosci. 37, 7049– 7062 27. Kaczmarek-Hajek, K. et al. (2018) Re-evaluation of neuronal P2X7 expression using novel mouse models and a P2X7-specific nanobody. eLife 7, e36217 28. Khan, M.T. et al. (2018) Astrocytic rather than neuronal P2X7 receptors modulate the function of the tri-synaptic network in the rodent hippocampus. Brain Res. Bull. 151, 164–173 29. Theis, M. and Giaume, C. (2012) Connexin-based intercellular communication and astrocyte heterogeneity. Brain Res. 1487, 88–98 30. Fujii, Y. et al. (2017) Astrocyte calcium waves propagate proximally by gap junction and distally by extracellular diffusion of ATP released from volume-regulated anion channels. Sci. Rep. 7, 13115 31. Pannasch, U. and Rouach, N. (2013) Emerging role for astroglial networks in information processing: from synapse to behavior. Trends Neurosci. 36, 405–417 32. Cheung, G. et al. (2014) Connexons and pannexons: newcomers in neurophysiology. Front. Cell Neurosci. 8, 348 33. Delekate, A. et al. (2014) Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer’s disease mouse model. Nat. Commun. 5, 5422 34. Reichenbach, N. et al. (2018) P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer’s disease model. J. Exp. Med. 215, 1649–1663 35. Bradley, S.J. and Challiss, R.A. (2012) G proteincoupled receptor signalling in astrocytes in health and disease: a focus on metabotropic glutamate receptors. Biochem. Pharmacol. 84, 249–259 36. Tang, W. et al. (2015) Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3–CA1 synapses of adult mice are modulated by glutamate and ATP. J. Neurosci. 35, 3016–3021 37. Pankratov, Y. and Lalo, U. (2015) Role for astroglial a1-adrenoreceptors in gliotransmission and control of synaptic plasticity in the neocortex. Front. Cell Neurosci 9, 230 38. Jennings, A. et al. (2017) Dopamine elevates and lowers astroglial Ca2+ through distinct pathways depending on local synaptic circuitry. Glia 65, 447–459 39. Gundersen, V. et al. (2015) Neuroglial transmission. Physiol. Rev. 95, 695–726 40. Araque, A. et al. (1999) Tripartite synapses: glia, the unacknowledged partner. Trends Neurosci. 22, 208–215 41. Pascual, O. et al. (2005) Astrocytic purinergic signaling coordinates synaptic networks. Science 310, 113–116 42. Halassa, M.M. and Haydon, P.G. (2010) Integrated brain circuits: astrocytic networks modulate neuronal activity and behavior. Annu. Rev. Physiol. 72, 335–355 43. Verkhratsky, A. and Nedergaard, M. (2014) Astroglial cradle in the life of the synapse. Philos. Trans. R. Soc. Lond B Biol. Sci. 369, 20130595 44. Wolosker, H. et al. (2016) The rise and fall of the D-serine-mediated gliotransmission hypothesis. Trends Neurosci 39, 712–721 45. Oliet, S.H. and Mothet, J.P. (2009) Regulation of N-methyl-D-aspartate receptors by astrocytic D-serine. Neuroscience 158, 275–283 46. Papouin, T. et al. (2017) Astrocytic control of synaptic function. Philos. Trans. R. Soc. Lond B Biol. Sci. 372

Please cite this article in press as: Illes et al., Astroglia-Derived ATP Modulates CNS Neuronal Circuits, Trends in Neurosciences (2019), https:// doi.org/10.1016/j.tins.2019.09.006

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47. Fujita, T. et al. (2014) Neuronal transgene expression in dominant-negative SNARE mice. J. Neurosci. 34, 16594–16604 48. Sun, W. et al. (2013) Glutamate-dependent neuroglial calcium signaling differs between young and adult brain. Science 339, 197–200 49. Savtchouk, I. and Volterra, A. (2018) Gliotransmission: beyond black-and-white. J. Neurosci. 38, 14–25 50. Bazargani, N. and Attwell, D. (2016) Astrocyte calcium signaling: the third wave. Nat. Neurosci. 19, 182–189 51. Zorec, R. et al. (2016) Astrocytic vesicles and gliotransmitters: slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture. Neuroscience 323, 67–75 52. Fiacco, T.A. and McCarthy, K.D. (2018) Multiple lines of evidence indicate that gliotransmission does not occur under physiological conditions. J. Neurosci. 38, 3–13 53. Pangrsic, T. et al. (2007) Exocytotic release of ATP from cultured astrocytes. J. Biol. Chem. 282, 28749– 28758 54. Pryazhnikov, E. and Khiroug, L. (2008) Sub-micromolar increase in [Ca2+]i triggers delayed exocytosis of ATP in cultured astrocytes. Glia 56, 38–49 55. Lalo, U. et al. (2014) Exocytosis of ATP from astrocytes modulates phasic and tonic inhibition in the neocortex. PLoS Biol. 12, e1001747 56. Rasooli-Nejad, S. et al. (2014) Cannabinoid receptors contribute to astroglial Ca2+-signalling and control of synaptic plasticity in the neocortex. Philos. Trans. R. Soc. Lond B Biol. Sci. 369, 20140077 57. Zhang, Z. et al. (2007) Regulated ATP release from astrocytes through lysosome exocytosis. Nat. Cell Biol. 9, 945–953 58. Li, D. et al. (2008) Lysosomes are the major vesicular compartment undergoing Ca2+-regulated exocytosis from cortical astrocytes. J. Neurosci. 28, 7648–7658 59. Liu, T. et al. (2011) Calcium triggers exocytosis from two types of organelles in a single astrocyte. J. Neurosci. 31, 10593–10601 60. Oya, M. et al. (2013) Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes. Biochem. Biophys. Res. Commun. 438, 145–151 61. Verkhratsky, A. et al. (2016) Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion. EMBO J. 35, 239–257 62. Xiong, Y. et al. (2018) Ca2+-dependent and Ca2+independent ATP release in astrocytes. Front Mol. Neurosci. 11, 224 63. Suadicani, S.O. et al. (2012) ATP signaling is deficient in cultured pannexin1-null mouse astrocytes. Glia 60, 1106–1116 64. Liu, H.T. et al. (2008) Oxygen-glucose deprivation induces ATP release via maxi-anion channels in astrocytes. Purinergic. Signal. 4, 147–154 65. Lee, S. et al. (2010) Channel-mediated tonic GABA release from glia. Science 330, 790–796 66. Lazutkaite, G. et al. (2017) Amino acid sensing in hypothalamic tanycytes via umami taste receptors. Mol. Metab. 6, 1480–1492 67. Dahl, G. (2015) ATP release through pannexon channels. Philos. Trans. R. Soc. Lond B Biol. Sci. 370 68. Chaumont, S. and Khakh, B.S. (2008) Patch-clamp coordinated spectroscopy shows P2X2 receptor permeability dynamics require cytosolic domain rearrangements but not Panx-1 channels. Proc. Natl. Acad. Sci. U. S. A. 105, 12063–12068 69. Pelegrin, P. and Surprenant, A. (2006) Pannexin-1 mediates large pore formation and interleukin-1b release by the ATP-gated P2X7 receptor. EMBO J. 25, 5071–5082

70. Sperlagh, B. and Illes, P. (2014) P2X7 receptor: an emerging target in central nervous system diseases. Trends Pharmacol. Sci. 35, 537–547 71. Harkat, M. et al. (2017) On the permeation of large organic cations through the pore of ATP-gated P2X receptors. Proc. Natl. Acad. Sci. U. S. A. 114, E3786– E3795 72. Di Virgilio, F. et al. (2018) The elusive P2X7 macropore. Trends Cell Biol. 28, 392–404 73. Qiu, F. and Dahl, G. (2009) A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am. J. Physiol. Cell Physiol. 296, C250–C255 74. Pelegrin, P. (2011) Many ways to dilate the P2X7 receptor pore. Br. J. Pharmacol. 163, 908–911 75. Huang, C. et al. (2012) Critical role of connexin 43 in secondary expansion of traumatic spinal cord injury. J. Neurosci. 32, 3333–3338 76. Beckel, J.M. et al. (2014) Mechanosensitive release of adenosine 5’-triphosphate through pannexin channels and mechanosensitive upregulation of pannexin channels in optic nerve head astrocytes: a mechanism for purinergic involvement in chronic strain. Glia 62, 1486–1501 77. Chever, O. et al. (2014) Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission. J. Neurosci. 34, 11228–11232 78. Prochnow, N. et al. (2012) Pannexin1 stabilizes synaptic plasticity and is needed for learning. PLoS One 7, e51767 79. Ardiles, A.O. et al. (2014) Pannexin 1 regulates bidirectional hippocampal synaptic plasticity in adult mice. Front. Cell Neurosci. 8, 326 80. Lopatar, J. et al. (2015) Pannexin-1-mediated ATP release from area CA3 drives mGlu5-dependent neuronal oscillations. Neuropharmacology 93, 219–228 81. Lalo, U. et al. (2014) Exocytosis of gliotransmitters from cortical astrocytes: implications for synaptic plasticity and aging. Biochem. Soc. Trans. 42, 1275– 1281 82. Boue-Grabot, E. and Pankratov, Y. (2017) Modulation of central synapses by astrocytereleased ATP and postsynaptic P2X receptors. Neural Plast. 2017, 9454275 83. Lalo, U. et al. (2018) Diversity of astroglial effects on aging- and experience-related cortical metaplasticity. Front. Mol. Neurosci. 11, 239 84. Gordon, G.R. et al. (2005) Norepinephrine triggers release of glial ATP to increase postsynaptic efficacy. Nat. Neurosci. 8, 1078–1086 85. Gordon, G.R. et al. (2009) Astrocyte-mediated distributed plasticity at hypothalamic glutamate synapses. Neuron 64, 391–403 86. Pougnet, J.T. et al. (2014) ATP P2X receptors downregulate AMPA receptor trafficking and postsynaptic efficacy in hippocampal neurons. Neuron 83, 417–430 87. Pougnet, J.T. et al. (2016) P2X-mediated AMPA receptor internalization and synaptic depression is controlled by two CaMKII phosphorylation sites on GluA1 in hippocampal neurons. Sci. Rep. 6, 31836 88. Wirkner, K. et al. (2007) Modulation of NMDA receptor current in layer V pyramidal neurons of the rat prefrontal cortex by P2Y receptor activation. Cereb. Cortex 17, 621–631 89. Crosby, K.M. et al. (2018) Cholecystokinin switches the plasticity of GABA synapses in the dorsomedial hypothalamus via astrocytic ATP release. J. Neurosci. 38, 8515–8525 90. Zhang, J.M. et al. (2003) ATP released by astrocytes mediates glutamatergic activity-dependent heterosynaptic suppression. Neuron 40, 971–982 91. Bowser, D.N. and Khakh, B.S. (2004) ATP excites interneurons and astrocytes to increase synaptic

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92.

93. 94. 95.

96. 97. 98. 99. 100. 101.

102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115.

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inhibition in neuronal networks. J. Neurosci. 24, 8606–8620 Shigetomi, E. et al. (2008) Two forms of astrocyte calcium excitability have distinct effects on NMDA receptor-mediated slow inward currents in pyramidal neurons. J. Neurosci. 28, 6659–6663 Tan, Z. et al. (2017) Glia-derived ATP inversely regulates excitability of pyramidal and CCKpositive neurons. Nat. Commun. 8, 13772 Shen, W. et al. (2017) An autocrine purinergic signaling controls astrocyte-induced neuronal excitation. Sci. Rep. 7, 11280 Matos, M. et al. (2018) Astrocytes detect and upregulate transmission at inhibitory synapses of somatostatin interneurons onto pyramidal cells. Nat. Commun. 9, 4254 Martin-Fernandez, M. et al. (2017) Synapse-specific astrocyte gating of amygdala-related behavior. Nat. Neurosci. 20, 1540–1548 Halassa, M.M. et al. (2009) Astrocytic modulation of sleep homeostasis and cognitive consequences of sleep loss. Neuron 61, 213–219 Blutstein, T. and Haydon, P.G. (2013) The importance of astrocyte-derived purines in the modulation of sleep. Glia 61, 129–139 Steriade, M. et al. (2001) Natural waking and sleep states: a view from inside neocortical neurons. J. Neurophysiol. 85, 1969–1985 Poskanzer, K.E. and Yuste, R. (2011) Astrocytic regulation of cortical UP states. Proc. Natl. Acad. Sci. U. S. A. 108, 18453–18458 Florian, C. et al. (2011) Astrocyte-derived adenosine and A1 receptor activity contribute to sleep lossinduced deficits in hippocampal synaptic plasticity and memory in mice. J. Neurosci. 31, 6956–6962 Illes, P. and Verkhratsky, A. (2016) Purinergic neurone–glia signalling in cognitive-related pathologies. Neuropharmacology 104, 62–75 Santello, M. et al. (2019) Astrocyte function from information processing to cognition and cognitive impairment. Nat. Neurosci. 22, 154–166 Adamsky, A. et al. (2018) Astrocytic activation generates de novo neuronal potentiation and memory enhancement. Cell 174, 59–71 Mouro, F.M. et al. (2018) Adenosine A2A receptors facilitate synaptic NMDA currents in CA1 pyramidal neurons. Br. J. Pharmacol. 175, 4386–4397 Henstridge, C.M. et al. (2019) Beyond the neuroncellular interactions early in Alzheimer disease pathogenesis. Nat. Rev. Neurosci. 20, 94–108 Verkhratsky, A. et al. (2010) Astrocytes in Alzheimer’s disease. Neurotherapeutics 7, 399–412 Zorec, R. et al. (2017) Astroglial vesicular trafficking in neurodegenerative diseases. Neurochem. Res. 42, 905–917 Erb, L. et al. (2015) P2Y receptors in Alzheimer’s disease. Biol. Cell 107, 1–21 Sperlagh, B. et al. (2006) P2X7 receptors in the nervous system. Prog. Neurobiol. 78, 327–346 Bhattacharya, A. and Biber, K. (2016) The microglial ATP-gated ion channel P2X7 as a CNS drug target. Glia 64, 1772–1787 Illes, P. et al. (2019) The P2X7 receptor: a new therapeutic target in Alzheimer’s disease. Expert. Opin. Ther. Targets. 23, 165–176 Krishnan, V. and Nestler, E.J. (2011) Animal models of depression: molecular perspectives. Curr. Top. Behav. Neurosci. 7, 121–147 Banasr, M. and Duman, R.S. (2008) Glial loss in the prefrontal cortex is sufficient to induce depressivelike behaviors. Biol. Psychiatry 64, 863–870 Rial, D. et al. (2015) Depression as a glial-based synaptic dysfunction. Front. Cell Neurosci. 9, 521

Trends in Neurosciences, --, Vol. --, No. --

116. Cao, X. et al. (2013) Astrocyte-derived ATP modulates depressive-like behaviors. Nat. Med. 19, 773–777 117. Jun, M. et al. (2018) Calhm2 governs astrocytic ATP releasing in the development of depression-like behaviors. Mol. Psychiatry 23, 883–891 118. Kinoshita, M. et al. (2018) Anti-depressant fluoxetine reveals its therapeutic effect via astrocytes. EBioMedicine 32, 72–83 119. Sperlagh, B. et al. (2012) The role of purinergic signaling in depressive disorders. Neuropsychopharmacol. Hung. 14, 231–238 120. Stokes, L. et al. (2015) Understanding the role of P2X7 in affective disorders – are glial cells the major players? Front. Cell Neurosci. 9, 258 121. Bhattacharya, A. and Jones, D.N.C. (2018) Emerging role of the P2X7–NLRP3–IL1b pathway in mood disorders. Psychoneuroendocrinology 98, 95–100 122. Frenguelli, B.G. and Wall, M.J. (2016) Combined electrophysiological and biosensor approaches to study purinergic regulation of epileptiform activity in cortical tissue. J. Neurosci. Meth. 260, 202–214 123. Beamer, E. et al. (2019) ATP release during seizures – a critical evaluation of the evidence. Brain Res. Bull. 151, 65–73 124. Kumaria, A. et al. (2008) ATP signalling in epilepsy. Purinergic Signal 4, 339–346 125. Wetherington, J. et al. (2008) Astrocytes in the epileptic brain. Neuron 58, 168–178 126. Jimenez-Pacheco, A. et al. (2016) Transient P2X7 receptor antagonism produces lasting reductions in spontaneous seizures and gliosis in experimental temporal lobe epilepsy. J. Neurosci. 36, 5920–5932 127. Engel, T. et al. (2016) ATPergic signalling during seizures and epilepsy. Neuropharmacology 104, 140–153 128. Jimenez-Mateos, E.M. et al. (2015) microRNA targeting of the P2X7 purinoceptor opposes a contralateral epileptogenic focus in the hippocampus. Sci. Rep. 5, 17486 129. Fischer, W. et al. (2016) Critical evaluation of P2X7 receptor antagonists in selected seizure models. PLoS One 11, e0156468 130. Alves, M. et al. (2019) Context-specific switch from anti- to pro-epileptogenic function of the P2Y1 receptor in experimental epilepsy. J. Neurosci. 39, 5377–5392 131. Nikolic, L. et al. (2018) Blocking TNFa-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis. Glia 66, 2673–2683 132. Parent, J.M. (2007) Adult neurogenesis in the intact and epileptic dentate gyrus. Prog. Brain Res. 163, 529–540 133. Bouilleret, V. et al. (1999) Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy. Neuroscience 89, 717–729 134. Shapiro, L.A. et al. (2007) Newly generated granule cells show rapid neuroplastic changes in the adult rat dentate gyrus during the first five days following pilocarpine-induced seizures. Eur. J. Neurosci. 26, 583–592 135. Rozmer, K. et al. (2017) Pilocarpine-induced status epilepticus Increases the sensitivity of P2X7 and P2Y1 receptors to nucleotides at neural progenitor cells of the juvenile rodent hippocampus. Cereb. Cortex 27, 3568–3585 136. Tang, Y. and Illes, P. (2017) Regulation of adult neural progenitor cell functions by purinergic signaling. Glia 65, 213–230