By means of their close juxtaposition to synapses, their expression of an extraordinary assortment of membrane transporters and receptors as well as their physical and functional coupling through gap junctions (GJs), astroglial networks provide a perfect spatial buffering for neural activity (Figure 2) (97–99). Astrocytes are key regulators of the extracellular K⁺ concentration. Their high K⁺ permeability mediated by inwardly-rectifying K_ir and two-pore-domain K_2P channels, Na⁺/K⁺ pumps and Na⁺/K⁺/Cl⁻ transporters associated with their extensive GJ coupling enables them to uptake and redistribute excessive extracellular K⁺ resulting from neuronal firing (100–103). Astroglial K⁺ and glutamate uptake is altered in cultured cortical astrocytes after K_ir4.1 channel downregulation (104) as well as in astroglial-specific K_ir4.1 knock-out mice (105, 106). Gain-of-function (107) as well as loss-of-function mutations (108, 109) in the human KCNJ10 gene encoding K_ir4.1 have been linked to forms of childhood epilepsies associated with ataxia and cognitive impairment, but not to CAE. Notably, artificially increasing extracellular K⁺ concentration ex vivo is associated with propagating epileptiform discharges induced by focal optogenetic activation of parvalbumin-expressing interneurons (110). In vivo though, K⁺ clearance impairment induced by blocking GJ was not sufficient to induce neocortical seizures (111). Interestingly, valproic acid (but not ethosuximide) induces K_ir4.1 overexpression in the cortex of healthy rats (112). Nevertheless, further research is required to address the actual contribution of K_ir4.1 overexpression in the anti-absence effects of valproic acid, as well as the putative role of astroglial K_ir4.1 itself in the development and propagation of SWDs. The combined use of cell-specific conditional knock-out of these channels and pharmacological models of SWDs could shine new light on the topic.

K⁺ uptake is associated with cellular swelling due to Na⁺/K⁺-pump dependent water influx (113). Although the exact molecular mechanisms are still under debate, water fluxes across the astrocytic membrane are associated with K⁺ homeostasis and they influence local interstitial osmolarity as well as seizure generation and progression (114–120). Given the accumulating evidence against the predominant contribution of the astroglial water channel aquaporin-4 (AQP4) in water homeostasis (113, 121), the use of AQP4 conditional knock-out as a model of disrupted water homeostasis has been recently challenged. Nevertheless, water homeostasis impairment and the resulting volume and osmolarity dysregulation should affect neural network excitability. Indeed, a recent structural MRI study on CAE showed significant gray matter volume abnormalities in both frontotemporal cortical region and posterior thalami compared to controls (122, 123).

GJ coupling, particularly mediated by connexins Cx30 and Cx43, provides the astroglial network with a high level of intercellular structural, metabolic and functional connectivity, enabling the exchange of ions and small molecules (124–131). In the context of epilepsy, connexins mediate ATP release (into the extracellular space through hemichannels), the spreading of intercellular Ca²⁺ waves (132) and are fundamental in the spatial buffering required for K⁺ and water homeostasis as well as glutamate clearance (133, 134). With respect to absence epilepsy, most advancement in unraveling the role of GJs has been obtained employing GJ blockers in well-established genetic animal models (135). The broad-spectrum GJ blocker carbenoxolone (CBX) decreased both amplitude and duration of 4-aminopyridine-induced seizure-like events (SLEs) in thalamocortical slices obtained from mice with spontaneous SWDs (136, 137) as well as the duration of SWDs seen in GAERS rats in vivo after systemic application (138). Interestingly, in vivo injection of CBX in the NRT of rats with atypical absence seizures and spontaneous SWDs decreased the duration of SWDs (139), whereas no alteration of SWD phenotype was observed if CBX was injected in the posterior thalami of WAG/Rij rats and the lethargic mouse genetic model of absence epilepsy (140). Recently, intraperitoneal injection of CBX was associated with absence seizures worsening in WAG/Rij rats (141), hinting at non-obvious and non-trivial differences across the absence epilepsy models. The endocannabinoids anandamide (N-arachidonoylethanolamine, ANA) and oleamide (cis-9,10-octadecenoamide, OLE) are specific Cx43 blockers (142, 143). Intracerebroventricular injection of ANA decreased in a dose-dependent manner the recurrence and duration of SWDs, although its mechanism of action likely involves type-1 cannabinoid (CB1) receptor activation (144) or even direct inhibition of T-type Ca²⁺ channels (145). Interestingly, although specific studies addressing the impact of OLE in absence epilepsy are still required, OLE has a sleep-inducing effect and enhances GABA_A receptor-mediated responses, thus possibly affecting the physiological, temporal-spatial pattern of cortico-thalamo-cortical oscillations (146, 147). CBX as well as ANA and OLE block both GJ activity and connexin hemichannels regulating water and solute (notably ATP) exchanges between the intra- and extracellular space, thus challenging the attribution of any observed phenotype to the sole GJ coupling (131, 148). Moreover, regional differences in connexin isoform expression may be at the basis of different contributions of GJ and hemichannel inhibition in different neural networks, and thus the net phenotypical outcome of the pharmacological manipulation (149). CBX is also known to block pannexin-1, which bears significant topological and pharmacological similarities with the connexins and forms single-membrane channels which have been linked to network hyperexcitability and hypersynchronization by mediating both ATP and glutamate release (150, 151). The use of antibodies or small peptides targeting specific amino acid sequences of different connexins (152–155) could shed new light into the differential contribution of GJ coupling and hemichannel function as well as into the role of different connexin isoforms and pannexin-1 channels in the generation and propagation of SWDs. Finally, ANA, but not OLE, can block Ca²⁺ wave propagation in astrocytes, which has to be taken into consideration in the interpretation of the results (142, 143).

In summary, astroglial networks contribute to the imbalance of neural excitation/inhibition through K⁺ and neurotransmitter (glutamate but also GABA) clearance under physiological conditions, thus counteracting network priming through aberrant shifts in the E/I balance possibly leading to network synchronization. Astrocytes rely on their extensive GJ coupling enabling effective spatial ionic, osmotic, and functional buffering. GJ hemichannels as well as pannexin-1 channels may be responsible for augmented synchronous activity through ATP and glutamate release and following Ca² spreading throughout the astroglial network. So far, we are still missing evidence for linking the astroglial fine-tuning of the extracellular ion and transmitter homeostasis to SWDs. However, as it is the case for other kinds of epileptiform activity, their role in regulating such network excitability is very likely.

Astrocytes do not only contribute to neural excitability and functioning by responding to neurotransmitter release and modification of extracellular ionic composition, they are also actively involved in neurotransmitter uptake and release, thus having a direct control of E/I balance (Figure 3) (86, 156–158). One of the key features of absence epilepsy are altered GABA levels (72, 159) and GABAergic tonic and phasic inhibition in the cortico-thalamo-cortical network (25, 73). In both GAERS rats and stargazer mice, astroglial GABA transporter GAT-1 malfunction leads to increased GABA levels in the thalamus resulting in altered tonic inhibition of GABA_A receptors on the thalamo-cortical neurons (72, 74, 75, 77). Notably, a number of human mutations in SLC6A1 encoding GAT-1 leads to reduced GABA transport activity, and some of the mutations are associated with CAE or clinical conditions associated with absence seizures (160–164). Moreover, GABA released by astrocytes was proven to activate GABA_A receptors on the membrane of thalamocortical neurons in rodents (165) and blocking astroglial GATs increased extrasynaptic GABA_A receptor-mediated tonic inhibition (166). On the other hand, thalamic astrocytes express GABA_A receptors themselves (167), whose specific role has not been fully resolved yet. Neuronal presynaptic GABA_B receptor expression and function is impaired in the neocortex of WAG/Rij rats, possibly contributing to network hyperexcitability (168, 169). There is plenty of evidence that GABA_B receptors contribute to network priming in absence seizures facilitating thalamo-cortical burst firing, as supported by the exacerbation of SWDs after baclofen or GHB treatment (170–176). Interestingly, the activation of extrasynaptic GABA_B receptors require GABA spillover resulting from an intense GABAergic stimulation, which is in accordance with a predominant role of astrocytic GAT-1 in regulating SWDs, given its expression in close proximity of neuronal synapses compared to a more distal location of GAT-3 (175). A further level of complexity is given by the fact that astrocytes themselves express GABA_B receptors and their activation leads to downstream Ca²⁺ signaling and possibly gliotransmission as shown in the thalamus upon local ex vivo baclofen and GHB application (177).

As expected, the injection of the GAT inhibitor tiagabine in the thalamus enhances SWDs (178), while its injection in the somatosensory cortex suppresses SWDs, as does the injection of positive allosteric modulators of glutamate metabotropic receptors mGluR1 and mGluR5 in both somatosensory cortex and thalamus (178). A line of experimental evidence suggests that possibly all metabotropic glutamate receptors, including the mGluR2/3 and mGluR5 expressed on the astroglial membrane, are involved in SWDs through modulation of NMDA receptors and GABA uptake (178–182). Indeed, a subpopulation of astrocytes in the thalamus expresses mGluR5 and respond to cortico-thalamic glutamatergic afferents via intracellular Ca²⁺ oscillations (183). Therefore, it is very likely that astrocytes contribute to SWD phenotype by processing glutamate signaling. Astroglial glutamate transporters EAAT1 (GLutamate ASpartate Transporter, GLAST) and EAAT2 (GLutamate Transporter-1, GLT-1) (184, 185) as well as astroglial glutamine-glutamate-GABA cycle impairment (186, 187) have already been associated with the development of various forms of epileptic activities. GAERS rats display decreased protein expression of both astroglial GLT-1 and GLAST proteins before the development of absence seizures (188). Notably, GLAST is overexpressed at the mRNA level, possibly due to a compensatory mechanism of gene transcription (189). Moreover, excessive neuronal firing is known to induce astroglial swelling and subsequent glutamate release (190). This may add a further level of complexity in the already complex temporal firing dynamics of the thalamo-cortical neurons and NRT neurons both ictally and at interictal-to-ictal transitions (25). Although not yet proven in the context of SWDs, astrocytes possess the extraordinary capability of converting intensive glutamatergic neuronal activity into tonic inhibition, by coupling the glutamate/Na⁺ symport with the glutamine and GABA/Na⁺ symport (191). Notably, the only ATP expenditure associated with this process relies on the replenishment of the intracellular GABA storage since the driving force of the glutamine and GABA release is the re-establishment of the physiological Na⁺ homeostasis altered by the glutamate/Na⁺ symport. Finally, a comprehensive study of metabolic alterations in GAERS rats provides further insight into the cortical and thalamic astroglial contribution to the pathology of SWDs. Most strikingly, cortical astroglial metabolism and glutamine-glutamate-GABA cycle are enhanced in GAERS rats, leading to increased glutamate and glutamine levels and decreased GABA labeling (192). Interestingly, the expression of astroglial glutamate dehydrogenase is increased, in the cortex before the development of absence seizures and in the thalamus before and after the development of absence seizures, thus possibly leading to a decreased glutamate availability and a shift to the thalamic GABAergic inhibition fundamental for the generalization of SWDs (193). In line with this hypothesis, the intraperitoneal injection of branched-chain amino acids and α-ketoisocaproate pushing the chemical equilibrium toward the synthesis of glutamine led to decreased thalamic glutamate levels and the worsening of absence seizures (194). Moreover, a gain-of-function mutation of the glutamate dehydrogenase gene leading to aberrant glutamate availability and hyperammonemia has been associated with myoclonic absence epilepsy (195). Although further research in the field of absence epilepsy is still required, this evidence supports the role of astrocytic metabolism and glutamine-glutamate-GABA cycle in providing adequate energy supply and network homeostasis required for epileptic activity generation and propagation (94, 196).

In situ hybridization and Western blot analysis showed reduced levels of CB1 receptor mRNA and protein in the NRT and of the CB1 receptor in the thalamus of WAG/Rij rats at the protein level, thereby suggesting an impaired depolarization-induced CB1-mediated suppression of inhibition (197). Indeed, acute systemic injection of the synthetic CB1 receptor agonist WIN55,212-2 resulted in a transient reduction in SWDs frequency, however surprisingly followed by an increase in SWD duration in subchronic treatment (144, 197–199). Since the beneficial effects of the endocannabinoid ANA, previously described, last longer than the transient reduction in SWD frequency induced by the synthetic CB1 agonist and since ANA does indeed shorten SWDs, its mechanism of action is likely not only dependent on CB1 activation but a more complex molecular process (144).

The release of ATP through connexin and pannexin-1 hemichannels and the resulting spread of Ca²⁺ waves largely contribute to the astrocyte-mediated purinergic signaling in epilepsy (200). However, the net impact on the neural network is often context-dependent and may include the conversion of ATP into adenosine. Adenosine levels depend on extracellular ectonucleotidases as well as on the astroglial adenosine kinase (ADK) and its contribution encompasses antiepileptic A1 receptor-mediated as well as proepileptic A2 and A3 receptor-mediated effects (200–203). Once again, most research results have been derived from the analysis of convulsive seizures. Nevertheless, there is a number of evidence suggesting that purinergic signaling is altered in SWDs, too. To which extent this is related to astroglial contribution is still elusive. With respect to SWDs, GAERS rats show lower expression of A1 receptors in the NRT (204) and WAG/Rij rats are characterized by altered expression of A2A receptors in the somatosensory cortex, NRT and thalamus (205). Absence epileptic activity in WAG/Rij rats increases after activation of A2A receptors directly by the specific synthetic agonist 2-[4-(-2-carboxyethyl)-phenylamino]-5′-N-ethylcarboxamido-adenosine (CGS21680) (205) or indirectly after intraperitoneal injection of guanosine (206) as well as of adenosine (207). Conversely, acute caffeine administration, which is a mixed non-specific A1 and A2A receptor antagonist, reduced both amplitude and duration of SWDs in GAERS rats (208). However, the administration of the specific A1 antagonist 1,3.dipropyl-8-cyclopentylxanthine (DPCPX) in WAG/Rij rats had a proepileptic effect on SWDs (209). Notably, a duplication in the chromosomal region containing the gene coding for the extracellular catabolic enzyme adenosine deaminase was associated with a case of early-onset absence epilepsy, possibly leading to an impairment in adenosine homeostasis (210, 211).

The neuropeptide Y (NPY) released by thalamic neurons promotes phase-specific long-term depression of neuronal excitability in the NRT as well as in the thalamus itself and thus possibly contributing to thalamocortical synchronization and the altered dynamics of T-type Ca²⁺ channel-mediated bursting activity in the thalamic nuclei (212). Interestingly, valproic acid treatment increases thalamic levels of NPY mRNA in GAERS rats (213). Moreover, NPY intracerebroventricular injection as well as focal administration of NPY in the somatosensory cortex of GAERS rats had a strong antiepileptic effect mediated by the NPY receptor Y2 (214–216). This was confirmed by the analysis of specific NPY receptor knock-out mice (217, 218) and injection of the specific Y2 receptor agonist Ac[Leu (28, 31)] NPY24-36 and the specific Y2 receptor antagonist BIIE0246 in GAERS rats (215). Notably, viral overexpression of NPY as well as the mRNA of its receptor Y2, both in thalamus and somatosensory cortex of GAERS rats, reduced the number of seizures and the time spent in seizure activity (219). Since astrocytes produce (220) and release (221) NPY and express NPY receptors, including Y2 receptor (222, 223), one can imagine that astrocytes may play a role in NPY signaling in the pathophysiology of cortico-thalamo-cortical networks.

Alterations of astroglial neurometabolic coupling and contribution to the glutamine-glutamate-GABA cycle may be at the basis of SWDs, possibly through enhanced metabolism and glutamate presentation to cortical neurons. Moreover, astroglial control of extracellular neurotransmitter level, based on the expression of glutamate and GABA transporters (EAATs and GATs, respectively) and receptors (both metabotropic and ionotropic) and direct and indirect release of glutamate and GABA, plays a fundamental role in maintaining the E/I balance in the cortex, thalamus and NRT. Astroglial ATP release and subsequent adenosine production seem to have context-dependent effects on neural excitability, but generally in line with observations derived from convulsive seizures pointing at an antiepileptic and proepileptic role of A1 and A2 receptors, respectively. Shifts in the E/I toward inhibition in the thalamus (possibly through altered endocannabinoid signaling) and toward excitation in the NRT and cortex have a pro-epileptic effect on SWDs. Unexpected net outcomes of pharmacological or genetic manipulation may be due to differential impact on different key nodes of the cortico-thalamo-cortical network and/or to astrocytic ability to both preserve and reverse the sign of the input signal.

Intracellular Ca²⁺ oscillations are one of the most studied indicators of astroglial activity and information coding mechanism at the core of the astroglial signaling cascade resulting, among others, in gliotransmission (86). In the context of convulsive epilepsy, excitotoxic spilling of glutamate, GABA and ATP resulting from excessive network activity as well as dying cells induce perturbation in astroglial Ca²⁺ signals (224, 225). Conversely, spontaneous as well as induced Ca²⁺ oscillations lead to gliotransmission thus influencing neuronal synchrony and E/I balance (226–234). Notably, astroglial Ca²⁺ elevations precede temporally neuronal engagement and their attenuation results in reduction of the epileptic activity in an in vivo model of temporal lobe epilepsy (TLE) (235). Moreover, astroglial Ca²⁺ activity is associated with spreading depolarization-mediated seizure termination (236). However, current research is far from understanding astroglial Ca²⁺ contribution to seizure generation, propagation, severity, and termination both in mechanistic and logical (sufficiency and/or necessity) terms. In particular, research on the contribution of astroglial Ca²⁺ signaling in seizure phenotype has not yet provided causative links to the SWD pathophysiology. Nevertheless, in the following paragraph we include some observations that encourage further research on the topic.

Thalamic astroglial networks display multi-cellular Ca²⁺ oscillations in absence of neuronal input and induce glutamate release and NMDA-receptor mediated long lasting inward currents in thalamocortical neurons as studied in acute brain slice preparations (226, 237). Thalamic astrocytes segregate into two groups: a first group with mGluR5-dependent and no voltage-dependent Ca²⁺ oscillations in response to cortico-thalamic activation, and a second group with no mGluR5- but voltage-dependent Ca²⁺ responses (183). Moreover, thalamic astroglial Ca²⁺ responses were recorded after acute ex vivo application of the weak GABA_B receptor agonist GHB (177), thus suggesting a putative role of astrocytes in the regulation of GABAergic signaling in the thalamus and possible in the phenomenology of SWDs. Notably, sustained GABA_B receptor activation led to a decrease in glutamate release from astrocytes (177). In addition, Ca²⁺ signaling and GABA seem to be connected since artificial inhibition of Ca²⁺ oscillation in striatal astrocytes leads to GAT3 functional upregulation and increased GABA uptake (238). Further evidence suggesting an integrative role of thalamic astrocytes in cortico-thalamic interactions comes from the observation that astroglial glutamate- and NMDA receptor- mediated slow inward currents (SICs) in the thalamo-cortical neurons are largely resistant to afferent cortico-thalamic inputs in their emergence but not in their frequency upon sustained input (239, 240). Moreover, cortico-thalamic glutamatergic input induced disinhibition of thalamo-cortical neurons through astroglial mGluR2 activation, Ca²⁺-dependent glutamate release and inhibition of presynaptic GABAergic projections from the NRT (241). In the NRT astrocytes also enhance GABA_A receptor signaling (242). Astrocyte-induced glutamate-mediated SICs of thalamo-cortical neurons seem to be dependent on extracellular glutamate levels, since exogenous exposure to the glutamate-mimetic D-aspartate increased the frequency of SICs (243). Although it is still unclear if abnormal or hypersynchronous astroglial Ca²⁺ signals could promote epileptiform network activity by itself, this evidence further supports an astroglial contribution to the propagation and self-sustain of seizure-like activity (244, 245).

The role of astrocytic Ca²⁺ signaling in epilepsy, and particularly in SWD-displaying epilepsies, is far from being understood. Yet, association studies on CAE and other idiopathic epileptic forms displaying SWDs as well as the evaluation of the genetic etiology of rodent absence epilepsy models point to a plethora of genes involved in voltage-gated Ca²⁺ channel signaling and G protein-coupled receptor signaling that is worth further assessment (7).

Astroglia display spontaneous Ca²⁺ oscillations responsible for gliotransmission and homeostatic control of the E/I balance as well as network synchronicity. Moreover, astrocytes respond to physiological network activity and pathological neurotransmitter spilling and release from dying cells by Ca²⁺ elevations, typically further contributing to network priming, seizure initiation and progression. Conversely, Ca²⁺ signaling-induced gliotransmitter release and modulation of astroglial neurotransmitter receptors and transporters may underlie putative (or potential) anti-epileptic roles of Ca²⁺ signaling. Notably, astroglial Ca²⁺ signaling may also contribute to seizure suppression. To which extent this applies to SWDs is still unclear.

Astroglial proliferation and morphological, biochemical, and functional changes associated with epilepsy as well as with other neurodegenerative diseases are commonly referred to as reactive astrogliosis (246–248). The term is misleading since it implies that the pathological phenotype of astrocytes results from the epileptiform activity and oversees the possible causative role of astrocyte modifications in its genesis (249–251). In GAERS rats, cortical as well as thalamic astrocytes display enhanced expression of the glial fibrillary acidic protein (GFAP) even before the onset of absence seizures (193). Similarly, increased levels of GFAP expression can be found in adult WAG/Rij rats, though to a lesser extent than in GAERS rats (252). Astonishingly, the number of glial cells in the somatosensory cortex is significantly decreased (253). This suggests that biochemical and functional changes may contribute to a greater extent to the pathology of absence seizures than morphological alteration or that the latter involves qualitative astroglial reorganization, e.g., overlap of the astroglial processes, astroglial domain reorganization, structural and quantitative alteration of synaptic contacts or blood-brain barrier dysfunction. Notably, valproic acid diminishes the overlap of astroglial processes observed in correspondence of epileptic foci in several pathological models of convulsive seizures (254). Nevertheless, it is not clear if the same is happening in the pharmacodynamics of valproic acid in the context of SWDs. The same is true for the alterations of the blood-brain barrier (BBB) which have been associated with many pathological scenarios, including epilepsy (255), but whose role in SWDs has not been extensively addressed yet.

Pathological stimulation of astrocytes during convulsive epileptiform activity leads to astrocytic upregulation and release of proinflammatory cytokines, with IL-1β, Il-6, and TNFα as the most prominent ones. These factors, in turn, can induce astroglial dysfunction leading to, among others, increased glutamate release, decreased glutamate uptake, down-regulation of K_ir4.1, AQP4, connexins, and glutamine synthetase as well as upregulation of adenosine kinase (256–258). IL-1β is induced in reactive astroglia in the somatosensory cortex (and not in other regions of the cortex) in adult GAERS rats with mature SWDs and interestingly also in some young GAERS in association with immature forms of SWDs (259). Furthermore, inhibition of IL-1β biosynthesis in adult GAERS reduced both the number as well as the duration of SWDs. Conversely, IL-1β intraperitoneal administration in WAG/Rij rats induced a significant increase in SWDs and worsened the proepileptic effects of the GABA reuptake inhibitor tiagabine (260). TNFα administration also aggravates SWDs but with kinetics incompatible with a direct effect and therefore possibly through de novo production of IL-1β itself. Moreover, before the onset of SWDs, young WAG/Rij rats showed increased TNFα blood levels, which gradually decreased with age and returned to physiological levels in adult rats displaying mature SWDs, thus possibly suggesting a neuroprotective role of TNFα (260). The precise mechanism of TNFα action in this scenario is not clear, although it is known that TNFα reduces astroglial glutamate uptake and decreases neuronal GABA_A receptor expression (261, 262). Notably, IL-1β-, TNFα-, and IL-6-inducing lipopolysaccharide (LPS) injection in WAG/Rij also promoted SWDs and the increase in the latter was prevented by blocking the inflammatory response with indomethacin (263, 264) as well as blockers of the mTOR pathway (265, 266). Similarly, LPS effects on SWDs were later confirmed in GAERS rats (267). Although IL-1β is believed to increase the levels of glutamate, co-administration of LPS and the NMDA receptor antagonist D-(-)-2-Amino-5-phosphonopentanoic acid (AP-5) did not counteract LPS effects as expected, but conversely prolonged them (264). Recently, it has been reported that IL-6 receptor (IL-6R) blockage via tocilizumab (a humanized monoclonal antibody against IL-6R) reduces SWDs in WAG/Rij rats and inhibits their LPS-induced worsening (268). In line with that, human CAE is known to be associated with detectable levels of IL-6 and IL-8 in the cerebrospinal fluid (269) and treatment with valproic acid reduces IL-6 serum levels in children with tonic-clonic generalized seizures (270).

Several lines of evidence support a role for a direct contribution of pro-inflammatory cytokines in the genesis and worsening of SWDs. Notably, astroglial alterations and cytokine release precede SWD onset, although it cannot be excluded that these cell responses may be due to subclinical epileptiform activities or genetic predispositions. IL-1β, IL-6 and TNFα may contribute to the pathology of SWDs, possibly through impaired K⁺ clearance, glutamine-glutamate-GABA cycle, adenosine metabolism, gliotransmission, and neurotransmitter reuptake. Other morphological alterations, such as astroglial overlap, connectivity, and synaptic coverage, may play a role as well.

Given the fact that both SWDs and NREM recruit the cortico-thalamo-cortical network, further insights into the role of astrocytes in the pathophysiology of SWDs may derive from evidence in their contribution to sleep (particularly sleep architecture and sleep/wake cycle) (Figure 5) as well as the circadian cycle. Astroglial impact on circadian clock mechanisms generated in deep structures may contribute to epilepsy in non-intuitive ways (335). In the hypothalamic primary timekeeping center, the suprachiasmatic nucleus (SCN), astrocyte-derived glutamate inhibits neuronal firing through presynaptic NMDA receptors specifically during the night (338). Moreover, astrocytes release adenosine in a CB1 receptor- and intracellular Ca²⁺ signaling-dependent manner and induce the disinhibition of SCN neurons (339). WAG/Rij rats are also characterized by astrogliosis and impaired GABAergic transmission in the thalamic intergeniculate leaflet, which coordinates inputs from the retina and outputs to the SCN (340). The research on the role of astrocytes on timekeeping is still at its early days, but several lines of evidence support astroglial contribution to sleep, namely the modulation of sleep homeostasis, sleep pressure, vigilance states and sleep-dependent cognitive function, brain energetics and network metabolic supply, network excitability and sleep-associated waste clearance (341, 342).

The sleep/wake cycle is associated with changes in interstitial fluid and of the ionic composition with increased extracellular space and decreased interstitial K⁺ concentration during sleep (343–345), a process that involves norepinephrine-mediated inhibition of the astroglial Na⁺/K⁺ pump during wakefulness (346). This process is responsible for widespread neuronal hyperpolarization and decreased firing rate particularly during NREM sleep (347, 348). In parallel, it was recently reported that children with an autism-associated epilepsy phenotype carrying a gain-of-function mutation in the K_ir4.1 coding KCHJ10 gene display abnormal slow-wave sleep with a significantly longer slow-wave period (349). Norepinephrine induces astroglial process elongation and astroglial synaptic coverage during wakefulness (345, 350). Conversely, decreased levels of norepinephrine may be responsible for reduction of direct and indirect astroglial release of ATP/adenosine and D-serine, thus contributing to the overall decreased synaptic failure and increased release probability during sleep. Interestingly, norepinephrine level is particularly low during NREM sleep, due to suppression of noradrenergic neuronal firing (351).

Astroglial-dependent cerebrospinal fluid (CSF) flow is responsible for waste and interstitial fluid clearance during sleep (352) and inward flow of CSF through astroglial AQP4 occurs mainly during NREM sleep (343, 353). The CSF flow is under circadian control mediated by changes of AQP4 polarization (354). Recently, a haplotype of AQP4 carrying several single nucleotide polymorphisms (SNPs), among which some associated with reduced AQP4 expression, has been linked to altered slow-wave NREM sleep modulation (355). Moreover, astroglial gap junction coupling is likely to contribute to the regulation of the sleep/wake by means of modulation of both CSF flow and waste clearance. To date, most studies addressing GJ coupling in astrocytes are focused on the altered metabolite trafficking (namely glucose and lactate) resulting from GJ manipulation that impairs the fundamental role of astrocytes in synaptic energy support and brain energy metabolism (356). Astrocyte-specific conditional knock-out of Cx43 in mice resulted in enhanced sleepiness, fragmented wakefulness, and impaired neuromodulation of the sleep/wake cycle (357). Conversely, astroglial neurometabolic coupling impairment results from sleep deprivation that leads to astroglial upregulation of the transporters GLUT1, GLT1, the Na⁺/K⁺ pump as well as other components of the astrocyte-neuron lactate shuttle (358). Sleep deprivation could therefore possibly contribute to increased network activity also through enhanced lactate delivery to neurons as suggested by the fact that the anticonvulsant stiripentol is a lactate dehydrogenase inhibitor (359) in addition to being a positive allosteric modulator of GABA_A receptors (360).

Impairment of astroglial exocytosis and gliotransmission using cell-specific expression of dnSNARE results in reduced tonic A1R adenosinergic signaling, altered sleep homeostasis and reduced slow-wave power, and reduced sleep pressure in mice (361), as confirmed by previous evidence suggesting the role of A1 receptors in augmented sleep pressure (362–364). The same genetic manipulation suppressed the LPS-induced increase in slow-wave power during NREM sleep, proving the astroglial contribution to inflammatory-derived increased sleep pressure (365). Although the role of A2 receptor (A2R) activation by astroglial adenosine is still controversial, A2Rs may play a role in sleep homeostasis through activation of A2AR-expressing neurons in the nucleus accumbens core involved in the induction of slow-wave sleep (366). In the cortex, altered gliotransmission resulted in reduced neuronal NMDA receptor activity and reduced slow oscillations (367), whereas astroglial specific activation induced neuronal transition to slow oscillations (368). In vivo characterization of Ca²⁺ signaling in both rat cortical astrocytes and neurons revealed that astroglial synchronized activity reliably precedes neuronal oscillations and that both astrocyte uncoupling and intracellular Ca²⁺ chelation reduced the fraction of astrocytes and neurons involved in the cortical slow waves. Remarkably, neurons closer to active astrocytes were more likely involved in the oscillations (369). Recently, simultaneously recording of BOLD fMRI and astroglial Ca²⁺ signaling in anesthetized rats revealed that a fraction of intrinsic cortical Ca²⁺ signals were associated with reduced EEG power and negative fMRI signal throughout the cortex (correlated with decreased neuronal activity) and that increased activity in the thalamus specifically preceded these signals (370). Conversely, reduction in the density of cortical astrocytes in the medial prefrontal cortex (and therefore putatively in their connectivity) has been linked to a decrease in δ (0.5–4 Hz) and α (8–12 Hz) spectrum power (371). Moreover, mice overexpressing an astrocyte-specific inositol triphosphate (IP₃) phosphatase, and therefore displaying reduced IP₃-dependent Ca²⁺ activity, spent more time in REM sleep and revealed more transitions to REM sleep from passive wakefulness (372). In line with that, mice lacking the inositol 1,4,5-triphosphate receptor type 2 exhibit reduced Ca²⁺ signaling, a more fragmented and shorter NREM sleep associated with decreased δ spectrum power and more frequent microarousals (373). Remarkably, astroglial intracellular Ca²⁺ increases precede the transition from NREM sleep to wakefulness but follow arousal from REM sleep (373), thus suggesting that astroglia could mediate norepinephrine-induced arousal from NREM sleep (374). Furthermore, it has been recently shown that in vitro application of an oscillatory electric field specifically in the slow-wave range (and not at higher frequencies) increased astroglial synaptic vesicle mobility (375), thus suggesting a positive feedback mechanism for slow-wave state perpetuation. Taken together, these data suggest that astroglial connectivity, astroglial Ca²⁺ waves and gliotransmitter release may initiate and/or support the initiation of cortical slow wave oscillations or favor this transition over others (e.g., passive wakefulness-REM).

Another piece of evidence supporting the contribution of astroglial synaptic plasticity in the modulation of sleep derives from studies focused on the extracellular matrix components in the synaptic cleft. In the mouse forebrain, sleep pressure after sleep deprivation decreased the expression of astroglial neuroligin-1 (376), a cell adhesion molecule binding to presynaptic neurexins (377, 378). Conversely, neuroligin-1 knock-out mice have increased slow-wave sleep and enhanced synchrony during sleep (379). Astrocytes contribute to glutamatergic synaptic plasticity of thalamo-cortical synapses through secretion of hevin, a synaptogenic protein inducing the interaction between non-canonical synaptic partners including neuroligin-1 (380). Knock-out mice lacking another member of the neuroligin family, neuroligin-2, develop SWDs and behavioral arrests, a phenotype blocked by ethosuximide and attenuated by expression of neuroligin-2 selectively in the thalamic neurons or optogenetic activation of GABAergic projections from the NRT (381). This could be due to an interaction with GABA_A receptors as suggested by previous studies on sleep-deprived mice (382). Mice with a missense mutation in neuroligin-3 exhibit an altered EEG power spectrum (383). Moreover, variations in the copy number of the gene encoding the postsynaptic scaffolding protein Shank3, which interacts with both neuroligin and glutamate receptors thus regulating synaptic plasticity (384, 385), have been linked to epileptiform activity specifically arising during slow-wave sleep (386). Shank3 loss-of-function mutations have been associated with several different epileptic forms but most commonly with atypical absence seizures (387).

Finally, as mentioned already above, inflammation and sleep are tightly intertwined. TNFα and IL-1β increase during wakefulness and decrease during sleep both at mRNA and protein level (388–390) and their systemic injection selectively increase slow-wave sleep (389, 391). Mice lacking IL-1β and TNFα receptors are characterized by less slow-wave and REM sleep (392, 393). Notably, agonists of both P2X- and P2Y-type purinergic receptors are known to activate the astroglial release of TNFα and IL-1β (394) and pannexin-1 knock-out mice with impaired ATP release display altered slow-wave sleep (395). Interestingly, unilateral cortical TNFα injection induces state-specific EEG asymmetries during NREM sleep (396) and ipsilateral increase in the number of IL-1β positive cells (mainly astrocytes) in the cortex, the NRT and in the thalamic nuclei (397). Along with TNFα and IL-1β, reactive astroglia also produce nitric oxide (NO) via inducible NO synthetase (iNOS) (398, 399) and NO has been linked to the pathology of epilepsy (400, 401). NO affects both NREM and REM sleep (402, 403) but inhibiting iNOS in sleep-deprived mice specifically impairs NREM recovery, whereas inhibiting neuronal NOS (nNOS) impairs the recovery of REM sleep (404), thus suggesting a specific role of astrocyte in NREM physiology.

Astroglial homeostatic control of the extracellular space and synaptic transmission through K⁺ clearance mediates widespread neuronal hyperpolarization and decreased firing activity during sleep and impacts NREM sleep architecture. Astroglial regulation of the extracellular volume controls both waste and cerebrospinal fluid clearance essential for correct functioning of the brain and relies on AQP4-mediated water influx and GJ coupling. Adenosine released by astrocytes governs sleep homeostasis, sleep pressure and slow-wave power and astroglial gliotransmission contributes to neuronal slow oscillations in the cortex. Moreover, artificial lowering of astroglial Ca²⁺ oscillations and connectivity leads to reduced slow-wave power and relative shortening and fragmentation of NREM sleep. Finally, astroglial TNFα, IL-1β, and NO are likely to impact sleep architecture by selective increase of slow-wave sleep. These findings support the fact that astroglia play a fundamental role in the physiology of NREM sleep and therefore represent a promising target to study pathophysiological alterations inducing or sustaining the abnormal recruitment of the cortico-thalamo-cortical network during SWDs.