Abstract
Dysregulation of excitatory and inhibitory signaling is commonly observed in major psychiatric disorders, including schizophrenia, depression, and bipolar disorder, and is often targeted by psychological and pharmacological treatment methods. The balance of excitation and inhibition is highly sensitive to severe psychological stress, one of the strongest risk factors for psychiatric disorders. The role of astrocytes in regulating excitatory and inhibitory signaling is now widely recognized; however, the specific involvement of astrocytes in the context of psychiatric disorders with a history of significant stress exposure remains unclear. In this review, we summarize how astrocytes regulate the balance of excitation and inhibition in the context of stress exposure and severe psychopathology, with a focus on the PFC, a brain area highly implicated in psychopathology. We first focus on preclinical models to demonstrate that the duration of stress (particularly acute vs chronic stress) is key to shaping astrocyte function and downstream behavior. We then provide a hypothesis for how astrocytes are involved in stress-associated cortical signaling imbalance, discuss how this directly contributes to phenotypes of psychopathologies, and provide suggestions for future research. We highlight that astrocytes are a key target to understand and treat the dysregulation of cortical signaling associated with stress-related psychiatric disorders.
Introduction
A common feature of severe psychiatric disorders, including depression, bipolar disorder, and schizophrenia, is an imbalance of excitatory and inhibitory signaling (Duman et al., 2016, 2019; Fogaça and Duman, 2019; Page and Coutellier, 2019). Recent evidence indicates that the balance of excitatory and inhibitory signaling is impacted by exposure to highly stressful experiences. This is significant as exposure to high levels of stress (e.g., physical and sexual abuse, neglect, accidents that are life-threatening, and high levels of chronic lifestyle stressors, such as financial, relationship, and work stress) is also the strongest transdiagnostic environmental risk factor for developing psychopathology (Albrecht et al., 2016; Duman et al., 2016, 2019; Radua et al., 2018; Fogaça and Duman, 2019; Page and Coutellier, 2019; Ohta et al., 2020). Consequently, there is significant interest in understanding the cellular mechanisms that contribute to the balance of excitatory and inhibitory signaling to shape brain function and behavior, as this may provide insight into how stress raises risk to psychopathology, and reveal novel targets for treatment (Duman et al., 2019; Page and Coutellier, 2019; Sohal and Rubenstein, 2019; Voineskos et al., 2019).
Both excitatory and inhibitory signaling are coordinated by complex networks of neurons and glia. Astrocytes are widely abundant glia in the mammalian brain, accounting for 8%-20% of total cells and 20%-40% of glia in humans (Verkhratsky et al., 2017; O'Leary and Mechawar, 2021) and are highly prevalent with a density of 1100 astrocytes per cm3 in the human cortex (DeFelipe et al., 2002). Once thought of only as passive supporting cells, astrocytes are now recognized for their essential and active roles in metabolism (Bélanger et al., 2011), neuroinflammation (Giovannoni and Quintana, 2020), blood–brain barrier formation and maintenance (Abbott et al., 2006), the regulation of synapse activity, formation, and elimination (Allen and Eroglu, 2017), and importantly, maintaining the balance of excitatory and inhibitory signaling (Fellin et al., 2006; Heja et al., 2012). It is estimated that one single astrocyte can modulate up to 2 million excitatory and inhibitory synapses (Oberheim et al., 2009). Therefore, disruptions to even a small fraction of astrocytes can have drastic effects to the balance of excitation and inhibition, and consequently brain function and behavior.
Rodent stress studies consistently report that astrocytes are highly sensitive to the molecular cascade caused by exposure to psychological stressors, and disruption to astrocytic function induces psychiatric-like symptoms (Murphy-Royal et al., 2019); while most of these rodent stress studies have focused on depression, it is important to note that stress-induced depression-like symptoms are a clinical feature of many major psychiatric disorders, including bipolar disorder and schizophrenia. Thus, the relevance of these studies extends beyond depression and is pertinent to the spectrum of severe psychiatric disorders. Preclinical rodent stress studies also largely focus on the PFC, a region that closely regulates the hypothalamic-pituitary-adrenal axis and is sensitive to the downstream consequences of exposure to severe stressors (Kaul et al., 2021). As a result, the PFC is highly impacted in psychiatric disorders, contributing to several symptoms, including cognitive dysfunction, anhedonia, negative process biases, and despair (Pizzagalli and Roberts, 2022).
Several cortical circuits, including frontostriatal, frontolimbic, and corticocortical, are heavily implicated in many hallmark symptoms of psychiatric disorders (Fales et al., 2008; Veer, 2010; Sacher et al., 2012; Cheng et al., 2016; Park et al., 2018). Disruptions at the circuit level can be induced by stress in preclinical psychopathology models, either through direct administration of glucocorticoids to activate the hypothalamic-pituitary-adrenal axis, or by stressing the animals through individual restraint, foot-shocks, social defeat, or social depravation. These preclinical stress tests produce a variety of behavioral phenotypes, such as depressive-like and anxiety-like behaviors, as well as cognitive changes affecting fear memory and spatial memory (for a synopsis of stress models, the phenotypes they induce, and their current limitations, see review by Gururajan et al., 2019). Although not the only phenotypes examined in these models, anhedonia and despair-like behavior (also commonly referred to as learned helplessness) are among the most consistently tested behavioral phenotypes following stress exposure. These behaviors are measured with the sucrose preference test and forced swim test, respectively, and have been linked to PFC circuits in rodents (Covington et al., 2010; Warden et al., 2012; Miller et al., 2017). Interestingly, these behaviors have also been associated with atrophy of astrocyte processes and their capacity to network via connexins, proteins essential to forming gap junctions (J. D. Sun et al., 2012; Huang et al., 2019). This suggests that astrocytes are involved in cortical circuit dysfunctions and behaviors related to psychiatric disorders, and that this process can be influenced by exposure to different levels (dependent on type, intensity, timing, and duration) of stress. While others have reviewed astrocytic regulation of excitatory and inhibitory signaling in psychiatric disorders (e.g., Kruyer et al., 2022), there has been little consideration given to the role that specific risk factors, such as stress exposure, play in this process. This is important given that decades of epidemiological studies demonstrate that exposure to adverse life events is a leading cause of psychiatric disorders (Albrecht et al., 2016; Page and Coutellier, 2019; Ohta et al., 2020).
The goal of this article is to review evidence regarding how stress exposure alters astrocytic regulation of excitatory and inhibitory signaling, and how this contributes to coordinating brain function and behavior relevant to psychiatric disorders. In line with this evidence, we hypothesize that exposure to highly stressful experiences causes a molecular cascade that affects astrocyte function to precipitate the development of major psychiatric disorders, including schizophrenia, depression, and bipolar disorder. Although stress has widespread effects across the brain, this review focuses on the PFC, which is highly sensitive to stress and with evidence of altered excitatory and inhibitory signaling in psychopathology (Pizzagalli and Roberts, 2022). With this review, we provide new insights into how exposure to highly stressful experiences affects astrocytic function, to cause the dysregulation of cortical signaling that is a hallmark of individuals with severe psychiatric disorders.
Astrocytic regulation of excitatory signaling in the context of stress
Astrocytic regulation of excitatory signaling and behavior
Glutamate is the primary excitatory neurotransmitter in the human brain, particularly in the cortex (Krebs et al., 1949; Erecińska and Silver, 1990). Astrocytes play an active role in coordinating glutamatergic signaling between neurons, through their ability to sense, regulate uptake or release, and metabolize glutamate (Fig. 1a) (for reviewed elsewhere in detail, see Farhy-Tselnicker and Allen, 2018; Lalo et al., 2021). Efficient regulation of glutamate is central to healthy brain function. Irregular activation of neuronal glutamate receptors, either by excess glutamate or oversensitive receptors, results in excitotoxicity. This has been associated with a wide range of brain pathologies, including neurodegenerative and psychiatric disorders (Dong et al., 2009; Olloquequi et al., 2018), in which astrocytes are key players (Siracusa et al., 2019). Excess extracellular glutamate has also been associated with reduced neuronal dendritic growth, increased dendritic and spine pruning, and synaptic loss (Monnerie et al., 2003; Parellada and Gassó, 2021), phenotypes that have been identified in rodents exposed to severe chronic stress and also in human psychopathologies (Kaul et al., 2021; Parellada and Gassó, 2021).
Astrocytic regulation of excitatory and inhibitory functions in health and following chronic stress. a, In typical conditions, astrocytes coordinate excitatory and inhibitory functions through a tightly regulated set of mechanisms. Transmembrane transporters (EAAT1/2 and GAT-3) clear glutamate and GABA, respectively, and have high uptake capacities, meeting the demands of extensive synaptically released neurotransmitters. Astrocytes are also the primary site of glutamate and GABA metabolism, which can occur either dependent of the tricarboxylic acid (TCA) cycle, via succinate and alpha-ketoglutarate (α-KG), or independently via glutamine synthetase. These functions are further tightly regulated by several ionotropic receptors for glutamate (NMDAR and AMPAR) and GABA (GABAAR) as well as G protein-coupled metabotropic receptors for glutamate (mGluR2/3) and GABA (GABABR). These receptors help coordinate clearance and metabolism and the release of gliotransmitters, which is largely consolidated through intracellular calcium signaling. b, Following chronic stress, a number of these mechanisms are impaired in the PFC. Evidence indicates that both clearance and metabolism of glutamate and GABA are impaired via TCA-dependent and -independent pathways. There are significant gaps in knowledge surrounding how chronic stress alters the expression and function of astrocytic ionotropic and metabotropic GABA and glutamate receptors and, overall, how this influences astrocytic control of excitation and inhibition in the PFC. In addition, there is limited understanding of how stress impacts intracellular calcium signaling pathways and the subsequent coordination of gliotransmitter release.
At the synapse, astrocytes transport glutamate across the cell membrane through sodium-dependent solute carrier transporters, which exist in two primary astrocytic isoforms: excitatory amino acid transporters 1 and 2 (EAAT1 and EAAT2; the rodent analogs are, respectively, GLAST and GLT-1, referred to here also as EAAT1 and EAAT2) (Fig. 1, blue). Astrocytic glutamate transporters have a large uptake capacity and are important for buffering synaptic glutamate, taking in over 90% of synaptically released glutamate and preventing glutamate spillover to extrasynaptic sites (Lehre and Danbolt, 1998; Zhou et al., 2014; Trabelsi et al., 2017; Henneberger et al., 2020). Blockade of EAAT2 using dihydrokainic acid in the rodent PFC is sufficient to induce anhedonia-like behavior (John et al., 2012). This is supported by the knockdown of EAAT2 and EAAT1 (20%-30% reduction) in the mouse infralimbic cortex using small interfering RNA, which increased cortical excitability and was associated with the presentation of depression-like behavior (i.e., anhedonia, time to defeat) (Fullana et al., 2019a,b). Similarly, conditional EAAT2 knockdown in C57BL/6J-background mice impaired extracellular calcium dynamics and modulated neuronal signal propagation across the cerebral cortex, increasing the susceptibility of neurons to morphologic deterioration via synaptic depression (Aizawa et al., 2020). Complete KO of EAAT2 in mice induced excitotoxicity, and caused spontaneous and lethal seizures by the third postnatal week (Tanaka et al., 1997), as well as atypical neocortex development (Matsugami et al., 2006). Conversely, complete KO of EAAT1 induced psychiatric-like symptoms, including anhedonia, social withdrawal, and impaired cognitive performance (Karlsson et al., 2009). Limiting astrocyte glutamate clearance in the cortex is thus closely tied to local circuit function, broader regulation of network activity, and subsequent behavioral outcomes relevant to psychiatry.
Another essential role for astrocytes involves the conversion of glutamate to glutamine, the sole precursor for both glutamate and GABA (Fig. 1, red). This process is mediated by direct enzymatic conversion by the highly astrocyte-enriched glutamine synthetase and by metabolism via the tricarboxylic acid (TCA) cycle (Hertz, 2013; Schousboe et al., 2014). Inhibition of glutamine synthetase is sufficient to acutely impair excitatory postsynaptic signaling in the hippocampus (Kam and Nicoll, 2007) and can persistently impair glutamatergic signaling when activity is reduced by the inhibitor methionine sulfoximine during postnatal synaptogenesis (Son et al., 2019). Indeed, methionine sulfoximine administration is associated with depressive-like symptoms (i.e., decreased sucrose preference and faster time to defeat) in male mice (Lee et al., 2013). This suggests that neurotransmitter cycling is also an important modulator of cortical circuits and associated behavior.
Cortical astrocytes also express several G-protein coupled mGluRs, which further coordinate excitatory signaling. They primarily express the Gi/Go coupled Class II receptors mGluR2/3, which are expressed in astrocytes at all developmental stages (W. Sun et al., 2013). mGluR3 (Grm3) KO mice demonstrate impaired working memory and hyperactivity, which are schizophrenia-like behaviors (Fujioka et al., 2014). Although there is no specific evidence of behavioral outcomes for knockdown of Grm3 specifically in astrocytes, some evidence from pan Grm3 knockdown suggests that this may be an important avenue to consider. In primary mouse astrocyte-neuron cocultures, the normally neuroprotective mGluR2/3 receptor agonist LY379268 failed to provide neuroprotection in cultures with astrocytes lacking mGluR3 (Corti et al., 2007). Broad agonism of mGluR3s also reduced the release of glutamate from astrocytes in situ (S. Wang et al., 2012). Together, this indicates that activation of astrocytic Class II metabotropic receptors may attenuate glutamate signaling, and may be neuroprotective. In summary, astrocytes are key coordinators of both the extrasynaptic glutamate environment and the pool of available neurotransmitters. Selective impairment of these functions, particularly in cortical circuits, is sufficient to induce depression-like (and probably schizophrenia-like) phenotypes in rodents.
Astrocyte regulation of excitatory signaling under the influence of acute stress
In response to acute stress exposure, rodents demonstrate a rapid increase in cortical excitatory signaling. Rats acutely restrained for as little as 20 min had increased extracellular glutamate, particularly in the PFC (Moghaddam, 1993). Similarly, rats exposed to a 40 min acute foot-shock stress had increased PFC depolarization events, stimulating elevated glutamate release; this effect was dependent on glucocorticoid receptors (Musazzi et al., 2010). It was also demonstrated that this foot-shock stress rapidly increased the pool of available glutamate vesicles in the presynaptic sites of neurons, although glutamate release into the synaptic space likely requires time periods >20 min (Treccani et al., 2014). A follow-up study found that increased glutamate release associated with this foot-shock paradigm lasted as long as 24 h after the removal of the stress, persisting long after circulating glucocorticoids had returned to baseline (Musazzi et al., 2017). Another multimodal single prolonged stress model consisting of restraint (2 h), forced swim (15 min), and ether exposure, led to decreased glutamate and glutamine in the mPFC of rats, which was measured 7 d following the removal of the stressor (Knox et al., 2010), indicating that acute stressors can have persistent effects on glutamatergic signaling.
Clear evidence for the impacts of acute stress on astrocytic control of excitation has emerged from studies of glutamate clearance, as this approach enables direct examination of a range of astrocyte-specific markers and functions. Recent evidence demonstrates that astrocytes are responsive to even very short stressor exposure (<30 min). For example, an in situ approach reported that astrocytes rapidly modulate excitatory signaling in response to acute swim stress; 20 min of acute forced swim stress was sufficient to prolong the duration of in situ calcium events in microdomains of cortical astrocyte processes, but not at the soma, and to reduce excitatory postsynaptic events within the domain of the astrocyte (Murphy-Royal et al., 2020). However, subsequent RNA sequencing detected no significant change in astrocyte-specific genes directly related to glutamate signaling. Instead, the authors demonstrated that these deficits were largely attributable to a degraded astrocytic syncytium and impaired delivery of readily mobilizable signaling molecules, such as L-lactate (Murphy-Royal et al., 2020). With a slightly longer duration of stress exposure (30-60 min of acute restraint stress), glutamate uptake was shown to further increase, measured in synaptosomal forebrain and hippocampus homogenate from rats immediately following dissection (Gilad et al., 1990). Although the extent to which this glutamate uptake is mediated by astrocytes is unclear, it can be hypothesized that it is largely mediated by astrocytes, given their large glutamate uptake capacity. Alternatively, this immediate increase in glutamate clearance could be a result of the high basal clearance capacity of astrocytes (Diamond, 2005).
As the stress paradigms move toward the hour timescale, astrocytic glutamate signaling begins to show signs of alterations. In vitro evidence from primary mouse astrocyte culture demonstrates that up to 6 h of exposure to dexamethasone (100 nm), a synthetic analog of cortisol, caused no significant change in solute carrier family 1 member 2 or 3 (Slc1a2/3), which encode for EAAT2 and EAAT1, respectively (Carter et al., 2012). In this study, a significant increase in the mRNA for Glul, encoding glutamine synthetase, increased up to 6 h (Carter et al., 2012). These findings were subsequently mirrored in vivo in mice, which did not demonstrate significant changes in Slc1a2, Slc1a3, or Glul following acute corticosterone injection (Carter et al., 2013). However, over longer time courses in vitro, both dexamethasone and corticosterone increased the mRNA and protein levels of EAAT2 alongside glutamate clearance in rat primary cortical astrocyte culture (Zschocke et al., 2005). These effects were both time- and dose-dependent, with increases observed from 24 to 72 h of exposure and 1-1000 nm dexamethasone. This may indicate that the first critical window in which astrocytes begin to transcriptionally adapt to meet the demands of elevated glutamate clearance and metabolism occurs within as little as a day of prolonged stress response. However, no behavioral testing was conducted in these studies, which would have been helpful to determine how these changes in astrocytic glutamate signaling is related to behavioral outcomes. These initial shifts could be pro-survival adaptations to meet the demands of increased neuronal activity (Yuen et al., 2009).
Astrocyte regulation of excitatory signaling under the influence of chronic stress
As the duration of the stress increases to weeks, glutamatergic signaling becomes further impaired. No change in basal glutamate concentrations were detected in PFC slices dissected from male Wistar rats exposed to multiple chronic restraint stress events over 28 d; however, the expected increase in glutamate response after application of BDNF, an important regulator of synaptic transmission and plasticity (Miranda et al., 2019), was impaired (Chiba et al., 2012). In contrast, significantly reduced PFC glutamine and glutamate levels were observed in male C57BL/6 mice that developed depression-like behavior after being exposed to chronic social defeat stress for 10 d; no change was seen in the rodents that did not develop depression-like behavior (Veeraiah et al., 2014; Mishra et al., 2018). A similar reduction in both glutamate and glutamine was observed in rats exposed to forced swim stress for 13 d (C. X. Li et al., 2008) or chronic restraint stress for 21 d (Liu et al., 2016). Glutamate signaling is key to the development and stability of dendrites and dendritic spines on pyramidal neurons (McKinney, 2010). Reduced complexity of these dendritic features in the PFC are among the most common cellular alterations associated with severe stress (Radley et al., 2004, 2006; Liston et al., 2006; Popoli et al., 2011; Yuen et al., 2012; Kaul et al., 2020, 2021). Astrocyte excitatory functions, such as glutamate clearance, regulate spine formation (Verbich et al., 2012), suggesting altered astrocytic regulation of excitatory signaling may contribute to these widely observed phenomena.
At these longer durations of stress exposure, astrocytic regulation of cortical glutamatergic signaling is largely impaired, as summarized in Figure 1. Chronic ingestion of corticosterone (10 mg/ml) over 2 weeks induced significant decreases in Slc1a2/3 (Carter et al., 2013). Likewise, rats exposed to chronic unpredictable or restraint stress for 21 d demonstrated impaired capacity for glutamate clearance in the PFC (Olivenza et al., 2000; Réus et al., 2015). This is likely because of related molecular alterations in the PFC, given that Slc1a2 mRNA and EAAT2 protein levels are reduced following either chronic social defeat or chronic immobilization stress in rodents (Veeraiah et al., 2014; Rappeneau et al., 2016; Baek et al., 2019). Following chronic defeat stress, female mice also demonstrated reduced glutamate clearance following cannula infusion of the neurotransmitter (Rappeneau et al., 2016). It is plausible that reduced capacity to clear glutamate in high-risk rodents sensitizes the cellular environment to glutamate release at the synapse, and increases the amount of neurotransmitter spillover. In the context of neuronal networks, this would impair the efficiency and specificity of local circuitry, contributing to a number of deleterious consequences, such as excitotoxicity (Popoli et al., 2011). However, the extent to which stress-induced changes in astrocytic functions are governed by neuronal signaling and how these changes inform neuronal signals remain unclear.
Studies of astrocyte metabolism support the hypothesis that these cells are active modulators of stress-associated behavioral phenotypes. Chronic unpredictable stress over 5 weeks reduced astrocytic glutamate metabolism to glutamine mediated by glutamine synthetase (Banasr et al., 2010). Similarly, glutamatergic metabolism was impaired in mice exposed to 3 weeks of chronic unpredictable mild stress that developed anhedonia- (reduced sucrose preference) and despair- (in the forced swim test) like phenotypes (Mishra et al., 2020). Importantly, direct inhibition of cortical glutamine synthetase with methionine sulfoximine induced the same depression-like behaviors (Lee et al., 2013), suggesting that the depression-like behaviors stem from glutamate deficits.
A large body of work suggests that ionotropic and metabotropic signaling systems are affected by stress and are associated with stress-associated psychopathology (Duman et al., 2019). To our knowledge, no studies have directly evaluated the expression or function of neither ionotropic (e.g., NMDA and AMPA) receptors nor metabotropic receptors (e.g., mGluR2/3/5) specifically in cortical astrocytes. One study performed RNA sequencing on isolated astrocytes isolated from the PFC of rodents exposed to chronic variable stress (Simard et al., 2018). Of 545 differentially expressed genes, they found upregulation of NMDA and AMPA receptor subunits, including NMDA receptor subunit 1 (Grin1) and AMPA receptor subunit 2 (Gria2). Genes encoding metabotropic receptors mGluR2 (Grm2) and mGluR7 (Grm7) were also upregulated, implicating both ionotropic and metabotropic glutamate signaling. Promisingly, following astrocyte depletion with gliotoxins, injection of the mGluR5 antagonist 3-((2-methyl-4-thiazolyl)ethynyl)pyridine into the PFC of rats restored GFAP levels, improved anhedonia and despair phenotypes, and prevented gliotoxin-induced degeneration (Domin et al., 2014). Modulation of astrocytic glutamate signaling may therefore be a promising target for both prevention and treatment of astrocyte imbalances caused by stressor exposure and associated behaviors. As strategies to characterize these systems in astrocytes are developed, determining how stressor exposures influence the function and downstream signaling pathways of each of these receptors in astrocytes will provide insight into how the regulatory capacity of astrocytes is shaped by stressor exposures. Key avenues important to explore will be the roles of NMDARs and mGluR3/5, given their particular importance in mature cortical astrocyte signaling and regulators of intracellular calcium dynamics (Lalo et al., 2021).
These findings suggest that astrocytic regulation of excitatory signaling is a phasic response to stress. Initial stress induces a delayed, but significant increase in glutamate uptake, but prolonged stress coincides with a gradual decline in the capacity of these cells to clear and metabolize glutamate in rodents (Fig. 2). The phasic response is in line with the glutamatergic activity and excitatory neuronal responses that have been previously suggested (Page and Coutellier, 2019), highlighting that stress-induced dysregulation of neurons and glia are likely closely linked. This response is most pronounced in rodents which exhibit psychiatric-like phenotypes, including impaired cognition, anhedonia, and despair. In tandem with genetic manipulation studies, this suggests that astrocytes play a key role in the development of psychiatric symptoms associated with stress rather than general stress adaptation. Although this is a promising avenue of exploration, linking these molecular findings with their functional correlates will be essential for understanding how stress impacts astrocytes, how this shapes neuronal circuits, and eventually how this influences behavior.
Modeling the impact of stress on cortical excitatory and inhibitory astrocyte signaling over time. At acute time points up to the timespan of hours, astrocytes have delayed, but upregulated, glutamatergic clearance and metabolism, with increases in function persisting up to 72 h. At more chronic time points, such as in chronic restraint and social defeat stress models, these functions are largely impaired. However, it is uncertain what timescale and events trigger this switch from increased function to impaired function. Inhibitory functions of cortical astrocytes are also impaired at chronic time points, which aligns with reduced GABA levels; however, it is unclear how these systems are affected at acute time points, as the earliest evidence is observed after 24 h of stress. Plotting these against levels of cortical glutamate and GABA, astrocytic regulation of excitatory and inhibitory signaling largely follows the trajectory of glutamate and GABA levels in the cortex following stress, although the significance of this is yet to be determined.
Astrocytic regulation of inhibitory signaling in the context of stress
Astrocytic regulation of inhibitory signaling and behavior
In the PFC, inhibitory signaling is largely moderated through the neurotransmitter GABA (Isaacson and Scanziani, 2011). Inhibitory signaling is an essential regulatory counterbalance to excitatory signaling, providing a reciprocal dampening control over neural circuits to ensure that signaling is temporally and spatially efficient. It is vital to the successful coordination of dynamic network activity through both feedforward and feedback mechanisms (Isaacson and Scanziani, 2011). Reduced tonic GABA levels significantly increase the incidence of seizures (Yang et al., 2001), with altered cortical inhibition impairing synaptic plasticity, information processing and behavior relevant to psychopathologies (Daskalakis et al., 2007; Perez et al., 2019). Cortical GABAergic signaling is thus an emerging target for the development of novel therapeutics for psychopathologies (Duman et al., 2016; Fogaça and Duman, 2019). Astrocytes are important mediators of inhibitory signaling through direct clearance and metabolism of GABA and modulation of indirect signaling pathways (e.g., ionotropic and metabotropic receptors) (Fig. 1). However, specific evidence of involvement of these astrocytic functions in downstream regulation of behavior is limited. Only one study has looked at how the astrocyte-enriched GABA transporter Type 3 (GAT-3) associates with behavior. This group demonstrated that administration of 1-(2-[tris(4-methoxyphenyl)methoxy]ethyl)-(S)-3-piperidinecarboxylic acid (SNAP 5114), a semi-selective GAT-3 inhibitor, to the lateral habenula improved depression-like symptoms (sucrose preference and immobility time) induced by lesions. It is important to explore the effects of this drug in the PFC (Lyu et al., 2020).
Recent evidence suggests that astrocytic GABA signaling regulates both circuit activity and behavior. In mice, knockdown of the Gabbr1 subunit of the GABA Type B receptor (GABABR) in PFC astrocytes impaired neuronal synchronization and firing, as well as cognitive performance (Mederos et al., 2021). At the circuit level, these mice had increased firing rates of both excitatory and inhibitory neurons during a T-maze task, strengthening the hypothesis that astrocytes play an important role in coordinating synaptic connectivity, both excitatory and inhibitory circuits, and maintaining homeostasis. They also demonstrated that astrocytic GABABRs are essential for the potentiation of inhibitory postsynaptic currents in pyramidal neurons driven by parvalbumin interneurons, implicating astrocytes as important coordinators of cortical inhibition. Earlier evidence indicates that astrocytes in the cortex also have unique responses to distinct interneuron classes via GABABR. Signaling from parvalbumin interneurons reduced astrocyte Ca2+ events over time; however, somatostatin interneurons induced increases in Ca2+ signaling (Mariotti et al., 2018). Astrocyte-specific responses on the stimulation of interneuron types may be important for synaptic coding in the cortex and may be involved in the presentation of cognitive symptoms of psychiatric disorders.
Astrocyte regulation of inhibitory signaling under the influence of acute stress
At acute stress time points, evidence regarding how stress affects GABA levels in the cortex is not as well defined as it is for glutamate. In mice, acute immobilization stress (5 min) failed to induce a change in GABA concentrations in the frontal cortex (Otero Losada, 1989), as did 3 h of cold stress (Losada, 1988). However, after 15 min of forced swimming, rats had reduced GABA levels measured 5 min after swim, which returned to baseline by 45 min (Borsini et al., 1988). Conversely, 1 h of acute immobilization stress increased GABA in the PFC of Sprague Dawley rats (Drouet et al., 2015). Clinical studies in humans have also provided inconclusive results. One study found that a multimodal acute performance stress did not affect GABA or glutamate levels in a human cohort, measured with magnetic resonance spectroscopy (n = 29) (Houtepen et al., 2017). However, another small cohort (n = 10) found that acute psychological stress (threat of shock) decreased GABA concentration in the PFC, measured by magnetic resonance spectroscopy (Hasler et al., 2010).
Similarly, there is a gap in our understanding of how cortical astrocytic GABAergic coordination occurs at acute time points. One study in male Sprague Dawley rats demonstrated that repeated unpredictable acute foot-shocks over a 24 h period reduced cortical GAT-3 expression (Zink et al., 2009). This effect was only observed in outbred rodents susceptible to developing learned helplessness. To our knowledge, no other study has assessed the effects of acute stress on cortical GABAergic astrocyte functions. It would be valuable to explore whether GABAergic functions are impaired at acute time points as it may contribute to the inhibition of cortical function associated with acute responses to stress (Arnsten, 2009) and may thus provide targets to mediate these effects.
Astrocyte regulation of inhibitory signaling under the influence of chronic stress
After longer stress exposure, evidence indicates that GABAergic signaling is largely disrupted in the cortex. Repeated immobilization stress for 2 weeks significantly reduced GABA levels and increased GABA turnover (Losada, 1988). Similarly, 3 weeks of chronic mild stress in male albino rats significantly reduced PFC GABA levels (Shalaby and Kamal, 2009). Evidence of a loss of tone is mirrored in inhibitory components of neuronal circuits, which demonstrate impaired morphology, receptor expression and synaptic function in response to stress Fogaça and Duman (2019).
Astrocytic regulation of inhibition is similarly impaired by stressor exposure spanning weeks. A transcriptome-wide RNA sequencing study of the mPFC indicated that pathways involved in GABAergic transmission were the most transcriptionally altered pathway in male mice exposed to chronic unpredictable mild stress over 21 d displaying depression-like behaviors, including anhedonia (sucrose preference test) and anxiety (Y-maze test) (Ma et al., 2016). In these mice, astrocytic GAT-3 was one of the most transcriptionally downregulated genes, with a 1.6-fold reduction compared with controls. miRNAs that are known to downregulate GAT-3 expression (miRNA-144-3p, miRNA-15b-5p, and miRNA-879-5p) were increased in the same animals Bath et al., 2016; Ma et al., 2016). This study highlights that GABAergic signaling is sensitive to stress exposure, and that the consequences of this stress exposure could be modulated via miRNAs. This is significant because miRNAs not only play a critical role in the pathogenesis of many psychiatric disorders, but also provide specific drug targets to upregulate or downregulate astrocytic control of excitation and inhibition via GAT-3.
GABA metabolism is also impaired by chronic stress. In male rats, chronic unpredictable stress reduced astrocytic GABA synthesis (Banasr et al., 2010), and this was rescued by the neuromodulator riluzole, a drug that blocks glutamate release by blocking voltage-gated sodium channels. Chronic social defeat stress over 10 d or chronic unpredictable mild stress for 21 d also impaired GABA metabolism and cycling in mice displaying depression-like phenotypes (Veeraiah et al., 2014; Mishra et al., 2020). Given recent evidence that GABA metabolism in astrocytes is an active process and essential to GABA recycling (Andersen et al., 2020), how these stress-induced deficits impact inhibitory neurotransmission will be a valuable avenue to explore to identify potential targets for treatment development.
Although broader changes in GABAARs or GABABRs in the cortex have been reviewed elsewhere (Fogaça and Duman, 2019), evidence of astrocyte-specific deficits are limited. One study performed RNA sequencing on astrocytes derived from the PFC of rodents exposed to chronic variable stress (Simard et al., 2018). Of 545 differentially expressed genes, they found that several GABA Type A receptor (GABAAR) α1 subunits 1/2/5 (Gabra1, Gabra2, Gabra5), GABAAR γ2 subunits (Gabrg2) were upregulated following stress exposure, but no change for GABA Type B receptor (GABABR) transcripts was detected. Given recent evidence that demonstrates GABABRs in cortical astrocytes are specifically associated with altered firing and behavior (Mederos et al., 2021), it is important to understand how these receptors are impacted by stress.
Astrocytic regulation of GABA uptake and turnover is reduced at chronic time points and associated with the development of depression-like phenotypes, as summarized in Figure 1. This suggests that these functions help determine how stress leads to psychiatric symptoms. Importantly, this aligns with the decreases in both inhibitory circuits and GABA levels observed in the cortex, suggesting that, as with excitatory circuits, the regulatory capacity of astrocytes in coordinating inhibition is reduced and is closely related to interneurons. Given the strong evidence that astrocytes distinctly engage with different interneuron types (Mariotti et al., 2018; Matos et al., 2018), impaired inhibitory regulation by astrocytes likely contributes to specific deficits in inhibitory circuits, such as impaired somatostatin interneuron signaling and increased tonic inhibition. As a result, astrocytes are likely key players in over-inhibition, as proposed by previous groups (Page and Coutellier, 2019) and likely contribute to reduced network function of the cortex, driving behavioral phenotypes associated with impaired prefrontal function, including emotional and cognitive phenotypes (Fig. 2).
Astrocytes, stress, and the imbalance of excitation and inhibition in the context psychiatric disorders
Alterations of astrocyte-specific transcripts or proteins that regulate glutamate and GABA signaling in psychopathology have been reported in several postmortem human studies. For example, decreased mRNA of EAAT2 and glutamine synthetase have been identified in the dorsolateral prefrontal and anterior cingulate cortex of individuals with major depression (Choudary et al., 2005), and increased GAT-3 protein density (measured by receptor autoradiography assays) in the dorsolateral PFC in schizophrenia (Schleimer et al., 2004). This evidence (extensively reviewed elsewhere, Q. Wang et al., 2017; Parkin et al., 2018; Blacker et al., 2020; Kruyer et al., 2022) aligns with studies from rodents, which show that astrocyte-specific glutamatergic and GABAergic functions are impacted by chronic stress exposure, a widely used preclinical model of depression. These rodent studies consistently show that astrocytic dysregulation of excitatory and inhibitory signaling is associated with the onset of psychopathological phenotypes, including faster time to social and psychological defeat and anhedonia, as well as impaired cognition and emotionality (Gururajan et al., 2019). In addition, the regulation of excitation and inhibition are responsive to common antidepressants (Shalaby and Kamal, 2009; Banasr et al., 2010; Malki et al., 2012; Chen et al., 2014). An important extension of this work will be to determine whether the restoration of astrocyte function is associated with antidepressant activity, or if this is an adaptive response triggered by the recovery of other systems.
Stress exposure has been hypothesized to cause both weakened and hypoactive neuronal circuits (Page and Coutellier, 2019), impairing top-down regulation from the cortex and the capacity of the cortex to respond to subsequent events. These dysfunctional circuits are an underlying driver of behavioral correlates of psychopathologies, one avenue through which stress can lead to psychopathology.
By considering stress duration in rodent models and building on previous hypotheses, we suggest that astrocyte involvement in coordinating excitation and inhibition is acutely heightened in response to stress and is largely controlled by autonomous cellular signaling, such as changes in intracellular calcium concentrations, shifts in membrane protein dynamics, and increased metabolism. As stress persists into a chronic timescale, and molecular and cellular landscapes are impacted by persisting stress exposure, long-term changes in the ability of astrocytes to regulate excitation and inhibition occurs. Currently, this is most robustly evidenced through neurotransmitter clearance and metabolism, which are largely coordinated by astrocytes (illustrated in Fig. 2). Given how integral neurotransmitter clearance and metabolism are in maintaining the balance of excitation and inhibition at the synapse, this is likely to drive dysfunctional PFC circuits and the subsequent impairment of behavior. However, astrocyte development is also regulated by neuronal circuits, particularly to drive the expression of the astrocytic systems which regulate excitation and inhibition, such as the expression of EAAT2 (Morel et al., 2014; Armbruster et al., 2016; Hasel et al., 2017). It is important to acknowledge that shifts in neuronal circuits likely reciprocally contribute to the identified impacts on astrocyte functions.
Limitations and future directions
It is important to note that there are limitations to the research conducted on astrocytes to date. Astrocytes demonstrate enormous regional and subregional heterogeneity, as summarized in several recent reviews (Schitine et al., 2015; Verkhratsky et al., 2017; Pestana et al., 2020). Human astrocytes are more morphologically complex than in rodents, and also have unique functional properties and distinct transcriptomes (Oberheim et al., 2006, 2009; Zhang et al., 2016). They also respond differently to environmental stressors, such as oxidative stress and hypoxia (J. Li et al., 2021). However, almost all knowledge of astrocyte function and responsiveness to stress has come from rodent models, presenting a significant limitation. Given that humans cannot be manipulated at the molecular level to study behavior, rodent models remain invaluable; however, it must be noted that evidence from rodents will not be identical to humans.
Another limitation of the current literature regards how astrocytes are identified for examination. The most used marker for astrocytes is the astrocyte-specific intermediate filament GFAP (Zhang et al., 2021). GFAP is commonly used as a pan-astrocyte marker, despite the fact that it does not demarcate all astrocytes; GFAP labels as few as 10% of astrocytes in the cortex (O'Leary and Mechawar, 2021), and is not evenly distributed through the cortical layers, with high localization in the upper and deeper layers only (Oberheim et al., 2009). Additionally, GFAP-positive astrocytes poorly colocalize with both the astrocyte-specific glutamate transporter EAAT2 (DeSilva et al., 2012; Bayraktar et al., 2020) and the astrocyte-enriched GABA transporter GAT-3 (Tatsumi et al., 2018). Several reports (albeit in rodents, which may differ from humans) suggest that different populations of astrocytes are enriched in either excitatory or inhibitory circuits. In one study, astrocytes emerging from Olig2 progenitors were largely GFAP-negative. This population had a similar expression pattern across the brain as GAT-3, but not GFAP (Tatsumi et al., 2018). In another single-cell RNA-sequencing study of rodent cortex, a population of astrocytes with low levels of GFAP had high expression of the GABA receptor Type A (GABAAR) subunit transcript Gabarg (Batiuk et al., 2020). The study also identified another distinct astrocyte population with low levels of GFAP and high levels of several glutamatergic transcripts, such as the glutamate symporter Solute Carrier Family 25 Member 18 (Slc25a18) and the AMPA receptor subunit Gria2. These newly identified astrocyte subpopulations have also emerged in another rodent spatial transcriptomics study to delineate novel astrocyte subpopulations (Bayraktar et al., 2020). Consequently, initial evidence from the cortex of rodents suggests that astrocytes are a molecularly and functionally heterogeneous class of cells differentially involved in excitatory and inhibitory signaling, and that GFAP alone is not a sufficient marker for studying astrocytes.
Although evidence from cell and rodent models robustly demonstrates that cortical astrocytes regulate the balance of excitation and inhibition, and this process can be disrupted by stress exposure, many research avenues remain. Many of these are related to the fundamental functions of astrocytes, where we still have gaps in knowledge. For example, the exact molecular mechanisms underpinning astrocytic modulation of glutamate and GABA clearance and metabolism are still being refined. In addition, the relationship between calcium signaling, astrocytic glutamate, and GABA receptors is not fully understood. Understanding their interactions may uncover novel drug targets within the glutamate and GABA signaling systems. For example, metabotropic receptors mGluR3 and GABABR, key players of glutamate and GABA signaling, have emerged as important astrocytic modulators of neuronal circuits and resulting behavior (S. Wang et al., 2012; Mederos et al., 2021). Other drugs, such as NMDAR antagonists and metabotropic receptor modulators, also hold promise to target these systems (Taylor et al., 2005; Kraus et al., 2019; Wilkinson and Sanacora, 2019). Gene targeting approaches are also extremely valuable in understanding the relationships between astrocyte function, the balance of excitation and inhibition, and behavioral output (Mederos et al., 2021). These approaches can be tailored to capture specific astrocyte subtypes, to determine their fundamental functions in circuits and microcircuits (Yu et al., 2018; Nagai et al., 2021). With this fundamental knowledge gap addressed, we can then study astrocytes within the context of stress and psychopathology to provide insight into their underlying pathogenesis. It will be important to consider how astrocyte subtypes immediately respond to and recover from stress with a special focus on stress duration, which clearly exerts differential effects. In addition, when pro-adaptive effects start becoming detrimental should be determined.
To address these questions, we will need to use the entire model and specimen toolbox, including human cell models and widely used preclinical rodent models, as well as human postmortem cohorts to anchor key preclinical findings in human-relevant context (Kaul et al., 2020; Cruceanu et al., 2021). Studying astrocytes from multiple angles, with human relevancy, is particularly critical as astrocytes are some of the most poorly evolutionarily conserved cell types in the brain (Pembroke et al., 2021). Combining this with the rapid developments in multi-omics analyses and the ability to map large-scale sequencing data onto spatial and morphologic features, will no doubt clarify and extend our understanding of the roles of astrocytes at an unprecedented resolution (Longo et al., 2021; Miao et al., 2021). This will also provide new genetic targets of astrocytes to probe more specific understanding of circuit and behavior. As astrocytes are highly sensitive to stress across transcriptomic, morphologic, and functional indicators, we conclude that they are a clear, opportune cellular target to halt or reverse the detrimental effects of stress that give rise to psychopathology.
Footnotes
N. Matosin was supported by the Al & Val Rosenstrauss Fellowship from the Rebecca L. Cooper Medical Research Foundation F2021971; Brain Behavior Research Foundation, National Alliance for Research on Schizophrenia and Depression Young Investigator Grant 26486; and Rebecca L. Cooper Medical Research Foundation PG2020645. L.O. was supported by National Health and Medical Research Council of Australia Boosting Dementia Research Leadership Fellowship APP1135720. N. Mechawar was supported by a Canadian Natural Sciences and Engineering Research Council Discovery Grant.
The authors declare no competing financial interests.
- Correspondence should be addressed to Natalie Matosin at nmatosin{at}uow.edu.au
