Review How Stress Physically Re-shapes the Brain: Impact on Brain Cell Shapes, Numbers and Connections in Psychiatric Disorders

Dominic Kaul1,2, Sibylle G. Schwab1,2, Naguib Mechawar4, Natalie Matosin1,2,3*

1 Illawarra Health and Medical Research Institute, Northfields Ave, Wollongong 2522, Australia

2 Molecular Horizons, School of Chemistry and Molecular Biosciences, University of Wollongong, Northfields Ave, Wollongong 2522, Australia

4 Douglas Mental Health University Institute, 6875 LaSalle blvd, Verdun (Qc), Canada, H4H 1R3

3 Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany

* Corresponding Author

Dr Natalie Matosin Faculty of Science, Medicine and Health University of Wollongong Northfields Avenue Wollongong 2522, Australia Phone: +61 2 4221 5150 Email: [email protected]

Conflict of interest: The authors declare no competing financial interests.

0 ABSTRACT

KAUL, D., S.G. Schwab, N. Mechawar and N. Matosin. How Stress Physically Re-shapes the Brain: Impact on Brain Cell Shapes, Numbers and Connections in Psychiatric Disorders…NEUROSCI BIOBEHAV REV XX(X) XXX-XXX, 2020.-Severe stress is among the most robust risk factors for the development of psychiatric disorders. Imaging studies indicate that life stress is integral to shaping the human brain, especially regions involved in processing the stress response. Although this is likely underpinned by changes to the cytoarchitecture of cellular networks in the brain, we are yet to clearly understand how these define a role for stress in human psychopathology. In this review, we consolidate evidence of macro-structural morphometric changes and the cellular mechanisms that likely underlie them. Focusing on stress-sensitive regions of the brain, we illustrate how stress throughout life may lead to persistent remodelling of the both neurons and glia in cellular networks and how these may lead to psychopathology. We support that greater translation of cellular alterations to human cohorts will support parsing the psychological sequalae of severe stress and improve our understanding of how stress shapes the human brain. This will remain a critical step for improving treatment interventions and prevention outcomes.

Keywords: Stress, adversity, cytoarchitecture, psychiatric disorder, neuron, glia, prefrontal cortex, hippocampus, amygdala, depression, schizophrenia, bipolar disorder

1 1. INTRODUCTION

The brain is centrally responsible for coordinating stress detection and response. In particular, the prefrontal cortex (PFC), hippocampus, and amygdala are areas critical for regulating the systemic effects of stress [1]. The sensitivity of these brain regions to stress may also precipitate changes to brain structure in an effort to adapt to stress and enhance survival by improving the ability to deal with similar future stress [2]. However, this is not always beneficial, as long-term brain adaptations in response to severe stress can have deleterious effects on crucial brain functions, including emotional regulation and cognitive capacity [3].

Stress is among the strongest risk factors for psychotic and mood disorders [4, 5]. This severe stress may be any environmental stress that is perceived to overwhelm the individual’s adaptive capacity [6]. The complexity and inherent individuality of stress and stress vulnerability, particularly in humans [7, 8] but also in rodent models [9, 10], has posed a major challenge in determining how specific psychological stressors contribute or lead to the development of distinct psychopathologies in human populations. Stress vulnerability is highly variable given the complex interaction between genetics and environment [11]. How stress physiologically contributes towards individual disorder trajectory under current nosology has been largely overlooked in much of the psychiatry literature [12]. Refining our understanding of how stress physiologically contributes to symptoms of psychiatric disorders is important to refine targets for prevention and intervention.

In this review, we summarise data from both rodent models and humans examining how stress impacts on the PFC, hippocampus, and amygdala, brain areas which have been the focus of extensive efforts to delineate a role for stress in psychopathology. First, we summarise evidence regarding how stress shapes the brain at the macrostructural level (e.g. entire brain region volume/shape). We then explore how these key regions are impacted at the cytoarchitectural

2 level, across both neurons and glia. With this information, we suggest that dysregulation at the cytoarchitectural level may underlie why distinct stress timings contribute differently towards psychiatric risk and that specific cellular contributions likely shape larger structural and functional deficits.

2. MACROSTRUCTURAL CHANGES TO A STRESSED BRAIN: IMPLICATIONS FOR PSYCHOPATHOLOGY

How stress shapes human brain morphology over the lifespan has by-and-large been conducted through macrostructural studies utilising structural and functional magnetic resonance imaging

(sMRI and fMRI). These studies have suggested significant association between stressful life experiences and altered brain shape. Alterations to the structure and function of specific regions of the brain likely contributes toward a range of emotional and cognitive phenotypes associated with psychopathology. For example, sMRI studies have associated volumetric changes in stress- sensitive regions, including the PFC, hippocampus, and amygdala, with emotional and cognitive symptoms in cases of depression [13-15]. Similarly, an fMRI study indicated that psychosocial stress is associated with reduced centrality, or communicability, of the PFC and hippocampus, and an increased centrality in amygdala connectivity [16], features also observed in psychiatric disorders [17-19]. This is perhaps not surprising given the PFC, hippocampus, and amygdala are strongly influenced by stress [20, 21] and subsequent stress regulation [7, 22]. It is however important to note that although this review focuses on these three brain regions, the impacts of stress are seen across the entire brain. For example, elevated stress is associated with increases in the volumes of some regions such as the fusiform cortex and parahippocampal gyrus, and volumetric reductions in the corpus callosum [21]. Lower volumes in the cerebellum and insular cortex have also been associated with experiencing adversity throughout life [23-25].

3 2.1. Prefrontal cortex (PFC)

The PFC plays an important role in the top-down control of behaviour, thought, and mood [26].

This regulatory role extends to the stress response. Through connectivity across the cortex and larger subcortical networks, including the hippocampus and hypothalamus, the PFC plays a complex role in the perception and response to stress. In particular, the PFC is important for modulating the neuroendocrine (hypothalamic pituitary adrenal [HPA] axis) stress response [27-

29], which is intimately involved in exerting the effects of stress across nearly every cell in the body [30]. However, the responsiveness of the PFC to stress appears to make the region highly sensitive to stress-induced remodelling. Several sMRI studies have associated psychological stress with reductions in cortical volume across the medial PFC (mPFC) [25, 31], orbitofrontal cortex (OFC) [32, 33], ventromedial PFC (VMPFC) [34, 35], dorsolateral PFC (DLPFC) [36,

37], cingulate cortex [38, 39] and the underlying white matter [40] (Table 1). An extensive review by Savitz and Drevets [41] also notes that the PFC, including the OFC, VMPFC, DLPFC, and cingulate cortex, generally exhibit decreased volume, and reduced functional activation in cases of depression and bipolar disorder. Accordingly, several subsequent neuroimaging studies have reported volumetric reductions in the PFC in several psychiatric disorders, including major depressive disorder, bipolar disorder and schizophrenia [42-44]. In addition, reduced volume has been associated with the speed of disorder progression and symptom severity [45, 46].

Consequently, it is hypothesised that persistent changes in the PFC structure induced by stress may play a role in the development of psychiatric symptoms.

A large number of fMRI studies corroborate the hypothesis that stress-induced deficits in the

PFC contribute to some extent in the dysregulation of functions commonly seen in psychiatric disorders (Table 1). For example, adults exposed to chronic psychosocial stress demonstrate reduced DLPFC function and connectivity [47]. Reduced DLPFC function in fMRI studies has been associated with deficits in behavioural and cognitive scoring in cases of schizophrenia [48].

4 Losses of mPFC volume due to chronic stress have also been associated with reduced synaptic output and reduced capacity to suppress the HPA axis and thus stress responsiveness [49]. Long- term reductions to PFC function may therefore be associated with the development of a range of symptoms expressed in severe psychiatric disorders, including hypercortisolemia [50] and anhedonia [51].

Early-life adversity poses a particularly strong and distinct risk for psychopathology [52].

Moreover, the type of stress during early life further distinguishes the risk towards psychiatric disorder development [52]. Early-life physical and emotional abuse have been closely associated with persistently reduced cortical thickness in the mPFC [53], OFC [31, 33], cingulate cortex

[38, 39], and DLPFC [36]. These effects are distinct from stress experienced in adulthood. In older adults, recent self-perceived stress was associated with reductions in the lateral regions of the PFC, with less significant effects seen in the medial regions [34]. Likewise, the recurrence of stress also contributes to distinct risk for psychopathology [54]. Repeated exposure to stress has been associated with reduced mPFC and cingulate cortex volume [25]. Smaller PFC volume also associates with increased susceptibility to perceived stress, suggesting that accumulative stress exposure is a key factor affecting PFC function, even in older adults [34]. As reduced PFC volume is also associated with impaired stress regulatory pathways [49], reducing the capacity of the PFC to regulate stress responses can sensitise the region to further harmful effects of stress.

Indeed, a recent fMRI study indicates that individuals exposed to childhood stress exhibit significant increases in activation in the DLPFC and decreases in the VMPFC when faced with psychosocial stress in adulthood, compared to those without a history of stress [55].

Consequently, the nature and timing of stress is intimately involved with the structural remodelling of the cortex, and suggests that further dynamics influence how stress contributes towards cortical psychopathology.

5 Although substantial efforts to resolve how stress shapes the PFC have been undertaken, several reports also suggest that the wider cortex is vulnerable to stress. Cortical thinning associated with childhood adversity is widespread across the cortex [38]. In particular, a number of studies suggest that the type of stress experienced is integral to how the cortex is reshaped. In the somatosensory cortex, thinning in the genital area is specifically associated with sexual assault, whereas emotional abuse thinned regions associated with self-awareness and reflection [38].

Thinning of the visual cortex after witnessing domestic violence during childhood [56] and the auditory association cortex after parental verbal abuse [57] have also been reported.

Consequently, the cortex is highly sensitive to specific kinds of stress, which may preferentially remodel specific circuits that process those stressors. Understanding how these specific circuit vulnerabilities precipitate across the brain is necessary to resolving the pathophysiological contributions of stress.

2.2. Hippocampus

The hippocampus is also intricately involved in regulation of the stress response. It is central to processing and contextualising memories including experiences of severe stress [58].

Functionally, increased activation of the hippocampus suppresses stress responses through direct projections from the ventral hippocampus (anterior hippocampus in humans) to the paraventricular nucleus of the mPFC, amygdala, and hypothalamus, where the HPA axis hormone cascade initiates [59-61]. Rodent models also indicate that the ventral hippocampus is distinctly vulnerable to stress across volumetric, cellular and molecular studies [62-64]. In humans and rodents, the hippocampus is particularly sensitive to stress mediated by the HPA axis as it contains high levels of corticosteroid receptors [65, 66]. As a result, it is closely tuned to stress responses and one of the most widely examined regions in macrostructural studies of stress.

6 As in the PFC, severe psychological stress is generally associated with reduced hippocampal volume [67-72] (Table 1). These effects are observed across a range of stress timings including both early-life, adolescence, and adulthood stress as well as both acute and chronic stress [67,

73-78], although some studies have also observed no change [79], or increases [80] in volume.

Additionally, the severity of stress (type, persistence, recurrence) during early-life has been an effective predictor of hippocampus volume, including events such as life-threatening illness, parental neglect, and physical, emotional, and sexual abuse [81]. Psychological stress also specifically reduces the total volume of the anterior hippocampus, further supporting that this region is particularly responsive to stress [82]. This is paralleled in functional neuroimaging studies. In an fMRI study, chronic stress associated with reduced connectivity across the CA2,

CA3, and dentate gyrus (DG) of the hippocampus in adults, which was in turn associated with impaired cognitive functions, such as memory [83]. Conversely, reduced hippocampal volume may confer vulnerability to stress and also stress regulation. Twin studies indicate that reduced hippocampal volume is associated with increased vulnerability to psychological stress, including the development of stress-associated psychopathology [84]. Stress, in all its heterogeneity, consistently impairs the hippocampus, both structurally and functionally.

Reduced hippocampal volume is also commonly observed in cases of psychiatric disorders, including mood and psychotic disorders [85-87], with disorder progression compounding loss of hippocampal volume [88-91]. Deficits in hippocampal morphology have also been strongly associated both the severity of psychotic [92] and depressive [93, 94] symptoms. However, changes to hippocampal volume are not solely associated with the development of psychiatric disorders. Severe stress can reduce hippocampal volume without associating with the development of psychiatric symptoms [95]. Likely, the interaction between the timing of stress exposure, individual susceptibility to stress, and recurrence of stress exposure all differentially impact on hippocampal development and function. Further study into the impact that specific life

7 stressors, such as early-life stress, have both acute and long-term are essential to resolve the role of the hippocampus in mediating the risk of stress in the development of psychiatric disorders.

2.3. Amygdala

The amygdala is another brain region that has become a focus of studies of stress neurobiology, as it has been widely implicated in emotional learning and memory, as well as fear aversion [96].

Unlike the PFC and hippocampus, the amygdala generally potentiates the stress response [97].

Morphologically, studies of amygdala have provided conflicting accounts how stress affects volume. Some accounts have noted stress in adulthood increases amygdala volume [99] while others report decreased volume [72, 100], as summarised in Table 1. In both contexts, these effects appear to be mostly recoverable after a period with stress-reduction activity [99].

Similarly, after psychological stress, the amygdala sustains increased connectivity [101, 102] and activity [103], and can also be improved with stress-intervention strategies [98]. This confers with evidence from PTSD studies which suggest that the presence of previous trauma did not associate with altered amygdala volume [104]. The amygdala may be sensitive to acute responsiveness to stress during adulthood, yet also responsive to treatment. However, it is unclear what effects may be seen in chronically stressed individuals and whether these effects are also largely recoverable in adulthood. Also unclear is how the short-term response of the amygdala drives dysregulation in other areas.

Early-life stress particularly impacts amygdala, being closely associated with increased amygdala size [105] and reactivity [106]. In pre-adolescence, psychosocial stress also associated with increased amygdala volume in later adolescence [107] adulthood [108]. However, as reviewed by Teicher and Samson [109], several studies in which individuals were exposed to adversity across adolescence display reduced amygdala volume, compared to controls [110-112].

The timing of stress therefore appears to be particularly important in determining how the

8 amygdala is shaped. Initial exposures to stress may sensitise the brain to subsequent stress, and the recurrence of stress throughout adolescence may be important to shaping the amygdala, contributing to a diversity of phenotypes in adulthood. This is particularly relevant as a meta- analysis of adults with a history of childhood adversity found no significant change in amygdala volume [113], although this may be owing to the diversity of stressors in included studies.

The morphological alterations to amygdala in psychiatric disorders has faced similar challenges.

Some studies have found no difference in amygdala volume between healthy human controls and cases of depression [114, 115], while others have identified reductions [116]. Reduced amygdala volume has also been observed in individuals with psychotic symptoms, compared to those without and healthy controls [115, 117]. Despite losses in volume, amygdala hyperactivity has still been noted in cases with psychotic symptoms [118]. Moreover, psychotic symptoms are associated with blunted amygdala-PFC connectivity [119]. The diversity of observations regarding the amygdala have made resolving a role for how the amygdala contributes towards stress risk in these conditions difficult. One hypothesis is that hyperactivity, combined with impaired connectivity, may lead to impaired stress regulation and increased stress vulnerability, which feed forward into regions of the brain where functional and morphological alterations are more persistent [120]. It is crucial to understand how specific stress types and timings contribute to distinct amygdala morphology before its role in shaping how stress contributes to psychopathology can be discerned.

3. CYTOARCHITECTURAL CHANGES INDUCED BY STRESS: DEFININING THE ROADMAP OF PATHOLOGICAL PROGRESSION Although macrostructural neuroimaging studies can shed light on what brain areas are affected by stress, and how their overall physical structure might change following stress (e.g. volume loss or cortical thinning), what these changes represent requires investigation at the cellular

9 level. Several recent efforts have suggested that altered volumes detected by neuroimaging are associated with altered neuropil density, in particular dendrites and synapses [8, 121-123], suggesting that macrostructural studies may indeed correlate with specific cellular changes.

However, we are only just beginning to explore the changes to local circuits at the level of cell distribution, density, and morphology (referred to collectively as cytoarchitecture) which underlie these larger volumetric changes in humans. Particularly relevant in this sense is determining the cellular contributions driving region-specific vulnerability and resilience to stress and what role this plays in psychopathology. The formidable task of resolving the cellular contributions towards regional alterations is important in enabling psychiatric subtyping, and may provide new avenues of pursuit for prevention, intervention and treatment strategies.

3.1.Stress at the centre of pre-clinical rodent models of psychiatric

disorders

How the brain is shaped by stress and how these stress-induced changes may contribute to psychopathology have been largely examined in pre-clinical rodent models. These models remain an essential tool in understanding fundamental brain functions, and how these functions may be impaired by factors such as genetics and environment [124]. The capacity to model both the specific and longitudinal effects of stress in controlled scenarios holds a distinct advantage in determining how stressful events contribute to the onset of specific symptoms and the cellular and molecular consequences which underlie these states. The focus on pre-clinical rodent models has also been driven by the very limited possibility to study the human brain in the living at a detailed cellular and molecular resolution, relying almost exclusively on postmortem brain samples which are often further limited to late-stage cases, and sometimes, poor quality.

Pre-clinical rodent models of depression principally rely on stress to induce depression-like behaviour in either wild-type rodent lines, such as Sprague-Dawley rats or C57BL/6 mice, or

10 stress-vulnerable rodent lines, such as Wistar-Kyoto rats (for review of such pre-clinical models and their limitations and relevancy to human psychopathology, see Gururajan et al. [125]). These stress paradigms vary from adult chronic restraint and social defeat, to early-life maternal separation, learned helplessness, and corticosterone administration, but all operate based on the principal that overcoming the organism’s ability to cope with adversity will ultimately induce emotional and cognitive dysregulation in vulnerable individuals. Stress has also been used to induce psychosis-like symptoms in rodent lines, however, less often [126]. Importantly, while these models induce similar behavioural and cognitive phenotypes, the nature of these stress paradigms (chronicity, repetition, intensity, age of exposure, type of stress) all likely contribute to underlying variability in the cellular and molecular effects over the life course. For the intent of this review, we will consider stress based on time-point of exposure, either during early life, adolescence, and adulthood, unless otherwise stated. However, underlying variability between models should be weighed when considering these consolidated findings.

When discussing rodent models of psychiatry, it is also important to note that these models are designed to induce certain psychiatric phenotypes, such as social avoidance and cognitive impairments. These models do not comprehensively model neither the symptoms nor the developmental trajectory of psychiatric disorders in humans. They are, however, highly valuable in facilitating the examination of specific stress effects, such as resolving the cell-type specific impacts, and dissecting how these contribute to psychiatric-like phenotypes, which remains difficult to study in humans.

The final caveat in these pre-clinical models is that research has been almost exclusively conducted in males, due to the presumption that hormonal variability over time would introduce extensive variability in results, despite meta-analyses since indicating that this is likely not the case [127, 128]. A growing body of evidence indicates that there are several variations between

11 males and females in response to stress and vulnerability to subsequent psychiatric disorders.

This has been extensively reviewed by Wellman et al. [129], and presents an important consideration as some stress-linked psychiatric disorders are either equally prevalent in females compared to males (e.g. bipolar disorder) [130] or twice as prevalent in females compared to males (e.g. depression) [131]. Consequently, what contributes to these sex-specific differences will remain an important consideration in ensuring equitable and effective prevention and treatment strategies moving forward.

3.2.Excitatory Neurons

A plethora of neuronal subtypes exist across the brain, varying on the basis of their morphological, molecular, and electrophysiological properties [132]. Broadly, the overwhelming majority of neurons can be classified as excitatory (e.g. glutamatergic neurons) or inhibitory (e.g.

GABAergic neurons), however, within these classifications exists enormous diversity. The recent explosion of single-cell transcriptomics studies of the brain indicate that excitatory neurons demonstrate highly diverse transcriptomes both across and within regions [133]. As the most populous excitatory neurons in the cortex, hippocampus, and amygdala, glutamatergic pyramidal neurons play an integral role in the consolidation of inputs and broader integration via projections, often between different regions of the brain [134]. As a result, a broad body of work has begun to unravel how these cells are shaped by stress and how this likely contributes to specific emotional and cognitive symptoms of psychiatry.

3.2.1. Excitatory neurons are highly shaped by stress: evidence from pre-clinical models Several seminal works have documented that chronic stress causes a loss of dendritic complexity across the PFC, particularly in the mPFC of adult rodents (mice and rats) [135-139] (Figure 1).

Similarly, in the hippocampus of adult rodents (mice and rats) and tree shrews, chronic restraint and psychosocial stress reduced dendritic complexity in CA3, despite very few effects being

12 noted in CA1 and the DG [140-144]. This reduced dendritic complexity is also more pronounced in the ventral aspects of CA3, further supporting that the ventral hippocampus is distinctly responsive to stress [143]. The mPFC and CA3 also demonstrate reductions in dendritic complexity in response to chronic corticosteroid administration [145-147], supporting that stress- associated remodelling may be in part associated with altered HPA axis signalling [148]. One hypothesis suggests this occurs in an effort to promote stress reactivity through both the suppression of inhibitory regions, such as the mPFC and hippocampus, as well as to de-centralise processing away from higher-cognitive areas to promote rapid innate responsiveness [149].

However, these alterations may contribute to secondary, unwanted changes in certain brain functions, such as decision making, memory, and behavioural responses [150].

Across rodent models, stress-induced dendritic retractions predominantly impact apical dendrites

[140, 142, 144, 145, 147, 151]. Apical dendrites have distinct inputs (as compared to basal dendrites [152]), being closely involved in the integration of projections from other brain regions, particularly at their distal sites [152, 153]. Impairment of these synapses again suggests that stress is contributing to specific short-term inhibition of communication between regions, such as the PFC and hippocampus, as a compensatory mechanism to increase stress responsiveness [149]. This is supported by findings from Radley and Sawchenko [154] wherein lesions in the rat PFC and hippocampus had additive effects in hyper-activation of HPA axis. In further support of this stress adaptation hypothesis, the retraction of dendrites appears to be mostly recoverable after the removal of the stress despite dendritic regrowth often occurring closer to the soma [123, 136, 155, 156] (Figure 1).

3.2.2. Early-life stress exerts persistent changes on excitatory neurons Unlike stress during adulthood, stress during early-life may exert more persistent effects on pyramidal neurons (Figure 1). In one study, rats exposed to interrupted maternal care throughout early-life displayed impaired dendritic complexity in CA1 after maturation to adulthood [138].

13 Another study in the rodent Octodon degus examining the OFC found that losses of both basal and apical dendrites were associated with parental neglect, and that these rodents had persisting impairments to apical dendrite complexity into adulthood [139]. Further adding to these temporal dynamics, early-life stress can sensitise the brain to subsequent stressors during adulthood [157]. In fact, rats exposed to early-life maternal separation also exposed to restraint stress in adulthood display greater depressive-like behaviour in a forced swim test than either maternal separation or restraint stress individually [158]. One hypothesis is that early-life stress causes persistent impairments to prefrontal and hippocampal circuits, such as seen in volumetric and fMRI studies, which increase the perception and vulnerability to stress in later life [159].

Underlying these changes may be cytoarchitectural changes to the neuropil, including dendrites and synapses [8, 121-123]. The double-hit model suggests that stress during key neurodevelopmental stages may prime the brain for vulnerability to subsequent stress, which persists throughout life [160]. As the brain loses synaptic plasticity with aging, the primed regions may acquire alterations which are then unable to be recovered, leading to the persistently impaired circuits and thus driving neurodevelopment of psychiatric disorders. This agrees with evidence in aged rodents which are less responsive to dendritic remodelling, but when they are affected, display a diminished ability to recover [139, 161, 162].

Unlike the PFC and hippocampus, the impacts of stress on dendritic morphology in the amygdala have been noted to be variable. Both increases [163-165] and decreases [166, 167] in dendritic complexity have been identified in response to chronic stress. In response to acute corticosterone administration, the amygdala displays dendritic hypertrophy in rats and induces anxiety-like behaviour [168]. In further disparity from the PFC and hippocampus, amygdala dendritic hypertrophy after chronic immobilisation of adult rats is persistent, even after stress recovery [169, 170]. Likewise, chronic maternal separation in early-life, has been shown to induce long-term dendritic hypertrophy in the BLA of rodents, but may be recoverable after

14 environmental enrichment in adulthood [171]. Chronic hyper-activation of the amygdala likely contributes towards both anxiety and emotional alterations associated with psychopathology.

The amygdala is vulnerable to stress-associated neural remodelling in both early-life and adulthood, although the discrimination of how these experiences contribute to psychiatric-like symptoms remains to be evaluated.

3.2.3. Alterations of dendritic spines and post-synaptic sites Dendritic spines are small (1-10 µm) protrusions from neuronal dendrites. Mature spines are sites of the postsynaptic density (PSD), containing several important postsynaptic proteins, neurotransmitter, neurotrophic, and hormone receptors [172]. The unique structure, localisation and motility of dendritic spines play a role in the compartmentalisation of neuronal signalling

[173]. Their integral role in synaptic signalling has made these structures a well-documented feature in studies of stress and are hypothesised to significantly contribute to volumetric changes in response to stress [123, 155].

In rodent models of chronic stress, alterations to dendritic spines are among the most consistently identified cellular changes. Significant losses of dendritic spines in response to chronic stress (restraint, corticosterone treatment) in adult rodents (rats and mice) have been identified in the PFC [139, 174-176], with the most striking losses observed on apical dendrites

[137, 177]. In the hippocampus, several studies in rats have also identified reduced dendritic spine density in response to chronic restraint stress, particularly on apical dendrites in CA3 [156,

178, 179], and findings in CA1 have been variable, reporting both increases and decreases in spine density in response to restraint stress [155, 178, 180]. Chronic corticosterone administration has also been shown to cause loss of dendritic spines in the mPFC of adult rats and the CA3 of adult mice [146, 177]. After recovery from stress in adulthood, however, it appears that mPFC and CA3 spine losses are mostly restored [176, 179, 181]. Together, it appears that not only are dendrites retracted by stress, but their constituent post-synaptic sites

15 may be eliminated in regions of the PFC and hippocampus associated with regulating the stress response (Figure 1). This two-fold inhibition of post-synaptic structures (i.e. the post-synaptic sites and the dendrites themselves) greatly reduces the capacity of these dendrites to form efficient synapses, particularly in these distal apical circuits. In agreement, following chronic stress, glutamatergic neurotransmission is impaired across the PFC and hippocampus, despite acute stress inducing transient increases, as reviewed by Popoli et al. [182]. When compared with fMRI data from humans that indicate the centrality of the PFC and hippocampus is reduced in response to stress [16], cytoarchitectural evidence suggests that the communication in pyramidal excitatory circuits in these regions is similarly inhibited, particularly in response to chronic stress.

In the amygdala, chronic stress (immobilisation, social defeat) in adult rodents (rats and mice) has been repeatedly shown to increase dendritic spine density, particularly in the BLA [165, 183-

187]. Similarly, chronic corticosterone administration over a 20 day period in adult mice also induces increases in spine density, although these increases return to normal after a washout period [146]. Moreover, these effects are delayed following traumatic experiences, taking up to

10 days for increases in both total spines and mushroom spines to be identified in rats after a single prolonged shock [188]. Interestingly, after one day, increases in stubby spines were identified, and persisted after 10 days, although their contribution to amygdala connectivity is not well-understood [188]. Increases in dendritic spine density in the amygdala such as those induced in adult rats by chronic immobilisation stress may also increase susceptibility to subsequent stressors through increasing synaptic activity and increasing responsiveness to fear conditioning [165]. The persistent hyperactivity of the amygdala induced by stress may contribute to increases in anxiety and impaired emotional regulation commonly seen across psychiatric disorders [189, 190].

16 Early-life and adolescence appear to be particularly vulnerable developmental stages for psychological stress to cause persistent alterations to dendritic spines. For example, early-life maternal separation reduces total spine density in the whole PFC and CA1 of the hippocampus of rats, which persist to adulthood [191]. Similarly in adolescent mice, social defeat stress reduces the density of stubby spines, but increases that of thin spines in the hippocampal CA1, and is accompanied by depression-like behaviour, as indicated by social interaction and tail suspension tests [192]. Juvenile restraint stress can also reduce dendritic spines in the PFC and

BLA of rats, which persist into adulthood [193]. In the BLA, reduced spine density induced in young rats by chronic social stress contradicts increases seen in adults [194].

These cytoarchitectural changes likely have functional consequences which may be sex-specific.

For example, juvenile male rats exposed to repeated stress display reductions in postsynaptic α- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate receptor (NMDA) glutamate receptor expression in the PFC, associated with cognitive impairment in temporal order recognition testing; these glutamate receptor changes were also recapitulated with corticosterone treatment in a cell culture model, highlighting that these glutamate receptor changes are mediated through glucocorticoid receptors [195]. Interestingly, female juvenile rats exposed to a repeated restraint stress paradigm did not demonstrate the same cognitive impairments or glutamate receptor reductions [196]. However, by blocking oestrogen receptors with injections of the receptor antagonist ICI182 780, stress-related deficits were again observed [196]. These studies collectively suggest that early-life and juvenile stress sex- specifically induces deficits in the structure and function of excitatory circuitry, specifically via glucocorticoid-induced alterations of post-synaptic glutamate receptors, leading to both cognitive and behavioural consequences.

17 Studies in rodent models have also suggested that restoring impaired dendritic spines may alleviate psychiatric-like symptoms, further implicating glutamatergic signalling in mediating stress in psychopathology. For example, treatment with ketamine appears to recover spine density and restore microcircuits identified by calcium imaging in the PFC following chronic corticosterone administration [175]. Recovery of spines associated with ketamine administration in the PFC also alleviated depression-like symptoms, as indicated by a tail suspension test [175].

The antidepressant desipramine similarly recovers reduced spine populations in the mPFC induced by a single foot shock in rats [197]. Consequently, targeting dendritic spines may prove to be an effective method of alleviating some symptoms associated with psychiatric disorders, however, how stress drives these synaptic alterations remains to be resolved.

3.2.4. Translating stress-induced excitatory neuron remodelling into human populations – implications for psychopathology Although rodent models have been valuable for better understanding how different types and timings of stress may shape principal neurons, it is unclear to what extent these pre-clinical findings translate to humans. Particularly relevant in this context is that stress experienced by humans is extremely variable and likely exerts distinct effects compared to rodents. The remodelling of dendrites and dendritic spines is highly implicated in human pathology across stress-associated psychiatric disorders [198]. Alterations to the structure and function of excitatory neurons in the human brain in psychopathology have been extensively reviewed across disorders including depression [199, 200], bipolar disorder [198], and schizophrenia [201,

202]. A number of trans-diagnostic pathologies have begun to emerge which may be shaped by stress. For example, a reduction in spine density has been observed in the DLPFC’s cortical layer

3 in schizophrenia [203], bipolar disorder [204], and depression [205]. The DLPFC is important both to cognitive functions such as attention and memory as well as in the regulation of negative emotion [206], symptoms associated with these disorders under DSM-5 diagnostics [207].

18 Reduced NMDA receptor binding in the DLPFC is associated with individuals with a history early-life adversity [208], suggesting that these circuits are indeed impaired by a history of stress, and may be associated with specific stress timings. However, the contributions of stress towards the remodelling of excitatory circuits, have been sparsely investigated in human cohorts at the cellular level.

A handful of studies of human cohorts with stress histories have evaluated how glutamatergic circuits are shaped by stress. One of the first human postmortem PTSD cohorts identified specific reductions in mature spines and an increase in stubby spines in the OFC, compared to healthy controls [209], which may indicate and impaired capacity to form effective synapses. As

PTSD shares a high co-morbidity with psychiatric disorders, such as depression [210], these disorders may share related aetiology, including traumatic experiences [211]. Another study noted that dendritic retraction and reduced spine density in CA3 of a non-psychiatric adult cohort were associated with anxiety and depressive symptoms [212]. We have recently identified that dendritic spines in individuals with psychiatric disorders and early life stress demonstrate persistent and pronounced losses of mature dendritic spines in the superficial layers of the OFC.

[213]. Although more moderate, these same effects were seen in individual previously exposed to severe chronic stress in adulthood. Importantly, no significant losses were seen in individuals with psychopathology and no distinct chronic severe stress history [213]. These effects suggest that stress exposures can persistently shape excitatory circuits within psychiatric cases and highlights the importance of assessing stress history in future cohort design. Further studies in larger sample sizes are needed to replicate these findings and to provide more details on how stress exposures can reshape brain cytoarchitecture in other, equally important brain areas.

19 3.3.Inhibitory Neurons

Mounting evidence supports that inhibitory GABAergic neurons are susceptible to stress and mediate contributions of stress towards psychopathology [214]. While GABAergic neurons only account for approximately 10-20% of the neuronal population across the brain [215, 216], the balance between inhibitory and excitatory signalling remains at the crux of regular brain function

[217]. Inhibitory neurons are intimately involved in modulating local circuits and are integral in tuning regional functions such as emotional regulation in the cortex [218].

A significant amount of work has been undertaken to delineate the diversity of interneurons within the CNS, which are now recognised to be enormously diverse [219]. Inhibitory neurons are classified largely based on their expression of calcium binding proteins (parvalbumin [PV], calbindin, and calretinin) as well as neuropeptides (neuropeptide Y, vasoactive intestinal protein

(VIP), somatostatin (SST), cholecystokinin) [220]. Three of the most abundant inhibitory neuron classifications are those that express PV, SST, and VIP [221]. These inhibitory neurons form distinct microcircuits to attenuate signals within and between regions. Within these circuits, PV- expressing interneurons predominantly mediate somatic inhibition of pyramidal neurons and the inhibition of other PV-expressing interneurons [222]. They are important to both feedforward and feedback inhibition and in the regulation of pyramidal cell signal propagation [223].

Alternatively, SST-expressing interneurons primarily control the inhibition of inputs to pyramidal neuron apical dendrites [220], although also inhibit other inhibitory neuron types, such as PV-expressing inhibitory neurons [224]. Finally, VIP-expressing interneurons typically inhibit SST-interneurons, which feeds forward to reduce inhibition of other inhibitory neurons

[224]. Although further diversity of inhibitory neurons based on morphology, function and transcriptome has been widely demonstrated at single-cell molecular resolution [133, 221, 225], an extensive analysis of the matching cell morphology has not yet been performed in the context of stress and psychopathology. Using markers derived from the single-cell sequencing studies

20 could provide an important basis for morphologically categorising functional GABAergic neurons, and understanding how inhibitory microcircuits are influenced by stress.

3.3.1. Stress shifts the excitatory/inhibitory balance – evidence from pre-clinical studies Several lines of evidence indicate that inhibitory neurons are sensitive to stress and that these effects modulate broader neural circuitry. In the mouse PFC, GABAergic signalling and metabolic activity are highly dysregulated by social stress and associated with depression-like behaviour, as indicated by decreased sucrose preference and social interaction [226]. Similarly, in the rat mPFC, chronic mild stress greatly impairs GABAergic signalling and is also associated with the development of depression-like anhedonia [227]. Destabilisation of the excitatory/inhibitory (E/I) balance is widely hypothesised to be one pathway through which psychiatric disorders develop [214, 228, 229], and has been hypothesised to underlie disorder trajectories throughout adolescence [230]. Resolving how this balance is impacted by stress may be essential to understanding how distinct stress timings contribute to the aetiology of psychiatric disorders.

A recent review has extensively detailed evidence for the dysregulation of cortical GABAergic signalling by stress in rodent models of depression [231]. The authors note that stress broadly impairs cortical inhibitory neuron GABA production, turnover, and responsiveness as well as reduces markers across several classes of inhibitory neurons including both PV and SST. These deficits also associate with the development of depression-like behaviour and impaired cognition

[231]. This is seen across the PFC, including the mPFC, DLPFC, cingulate cortex, and OFC (see

Fogaça and Duman [231] for this extensive review) (Figure 2). Cortical inhibitory neurons are important in coordinating functions involved in emotional and behavioural processing [217] and losses of this control may be important in the development of associated psychiatric symptoms.

In mice, knockout of cortical PV interneuron transcription factors Dlx5 and Dlx6 reduced

21 cognitive flexibility, and optogenetic activation of these same interneurons recovered cognitive flexibility to levels comparable to controls [232]. Similarly, chemogenetic suppression of PV interneurons in the mouse mPFC induces helplessness [233]. Consequently, deficits in cortical inhibitory circuits induced by stress are likely involved in both behavioural and cognitive impairments as are seen in psychiatric disorders.

However, several further dynamics that influence inhibitory circuits are shaped by stress. First, the duration and intensity of stress appears to influence the cytoarchitecture of inhibitory neurons. Both male and female mice exposed to chronic mild stress for 2 or 4 weeks have increased numbers of PV-expressing interneurons across the PFC [234, 235]. Alternatively, male adult rats exposed to mild stress for 9 weeks [227] or more severe social isolation for 3 weeks

[236] exhibited losses of PV-expressing interneurons in the mPFC and displayed anhedonia-like symptoms. It is likely that there is a phasic response to stress, and that short-term increases may be followed by more gradual, yet persistent reductions in inhibitory neurons. Second, sex not only the impacts of stress on interneuron cytoarchitecture, but may contribute to distinct psychiatric-like symptoms. For example, chronic but not acute chemogenetic activation of PV- expressing interneurons in the mPFC was sufficient to induce anxiety-like behaviour in adult female rats, but not in males [235]. Alternatively, male rats exposed to chronic variable stress at postnatal day 60 demonstrated increased inhibitory connectivity between these neurons and excitatory neurons, dampening excitatory potential within PFC networks [49]. These male rats displayed reduced cognitive performance in a delayed spatial win-shift task [49].

Inhibitory circuits in subcortical regions also appear to be vulnerable to stress. In the hippocampal CA3 and DG, chronic social stress in adult tree shrews reduces the number of PV- expressing interneurons [237]. This is similar to chronic restraint of adult rats where reduced PV immunoreactive neurons across the whole hippocampus has been noted [238-240]. In rodents

22 susceptible to stress (displaying depressive-like symptoms), several additional interneuron types, including SST interneurons are also significantly reduced in number across CA1 and CA3 [239].

However, both acute corticosterone administration and chronic restraint stress of rats also increased GABAergic signalling, measured by postsynaptic currents, in the region [240]. In the amygdala chronic stress in a number of adult rodent models (unpredictable, water deprivation, corticosterone administration) broadly increase the inhibitory capacity of GABAergic neurons

(see review by Jie et al [241] for comprehensive detail), however, cytoarchitectural changes to cell populations and morphology are not well characterised in the region.

Evidently, one of the most notable challenges in defining a role for inhibitory neurons in response to stress are the seemingly contrary responses seen in the structure and function of inhibitory circuits. One hypothesis is that although both glutamatergic and GABAergic signalling are suppressed by stress, a net hypoactivity is established, contributing towards functional deficits across the PFC and hippocampus [214]. This may be driven both by specific alterations to the connectivity of inhibitory neuron microcircuits, such as reduced inhibition of

PV-expressing interneurons due to reductions in somatostatin interneuron populations and functions [224], as well as an over-representation of inhibitory signals exerted on excitatory neurons [214]. However, it will be important to resolve structural and functional abnormalities to determine how this informs the E/I balance across regions of the brain.

3.3.2. Excitatory/inhibitory imbalance: the path to resolving the developmental trajectory of psychopathology? One emerging hypothesis regarding how the developmental trajectory of psychiatric disorders is established, reasons that the balance of excitatory/inhibitory balance throughout development is an important factor in the eventual dysregulation of neural circuits [214]. Converging evidence from pre-clinical models suggests that inhibitory neurons are vulnerable to early-life stress, particularly in stress-sensitive regions. In rats, early-life isolation increases the number of PV-

23 expressing interneurons in the adult PFC, despite no changes in the amygdala [242]. Although no difference in total cell numbers were identified, the authors noted that the ratio of excitatory and inhibitory receptors (VLUGT1/VGAT) was decreased in the BLA [242], indicating a tendency towards over-representation of inhibitory signalling and subsequent hypoactivation of this brain area. Impaired early-life maternal care has also been shown to increase the number of PV- expressing interneurons in the hippocampus of male mice [243]. Recent evidence suggests that these changes are timing-dependent. Mice with early life fragmented maternal care have an increased rate of development of PV-immunoreactive neurons in the BLA and hippocampus, but not the mPFC; however by adolescence, no difference is seen in the BLA and hippocampus, yet increased numbers are seen in the mPFC [243, 244]. This is coupled with increased inhibitory postsynaptic currents in the mPFC during adulthood [245]. However, conflicting reports suggest that stress during early-life and adolescence impairs inhibitory signalling, particularly in the mPFC. Early-life maternal separation has been associated with reduced PV mRNA in the mPFC of male rats in adolescence [246], which were not recoverable with social reintegration [247].

Interestingly, maternal separation also reduces PV mRNA in the PFC of female rats, however, at an earlier juvenile time point [248]. In agreement, functional activation of PV inhibitory neurons following unpredictable chronic mild stress in adolescence can prevent the losses of pyramidal dendritic spines and improve behaviour [249], suggesting that improving inhibitory tone throughout development can alleviate stress induced structural deficits. It is likely that this highly vulnerable and plastic window presents an important impasse in stress-associated remodelling. Evidence from pre-clinical rodent models strongly suggests that stress induces deficits in inhibitory neurons and signalling, although these may exhibit phasic changes dependent on the timing of exposure (Figure 3). Understanding how inhibitory circuits are shaped, and how they shape connectivity across the brain, are important considerations in psychiatry.

24 3.3.3. Inhibitory signalling and a role in human psychopathology As mentioned above, the E/I balance has been purported to be involved in the trajectory of psychiatric disorders (as reviewed in [214]), and is quickly gaining traction for its likely contribution towards the development of cognitive and emotional symptoms in psychiatric disorders [214, 228, 229]. Evidence from postmortem studies largely suggests that several classes of interneurons are inhibited in psychiatric disorders and contribute to a loss of cognitive control [250], although matching these effects to those seen in response to stress has been difficult. In major depressive disorder, the density of calbindin-expressing neurons is reduced

DLPFC and OFC compared to healthy controls, despite no changes in the distribution of PV- expressing interneurons [251]. Decreased somatostatin and somatostatin-associated mRNA and proteins have also been reported in the PFC of cases of depression, despite no changes in PV- related expression [252]. Alternatively, in cases of schizophrenia, PV-expressing interneurons are decreased in the DLPFC [253], coupled with reduced expression of GABA synthesizing enzyme, GAD-67 [250, 254]. A reduction in GAD-67 has also been identified in major depression [255]. Likewise the number of PV and somatostatin-positive neurons are reduced across all subfields of the hippocampus in cases of schizophrenia [256, 257]. Although a wide range of evidence suggests that inhibitory neuron populations are decreased and also that their inhibitory functions are impaired in psychiatry, how these are influenced by stress remains to be clearly understood.

One of the most prominent gaps in our knowledge regarding how stress shapes the human brain is how the E/I balance shifts throughout life, both in health and disease. It remains important to resolve what are the consequences of deficits in both inhibitory and excitatory signalling on the function and communication of the brain. Rodent models suggest that stress timing is important to shaping this balance across several brain areas. Resolving how this balance is not only formed throughout life but also how environmental challenges such as stress shift the dynamic E/I

25 balance may provide important critical timings for intervention, treatment and prevention strategies.

3.4.Astrocytes

As the most abundant glia in the human brain, astrocytes are important regulators of the synaptic microenvironment. In the healthy adult brain, they account for 10-20% of total cells and 20-40% of glia in the brain, with this proportion changing between regions [258]. Once thought to be solely supporting cells, they are now known to be integral in many functions including ion/neurotransmitter homeostasis, metabolic support, and synaptic development/stabilisation and transmission [259]. As a consequence of this diversity, the heterogeneity of astrocytes has been widely identified across morphological [260], functional [261, 262], electrophysiological [263], and transcriptomic [264, 265] studies. Astrocytic heterogeneity is also evident across the whole brain [266], within individual regions, and even within local circuits [267]. A consideration that underlies studies of astrocytes is the difficulty in effective methods to both delineate astrocytes from other cellular populations, as well as delineate subpopulations involved in specific functions. For example, the most widely used astrocyte marker, glial fibrillary acidic protein

(GFAP), is not constitutively expressed in all astrocytes and shows both regional and subregional localisation [268-270]. In the hippocampus, approximately 80% of all astrocytes are GFAP positive [271], whereas only 10-20% are marked in the PFC [272], in which this expression is most pronounced in the superficial layers and white matter [273]. A wide diversity of “pan”- astrocyte markers have also emerged, including, ALDH1L1, GLT-1, SOX9, however, these markers still demonstrate some degree of either regional heterogeneity or subcellular localisation

[274, 275]. One of the primary challenges facing studies of astrocytes is defining what constitutes an astrocyte and how can these populations be effectively, and most importantly, comparatively evaluated across the brain and in disease states.

26 Another major obstacle for discerning a role for astrocytes in stress-mediated psychopathology is that astrocytic complexity is linked with species complexity. Astrocytes in the two most commonly evaluated systems, rodent and human, differ in several metrics with human astrocytes being more morphologically diverse and complex [260, 272, 276], and astrocyte transcriptomic responses to environmental stimuli differing in rodents compared to humans [277]. Although these considerations must be recognised, studies in rodent models have formed the basis of our knowledge regarding how stress shapes astrocytes and still provide an important framework for determining the contributions of stress to shaping astrocytes. Given how vital astrocytes are in maintaining and modulating numerous functions across the brain and their capacity to interact and influence a wide range of cells in the brain, including interacting with the blood-brain barrier, neurons, oligodendrocytes and microglia in the brain, astrocytes are uniquely positioned at the frontline of systemic stress, while also baring the capacity to transduce responses across cells of the brain.

3.4.1. Astrocytes at the interface of stress: evidence from pre-clinical rodent studies The transcriptional profile of rodent astrocytes is highly responsive to glucocorticoids both in vitro and in vivo [278]. In fact, astrocytes are particularly vulnerable to stress being more transcriptionally responsive to glucocorticoids than neurons [279]. Cytoarchitectural studies also note that astrocytes are vulnerable to glucocorticoids as chronic exposure reduces the number of

GFAP-positive cells both in vitro in primary rodent culture [280] and in vivo in the rat PFC

[281]. In primary hippocampal astrocyte culture, this GFAP reduction is coupled with reduced

S100β, a protein widely expressed by brain astrocytes, however, in vivo, only GFAP is reduced

[282].

In chronic stress models, such as restraint and variable stress, reduced GFAP immunoreactivity in the PFC of adult rodents (rats and mice) is well-documented, despite no changes in total astrocyte number [283, 284]. This is paired with reduced complexity of GFAP-positive processes

27 [283], however, may due to activity-dependent decreases in the protein rather than process retraction [285]. Alternatively, acute shock stress in adult mice causes astrocytic hypertrophy in the neocortex marked with ALDH1L1 as an astrocyte marker [286]. This acute stress was sufficient to induce reduced functional coupling between neurons and astrocytes as indicated by reduced astrocyte gap junction proteins [286]. The astrocytic gap junction proteins, connexin

(Cx) 30 and 43, are rapidly becoming a focus of attempts to resolve a role for astrocytes in shaping stress responses. These gap junctions are important in cell-cell communication both within the astrocytic syncytium, as well as between astrocytes and other glia and neurons [287].

As with acute stress, chronic unpredictable stress in adult rats reduces Cx30 and Cx43 punctae across the PFC, including the OFC, and ACC [288, 289]. This has also been observed in the mPFC of adult mice exposed to chronic social defeat stress [290] and is associated with reduced synaptic activity [288, 290] and myelination [288]. Reduced neuronal coupling with astrocytes is also sufficient to impair the synaptic plasticity of neurons [286] and suggests that stress-induced deficits in astrocyte function may mediate impaired synaptic plasticity across the PFC seen in response to stress. Indeed, these gap junctions are also important to the development of psychiatric-like symptoms in rodents. Blocking astrocytic gap functions is sufficient to induce depressive-like behaviour in rats, as indicated by decreased sucrose preference [289]. Given the role that astrocytes play in both mediating synaptic transmission and myelination, the vulnerability of these cells to stress is likely important to transducing stress across the brain and in precipitating these effects into psychopathology (Figure 2).

Similar deficits are observed in several subcortical regions, such as the hippocampus and amygdala. In the hippocampus, the density of GFAP immunoreactive astrocytes is typically reduced in adult rats exposed to either acute (foot shock) [291] or chronic stress (unpredictable and repeated shock) [292, 293], although some studies failed to identify changes, such as in response to chronic immobilisation [294]. Likewise, the number of GFAP-positive cells, and

28 their occupied volume, decreases in the amygdala of adult rodents exposed to chronic immobilisation stress [294]. This is paired with decreased volume occupied by astrocytes and reduced neuropil volume [294]. Several of these effects translate into primates. In adult tree shrews, both the number and soma size of GFAP-positive astrocytes were reduced by psychosocial stress in the whole hippocampus [295]. Although in neurogenic regions, such as the

DG, chronic unpredictable stress coupled with social isolation increases number of GFAP and

S100β-positive astrocytes, as well as branching complexity in male mice [296]. Stress, such as chronic social defeat, also reduces Cx30 and Cx43 expression in the hippocampus [290]. Indeed, this is mediated by the HPA axis as chronic corticosterone administration increases the proportion of phosphorylated Cx43 in the hippocampus of adult mice, which associated with anxiety and depression-like behaviour [297]. Consequently, astrocyte stress sensitivity is evident across many regions of the brain, particularly the brain areas closely involved in stress regulation.

Importantly, astrocytes are responsive to antidepressants and may be leveraged for the treatment of psychopathology. Across rodent and primate models of depression, stress-induced changes to astrocytes in the PFC and hippocampus during adulthood, such as reduced gap junctions and reduced GFAP-positive cell density, are recoverable with the use of antidepressants, including fluoxetine, duloxetine and clomipramine [295, 297-300]. Reversal of these astrocytic impairments was shown to rescue depression-like phenotypes [289, 295, 298, 299]. Chronic antipsychotics can also increase the density of astrocytes, such as in the rat PFC [301].

Normalising astrocyte populations and function thus provide a putative target for reversing stress-induced psychopathological phenotypes, at least in a subset of psychiatric patients.

Delineating which patients have astrocyte-related pathologies and therefore would benefit from these interventions remains an avenue of exploration.

29 3.4.2. Astrocytes and a role in early-life vulnerability to stress As with neurons, astrocytes are also highly vulnerable to stress in a timing-specific manner.

Octodon degus exposed to maternal separation for the first three postnatal weeks demonstrate an increased total number of S100β-positive astrocytes as well as decreased total GFAP-positive cells across the mPFC, anterior cingulate cortex, and prelimbic cortex [302]. Alternatively, rats exposed to maternal separation in adolescence (14 days postnatal) exhibited increased numbers of GFAP positive astrocytes in the PFC [303], although whether this effect is sustained into adulthood has not been considered. These timing-dependent increases following early-life stress have also been observed in the hippocampus of mice [304]. Following 7 days of postnatal maternal separation, wild-type mice exhibit increased GFAP coverage in the hippocampus, however, by adulthood (10 months), GFAP coverage is reduced to lower than that of controls

[304], in line with adult rodent stress models. Regular astrocyte function is essential to the maintenance of numerous developmental processes including in the development and diversification of synapses [305], regulating neuroinflammation [306], and establishment of myelination [307]. Homeostasis of these functions is essential to the development of a healthy brain and so astrocytes are distinctly positioned to precipitate the effects of environmental cues, such as stress, across the brain. It will be important to determine how these transient shifts in astrocytes contribute to cellular remodelling throughout development. While a consensus on the specific cytoarchitectural changes to astrocytes have not yet been reached, there is robust indication that stress-induced changes to astrocytes are involved in psychiatric disorder pathology contributing to synaptic and network abnormalities, common to psychiatric disorders.

3.4.3. Bridging the gap between astrocyte models and human psychopathology There is little literature translating rodent studies of astrocytes to the human brain. To our knowledge, there have been no studies focusing on assessing the impact of stress on astrocyte numbers or their morphology in the postmortem human brain, although several studies have

30 examined postmortem brain tissues derived from individuals who lived with stress-associated psychiatric disorders. These studies indicate that astrocytes are involved in the pathology of at least a subset of psychiatric patients. For example, alterations to GFAP expression in cases of major depressive disorder are not seen globally across the brain [268]. Reductions in this marker are limited to networks associated with mood disorders, including the caudate nucleus and mediodorsal thalamus [268].

Across the PFC, Cx30 and Cx43, are reduced in cases of depression [308]. In the ACC of depressed suicides, reduced Cx30 and Cx43 are coupled with reduced communication between astrocytes and oligodendrocytes compared to healthy controls, as indicated by reduced gap junction coupling [309]. Gap junctions between astrocytes and oligodendrocytes are essential to the transportation of small ions and metabolites and losses of these junctions have been associated with demyelination [310, 311]. Further studies in mood disorders have indicated that

GFAP reductions are exacerbated in cortical white matter [312], however, morphological studies note astrocytic hypertrophy in the white matter of depressed suicides, as measured by Golgi staining [313]. These regional effects may indicate that astrocyte losses are closely involved in mediating demyelination associated with mood disorders (see oligodendrocyte section below).

There are also several studies indicating that astrocyte pathology in the PFC of psychiatric disorders exhibits age-dependent effects. Reduced GFAP-positive cells have been reported in the

DLPFC and mPFC of adult (<45 years old) depressed individuals [314, 315]. Likewise, reduced glial density has also been observed in the ACC of depressed individuals [316]. This effect was not observed in cases later in life (>60 years old), as measured by the number GFAP-positive cells [317]. GFAP-positive astrocytes are also reduced in the deeper layers of grey matter in the

DLPFC of psychotic cases [318] and may be also in part attributable to age [319]. However, this phenomenon is not seen across all major psychiatric disorders. In cases of bipolar disorder older

31 than 60, reduced astrocytic GFAP immunoreactivity and process complexity has been identified

[317]. It is probable that cortical astrocyte dysregulation is involved in a subset of psychiatric pathology and is age-dependent, yet the role that stress exposure plays in mediating/exacerbating these effects is not clear.

Astrocyte alterations have also been reported in subcortical areas of the postmortem brain in psychiatric cases, although are not as well documented as prefrontal effects. Losses of GFAP positive astrocyte density have been reported in the hippocampus of cases of depression and bipolar disorder [320] and more specifically in CA1 in individuals with no history of antidepressants [321]. Furthermore, the number of GFAP-positive cells is decreased in the CA1 of depressed individuals compared to controls, despite no effects being identified in the CA3 or

DG [322]. A decrease in GFAP-positive astrocytes has also been identified in the amygdala of depressed individuals, although was not observed in cases of bipolar disorder or schizophrenia

[323]. In both in cortical and subcortical regions of the brain central to the processing of stress, a common phenotype in psychiatric disorders is a loss of astrocyte function. Although we have evidence that astrocytes are indeed implicated in psychiatric disorders across the brain, as of yet, we do not know what the contribution of astrocytes in mediating stress within these disorders.

3.5.Microglia

Microglia are the primary immune constituents of the central nervous system. In addition to their immune functions, they mediate synaptic plasticity, most notably through regulating synaptic pruning [324, 325], and participate in the pruning of excess synapses throughout neurodevelopment [326]. Microglia are also highly motile cells [327] which facilitates their high responsiveness to changes in the cellular environment as well as their ability to influence the functions of neurons and other glia [328]. As with the other glial cell types, microglia contain a

32 high abundance of glucocorticoid and mineralocorticoid receptors [329], priming them to detect and respond to stress.

3.5.1. Neuroinflammation at the centre of dysregulation in response to stress – evidence from pre-clinical models Rodent models have shown that stress is central to the potentiation of the microglial immune response [330, 331]. Several rodent psychological stress protocols (restraint, social defeat, foot shock) dramatically increase mRNA and protein levels for the activated microglia marker ionised calcium binding adaptor molecule 1 (Iba-1) [332]. This has been observed across the amygdala [333], hippocampus [334], and PFC [335, 336]. Reports are conflicting about whether these changes are associated with increased numbers of reactive cells, or with increases in already reactive populations [337-339], although microglia-mediated neuroinflammation is consistently increased nevertheless. Morphologically, the activation of microglia in response to stress typically increases microglia cell soma size, process thickness, and reactive density of Iba-

1, transitioning to an ameboid-like shape [340, 341]. These morphological changes have been noted in both the PFC and hippocampus in response to social defeat stress in mice and associated with the development of depression-like symptoms [334, 342] (Figure 2). As part of the neuroinflammation response, reactive microglia synthesise and release a number of pro- inflammatory molecules, including cytokines and chemokines [343]. These pro-inflammatory molecules, including Interleukin 1 and Tumour Necrosis Factor α, hold the potential to reshape both neurons and glia by impairing synaptic plasticity [344], myelination [345], and astrocyte functions [346]. Increased reactivity of microglia may assist in driving cellular dysregulation in response to stress.

Microglia are also important facilitators of increasing vulnerability to subsequent stress.

Following recovery from stress, microglia remain primed to subsequent stimuli including psychological stress [347] and facilitate increased reactiveness to repeated stress [334]. In adult

33 mice, primed microglia have been observed in response to social defeat stress 24 hours after exposure [348]. Chronic unpredictable stress in rats/mice adult/teen/juvenile also increases contact between microglia and neuronal processes in both the mPFC [163] and hippocampal

CA1 [349]. Morphologically, these changes prime increased microglial activation to change the cellular environment, facilitating upregulated cytokine release and synaptic remodelling at both the presynaptic and postsynaptic sites [350].

However, not all microglia appear to remain sensitised following stress (Figure 2). Restraint stress paradigms indicate that, although exposure to chronic stress increases Iba-1 reactivity in adult rats/mice, this is coupled with populations of hyper-ramified microglia [336, 337]. Stress- induced hyper-ramification of microglia has been associated with synaptic deficits in the mPFC

[339] and may contribute to depressive-like behaviour [351]. Classically, microglial hyper- ramification has been more closely associated with quiescent states [352], although ramification does not directly correlate to reactivity [353]. It has been suggested that, in response to adversity, populations of microglia are “sensitised” while other become “de-sensitised”, exhibiting hyper- ramification [354], although the consequences of these diverging populations remains unknown.

Exploring the subpopulations of microglia may be important to understanding why certain regions of the brain are more closely affected by stress, and the particular vulnerability that recurrent stress associates with.

3.5.2. Neuroinflammation and early-life vulnerability to stress Psychological stress and the onset of increased activation of microglia is particularly notable during key neurodevelopmental stages, such as in adolescence. Microglial functions, including the maintenance of synaptic pruning, astrocyte maturation, and myelination [355] are essential to typical neural development [324, 356]. Dysregulation of these functions during development can result in atypical and potentially deleterious cellular networks [349] which persist into adulthood

[357]. Moreover, alterations in microglial functions caused by stress have been associated with

34 the development of depression-like symptoms [163, 351, 358], and can be diminished via reversal of microglia-mediated neuroinflammation [359]. It has been hypothesised that alterations in microglial functions throughout early-life and adolescence shift the trajectories of neuronal and glial maturation which lead to persistent dysregulated circuits which underlie psychiatric symptoms [360]. Microglial priming may also underlie why the brain becomes sensitised to subsequent stress after early-life adversity and why stress at this critical time point leads to such a high risk for psychiatric disorder development. Primed microglia hold a greater potential to alter neurons and glia [361] and thus are likely involved in enacting the first line of response to stress in the brain. While we are now beginning to understand how microglia can influence neural circuits in a development-specific manner, we are yet to resolve the contributions of these cells towards the dysregulation of neural circuits which may underlie psychopathology. The next major hurdle will be to resolve exactly how microglia mediate stress and how this influences the developmental trajectories of neurons and other glia, as well as the development of psychiatric symptoms.

3.5.3. Neuroinflammation at the interface of stress and human psychopathology? Although rodent models suggest that microglia may mediate the effects of stress across the brain, evidence supporting a role for microglia in human psychopathology is not widely agreed upon.

Developments in positron emission tomography imaging of the activated microglial marker translocator protein (TSPO), a translocator protein, have facilitated in vivo analysis of microglial quantity and activity. In individuals with a current episode of depression, increased TSPO binding activity indicative of neuroinflammation, are seen in the PFC and ACC [362]. TSPO appears to be particularly high in depressed individuals with suicidal thoughts [363]. Increased activity also persists in recovery periods following depression later in life [364]. These imaging studies, however, do not provide the resolution needed to understand detailed cytoarchitectural

35 changes that might occur beyond general microglial activity, but support that microglia indeed mediate neuropathology of mood disorders, meriting further human research.

In postmortem studies, it has been difficult to disentangle the contributions of suicide facilitation from neuroinflammatory risk in depression. Increased Iba-1 as well as increased microglial priming have been reported in the white matter of the ACC of depressed individuals having died by suicide [365]. Although increased microglial activation is also seen in the grey matter of the

ACC, this may be associated with the risk of suicide within the condition, rather than the condition itself [366]. Indeed, increased microglial density appears to be an important consideration for suicide facilitation in depression [366, 367]. Similarly, postmortem studies of bipolar disorder have had limited success identifying increased microglial populations or activation across the DLPFC, ACC, and hippocampus, owing to small sample sizes, influences of medications, and variability in sample demographics (see systemic review by Giridharan et al.

[368] for limitations of current postmortem bipolar studies).

Microglia-mediated inflammation is also a hypothesised mechanism contributing to abnormal neural connectivity in schizophrenia [369, 370], however, resolving a defined microglial pathology in schizophrenia has been as contentious. Several postmortem studies have identified increased expression of microglial markers as well as number and density across the PFC and in the hippocampus and amygdala, yet almost as many studies have noted the opposite or null trends (as reviewed by Laskaris et al.[371] and Trépanier et al. [372]). However, markers of other functions, such as microglial-associated phagocytosis in synaptic pruning are elevated in cases of schizophrenia [373]. Indeed, a schizophrenia patient-derived cell model demonstrated increased microglial-mediated synaptic pruning [374]. Consequently, microglial pathology in psychotic disorders may be driven by activity-dependent increases in functions such as synaptic pruning, rather than limited to states of increased activated microglia associated with

36 neuroinflammation. Determining how the functions of microglia are influenced within these disorders may be important in resolving why stress is a highly common sequelae for psychiatric disorders.

3.6.Oligodendrocytes

Oligodendrocytes are essential myelinating cells of the central nervous system that consist of a small cell body and a few branching processes able to myelinate dozens of axons. They are also important in maintaining axonal integrity [375], metabolic and trophic support of neurons [376] and in the support of the blood-brain barrier [377]. Myelinating oligodendrocytes are largely restricted to white matter [378], while those in the grey matter, considered satellite oligodendrocytes, are less intimately involved in myelination [379]. These functions are important in coordinating neural connectivity, and thus are an important, but often overlooked contributor towards psychiatric disorders [380]. The distribution of oligodendrocytes varies notably between brain regions e.g. low in the cerebellum and higher in the cortex [378] and decreases with age [381].

3.6.1. Stress impairs myelination: evidence from pre-clinical studies As with other glia, oligodendrocytes are highly susceptible to stress. In adult mice, chronic social stress downregulates a number of oligodendrocyte proteins involved in myelination in the amygdala and PFC [382]. This stress paradigm also decreases myelin thickness and internodal length alongside the total number of mature oligodendrocytes in the mouse mPFC [383] (Figure

2). Adult mice more susceptible to the effects of stress, demonstrated by increased social avoidance behaviour, had even greater reductions in myelin thickness, internodal length and number of oligodendrocytes [383]. A reduction in myelination in the mouse PFC also accompanies the development of depression-like symptoms [384]. Conversely, both restraint stress and corticosteroid administration increase oligodendrogenesis in the DG of the hippocampus [385], potentially as a compensatory effect for the loss of oligodendrocyte 37 function. Loss of oligodendrocyte function, especially loss of myelination is intimately involved with neuronal damage [386] and impairment of synaptic signal conductance [387], likely contributing towards deficits in regional functions such as in cognition and behaviour [388].

3.6.2. Persistent shaping of oligodendrocytes by early-life stress As seen for other glia, the timing of stress exposure is important to the long-term effects on oligodendrocyte cytoarchitecture. Exposing adult mice to social isolation for eight weeks reduces myelination in the PFC [389]. Upon social reintegration for four weeks following this adversity, myelination is recoverable to levels similar to controls, suggesting a robust level of glial plasticity [389]. However, when exposed during juvenile stages, myelination is not recoverable even after maturation to adulthood is completed [390]. This may contribute towards degraded neuronal signalling in the PFC and may help explain why the effects of stress in early life are not only persistent, but notably damaging for the brain. Both astrocytes and microglia are important facilitators of myelination, and affect the migration, proliferation and mature functions of these cells [391]. For example, microglial activation stimulates the production of a number of pro- inflammatory molecules (e.g. reactive oxygen species, cytokines, chemokines) which oligodendrocytes are particularly sensitive to [392]. In fact, activation of microglia by lipopolysaccharide injection into rats is sufficient enough to delay the development of oligodendrocytes and induce hypomyelination [393]. As discussed in previous sections, both astrocytes and microglia are closely involved in timing-dependent vulnerability to stress and may contribute to myelin and oligodendrocyte impairment throughout development. This may facilitate the persistent dysregulation of neuronal connectivity and thus may be important in resolving how different stress histories contribute towards the dysregulation of brain functions.

3.6.3. Oligodendrocytes are sensitive to stress in the human brain: cause or effect? It is not yet clear whether oligodendrocytes are impacted as a consequence of stress, or as effectors of stress and how this contributes towards psychopathology. At the cytoarchitectural

38 level, markers of apoptosis and necrosis of oligodendrocytes are seen in major depressive disorder, bipolar disorder, and schizophrenia [394, 395]. These findings indicate that oligodendrocytes and their capacity to myelinate other cells are both reduced in these disorders.

This would suggest that oligodendrocytes are impacted downstream in the progress of cellular dysregulation. However, recent single-nucleus RNA sequencing study of the DLPFC implicated oligodendrocyte precursors, alongside excitatory neurons, as being the most transcriptionally dysregulated cellular population in depression [396]. In psychiatric disorders, alterations in gene expression associated with myelination are most pronounced in the hippocampus and PFC [397,

398], and favour that oligodendrocytes are particularly vulnerable in stress responsive regions. In further support that oligodendrocytes are actively involved in stress responsiveness, in the human ACC early-life adversity has a robust and lasting effect on the oligodendrocyte transcriptome, being associated with decreased myelination of fine axons [399]. This adversity also impairs the maturation of oligodendrocytes in the VMPFC [400]. However, this still bares the question of whether these changes are induced by stress or by changes in the cellular environment. As mentioned previously, regular myelination and communication between oligodendrocytes and neurons remains central to the maintenance of a healthy brain [401]. The communicability of oligodendrocytes with both neurons and other glia suggests that these deficits may be driven by complex interactions with the cellular environment, for example, reduced metabolic support from astrocytes, and increased neuroinflammation.

39 4. CONCLUSIONS AND FUTURE DIRECTIONS

An extensive body of work has contributed to our understanding of how stress shapes the brain and how this likely contributes to psychopathology, yet we are still a distance away from understanding these effects with clarity. Progress has largely been achieved through rodent preclinical models. These models remain essential to advancing our understanding of the neurobiology of stress, particularly with de-convoluting the complexity of stress in a controlled manner (often not possible in human studies where stress exposures are varied and complex).

Animal model studies have particularly provided valuable knowledge about how different types, timings and durations of stress shape the cellular environment through a complex inter- relationship between cells, as all major cell types are in some regard affected by stress and also associated with psychopathology. Although the data provided by rodent models have provided fundamental understanding and insight into the effects of stress, a challenge in the field has been translating the effects identified in pre-clinical rodent models into comparable effects in humans, as very little consideration has been given to stress history in human cohorts. To further progress this research area, alongside rodent models, the study of both living (molecular brain imaging methods such as PET) and postmortem (cellular and molecular) human cohorts is important.

At the macrostructural level, human studies have offered insight into how the brain is shaped by stress and potential roles this may play in psychopathology. However, several questions remain to be answered by future studies:

Firstly, how do individual experiences of stress contribute to individual psychiatric disorder trajectories? Macrostructural studies highlight that the recurrence [54], timing, and chronicity of stress are an important consideration as to how the brain is shaped [402], yet these factors are often not considered in clinical psychiatry research despite stress being a leading risk factor for psychopathology. Curation of specialised cohorts that consider the individual sequalae shaping

40 psychopathology – accounting for stress history, treatment strategies and disorder progression – may be crucial to accurately understand the complex trajectory of how psychiatric disorders develop.

Secondly, how does stress differentially contribute to structural changes in the brain throughout development? Determining which aspects of stress cause persistent effects and influence developmental trajectories is important in being able to identify at-risk individuals and design the interventions that prevent them from developing psychiatric disorders. Finally, what are the molecular and cellular processes that underlie these macrostructural changes? Characterising the molecular and cellular impacts of stress and how these influence psychopathology will enable development of new targets for prevention and treatment.

At the cytoarchitectural level, several additional questions concerning the temporal sequence in which morphological and functional changes occur in response to stress are important for future studies to address. For example, is there a domino effect (in which case, what is the first cell type to “fall”?), or are all cell types affected equally in parallel? To what extent are cellular changes recoverable and when do cellular deficits begin to contribute to the development of behavioural and cognitive dysfunction? We suggest that, while stress affects cellular populations at any life stage, brain-cell changes induced during periods when cellular population trajectories are being established, could lead to sustained atypical connectivity contributing to psychopathology. This is likely compounded by an increased responsiveness of different brain cells to stress, as well as alterations to stress responsiveness through cell-to-cell communication. Thus persistent dysregulation of these circuits via these mechanisms during early-life and adolescence could increase the risk of developing behavioural and cognitive symptoms throughout development.

Another important question is, why do certain brain areas appear to be more vulnerable to stress, and what are the cellular and molecular drivers of this vulnerability? The recent explosion of

41 single-cell sequencing and high-resolution molecule/cell visualisation techniques being applied to the brain indicate that the brain consists of an enormously diverse profile of cells that varies according to brain region [403]. It is likely that subpopulations of cells are uniquely vulnerable to stress [265], and this precipitates specific effects across cellular networks that persistently impact on brain circuitry. Exploring this possibility in cases of psychopathology, especially in the context of stress exposure or history, will assist in improving current understandings of cellular pathologies in psychiatry that are induced by stress. This approach can also assist in the identification of specific patient subgroups characterised by similar underlying pathology, the biomarkers for identification of these patient subgroups, and the biological targets for their treatment.

42 Acknowledgements: This work was supported by an NHMRC Early Career Fellowship

(APP1105445) awarded to Dr Matosin and grants from the Brain Behavior Research Foundation

(BBRF/NARSAD Young Investigator Grant) as well as the Rebecca L. Cooper Medical

Research Foundation. Dr Mechawar’s research is supported by grants from CIHR, HBHL and

ERA-NET NEURON.

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64 FIGURES

Figure 1. Early-life/juvenile stress vs. adulthood stress impacts on pyramidal neurons. Stress-induced changes are highlighted by the dotted outline. Stress in adulthood causes a two- phase retraction; both of the dendrites themselves and of their constituent dendritic spines. With time, these changes are largely recoverable, albeit with regrowth occurring closer to the soma. However, if stress is experienced during early-life/adolescence, retraction of dendrites and dendritic spines can persist into adulthood. This is seen in both the PFC and hippocampus.

65 Figure 2. Model of cellular impacts of stress based off of evidence from the prefrontal cortex. Stress-induced changes are highlighted by the dotted outline. In control, non-stressed individuals, excitatory and inhibitory balance is maintained through attenuation of inhibitory and excitatory neurons. Glia provide important and diverse roles and form a closely communicative and tightly coordinated network which maintains homeostatic control. Following severe psychological stress, broad structural and functional consequences are seen across cellular networks (highlighted by dotted outlines). Changes to morphology are seen after several different stress exposures but are most substantial in stress-susceptible individuals, during key neurodevelopmental stages (e.g. early-life). Chronic dysregulation of cell-to-cell communication due to cell morphology changes can contribute to the development of cognitive and emotional impairments, which are hallmark symptoms of psychiatric disorders. Specifically: i) Excitatory (pyramidal) neurons display decreased synaptic number and activity as well as reduced process branching, impairing top-down regulation of other brain areas with their projections, as well as integrative capacity of the PFC; ii) Although parvalbumin (PV)-expressing interneurons display

66 decreased branching, they maintain increased number and connectivity with excitatory neurons, dampening the capacity for top-down control of the prefrontal cortex (PFC); iii) Somatostatin (SST)-expressing interneurons display decreased branching and also reduced connectivity with neurons and other interneurons, promoting greater inhibitory actions; iv) Astrocytes display impaired capacity to modulate synaptic environments, further impairing neuronal function and can also impair the function of other glia, such as oligodendrocytes; v) Chronic neuroinflammation caused by increased reactivity and potential reactive capacity of microglia, in conjunction with increased interaction with other cells, contributes to impairments seen in other cellular populations. A population of microglia are also hyper-ramified following stress; vi) Reduced myelination via oligodendrocyte changes further impairs the potential for neuronal signal transduction, as well increases susceptibility of neurons to damage. This may be due to both direct effects of stress on oligodendrocytes as well an impairment of astrocytes and microglia which are important to oligodendrocyte maintenance.

Figure 3. Model of excitatory/inhibitory balance shifts over the life course in response to stress. During key developmental periods, such as early life and adolescence (left panels, x- axis), the excitatory/inhibitory balance (y-axis) is sensitive to stress (A). Persisting stress, such as neglect or trauma, then establishes shifts across the cellular network including altering microglial

67 and astrocyte functions in synapse regulation, increasing neuroinflammation, decreasing myelination and establishing a net over-inhibition (B). These changes to the cytoarchitecture and function of neurons and glia induced by stress likely impact the developmental trajectory of circuits, with lasting consequences across the life-course (C). In adulthood (right panel, x-axis), the excitatory/inhibitory balance is also sensitive to stress, but the consequences are less persistent and can often be restored to homeostasis if the stress is removed for a period of time (D). The exception appears when stress is experienced earlier in life, and again in adulthood (E), with the earlier stress priming microglia and astrocytes to subsequent stress (F). This likely precipitates dysregulation across excitatory/inhibitory cells, leading to persistent dysfunction of circuits, as seen in (B/C).

68 Table 1. Summary of structural and functional magnetic resonance imaging studies evaluating the structure and function of the prefrontal cortex, hippocampus, and amygdala following stress exposure. Studies were detected in a PudMed search using the terms “adversity”, “stress”, “MRI”, “magnetic resonance imaging”, combined with either “prefrontal cortex”, “hippocampus” or “amygdala”. Unless otherwise stated, stress is compared to healthy controls without a history of stress or adversity. Studies of cohorts limited to cases of PTSD compared to healthy no stress controls were not included in this summary.

Referenc Type of Stress Timing of Stress Time of Measurement Method Impact of stress e Prefrontal Cortex [32] Severe poverty Early life Adulthood sMRI ↓ OFC volume [33] Chronic adversity (multimodal e.g. Infancy/childhood/adolescence Adulthood sMRI ↓ right OFC volume (infancy) socioeconomic status, familial stress) [31] Physical abuse Childhood Childhood sMRI ↓ mPFC volume ↓ OFC volume ↓ DLPFC volume ↓ VMPFC volume [35] Abuse Childhood Adolescence sMRI ↓ VMPFC volume ↓ OFC volume [53] Emotional maltreatment (various) Childhood Adolescence sMRI ↓ mPFC volume [38] Sexual assault Childhood Adulthood sMRI ↓ ACC volume (emotional abuse) Emotional abuse ↓ PCC volume (emotional abuse) [37] Childhood adversity Childhood Adulthood1 sMRI ↓ left DLPFC volume [103] Cumulative adversity Childhood through to Adulthood fMRI ↑ lateral PFC activity adulthood [39] Self-perceived social status Adulthood Adulthood sMRI ↓ ACC volume [25] Cumulative adversity Adulthood Adulthood sMRI ↓ mPFC volume ↓ ACC volume [47] Self-perceived stress Adulthood Adulthood fMRI ↓ DLPFC functional connectivity with frontoparietal network

[34] Self-perceived stress Older adulthood (65-90 years) Adulthood sMRI ↓ lateral PFC volume (dorsolateral and ventrolateral)

Hippocampus [77] Early life adversity Early life Childhood sMRI ↓ left and right hippocampus volume ↓ CA1 volume ↓ CA3 volume ↓ DG volume

0 [76] Early life adversity Early life Childhood sMRI ↓ in total hippocampus volume (only in cases where stress onset during early childhood (<5 years)) [81] Early life adversity (e.g. neglect/abuse), Early life/adolescence Adolescence sMRI ↓ left and right hippocampus volume adolescent chronic stress (socioeconomic status, social, health) [74] Childhood maltreatment Early life Adulthood sMRI ↓ left CA2 volume ↓ left CA3 volume ↓ DG volume [78] Threat (e.g. abuse) Childhood Adolescent/Adulthood sMRI ↓ right hippocampus volume Deprivation (e.g. neglect) (deprivation) ↔ hippocampus volume (threat) [70] Childhood violence Childhood Childhood/Adolescence sMRI ↓ total hippocampus volume (8-17 years) [80] Childhood adversity Childhood Adulthood2 sMRI ↑ CA3 volume [37] Childhood adversity Childhood Adulthood1 sMRI ↓ left and right hippocampus volume [75] Abuse/maltreatment Childhood Adulthood sMRI ↓ left, right and total hippocampus volume ↓ only in men (not women) [79] Childhood adversity Childhood Adulthood sMRI ↔ hippocampus volume [113] Childhood adversity (meta-analysis) Childhood Adulthood sMRI ↓ hippocampus volume [67] Meta-analysis (including Childhood/adulthood Adulthood sMRI ↓ left, right, and total hippocampus childhood/adulthood abuse and combat volume across 12 studies trauma) [103] Cumulative adversity throughout life Childhood through to Adulthood fMRI ↑ hippocampus activity adulthood [73] Self-perceived stress Adolescence Adolescence sMRI ↓ left and total hippocampus volume [71] Emotional trauma Adolescence Adolescence sMRI ↓ CA3 volume [83] Recent life stress Young Adulthood Young Adulthood fMRI ↓ CA2/CA3/DG connectivity with the caudate [68] Self-perceived stress Adulthood Older Adulthood sMRI ↓ left and right hippocampus volume Number of negative stressful life events [72] Financial hardship Adulthood Adulthood sMRI ↓ hippocampus volume [69] Stressful military service Adulthood Adulthood sMRI/fMRI ↓ total hippocampus volume in vulnerable individuals ↓ connectivity with VMPFC [82] Self-perceived stress Adulthood Adulthood sMRI ↓ anterior hippocampus volume 1 Amygdala [106] Early life stress Early life Adulthood fMRI ↑ reactivity in response to mood change [100] Sexual/Physical abuse Early life Adulthood sMRI ↓ left amygdala volume (recent life Recent life stress Adulthood stress) ↔ volume (childhood abuse)

[404] Childhood violence Childhood Adolescence fMRI ↔ amygdala PFC connectivity Social deprivation independently ↓ amygdala-PFC connectivity when exposed to both stress [107] Childhood trauma Childhood Adolescence sMRI ↓ left amygdala development (early to late adolescence)

[70] Childhood violence Childhood Childhood/Adolescence sMRI ↓ total amygdala volume (8-17 years) [108] Childhood adversity Childhood Adulthood sMRI ↑ amygdala volume ↑ right amygdala volume with adversity severity ↑ right amygdala volume with verbal abuse, emotional abuse, and witnessing sibling assault [110] Childhood trauma Childhood Adulthood sMRI ↓ amygdala volume [111] Childhood trauma Childhood Adulthood sMRI ↓ amygdala volume [113] Childhood adversity (meta-analysis) Childhood Adulthood sMRI ↔ amygdala volume [112] Childhood adversity Childhood Adulthood sMRI ↓ right amygdala volume ↓ BLA volume [103] Cumulative adversity throughout life Childhood through to Adulthood fMRI ↑ amygdala activity adulthood [98] Financial stress (unemployment) Adulthood Adulthood fMRI ↑ amygdala functional coupling to ACC

[99] Self-perceived stress Adulthood Adulthood sMRI ↑ right amygdala volume (reduced PSS over 8-week period associated with reduced volume) [72] Financial hardship Adulthood Adulthood sMRI ↓ amygdala volume [105] Various (childhood abuse, domestic Various Adulthood sMRI ↑ left lateral amygdala volume 2 violence, loss of loved one) Abbreviations: fMRI, functional magnetic resonance imaging; sMRI, structural magnetic resonance imaging PFC, prefrontal cortex; mPFC, medial prefrontal cortex; OFC, orbitofrontal cortex; DLFPC, dorsolateral prefrontal cortex; VMPFC ventromedial prefrontal cortex; ACC anterior cingulate cortex; PCC, posterior cingulate cortex, CA, Cornu Ammonis; DG, dentate gyrus; BLA, basolateral amygdala.

1cases diagnosed with schizophrenia 2cases diagnosed with major depressive disorder

3