Chronic Stress Alters Astrocyte Morphology in Mouse Prefrontal Cortex.

BACKGROUND: Neuromorphological changes are consistently reported in the prefrontal cortex of patients with stress-related disorders and in rodent stress models, but the effects of stress on astrocyte morphology and the potential link to behavioral deficits are relatively unknown. METHODS: To answer these questions, transgenic mice expressing green fluorescent protein (GFP) under the glial fibrillary acid protein (GFAP) promotor were subjected to 7, 21, or 35 days of chronic restraint stress (CRS). CRS-induced behavioral effects on anhedonia- and anxiety-like behaviors were measured using the sucrose intake and the PhenoTyper tests, respectively. Prefrontal cortex GFP+ or GFAP+ cell morphology was assessed using Sholl analysis, and associations with behavior were determined using correlation analysis. RESULTS: CRS-exposed male and female mice displayed anxiety-like behavior at 7, 21, and 35 days and anhedonia-like behavior at 35 days. Analysis of GFAP+ cell morphology revealed significant atrophy of distal processes following 21 and 35 days of CRS. CRS induced similar decreases in intersections at distal radii for GFP+ cells accompanied by increased proximal processes. In males, the number of intersections at the most distal radius step significantly correlated with anhedonia-like behavior (r = 0.622, P < .05) for GFP+ cells and with behavioral emotionality calculated by z-scoring all behavioral measured deficits (r = -0.667, P < .05). Similar but not significant correlations were observed in females. No correlation between GFP+ cell atrophy with anxiety-like behavior was found. CONCLUSION: Chronic stress exposure induces a progressive atrophy of cortical astroglial cells, potentially contributing to maladaptive neuroplastic and behavioral changes associated with stress-related disorders.

Behavioral Deficits Induced by Somatostatin-Positive GABA Neuron Silencing Are Rescued by Alpha 5 GABA-A Receptor Potentiation.

INTRODUCTION: Deficits in somatostatin-positive gamma-aminobutyric acid interneurons (SST+ GABA cells) are commonly reported in human studies of mood and anxiety disorder patients. A causal link between SST+ cell dysfunction and symptom-related behaviors has been proposed based on rodent studies showing that chronic stress, a major risk factor for mood and anxiety disorders, induces a low SST+ GABA cellular phenotype across corticolimbic brain regions; that lowering Sst, SST+ cell, or GABA functions induces depressive-/anxiety-like behaviors (a rodent behavioral construct collectively defined as "behavioral emotionality"); and that disinhibiting SST+ cells has antidepressant-like effects. Recent studies found that compounds preferentially potentiating receptors mediating SST+ cell functions, α5-GABAA receptor positive allosteric modulators (α5-PAMs), achieved antidepressant-like effects. Together, the evidence suggests that SST+ cells regulate mood and cognitive functions that are disrupted in mood disorders and that rescuing SST+ cell function via α5-PAM may represent a targeted therapeutic strategy. METHODS: We developed a mouse model allowing chemogenetic manipulation of brain-wide SST+ cells and employed behavioral characterization 30 minutes after repeated acute silencing to identify contributions to symptom-related behaviors. We then assessed whether an α5-PAM, GL-II-73, could rescue behavioral deficits. RESULTS: Brain-wide SST+ cell silencing induced features of stress-related illnesses, including elevated neuronal activity and plasma corticosterone levels, increased anxiety- and anhedonia-like behaviors, and impaired short-term memory. GL-II-73 led to antidepressant- and anxiolytic-like improvements among behavioral deficits induced by brain-wide SST+ cell silencing. CONCLUSION: Our data validate SST+ cells as regulators of mood and cognitive functions and demonstrate that bypassing low SST+ cell function via α5-PAM represents a targeted therapeutic strategy.

Lasting changes induced by mild alcohol exposure during embryonic development in BDNF, NCAM and synaptophysin-positive neurons quantified in adult zebrafish.

Fetal alcohol spectrum disorder is one of the leading causes of mental health issues worldwide. Analysis of zebrafish exposed to alcohol during embryonic development confirmed that even low concentrations of alcohol for a short period of time may have lasting behavioral consequences at the adult or old age. The mechanism of this alteration has not been studied. Here, we immersed zebrafish embryos into 1% alcohol solution (vol/vol%) at 24 hr post-fertilization (hpf) for 2 hr and analyzed potential changes using immunohistochemistry. We measured the number of BDNF (brain-derived neurotrophic factor) and NCAM (neuronal cell adhesion molecule)-positive neurons and the intensity of synaptophysin staining in eight brain regions: lateral zone of the dorsal telencephalic area, medial zone of the dorsal telencephalic area, dorsal nucleus of the ventral telencephalic area, ventral nucleus of the ventral telencephalic area, parvocellular preoptic nucleus, ventral habenular nucleus, corpus cerebella and inferior reticular formation. We found embryonic alcohol exposure to significantly reduce the number of BDNF- and NCAM-positive cells in all brain areas studied as compared to control. We also found alcohol to significantly reduce the intensity of synaptophysin staining in all brain areas except the cerebellum and preoptic area. These neuroanatomical changes correlated with previously demonstrated reduction of social behavior in embryonic alcohol-exposed zebrafish, raising the possibility of a causal link. Given the evolutionary conservation across fish and mammals, we emphasize the implication of our current study for human health: even small amount of alcohol consumption may be unsafe during pregnancy.

Prophylactic Efficacy of Riluzole against Anxiety- and Depressive-Like Behaviors in Two Rodent Stress Models.

Introduction: Chronic stress-related illnesses such as major depressive disorder and post-traumatic stress disorder share symptomatology, including anxiety, anhedonia, and helplessness. Across disorders, neurotoxic dysregulated glutamate (Glu) signaling may underlie symptom emergence. Current first-line antidepressant drugs, which do not directly target Glu signaling, fail to provide adequate benefit for many patients and are associated with high relapse rates. Riluzole modulates glutamatergic neurotransmission by increasing metabolic cycling and modulating signal transduction. Clinical studies exploring riluzole's efficacy in stress-related disorders have provided varied results. However, the utility of riluzole for treating specific symptom dimensions or as a prophylactic treatment has not been comprehensively assessed. Methods: We investigated whether chronic prophylactic riluzole (∼12-15 mg/kg/day p.o.) could prevent the emergence of behavioral deficits induced by unpredictable chronic mild stress (UCMS) in mice. We assessed (i) anxiety-like behavior using the elevated-plus maze, open-field test, and novelty-suppressed feeding, (ii) mixed anxiety/anhedonia-like behavior in the novelty-induced hypophagia test, and (iii) anhedonia-like behavior using the sucrose consumption test. Z-scoring summarized changes across tests measuring similar dimensions. In a separate learned helplessness (LH) cohort, we investigated whether chronic prophylactic riluzole treatment could block the development of helplessness-like behavior. Results: UCMS induced an elevation in anhedonia-like behavior and overall behavioral emotionality that was blocked by prophylactic riluzole. In the LH cohort, prophylactic riluzole blocked the development of helplessness-like behavior. Discussion/Conclusion: This study supports the utility of riluzole as a prophylactic medication for preventing anhedonia and helplessness symptoms associated with stress-related disorders.

Brain structure and working memory adaptations associated with maturation and aging in mice.

Introduction: As the population skews toward older age, elucidating mechanisms underlying human brain aging becomes imperative. Structural MRI has facilitated non-invasive investigation of lifespan brain morphology changes, yet this domain remains uncharacterized in rodents despite increasing use as models of disordered human brain aging. Methods: Young (2m, n = 10), middle-age (10m, n = 10) and old (22m, n = 9) mice were utilized for maturational (young vs. middle-age) and aging-related (middle-age vs. old mice) comparisons. Regional brain volume was averaged across hemispheres and reduced to 32 brain regions. Pairwise group differences in regional volume were tested using general linear models, with total brain volume as a covariate. Sample-wide associations between regional brain volume and Y-maze performance were assessed using logistic regression, residualized for total brain volume. Both analyses corrected for multiple comparisons. Structural covariance networks were generated using the R package "igraph." Group differences in network centrality (degree), integration (mean distance), and segregation (transitivity, modularity) were tested across network densities (5-40%), using 5,000 (1,000 for degree) permutations with significance criteria of p < 0.05 at ≥5 consecutive density thresholds. Results: Widespread significant maturational changes in volume occurred in 18 brain regions, including considerable loss in isocortex regions and increases in brainstem regions and white matter tracts. The aging-related comparison yielded 6 significant changes in brain volume, including further loss in isocortex regions and increases in white matter tracts. No significant volume changes were observed across either comparison for subcortical regions. Additionally, smaller volume of the anterior cingulate area (χ2 = 2.325, pBH = 0.044) and larger volume of the hippocampal formation (χ2 = -2.180, pBH = 0.044) were associated with poorer cognitive performance. Maturational network comparisons yielded significant degree changes in 9 regions, but no aging-related changes, aligning with network stabilization trends in humans. Maturational decline in modularity occurred (24-29% density), mirroring human trends of decreased segregation in young adulthood, while mean distance and transitivity remained stable. Conclusion/Implications: These findings offer a foundational account of age effects on brain volume, structural brain networks, and working memory in mice, informing future work in facilitating translation between rodent models and human brain aging.

Chronic Stress Induces Coordinated Cortical Microcircuit Cell-Type Transcriptomic Changes Consistent With Altered Information Processing.

BACKGROUND: Information processing in cortical cell microcircuits involves regulation of excitatory pyramidal (PYR) cells by inhibitory somatostatin- (SST), parvalbumin-, and vasoactive intestinal peptide-expressing interneurons. Human postmortem and rodent studies show impaired PYR cell dendritic morphology and decreased SST cell markers in major depressive disorder or after chronic stress. However, knowledge of coordinated changes across microcircuit cell types is virtually absent. METHODS: We investigated the transcriptomic effects of unpredictable chronic mild stress (UCMS) on distinct microcircuit cell types in the medial prefrontal cortex (cingulate regions 24a, 24b, and 32) in mice. C57BL/6 mice, exposed to UCMS or control housing for 5 weeks, were assessed for anxiety- and depressive-like behaviors. Microcircuit cell types were laser microdissected and processed for RNA sequencing. RESULTS: UCMS induced predicted elevations in behavioral emotionality in mice. DESeq2 analysis revealed unique differentially expressed genes in each cell type after UCMS. Presynaptic functions, oxidative stress response, metabolism, and translational regulation were differentially dysregulated across cell types, whereas nearly all cell types showed downregulated postsynaptic gene signatures. Across the cortical microcircuit, we observed a shift from a distributed transcriptomic coordination across cell types in control mice toward UCMS-induced increased coordination between PYR, SST, and parvalbumin cells and a hub-like role for PYR cells. Finally, we identified a microcircuit-wide coexpression network enriched in synaptic, bioenergetic, and oxidative stress response genes that correlated with UCMS-induced behaviors. CONCLUSIONS: These findings suggest cell-specific deficits, microcircuit-wide synaptic reorganization, and a shift in cells regulating the cortical excitation-inhibition balance, suggesting increased coordinated regulation of PYR cells by SST and parvalbumin cells.

Reduced anterior cingulate cortex volume induced by chronic stress correlates with increased behavioral emotionality and decreased synaptic puncta density.

Clinical and preclinical studies report that chronic stress induces behavioral deficits as well as volumetric and synaptic alterations in corticolimbic brain regions including the anterior cingulate cortex (ACC), amygdala (AMY), nucleus accumbens (NAc) and hippocampus (HPC). Here, we aimed to investigate the volumetric changes associated with chronic restraint stress (CRS) and link these changes to the CRS-induced behavioral and synaptic deficits. We first confirmed that CRS increases behavioral emotionality, defined as collective scoring of anxiety- and anhedonia-like behaviors. We then demonstrated that CRS induced a reduction of total brain volume which negatively correlated with behavioral emotionality. Region-specific analysis identified that only the ACC showed significant decrease in volume following CRS (p < 0.05). Reduced ACC correlated with increased behavioral emotionality (r = -0.56; p = 0.0003). Although not significantly altered by CRS, AMY and NAc (but not the HPC) volumes were negatively correlated with behavioral emotionality. Finally, using structural covariance network analysis to assess shared volumetric variances between the corticolimbic brain regions and associated structures, we found a progressive decreased ACC degree and increased AMY degree following CRS. At the cellular level, reduced ACC volume correlated with decreased PSD95 (but not VGLUT1) puncta density (r = 0.35, p < 0.05), which also correlated with increased behavioral emotionality (r = -0.44, p < 0.01), suggesting that altered synaptic strength is an underlying substrate of CRS volumetric and behavioral effects. Our results demonstrate that CRS effects on ACC volume and synaptic density are linked to behavioral emotionality and highlight key ACC structural and morphological alterations relevant to stress-related illnesses including mood and anxiety disorders.

Chronic Stress-induced Behaviors Correlate with Exacerbated Acute Stress-induced Cingulate Cortex and Ventral Hippocampus Activation.

Altered activity of corticolimbic brain regions is a hallmark of stress-related illnesses, including mood disorders, neurodegenerative diseases, and substance abuse disorders. Acute stress adaptively recruits brain region-specific functions for coping, while sustained activation under chronic stress may overwhelm feedback mechanisms and lead to pathological cellular and behavioral responses. The neural mechanisms underlying dysregulated stress responses and how they contribute to behavioral deficits are poorly characterized. Here, we tested whether prior exposure to chronic restraint stress (CRS) or unpredictable chronic mild stress (UCMS) in mice could alter functional response to acute stress and whether these changes are associated with chronic stress-induced behavioral deficits. More specifically, we assessed acute stress-induced functional activation indexed by c-Fos+ cell counts in 24 stress- and mood-related brain regions, and determined if changes in functional activation were linked to chronic stress-induced behavioral impairments, summarized across dimensions through principal component analysis (PCA). Results indicated that CRS and UCMS led to convergent physiological and anxiety-like deficits, whereas working and short-term memory were impaired only in UCMS mice. CRS and UCMS exposure exacerbated functional activation by acute stress in anterior cingulate cortex (ACC) area 24b and ventral hippocampal (vHPC) CA1, CA3, and subiculum. In dysregulated brain regions, levels of functional activation were positively correlated with principal components reflecting variance across behavioral deficits relevant to stress-related disorders. Our data supports an association between a dysregulated stress response, altered functional corticolimbic excitation/inhibition balance, and the expression of maladaptive behaviors.

Residual avoidance: A new, consistent and repeatable readout of chronic stress-induced conflict anxiety reversible by antidepressant treatment.

Stress-related illnesses such as major depressive and anxiety disorders are characterized by maladaptive responses to stressful life events. Chronic stress-based animal models have provided critical insight into the understanding of these responses. Currently available assays measuring chronic stress-induced behavioral states in mice are limited in their design (short, not repeatable, sensitive to experimenter-bias) and often inconsistent. Using the Noldus PhenoTyper apparatus, we identified a new readout that repeatedly assesses behavioral changes induced by chronic stress in two mouse models i.e. chronic restraint stress (CRS) and chronic unpredictable mild stress (UCMS). The PhenoTyper test consists of overnight monitoring of animals' behavior in home-cage setting before, during and after a 1hr light challenge applied over a designated food zone. We tested the reproducibility and reliability of the PhenoTyper test in assessing the effects of chronic stress exposure, and compared outcomes with commonly-used tests. While chronic stress induced heterogeneous profiles in classical tests, CRS- and UCMS-exposed mice showed a very consistent response in the PhenoTyper test. Indeed, CRS and UCMS mice continue avoiding the lit zone in favor of the shelter zone. This "residual avoidance" after the light challenge, lasted for hours beyond termination of the challenge, was not observed after acute stress and was consistently found throughout stress exposure in both models. Chronic stress-induced residual avoidance was alleviated by chronic imipramine treatment but not acute diazepam administration. This behavioral index should be instrumental for studies aiming to better understand the trajectory of chronic stress-induced deficits and potentially screen novel anxiolytics and antidepressants.

Novel Benzodiazepine-Like Ligands with Various Anxiolytic, Antidepressant, or Pro-Cognitive Profiles.

Altered gamma-aminobutyric acid (GABA) function is consistently reported in psychiatric disorders, normal aging, and neurodegenerative disorders and reduced function of GABA interneurons is associated with both mood and cognitive symptoms. Benzodiazepines (BZ) have broad anxiolytic, but also sedative, anticonvulsant and amnesic effects, due to nonspecific GABA-A receptor (GABAA-R) targeting. Varying the profile of activity of BZs at GABAA-Rs is predicted to uncover additional therapeutic potential. We synthesized four novel imidazobenzodiazepine (IBZD) amide ligands and tested them for positive allosteric modulation at multiple α-GABAA-R (α-positive allosteric modulators), pharmacokinetic properties, as well as anxiolytic and antidepressant activities in adult mice. Efficacy at reversing stress-induced or age-related working memory deficits was assessed using a spontaneous alternation task. Diazepam (DZP) was used as a control. Three ligands (GL-II-73, GL-II-74, and GL-II-75) demonstrated adequate brain penetration and showed predictive anxiolytic and antidepressant efficacies. GL-II-73 and GL-II-75 significantly reversed stress-induced and age-related working memory deficits. In contrast, DZP displayed anxiolytic but no antidepressant effects or effects on working memory. We demonstrate distinct profiles of anxiolytic, antidepressant, and/or pro-cognitive activities of newly designed IBZD amide ligands, suggesting novel therapeutic potential for IBZD derivatives in depression and aging.

Shifting priorities: highly conserved behavioral and brain network adaptations to chronic stress across species.

Parallel clinical and preclinical research have begun to illuminate the biological basis of stress-related disorders, including major depression, but translational bridges informing discrete mechanistic targets for intervention are missing. To address this critical need, we used structural MRI in a mouse model and in a large human sample to examine stress effects on brain structure that may be conserved across species. Specifically, we focused on a previously unexplored approach, whole-brain structural covariance, as it reflects synchronized changes in neuroanatomy, potentially due to mutual trophic influences or shared plasticity across regions. Using the unpredictable chronic mild stress (UCMS) paradigm in mouse we first demonstrate that UCMS-induced elevated behavioral emotionality correlates with increased size of the amygdala and other corticolimbic regions. We further identify focal increases in the amygdala's 'hubness' (degree and strength) set against the background of a global stress-related loss of network clustering and modularity. These macroscopic changes are supported on the molecular level by increased postsynaptic density-95 protein in the amygdala, consistent with stress-induced plastic changes and synaptic strengthening. Finally, we provide clinical evidence that strikingly similar structural network reorganization patterns exist in young adults reporting high childhood trauma and increased mood symptoms. Collectively, we provide initial translational evidence for a conserved stress-related increase in amygdala-centered structural synchrony, as measured by enhanced structural covariance, which is paralleled by a decrease in global structural synchrony. This putative trade-off reflected in increased amygdala-centered plastic changes at the expense of global structural dedifferentiation may represent a mechanistic pathway for depression and related psychopathology.

Increased Neuronal DNA/RNA Oxidation in the Frontal Cortex of Mice Subjected to Unpredictable Chronic Mild Stress.

BACKGROUND: Chronic stress is implicated in the development of various psychiatric illnesses including major depressive disorder. Previous reports suggest that patients with major depressive disorder have increased levels of oxidative stress, including higher levels of DNA/RNA oxidation found in postmortem studies, especially within brain regions responsible for the cognitive and emotional processes disrupted in the disorder. Here, we aimed to investigate whether unpredictable chronic mild stress in mice induces neuronal DNA/RNA oxidation in the prelimbic, infralimbic, and cingulate cortices of the frontal cortex and the basolateral amygdala and to explore potential associations with depressive-like behaviors. We expected that animals subjected to unpredictable chronic mild stress will present higher levels of DNA/RNA oxidation, which will be associated with anxiety-/depressive-like behaviors. METHODS: C57BL/6J mice were assigned to unpredictable chronic mild stress or nonstress conditions (n = 10/group, 50% females). Following five weeks of unpredictable chronic mild stress exposure, mice were tested in a series of behavioral tests measuring anxiety- and depressive-like behaviors. Frontal cortex and amygdala sections were then immunolabeled for neuronal nuclei, a marker of post-mitotic neurons and anti-8-hydroxy-2-deoxyguanosine/8-oxo-7,8-dihydroguanosine, which reflects both DNA and RNA oxidation. RESULTS: Levels of neuronal DNA/RNA oxidation were increased in the frontal cortex of mice subjected to unpredictable chronic mild stress (p = 0.0207). Levels of neuronal DNA/RNA oxidation in the frontal cortex were positively correlated with z-emotionality scores for latency to feed in the novelty-suppressed feeding test (p = 0.0031). Statistically significant differences were not detected in basolateral amygdala levels of neuronal DNA/RNA oxidation between nonstress- and unpredictable chronic mild stress-exposed mice, nor were correlations found with behavioral performances for this region. CONCLUSION: Our results demonstrate that unpredictable chronic mild stress induces a significant increase in neuronal DNA/RNA oxidation in the frontal cortex that correlate with behavioral readouts of the stress response. A lack of DNA/RNA oxidation alterations in the basolateral amygdala suggests greater vulnerability of frontal cortex neurons to DNA/RNA oxidation in response to unpredictable chronic mild stress. These findings add support to the hypothesis that chronic stress-induced damage to DNA/RNA may be an additional molecular mechanism underlying cellular dysfunctions associated with chronic stress and present in stress-related disorders.