StressMatic: A Novel Automated System to Induce Depressive- and Anxiety-like Phenotype in Rats.

Major depressive disorder (MDD) is a multidimensional psychiatric disorder that is estimated to affect around 350 million people worldwide. Generating valid and effective animal models of depression is critical and has been challenging for neuroscience researchers. For preclinical studies, models based on stress exposure, such as unpredictable chronic mild stress (uCMS), are amongst the most reliable and used, despite presenting concerns related to the standardization of protocols and time consumption for operators. To overcome these issues, we developed an automated system to expose rodents to a standard uCMS protocol. Here, we compared manual (uCMS) and automated (auCMS) stress-exposure protocols. The data shows that the impact of the uCMS exposure by both methods was similar in terms of behavioral (cognition, mood, and anxiety) and physiological (cell proliferation and endocrine variations) measurements. Given the advantages of time and standardization, this automated method represents a step forward in this field of preclinical research.

Beyond New Neurons in the Adult Hippocampus: Imipramine Acts as a Pro-Astrogliogenic Factor and Rescues Cognitive Impairments Induced by Stress Exposure.

Depression is a prevalent, socially burdensome disease. Different studies have demonstrated the important role of astrocytes in the pathophysiology of depression as modulators of neurotransmission and neurovascular coupling. This is evidenced by astrocyte impairments observed in brains of depressed patients and the appearance of depressive-like behaviors upon astrocytic dysfunctions in animal models. However, little is known about the importance of de novo generated astrocytes in the mammalian brain and in particular its possible involvement in the precipitation of depression and in the therapeutic actions of current antidepressants (ADs). Therefore, we studied the modulation of astrocytes and adult astrogliogenesis in the hippocampal dentate gyrus (DG) of rats exposed to an unpredictable chronic mild stress (uCMS) protocol, untreated and treated for two weeks with antidepressants-fluoxetine and imipramine. Our results show that adult astrogliogenesis in the DG is modulated by stress and imipramine. This study reveals that distinct classes of ADs impact differently in the astrogliogenic process, showing different cellular mechanisms relevant to the recovery from behavioral deficits induced by chronic stress exposure. As such, in addition to those resident, the newborn astrocytes in the hippocampal DG might also be promising therapeutic targets for future therapies in the neuropsychiatric field.

Suppression of adult cytogenesis in the rat brain leads to sex-differentiated disruption of the HPA axis activity.

OBJECTIVES: The action of stress hormones, mainly glucocorticoids, starts and coordinates the systemic response to stressful events. The HPA axis activity is predicated on information processing and modulation by upstream centres, such as the hippocampus where adult-born neurons (hABN) have been reported to be an important component in the processing and integration of new information. Still, it remains unclear whether and how hABN regulates HPA axis activity and CORT production, particularly when considering sex differences. MATERIALS AND METHODS: Using both sexes of a transgenic rat model of cytogenesis ablation (GFAP-Tk rat model), we examined the endocrinological and behavioural effects of disrupting the generation of new astrocytes and neurons within the hippocampal dentate gyrus (DG). RESULTS: Our results show that GFAP-Tk male rats present a heightened acute stress response. In contrast, GFAP-Tk female rats have increased corticosterone secretion at nadir, a heightened, yet delayed, response to an acute stress stimulus, accompanied by neuronal hypertrophy in the basal lateral amygdala and increased expression of the glucocorticoid receptors in the ventral DG. CONCLUSIONS: Our results reveal that hABN regulation of the HPA axis response is sex-differentiated.

Impact of COVID-19 Pandemic on In-Hospital Mortality in Patients Without SARS-CoV-2 Infection in an Internal Medicine Ward of a Tertiary Care Hospital in Portugal.

INTRODUCTION: Despite the emergence of a new worldwide cause of death related to COVID-19, several studies have hypothesized that the international mortality rate attributed to non-COVID-19 causes was significantly higher during the COVID pandemic, questioning whether this excess in mortality is related only to COVID-19 or to the difficulties that the healthcare systems faced during the pandemic. Therefore, understanding the impact of the COVID-19 pandemic on the prognosis of patients without severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a major unmet need as this was overshadowed by the overwhelming number of patients with SARS-CoV-2. METHODS: This is a retrospective, cross-sectional, observational study in the internal medicine non-COVID-19 wards of a tertiary care hospital in Portugal. A total of 2021 patients without SARS-CoV-2 infection admitted between March and May of 2019 and 2020 were included. For each patient, we collected information regarding demographic characteristics, emergency department admission information, hospitalization information, date of discharge or death, health comorbidities, and current medication. RESULTS: Data from 1013 patients in 2019 and 1008 patients in 2020 was analyzed. The patients' demographic characteristics, health comorbidities, and current medications were distributed in similar patterns in the two studied periods. There was a statistically significant difference in the in-hospital mortality in patients without SARS-CoV-2 infection between 2019 and 2020 (12% vs 17%, p-value < 0.001) and in admission severity in hospitalized patients without SARS-CoV-2 infection between 2019 and 2020 (0.9 vs 0.6, p-value < 0.001). CONCLUSION: Our work showed a statistically significant increase in in-hospital mortality during the COVID-19 pandemic in patients without SARS-CoV-2 infection, which was not apparently explained by differences in the characteristics of hospitalized patients. As this is one of the first works describing the silent impact of the COVID-19 pandemic in Portugal, we believe it holds an important value in the provision of bases for building up future health policies in case of new COVID-19 outbreaks or other medical emergencies.

Constitutive deficiency of the neurogenic hippocampal modulator AP2γ promotes anxiety-like behavior and cumulative memory deficits in mice from juvenile to adult periods.

The transcription factor activating protein two gamma (AP2γ) is an important regulator of neurogenesis both during embryonic development as well as in the postnatal brain, but its role for neurophysiology and behavior at distinct postnatal periods is still unclear. In this work, we explored the neurogenic, behavioral, and functional impact of a constitutive and heterozygous AP2γ deletion in mice from early postnatal development until adulthood. AP2γ deficiency promotes downregulation of hippocampal glutamatergic neurogenesis, altering the ontogeny of emotional and memory behaviors associated with hippocampus formation. The impairments induced by AP2γ constitutive deletion since early development leads to an anxious-like phenotype and memory impairments as early as the juvenile phase. These behavioral impairments either persist from the juvenile phase to adulthood or emerge in adult mice with deficits in behavioral flexibility and object location recognition. Collectively, we observed a progressive and cumulative impact of constitutive AP2γ deficiency on the hippocampal glutamatergic neurogenic process, as well as alterations on limbic-cortical connectivity, together with functional behavioral impairments. The results herein presented demonstrate the modulatory role exerted by the AP2γ transcription factor and the relevance of hippocampal neurogenesis in the development of emotional states and memory processes.

Cell Cycle Regulation of Hippocampal Progenitor Cells in Experimental Models of Depression and after Treatment with Fluoxetine.

Changes in adult hippocampal cell proliferation and genesis have been largely implicated in depression and antidepressant action, though surprisingly, the underlying cell cycle mechanisms are largely undisclosed. Using both an in vivo unpredictable chronic mild stress (uCMS) rat model of depression and in vitro rat hippocampal-derived neurosphere culture approaches, we aimed to unravel the cell cycle mechanisms regulating hippocampal cell proliferation and genesis in depression and after antidepressant treatment. We show that the hippocampal dentate gyrus (hDG) of uCMS animals have less proliferating cells and a decreased proportion of cells in the G2/M phase, suggesting a G1 phase arrest; this is accompanied by decreased levels of cyclin D1, E, and A expression. Chronic fluoxetine treatment reversed the G1 phase arrest and promoted an up-regulation of cyclin E. In vitro, dexamethasone (DEX) decreased cell proliferation, whereas the administration of serotonin (5-HT) reversed it. DEX also induced a G1-phase arrest and decreased cyclin D1 and D2 expression levels while increasing p27. Additionally, 5-HT treatment could partly reverse the G1-phase arrest and restored cyclin D1 expression. We suggest that the anti-proliferative actions of chronic stress in the hDG result from a glucocorticoid-mediated G1-phase arrest in the progenitor cells that is partly mediated by decreased cyclin D1 expression which may be overcome by antidepressant treatment.

Hippocampal cytogenesis abrogation impairs inter-regional communication between the hippocampus and prefrontal cortex and promotes the time-dependent manifestation of emotional and cognitive deficits.

Impaired ability to generate new cells in the adult brain has been linked to deficits in multiple emotional and cognitive behavioral domains. However, the mechanisms by which abrogation of adult neural stem cells (NSCs) impacts on brain function remains controversial. We used a transgenic rat line, the GFAP-Tk, to selectively eliminate NSCs and assess repercussions on different behavioral domains. To assess the functional importance of newborn cells in specific developmental stages, two parallel experimental timeframes were adopted: a short- and a long-term timeline, 1 and 4 weeks after the abrogation protocol, respectively. We conducted in vivo electrophysiology to assess the effects of cytogenesis abrogation on the functional properties of the hippocampus and prefrontal cortex, and on their intercommunication. Adult brain cytogenesis abrogation promoted a time-specific installation of behavioral deficits. While the lack of newborn immature hippocampal neuronal and glial cells elicited a behavioral phenotype restricted to hyperanxiety and cognitive rigidity, specific abrogation of mature new neuronal and glial cells promoted the long-term manifestation of a more complex behavioral profile encompassing alterations in anxiety and hedonic behaviors, along with deficits in multiple cognitive modalities. More so, abrogation of 4 to 7-week-old cells resulted in impaired electrophysiological synchrony of neural theta oscillations between the dorsal hippocampus and the medial prefrontal cortex, which are likely to contribute to the described long-term cognitive alterations. Hence, this work provides insight on how newborn neurons and astrocytes display different functional roles throughout different maturation stages, and establishes common ground to reconcile contrasting results that have marked this field.

Astrocytic plasticity at the dorsal dentate gyrus on an animal model of recurrent depression.

Astrocytes are now known to play crucial roles in the central nervous system, supporting and closely interacting with neurons and therefore able to modulate brain function. Both human postmortem studies in brain samples from patients diagnosed with Major Depressive Disorder and from animal models of depression reported numerical and morphological astrocytic changes specifically in the hippocampus. In particular, these studies revealed significant reductions in glial cell density denoted by a decreased number of S100B-positive cells and a decrease in GFAP expression in several brain regions including the hippocampus. To reveal plastic astrocytic changes in the context of recurrent depression, we longitudinally assessed dynamic astrocytic alterations (gene expression, cell densities and morphologic variations) in the hippocampal dentate gyrus under repeated exposure to unpredictable chronic mild stress (uCMS) and upon treatment with two antidepressants, fluoxetine and imipramine. Both antidepressants decreased astrocytic complexity immediately after stress exposure. Moreover, we show that astrocytic alterations, particularly an increased number of S100B-positive cells, are observed after recurrent stress exposure. Interestingly, these alterations were prevented at the long-term by either fluoxetine or imipramine treatment.

miR-409 and miR-411 Modulation in the Adult Brain of a Rat Model of Depression and After Fluoxetine Treatment.

Depression is a chronic debilitating disorder predicted to affect around 20% of the world population. Both brain and peripheral changes, including neuroplastic changes have been shown to occur in the brains of depressed individuals and animal models of depression. Over the past few decades, growing evidence has supported the role of miRNAs as regulators of critical aspects of brain plasticity and function, namely in the context of depression. These molecules are not only highly expressed in the brain, but are also relatively stable in bodily fluids, including blood. Previous microarray analysis from our group has disclosed molecular players in the hippocampal dentate gyrus (DG), in the context of depression and antidepressant treatment. Two miRNAs in particular-miR-409-5p and miR-411-5p-were significantly up-regulated in the DG of an unpredictable chronic mild stress (CMS) rat model of depression and reversed by antidepressant treatment. Here, we further analyzed the levels of these miRNAs along the DG longitudinal axis and in other brain regions involved in the pathophysiology of depression, as well as in peripheral blood of CMS-exposed rats and after fluoxetine treatment. The effects of CMS and fluoxetine treatment on miR-409-5p and miR-411-5p levels varied across brain regions, and miR-411-5p was significantly decreased in the blood of fluoxetine-treated rats. Additional bioinformatic analyses revealed target genes and pathways of these miRNAs related to neurotransmitter signaling and neuroplasticity functions; an implication of the two miRNAs in the regulation of the cellular and molecular changes observed in these brain regions in depression is worth further examination.

Region-specific control of microglia by adenosine A2A receptors: uncoupling anxiety and associated cognitive deficits in female rats.

Epidemiologic studies have provided compelling evidence that prenatal stress, through excessive maternal glucocorticoids exposure, is associated with psychiatric disorders later in life. We have recently reported that anxiety associated with prenatal exposure to dexamethasone (DEX, a synthetic glucocorticoid) correlates with a gender-specific remodeling of microglia in the medial prefrontal cortex (mPFC), a core brain region in anxiety-related disorders. Gender differences in microglia morphology, the higher prevalence of anxiety in women and the negative impact of anxiety in cognition, led us to specifically evaluate cognitive behavior and associated circuits (namely mPFC-dorsal hippocampus, dHIP), as well as microglia morphology in female rats prenatally exposed to dexamethasone (in utero DEX, iuDEX). We report that iuDEX impaired recognition memory and deteriorated neuronal synchronization between mPFC and dHIP. These functional deficits are paralleled by microglia hyper-ramification in the dHIP and decreased ramification in the mPFC, showing a heterogeneous remodeling of microglia morphology, both postnatally and at adulthood in different brain regions, that differently affect mood and cognition. The chronic blockade of adenosine A2A receptors (A2A R), which are core regulators of microglia morphology and physiology, ameliorated the cognitive deficits, but not the anxiety-like behavior. Notably, A2A R blockade rectified both microglia morphology in the dHIP and the lack of mPFC-dHIP synchronization, further heralding their role in cognitive function.

AP2γ: A New Player on Adult Hippocampal Neurogenesis Regulation.

Since the recognition that the mammalian brain retains the ability to generate newborn neurons with functional relevance throughout life, the matrix of molecular regulators that govern adult neurogenesis has been the focus of much interest. In a recent study published in Molecular Psychiatry, we demonstrate Activating Protein 2γ (AP2γ), a transcription factor previously implicated in cell fate determination in the developing cortex, as a novel player in the regulation of glutamatergic neurogenesis in the adult hippocampus. Using distinct experimental approaches, we showed that AP2γ is specifically present in a subpopulation of transient amplifying progenitors, where it acts as a crucial promoter of proliferation and differentiation of adult-born glutamatergic granule neurons. Strikingly, deficiency of AP2γ in the adult brain compromises the generation of new glutamatergic neurons, with impact on the function of cortico-limbic circuits. Here, we share our view on how AP2γ integrates the transcriptional orchestration of glutamatergic neurogenesis in the adult hippocampus, and consequently, how it emerges as a novel molecular candidate to study the translation of environmental pressures into alterations of brain neuroplasticity in homeostatic, but also in neuropathological contexts.

Chronic stress targets adult neurogenesis preferentially in the suprapyramidal blade of the rat dorsal dentate gyrus.

The continuous generation of new neurons and glial cells in the adult hippocampal dentate gyrus (DG) represents an important form of adult neuroplasticity, involved in normal brain function and behavior but also associated with the etiopathogenesis and treatment of psychiatric disorders. Despite the large number of studies addressing cell genesis along the septotemporal axis, data on the anatomical gradients of cytogenesis along the DG transverse axis is scarce, especially after exposure to stress. As such, in this study we characterized both basal proliferation and survival of adult-born neural cells along the transverse axis of the rat dorsal DG, and after stress exposure. In basal conditions, both proliferating cells and newborn neurons and glial cells were preferentially located at the subgranular zone and suprapyramidal blade. Exposure to chronic stress induced an overall decrease in the generation of adult-born neural cells and, more specifically, produced a regional-specific decrease in the survival of adult-born neurons at the suprapyramidal blade. No particular region-specific alterations were observed on surviving adult-born glial cells. This work reveals, for the first time, a distinct survival profile of adult-born neural cells, neurons and glial cells, among the transverse axis of the DG, in both basal and stress conditions. Our results unveil that adult-born neurons are preferentially located in the suprapyramidal blade and suggest a regional-specific impact of chronic stress in this blade with potential repercussions for its functional significance.

Mesenchymal stem cells secretome as a modulator of the neurogenic niche: basic insights and therapeutic opportunities.

Neural stem cells (NSCs) and mesenchymal stem cells (MSCs) share few characteristics apart from self-renewal and multipotency. In fact, the neurogenic and osteogenic stem cell niches derive from two distinct embryonary structures; while the later originates from the mesoderm, as all the connective tissues do, the first derives from the ectoderm. Therefore, it is highly unlikely that stem cells isolated from one niche could form terminally differentiated cells from the other. Additionally, these two niches are associated to tissues/systems (e.g., bone and central nervous system) that have markedly different needs and display diverse functions within the human body. Nevertheless they do share common features. For instance, the differentiation of both NSCs and MSCs is intimately associated with the bone morphogenetic protein family. Moreover, both NSCs and MSCs secrete a panel of common growth factors, such as nerve growth factor (NGF), glial derived neurotrophic factor (GDNF), and brain derived neurotrophic factor (BDNF), among others. But it is not the features they share but the interaction between them that seem most important, and worth exploring; namely, it has already been shown that there are mutually beneficially effects when these cell types are co-cultured in vitro. In fact the use of MSCs, and their secretome, become a strong candidate to be used as a therapeutic tool for CNS applications, namely by triggering the endogenous proliferation and differentiation of neural progenitors, among other mechanisms. Quite interestingly it was recently revealed that MSCs could be found in the human brain, in the vicinity of capillaries. In the present review we highlight how MSCs and NSCs in the neurogenic niches interact. Furthermore, we propose directions on this field and explore the future therapeutic possibilities that may arise from the combination/interaction of MSCs and NSCs.

Differential and converging molecular mechanisms of antidepressants' action in the hippocampal dentate gyrus.

Major depression is a highly prevalent, multidimensional disorder. Although several classes of antidepressants (ADs) are currently available, treatment efficacy is limited, and relapse rates are high; thus, there is a need to find better therapeutic strategies. Neuroplastic changes in brain regions such as the hippocampal dentate gyrus (DG) accompany depression and its amelioration with ADs. In this study, the unpredictable chronic mild stress (uCMS) rat model of depression was used to determine the molecular mediators of chronic stress and the targets of four ADs with different pharmacological profiles (fluoxetine, imipramine, tianeptine, and agomelatine) in the hippocampal DG. All ADs, except agomelatine, reversed the depression-like behavior and neuroplastic changes produced by uCMS. Chronic stress induced significant molecular changes that were generally reversed by fluoxetine, imipramine, and tianeptine. Fluoxetine primarily acted on neurons to reduce the expression of pro-inflammatory response genes and increased a set of genes involved in cell metabolism. Similarities were found between the molecular actions and targets of imipramine and tianeptine that activated pathways related to cellular protection. Agomelatine presented a unique profile, with pronounced effects on genes related to Rho-GTPase-related pathways in oligodendrocytes and neurons. These differential molecular signatures of ADs studied contribute to our understanding of the processes implicated in the onset and treatment of depression-like symptoms.

The effects of chronic stress on hippocampal adult neurogenesis and dendritic plasticity are reversed by selective MAO-A inhibition.

There is accumulating evidence that adult neurogenesis and dendritic plasticity in the hippocampus are neuroplastic phenomena, highly sensitive to the effects of chronic stress and treatment with most classes of antidepressant drugs, being involved in the onset and recovery from depression. However, the effects of antidepressants that act through the selective inhibition of monoamine oxidase subtype A (MAO-A) in these phenomena are still largely unknown. In the present study, adult neurogenesis and neuronal morphology were examined in the hippocampus of rats exposed to chronic mild stress (CMS) and treated with the selective reversible MAO-A inhibitor (RIMA) drug, pirlindole and the selective serotonin reuptake inhibitor (SSRI), fluoxetine. The results provide the first demonstration that selective MAO-A inhibition with pirlindole is able to revert the behavioural effects of stress exposure while promoting hippocampal adult neurogenesis and rescuing the stress-induced dendritic atrophy of granule neurons.

The Sweet Drive Test: refining phenotypic characterization of anhedonic behavior in rodents.

Measuring anhedonic behavior in rodents is a challenging task as current methods display only moderate sensitivity to detect anhedonic phenotype and, consequently, results from different labs are frequently incongruent. Herein we present a newly-developed test, the Sweet Drive Test (SDT), which integrates food preference measurement in a non-aversive environment, with ultrasonic vocalizations (USVs) recording. Animals were placed in a soundproofed black arena, under red light illumination, and allowed to choose between regular and sweet food pellets. During the test trials, 50 KHz USVs, previously described to be associated with positive experiences, were recorded. In a first experimental approach, we demonstrate the ability of SDT to accurately characterize anhedonic behavior in animals chronically exposed to stress. In a subsequent set of experiments, we show that this paradigm has high sensitivity to detect mood-improving effects of antidepressants. The combined analysis of both food preference and the number of 50 KHz vocalizations in the SDT provides also a valuable tool to discriminate animals that responded to treatment from non-responder animals.

Re-cycling paradigms: cell cycle regulation in adult hippocampal neurogenesis and implications for depression.

Since adult neurogenesis became a widely accepted phenomenon, much effort has been put in trying to understand the mechanisms involved in its regulation. In addition, the pathophysiology of several neuropsychiatric disorders, such as depression, has been associated with imbalances in adult hippocampal neurogenesis. These imbalances may ultimately reflect alterations at the cell cycle level, as a common mechanism through which intrinsic and extrinsic stimuli interact with the neurogenic niche properties. Thus, the comprehension of these regulatory mechanisms has become of major importance to disclose novel therapeutic targets. In this review, we first present a comprehensive view on the cell cycle components and mechanisms that were identified in the context of the homeostatic adult hippocampal neurogenic niche. Then, we focus on recent work regarding the cell cycle changes and signaling pathways that are responsible for the neurogenesis imbalances observed in neuropathological conditions, with a particular emphasis on depression.

Immuno-Golgi as a tool for analyzing neuronal 3D-dendritic structure in phenotypically characterized neurons.

Characterization of neuronal dendritic structure in combination with the determination of specific neuronal phenotype or temporal generation is a challenging task. Here we present a novel method that combines bromodioxyuridine (BrdU) immunohistochemistry with Golgi-impregnation technique; with this simple non-invasive method, we are able to determine the tridimensional structure of dendritic arborization and spine shape of neurons born at a specific time in the hippocampus of adult animals. This analysis is relevant in physiological and pathological conditions in which altered neurogenesis is implicated, such as aging or emotional disorders.

Epigenetic (de)regulation of adult hippocampal neurogenesis: implications for depression.

Adult neurogenesis represents a dynamic level of modulation upon the neuroplastic properties of the mature nervous system, that is essential to the homeostatic brain function. The adult neurogenic process comprises several sequential steps, all of which subjected to an assortment of cell-intrinsic and neurogenic-niche complex regulatory mechanisms. Among these, epigenetic regulation is now emerging as a crucial regulator of several neurogenesis steps. In particular, the active regulation of hippocampal neurogenesis and its repercussions in global hippocampal function are of special interest for the biomedical field, since imbalances at this level have been strongly related to the precipitation of several neuropsychyatric disorders, such as depression. Indeed, growing evidence supports that the detrimental effects on adult hippocampal neurogenesis, that have been associated with depression, might be epigenetically-mediated. Therefore, understanding the epigenetic regulation of the neurogenic process may provide a link between neurogenesis imbalances and the deterioration of the behavioural and cognitive domains frequently affected in depression, thus contributing to unravel the complex pathophysiology of this disorder.Here, we outline some of the major epigenetic mechanisms contributing to the regulation of hippocampal neurogenesis and discuss several lines of evidence supporting their involvement on the development of imbalances in the neurogenic process, often correlated to behavioural and cognitive deficits commonly observed in major depressive disorder.