Microglial and neurogenic alterations in hypothalamus due to acute stress

This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to Pedraza C.), and Ministerio de Educación, Cultura y Deporte from Spain (FPU16/05308 to Nieto-Quero A).Microglial cells are an important glial population known to be involved in several biological processes such as stress response. These cells engage an activated state following a stress insult that may lead to nervous tissue damage, including new cell generation impairment. This has been widely studied in regions with notable neurogenesis such as de hippocampus, however, the effect in other regions with fewer yet relevant neurogenesis remains partially unknown. One of them is the hypothalamus, a key vegetative control center playing an important role in stress response. Moreover, most of the stress models studied concern neuroinflammatory and neurogenic changes due to a chronic stressor but not a single stress event. Given the repercussion of these processes alone, it would be interesting to elucidate the relationship between microglial response, hypothalamic neurogenesis, and acute stress. This project focuses on studying acute stressed C57BL/6J mice, both at the histological and molecular level. An intense stressor combining water immersion and movement restriction was performed. Using immunohistochemical and molecular analysis with Luminex, we could analyze microglial distribution and morphology, neurogenesis, and inflammatory environment in the hypothalamic parenchyma (paraventromedial, ventromedial and arcuate nucleus).Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sexual differences in hippocampal microglia of adult mice subjected to maternal separation stress.

Introduction: It is well known that early life adversities could a"ect brain development and increase the vulnerability to stress-related disorders later in adulthood. Nevertheless, the neurobiological mechanisms underlying this susceptibility have been poorly characterized and sex could be an important variable. Recently, microglia, which is involved in many neurodevelopmental processes such as neurogenesis and synaptic plasticity, has been proposed as a mediator of this stress response and early life stress could “prime” microglia to be over- responsive in future challenges. Objective: The analysis of hippocampal microglia morphology and distribution in the dentate gyrus (DG) of mice subjected to early stress. Methods: Female and male C57BL/6J mice were subjected to 3h daily maternal separation (MS) for 21 days. In postnatal day 60, adult mice undertook a single 2h restriction stress (RS). Accordingly, the experimental groups were as follows: CTRL, RS, MS, MS+RS. The DG was analyzed using immunohistochemistry techniques against Iba1 (microglia) following image analysis (ImageJ) to obtain morphological and distribution data of microglial somas and DG surface area. Results: Smaller DG surface area was observed in MS male mice compared with the CTRL group, but not in female. Furthermore, microglial soma area changed in a sex-dependent manner, having female mice from MS group an increased soma area than those of MS male mice. This was also observed to be region-specific, with a larger microglia soma in DG subgranular zone (SGZ) of MS female compared to MS male. Since microglia in this DG zone is involved in neurogenesis, this might suggest a possible change in the formation of new born neurons. Conclusion: These results revealed a di"erent microglial response to stress depending on the animal sex and open the door to a better understanding of neurobiological basis in pathologies like depression. .University of Málaga, the project PID2020-117464RB-I00 from Ministerio de Ciencia e Innovación (MCIN/AEI) Spain, awarded to Pedraza, C. and Pérez-Martín, M. ; the project P20_00460 from Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía awarded to Pedraza, C. and predoctoral fellowship FPU21/01318 awarded to Munoz- Martin, J. funded by MCIN/AEI, Spain. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Mild juvenile stress increases resilience to the development of anxious behaviors and prevents neurogenic reduction after stress exposure in adulthood.

Stress, especially during sensitive periods of development, can induce neuroplastic changes in brain regions such as the hippocampus, which increases vulnerability to the negative effects of a second stressor during adulthood, precipitating the development of depressive symptoms. For this reason, C57BL/6J mice were subjected to two stress protocols, the first in the juvenile period and the second in adulthood. Neurogenic and behavioral changes (saccharin preference test and social behavior test) were analyzed. The results revealed that juvenile stress increased basal saccharin preference in adulthood. However, animals subjected to stress in both juvenile and adulthood showed anhedonic behavior. In addition, stress in adulthood resulted in increased anxious behavior without affecting interest in social relationships. Stress in adulthood reduced neurogenesis. In contrast, juvenile stress prevented the development of anxious behavior and the reduction of hippocampal neurogenesis induced by stress in adulthood. In conclusion, juvenile stress increases the risk of developing anhedonia after exposure to a second stress, but, in contrast to our expectations, mild stress during the juvenile period increases resilience to the development of anxious behaviors and prevents neurogenic reduction after stress exposure in adulthoodUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects on the hippocampal microglia after acute treatment of a psychological stressor associated with depressive-like behaviours

Funding: This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to C. Pedraza), and Ministerio de Universidades from Spain (FPU16/05308 to A. Nieto-Quero and FPU19/03629 to M.I. Infantes-López).Stressful life events may have a negative impact on mental health compromising people's well-being, so knowing the neurobiological changes that occur after psychosocial stressors can have an impact on overall health. However, the neurobiological mechanisms responsible for the negative effects are not known in detail, and the initial changes that take place after the initiation of a stress protocol are much less well understood. Hippocampus constitutes a target structure of the adverse effects of stress. Among the possible mechanisms involved, the response of microglia to stress is receiving increasing interest. For this reason, after 1 and 24 hours of submitted C57BL/6J mice to acute and intense stress procedure denominated WIRS (water immersion restraint stress), the microglial response were analysed using a set of morphofunctional parameters. Then, the levels of the cytokines: IL-6, IFN-gamma and TNF-alpha cytokine were measured. Furthermore, a complementary proteomic analysis based on the principle of mass spectrometry was carried out. Results reveal that acute stress increased the number of microglia and induced microglial morphofunctional changes. Regarding cytokines, acute stress only increased IL-6 levels, which remained elevated at 24 h. Proteomic analysis, over time (in 24 h post-stress), showed an increase in proteins associated with the intracellular calcium metabolism. These findings suggest a neuroinflammatory response after acute stress observed at one hour after the application of the WIRS protocol and maintained at least 24 hours after the end of the stressor.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Social defeat stress induces microglial alterations and impaired cell survival in the hypothalamus according to behavioral phenotype

Stress is the main environmental cause for depression, known to cause brain immune alterations. As major brain immune cells, microglia undergo transcriptional and, consequently, morphological changes that result in tissue damage, including new cell generation impairment. Even so, few brain regions have been thoroughly studied, excluding key regulators as the hypothalamus, in which this process remains partially unknown. Moreover, there is a poor understanding in physiology related to behavioral outcome. Therefore, it would be interesting to study the relationship between microglia and cell proliferation in stressed mice while controlling for behavior. Here, we used the social defeat stress (SDS) paradigm as a depression-inducing protocol in 8-week-old male C57BL/6J mice for 10 consecutive days. Intruder mice behavior was analyzed to assess depression using behavioral tests and K-means clustering. By immunohistochemical and imaging procedures, microglial morphology, and distribution, as well as cell survival, were analyzed in the hypothalamic paraventricular, ventromedial and arcuate nucleus. Finally, statistical mediation analysis was conducted to evaluate the relationship among variables. Results show mice response to SDS was different, being half the mice resilient and half sensitive to depressive-like symptoms. Microglial morphological activation was enhanced in the ventromedial and arcuate nucleus, especially in stress sensitive animals. Similar results were observed in cell survival, which was particularly affected in sensitive mice. Strikingly, these cell survival changes were statistically mediated by microglial activation. As a conclusion, hypothalamic regions were found to respond differently to stress, accordingly to behavioral outcome, in terms of microglial activation and subsequent decrease in cell survival.This study was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación from Spain (PSI2017-83408-P to Pedraza C.), FEDER/Junta de Andalucía from Spain (UMA20-FEDERJA-112 to Pedraza C. and Pérez- Martín M), and Ministerio de Educación, Cultura y Deporte from Spain (FPU19/03629 to Infantes-López MI and FPU16/05308 to Nieto-Quero A). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Effects of chronic stress on hippocampal microglia and neurogenesis of mice under social defeat stress.

Introduction: Chronic stress is the main environmental factor in the aetiology of depression and it is known that this type of stress may cause alterations in brain regions such as the hippocampus. Nevertheless, changes in a cellular basis are still a subject of study. Objective: The analysis of microglial cells and immature neurons in the dentate gyrus (DG) of stressed mice. Methods: C57BL/6J mice were subjected to Social Defeat Stress model (SDS), consisting of 6 days of social isolation prior to 10 days of stressor. The DG was analysed using immunohistochemistry techniques against Iba1 (microglia) and DCX (immature neurons) following image analysis (ImageJ) to obtain morphological and distribution data of microglial somas. Furthermore, hippocampal neurogenesis was assessed through stereological quantification of DCX+ cells (Visiopharm). Results: An increase in soma size under chronic stress conditions was shown, as well as a less circular and more ameboid soma. These changes were observed mainly in the infrapyramidal blade of the DG. According to microglial cells distribution parameters, the granular cell layer (GCL) was the region which presented the highest microglial density under SDS. Regarding hippocampal neurogenesis, a decrease in the number of DCX+ Type 2-3 cells was observed in the whole DG. Conclusion: All these results o!er a more profound insight of stress changes at a cellular level and could contribute to a better understanding of neurobiological basis in pathologies such as depression. Projects: PSI2017-83408-P (MINECO) and P20 00460 (Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía).University of Málaga and the projects PID2020-117464RB-I00 (Ministerio de Ciencia e Innovación, Spain) to Pedraza C. and Pérez-Martin M., PSI2017-83408-P (FEDER/Ministerio de Ciencia, Innovación y Universidades, Spain) to Pedraza C., UMA20- FEDERJA-112 (FEDER/Junta de Andalucía) to Pedraza C. and Pérez-Martin M. and P20-00460 (Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía) to Pedraza C. Predoctoral Fellowship: FPU16/05308 to Nieto-Quero A. and FPU19/03629 to Infantes-López MI., Ayuda A.2 para Contrato Predoctoral Del I Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, Convocatoria 2021 to Munoz-Martin J. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Do changes in microglial status underlie neurogenesis impairments and depressive-like behaviours induced by psychological stress? A systematic review in animal models

Stress may have a negative effect on mental health and is the primary environmental risk factor in the aetiology of depression. Nevertheless, the neurobiological mechanisms underlying this mood disorder remain poorly characterized. The hippocampus is a target structure of the adverse effects of stress, and hippocampal neuro- genesis plays a crucial role. However, we do not know the mechanisms by which stress impacts neurogenesis. Recent studies indicate that changes in neuroinflammation, primarily via microglial cells, may play an essential role in this process. However, the relationship between stress, microglial changes, and alterations in neurogenesis and their involvement in the development of depression is poorly characterized. For this reason, this systematic review aims to synthesise and evaluate current studies that have investigated the relationship between these variables. Taken together, the revised data, although not entirely conclusive, seem to suggest that microglial changes induced by psychological stress regulate neurogenesis and in turn may be responsible for the devel- opment of depressive-like behaviours, but other factors that influence these stressful experiences should not be dismissed.We would like to thank the University of Malaga/CBUA for the contribution to the payment of the open access charge. This study was supported by FEDER/Ministerio de Ciencia, Innovacion ´ y Universidades – Agencia Estatal de Investigacion ´ (PSI 2017-83408-P) to C.P. and Ministerio de Universidades (FPU16/05308 to AN-Q). PC-P has been funded by the research project PSI 2017-83408- P as technician (CI-19-082)

Acute psychological stress: effects on hippocampal neurogenesis and the role of microglia

Among all the factors that can contribute to the onset of psychopathological disorders, stress is the main environmental factor. The hippocampus is one of the most sensitive regions to the harmful effects of stress, in which the neurogenic process is impaired. On the other hand, under stress situations, microglia are also affected and can trigger a proinflammatory response, acting as anti-neurogenic cells and releasing cytokines and other proinflammatory molecules. Knowing what happens in the early stages of stress may be relevant to investigate the temporal aspects of the development of stressassociated psychopathological disorders, and even their possible treatment. Therefore, after subjecting C57BL/6J mice for 2 hours to an acute and intense stress procedure called WIRS (water immersion restraint stress), data were analyzed to study microglia, cell proliferation and neuronal maturation. In addition, a mediation analysis study was conducted for data integration. The results revealed that the applied acute stress is sufficiently intense to induce an increase in the number of microglia, accompanied by morphometric changes, as well as negatively affecting the neuronal maturational process. Furthermore, these data suggest that effects on the neurogenic process mediate the microglial response to an intense acute stressor. This leads to the conclusion that this may be the initial mechanism for any intense stress response, or may even be the first steps in the development of the response to a chronic stressor.Ministerio de Ciencia e Innovación – Plan Nacional I+D+i from Spain: PID2020- 117464RB-I00 to C. Pedraza and M. Pérez-Martín - FEDER/Ministerio de Ciencia e Innovación – Agencia Estatal de Investigación from Spain: PSI2017-83408-P to C. Pedraza -- FEDER/Junta de Andalucía – Proyectos I+D+I en el marco del Programa Operativo FEDER Andalucía 2014-2020: UMA20-FEDERJA-112 to C. Pedraza and M. Pérez- Martín - Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía: P20_00460 to C. Pedraza - Contract in charge of the project P20_00460 to P. Chaves-Peña - Ministerio de Universidades from Spain: FPU16/05308 to A. Nieto-Quero and FPU19/03629 to M.I. Infantes-López - I Plan Propio de Investigación, Transferencia y Divulgación Científica (convocatoria 2021) from University of Malaga: predoctoral fellowship to J. Munoz-Martin. II Plan Propio de Investigación, Transferencia y Divulgación Científica; Universidad de Málaga. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

New insights into hypothalamic neurogenesis disruption after acute and intense stress: implications for microglia and inflammation

In recent years, the hypothalamus has emerged as a new neurogenic area, capable of generating new neurons after development. Neurogenesis-dependent neuroplasticity seems to be critical to continuously adapt to internal and environmental changes. Stress is a potent environmental factor that can produce potent and enduring effects on brain structure and function. Acute and chronic stress is known to cause alterations in neurogenesis and microglia in classical adult neurogenic regions such as the hippocampus. The hypothalamus is one of the major brain regions implicated in homeostatic stress and emotional stress systems, but little is known about the effect of stress on the hypothalamus. Here, we studied the impact of acute and intense stress (water immersion and restrain stress, WIRS), which may be considered as an inducer of an animal model of posttraumatic stress disorder, on neurogenesis and neuroinflammation in the hypothalamus of adult male mice, focusing on three nuclei: PVN, VMN and ARC, and also in the periventricular area. Our data revealed that a unique stressor was sufficient to provoke a significant impact on hypothalamic neurogenesis by inducing a reduction in the proliferation and number of immature neurons identified as DCX+ cells. These differences were accompanied by marked microglial activation in the VMN and ARC, together with a concomitant increase in IL-6 levels, indicating that WIRS induced an inflammatory response. To investigate the possible molecular mechanisms responsible for neuroplastic and inflammatory changes, we tried to identify proteomic changes. The data revealed that WIRS induced changes in the hypothalamic proteome, modifying the abundance of three and four proteins after 1 h or 24 h of stress application, respectively. These changes were also accompanied by slight changes in the weight and food intake of the animals. These results are the first to show that even a short-term environmental stimulus such as acute and intense stress can have neuroplastic, inflammatory, functional and metabolic consequences on the adult hypothalamus