Primed microglia after acute neuroinflammation may drive an enhanced stress response.

Microglial cells become activated during acute neuroinflammation and usually they return to their basal surveillant state in a few days. However, sometimes microglia evolve towards a primed state characterized by an exacerbated response to new stimuli, which may jeopardize brain functions. Here we aimed to explore microglial priming in the hypothalamus and its consequences on the neuroendocrine regulation of the stress response. To induce priming we used a model of acute ventricular neuroinflammation by intracerebroventricular (ICV) injection of the enzyme neuraminidase (NA). Three months later, an acute stressor (consisting in forced swimming) was applied to investigate the activation of the hypothalamic-pituitary-adrenal axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. Stressed rats previously injected with NA had increased plasma levels of corticosterone compared to control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies revealed that NA-treated rats presented an increased expression of the microglial marker IBA1 and of the inflammasome protein NLRP3. Concomitantly, the morphological analysis of hypothalamic microglial cells showed a morphological bias towards a slightly activated state in microglia of NA injected rats compared to those of saline injected controls. Furthermore, in the open field test NA-treated rats displayed increased locomotor activity. These results suggest that prior neuroinflammatory episodes might result in subtle but persistent changes in microglial cells that could determine the response to future challenges such as stressful events.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Neuraminidase-induced neuroinflammation causes anxiety and microgliosis in the amygdala

An intracerebroventricular (ICV) injection of neuraminidase (NA) within the lateral ventricles originates an acute event of neuroinflammation, which is solved to a great extent after two weeks. Recently, neurological problems or behavioral alterations have been associated with neuroinflammation. Although the majority of them fade along with inflammation resolution, the possibility of long-term sequelae should be taken into consideration. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Initially, rats were ICV injected with NA or saline. Two or 10 weeks later they were made to perform a series of neurological tests and behavioral evaluations (open field test). The neuroinflammation status of the brain was studied by immunohistochemistry and qPCR. While no neurological alterations were found, the open field test revealed an increased anxiety state 2 weeks after NA administration, which was not observed after 10 weeks. In accordance with this behavioral findings, an overexpression of the molecular pattern receptor TLR4 was revealed by qPCR in hypothalamic tissue in NA treated animals after 2 weeks of ICV, but not after 10 weeks. Moreover, histological studies showed a microgliosis in the amygdala of NA injected rats 2 weeks post-ICV, as well as a slightly activated state evidenced by morphometric parameters of these cells. These histological findings were not present 10 weeks after the ICV injection. These results suggest that NA-induced neuroinflammation might cause anxiety, with no neurological manifestations, in the medium term, along with a mild microglial activation in amygdala. Such symptoms seem to revert, as they were not detected 10 weeks after NA administration.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Enhanced stress response in rats that suffered acute neuroinflammation induced by neuraminidase three months before

Microglial cells are protagonists in neuroinflammatory processes and their activation is a notorious feature of such events. In acute inflammation, microglial cells return to their basal surveillant state in few days. However, sometimes they evolve towards a primed state, characterized by hypersensitivity to new stimuli and an exacerbated response which may jeopardize brain functions. Because the hypothalamus is a pivotal hub for neuroendocrine and autonomic functions, we have been exploring evidences of microglial priming in this region and its consequences. We used a model of acute ventricular neuroinflammation consisting in the intracerebroventricular (ICV) injection of neuraminidase (NA). This enzyme is found in the cover of neurotropic bacteria and viruses, e.g. influenza, mumps or measles viruses, thus mimicking a brain infection. Three months after inducing neuroinflammation with NA to rats, an acute stressor was applied to investigate the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the stress response elicited, as well as the inflammatory activation of hypothalamic microglial cells. The acute stressor was forced swimming for 6 minutes. Afterwards, blood samples were retrieved to determine corticosterone levels by ELISA, and the brains extracted to analyze microglial cells in histological sections by immunohistochemistry with IBA1 and inflammatory markers by qPCR. Stressed rats previously injected with NA had increased levels of corticosterone compared with control rats that were equally stressed but had been ICV injected with saline. Also, qPCR studies in hypothalamic tissue revealed that NA treated rats presented an increased expression of the genes for the inflammasome protein NLR family pyring domain containing 3 (NLRP3) and the microglial marker IBA1. Concomitantly, the morphological analysis of microglial cells located in the paraventricular nucleus (PVN) showed a morphological bias towards a slightly activated state in microglia...Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Sexual differences in depressive-like behaviours after juvenile and adult stress.

Early stress episodes affect brain development and is related to increased risk of developing stress-induced depression in adulthood. However, little is known about the sexual differences in this process. Our goal is to study how stress in different developmental periods affects depression-like behaviours in male and female mice. For this purpose, four experimental groups of both male and female mice of strain C57BL/6J were used: control (C) Juvenile (JE), adult (AE), and double stress JE+AE (DE). Depression-like behaviours were evaluated in the adult period. Nest building test was used to assess motivation and fatigue, the open field test to study exploratory behaviour, and the tail suspension test for learned helplessness. Taken together, the results suggest that male mice behaviours are more affected by adult stress (AE, DE), while female mice behaviours are more affected by juvenile stress (JE, DE). A PCA was performed to integrate the results. It revealed three principal components that account for 64,62% of the variance. The first is related to exploratory behavior, the second to active coping mechanisms and the third is the motivation for basic behaviors. Exploratory behaviour seems to be affected by all types of stress, while motivation is specially hindered in the female DE. Taken together, these results suggest that stress in different developmental periods affects differently male and female mice, with DE females exhibiting more depressive-like behaviours.Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía P20_00460-Co-financing by the European Regional Development Fund (ERDF/FEDER) ; Ministerio de Ciencia e Innovación-Plan Nacional I+D+I from Spain Grant PID2020-117464RB-I00 funded by MCIN/AEI/10. 13039/501100011033 ; ERDF/Junta de Andalucía-Proyectos I+D+I en el marco del Programa Operativo FEDER Andalucía 2014-2020 (UMA20-FEDERJA-112). II plan propio de investigación, transferencia y divulgación científica de la Universidad de Málaga. 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

Gut microbiome specific changes in different behavioral profiles in a mouse social defeat stress model.

Comunicación de tipo PósterThe gut microbiome has arisen as one important modulator of general health, including brain function. In fact, disturbances in brain health are commonly mirrored in the microbiome, which could be contributing to pathology. One of the most common brain disorders is depression, which is tightly linked to environmental factors such as stress and drives alterations in regular behavior. However, not much is known about the role of the gut microbiome in response to stress and its relationship to behavior. In this study, the social defeat stress (SDS) paradigm was used as a depressive-like symptoms inducer in 8 w.o. male C57BL/6J mice for 10 days. Mice were segregated in stress resilient and sensitive according to behavior using K-means clustering and behavioral data was interpreted using principal component analysis. Then, the mice microbiome was extracted from fecal pellets after the stress protocol. DNA was extracted and purified followed by 16S (V3-V4) region amplification for sequencing. These data were analyzed to obtain diversity indexes and identify bacterial taxa within samples and groups. Data revealed that mice responded differently to the same stressor. Half the mice were found to have mild depressive-like symptoms whereas the other half showed profound alterations. Behavioral data was found to be explained in three factors: anhedonia, exploration, and motility. Stressed mice showed overall differences in their microbiome, being less diverse and populations associated with higher inflammation. Moreover, the healthy gut associated Verrucomicrobiae class was only identified in stress resilient mice, suggesting a possible relationship with their behavioral phenotype. Altogether, these results show a different behavioral response to stress in animals that reflects in their microbiome, which could be a key factor in determining stress resilience.This study was supported by Ministerio de Ciencia e Innovación - Plan Nacional I+D+I from Spain (PID2020-117464RB-I00) to CP and MP-M; FEDER/Junta de Andalucía - Proyectos I+D+I en el marco del Programa Operativo FEDER Andalucía 2014-2020 (UMA20-FEDERJA-112) to CP and MP-M; Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía (P20_00460) to CP. PC-P has been funded by the research project PID2020-117464RB-I00. Ministerio de Educación, Cultura y Deporte from Spain (FPU19/03629 to Infantes-López MI). Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Hippocampal neurogenesis changes in a sex and region-specific manner in adult mice subjected to maternal separation as an early life stress.

Introduction: Early life stress (ELS) might produce long lasting changes in hippocampal neurogenesis and increase the vulnerability to stress-related disorders later in adulthood. Objective: The analysis of immature granule cell densities in the hippocampal dentate gyrus (DG) between male and female mice subjected to ELS. Methods: Female and male C57BL/6J mice were subjected to 3h daily maternal separation (MS) for 21 days since birth. In day 60, mice underwent a single 2h restriction stress (RS) and sacrificed 24 hours after. The experimental groups were: Control, RS, MS, MS+RS. The DG was analyzed using immunohistochemistry against DCX following cell stereology to obtain cell densities according to their developmental morphology (Type A: round DCX+ cells, Type B: one-process DCX+ cell, Type C: ramified DCX+ cell). Results: DG neurogenesis changes in a sex-dependent manner, being altered in female mice but not male MS+RS. Female DCX+ Type C density (latest stages of cell immaturity) increases after ELS, significantly in the MS+RS group and with a tendency in the MS alone, compared to the Control. This was also observed to be region-specific in females, with the ventral DG beholding these changes but not the dorsal part. In the ventral DG, female and male MS groups present differences within each other, but not between their controls, which may indicate sex differences in the effects of stress. Conclusion: These differential neurogenic responses to stress depending on the sex and region could explain neurobiological basis behind pathologies like depression. Funding: PID2020-117463RB-I00, P20-00460, UMA20-FEDERJA-112, Ayuda predoctoral 'Plan propio de Investigación de la Universidad de Málaga' (convocatoria 2021) and FPU21/01318. University of Málaga.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Esta investigación ha sido financiada por los proyectos: PID2020-117464RB-I00 del MCIN/AEI/10.13039/501100011033 a Pedraza, C. y Pérez-Martín, M. El proyecto P20_00460 de la Consejería de Conocimiento, Investigación y Universidades, Junta de Andalucía concedido a Pedraza, C. El proyecto UMA20-FEDERJA-112 de FEDER/Junta de Andalucía—Proyectos I + D + I en el marco del Programa Operativo FEDER Andalucía 2014-2020 concedido a Pedraza, C. y Pérez-Martín, M. La financiación de Munoz-Martin J. proviene de los contratos predoctorales del I Plan Propio de Investigación, Transferencia y Divulgación Científica (convocatoria 2021) de la Universidad de Málaga y del Programa de Formación de Profesorado Universitario (FPU21/01318) del Ministerio de Universidades

Exploratory, anxiety and spatial memory impairments are dissociated in mice lacking the LPA1 receptor

Lysophosphatidic acid (LPA) is a new, intercellular signalling molecule in the brain that has an important role in adult hippocampal plasticity. Mice lacking the LPA1 receptor exhibit motor, emotional and cognitive alterations. However, the potential relationship among these concomitant impairments was unclear. Wild-type and maLPA1-null mice were tested on the hole-board for habituation and spatial learning. MaLPA1-null mice exhibited reduced exploration in a novel context and a defective intersession habituation that also revealed increased anxiety-like behaviour throughout the hole-board testing. In regard to spatial memory, maLPA1 nulls failed to reach the controls’ performance at the end of the reference memory task. Moreover, their defective working memory on the first training day suggested a delayed acquisition of the task’s working memory rule, which is also a long term memory component. The temporal interval between trials and the task’s difficulty may explain some of the deficits found in these mice. Principal components analysis revealed that alterations found in each behavioural dimension were independent. Therefore, exploratory and emotional impairments did not account for the cognitive deficits that may be attributed to maLPA1 nulls’ hippocampal malfunctio

Absence of LPA1 signaling results in defective cortical development

Lysophosphatidic acid (LPA) is a simple phospholipid with extracellular signaling properties mediated by specific G protein-coupled receptors. At least 2 LPA receptors, LPA(1) and LPA(2), are expressed in the developing brain, the former enriched in the neurogenic ventricular zone (VZ), suggesting a normal role in neurogenesis. Despite numerous studies reporting the effects of exogenous LPA using in vitro neural models, the first LPA(1) loss-of-function mutants reported did not show gross cerebral cortical defects in the 50% that survived perinatal demise. Here, we report a role for LPA(1) in cortical neural precursors resulting from analysis of a variant of a previously characterized LPA(1)-null mutant that arose spontaneously during colony expansion. These LPA(1)-null mice, termed maLPA(1), exhibit almost complete perinatal viability and show a reduced VZ, altered neuronal markers, and increased cortical cell death that results in a loss of cortical layer cellularity in adults. These data support LPA(1) function in normal cortical development and suggest that the presence of genetic modifiers of LPA(1) influences cerebral cortical development