Role of the LPA1 receptor in mood and emotional regulation

Depression is a debilitating psychiatric condition characterized by anhedonia and behavioural despair among others symptoms. Despite the high prevalence and devastating impact of depression, underlying neurobiological mechanisms of mood disorders are still not well known. Regardless its complexity, central features of this disease can be modelled in rodents in order to better understand the potential mechanisms underlying. On the other hand, the lack of LPA1 receptor compromises the morphological and functional integrity of the limbic circuit and the neurogenesis in hippocampus, induces cognitive alterations on hippocampal-dependent tasks and dysfunctional coping of chronic stress, provokes exaggerated endocrine responses to emotional stimuli and impairs adaptation of the hypothalamic-pituitary-adrenal axis after chronic stress. Factors, which all have been related with depression. Here, we sought to establish the involvement of the LPA1 receptor in regulation of mood and emotion. To this end, in wild-type and maLPA1-null mice active coping responses to stress were examined using the forced swimming test (FST). To assess hedonic behaviour saccharine preference test and female urine sniffing test were used. Our data indicated that the absence of the LPA1 receptor significantly affected to coping strategies. Thus, while null mice displayed less immobility than wt in FST, exhibited more climbing and less swimming behaviour, responses that could be interpreted as an emotional over-reaction (i.e., a panic-like response) to stress situations. Concerning hedonic behaviour, the lack of the LPA1 receptor diminished saccharin preference and female urine sniffing time. Overall, these data supports the role of LPA1 receptor in mood and emotional regulation. Specially, the lack of this receptor induced emotional dysregulation and anhedonic behaviour, a core symptom of depression.Universidad de Málaga, Campus de Excelencia Andalucía Tech. Andalusian Regional Ministries of Economy, Innovation, Science and Employment (SEJ-1863; CTS643) and of Health (PI-0234-2013; Nicolas Monardes Programme), MINECO (PSI2013-44901-P) and National Institute of Health Carlos III (Sara Borrel)

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

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

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

Anxiety and mild microglial activation in the amygdala two weeks after NA-induced neuroinflammation

A single injection of neuraminidase (NA) within the cerebral ventricles (ICV) triggers an acute neuroinflammation. Neurological complications or behavioral alterations have been associated to neuroinflammation. While some of these symptoms decline with time along with inflammation, the possibility of long-term sequelae should be considered. Thus, we aimed to explore if NA-induced neuroinflammation provokes behavioral or neurological disturbances at medium (2 weeks) and long (10 weeks) term. Rats were ICV injected with NA or saline. First, neurological alterations of the sensorimotor reflexes were not found, suggesting that NA does not cause disturbances in major brain functions. While the open field test revealed normal locomotor capacity in the animals injected with NA, however the evaluation of specific behaviors (rearing and rearing with support) pointed out an increased anxiety state 2 weeks after NA administration, but not at long term (10 weeks). A histological study of brain areas related to emotions (amygdala) and stress response (hypothalamic PVN) revealed no significant differences in the number of microglia or astrocytes. Nevertheless, the morphological analysis of microglial cells demonstrated that, in the amygdala of NA injected rats, microglia presented a morphology consistent with a slightly activated state. Such morphological change, which was evident 2 weeks after NA injection, was virtually reverted 10 weeks post-ICV. These results point out that NA injected ICV may cause anxiety in the medium term (while not affecting other functions like sensorimotor functions or the locomotor capacity), a behavioral alteration that is transient and that concurs with a mild inflammation, evidenced by the overexpression of certain genes and, more notably, by the morphological bias of microglial cells located in the amygdala towards an activated profile.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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