Chronic psychosocial stress has been implicated in the etiology and progression of psychiatric disorders such as major depression and post-traumatic stress disorder (PTSD). In mice, we study the effects of psychosocial stress in a chronic social defeat (CSD) paradigm that leads to behavioral alterations manifested as social avoidance, anhedonia, anxiety, and depressive-like states. In CSD, two male mice are placed in a continuous dyadic living relationship in which the subordinate experimental mouse is chronically exposed to and periodically defeated by a dominant mouse of a different strain. Over the course of weeks in this living situation, the experimental mouse develops anti-social, anhedonic, and anxious behaviors that can be experimentally linked with neurochemical and physical alterations in identified stress-related, limbic brain circuits. For example, our past research showed that animals undergoing CSD have reduced hippocampal new-cell proliferation and reduced medial prefrontal cortical myelination. These kinds of changes have been associated with anxiety-like and depressive-like behaviors in animal and human studies. Thus, the paradigm allows us to examine mechanistic bases for mental illness in humans. We hypothesize that there is a bidirectional dialog between the brain and the immune system that serves to maintain homeostasis in healthy states, but disturbances within this dialog lead to homeostatic deviations that contribute to the onset and course of psychiatric disease. Therefore, we measure immune system parameters as we manipulate psychosocial processes that affect brain states. Two projects investigate immune changes in rodents undergoing CSD stress; one focuses on the role of the peripheral immune system in maintaining homeostatic balance during chronic stress, and the second investigates stress-induced changes in immune cells and molecules within the brain itself. 1) We conduct experiments aimed at addressing the role of the peripheral immune system in controlling affective behaviors in chronic psychological stress. We are particularly interested in particular leukocyte cell types that may contribute to altered mood states. These include T and B lymphocytes of the adaptive immune system and neutrophils and monocytes of the innate immune system. Leukocytes do not enter the brain in significant numbers in non-disease conditions, and we do not observe elevated numbers in the brain in the CSD stress paradigm. Consequently, we investigate molecular and cellular interactions between the periphery and the brain taking place at the blood-brain interfaces. We are particularly focused on the meninges, a special component of the blood-brain barrier (BBB), where leukocytes normally reside sequestered in the subarachnoid spaces, i.e., on the brain side of the BBB. Cells in this compartment can release bioactive molecules that circulate throughout the brain in the cerebrospinal fluid to potentially influence brain activity. We track leukocytes in the meninges using whole-mount meningeal preparations for histochemical determination of cellular identity and flow cytometry for molecular analysis of cell types and activation states. In addition, cells recovered from the meninges and brain by cell sorting were applied to microarray chips and single-cell RNAseq platforms to interrogate gene expression profiles. Our analysis of cells in CSD-stressed versus non-stressed animals has shown that stress alters the make-up of meningeal leukocytes in complex ways best unraveled by bioinformatics analyses. Flow cytometry indicated altered numbers and profiles of lymphocytes, monocytes, and neutrophils in meninges from stressed mice. Single-cell RNAseq analysis showed unique gene ontology classifications for each cell type from control and stressed mice. Similar studies are directed at lymphopenic Rag2-/- mice that received adoptive transfer of lymphocytes programmed in vitro towards Th0, Th1, Th2, or Th17 phenotypes. We previously showed in this adoptive transfer paradigm that lymphocytes transferred from stressed mice conferred antidepressant-like effects on the Rag2-/- host mice, and current studies with lymphocyte subsets are aimed at disclosing the particular mechanism for this effect. Such studies may lead to insights into new targets for therapeutic interventions in psychiatric disorders. 2) Microglia are specialized immune cells within the brain, sharing many properties with peripheral monocytes, though they have different hematopoietic origins. Microglia are activated by stress, and the nature of this response has been the subject of much research and debate. The causes of activation and subsequent effects on neuronal and stress circuit function are not well understood. We isolated microglia from stressed and unstressed mice and characterized microglial gene expression patterns by microarray. The data indicated that microglia from mice that had been susceptible (CSD-S) to the depressive effects of defeat were functionally distinct from microglia taken from mice that showed behavioral resilience (CSD-R) to the stress procedure. The microglial gene expression characteristics of the CSD-R group were more like those from unstressed home-cage (HC) control mice, suggesting that microglial activity contributes to the brain states that afford resilience to the effects of psychosocial stress. Gene expression profiles in the microglia from CSD-S mice showed evidence of inflammation, phagocytosis, extracellular matrix breakdown, oxidative stress, and extravasation. The microarray findings were confirmed by histochemical and ex vivo assay methods. In the CSD-S mice only, local breeches of the BBB and brain microbleeds were found. This finding turned our focus to stress effects on the cerebral vasculature and the possible role of blood pressure surges during the defeat sessions, which we documented by blood pressure telemetry. We hypothesized that microglia might be responding to vascular microbleeds by phagocytosing blood products and participating in wounding and repair processes. We developed a number of histochemical markers to visualize the altered status of the vasculature and BBB breakdown in the CSD model. Using dihydroethidium (DHE) fluorescence staining for reactive oxygen species (ROS), we found that microglia from stressed mice had elevated production of ROS, indicating increased levels of oxidative stress. We found that intracerebroventricular administration of a ROS inhibitor N-acetyl cysteine protected against CSD stress effects. We then found that complete depletion of microglia by administering the CSF-1R antagonist drug PLX5622 also protected against stress effects. Microglial repopulation of the brain post-CSD reintroduced adverse stress effects, and ROS inhibition in this phase protected against the effects. The combined data suggest that stress-induced microglial ROS production drives a central state that supports dysregulated affective behavior. Additional studies of the microbleeds using markers of inflammatory blood components such as fibrinogen are underway to verify the causes of the microglial activation. Frontal cortical brain tissue sections obtained from the NIMH Human Brain Collection Core are also being examined with these histochemical stains.
- National Institute of Mental Health
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