Studies of Central Nervous System Functional Anatomy

National Institute Of Mental Health

Investigators

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Abstract

Chronic psychosocial stress has been implicated in the etiology and progression of psychiatric disorders such as major depression and post-traumatic stress disorder (PTSD). We study the effects of psychosocial stress in a chronic social defeat (CSD) paradigm in mice that generates 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 briefly defeated by a dominant mouse of a different strain. Over the course of weeks in this living situation, the experimental mouse develops asocial, 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 bi-directional dialog between the brain and the periphery 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. Peripheral interactions with the brain can occur at the blood-brain barrier (BBB). CSD can result in compromise to the integrity of the BBB. Immune cells that normally reside at the BBB alter their composition and activity during CSD. We examine these changes using histochemical, cellular, and molecular techniques. One project focuses on vascular and microglial changes in rodents undergoing CSD stress. A second project focuses on the role of the peripheral immune system in maintaining homeostatic balance during CSD. 1) Stressful events engage the periphery via well-characterized activation of the hypothalamic-pituitary-adrenal (HPA) axis and by activity within the sympathetic nervous system (SNS). Thus, hormonal and neural activity affects all peripheral organs, and these organs, in turn, generate neuronal, cellular, and chemical signals that can reach the brain to adjust the homeostatic balance in response to challenges. Many of these signals must first encounter the BBB, which largely excludes blood-borne signals generated by the peripheral organs. However, numerous bypass mechanisms have evolved to permit information transfer into the brain. Furthermore, the BBB at a cellular level is highly dynamic, and transient breeches can occur under certain circumstances. We were surprised to discover that animals susceptible to CSD (called CSD-S mice), i.e., mice that showed asocial and anxiety-like behaviors, had microglial gene expression changes that suggested breakdown of the extracellular matrix, phagocytic activity, and BBB extravasation. To investigate this further, we used histochemical methods to discover that indeed, CSD-S animals showed scattered, rare microhemorrhages throughout the brain. These small BBB breaches allowed blood products to gain access to perivascular spaces and the brain parenchyma. At the level of the blood vessel, the main constituent of the BBB is the vascular brain endothelial cell, so we endeavored to characterize the response of these cells to CSD. Microarray transcriptomics analysis revealed that CSD induced in endothelial cells a dynamic temporal pattern of gene activation reflecting the biological process of wounding and repair. In current experiments, the recovery period following the cessation of CSD is being examined. We have found that peripheral monocytes, which are not involved in the CSD and therefore do not accumulate in the 14-day period of CSD, do accumulate at perivascular areas during a two-week recovery period. Here, they phagocytose fibrinogen, an inflammatory blood product. They also appear to participate in vascular repair and behavioral recovery in this period. Elimination of the peripheral monocyte population hinders fibrinogen clearance and recovery from CSD-induced behavioral deficits in the urine scent marking (USM) task. The appearance of scattered microbleeds during CSD suggested that stress causes changes in cardiovascular function in the form of elevated blood pressure and/or heart rate. Mice were equipped with transponders, and these parameters were tracked during the defeat sessions. Dramatic biphasic drops and elevations in blood pressure were observed, and they did not habituate over the course of the CSD session. Because environmental enrichment (EE) was previously shown by us to protect against the deleterious effects of chronic stress, we employed it again and found that EE also protected against the blood pressure drops and surges. Anti-hypertensive drug administration also offered protection. 2) 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 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. 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, where leukocytes normally reside sequestered in the subarachnoid spaces. 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 1) whole-mount meningeal preparations for histochemical determination of cellular identity and 2) 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 RNA-Seq platforms to interrogate gene expression profiles. Flow cytometry and immunohistochemistry of leukocytes indicated that CSD elevates neutrophil numbers in meninges and brain. Single-cell RNA-Seq analysis showed unique gene ontology classifications for each cell type from control and stressed meninges. We found that CSD increased splenic B cell activation and secretion of the immunoregulatory cytokine interleukin (IL)-10. Meningeal B cells were prevalent in homeostasis but substantially decreased during CSD, whereas activated monocytes increased. Single-cell RNA sequencing of meningeal B cells demonstrated the induction of innate immune transcriptional programmes following stress, including genes encoding antimicrobial peptides that are known to alter myeloid cell activation. CD19 knockout mice with reduced B cells showed baseline meningeal myeloid cell activation and decreased exploratory behaviour. Together, these data suggest that B cells may influence behavior by regulating meningeal myeloid cell activation. Similar studies are directed at lymphopenic Rag2 knockout mice that received adoptive transfer of lymphocytes programmed in vitro towards Th0, Th1, Th2, or Th17 T helper cell phenotypes. We previously showed in this adoptive transfer paradigm that lymphocytes transferred from stressed mice conferred antidepressant-like effects on the Rag2 knockout host mice, and current studies with lymphocyte subsets are aimed at disclosing the mechanism for this effect. Such studies may lead to insights into new targets for therapeutic interventions in psychiatric disorders.