Neurobiology of loss: role of microglia

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Project Summary Psychological loss occurs when one is deprived of something valuable, such as social relationships, physical health, or financial stability. These experiences often lead to depression-like symptoms such as amotivation, social withdrawal, weight gain and hypoactive stress responses. While loss experiences are common, little is known about the molecular mechanisms driving loss symptomology. Our laboratory uses enrichment removal (ER) to simulate the experience of loss in rats. In this protocol, rats are first housed in positive, highly stimulating environmental enrichment (EE) for a month, and then moved to single housing with no further stimuli. This removal alters how the rats respond to their environment, producing behaviors and physiology that specifically resemble loss in humans, including increased passive coping, weight gain, and hypoactive stress responses. We used an innovative discovery-based multi-omics approach to investigate ER molecular mechanisms in the basolateral amygdala (BLA), a traditionally stress promoting region that is differentially activated in EE and ER. Using a combination of RNA sequencing, proteomics, and kinomics, we identified a unique microglia phenotype in ER. We hypothesize that ER decreases signaling in BLA microglia, impairing their ability to regulate nearby neurons and dampen stress-promoting BLA signals. This dysregulated microglia signaling is supported on all three omics platforms and is expected to contribute to ER behavioral and physiological phenotypes. Additional analyses revealed that MAP3K8, a microglia-specific kinase, is a signaling hub in this phenotype and could be a potential target to ameliorate ER phenotypes. The objective of this proposal is to further define BLA microglia phenotypes in ER and investigate the role of the signaling hub MAP3K8 in ER behavioral and physiological phenotypes. Specific Aim 1 will use fluorescence activated cell sorting (FACS) to isolate BLA microglia, followed by qPCR to determine microglia-specific expression of key genes identified in our preliminary analyses. Aim 1 will also use confocal microscopy and quantitative morphometry to determine ER-related microglia morphology and expression of MAPK activity markers. These well-established, cell type specific studies are necessary to clearly define the specific role of microglia, as opposed to other cell types, in ER phenotypes. Specific Aim 2 will use behavioral pharmacology to examine the role of MAP3K8 in ER behavioral and physiological phenotypes. We will implant BLA-targeted cannula attached to osmotic minipumps containing a MAP3K8 antagonist prior to the ER paradigm and behavioral testing. Differential timing of drug delivery will allow us to investigate both the necessity and sufficiency of MAP3K8 signaling. Overall, the findings of these studies will define the role of BLA microglia and MAP3K8 signaling in ER phenotypes, and provide possible insight into the negative consequences of loss in humans.