Project Summary Chronic stress is a detrimental condition affecting brain physiology, cognition and mental health. Altered cerebrovascular network has long been recognized as a potential modulator of neuronal function in response to stress. However, key molecular substrates conveying signals from cerebrovascular input to functional pathways in specific types of neurons are not fully understood. Our recent study with a chronic social defeat stress (CSDS) paradigm indicates that a) hippocampal parvalbumin (PV)-expressing interneurons are a determinant of CSDS- induced behavioral adaptations, b) hemoglobin (Hb) α- and β-chain levels are drastically upregulated in hippocampal PV neurons in stress-susceptible mice, and c) mitochondrial oxidative phosphorylation in PV neurons is the most significant functional pathway associated with stress susceptibility. Because chronic stress is known to cause cerebrovascular dysfunction and dysregulated blood flow in the brain, we hypothesize that PV interneurons are a contiguous target of chronic stress, and Hb expressed in PV neurons is a critical node sensing vascular input and regulating mitochondrial adaptation in PV neurons. The overarching goal of this study is to establish a cell-type-specific deletion model of Hb and determine the role of Hb in chronic stress-induced PV neuron adaptation and behavioral responses to chronic stress. First, we will use the CSDS model, and examine the effect of an antihypertensive drug (captopril) on CSDS-induced microbleeds, BBB disruption, Hb induction in PV neurons and behavioral outcomes to determine the relationship between vascular disruption and Hb induction in PV neurons (Aim 1a). Hippocampal PV neuron-selective deletion of Hb β-chain genes will be achieved by injecting AAV-sgRNAs vector into the hippocampus of transgenic mice expressing Cas9 selectively in PV neurons. The control and β-chain-deleted mice will be subjected to CSDS, and behavioral responses to CSDS will be measured (Aim 1b). After behavioral assays, the hippocampal tissues will be analyzed using a PV- neuron-selective Translating Ribosome Affinity Purification (TRAP)/RNA-seq approach (Aim 2). Differentially expressed genes (DEGs) will be analyzed by bioinformatic approaches to find altered functional pathways including mitochondrial pathways (Aim 2). We aim to determine whether neuronal Hb is required for chronic stress-induced behavioral changes (Aim 1b) and identify downstream genes and functional pathways influenced by Hb deletion (Aim 2). Cell-type-specific deletion tools for neuronal Hb in conjunction with a cell-type-specific TRAP/RNA-seq approach established in this study can be applicable to the future studies of Hb in other cell types in different brain regions under various stress or disease models. The research paradigm established in this study will facilitate an array of new studies paving the gap between stress-induced cerebrovascular deficit and brain-cell-type-specific responses. Thus, t...