Functional and molecular organization of lateral septum in threat processing - PROJECT SUMMARY The lateral septum (LS) is an inhibitory limbic brain structure long thought to be essential for threat processing and defensive behavior. In part due to its anatomical and molecular heterogeneity, the precise means by which the LS mediates these processes remains elusive. To this end, I have focused my graduate work on LS neurons expressing the stress-related Corticotropin releasing hormone type II receptor (LS-Crhr2). CRH-family G-protein coupled receptors (GPCRs) are critical for behavioral and physiological stress responses and are implicated in mood and anxiety disorders, providing a direct link between this subset of LS neurons and a central stress system. Moreover, LS-Crhr2 neurons are activated by innate threats, and its activity modulates concomitant defensive responses, evokes physiological arousal, and elicits aversion. The overall goal of these studies is to determine the molecular and functional organization of LS-Crhr2 neurons in threat processing. Threat processing, however, is multifaceted and largely involves the ability to rapidly detect and evaluate sensory stimuli as potential threats in order to respond with the appropriate behavioral defenses. To capture this complexity, I use two-way signaled active avoidance, a behavioral paradigm in which mice learn to perform goal-directed avoidance behaviors in response to punishment-predictive cues. Using microendoscopic calcium imaging, optogenetics, and single-nuclei RNA sequencing, I have thus far established the following: 1) LS-Crhr2 population activity is required for executing learned avoidance; 2) LS-Crhr2 population activity dynamically codes threat cues in a manner predicting defense decisions and upcoming action initiation; 3) LS-Crhr2 comprises of ten functional subclasses (defined by calcium dynamics) specialized in distinct threat-related processes; and 4) LS-Crhr2 comprises of ten molecular subclasses (defined by transcriptomics) with unique anatomical organization. Building on these observations, the central goal of this study is to identify how these molecular LS- Crhr2 subsets mediate specific threat-related processes and how they are dynamically recruited. In the following aims, we will use viral-genetic strategies to monitor and manipulate the activity of these molecularly-defined subclasses, which together comprise of roughly 84% of the total LS-Crhr2 population. Aim 1 will use microendoscopic calcium imaging of single neurons to determine whether each molecular subclass maps to functional LS-Crhr2 clusters whose neural activity dynamics resemble discrete threat-related processes during active avoidance. Aim 2 will use optogenetics to directly inhibit the activity of each subset to test their role in these distinct processes. The successful completion of these aims will capture how the molecular diversity of LS-Crhr2 neurons maps to function, thereby providing a framework for understanding the role of LS in threat processing and, more generally, for studying the role of discrete functional units of cells that express druggable GPCRs of interest.
