Avishek Adhikari, Ph.D - US grants

? DESCRIPTION (provided by applicant): Avoidance of potential threats is highly adaptive, and decreases unnecessary exposure to risks. However, excessive anxiety and fear leads to anxiety disorders, which impact many aspects of life, from the interpersonal to professional spheres. Although each anxiety disorder has different symptoms, they all share a core feature: mal-adaptive expression of high levels anxiety. Here, we will study how the brain suppresses anxiety. Prior studies showed the amygdala is largely responsible for generating high anxiety and fear, while the ventral medial prefrontal cortex (vmPFC) decreases these behaviors, possibly by inhibiting amygdala output. Indeed, in humans higher vmPFC activation correlates with lower amygdala activation and decreased anxiety. These data suggest the vmPFC-amygdala pathway may decrease anxiety and fear, but they rely on correlative measures, and can't directly test this hypothesis. I used optogenetics to directly test if the vmPFC-amygdala projection suppresses anxiety and fear. Remarkably, optogenetic activation of the vmPFC-amygdala pathway robustly inhibits innate anxiety and learned fear, while inhibition of this pathway increases anxiety. Intriguingly, these behavioral effects were mediated by a poorly studied region of the amygdala called the basomedial amygdala (BMA), as direct activation of the BMA also decreases anxiety. Now, I will map neural activity in the vmPFC-BMA circuit and dissect how activation of this circuit decreases anxiety. I will first investigate how vmPFC activiy affects the BMA in vitro (Aim 1), uncovering the microcircuit-level dynamics underlying our behavioral findings. Next, to map the activity of the vmPFC-BMA projection, I will monitor calcium transients in the vmPFC terminals in the BMA during exploration of control and anxiogenic environments (Aim 2), revealing how activity of this projection differs in animals with high and low anxiety. Lastly, during the independent phase, in Aim 3, I will use the skills acquired during the mentored year to characterize activity of the BMA and of its output projections during anxiety and fear. Completion of these aims will make me proficient in patch-clamping and in vivo calcium monitoring. I will learn these skills under the guidance of a mentoring (Profs. K. Deisseroth and R.C. Malenka) and consulting (Profs. A. Losonczy and M.R. Warden) teams who have pioneering experience in using these methods and in training other researchers to employ them. Combining these new skills with my prior expertise in vivo electrophysiology will ensure a methodologically strong foundation to launch an independent lab, while dissecting how the poorly-studied BMA decreases anxiety will provide new research avenues. Importantly, my mentors have a remarkable track record in training independent researchers. This project involves experiments ranging from microcircuit dissection to optogenetic control of behavior, and will give us critical insight about how the brain dampens anxiety, and how and when it fails to do so.

Project Summary/Abstract Panic attacks are a common symptom in patients suffering from numerous anxiety disorders. These overwhelming attacks are particularly common populations with severe trauma, such as rape victims or veterans. Circuits mediating escape from imminent threats such as asphyxiation are strongly implicated in the generation of panic attacks. Naturalistic escape from threats occur in complex environments in which animals must quickly flee through the most efficient route. A single incorrect choice of context-specific escape plan may result in death. Prior studies have identified regions that produce escape movements during neural stimulation, such as jumping. However, these movements often do not result in choice of optimal escape routes. To date, circuits inducing context-specific choice of escape routes have not been identified. Now, we show that optogenetic stimulation of nitric oxide synthase1 (nos1)+ cells in the dorsal premammillary nucleus (PMd) creates context-specific escape, similarly to naturalistic escape. In an empty box, PMD stimulation causes jumping, but after adding a climbing rope escape, stimulation causes escape by climbing the rope. In contrast, stimulation of the dorsolateral periaqueductal gray (dlPAG), which is the region most deeply studied in panic-related escape, causes jumping and running in all situations, even when these actions do not allow escape. Intriguingly, the PMd is the densest input to the panic-inducing dlPAG, but it has never been activated directly. Activation of the nos1+PMd-dlPAG projection also led to the same panic-related symptoms as stimulation of nos1 PMd cell bodies, including escape and aversion. This finding suggests the PMd is creating context-specific escape by acting on the dlPAG. To study this circuit, we developed two novel paradigms with escape-provoking threats: a corridor containing a live predator (an awake rat that is not separated by a barrier) and a chamber for exposure to 15% CO2, a stimulus known to cause panic in humans. In both paradigms threat exposure can only be maximized with context-specific escape plans requiring coordinated action. These paradigms produce a full range of defensive behaviors (risk- assessment, freezing, jumping/running and planned escape using optimal routes) depending on threat intensity (distance to rat or CO2 concentration), allowing us to precisely identify which behaviors are controlled by the PMd-dlPAG circuit. Our aims are to: 1) Optogenetically dissect how the PMd-dlPAG circuit produces these symptoms, 2) Characterize how panicogenic threats affect PMd activity and synchrony in the PMd-dPAG circuit and 3) Examine how PMd input influences threat-encoding in the dlPAG and how it synaptically affects dlPAG cells. Since PMd-dlPAG activation selectively induced escape, but not other defensive behaviors, we hypothesize that the nos1+PMd-dlPAG circuit specifically affects planned context-specific escape. We also predict that neural activity in this circuit is most strongly correlated with escape. These aims will reveal novel circuit mechanisms underlying panic-related escape from threat.