Periodic Reporting for period 4 - InsularAnxiety (Insular cortical circuits controlling fear and anxiety) Reporting period: 2022-09-01 to 2023-02-28 Summary of the context and overall objectives of the project The general goal of the project ‘InsularAnxiety’ was to investigate the role of the insular cortex in fear- and anxiety-related behaviors. While human functional imaging data have suggested that the insular cortex is a key regulator of emotional responses and especially plays an important role in regulating fear and anxiety, how precisely the insular cortex processes threatening stimuli and alters behavioral outcomes is not known. We addressed this problem by breaking it down into several questions: (i) What is the precise architecture of insular cortex microcircuits? (ii) How are stimuli processed within the insular microcircuits? (iii) How does activity in defined insular cortex microcircuits change behavior?Since the insular cortex is one of the major brain regions implicated in severe human psychiatric diseases, such as anxiety disorders, addiction, and major depression, a better mechanistic understanding of the insular cortex may contribute to a better understanding of human psychiatric disorders and guide the way into novel therapeutic strategies. Therefore, the overarching aim of this project was to provide a better basic understanding of the neuronal circuit underpinnings of the insular cortex’s contribution to fear and anxiety and their regulation. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The goal of the project ‘InsularAnxiety’ was to elucidate the largely overlooked role of the insular cortex in emotion processing in general terms, and in fear- and anxiety-related behaviors in particular. Aim 1 of our project was to study the architecture of microcircuits with the insular cortex and how they connect to anxiety-related brain regions. By combining measurements and targeted manipulations of insular cortex activity, we uncovered a critical role for the posterior insular cortex in processing aversive sensory stimuli and emotional and bodily states, as well as in exerting prominent regulation of ongoing behaviors. Specifically, we could demonstrate that the insular cortex mediates behavioral effects of persistent anxiety but also of sustained bodily states such as malaise. In addition to these functional results, we were also able to establish a comprehensive whole-brain connectivity map of the mouse insular cortex. These data provide an anatomical framework to guide future functional investigations of the complex functions of the insular cortex. Aim 2 of our project was to address the role of the insular cortex in regulating fear. Interestingly, we found that the posterior InsCtx serves as a state-dependent regulator of emotion, necessary to establish a balance between the extinction and maintenance of fear memories in mice. We further found that insular activity is potently modulated via bodily feedback signals arising from heart rate changes. Indeed, heart rate decelerations during freezing seemed to provide negative feedback on insular activity. Perturbation of body-brain communication by Vagus Nerve stimulation disrupted the balance between fear extinction and maintenance similar to InsCtx inhibition. Our data revealed that the InsCtx integrates predictive sensory and interoceptive signals to provide graded and bidirectional teaching signals that gate fear extinction and illustrate how bodily feedback signals are used to maintain fear within a functional equilibrium. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) One of the overarching aims of this project became relating insular activity to the processing of diverse emotion states, including fear and anxiety, but also encompassing other emotion states, such as pleasure or disgust. Toward this goal, we developed objective readouts of the emotion states in mice. Using machine vision and machine learning tools, we could show that mice exhibit stereotyped facial expressions that can be classified into distinct emotion categories and that offer temporary and quantitative precise ways to “measure” emotions in mice. These unexpected results allowed us to correlate facial expressions directly to brain activity and provide an unprecedented means to study the neuronal underpinnings of emotion. Further, our results on the insular microcircuits establish the insular cortex as a potent mediator of aversive states such as anxiety. Our data support a model in which the posterior insular cortex can shift behavioral strategies upon the detection of aversive internal states, providing a new entry point to understand how alterations in insular circuitry may contribute to neuropsychiatric conditions. Furthermore, we expect that our findings concerning the powerful role of bodily feedback to the insular cortex and their potent role in regulating fear may serve as an entry point to the development of novel treatment strategies for fear and anxiety disorders in humans. Neurons in the posterior i.c. process neg. emotions+states+can consequently affect the behavior
