Molecular and neuronal correlates of social fear in mice

Fear is a basic adaptive emotional response to threatening environmental stimuli. From an evolutionary standpoint, presence and efficient functionality of the neural substrates of fear are imperative for an organism survival. Human anxiety disorders are caused by the impaired functionality of systems within the brain that code for and regulate our responses to fearful and anxiogenic stimuli. Anxiety and fear-based psychopathologies include social anxiety disorder (SAD), generalized anxiety disorder, panic disorders, obsessive-compulsive disorders. SAD is characterized by excessive fear and avoidance of social situations and severely deteriorates the quality of life of the afflicted individual. Treatment for SAD is majorly phenomenological which is mostly caused by the sparse understanding of the neural and molecular underpinnings of this disorder. Another problem is that although these psychopathologies are twice as prevalent in women in comparison to men, most of the current research uses males as primary subjects. To reveal the molecular and neuronal underpinnings of SAD, we have established a model of social fear using a social fear conditioning (SFC) paradigm in male mice which resembles SAD in humans. Using this model we were able to show that local infusion of neuropeptide oxytocin (OXT) which is known for its prosocial and anxiolytic properties into the lateral septum (LS) reverses social fear in male mice. Social fear conditioned (SFC+) mice showed an increase in OXT receptor (OXTR) binding in the LS which normalized after social fear extinction, while local OXT release in response to social stimuli was found to be blunted in LS of SFC+ mice. In lieu of these findings, and to address the abovementioned concerns, I used the SFC paradigm to: (1) Reveal the role of endogenous OXT system in the regulation of social fear in female mice, and (2) assess the contribution of epigenetic mechanisms in the regulation of social fear memory in male mice. In order to study the endogenous OXT system in females, I chose the state of lactating mice which have an activated brain OXT system as a model. SFC+ lactating mice did not show any SFC-induced fear in comparison to virgin females. This lack of SFC-induced social fear could be reinstated by intracerebroventricular (icv) infusion of OXTR antagonist (OXTR-A). Conversely, icv infusion of OXT reversed SFC-induced social fear in virgin females. cFos immunohistochemistry revealed increased activation of the LS in SFC+ virgin mice in comparison to the SFC- controls, and this returned to baseline levels after extinction, whereas LS-activity remained dampened throughout SFC in lactating mice. I also found an increased in the number of OXT-positive fibers within the LS of lactating mice along with increased OXT release in the LS of lactating mice in response to the extinction of social fear. Moreover, calbindin staining of OXTR-Venus mice revealed most of the OXTR-expressing neurons within the LS to be GABAergic interneurons. Corroborating this, local-LS application of the OXTR-A revived, and OXT reversed SFC-induced social fear in lactating and virgin mice respectively implicating LS-OXT system in the reversal of SFC-induced social fear in lactating mice. In line with the pharmacological manipulations, viral activation of the OXTR-positive neurons within the LS facilitated extinction of social fear whereas constitutive genetic knockdown of OXTR in the mouse brain impaired extinction of social fear. Finally, I was also able to show that specific chemogenetic silencing of magnocellular OXTergic SON afferents to the LS completely blocked social contact in lactating mice. In the second half of my project, I focused on delineating the epigenetic mechanisms which could underlie the formation of social fear and social fear extinction memory. cFos immunohistochemistry revealed increased activity within the LS of SFC+ male CD1 mice post-acquisition of social fear which reverted to baseline after extinction while such an effect was absent in the case of cued fear conditioning. Following this, I checked for mRNA levels of class I HDACs and found an increase in Hdac1 in SFC+ mice which again went back to baseline after the extinction of social fear. Pre-extinction pharmacological blockade of HDAC1 within the LS using MS275 led to facilitation of extinction only in the case of social fear. Finally, I performed a microarray to identify the set of genes which are differentially expressed in the LS of SFC+ and SFC- mice. Cross-referencing these genes with the set of putative HDAC1 regulated genes led me to a final set of genes which could underlie the HDAC1-mediated regulation of social fear extinction. Taken together, my data show that molecular mechanisms within the LS are crucial for regulation of traumatic events associated with a social context in male and female mice. In the case of female mice, I was able to convincingly show that endogenous OXT-mediated activation of OXTR-positive GABAergic neurons within the LS is essential for countering SFC-induced social fear. In the case of males, I was able to show that HDAC1 regulates social fear extinction memory formation within the LS. Such molecular and neuronal mechanism probably help define the emotional response of an individual towards socially relevant environmental stimuli and form the neuronal correlates of social fear in mice. Thus, their better understanding might help us develop efficient therapeutic strategies for emotionally crippling psychopathologies such as SAD.