Indiana University-Purdue University Indianapolis (IUPUI) / Mental health is crucially linked to social behavior. A crucial aspect of healthy social behavior involves learning to adapt emotional responses to social cues, for example learning to suppress anxiety through social familiarity, or social familiarity induced anxiolysis (SoFiA). SoFiA is well documented; however, the neural mechanisms of SoFiA are unclear. SoFiA is modeled in rats by employing a social interaction habituation (SI-hab) protocol. Using SI-hab protocol it has been determined that SoFiA represents social safety learning, which requires both anxiogenic stimulus (Anx) and social familiarity (SF) during training sessions (5-6 daily SI sessions), and SoFiA expression is dependent on infralimbic cortex (IL). Based on these findings we hypothesize that Anx and SF are processed by unique neural systems, and repeated convergence of these signals interact within IL to induce plasticity, resulting in social safety learning and anxiolysis. Following SoFiA expression, rats were either sacrificed 30 minutes {for gene expression or Neural Activity Regulated Gene (NARG) analysis} or perfused 90 minutes (for cFos immunoreactivity analysis) after SI session on social training day 5. This led to gaining insights into regions of brain involved in SoFiA response as well as the underlying molecular mechanisms. We identified amygdala, specifically the central amygdala (CeA), basomedial amygdala (BMA) and basolateral amygdala (BLA) as potential candidate regions in SoFiA response. Next, we investigated the role of IL and its efferent pathways in SoFiA expression using inhibitory DREADDs and intersectional chemogenetics to inhibit IL projection neurons and/or axons. We identified that specific projection neurons within the IL are pivotal for SoFiA expression, and that within these projections, the ones that specifically projected to the amygdala are most crucial for expression of SoFiA. / 2021-07-01

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

The placebo effect, a beneficial effect attributable to a treatment containing no specific properties for the condition being treated, has been demonstrated in a variety of medical conditions. This thesis includes four studies aimed at increasing our knowledge on the neurobiology of placebo. Study I, a review of the placebo neuroimaging literature, suggested that the anterior cingulate cortex (ACC) may be a common site of action for placebo responses. However, because placebo neuroimaging studies in clinical disorders are largely lacking, the clinical relevance of this needs further clarification. The subsequent three empirical studies were thus designed from a clinical perspective. Using positron emission tomography (PET) these studies investigated the underlying neurobiology of sustained placebo responses in patients with social anxiety disorder (SAD), a disabling psychiatric condition that nonetheless may be mitigated by placebo interventions. Study II demonstrated that serotonergic gene polymorphisms affect anxiety-induced neural activity and the resultant placebo phenotype. In particular, anxiety reduction resulting from placebo treatment was tied to the attenuating effects of the TPH2 G-703T polymorphism on amygdala activity. Study III further compared the neural response profile of placebo with selective serotonin reuptake inhibitors (SSRIs), i.e the first-line pharmacological treatment for SAD. A similar anxiety reduction was noted in responders of both treatments. PET-data further revealed that placebo and SSRI responders had similar decreases of the neural response in amygdala subregions including the left basomedial/basolateral (BM/BLA) and the right ventrolateral (VLA) sections. To clarify whether successful placebo and SSRI treatments operate via similar or distinct neuromodulatory pathways, study IV focused on the connectivity patterns between the amygdala and prefrontal cortex that may be crucial for normal emotion regulation. In responders of both treatment modalities, the left amygdala (BM/BLA) exhibited negative coupling with the dorsolateral prefrontal cortex and the rostral ACC as well as a shared positive coupling with the dorsal ACC. This may represent shared treatment mechanisms involving improved emotion regulation and decreased rumination. This thesis constitutes a first step towards better understanding of the neurobiology of placebo in the treatment of anxiety, including the neural mechanisms that unite and segregate placebo and SSRI treatment.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

Indiana University-Purdue University Indianapolis (IUPUI) / Social support is a powerful therapeutic against fear and anxiety and is utilized in many psychotherapies. The concept that a familiar or friendly presence helps a person learn to overcome anxiety has been well-known for decades, yet, the basic neural mechanisms that regulate this psychosocial learning remain unknown. A first step towards elucidating these basic mechanisms is the development of a valid preclinical animal model. However, preclinical behavioral models exploring the use of a social presence in reducing anxiety have not been fully characterized. Therefore, it was our goal to identify a useful way in which to study the mechanisms of how a social presence can induce anxiolysis (the reduction of anxiety). We accomplished this goal by characterizing and validating a preclinical model, as well as demonstrating that the model was capable of measuring deficits in rats given a mild traumatic brain injury. To this end, we identified an existing, but uncharacterized model, the social interaction-habituation model, as an effective model of social familiarity-induced anxiolysis (SoFiA), which demonstrates socially enhanced safety learning, or psychosocial learning. We find that as social familiarity develops across time, anxiolysis develops. We identified that the use of a Bright Light Challenge is a useful anxiogenic stimulus to use during SI-habituation training. The anxiolysis acquired following SI-habituation testing is partner specific, and can be blocked by an inhibition of the medical prefrontal cortex, while it can be enhanced by D-cycloserine. We found that this model identified deficits in SoFiA acquisition in rodents exposed to a mild traumatic brain injury, which, in humans, has been linked to psychosocial deficits. This work is a step in creating ways in which we can study and better understand the regulatory processes of emotions mediated by social behavior.