Investigating the Functional Connectivity of the Bed Nucleus of the Stria Terminalis During Conditions of Threat and Safety Using High Resolution 7 Tesla FMRI

An influential model of the extended amygdala defines fear as the immediate response to phasic threat and anxiety as the prolonged response to unpredictable or sustained threat (Davis, Walker, Miles & Grillon, 2010). This model proposes that in response to unpredictable threat, the centromedial amygdala (CeA) activates the bed nucleus of the stria terminalis (BNST), which coordinates the anxiety response, and, in turn, inhibits the CeA. Connectivity between the BNST and both the basolateral amygdala (BLA) and hippocampus may also play an important role in the coordination of the anxiety response (Davis et al., 2010; Herman et al., 2003; Zhu, Umegaki, Suzuki, Miura & Iguchi, 2001). However, there is a dearth of human research investigating whether state anxiety is accompanied by increased connectivity between the BNST and CeA, BLA and hippocampus. To test whether sustained threat elicits increases connectivity between the BNST and these areas, I monitored participants’ resting brain activity via high resolution 7 tesla fMRI during two five minute resting state scans, one while under threat of unpredictable shock and one while safe. I predicted that each of these areas would exhibit greater connectivity with the BNST during periods of threat vs. safety. To test whether BNST connectivity during periods of threat is altered in anxiety prone individuals, I collected self-reported behavioral inhibition. I predicted that greater behavioral inhibition would predict increased connectivity during periods of threat (vs. safety) between the BNST and the CeA, BLA and hippocampus. I also tested whether connectivity in these areas changes over time following the onset of threat by examining connectivity for three time windows, corresponding roughly to the first, second and fifth minute following threat onset. I predicted positive threat vs. safe BNST-CeA connectivity during the first time window, negative threat vs. safe BNST-CeA connectivity during the second time window, and no difference in BNST-CeA connectivity during the final time window. I found a marginally significant trend toward greater BNST-BLA connectivity during threat vs. safety. I found no evidence for increased BNST-hippocampus connectivity during threat, or that BNST connectivity with either the BLA or hippocampus is modulated by behavioral inhibition. Threat condition, behavioral inhibition, and time window interacted to affect BNST-CeA connectivity, although a lack of significant follow-up tests makes interpreting this interaction challenging. Further research is needed to characterize how individual differences alter the time course of BNST-CeA connectivity during conditions of threat and safety, and the conditions under which threat may elicit BNST connectivity with the hippocampus and BLA

Cannabidiol Administered via Vapor Inhalation Restores Social Interaction Deficits in a Mouse Model of Social Anxiety

Cannabidiol (CBD), the main non-intoxicating component of the plant cannabis, has shown various promising therapeutic effects in treatment of anxiety and depression in both humans and animals. One potential beneficial effect of CBD is restoration of social interaction deficits following chronic stress. Here I investigate the potential for CBD to be used as a treatment in animal models of Social Anxiety Disorder (SAD), as well as potential mechanisms of action by which CBD may produce these effects. Mice exposed to 10 days of chronic social-defeat stress were administered vaporized CBD in a single 30 minute session before being tested behaviorally. Mice exposed to CBD showed significantly higher levels of social interaction as measured by the three-chamber test compared to mice exposed to vehicle VG/PG vapor, and comparable levels of interaction to unstressed mice. Contrary to my hypothesis, restorative effects of CBD on social interaction were unrelated to levels of BDNF in the hippocampus. These results provide a foundation for the development of novel and improved treatments for chronic stress-related disorders, including social anxiety disorder. These studies also contribute to a growing body of literature on the potential therapeutic effects of CBD and its targeting of various neurobiological pathways in the treatment of chronic stress-related disorders

Evaluating Changes in Error-Monitoring Electrocortcial Responses as an Outcome of Attention Bias Modification Training

Anxiety disorders are among one of the most debilitating and prevalent mental disorders. Maladaptive anxiety has been associated with enhanced attention bias to threat as well as heightened error-monitoring following an erroneous response. In an effort to reduce an anxious individual’s attention bias to threat, an attention training paradigm known as attention bias modification (ABM) was developed. While ABM training has demonstrated the ability to reduce attention bias and anxiety symptoms, there are inconsistencies in the magnitude of symptom reduction and there is a lack of neuroimaging support in regards to ABM outcome. Therefore, this study evaluated the outcome of ABM training using error-related negativity (ERN) an event-related potential (ERP) that is associated with an error-monitoring response after an individual commits an error. To elicit an erroneous response a modified flanker task paradigm was used. The ERN has the potential to be used as a measure of ABM outcome due to the common neural structures that both processes recruit – in particular, the anterior cingulate cortex (ACC). The results demonstrate no reduction in anxiety following ABM, but reductions in attention bias in both the ABM and control groups. There were also no significant relationships between ERN and ABM outcome, suggesting that ERN is not an effective measure of functional outcome. Limitations and future directions involving multi-session ABM and functional outcomes are discussed

Brain Systems Coordinating Fear To Uncertain Threats

Anxiety disorders are among the most common and debilitating forms of mental illnesses in society. Through greater understanding of the fundamental mechanisms of anxiety, as well as of the factors that lead to the persistence and relapse of fear- and anxiety-related symptoms, we may develop novel behavioral and brain techniques for intervention. Emerging evidence in humans and rodent models suggests that the bed nucleus of the stria terminalis (BNST) is a critical brain structure in the regulation and expression of fear and anxious behaviors. However, the precise contributions of the BNST to the expression and relapse of aversive learning and memory are poorly understood. Uncertainty is a key feature in anxiety disorders, and laboratory experiments suggest that the BNST may be required for processing ambiguous signals. Utilizing various modern neuroscientific techniques, including behavioral analyses, intracranial pharmacology, and immunohistochemistry, the current work explored the critical factors and boundary conditions that control BNST-dependent learning and memory. In particular, we utilized an important and clinically relevant animal model—known as Pavlovian fear conditioning, extinction, and relapse—to probe contributions of the BNST to fear- and anxiety-related defensive behaviors. These processes involved exposing rats to pairings of discrete auditory and environmental stimuli (tones and static contexts) with an aversive stimulus (footshock). Animals will come to express conditioned fear responses (defensive immobility) to the conditioned auditory and contextual stimuli alone. These fear behaviors can be extinguished by presenting the conditioned stimuli in the absence of the aversive outcome till fear subsides—relapse of conditioned behaviors can occur after after a variety of aversive triggers. In the current work, we explored the contextual factors that regulate the relapse of extinguished fear, and we identified the BNST as a critical regulator of relapse, particularly in cases where there is uncertainty of when an aversive stimulus might occur. Temporal uncertainty of an aversive outcome as an overarching factor was tested in detail, revealing a critical role for timing in the recruitment of BNST afferents to learned fears. Primary contributions of the BNST and its neural circuits to conditioned behaviors are analyzed and discussed. In total, this work suggests that temporal and contextual mechanisms, involving the BNST, may contribute to anxious symptoms and relapse. Accordingly, the BNST should be a target of possible therapeutic intervention for anxiety disorders

Dissecting the neuronal basis of threat responding in mice

Environmental threats demand adaptive defensive responses of an organism that ensure its survival. Extreme stressors, however, can unbalance stress homeostasis and lead to long-term changes that impair appropriate defensive behaviors and emotional responses. In my thesis, I assessed (1) the interaction of two stress-related neuromodulatory systems, (2) the effects of a traumatic incident on brain volume and hyperarousal, and (3) sonic vocalization as a defensive behavior in mice, and discussed the topics in three independent studies.In the first study, I evaluated the interaction of two regulatory systems with respect to fear, anxiety, and trauma-related behaviors. Although the endocannabinoid and the corticotropin-releasing factor (CRF) systems are well described in modulating stressrelatedresponses, the direct interaction of both systems remained poorly understood. The generation of a new conditional knockout mouse line that selectively lacked the expression of the cannabinoid type 1 (CB1) receptor in CRF-positive neurons presented no differences in various tests of fear and anxiety-related behaviors under basal conditions or after a traumatic event. Also stress hormone levels were unaffected. However, male knockout animals exhibited a significantly increased acoustic startle response thus suggesting a specific involvement of CB1-CRF interactions in controlling arousal.In the second study, I assessed the consequences of a traumatic experience on behavior and grey matter volume in mice. Whole-brain deformation-based morphometry (DBM) by means of magnetic resonance imaging (MRI) after incubation of a traumatic incident showed changes in the dorsal hippocampus and the reticular nucleus. Using the severity of hyperarousal as regressor for cross-sectional volumetric differences between traumatized mice and controls revealed a negative correlation with the dorsal hippocampus. Further, longitudinal analysis including volumetric measurements before and after the traumatic incident showed that volume reductions in the globus pallidus reflect trauma-related changes in hyperarousal severity.In the third study, I characterized sonic vocalization as a defensive behavior in mice. Mice bred for high anxiety-related behavior (HAB) were found to have a high disposition to emit audible squeaks when taken by the tail which was not the case for any of the other five mouse lines tested. The calls emitted had a fundamental frequency of 3.8 kHz and were shown to be sensitive to anxiolytic but not panicolytic compounds. Manganese-enhanced MRI (MEMRI) scans pointed towards an increased tonic activity, among others, in the periaqueductal grey (PAG). Inhibition of the dorsal PAG by muscimol not only completely abolished sonic vocalization, but also reduced anxiety-like behavior. This suggests that sonic vocalization of mice is related to anxiety and controlled by the PAG. To explore the ecological relevance of defensive vocalization, I performed playback experiments with conspecifics and putative predators. Squeaks turned out to be aversive to HAB mice but became appetitive to both mice and rats when a stimulus mouse was present during playback.Collectively, the results of this thesis provide novel insights into fear and anxiety-related behaviors and shine light onto their mechanistic basis and ecological relevance

IMAGING EMOTIONAL SOUNDS PROCESSING AT 7T

Emotional sounds and their localization are influential stimuli that we need to process all along our life. Affective information contained in sounds is primordial for the human social communications and interactions. Their accurate localization is important for the identification and reaction to environmental events. This thesis investigate the encoding of emotional sounds within auditory areas and the amygdala (AMY) using 7 Tesla fMRI. In a first experiment, we studied the encoding of emotion and vocalization and their integration in early-stage auditory areas, the voice area (VA) and the AMY. We described that the response of the early-stage auditory areas was modulated by the vocalization and by the affective content of the sounds, and that this affective modulation is independent of the category of sounds. In contrast, AMY process only the emotional part, while VA is responsible for the processing of the emotional valence specifically for the human vocalization (HV) categories. Finally, we described a functional correlation between VA and AMY in the right hemisphere for the positive vocalizations only. In a second experiment, we investigated how the spatial origin of an emotional sound (HV or non- vocalizations) modulated its processing within early-stage auditory areas and VA. We highlighted a left hemispace preference for the positive vocalizations encoded bilaterally in the primary auditory cortex (PAC). Moreover, comparison with the first study indicated that the saliency of emotional valence could be increased by spatial cues, but that the encoding of vocalization is not impacted by the spatial context. Finally, we examined the functional correlations between early-stage auditory areas and VA and how they are modulated by the sound category, the valence and the lateralization. We documented a strong coupling between VA and early-stage auditory areas during the presentation of emotional HV, but not for other environmental sounds. The category of sound modulated strongly the functional correlations between VA, PAC and auditory belt areas, while the spatial positioning induced only a weak modulation and no modulation was caused by the affective content. Overall, these studies demonstrate that the affective load modulates the processing of sounds within VA only for HV, and that this preference for vocalizations impacts the functional correlations of VA with other auditory regions. This strengthens the importance of VA as a computation hub for the processing of emotional vocalizations. -- Les sons émotionnels ainsi que leur localisation sont des stimuli importants que nous devons traiter tout au long de notre vie. L’information affective contenue dans les sons est primordiale pour les communications et interactions sociales. Leur localisation correcte est importante pour l’identification et la réaction par rapport aux événements nous entourant. Cette thèse étudie l’encodage des sons émotionnels dans les aires auditives et l’amygdale (AMY) en utilisant l’IRM fonctionnel à 7 Tesla. Dans une première expérience, nous avons étudié l’encodage des émotions et des vocalisations, ainsi que leur intégration dans les aires auditives primaires et non-primaires, dans l’aire des voix (VA) et dans AMY. Nous avons décrit que la réponse des aires auditives primaires et non-primaires étaient modulées par les vocalisations ainsi que par le contenu affectif des sons, et que cette modulation affective était indépendante de la catégorie sonore. En revanche, AMY traite uniquement la partie émotionnelle, tandis que la VA est responsable du traitement de la valence émotionnelle spécifiquement pour les vocalisations humaines (HV). Finalement, nous avons décrit une corrélation fonctionnelle entre VA et AMY dans l’hémisphère droit pour les vocalisations positives uniquement. Dans une seconde expérience, nous avons cherché à comprendre de quelle manière l’origine spatiale d’un son émotionnel (HV et non-vocalisations) modulait son traitement dans les aires auditives, primaires et non-primaires, et VA. Nous avons mis en évidence une préférence de l’hémi-champ gauche pour les vocalisations positive encodées bilatéralement dans le cortex auditif primaire (PAC). De plus, une comparaison avec la première étude a indiqué que l’importance de la valence émotionnelle pourrait être augmentée grâce aux indices spatiaux, mais que l’encodage des vocalisations n’étaient pas impacté par le contexte spatial. Finalement, nous avons examiné les corrélations fonctionnelles entre les aires auditives primaires, non-primaires et VA afin d’évaluer de quelle manière elles étaient modulées par la catégorie sonore, la valence et la latéralisation. Nous avons mis en évidence un fort couplage entre VA et les aires auditives primaires et non-primaires durant la présentation des HV émotionnelles, mais cet effet n’était pas présent pour les autres sons environnementaux. La catégorie sonore modulait fortement les corrélations fonctionnelles entre VA, PAC et les régions auditives latérales, alors que le positionnement spatial n’influençait que faiblement leur modulation. De plus, il n’y avait pas de modulation causée par le contenu affectif. En résumé, ces études démontrent que le contenu affectif module le traitement des sons dans VA uniquement pour les HV, et que cette préférence pour les vocalisations a un impact sur les corrélations fonctionnelles de cette région avec les autres régions auditives. Cela souligne l’importance de VA comme centre computationnel pour le traitement des vocalisations émotionnelles

Brain oscillations and novelty processing in human spatial memory

Hippocampal activity in rodent model systems is commonly associated with movement and exploratory behaviour, while human hippocampal research has traditionally focused on mnemonic function. I attempted to bridge this gap with a set of experiments where human participants performed an interactive virtual navigation paradigm that resembled rodent spatial exploration tasks, in conjunction with neuroimaging techniques such as functional magnetic resonance imaging (fMRI) and magnetoencephalography (MEG). I then used this interactive paradigm to examine the oscillatory correlates of memory, novelty and the behavioural relevance of the default mode network. The first experiment used MEG and fMRI to examine whether the movement-related theta rhythm (4-8 Hz) recorded from the rodent hippocampus has a measurable human analog. I found that the human hippocampal theta rhythm supports memory, and may coordinate exploratory movements in the service of self-directed learning. In further analyses in Experiment 2, during cued spatial memory retrieval, I observed that medial prefrontal cortex theta phase couples with ongoing theta oscillations in the right anterior medial temporal lobe and with neocortical gamma (65-85 Hz) amplitude. In Experiment 3, with fMRI I investigated the effect of environmental novelty versus object novelty during the navigation task and found that hippocampal activity is modulated only by environmental novelty, while the fusiform gyrus/posterior parahippocampal cortex responded to object novelty. Finally, in Experiment 4 using 3T and high-field 7T fMRI, I investigated endogenous (task-free) periods that flanked different stages of a spatial navigation paradigm to determine how endogenous slow oscillations in the default mode network correlate with subsequent spatial memory performance and found mixed evidence that default mode network activity predicts individual performance. Finally, I discuss my results in the context of recent findings in spatial memory and novelty processing, and consider the relationship between the human hippocampus and rodent model systems

Hippocampal regulation of encoding and exploration under the influence of contextual reward and anxiety

Hippocampal researchers have recently turned their attention to the computations that may be implemented by the hippocampal circuit (e.g. pattern separation and pattern completion). This focus on the representational and information-processing capabilities of the hippocampus is likely to be important in resolving on-going debates regarding the nature of hippocampal contributions to perception, anxiety and exploration. A first aim of my research was to examine how context representations interact with reward to influence memory for embedded events. In my first experiment, I show that recollection for neutral objects is improved by sharing a context with other rewarding events. To further examine contextual influences on memory, I conducted a second experiment that examined the effect of contextual reward itself on object memory. Additionally, I manipulated the extent to which disambiguation should rely on hippocampal computations, by varying the perceptual similarity between the rewarding and neutral contexts. Improved object memory was only observed when the rewarding and neutral contexts were perceptually similar, and this contextual memory effect was further linked to co-activation of the hippocampal CA3/dentate gyrus and substantia nigra/ventral tegmental area. A second major aim of my work was to characterize hippocampal contributions to anxiety. In my third experiment, I combine a novel experiment with fMRI to show that hippocampal activation is associated with behavioural inhibition rather than exploratory risk assessment. This insight is important because a major theoretical perspective in the literature conflates these two psychological processes. In my final experiment, I employ this novel experimental paradigm to examine the effect of exploration on memory, and find that the propensity to explore (rather than the act of exploring per se) leads to better memory at subsequent recall

Quantification of appetite-regulating hormones in children with hypothalamic and common obesity

Background. Current understanding of the appetite-regulating neuroendocrine circuitry remains incomplete, and efficacious treatments for both common and hypothalamic obesity (HyOb) are lacking. Concurrently, the expanded role of oxytocin (OXT) in energy homeostasis and human behaviour is beginning to be understood. Objectives. To optimise and translate an OXT enzyme immunoassay (EIA) to elucidate whether there were any unique differences in the plasma endocrine milieu in patients with HyOb. Methods. Optimisation work was carried out using EIAs with polyclonal and monoclonal secondary antibodies. Obese (BMI>+2 SDS) and lean (BMI≤+2 SDS) children with (HyOb and HyLean) and without (Ob and Lean) hypothalamic disorders (septo-optic dysplasia or suprasellar tumours) were phenotyped using the Dykens’ Hyperphagia Questionnaire Score (DHQS). Plasma concentrations of leptin, insulin, OXT, BDNF, αMSH, acylated ghrelin, AgRP and copeptin were measured. Results. Solid phase extraction demonstrated markedly variable OXT recovery, and potentially increased rather than decreased interference. A polyclonal secondary antibody-containing EIA showed significant cross-reactivity with several peptides in human plasma compared to a monoclonal secondary antibody-containing EIA. Of the 122 children recruited (50 HyOb, 29 HyLean, 24 Ob, 19 Lean, mean age 11.3±3.9 years) there were no differences in DHQS or hormone concentrations between HyOb and Ob groups. Obesity was associated with compensatorily increased leptin and insulin, and decreased ghrelin and AgRP concentrations. More rapidly increasing BMI was independently associated with a younger age and lower plasma αMSH concentrations. OXT concentrations did not show any correlation with BMI or DHQS. Conclusion. The use of plasma extraction processes and EIAs in the literature needs re-examination. The plasma endocrine milieu in HyOb vs. common obesity does not differ, with a compensatory increase in anorexigens and decrease in orexigens. Lower plasma αMSH was associated with more rapid weight gain, suggesting that MC4R agonists may be a therapeutic option in all forms of obesity