When a human or animal is threatened or confronted with a stimuli signalling danger, internal defence mechanisms are activated that evoke feelings of fear and anxiety. These emotional responses promote the behaviour patterns necessary for an organism's survival. Animal research seeks to understand how these emotions affect behaviour both physiologically and neurologically in order to develop effective treatment for those suffering from severe anxiety disorders. The aim of this thesis was to examine the role of the amygdala, and dorsal and ventral hippocampus in relation to immediate fear arousal brought on by footshock. This was assessed by examining whether muscimol would interfere with the acoustic startle response before or after footshock presentation, and then comparing these reactions to a control group that received saline infusions. The results of this research are extremely important because they identify various brain structures involved in the fear-arousing effects of footshock as measured by the shock sensitization of acoustic startle. Laboratory rats received muscimol (0.1ug and 0.01ug) infusions into the basolateral amygdala, dorsal and ventral hippocampus. These three brain regions have been identified as playing a prominent role in fear neurocircuitry. The results demonstrated that the GABA A receptor agonist muscimol in doses of 0.1ug and 0.01ug reliably blocked shock sensitization of the acoustic startle response. The muscimol doses did not alter the shock reactivity amplitudes therefore indicating a normal perception of the fear arousing properties of footshock. Therefore, the present study's results suggest that a decrease of GABA activity in the amygdala, dorsal and ventral hippocampus may be essential for the neuronal basis of fear acquisition and expression of unconditioned and conditioned stimuli.

When a human or animal is threatened or confronted with a stimuli signalling danger, internal defence mechanisms are activated that evoke feelings of fear and anxiety. These emotional responses promote the behaviour patterns necessary for an organism's survival. Animal research seeks to understand how these emotions affect behaviour both physiologically and neurologically in order to develop effective treatment for those suffering from severe anxiety disorders. The aim of this thesis was to examine the role of the amygdala, and dorsal and ventral hippocampus in relation to immediate fear arousal brought on by footshock. This was assessed by examining whether muscimol would interfere with the acoustic startle response before or after footshock presentation, and then comparing these reactions to a control group that received saline infusions. The results of this research are extremely important because they identify various brain structures involved in the fear-arousing effects of footshock as measured by the shock sensitization of acoustic startle. Laboratory rats received muscimol (0.1ug and 0.01ug) infusions into the basolateral amygdala, dorsal and ventral hippocampus. These three brain regions have been identified as playing a prominent role in fear neurocircuitry. The results demonstrated that the GABA A receptor agonist muscimol in doses of 0.1ug and 0.01ug reliably blocked shock sensitization of the acoustic startle response. The muscimol doses did not alter the shock reactivity amplitudes therefore indicating a normal perception of the fear arousing properties of footshock. Therefore, the present study's results suggest that a decrease of GABA activity in the amygdala, dorsal and ventral hippocampus may be essential for the neuronal basis of fear acquisition and expression of unconditioned and conditioned stimuli.

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Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects. We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation. We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear. / Doctor of Philosophy / Changes in epigenetic mechanisms, processes that control the expression of genes without changing the original sequences, play a crucial role in the formation and maintenance of memory. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, helping to understand post-traumatic stress disorder (PTSD). However, these majorly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. In the present study, we investigated some of these epigenetic mechanisms called histone modifications. In the brain, histone 3 lysine 4 trimethylation (H3K4me3), a histone modification, is critical for memory formation in the hippocampus, a key area for memory storage. However, it is still not fully understood how H3K4me3 is coordinated during memory formation. Another histone modification called H2B monoubiquitination (H2Bubi) helps recruit H3K4me3 to DNA and so is also crucial for memory formation. Here, using rat models, we found that the expression of 86 genes is changed during memory formation in the hippocampus and that this result is almost entirely dependent on the presence of H2Bubi. We also discovered that H2Bubi is critical for longterm memory formation only in neurons of the hippocampus, and not astrocytes (another type of brain cells). Additionally, there is a connection between H3K4me3 and the phosphorylation of histone H2A.X, another epigenetic mechanism that co-occurs with DNA breaks and may serve as a markerfor studying these breaks. DNA breaks play a vital role during gene expression and could be triggered by neuronal activity during memory formation. We observed an increase in H2A.X phosphorylation and H3K4me3 levels in a memory-permissive gene five minutes after memory retrieval. Inhibition of DSBs, prior to retrieval abolished these changes, and impaired long-term memory. This suggests a critical role for DSBs in memory maintenance and that H2A.X phosphorylation is necessary for the recruitment of H3K4me3 to DNA. Lastly, our data demonstrated that both males and females could learn fear indirectly from either sex by observing them undergoing auditory-cued fear conditioning. Additionally, we found distinct molecular patterns in the amygdala, a brain region involved in fear processing, depending on whether fear was directly or indirectly acquired, and it varied between sexes. Collectively, data from this dissertation reveals novel roles for histone modifications in memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.

Thesis advisor: John P. Christianson / Trauma and exposure to extreme stressors greatly increases a person’s vulnerability to developing mental illnesses like post-traumatic stress disorder (PTSD). Patients with PTSD often have impaired fear and safety learning, and despite the fact that women are more than twice as likely to develop PTSD, much of the research on this disorder has relied on the use of male subjects. This paper will review potential contributors to the sex differences seen in PTSD and fear-related learning. Our group has found that female rats show greater fear discrimination abilities than their male counterparts, but show no difference in levels of safety learning. Analysis of specialized extracellular matrix structures called perineuronal nets (PNNs) revealed that females displayed a much higher density of PNNs in the basolateral amygdala (BLA) than males, but not in the prefrontal cortex (PFC). / Thesis (BS) — Boston College, 2017. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Psychology.

Fear conditioning and extinction are central in the cognitive behavioral model of obsessive-compulsive disorder (OCD), which underlies exposure-based cognitive behavioral therapy (CBT). Youth with OCD may have impairments in conditioning and extinction that carries treatment implications. The present study examined these processes using a differential conditioning paradigm. Forty-one youth (19 OCD, 22 community controls) and their parents completed a battery of clinical interviews, rating scales, and a differential conditioning task. Skin conductance response (SCR) served as the primary dependent measure across all three phases of the conditioning procedure (habituation, acquisition, and extinction). During habituation, no meaningful differences were observed between groups. During acquisition, differential fear conditioning was identified across groups evidenced by larger SCRs to the CS+ compared to CS-, with no significant group differences. During extinction, a three-way interaction and follow-up tests revealed youth with OCD failed to exhibit differential fear conditioning during early fear extinction; whereas community controls consistently exhibited differential fear conditioning throughout extinction. Across participants, the number and frequency of OCD symptoms was positively associated with fear acquisition and negatively associated with fear extinction to the conditioned stimulus. OCD symptom severity was negatively associated with differential SCR in early extinction. Youth with OCD exhibit a different pattern of fear extinction relative to community controls that may be accounted for by impaired inhibitory learning in early fear extinction. Findings suggest the potential benefit of augmentative retraining interventions prior to CBT. Therapeutic approaches to utilize inhibitory-learning principles and/or engage developmentally appropriate brain regions during exposures may serve to maximize CBT outcomes.

Neural activity within the brain underlies complex behavior that allows us to interact with our environment. The anterior cingulate cortex (ACC) is believed to mediate appropriate behavioral responses by integrating emotional and cognitive information about external stimuli. If this understanding is correct, then neural activity within the ACC must therefore correlate with behavioral output in response to external experience. The aim of this thesis is to bridge mechanisms identified in vitro with behaviors observed in vivo to determine the neural substrates of ACC mediated behavior. This thesis focuses on glutamatergic receptors that have been established as mediators of excitatory transmission in the ACC. Through a combination of behavioral, pharmacological, biochemical, and electrophysiological methods, this thesis examined how behaviors observed in mouse models of fear learning, chronic pain, and itch correspond with in vitro observations of ACC neuronal activity. Three sets of experiments are presented. The first set investigated cortical LTP-like mechanisms, and assessed whether they could mediate fear learning. These sets of experiments provide in vivo evidence that trace fear learning requires rapid, NMDA receptor dependent, cortical AMPA receptor insertion. The second set of experiments investigated the contribution of forebrain CREB-mediated transcription in behavioral manifestations of chronic pain. These experiments show that forebrain overexpression of CREB is sufficient to enhance mechanical allodynia in animal models of chronic inflammatory or neuropathic pain. Lastly, the final set of experiments show that pruritogen-induced scratching corresponds with enhanced excitatory transmission in the ACC through KA receptor modulation of inhibitory circuitry. Through investigations of multiple behaviors linked to ACC activity, this thesis presents evidence that manifestations of behavior can be observed at the molecular level, and indicates that molecular mechanisms involved in ACC synaptic activity are a good target for translational research into pathological conditions that are related to abnormal ACC activity.

Due to difficulties in elucidating neurobiological aspects of psychological disorders, the National Institute of Mental Health (NIMH) created the Research Domain Criteria (RDoC), which encourages novel conceptualizations of the relationship between neurobiological circuitry and clinical difficulties. This approach is markedly different from the Diagnostic and Statistical Manual of Mental Disorders (DSM) based approach that has dominated clinical research to date. Thus, RDoC necessitates exploration of novel experimental and statistical approaches. Fear learning paradigms represent a promising methodology for elucidating connections between acute threat (“fear”) circuitry and fear-related clinical difficulties. However, traditional analytical approaches rely on central tendency statistics, which are tethered to a priori categories and assume homogeneity within groups. Growth Mixture Modeling (GMM) methods such as Latent Class Growth Analysis (LCGA) may be uniquely suited for examining fear learning phenotypes. However, just three extant studies have applied GMM to fear learning and only one did so in a human population. Thus, the degree to which classes identified in known studies represent characteristics of the general population and to which GMM methodology is applicable across populations and paradigms is unclear. This preliminary study applied LCGA to a fear learning lab study in an attempt to identify heterogeneity in fear learning patterns based on a posteriori classification. The findings of this investigation may inform efforts to move toward a trans-diagnostic conceptualization of fear learning. Consistent with the goals laid out in RDoC, explication of fear learning phenotypes may eventually provide critical information needed to spur innovation in psychotherapeutic and psychopharmacological treatment. / Master of Science / To date, most clinical psychology research has been based on the Diagnostic and Statistical Manual of Mental Disorders (DSM), which is a catalog of mental health disorders that was originally designed to facilitate communication among clinicians. Many experts contend that this approach has hampered progress in the field of biological clinical psychology research. Thus, the National Institute of Mental Health (NIMH) created a new template for biological clinical psychology research called the Research Domain Criteria (RDoC). Since RDoC calls for a complete overhaul in the conceptualization of clinical dysfunction, this approach requires statistical and experimental innovation. One traditional experimental approach that may be helpful in understanding the RDoC topic of acute threat (“fear”) is called Pavlovian Fear Learning (PFL). However, traditional PFL studies have utilized statistical methods that are based on comparing group averages and require researchers to determine groups of interest based on theory before the study begins. This is problematic because RDoC calls for research that begins with evidence rather than theory. Growth Mixture Modeling (GMM) is a statistical methodology that may allow researchers to analyze fear learning data without having to begin with theoretically determined categories such as DSM disorders. However, little research has tested how well this approach would work. This study is just the second to apply a GMM approach to a human PFL study. The findings from this investigation may inform efforts to develop a statistical technique that is well suited for RDoCian research and may also spur innovation in psychotherapeutic and psychopharmacological treatment.

Hypothermia therapy (HT) is used clinically following global cerebral ischemia (GCI) but its therapeutic mechanisms are not completely understood. An elucidation of such mechanisms may lead to novel therapeutic approaches that improve patient outcome. Using a murine model of GCI, we determined the effect of HT on the expression of inflammatory proteins in the hippocampus and serum. We also examined its effect on microglia/macrophage activation and neurodegeneration in the brain at 72 hours following ischemia, and its effect on long-term spatial memory/learning and contextual fear response. GCI led to increased neurodegeneration and microglia/macrophage activation in the hippocampus, and increased IL-1β and KC protein expression in the hippocampus at 72 hours. Hypothermia therapy attenuated these inflammatory responses. It also improved spatial learning/memory at 7 and 21 days, and preserved contextual fear response 21 days post-ischemia. Hypothermia therapy attenuated the post-ischemic inflammatory response, protected hippocampal neurons, and preserved long-term memory and learning.

Hypothermia therapy (HT) is used clinically following global cerebral ischemia (GCI) but its therapeutic mechanisms are not completely understood. An elucidation of such mechanisms may lead to novel therapeutic approaches that improve patient outcome. Using a murine model of GCI, we determined the effect of HT on the expression of inflammatory proteins in the hippocampus and serum. We also examined its effect on microglia/macrophage activation and neurodegeneration in the brain at 72 hours following ischemia, and its effect on long-term spatial memory/learning and contextual fear response. GCI led to increased neurodegeneration and microglia/macrophage activation in the hippocampus, and increased IL-1β and KC protein expression in the hippocampus at 72 hours. Hypothermia therapy attenuated these inflammatory responses. It also improved spatial learning/memory at 7 and 21 days, and preserved contextual fear response 21 days post-ischemia. Hypothermia therapy attenuated the post-ischemic inflammatory response, protected hippocampal neurons, and preserved long-term memory and learning.