Michael Fanselow - US grants

The present proposal seeks to understand how fear maps into behavior from a functional perspective. In addition, it seeks to uncover mechanisms that underlie the performance of such fear motivated behavior. In order to survive, animals must be able to react to a variety of environmental dangers with swift but effective patterns of defensive behavior. The animal may react with any one of a variety of motor behaviors such as changes in feeding pattern, freezing, or vigorous escape, with the particular behavior selected being a function of the degree of threat. Besides motor behaviors the fear reaction is accompanied by autonomic modulation and analgesia that support the motor behaviors. This coordination of environmental stimuli and behavior serving the function of defense against environmental dangers is referred to as the defensive behavior system. Within this framework, fear and anxiety are convenient labels to refer to the activation of this functional behavior system. Obviously, this elaborate orchestration of stimuli and responses must be performed by an efficient and well integrated neural circuitry. The evidence reviewed suggests that the periaqueductal gray matter is in an ideal position to coordinate these various response components with the degree of threat. The experiments proposed in this grant application are designed to build on this relationship of the PAG to the organization of defensive behavior and also to further develop our knowledge of the organization of defensive behavior in response to manipulations of relevant environmental variables. The experiments described attempt to determine how differing levels of fear map into different modes of behavioral action. We will attempt to find the brain sites corresponding to performance of conditional and unconditional defensive responses to further clarify how these responses interact. It will be determined if activity in these brain regions is necessary and sufficient for performance fear of related defensive behaviors and particular attention will be paid to the separation of learning and performance factors. We will also conduct an analysis of the mechanisms responsible for conditional fear mediated by emotional as opposed to representational associations.

In many situations fear is a learned response. This research project seeks to understand the neurobiological mechanisms underlying these learned fears. One line of research examines manipulations of N-methyl-d-aspartate receptors to see if they are involved in encoding of fear memory as opposed to memory consolidation, retrieval or behavioral performance. This receptor is interesting because it has been implicated in certain changes in neurons that may provide a way for the nervous system to store information. The second line of research explores the role of endogenous opioids (endorphins) in fear learning. Fear causes release of endorphins and these endorphins in turn limit or regulate additional learning. This regulation normally results in controlling the magnitude of fear, so that the amount of fear is appropriate for the particular circumstance.

Lay Abstract PI: Fanselow, Michael S. Proposal Number: IBN-9723295 When information is first learned, the brain stores this new memory in a form that is very susceptible to disruption. Over time, one particular part of the brain, the hippocampus, acts to consolidate these memories into a more indelible form. If the hippocampus is damaged or disrupted before consolidation of a particular experience is complete, there is an amnesia for that experience. While it is known that this memory consolidation goes on for a very long time, very little is known about what causes consolidation to occur or what factors control the duration of this process. This project provides crucial information about how memory consolidation is accomplished. There are two lines of investigation. The first examines several environmental manipulations that prevent this amnesia or shorten or extend the period of consolidation. These experiments provide information on the mental processes that cause the brain to consolidate its experiences. The second line of research determines how the various parts of the hippocampus are involved in the consolidation process. Together the two parts of this project provide information about how the brain operates on our experiences to create permanent memories.

[unreadable] DESCRIPTION (provided by applicant): We believe that the greatest progress toward understanding neural and genetic processes that contribute to normal and abnormal behavior, cognition and emotion will occur in a context where expertise in neuroscience goes hand in hand with a background in behavioral and cognitive psychology. We propose to educate and support the research of 4 predoctoral trainees working toward a Ph.D. degree and 2 postdoctoral trainees, in an integrative approach to Behavioral Neuroscience. Toward this end, the training program is centered in one of the foremost Psychology Departments in the world but combines that strength with the Brain Research Institute, one of the first and largest interdisciplinary neuroscience organizations in the world. Both are in close proximity on the UCLA campus offering state of the art facilities in all aspects of neural and behavioral science. Our 31 preceptors are organized into four areas of concentration: Learning and Memory, Neuroscience of Mental Disorders, Vision, and Cognitive Neuroscience. The predoctoral program draws from the Ph.D. programs in Psychology and the Interdepartmental Program in Neuroscience. Our goal is provide students with experience specifically designed to make them better able to do psychologically informed neuroscience research and neuroscientifically informed behavioral research because those will be the future scientists who are most likely to advance approaches to mental health. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): While the necessity of the hippocampal formation for some types of learning and memory largely is agreed upon, the exact role that the hippocampus plays in contextual fear conditioning is unknown. The proposed project is based on a theoretical model of how the hippocampus interacts with the amygdala in fear conditioning. The model is used to generate a set of predictions about the circumstances under which the hippocampus becomes involved in context conditioning. Furthermore, predictions are made regarding the effects of hippocampal manipulations (anatomical and pharmacological) on the establishment and performance of conditioned fear. With a cohesive set of manipulations and training parameters, the proposed studies will evaluate the model's applicability to fear conditioning phenomena. Firstly, the experiments will establish a temporal profile of hippocampal involvement. Pre- and post-training manipulations of the chemical and anatomical integrity of the hippocampus will reveal the points at which the hippocampus is necessary for learning. Secondly, a direct comparison of electrolytic and excitotoxic lesions will lend insight into the relationship between environmental explorations and context processing. Thirdly, the experiments will examine the effects of hippocampal manipulations on the immediate shock deficit, an exploration-related impairment in context fear conditioning. Finally, the cholinergic innervation of the hippocampus will be manipulated to determine its role in retrograde and anterograde amnesia produced by hippocampal lesions. At each stage in the experimentation, specific predictions are made based upon manipulations of hippocampal integrity and the disruption of hippocampus-amygdala interactions. It is hoped that, in addition to providing crucial information for the investigation of learned fear, the experiments will provide insight into the condition of those suffering from hippocampal damage.

How the brain extracts information to from the environment and stores that information as a memory is a fundamental but unanswered question in behavioral neuroscience. The brain structure that is most intimately associated with memory in humans and other animals is the hippocampus. Damage to this brain area produces amnesia, or loss of memory, that shares many features in all mammals. This project focuses on two of these commonalties. One is that memories that were acquired shortly before hippocampal damage are lost but older memories remain. This pattern of memory loss is called temporally graded retrograde amnesia. Retrograde amnesia in rats will be examined by making very precise damage to the hippocampus after various times after rats are trained on a task that is not normally forgotten. The task is fear conditioning, where rats show an emotional reaction to stimuli that have previously predicted that they will receive a brief mild electric footshock.
Another feature of the amnesia that stems from hippocampal damage is that certain types of memories are lost and others are completely spared. For example, if people with hippocampal damage are taught a new motor skill like tennis, their tennis playing ability will continual improve but they will have no memory of ever playing tennis. They remember how to play without being able to remember that they played. One form of this selective memory loss will be examined. If a tone predicts shock, hippocampal lesions may or may not cause amnesia for the relation between tone and the learned emotion. If the tone and shock overlap in time the hippocampus is not necessary. However, if a brief period of time is placed between tone termination and shock onset the hippocampus is necessary for memory. This type of conditioning is called trace conditioning and retrograde amnesia for trace conditioning following hippocampal damage will be examined.
Amnesia for trace conditioning is unusual because it differs from other forms of memory for which the hippocampus is needed. Usually rats with hippocampal damage have problems with memory for spatial locations or for stimuli that are composed of several different components. In this project it will be determined whether retrograde amnesia for trace conditioning and complex configurations stem from the same or different functions of the hippocampus. It will also be determined if various manipulations that alter the ability of normal animals to learn trace conditioning can alleviate the amnesia for trace conditioning. By investigating and contrasting these forms of memory it is hoped to find exactly what the hippocampus is extracting from experience and more precisely define what is lost and preserved in amnesia.

Amnesia is a consequence of enhancing GABAergic neurotransmission in clinical settings and GABA systems play a role in some forms of mental retardation but the precise role of GABA receptors in well-defined learning circuits is not well characterized. Stress and gonadal hormones also influence GABA function and learning processes but even less is known about the extent to which these factors converge on common brain mechanisms. Using learning preparations that have well characterized neural circuitry we will determine how stress and other alterations in the hormonal milieu act through GABA receptors to influence learning. A key focus of this work will be how hormonal factors impact learning and memory via neurosteroid action at d subunit containing GABA receptors. While d-subunits have limited distribution in the brain, two regions with especially high expression, the dentate gyrus of the hippocampus and granule cells of the cerebellum play a fundamental role in learning and memory. Therefore d subunit containing GABA receptors offer a site at which factors such as stress, can modulate learning systems. Our central hypothesis is that hormonal factors impact learning and memory via neurosteroid action at d subunit containing GABA receptors within the neural circuits mediating specific forms of memory. This hypothesis is based on 4 key findings: 1) Neurosteroids act on dGABARs. 2) dGABARs are strategically placed within well-defined learning/memory circuits. 3) Factors such as stress and estrus cycle influence both hormonal background and learning and memory. 4) d subunit knockout mice are not responsive to neurosteroids. Hormonal variation associated with the estrus cycle will provide one avenue for addressesing these issues. A second will be an animal model of past-traumatic stress disorder, where prior stress enhances certain forms of learning. We seek to determine how both of these factors converge to modulate learning and memory by action an delta containing GABAA receptors.

Principal Investigator/Program Director: Evans, Christopher J., Ph.D., CSORDA Mouse Core CORE: CSORDA Mouse Core ABSTRACT: The CSORDA Mouse Core (CMC) will coordinate and oversee the animal needs of all projects in the Center conducted at UCLA. It will maintain all mutant animal lines needed by the center and make a reliable supply of these animals available to all Center research components. The CMC will also genotype mutant animals used in by the Center for breeding and research. All the Projects of CSORDA will make extensive use of mice, particularly those coming from genetically modified lines. The purpose of the CSORDA Mouse Core (CMC) is to provide these research subjects to all the Components of the Center. The CMC will establish and maintain lines of genetically manipulated mice for use by the Components. The CMC was established in the previous finding cycle and developed a solid set of standardized procedures to accomplish this goal. The CMC continually consults with all Components in the Center about which particular strains of mice are needed and the numbers required to meet the demands of proposed experiments within those Components.

DESCRIPTION (provided by applicant): Following exposure to traumatic stress individuals often go on to develop Post-Traumatic Stress Disorder (PTSD), which is characterized by enhanced anxiety to reminders of the trauma, nightmares, reliving the traumatic experience and a propensity to acquire new fears. PTSD is characterized by excessive drinking, anxiety, and depression that lasts for a prolonged period after the initial trauma. The result is that PTSD has a significant impact on the lives of individuals as well as on society as a whole. One challenge is to find the common link between stress-induced changes in anxiety, alcohol intake and depression. Considerable data suggest that neuroimmune factors may be that link. Once thought to be merely the packing peanuts of the brain, glial cells in the brain and spinal cord are integral to the proper functioning, signaling, and neuronal reparation in the brain. Microglia specifically are involved in neuroinflammation, which is associated with destructive chronic neuroimmune response. Activation of microgila produces pro-inflammatory cytokines. Increases in levels of pro-inflammatory cytokines are observed in PTSD, and major depressive disorder (MDD). Microglia as well as pro-inflammatory cytokines are increased in the brains of alcoholics. Thus, neuroimmune factors lay at a nexus of PTSD, depression and alcoholism. It is not known what mediates these long-term stress-induced changes, but stress can produce a neuroimmune response that significantly alters behavior. To address this challenge, we have assembled a team with demonstrated expertise in emotion related behavior (Fanselow), stress-induced neuroimmune function (Bradesi &Mayer), and the neurobiology of alcohol (Spigelman). Using a behavioral model of PTSD, our aims are to determine if stress is associated with a neuroimmune response within the amygdala and to determine if this response predicts changes in behavior, as well as determine if blockade of glia activation/neuroimmune response will reverse the behavioral sequelae of stress. Preliminary data with this stress model indicates exaggerated reactions to novel startling stimuli, increased anxiety and enhanced learning of new fears, which persist at least 3 months after stress without diminution. The same stress increased voluntary alcohol consumption relative to an unstressed control group. The model also produces prolonged alterations in gene expression patterns in the amygdala with up-regulation of several genes related to cytokines. Although stress-induced activation of central nervous system pro- inflammatory responses have been reported, there are significant gaps in knowledge about how they contribute to long term emotional disturbances and influence the amygdala. PUBLIC HEALTH RELEVANCE: Post-Traumatic Stress Disorder is characterized by excessive drinking, anxiety, and depression that develop and last for a prolonged period after the initial trauma. We will test the hypothesis that neuroimmune factors, such as cytokines and chemokines released in the CNS that are activated by stress mediate these long-lasting changes in behavior by acting like neurotransmitters, neuromodulators, or neurohormones in the brain. We will also determine if pharmacologically interfering with these neuroimmune pathways mitigate the symptoms of PTSD.

DESCRIPTION (provided by applicant): The Behavioral Testing Core (BTC) Facility is a valuable resource for the UCLA scientific community. The UCLA Brain Research Institute membership consists of over 300 faculty members from 26 academic departments. The BTC provides the space, equipment, and instruction necessary for the behavioral testing needs of UCLA researchers. The type of assistance provided by the BTC enables behavioral testing for the researchers that cannot form collaborations with one of the few labs on campus that specialize in behavioral research. The BTC is requesting one-time equipment funding to greatly improve the fear conditioning setup which is widely used to study learning and memory mechanisms, drug and gene effects on fear and anxiety. The current BTC fear conditioning equipment requires replacement. The Med Associates equipment currently in use is outdated and unreliable. The automated scoring programs are first-generation "home-made" systems that do not have the power, flexibility and accuracy of state-of- the-art systems. In order to ensure that researchers get usable results, the BTC would like to provide better equipment for this task. The requested funds would be used to replace the ineffective equipment with state of-the -art instrument from Med Associates, the most-cited manufacturer of fear equipment. Their new Video Freeze software is highly reliable for automated freezing studies. It is the only system that was developed with validation of an expert in fear conditioning (Dr.Stephan Anagnostaras, UCSD). The requested funds cover 12 fear conditioning setups (boxes), controlling software, and computer. Crucial for research in fear extinction is the use of more than 2 contexts to understand how fear can be renewed, re-instated and spontaneously recovered following extinction. These boxes have interchangeable parts that allow the formation of multiple contexts and testing for both rats and mice. In addition, we plan to use 6 of these boxes within the mouse housing room suite to cut the quarantine time (normally 6- 8 weeks) of animals bred off-site (e.g., knock-out mice from a collaborator). This would enable PIs to conduct experiments that are time sensitive (e.g., age and time-dependent gene expression studies). This approach will also allow PI's to cut costs on animal per diems from a prolonged quarantine period. This plan has been developed in conjunction with campus veterinarian Dr.Marcelo Couto and the UCLA Division of Laboratory animals and Medicine. PUBLIC HEALTH RELEVANCE: Fear conditioning is a robust model of fear and anxiety in rats and mice. The use of such a model will allow for a more defined understanding of the neural, genetic, behavioral components underlying human anxiety disorders. Such knowledge would allow for the developments of better treatments.

DESCRIPTION (provided by applicant): Fear, whether it occurs in humans suffering from an anxiety disorder or in experimental models with rodents, is reduced by exposing the frightened organism to the fearful stimulus in the absence of any negative consequences (i.e., extinction, or exposure therapy). However, fear often renews when the feared stimulus is encountered in a context different from the exposure context. In rats, we found that interfering with the animal's ability to process contexts during extinction by administering an anticholinergic drug prevented fear renewal. This proposal will determine if the beneficial effect of this drug translates to exposure therapy in socially anxious humans. To this end, 100 individuals with Social Phobia who fear public speaking will undergo repeated sessions of exposure to public speaking, within a virtual reality context. Participants will be randomized to either drug placebo, .2mg/.01 mL Scopolamine, .3mg/.01 mL Scopolamine or .4mg/.01 mL Scopolamine, administered via nasal drops, prior to each session of exposure therapy. One month after completion of exposure therapy, context renewal will be tested by comparing physiological and subjective responses to public speaking in the same virtual context as used during exposure therapy versus a context different than the one used during exposure therapy. The goal is to identify the dose of Scopolamine associated with the greatest reduction in context renewal. In addition, a secondary analysis will attempt to identify those individuals who benefit most from Scopolamine-augmentation of exposure therapy.

DESCRIPTION (provided by applicant): Understanding the biological mechanisms and biomarkers of psychiatric disease is critical for understanding, assessing risk, and designing treatments of disorders such as post traumatic stress disorder (PTSD). In this regard, it was recently reported that the neuropeptide PACAP and its plasma membrane receptor PAC1 are linked to PTSD at both genetic and epigenetic levels. These findings complement a considerable set of prior evidence implicating PACAP/PAC1 signaling in stress and fear circuitries. Experiments in the proposal will use gene targeting approaches to dissect at the cellular, molecular, and behavioral levels, the involvement of PACAP-PAC1 signaling in the circuitry regulating fear in mice. The results are hoped to lay a mechanistic foundation for the development of an entirely new set of therapeutic targets for PTSD based on PAC1 signaling and downstream actions, and may also lead to the discovery of novel and robust biomarkers associated with this pathway.

Fear in nonthreatening contexts is a hallmark of anxiety disorders. The hippocampus' plays a critical role in recognizing a context as a previously experienced dangerous one or a safe one. We will study the ability to differentiate such contexts in rats. Rats have proven to be an excellent model of human anxiety disorders. Rats need a period to explore a context in order to learn that it is dangerous. Our data indicate that a period just long enough to support robust fear learning results in generalizing that fear to any similar context. They need a longer period to learn a specific fear that allows them to distinguish between danger and safety. We will study the biology of this type of learning. We will examine how a particular neuromodulator, acetylcholine (ACh), influences the ability for this learning and have discovered that stimulating this neuromodulatory system enhances the learning. Anxiety disorders are much more prevalent in women and we will test the hypothesis that at certain times during the female cycle, when metabolites of progesterone are high, they act to dampen the brain's response to this neuromodulator. This results in a state where fear learning will be more nonspecific and generalize to safe situations. To study this we will use optogenetics to selectively stimulate ACh and measure release of this neuromodulator with a novel biosensor implanted in a brain region responsible for learning. We will also measure the rhythms of brain activity produced by the neuromodulator, the ability to learn fear and to differentiate safe and dangerous environments. We will compare male and female rats and also compare females in different stages of their cycle. This will provide a deeper understanding of why anxiety disorders develop and why they are more prevalent in women.

ABSTRACT People suffering from anxiety disorders such as post-traumatic stress disorder (PTSD) very often use and abuse reinforcing drugs such as opioids. Although the comorbidity of PTSD and substance abuse is exceedingly high, little is known about the neurobiological mechanisms that result in this comorbidity. By leveraging the Fanselow laboratory's development of a preclinical model of PTSD with the expertise within the Center for Study of Opioid Receptors and Drugs of Abuse (CSORDA) we plan to attack this question. Based on findings with our model we hypothesize that, during a single traumatic event, stress hormones (corticosterone/cortisol) and neuromodulators (acetylcholine) act in concert on amygdala neurons resulting in changes in neural plasticity within this brain structure. These changes result in the altered fear responses that characterize several aspects of PTSD. The amygdala also contains neurons that participate in the rewarding property of drugs and we hypothesize that trauma causes similar changes in those neurons and this leads to increased drug reward learning. Therefore, in Aim 1 of this proposal we will determine if manipulations that mitigate the potentiation of fear learning caused by trauma also mitigate alterations in drug responsivity following trauma. Aim 2 seeks to identify a set of amygdala neurons that are activated by BOTH trauma and drug exposure and using optogenetics tests if activity in these neurons is necessary for comorbidity. We will also assess how stress and drug experience alter gene expression patterns in the amygdala. While most of the focus on drug use/anxiety disorder comorbidity has focused on stress as a causal factor in altered drug taking, we have obtained preliminary data that the converse is also true. A history of drug use and withdrawal increases the impact that a future stressor has on fear processes. Aim 3 investigates potential mechanisms for the ability of drug exposure to adversely impact fear behavior. We will determine if mu-opioid or kappa-opioid receptors in the amygdala are necessary for morphine's effects on future stress reactivity. We will also use resting state fMRI, with an amygdala seed, to determine the overlap in the modifications of whole brain connectivity caused by drug exposure and by stress.

PROJECT ABSTRACT A small but significant percentage of people that experience a traumatic, life-threatening, event develop a condition called Post-Traumatic Stress Disorder (PTSD), which is characterized by heightened anxiety and disturbed fear processing. The condition is more prevalent in women. Additionally, PTSD is highly comorbid with other psychiatric conditions notably alcohol abuse. We have developed an animal model that captures many of the features of PTSD and as such should be helpful in developing an understanding of the biological changes that produce the disorder and suggesting novel treatments. Here we propose to use a milder stress than we have used in our published work because it produces enhanced fear learning in a smaller proportion of animals (18%) that better approximates the rate of PTSD in humans. The rats subjected to this stress will be tested on a number of measures of anxiety, altered fear processing and alcohol intake. We will use this as a tool to understand how PTSD symptoms group together to form subtypes and how these subtypes relate to the brain changes that cause maladaptive behavior.

Project Abstract Stress can have a profound detrimental impact on our behavior, predisposing us to anxiety disorders such as post-traumatic stress disorder and major depression. Given the high prevalence of these disorders understanding the basic biological and psychological processes is vitally important if we wish to address these significant problems and develop effective treatments. One question in the literature is whether a single (acute) exposure to stress or repeated (chronic) stress has similar effects. A second question is how severe must stress be in order to produce detrimental effects. Do repeated mild stressful experiences have an added impact that equals that of a more severe acute stressor? Does chronic stress exposure have a greater impact than an equally intense acute stressful experience? Surprisingly, while there is a large basic science literature on stress, that literature simply does not answer these questions. This is because the methods employed have totally confounded the chronic nature of stress with its severity. Typically what is compared are a series of repeated stressors to a single instance of the same stress. But repeated instances mean that the stress is not only chronic it is also, in total, more severe. We have developed a method were we can independently manipulate the chronicity and severity of stress that will for the first time allow us to accurately answer these important questions. The work begins from our extensive use of an intense acute stressor and the database we have collected on its effects on physiology and behavior. The design of our acute stressor allows us to break it into 15 ?bits? than can be administered at one bit a day for 15 days because the acute stress repeats the same aversive stimulus 15 times over 90 min. We can also systematically vary the intensity of our aversive experience. Our preliminary data already indicate that chronic and acute stress provokes different behavioral responses. Our first aim tests the hypothesis that the differences occur because acute stress taps into a set of automatic ?nonassociative? processes while chronic stress taps into learning or associative conditioning processes. Our second aim focuses on the physiological and neural changes that are differentially provoked by chronic vs acute stressors. Additionally, we will determine to what extent stress severity and not chronicity is the culprit. The third aim focuses on manipulations designed to block the effects of stress to help elucidate how stress chronicity and severity produce their effects via different biological mechanisms. We also hypothesize that males and females have different thresholds for how severe a stressor must be to provoke maladaptive changes in behavior. That hypothesis if true can begin to help explain why anxiety disorders and depression are more prevalent in females.