Fred J. Helmstetter - US grants

This project is intended to improve the quality of undergraduate instruction and research at all levels within our program in Physiological Psychology. Modernization of an existing advanced laboratory course in Physiological Psychology is proposed which would provide students with invaluable "hands on" experience with important concepts and methodologies through a series of demonstrations, interactive computer simulations and structured experiments. Most laboratory exercises are designed around two "work-stations" composed of a microcomputer, interface and electrophysiological amplifiers. This equipment, when combined with the other standard scientific apparatus detailed in the proposal, provide a modern and highly flexible resource for laboratory instruction. Selected demonstrations and computer simulations developed in this context will be used to help students in lower-division Physiological Psychology courses better understand some of the dynamic aspects of physiology and behavior. The resources requested would also dramatically improve the ability of undergraduates to gain meaningful independent research experience in Physiological Psychology.

biomedical equipment purchase;

The long-term goal of this project is to understand how endogenous opioid peptides in the mammalian forebrain contribute to the regulation of stress-related behavior and hypoalgesia. Neural systems exist within the brain and spinal cord which are capable of modulating the transmission of pain, and the amygdaloid complex appears to be a critical forebrain structure involved in the activation of these systems in response to environmental stressors. The objective of the present series of experiments is to evaluate in detail our recent observation that microinjection of kappa opioid receptor agonists into the amygdala results in the modulation of a spinally-mediated nociceptive reflex in the pentobarbital-anesthetized rat. Animals will be anesthetized and prepared for measurement of heart rate and mean arterial blood pressure. Cannulae will be stereotaxically positioned over target brain structures and opioid receptor agonists will be applied to the amygdala. The latency of the animals reflex response to radiant heat applied to the tail is measured at 2 min intervals throughout the procedure. Specifically, we propose to [1] determine the type(s) of opioid receptors involved by characterizing the dose-response functions of 3 highly selective opioid receptor agonists (DAGO, DPDPE, U50,488H), [2] correlate dose-related elevations in tail flick latency with changes in heart rate and blood pressure, [3] localize the critical population of opioid receptors within the amygdala and [4] assess the involvement of descending antinociceptive systems by using lidocaine to block neural activity at critical brainstem sites (periaqueductal gray, nucleus raphe magnus) following opioid microinjection in the amygdala. These experiments will provide very important information about the function of forebrain opioid receptors and the mechanisms through which endogenous antinociceptive systems may be activated in the behaving animal.

The long-term goal of the project is to understand the neural systems responsible for anxiety, fear and aversive motivational/emotional processes. A model system is proposed in which the hypoalgesic reactions of rats briefly exposed to intense white noise is measured. This will allow us to explore the functional relationship between forebrain structures known to be important for certain forms of fear and emotional processing and lower brainstem systems involved in the expression of hypoalgesia and defensive behavior. Importantly, this will be done using a paradigm which is widely accepted as a laboratory model of acute anxiety for both human psychophysiological studies and more basic research with laboratory animals. A series of five experiments has been designed to determine: 1) the effects of manipulating the intensity and duration of noise stress on the magnitude and duration of hypoalgesia, 2) the relationship between hypoalgesia and noise stress-induced increases in arterial blood pressure, 3) the conditions under which endogenous opioid peptides are involved, 4) if the specific nuclei within the amygdaloid complex which are known to be critical for other forms of fear and anxiety contribute to hypoalgesia in this paradigm, 5) if noise stress hypoalgesia represents the activation of documented endogenous antinociceptive systems which include the periaqueductal gray and nucleus raphe magnus. This paradigm may prove to be an interesting alternative to many of the similar currently accepted models which employ electric shock since noise stress appears to have fewer non-specific long-term effects. The fact that identical procedures that do not require the application of nociceptive stimuli may be used to produce anxiety and hypoalgesia in humans and rats may also prove useful eventually.

The long-term goals of this project are to understand how endogenous opioid peptides and their receptors contribute to the regulation of stress-related behavior and nociception. Neural systems exist within the brain and spinal cord that can, when activated, modulate the transmission of information about noxious stimuli. Recent studies we have conducted indicate that the amygdaloid complex appears to be a critical forebrain region involved in the activation of these systems in response to environmental stress. We have proposed that efferent projections from the central nucleus of the amygdala to the ventrolateral periaqueductal gray (PA~)are critical for certain forms of stress-related hypoalgesia and cardiovascular alterations in the rat and have developed a model system using a spinally-mediated nociceptive reflex to test this idea. This proposal describes a series of experiments that are focused on understanding the functions of endogenous opioid peptides in the amygdala and PAG. In all experiments we will simultaneously measure arterial blood pressure and heart rate along with nociception while microinjecting selective opioid agonists and antagonists into these two brain structures. In the first set of studies, we will use a barbiturate- anesthetized rat preparation to address opioid modulation of the radiant heat tail flick reflex. We have recently shown that microinjection of mu, but not delta or kappa, opioid receptor agonists into the anterior basolateral nucleus of the amygdala in anesthetized rats will result in a dramatic inhibition of this nociceptive reflex. We will explore the neuropharmacology of this effect within the amygdala in greater detail and determine if chemical stimulation of the amygdala in this preparation causes the release of opioid peptides in the PAG. The second set of experiments will determine if opioid microinjection into the amygdala results in a dose-related inhibition of nociception in awake animals comparing both tail flick and the formalin test as behavioral assays. The final set of experiments will provide important information about endogenous opioid release in the amygdala and PAG during stress-induced hypoalgesia. Using a highly reliable Pavlovian conditioning procedure to produce stimulus-dependent opioid-mediated inhibition of the tail flick response, we will be able to directly compare patterns of endogenous opioid peptide involvement in nociceptive inhibition and cardiovascular alterations in awake and in anesthetized rats. The knowledge gained from these studies should have an appreciable impact on our understanding of endogenous opioid mechanisms of antinociception and thus may eventually aid the development of safer and more effective analgesic compounds for clinical use.

The long-term goal of this project is to understand the neural mechanisms involved in associative learning and specifically in the acquisition and expression of fear and anxiety in mammals. The project employs a model system approach using aversive Pavlovian conditioning in the rat. Presentation of an auditory stimulus that has been paired with an aversive event such as foot shock typically results in a constellation of behavioral and physiological responses which includes elevations in arterial blood pressure (ABP) and an attenuation of the organism's sensitivity to pain (hypoalgesia). This project will focus on a detailed analysis of the brain structures and connections that are essential for the learning and performance of hypoalgesic and cardiovascular conditional responses. These experiments will provide important new information about how biological processes occurring in the central nervous system give rise to psychological states like fear and anxiety. The project will also investigate the mechanisms and locations of plastic changes occurring within the nervous system during associative learning. Based on recent data from our laboratory, a functional neural system which includes the amygdala, periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) is proposed as being critical for the expression of conditional fear-induced hypoalgesia (CHA). Rats will be prepared for intracranial microinjection and chronic measurement of ABP. After auditory fear conditioning animals will be tested for simultaneous changes in ABP and nociception in response to the aversive CS. The first set of experiments will compare the relative contribution of the central, lateral and basolateral nuclei of the amygdala and the PAG, RVM and lateral parabrachial region to response expression (performance) versus response acquisition (learning) by using selective reversible inactivation techniques. The second set of experiments will test our hypothesis that corticotropin releasing factor (CRF) plays an important neuromodulatory role in the synaptic connection between the amygdala and ventrolateral PAG during learning. The final set of studies will expand on our recent work which indicates that calcitonin gene-related peptide (CGRP) is released in the amygdala during conditioning and may be involved in plastic changes that occur during auditory fear conditioning.

DESCRIPTION: (Adapted from the Investigator's Abstract) Benzodiazepines and related compounds bind to unique receptor sites in the brain and modify the postsynaptic effects of GABA. A large amount of evidence suggests that these ligands can exert profound effects on anxiety and on the formation of memory in humans and laboratory animals. There are several forms of the GABA-A/benzodiazepine receptor based on differential assembly of a series of subunits. Recent studies have suggested that receptors containing the alpha5 subunit are primarily localized in the hippocampus and may be specifically involved in the regulation of synaptic events underlying learning and memory. Our preliminary results indicate that inverse agonists with a high degree of selectivity for alpha5-containing receptor isoforms can enhance certain forms of memory in rats. The present study involves the design and synthesis of a series of novel alpha5-selective ligands that we will evaluate for their ability to modulate hippocampal-dependent and hippocampal-independent forms of memory using a Pavlovian fear conditioning paradigm with mice and rats. A subset of these compounds will be applied directly to the dorsal hippocampus to evaluate possible mechanism of action within this structure. The development of subsite selective agonists and antagonists at GABA-A/BzR receptors will provide important new tools in the investigation of the basic neural mechanisms of memory and anxiety and improve our understanding of benzodiazepine pharmacology. Drugs that enhance memory in the absence of unwanted side effects may prove to be important therapeutic agents in treating disorders of memory due to illness or aging.

[unreadable] DESCRIPTION (provided by applicant): Multiple neural systems exist within the human brain that allow us to learn and remember. At the present time it is not entirely clear how the many different forms of human memory relate directly to structures and circuits in the brain. The long-term goal of this study is to understand the neural mechanisms underlying a specific form of memory, aversive Pavlovian conditioning, in human subjects. The ability to learn associations through Pavlovian conditioning is shared by a wide variety of organisms but very little is known about the neurobiological substrates of this process in humans. [unreadable] [unreadable] Our general approach is to use state of the art whole brain functional magnetic resonance imaging (fMRI) to identify brain regions and circuits that contribute to the acquisition and performance of fear conditioning. Functional maps of brain areas in which activity is directly related to exposure to the CS+ or CS- in a differential conditioning paradigm will be constructed. Similar techniques are used to characterize brain areas in which activity is best related to autonomic fear responses versus cognitive awareness of the relationship between programmed stimuli. [unreadable] [unreadable] This phase of the project will focus on a series of predictions regarding the roles of the amygdala, hippocampus, frontal cortex, and cingulate cortex based on our progress during the last funding period and current knowledge from laboratory animal studies and other human memory paradigms. We will further evaluate our findings related to localized frontal lobe activity specifically related to contingency awareness and declarative memory. We will explore the dynamics of learning-related stimulus representations in primary sensory cortex. We will compare an auditory fear conditioning procedure with visual stimulus conditioning, and we will conduct studies related the mechanisms of fear extinction and long-term memory retrieval. [unreadable] [unreadable] The results will enable significant advancement in understanding; 1) the relationship between declarative and procedural memory systems, 2) the role of awareness in learning, 3) the comparative neuroanatomy of memory, and 4) the brain mechanisms of fear and anxiety. Our work should be particularly useful in improving the ability to translate fundamental results from laboratory animal models of memory and emotion directly to healthy humans as well as patient populations. The research described here has direct implications for understanding basic brain mechanisms of memory and anxiety in humans. This work may provide valuable insights into new treatments for diseases and disorders that affect memory and emotion. Our approach to these issues represents an important step toward the application of a large body of basic research in laboratory animals to clinical and experimental questions in people. [unreadable] [unreadable] [unreadable]

.DESCRIPTION (provided by applicant): Pavlovian fear conditioning in rats is a powerful model system currently being used to study memory at the molecular, cellular, and systems level. Recent work with this procedure indicates that the formation of long-term memory for fear conditioning requires the synthesis of new mRNA and protein in the amygdala and hippocampus. Furthermore, it appears that after these memories are retrieved from storage they go through a second time period during which protein synthesis is required. Our data indicate that while initial learning requires both mRNA and protein synthesis in amygdala neurons, the "stabilization" of memory after retrieval does not require mRNA transcription. This suggests that while initial memory formation requires nuclear transcription, the modification of synapses after retrieval may involve dendridic protein synthesis using local mRNA. We also provide evidence showing that mRNA and/or protein synthesis is required for new learning not only in the amygdala, but also in structures throughout the neural circuit active during fear conditioning. The experiments proposed here will evaluate the relative contribution of mRNA transcription and protein translation to memory consolidation and "reconsolidation" at multiple brain sites including the amygdala, hippocampus, medial geniculate nucleus of the thalamus, and periaqueductal gray region of the midbrain. We will describe the relative post-training time course of these processes at several sites and begin to explore specific functional contributions of each brain area to learning. The results will provide important new insights into the mechanisms of memory formation and long-term stability at the molecular and systems level.

DESCRIPTION (provided by applicant): The prefrontal cortex (PFC) plays a critical role in multiple cognitive processes. Dysfunction in the PFC has been implicated in a wide range of disorders including Alzheimer's disease, post-traumatic stress disorder, schizophrenia, and attention deficit hyperactivity disorder. Recent research has begun to focus on understanding how the PFC operates within functionally defined networks of brain structures. In particular, the PFC and medial temporal lobe areas such as the hippocampus may work together during the formation and retrieval of memory. This project will use trace fear conditioning (TFC) in laboratory rats as a model to understand the relative contributions of the PFC and hippocampus to the formation of new long-term memories. TFC differs from standard fear conditioning in that a small temporal gap, or "trace interval", is inserted between an auditory signal and an aversive outcome such as foot shock. Numerous studies have shown that normal learning in trace conditioning requires both the PFC and hippocampus, but the specific contributions of each structure as well as the principles by which they interact are currently not known. We will quantify the activity of intracellular signaling pathways known to be important for neural plasticity and learning within the PFC, hippocampus and amygdala in response to TFC training and determine how neural activity in one structure within this network affects responses in those connected to it. We will also determine the degree to which de novo protein synthesis within each brain area is required for the consolidation of a new TFC memory. This work represents the first systematic investigation of the molecular events required for memory formation in this network and one of very few studies to address the functional interactions between the PFC and hippocampus. PUBLIC HEALTH RELEVANCE: The prefrontal cortex (PFC) plays a critical role in multiple cognitive processes, and dysfunction in the PFC has been implicated in a wide range of disorders including Alzheimer's disease, post-traumatic stress disorder, schizophrenia, and attention deficit hyperactivity disorder. This study will provide important new data about how the PFC interacts with medial temporal lobe structures and about how molecular events in PFC neurons contribute to normal cognition.

Project abstract: The cognitive deficits that occur in normal aging as well an in age-related neurodegenerative disease are a major public health challenge. Recent work has highlighted the importance of protein homeostasis in the aging process in general, and in maintaining the functional integrity of neurons in particular. Deficits in protein degradation and targeted autophagy have been linked to several neurodegenerative disorders including Alzheimer's disease. The ubiquitin- proteasome system (UPS) is a major mechanism for protein regulation in all eukaryotic cells. Importantly, our research over the last several years has highlighted the key role of protein degradation driven by specific cellular activity in the induction and stability of synaptic plasticity related to memory in several key brain areas. The formation as well as the subsequent retrieval and use of simple associative memories involve increased polyubiquitination of key regulatory and structural proteins. Both learning and memory recall also trigger a CaMKII-dependent increase in the activity of the 20S proteasome. This proposal describes work that will evaluate our recent preliminary observation that alterations in UPS function may be a critical factor underlying memory impairments in aged rats. Under Aim 1 we will quantify proteasome activity and key activity related signals (e.g., phosphorylation of Rpt6) in young adult (3 mo), middle aged (15 mo) and aged (22 mo) animals. We will compare unstimulated values to those seen after retrieval of previously learned information and compare data from the hippocampus, amygdala, and prefrontal cortex. Aim 2 will test whether dietary pretreatment with methylene blue, which should upregulate metabolic and proteolytic capacity in neurons, will rescue age- related deficits in memory and UPS activity. Together, this set of experiments could provide the basis for several new lines of work related to the neurobiology of age-related cognitive impairment.        

Fear conditioning is an excellent model system for understanding how the brain responds to threat. When organisms learn that an auditory cue predicts danger, the formation of this emotional memory requires plastic changes at synapses in the amygdala. Importantly, the subsequent retrieval and use of this memory involves activity-dependent synaptic destabilization. The functional significance of memory destabilization at retrieval is yet to be fully understood, but this process is likely to be important for memory updating and flexibility under normal conditions. Understanding and control of memory destabilization may open new avenues for clinical interventions in anxiety disorders. Our recent work has focused on the critical role of the ubiquitin-proteasome system (UPS) in controlling synaptic stability when existing memories are recalled. While destabilization is well documented at amygdala synapses, very few data exist related to the factors that control this process. In this project we use optogenetic silencing and stimulation to control neural activity during memory retrieval in behaving animals while quantifying biochemical signals related to destabilization in the amygdala. These signals include proteasome activity and activity-driven phosphorylation of Rpt6 regulatory subunits. Aim 1 is focused on altering activity within specific amygdala nuclei or connections. Studies in Aim 2 assess the role of prelimbic medial prefrontal cortex (PL) activity in triggering memory destabilization and address functional interactions between PL and the amygdala. In Aim 3 we address the role of the ventral periaqueductal gray and some of its reciprocal connections with the amygdala. The knowledge gained here may ultimately be applied to the targeted destabilization and erasure of traumatic memories.