Marieke R. Gilmartin, Ph.D. - US grants

Auditory trace fear conditioning requires animals to associate a tone conditioned stimulus and ia shock unconditioned stimulus that are separated by an empty 20-second trace interval. This learning task is dependent upon both the hippocampus and the amygdala, which are two of the most important structures in learning and memory. These structures have been studied extensively, but relatively few studies have examined how the hippocampus and amygdala interact. Recently, a process known as reconsolidation has been described where established memories can be lost if protein synthesis is inhibited immediately after the memory is recalled. It is not known, however, whether simultaneously acquired memories are recalled independently of one another or if the recall of one memory reactivates multiple related memories. Understanding how multiple memories are recalled and reconsolidated would provide insights into how neurodegenerative processes, such as natural aging and Alzheimer's disease, could disrupt established memories. This proposal will examine the roles of the hippocampus and amygdala in the consolidation of trace and contextual fear memories and determine if these simultaneously acquired memories can be recalled and disrupted independently of one another. This proposal will also determine if these two important structures have differential roles in encoding information about trace fear conditioning.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this proposal is to determine how the hippocampus, medial prefrontal cortex (mPFC), and amygdala interact at the systems, cellular, and molecular levels to support the formation of long-term memory. The experiments in this project use a hippocampus-dependent task called trace fear conditioning (TFC), which has a temporal component for which the mPFC may be important. The findings from my doctoral work, which recorded single neurons in the hippocampus and mPFC during TFC, suggest that these structures work together to form trace fear memories (Gilmartin & McEchron, 2005 a,b), but a functional interaction has not been tested. Specific Aim 1 will directly test for a hippocampal-prefrontal interaction during TFC. This aim will combine pharmacological inactivation of the hippocampus with protein immunostaining in the mPFC to determine whether hippocampal activity is necessary for plasticity-related signaling mechanisms in the mPFC. Aim 1 will also include the amygdala. Aim 2 will use targeted infusions of a selective protein translation inhibitor to examine the contribution of translations! mechanisms to trace fear memory formation. Aim 3 will provide new information about whether plasticity-related signaling and protein translation are mediated by a common signaling mechanism during TFC. In addition to western-blot analysis, several experiments will also use immunofluorescence and confocal microscopy to examine differences in protein localization (e.g. prelimbic vs. infralimbic mPFC; dendrites vs. soma). The results of this project will provide new insights into hippocampal-prefrontal function, applicable to several clinically relevant issues. The hippocampus is important for declarative and episodic memories, and the mPFC is implicated in higher-order processes such as decision-making and situation-appropriate behavior. As learned experiences influence our decisions and guide our actions, an interaction between hippocampal and prefrontal networks is critical to a number of different cognitive processes. The results of this proposal will have the potential to guide further research into how these two structures work together during normal as well as aberrant neural processes that affect a significant subset of the human population, such as anxiety and post-traumatic stress disorders. [unreadable] [unreadable] [unreadable]

The ability to lay down long-lasting memories of events is crucial for survival. Being able to predict a threat that occurs seconds or minutes after an environmental cue allows for evasive or defensive action. Despite considerable progress towards understanding how memories are formed, many questions remain, such as how the brain learns about events separated in time. This project uses cutting-edge tools in neuroscience to address a previously intractable problem about how brain systems function on a second-by-second basis to link events in memory. This contribution is significant because it is expected to identify key principles of brain function that will advance our understanding of more complex forms of learning and memory. Undergraduate students, including underrepresented groups in science (women and minorities), participate in data collection, analysis, and dissemination. This project also provides an opportunity to develop educational tools to help stimulate interest in Science, Technology, Engineering and Mathematics (STEM) fields within the broader community. These tools include an interactive demonstration of neuronal activity and brain stimulation for annual outreach efforts to high schools in the greater Milwaukee, Wisconcin area.

Nearly all forms of motivated behavior require the association of events that are separated in time, but very little is known about the underlying mechanisms supporting these associations, highlighting a critical gap in the study of memory: how are sensory inputs integrated in memory when they do not overlap in time? The principal investigator recently revealed a causal link between a neural signature of working memory in the prefrontal cortex and the formation of long-term fear memory. The objective of this project is to determine how an auditory cue held in short-term working memory systems within the prefrontal cortex and hippocampus is associated with shock inputs in the amygdala to drive plasticity and adaptive fear responses. With the recent development of optogenetic tools, it is now possible to interrogate the function of discrete patterns of firing in specific neural connections. Here, the investigators leverage this tool in combination with electrophysiology in awake, behaving animals to determine when short-term mnemonic information is integrated with sensory input during memory formation. Given the importance of the prefrontal cortex and the hippocampus for the adaptive use of memory to guide behavior, a hallmark of executive function, determining how mnemonic input from these structures is integrated in downstream brain areas informs the understanding of a broad range of adaptive and maladaptive behaviors.

Project Summary/Abstract Post-traumatic stress disorder (PTSD) affects nearly 8% of the population and is more prevalent in women than men. The neurobiological factors that contribute to this sex bias are largely unknown. This project addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how transcriptional regulation of memory-related genes differs in males and females in support of fear memory. Recently our group demonstrated that male and female rats differentially engage signaling mechanisms within the prefrontal cortex (PFC) during the formation of a fear memory, and our preliminary findings point to sex differences in epigenetic modification of prefrontal genes. Currently, the transcriptional regulation of episodic memory within cognitive systems is poorly understood, and the impact of sex on this dynamic process is all but unknown. This represents a significant challenge to developing effective treatment for disorders such as PTSD. The objective of this proposal is to determine how DNA hydroxymethylase TET enzymes functionally regulate fear memory formation and to identify sex-specific methylomic signatures of aversive experience. DNA 5- hydroxymethylation (5-hmC), a major regulator of active (increased) transcription in cells, has been recently implicated in fear memory formation in the brain. However, whether sex differences exist in the engagement of DNA 5-hmC mechanisms during fear memory formation remain equivocal. In our preliminary studies we found that in the rat prefrontal cortex the DNA hydroxymethylase Tet2 increased in females while Tet1 decreased in males following learning in a trace fear conditioning paradigm. The central hypothesis is that TET-mediated DNA 5-hydroxymethylation of sex-specific gene targets in the prefrontal cortex facilitates the formation of fear memory. The approach uses unbiased genome-wide analysis (NIMH Strategy 1.2, Research Priority B) in combination with viral-mediated transcriptional control to test the functional link between TET activity and memory (NIMH Strategy 1.1, Research Priority D; NIMH Strategy 1.2, Research Priority B). Aim 1 will identify the sex-specific gene targets of TET1 and TET2-mediated DNA 5-hmC in the prefrontal cortex following fear learning, using next generation whole genome chromatin immunoprecipitation (ChIP) and hydroxymethylated DNA immunoprecipitation (hmeDIP) sequencing methods. Aim 2 will use cutting-edge CRISPR-dCas9 technology to manipulate the expression of Tet1 and Tet2 in the prefrontal cortex of males and females during trace fear conditioning, which will determine the sex-dependent functional role of cortical TET1 and TET2 in fear memory formation. The proposed research is significant because it is expected to provide a (epi)genomic map of memory formation in a sexually dimorphic brain region, the medial prefrontal cortex, that is needed for understanding and even identifying new genetic biomarkers of sex-biased emotional and cognitive deficits in psychiatric illness.

Project Summary The prevalence of post-traumatic stress disorder (PTSD) is nearly 2:1 in women compared with men, yet the neurobiological basis of this sex difference is unknown. This project addresses this gap, in accordance with NIMH Strategic Objective 1, by determining how the stress neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) functionally modulates fear circuitry and complex fear behavior, differentially in females and males. The central hypothesis driving this work is informed by clinical data linking this peptide system with PTSD. Women, but not men, with a genetic polymorphism within the gene encoding the type 1 receptor for PACAP (PAC1R) exhibit maladaptive responding to threat-related stimuli: heightened reactivity to threatening cues and deficits in the ability to learn to discriminate fear and safe cues. Recent work by the PI showed that interfering with PAC1R signaling within the prefrontal cortex impairs the formation of cued fear memory in female but not male rats, using a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. Females also had higher levels of PAC1R compared with males in that study. The objective of this proposal is to determine how PACAP-PAC1R signaling modulates activity within the fear circuitry needed for learning about threat and safety: the prefrontal cortex and its connection with the amygdala. The approach uses multiple levels of analysis to elucidate the basic biology linking sex-driven changes in peptidergic signaling and physiological changes in prefrontal-amygdala circuits to alterations in complex fear behavior (NIMH Strategy 1.1, Research Priority D). The central hypothesis is that PACAP-PAC1R signaling in the prefrontal cortex is dynamically regulated by estradiol and promotes both the strength and accuracy of fear memories in females, in part by increasing the excitability and synaptic regulation of amygdala-projecting prefrontal neurons. In Aim 1, the capacity for PACAP-PAC1R signaling to modulate intrinsic excitability and synaptic regulation of prefrontal cells connected with the basolateral amygdala will be assessed using ex vivo electrophysiology. In Aim 2, the ability of PACAP to enhance fear memories yet protect against fear generalization will be tested with local manipulation of PACAP signaling in the prefrontal cortex during trace conditioning and fear discrimination learning. In Aim 3, the capacity for estradiol to drive the expression of PAC1R and the consequence on memory of knocking down PAC1R locally in the prefrontal cortex using shRNA will be examined. Importantly, males and females will be included in all experiments to determine how PACAP contributes to the sexual dimorphism of the prefrontal cortex and the degree to which PACAP has the capacity to influence fear learning and behavior in males. The proposed work is significant because it is expected to advance our current understanding of sexual dimorphism in the prefrontal cortex as well as to provide a neurobiological substrate for maladaptive fear learning observed in women with PTSD.