Christine Ann Denny - US grants

[unreadable] DESCRIPTION (provided by applicant): The broad goal of this project is to determine the involvement of adult hippocampal neurogenesis in hippocampal-dependent learning and memory. It has been shown that the dentate gyrus of the hippocampus has the ability to produce new neurons into adulthood. Many of these newborn cells mature into dentate granule neurons with functional synapses. Previous literature showed that ablation of hippocampal neurogenesis in rodents inhibits the behavioral response to antidepressant drugs and led to the hypothesis that neurogenesis is required for the therapeutic action of antidepressant drugs. Conversely, arresting neurogenesis impairs several forms of hippocampus-dependent learning. We have shown that blocking hippocampal neurogenesis using x-irradiation or an inducible genetic ablation method impairs contextual fear conditioning but improves a working memory task, novel object recognition. Furthermore, arrest of neurogenesis using these methods eliminates a form of long term potentiatioh (LTP) in the medial perforant path, termed ACSF-LTP. In consideration of evidence that developing adult-born neurons exhibit unique physiological properties, the proposed research will use contextual fear conditioning, novel object recognition, and electrophysiology (ACSF-LTP) to determine at what age adult-bom neurons become necessary for these hippocampal-dependent tasks. PUBLIC HEALTH RELEVANCE: Hippocampal neurogenesis has been shown to influence specific learning paradigms as well as antidepressant responses. Strategies aimed at stimulating hippocampal neurogenesis could provide novel avenues for the treatment of cognitive and mood disorders. [unreadable] [unreadable] [unreadable]

7. PROJECT SUMMARY / ABSTRACT It is of utmost importance to identify the circuits underlying learning and memory in order to understand not only the mechanisms of memory but also the how these mechanisms become dysregulated in memory disorders, such as Alzheimer's disease (AD). Human and rodent lesion studies have suggested a role for the hippocampus (HPC) in long-term memory, specifically the subregion CA1. CA1 is preferentially activated when a memory must be retained over a long period of time, and studies have shown that a large proportion of CA1 neurons are reactivated in repeated exposures to the same environment. However, no previous studies have been able to assess the long-term (> 1 month) involvement of individual CA1 neurons in learning and memory, or in AD, since all previous transgenic lines have lacked an indelible label. In this proposal, the contribution of individual CA1 neurons to the encoding of an experience and to the retrieval of a corresponding memory will be investigated by utilizing a transgenic line, the ArcCreERT2 mice. This mouse line allows for the indelible labeling of cells expressing the immediate early gene Arc/Arg3.1 and allows for a comparison between the cells that are activated during the encoding of an experience and those that are activated during the retrieval of the corresponding memory. In combination with optogenetic reporter lines, these studies will assess the long- term involvement of CA1 neurons in memory encoding and retrieval. To fully characterize the role of CA1 neurons in memory, we will selectively express the blue light activated cation channel channelrhodopsin-2 (ChR2) or the yellow light activated outward proton pump archaerhodopsin (Arch) in populations of CA1 neurons during encoding. Using optogenetics, we will then test the hypothesis that a subpopulation of CA1 neurons is sufficient and necessary for the retrieval of a corresponding long-term memory. Next, the role of CA1 in memory encoding and retrieval will be delineated in AD mice by utilizing a triple transgenic design in which CA1 neurons, initially labeled during encoding, can be optogenetically modulated in control and AD mice. In vivo, we will test the hypothesis that optogenetic stimulation or inhibition of CA1 pyramidal neurons during memory retrieval will improve expression of a memory in AD mice. Finally, optogenetic manipulations will be used in order to mimic a deep brain stimulation-like protocol in control and AD mice in order to improve overall cellular function, cell survival, and memory retrieval in AD mice.

PROJECT SUMMARY / ABSTRACT Circadian rhythms are everywhere in living matter from protozoans to human beings. Every cell, organ, and biological system works on an endogenous 24-hour rhythm. However, in patients with Alzheimer's disease (AD), a neurodegenerative disorder affecting memory, this rhythm is dampened. In addition to cognitive decline, many AD patients (~50%) also exhibit agitation and increased anxiety in the evening or nocturnal hours, often referred to as sundowning. Here, we aim to dissect the neural circuitry underlying sleep disturbances, circadian rhythm abnormalities, and increased anxiety-like behavior representing sundowning and how it relates to cognitive decline and AD pathology. We will utilize behavioral assays, optogenetics, whole-brainmicroscopy, and in vivo Ca 2+ imaging. Our preliminary evidence suggests that AD mice exhibit increased anxiety-like (e.g., sundowning) behavior compared to control (Ctrl) mice immediately before their sleep cycle but not before their wake cycle. Therefore, in Aim 1A, we will use the PiezoSleep mouse behavioral tracking system to monitor sleep/wake cycles and circadian homecage locomotor activity in Ctrl and AD mice at different ages in order to better characterize AD mice throughout their wake/sleep cycle. We will also measure anxiety-like behavior at various points throughout their sleep/wake cycles. Using an activity- dependent tagging mouse, the ArcCreERT2 x enhanced yellow fluorescent protein (EYFP) mice crossed with the APP/PS1 (AD) model, we will then identify whole-brain neural ensembles and therefore, neural networks that contribute to sundowning in Aim 1B. This mouse line allows for the indelible labeling of cells expressing the immediate early gene (IEG) Arc/Arg3.1 and allows for a comparison between the cells that are activated during one experience and those that are activated during a second experience. Here, we will compare and contrast the neural ensembles within subjects that are active during anxiety-like behaviors prior to the sleep and wake cycles. In Aim 2A, after identifying which neuronal ensembles are altered during sundowning, we will use nVoke minimicroscopes from Inscopix, to image Ca2+ transients in freely moving AD x ArcCreERT2 mice, injected with a GCaMP6f virus, during the dark versus light cycle. Here, we will be able increase resolution and the time points at which we can correlate neural activity with increased anxiety-like (e.g., sundowning) behavior. In Aim 2B, we will then test the hypothesis that optogenetic modulation of these neural ensembles drives/inhibits sundowning behavior. The neural circuitry underlying sundowning has not yet been elucidated, but this project will inform us of novel brain regions and the neural circuitry affected by sundowning and how we can manipulate these systems to rescue this phenotype in AD.

It is of utmost importance to identify the circuits underlying learning and memory in order to understand not only the mechanisms of memory but also how these mechanisms become dysregulated during age-related cognitive decline (ARCD) and Alzheimer's disease (AD). Here, we will characterize the neural circuitry underlying brain plasticity and resilience as it occurs during cognitive loss in ARCD and AD with single-cell resolution. In this proposal, the individual neurons corresponding to an individual memory will be identified by using an activity-dependent transgenic line, the ArcCreERT2 mice. This mouse line allows for the indelible labeling of cells expressing the immediate early gene (IEG) Arc/Arg3.1 and allows for a comparison between the cells that are activated during the encoding of a memory and those that are activated during the retrieval of the corresponding memory. In combination with optogenetic reporter lines and/or viral injection strategies, these studies will assess the involvement of individual neurons in memory encoding and retrieval in naturally aged or AD (APP/PS1 or 3xTg-AD) ArcCreERT2 mice. To visualize and manipulate these neural ensembles, and thus, the corresponding circuits in memory, we will selectively express the blue light activated cation channel channelrhodopsin-2 (ChR2) in Arc+ cells activated during memory encoding. Whole-brain memories will first be visualized in order to determine which neural ensembles become dysregulated following aging and AD development. Identification of similarities and differences and thus, susceptibility and resiliency, between the ensembles will be performed using neuronal modeling developed in the Denny laboratory. In Aim 2, we will use in vivo Ca2+ imaging to better understand the dynamics (e.g., Ca2+ activity) of neural ensembles as they participate in memory encoding and retrieval in aged and AD mice. In Aim 3, the dysregulated neural ensembles in aged and AD mice will be restored using optogenetic modulation. In addition, in vivo Ca2+ imaging will occur during this stimulation in order to determine the mechanism by which modulation rescues cognitive decline. Comprehensive immunohistochemistry, network modeling, Ca2+ imaging, and optogenetic techniques will be utilized. As most studies have narrowed their analyses to a single brain structure, these studies will expand this scope exponentially by analyzing whole-brain memory traces mediating cognitive loss in AD and aging. This proposed study of whole brain memory traces will be conducted in a manner aligned with human imaging studies, and thus, is likely to provide novel and translational insights into the neural networks mediating memory loss in aging and AD.

PROJECT SUMMARY / ABSTRACT Traumatic brain injury (TBI) is a complex brain dysfunction caused by an outside force, usually a violent blow to the head and can result in physical, cognitive, social, and behavioral symptoms. A core symptom observed following TBI is increased fear generalization, as defined by the overgeneralization of fear from a conditioned, fear-inducing stimulus to novel, neutral stimuli. Fear generalization can lead to heightened, debilitating anxiety and maladaptive responses in a safe environment. A previous study has shown that mild TBI (mTBI) in rats results in generalized learned fear to both conditioned and novel stimuli. However, the neural ensembles mediating this increased fear generalization have yet to be identified. My laboratory uses a behavioral assay called contextual fear discrimination (CFD) to assess fear generalization in mice. We shock a mouse in one context (context A), and then we can measure whether they can discriminate between this aversive, shock- paired context and a similar, but safe context (context B) over approximately 10 days of discrimination learning. In this grant proposal, I will use this CFD paradigm, in combination with an activity-dependent tagging genetic mouse line to identify and pharmacologically manipulate the neural ensembles that underlie altered fear generalization following TBI. In Aim 1, we will identify and quantify how TBI alters the neural ensembles mediating fear generalization by utilizing the ArcCreERT2 x EYFP mice. This mouse line allows for the indelible labeling of cells expressing the immediate early gene (IEG) Arc/Arg3.1 and allows for a comparison between the cells that are activated during the encoding of a memory and those that are activated during the retrieval of the corresponding memory. Memory recall or expression is mediated by reactivation of the same neurons that were active during memory acquisition. Therefore, using our activity-dependent tagging mouse line, we can determine where and how TBI impacts fear generalization neural ensembles throughout the brain. In Aim 2, we will pharmacologically manipulate sham and TBI mice with the goal of improving behavioral fear overgeneralization and the corresponding neural circuits. Recently, ketamine has emerged as an anesthetic and sedative agent for TBI injuries with promising results. Furthermore, we have preliminary data indicating ketamine is effective at decreasing fear generalization in TBI mice. Here, both male and female mice will be administered a single dose of saline, (R,S)-ketamine, or one of its metabolites immediately after sham or TBI surgery. CFD and ex vivo whole-brain imaging will be utilized in order to determine the effectiveness of these treatments on fear overgeneralization behavior and on the underlying neural ensembles. The premise of this grant proposal is to identify and quantify fear overgeneralization neural ensembles in a mouse model of TBI with the goal of developing novel treatments. The outcome of this targeted rescue will provide direct evidence that disrupted neural ensembles result in fear overgeneralization in a mouse model of TBI. !

PROJECT SUMMARY / ABSTRACT A tremendous amount of research has provided us with an understanding of how neurons work in concert during the formation and retrieval of individual memories. While we understand how memories are stored in a limited number of brain regions, we do not yet understand how multiple memory traces are stored across whole-brain neural networks, as well as their real-time physiological dynamics, genetic landscape, and preferential wiring. What is needed now is technology to bridge the gap in our understanding between microscopic interactions at the neuronal level and macroscopic structures that perform computations across networks involved in learning and memory. Using a combination of two activity-dependent tagging systems that utilize the immediate early genes (IEG) Arc and c-fos, the aim of this proposal is to address the critical need for obtaining a map of multiple memories and provide the dynamic states of the brain in the context of behavioral performance and memory expression. We will first utilize behavioral assays and whole-brain imaging to provide unprecedented insight on how multiple memories (e.g., positive and negative memories) are stored with single-cell resolution in a brain-wide manner. Identification of similarities and differences between populations and projections of positive and negative memory ensembles will be quantified and correlated with behavioral performance by using neuronal modeling developed in the Denny laboratory. Tagged cells will also be pulled down and sequenced to delineate the genetic landscape differentiating positive and negative memories. We will then use in vivo Ca2+ imaging to resolve the real-time dynamics (e.g., Ca2+ activity) of neural ensembles as they participate in positive and negative memory encoding and retrieval. Moreover, we will use optogenetic modulation to manipulate the positive or negative ensembles in a within-subject manner during behavioral performance to identify key nodes involved in memory expression. Finally, we will use viral tracing strategies to determine how these ensembles are structurally wired across brain, thereby providing a wiring diagram for multiple experiences in the brain. In summary, comprehensive molecular biology, immunohistochemistry, network modeling, Ca2+ imaging, and optogenetic techniques will be utilized. As most studies have narrowed their analyses to a single brain structure, these studies will expand this scope exponentially by analyzing whole-brain memory traces mediating multiple memories. This combinatory system will result in a whole-brain atlas for individual memories, including positive and negative memories, with single- cell resolution.