Carol Ann Barnes - US grants

The proposed research will examine, in rats, the relationship between neurological change in the hippocampal formation which is associated with senescence, and the accompanying decline of spatial learning-memory performance. Three main areas of neurophysiological study will be covered: synaptic transmission and its modification through experience; postsynaptic integration and electrical excitability; and analysis of single unit activity in the intact freely moving animal. The methods will involve extra- and intracellular stimulation and recording in the in vitro hippocampal slice and extracellular techniques in both the acute (anesthetized) and chronically prepared (unrestrained) animal. Behavioral tests of spatial perception and memory (known to require an intact hippocampus for their proper performance) will be carried out in conjunction with some of the neurophysiological experiments. An RCDA would provide the essential training and time for additional neurochemical and neuropharmacological studies to be carried out in conjunction with the above program. Specifically, the extended project will examine the effects of aging on neuronal sensitivity to amino acids which are putative transmitters in the major pathway studied above (perforant path - fascia dentata) and its associated inhibitory interneurons. These studies would involve microiontophoresis and micropressure injections of these substances onto single neurons, both in vivo and in vitro. A second goal will be a search for the underlying biochemical basis of the decline in ability of older animals to retain stimulus-induced long-term enhancement of perforant path synapses. This investigation will primarily involve an examination of the effects of enhancing stimulation on protein phosphorylation in fascia dentata. And finally a number of pharmacological agents will be tested for their effectiveness in alleviating certain of the neurophysiological changes found normally to accompany advanced age. The long-term goal of this project is a more complete understanding of the biological basis for the deterioration of cognitive function known to occur in the elderly; a prerequisite for development of effective therapeutic measures.

? DESCRIPTION (provided by applicant): Declines in spatial cognition and function of brain circuits responsible for these behaviors are among the hallmark signs of normative biological aging across species. The objective of this research program is to understand the basis of these memory impairments, and rodent and nonhuman primate models each can provide unique windows into understanding how age impacts networks critical for cognition, at cellular resolution. The experiments proposed in the present application are guided by two primary aims. Aim 1 is to understand how brain circuits responsible for spatial cognition are altered in the aged rat. While empirical focus on the hippocampus is justified because of this structure's critical role in memory, the extent to which changes in upstream cortico-hippocampal inputs, such as the entorhinal cortex, contribute to these age- related behavioral deficits is unknown. Two approaches are taken in this aim to answer these questions. We have developed a novel spatial task (the Instantaneous Cue Rotation arena, ICR) that enables precise measurement of spatial behavior accuracy and flexibility in the rat. Additionally, the hippocampus and its primary afferent input, the entorhinal cortex, are recorded simultaneously in order to examine age-related changes in bi-directional interactions between these structures. The dual-structure recordings will enable identification of changes within the hippocampus proper that are driven by upstream entorhinal cortical inputs, as well as changes in the entorhinal cortex driven by degraded hippocampal feedback signals. This multi-site recording method also allows examination of neural dynamics thought to be involved in memory consolidation. Aim 2 is to understand how hippocampal representations are altered in aged freely-behaving primates. While other important questions about circuit dysfunction in aging remain to be addressed in rodent models of aging, recent advances in wireless recording technologies enable new experimental designs for primates that have the potential to test directly assumptions that our discoveries in the rat will find an analogue in the aging human brain. Free locomotion is an important missing link between the behavioral conditions employed to study place cells in rodents, and more constrained conditions under which human studies must be conducted. The experiments under this aim will implement the behavior and electrophysiological tools that allow us to determine the neural impact that aging and level of restraint has on the function of hippocampal networks in the nonhuman primate. Our hypotheses are that old monkeys will show faulty retrieval of hippocampal network patterns (similar to map retrieval failures in old rats) and that the global network activity state will be altered in both age groups when the animals are restrained, compared to when completely unrestrained and free to move. Taking advantage of new behavior and recording approaches in rodents and primates, we believe significant advances will be made in our understanding of the aging brain that will contribute substantively to the development of therapeutic or preventative treatments for cognitive decline in the elderly.

My general research program is directed towards an understanding of the electrophysiological, neurochemical and behavioral correlates of the decline in spatial cognition and memory with age. The ultimate goal is the development of preventative or ameliorative treatments for this decline in mental health. The investigations focus on aging of the rodent hippocampal system, including its intrinsic connections and information processing characteristics, and those of its major cortical and subcortical connections, in relation to the contribution that these neural structures make to cognitive changes that occur in old age. The first experimental question concerns the degree to which the age- related loss of spatial selectivity in hippocampal unit activity that we have found might be accounted for by disruption in information processing characteristics of its cortical afferent, and what the consequence of this loss might be for information processing at neural levels receiving hippocampal output. This problem will be addressed using a new technique developed in this laboratory (the stereotrode) that enables the simultaneous recording from several single neurons in conscious animals moving freely in a spatially extended environment. The second major question is whether information storage throughout the lifespan results in a significant rearrangement in the statistical distribution of connection strengths in old neurons, which might account for the restriction in the range of behavioral adaptability of older animals. The answer to this question requires the analysis of cellular interactions at the level of single neurons rather than the population techniques as employed in previous studies. The third major experimental question concerns the possibility that modulatory influences in the hippocampus from subcortical afferent systems change with age, and contribute to the observed cognitive inactivation of discrete subcortical projection nuclei in conscious young and old animals.

These studies are designed to investigate the effects on learning and memory of the selective loss of specific neurons in the basal forebrain of rats. The basal forebrain may play an important role in many cognitive functions related to attention, learning and memory. The lesions will be produced by injection of two different neurotoxins. These studies will provide valuable information on the selective vulnerability of selected basal forebrain cells and the biochemical and behavioral consequences of their loss.

This research program is directed towards an understanding of the electrophysiological, neurochemical and behavioral correlates of the decline in spatial cognition and memory with age. The ultimate goal is the development of preventative or ameliorative treatments for this decline in mental health. The investigations focus on aging of the rodent hippocampal system, including its intrinsic connections and information processing neural structures make to cognitive changes that occur in old age. There are three main experimental questions addressed in this proposal, and five aims for professional growth. The first experimental question concerns the degree to which the age- related loss of spatial selectivity in hippocampal unit activity that we have found might be accounted for by disruption in information processing characteristics of its cortical afferents, and what the consequence of this loss might be for information processing at neural levels receiving hippocampal output. This problem will be addressed using a new techniques developed in this laboratory (the stereotrode) that enables the simultaneous recording from several single neurons in conscious animals moving freely in a spatially extended environment. The second major question is whether information storage throughout the lifespan results in a significant rearrangement int he statistical distribution of connection strengths in old neurons, which might account for the restriction in the range of behavioral adaptability of older animals. The answer to this question requires the analysis of cellular interactions at the level of single neurons rather than the population techniques as employed in previous studies. The third major experimental question concerns the possibility that modulatory influences in the hippocampus from subcortical afferent systems change with age, and contribute to the observed cognitive deficits. This question will be addressed using local electrical and chemical excitation or reversible chemical inactivation of discrete subcortical projection nuclei in conscious young and old animals.

The program of research outlined here is directed towards an understanding of the mechanisms responsible for durable synaptic change in the mammalian central nervous system, and how these processes might be altered with age. The investigations focus on the neural plasticity found in the rodent hippocampal formation, referred to as long-term potentiation or enhancement (LTE), that may reflect processes normally involved in information storage in the brain. Although a rather large body of literature describes the phenomenological properties of LTE, and there is a rather good understanding of LTE's requirements for induction, there is as yet no clear understanding of the processes responsible for its maintenance. Recent evidence suggests that certain of the transcription factor genes (zif/268) can be rapidly activated by LTE-inducing stimulation (Cole et al., 1989). These data suggest the exciting possibility that transcription factor genes may regulate other proteins that play a critical role in the maintenance of neural plasticity in brain. Because the mechanisms of LTE induction appear to be normal while the maintenance of LTE is deficient in aged rats (Barnes and McNaughton, 1985), it is of interest to determine whether transcription factor activation mechanisms are defective in old animals. Because the rate of decay of LTE and spatial behavioral forgetting rates are correlated within age groups, such investigations may provide insights into why older organism show faster forgetting. Understanding such changes in neuronal plasticity may have an impact on therapeutic strategies for memory disorders in both normal and pathological conditions of aging. The experimental goals of the project can be broken into two main aims: 1) Experiments to identify synaptic mechanisms that regulate transcription factor genes in brain, and in the same preparation to correlate transcription factor responses with synaptic enhancement (LTE). Zif/268 mRNA and protein will be assayed by in situ hybridization and immunohistochemistry, respectively, and will be correlated with the degree of resultant plasticity. Additionally, we will examine the responsiveness of other transcription factors. These studies will refine our understanding of the relationship between LTE and transcription factor activation and will form the basis for comparison of transcription factor responses in young and old rats. 2) Experiments designed to determine whether there are changes in regulation of transcription factor activation in old animals that may contribute to their altered synaptic plasticity and behavior. All studies proposed will use chronic in vivo electrophysiological methods that offer important advantages over acute or in vitro preparations for this work. The necessity for this collaboration is highlighted by the divergent expertise of the two principal investigators: C.A.B. has experience with chronic electrophysiological techniques, and with behavior and physiology in young and old rats; P.F.W. has experience with the anatomical and in molecular techniques necessary to examine the transcription factors of interest. We hope to combine our different techniques and apply the most powerful available methods to test our hypotheses.

The potential functional significance of a newly discovered form of synaptic plasticity observed in the mammalian hippocampal formation will be examined specifically with regard to its possible direct or indirect role in learning and memory operations. This phenomenon, which we call short-term exploratory modulation (STEM), results from behaviors that can roughly be categorized as exploratory in nature, but is independent of the motor components of this behavior. The primary aim of the proposal is to determine whether the STEM mechanism is one that could plausibly be used for high capacity information storage, or whether it represents a form of global system modulation, perhaps important for the storage process, but not directly involved in it. The proposed approach to these questions include: a determination of the extent to which STEM is related to the relatively-well-studied but artificial form of synaptic enhancement (LTE/LTP) that can be induced by convergent high- frequency activity on hippocampal excitatory pathways; an assessment of the role of known heterosynaptic modulation systems in the hippocampus; the correlation of STEM with actual learning; and an investigation of the information content of the STEM phenomenon. Specifically, five different lines of experimental questions will be addressed: 1) Is STEM found only in the temporal lobe afferents to the fascia dentata, or do other extrinsic or intrinsic hippocampal synapses show this form of spontaneous plasticity? 2) Do STEM and LTE occlude, is STEM regulated by NMDA receptor dependent mechanism, and does STEM require spontaneous activity on the perforant path fibers on which it is expressed? 3) Do heterosynaptic transmitter systems cause STEM or contribute permissively in some way? 4) Is STEM related to explicit or incidental spatial learning? Are STEM and learning ability positively correlated? 5) Does STEM reflect information-specific changes in different synaptic subsets? It is expected that these studies will further our understanding of the types of plasticity that occur in the mammalian hippocampus, their relation to learning and memory operations, and their potential contribution to normal cognition.

In the last several decades, analyses of the neurobiological sequelae of the aging process has led to increasingly sophisticated understanding of both the extent and the specificity of age-related changes in brain structure and physiology. In spite of these achievements, there remains a relatively poor understanding of which age-related biological changes directly underlie the accompanying loss of functional capacity, and why. During the same era, there has been a revolution in the conceptual foundations of cognitive science. This revolution has lead to the development of network models at various levels of abstraction which have achieved extraordinary success in accounting for the fundamental data of human and animal cognition, in particular, associative memory processes. Surprisingly, there has been little attempt to apply this powerful conceptual framework to the design of experiments that might shed light on the manner in which neurobiological alterations contribute to functional impairments in the aging brain. It is the intent of the proposed research to begin to bridge the gap between these two disciplines with experiments that exploit recent advances in neurophysiological recording methods, permitting simultaneous recording from large populations of neuronal elements, within a conceptual framework provided by the theory of attractor neural networks and autoassociative pattern completion. Specifically, we will use the method of cross-correlation analysis to investigate the magnitude and duration of experience-dependent changes in synaptic coupling in young and old animals, and to test the hypotheses that synaptic loss during aging creates conditions of instability of neuronal cell assemblies. In addition, we will use temporary inactivation of hippocampal commissural connections to test the theory that the age-related loss of neurobiological resources places it near the critical point of the graceful degradation function that characterizes the initially slow, but subsequently catastrophic manner in which increasing loss of synaptic connections leads to functional degradation in neural networks. Finally, we will investigate the possibility that neural activity related to an impending goal is maintained in the hippocampus during a delayed conditional spatial response task, and how the quality of such goal representations change with age.

memory; neural plasticity; synapses; aging; brain electrical activity; age difference; hippocampus; estrogens; long term potentiation; growth hormone releasing hormone; female; laboratory rat;

DESCRIPTION: This program of research is directed towards an understanding of the mechanisms responsible for durable synaptic change in the mammalian central nervous system, and how these processes might be altered with age. The investigations focus on neural plasticity in rodent hippocampus, referred to as long-term potentiation or enhancement (LTP/LTE), that may reflect processes normally involved in information storage in the brain. Although there is good understanding of LTP's requirements for induction, there is as yet no clear understanding of the processes responsible for its maintenance. Recent evidence suggests that immediately early genes (IEGs) are rapidly activated by LTP- inducing stimulation (e.g., Cole et al., 1989), and raise the exciting possibility that specific IEGs play a critical role in the maintenance of neural plasticity in brain. The observations that LTP maintenance is reduced in aged rats, and that this reduction is correlated with behavioral deficits in spatial learning (Barnes and McNaughton, 1979), emphasizes the need to understand mechanisms that underlie the persistence of LTP, as such knowledge may contribute to selective therapeutic strategies for memory disorders in both normal and pathological conditions of aging. The experimental goals of the project are described by two principal aims: AIM 1 - To examine cellular mechanisms that are rapidly induced by LTP stimulation and that may be involved in LTP maintenance; AIM 2 - To determine whether the failure in old rats to maintain LTP and to exhibit deficits in spatial cognition can be explained, at least in part, by an alteration of signaling pathways involved in IEG expression. 1) Experiments in Aim 1 explore three different aspects of cell signaling that are likely to be involved in LTP maintenance. Experiment 1 will identify phosphoprotein signaling molecules that are rapidly phosphorylated following LTP. Experiment 2 will analyze a novel IEG that is induced in hippocampal granule cells by LTP stimulation. Experiment 3 will develop an in vivo technique to deliver antisense oligonucleotides to selectively block expression of IEGs and to assess the effect of this blockade on LTP maintenance. 2) Experiments in Aim 2 build upon the discovery made in the past grant period that the induction of the IEG c-fos , by a repetitive LTP stimulus, is selectively increased in aged, behaviorally- impaired rats. Experiment 4 examines whether there is an association between the memory ability of an individual rat and IEG mRNA induction. Experiment 5 examines the hypothesis that aging is associated with changes in LTP- induced signaling path- ways that lead to phosphorylation of specific proteins. Experiment 6 examines the hypothesis that the age-dependent reduction of LTP maintenance is associated with altered expression of specific IEG proteins. All studies proposed will use chronic in vivo electrophysiological methods that offer important advantages over acute or in vitro preparations for this work. The necessity for this collaboration is highlighted by the divergent expertise of the two principal investigators: C.A.B., chronic electrophysiological recording techniques and behavior in young and old rats; P.F.W., anatomical and molecular techniques necessary to examine the IEGs of interest. The strength of this interaction is the ability to combine and apply the most powerful available methods to test the hypotheses under study, which could not be accomplished in either laboratory in isolation.

DESCRIPTION (provided by applicant): Investigations of the neurobiological sequelae of aging have led to an increasingly sophisticated understanding of the extent and specificity of age-related changes in neuronal connectivity and cellular physiology. How these changes mediate alterations in behavior remain less well understood. In part, this is true because of the difficulties in bridging the gaps between behavioral, network and cellular levels of analysis. Dramatic advances have been made in recent years in the theory of how information may be represented, stored and retrieved in neural networks, and in the methodology for studying interactions among groups of neurons. Neurons in the hippocampus, for example, convey sufficient information within a given behavioral context about an animal's location in space, to enable the position of an animal to be derived from the firing pattern of 50 or more simultaneously recorded cells. The hippocampal firing pattern for a given environment is map-like in a formal sense, but questions remain about whether and how the rest of the brain makes use of these 'maps', and the extent to which the neurobiological changes known to occur during aging impact the dynamics of these network properties. A number of recent experiments have begun to examine these issues. For example, in young rats, the map for a given environment is usually stable from one experience to another, but can exhibit dramatic rearrangements when the sensory or behavioral context changes (remapping). For old rats, remapping can occur with no change in external stimuli, but, paradoxically, maps can remain stable under conditions of dramatic change in sensory context. This suggests that the old hippocampus can fail to retrieve consistent maps, and can also fail to generate new maps appropriately. In young rats, there is a rapid increase in the spatial information content of the map with experience, as predicted by theories of sequence learning, and this phenomenon is NMDA receptor-dependent. This effect is much less robust in old rats, suggesting a failure of encoding mechanisms at the biophysical level (such as associative storage mechanisms and synaptic connectivity). The experiments outlined in this proposal are designed with three goals in mind: 1) to clarify the mechanisms by which the population properties of hippocampal ensembles, within individual young and aged animals, may contribute to the accuracy of spatial memory; 2) to test hypotheses concerning the fundamental principle of attractor dynamics as it relates to hippocampal network behavior during aging; and 3) to examine neural correlates of sequence learning, and how behavioral deficits may arise in sequential associations in old animals. The ultimate result of such investigations will be an improved understanding of emergent ensemble properties of the aged brain that contribute to memory impairment.

This program of research is directed towards an understanding of the mechanisms responsible for durable synaptic change in the mammalian central nervous system, and how these processes might be altered with age. The investigation focus on neural plasticity in rodent hippocampus, referred to as long-term potentiation (LTP), that may reflect processes normally involved in information storage in the brain. The observations that LTP maintenance is reduced in aged rats, and that this reduction is correlated with behavioral deficits in spatial learning (Barnes and McNaughton, 1979), emphasizes the need to understand mechanisms that underlie the persistence of LTP. Because immediately early genes (IEGs) are rapidly activated by LTP-inducing stimulation (e.g., Cole et al., 1989), the hypothesis that specific IEGs play a critical role in the maintenance of neural plasticity in brain is proposed. Such knowledge may contribute to selective therapeutic strategies for memory disorders in both normal and pathological conditions of aging. The experimental goals of the project are described by four principle aims, each of which build upon the progress and new discoveries made during the past grant period. AIM 1- To analyze differentially activated immediate early gene (IEG) expression to hippocampus of young compared with old memory- impaired rats following LTP-inducing stimulation; AIM 2- To identify new delayed early genes (DERGs) that are associated with durable synaptic plasticity and to select those that are differentially regulated in young compared with old rats for further study; AIM 3- To use a new method that allows unprecedented cellular and temporal resolution of the onset, duration and pattern of transcriptional responses (compartment analysis of temporal activity by fluorescent in situ hybridization or catFISH), to systematically analyze the expression of genes in young and old rats that may be involved in the LTP maintenance deficits during aging; AIM 4- We were also able to show that the catFISH method allows the detection of IEGs activity in hippocampal cells as a result of a behavioral experience, and that it is possible to distinguish at least two separate episodes of experience with this method. This behavioral activation will also be compared in young and old rats. The necessity for this collaboration is highlighted by the divergent expertise of the two principle investigators: C.A.B., in vivo, chronic electrophysiology and behavioral; P.F.W.., molecular techniques necessary to examine the IEGs or DERGs of interest. The strength of this interaction is the ability to combine and apply the most powerful available methods to test the hypothesis under study, which could not be accomplished in either laboratory in isolation.

DESCRIPTION (Adapted from applicant's abstract): The principal goal of this application is to accelerate the development of a whole brain imaging method that can link complex gene expression dynamics in single cells, to the function of populations of neurons known to be responsible for cognition. For the first time, whole brain imaging, analogous to PET or fMRI methods, can be conducted at the multiple single cell and multiple gene levels. The new method described in this proposal provides a bridge between what we know about the activity characteristics of cell ensembles recorded during behavior, and what we know about multiple genes that are activated by these behaviors. This method uses fluorescent in situ hybridization (FISH) to monitor the subcellular distribution of RNA of immediate-early genes such as Arc. In a recent report (Guzowski et.al., Nature Neuroscience), we demonstrate that Arc RNA first appears in the nucleus at discrete foci that appear to be sites of transcription. The processed mRNA then accumulates in the cytoplasm. Because the time course of FISH signal in nuclear and cytoplasmic compartments is distinct, we are able to infer the activity history of individual neurons at two different times. This method, termed cellular compartment analysis of temporal activity by FISH (catfish), allows visualization of gene expression, activated by discrete behaviors or experiences, in the populations of the single cells responsible for encoding these events. Because of its temporal and cellular resolution properties, catfish offers a unique insight into the cellular basis of information processing for a large number of cells across distal brain structures. Currently, the application of the catfish approach is limited by the time-consuming manual counting methods that are used. Recent advances in computer-assisted cell counting algorithms promise to increase objectivity and accelerate the data analysis so that large numbers of samples or braod regions of the brain can be examined. The co-P.I.'s of this application represent the laboratories from which the molecular imaging and parallel electrophysiological recording methods were developed. Additionally, Dr. B.Roysam and S.J.Lockett, who are experts in computer-assisted analyses of cell counts, will join the research program. The goal of Aim 1 is to develop an automated system to rapidly convert data acquitted from the confocal fluorescence microscope into quantitative counts of cells with intracellular IEG FISH signal. Aim 2 will develop methods to assess the presence of cytoplasmic IEG FISH signal, and associate this staining to defined nuclei. Aim 3 will use methods from Aims 1 and 2 to compare the pattern of gene activation in multiple brain regions (hippocampus and neocortex) of young rats, and hippocampal regions in young and memory-impaired old animals. These studies will provide an assessment of the general utility of the catfish method for imaging gene activation induced by behavior when explicit hypotheses for cellular activation can be made from electrophysiological experiments. Our long-term goal is to create a novel image analysis tool that links behavioral systems, and molecular neuroscience approaches to a variety of behaviors and brain regions.

DESCRIPTION: The ultimate goal of this new line of research is to understand the neurophysiological basis of memory impairments that occur during the course of normal aging in all mammals, including humans. An extensive series of electrophysiological and behavioral studies in this and other laboratories has provided considerable insight into the pattern of neuronal alterations that occur in the hippocampus of old rats in relation to memory impairments. Over the last 15 years, this research group has been among the leaders in the development of methods for simultaneously recording from many single neurons in the hippocampus and neocortex of behaving rats. With this method, a principal neuronal population correlate of memory retrieval failure during aging has been recently discovered. This finding makes specific predictions for how the dynamics of neural coding between the hippocampus and neocortical structures may be altered in aging mammals. At the present time, there have been no electrophysiological studies of cognitive processes in mature and old nonhuman primates. Neurophysiological studies in the awake, behaving aged monkey will be technically demanding, time consuming and will require significant resources. With the collaboration of the staff at the California Regional Research Primate Center at Davis, and other scientists affiliated with the Center, the present project is designed to demonstrate the feasibility and scientific value of neuronal ensemble recording in young and old monkeys. The specific hypothesis to be tested is that changes in single cell responses in inferotemporal cortex (perirhinal) that occur as a result of associative learning, will be altered in aged memory-impaired primates. Specifically, altered synaptic plasticity of the backward projections from hippocampal formation structures to inferior temporal cortical areas should lead to reduced associative modifications in old neural networks. The extension of these ensemble recording methods to the aged nonhuman primate brain has the potential to lead to important direct insights into the mechanisms of cognitive impairment in aged humans and into possible treatment strategies.

DESCRIPTION (provided by applicant): The principal goal of this application is to accelerate the development of brain imaging methods that can link complex gene expression dynamics in single cells to the function of populations of neurons known to be responsible for cognition. With new technology developed by the applicants (termed cellular compartment analysis of temporal activity by FISH, or catFISH), it is possible to detect and compare the neural populations activated by two distinct behavioral experiences within the same brain. The temporal resolution properties of catFISH derive from the temporally precise transcriptional regulation of different immediate-early genes (lEGs) following periods of neuronal activity. Using fluorescence in situ hybridization (FISH) and confocal microscopy to reveal the subcellular location of specific lEG RNAs, the activity history of individual neurons throughout the brain can be determined at two or more time points. catFISH has the promise of providing a connectivity map that bridges what we know about the activity characteristics of cell ensembles recorded during behavior, and what we know about multiple genes that are activated by these behaviors. This proposal exploits the potential of the temporal and cellular precision of the catFISH method, combined with novel transgenic mouse models, to offer unique insights into the cellular basis of information processing for large numbers of cells across widely-distributed brain structures. We propose three aims to achieve these goals. Aim 1 combines the catFISH method in cell-specific GFP-expressing mice, along with circuit tracing techniques, to provide functional maps of cells in circuits activated by specific behaviors. Aim 2 uses Arc promoter BAC - GFP transgenic mice in combination with catFISH to increase the number of behaviorally-activated neural networks that can be compared within a single mouse brain. Aim 3 takes two approaches to further our ability to achieve large-scale neural circuit visualization and quantification: development of state-of-the-art, computer-assisted, 3-dimensional cell segmentation and classification software for confocal images; and development of hyperspectral slide scanning technologies for application to large regions of brain tissue. The co-P.I.s of this grant represent the laboratories from which the molecular imaging (JG, CB, PW, BM), genetic methodologies (PW), parallel electrophysiological recording methods (CB, BM), computer-assisted confocal quantitative image analysis (BR) and hyperspectral analysis (JT) were developed. This interdisciplinary team has a history of productive collaboration, and our goals are driven by the fundamental question posed in this Program Announcement: how to visualize large-scale, behavior-driven, genetically-manipulated, functional connectivity maps of the mammalian central nervous system.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and Specific Challenge Topic, 06-AG-101: Neuroscience Blueprint: Development of non-invasive imaging approaches or technologies that directly assess neural activity. As the title of the proposal implies (Functional Activity Mapping of Brain Networks), the goal of this proposal is to enable novel technologies for routinely and rapidly mapping behaviorally-driven neural circuits over large regions of the mammalian brain with single cell precision and subcellular detail. Although a number of methodologies have been developed that can map the regional distribution of neural activation within the brain during specific behaviors, knowledge of the cellular participation of circuits involved in behavior-driven activation has been lacking. Because it is known that neural encoding in the cortex involves primarily variations in which neurons are activated by a given event, rather than just how many, it is important to develop methods that enable cellular specification within active circuits. Thus, while non-invasive imaging techniques such as PET and fMRI permit repeated observations of the same subjects and can identify regions exhibiting enhanced activation when learning occurs, what has been missing, until recently, is a method that differentially images functional activity of multiple experiences at cellular resolution. We and our collaborators have developed an approach that allows the determination not only of how many neurons were activated in each of two distinct behavioral episodes in the same animal, but which individual neurons contributed to the encoding of each experience. The method involves a sensitive fluorescence in situ hybridization technique, combined with high resolution confocal microscopy. It also relies on the observations that the expression of the immediate early gene Arc is closely and dynamically coupled to neural activity associated with active information processing, and that the cellular compartmentalization of Arc mRNA varies as a function of time since the activation event (Guzowski et al., 1999). We have called this method "catFISH" (cellular compartment analysis of temporal activity by Fluorescence In Situ Hybridization;Guzowski et al., 1999), because the differential time-course of the post-activity appearance of specific immediate early gene products identifies which neurons were active in each of two separate sessions of behavior. Thus, the activity history of individual cells in a population can be determined for two different time points within the same animal. To achieve our long range goal of whole brain imaging of multiple discrete experiences, with single cell resolution, the present Challenge Grant proposal is focused on overcoming one major impediment for this technology: namely the issue of going from small-scale analysis to more large-scale fully-automated, objective and rapid procedures that will be necessary to implement functional anatomically linked maps of large brain regions. Thus, this proposal focuses on one major Technical Aim, and one Experimental Aim. The first is to develop the software to improve analysis efficiency and objectivity required for projects examining wide areas of the brain, to validate the automated analysis with manual verification to ensure that the methods are robust across brain regions, and to develop novel 3-dimensional montaging methods that will enable automated cytoarchitectonic analysis. These methods should provide a significant step towards the long-range goal of whole brain imaging with cellular resolution, and have the potential to make a significant impact on the range of questions that can be asked about brain circuits by the neuroscience community. Second, the implementation of these automated methods will allow us to examine both hippocampus and large areas of neocortex that would not have been possible within this timeframe with manual methods. Thus, with this tool, we will be able to determine, within an individual animal, whether the degree of context discrimination exhibited by the hippocampus influences the neocortical layers to which the hippocampus projects in young and aged rats. We expect that the outcome of the proposed project will have an important impact on our understanding of the neural basis of episodic memory and how this function changes with normal aging, as well as will benefit the larger neuroscience community's ability to interpret in more detail the activation patterns that are generated by non-invasive imaging methods such as fMRI. PUBLIC HEALTH RELEVANCE: We expect that the outcome of the proposed project will have widespread impact on our understanding of the neural basis of episodic and semantic memory and how this function changes with normal aging. Success of this study will also provide new methods to study memory disorders arising from multiple sources. This technology will be widely disseminated to other investigators in the field.

of observed states of the network, that is normally tightly coupled with corresponding relationships in physical space. Thus, under constant conditions, an external observer can reconstruct a rat's position from the collective firing pattern (e.g., Wilson and McNaughton, 1993;Zhang et al., 1998). The expression, storage, and retrieval of these neuronal 'maps'provides an excellent forum in which to explore the extent to which age-related alterations in neural system level dynamics can be linked at lower levels to changes in cellular physiology, and at higher levels to changes in the behavioral expression of memory 16

PI: Barnes, Carol A. PROJECT SUMMARY {See instructions): This proposal applies a novel set of cognitive, neurobiological and molecular profiling approaches to the question of how to understand the concept of cognitive reserve in a rodent model of aging. An integrated set of experiments is proposed designed lo assess, mechanisms underlying differential cognitive trajectories observed over the lifespan of the rat at three different phases of the lifespan, young adult, middle age, and old. The experiments take advantage of technologies that cannot be used in humans at present, as well as those that can (such as cognitive tests and MRl imaging methods). These methods have not been applied in combination in the rodent previously, and involve high resolution MRl Imaging ofthe entire brain, cognitive tests that examine domains relevant to 8 brain regions in the temporal and frontal lobes (hippocampal regions CAI, CA3, dentate gyrus and the subiculum; temporal lobe regions perirhinal cortex and medial entorhinal cortex; frontal lobe regions anterior cingulate and prelimbic cortex), behavior-induced, single cell gene expression imaging in these 8 regions, and in 4 hippocampal subregions cell-specific whole transcriptome analyses. These procedures will be conducted in rats that are chosen on the basis of possessing behavioral performance scores that are high, average or low with respect to performance distributions from young, middle-aged and old rats. The present experiments have the potential to identify cell-specific RNA transcripts and behavior-activated circuits in brain regions that are critical for cognition, and that define how individuals segregate along a cognitive competence continuum throughout life. The combination of these methods will enable identification of those variables that are associated with more or less successful cognitive aging trajectories - a fundamental necessity if effective treatments are to be developed. The specific goals of this project are approached experimentally under two main aims. AIM 1 is to identify transcriptional patterns in hippocampal subregions that are associated with differential cognitive aptitudes across the lifespan. AIM 2 is to identify behavior-induced activity patterns in temporal and frontal lobe circuits associated with differential cognitive abilities, to identify what circuit characteristics are associated with successful cognitive outcomes during aging and the extent to which temporal and frontal lobes age independently. Together, the data collected in these experiments should identify targets useful in the development of strategies to optimize cognition during aging. RELEVANCE (See instructions): The goal of this project Is to uncover critical genetic and circuit characteristics that increase the probability that an individual will maintain high levels of cognition across the lifespan. When the mechanisms conducive to high levels of behavioral function are well enough understood to be effectively manipulated, strategies for altering the trajectories of age-related declines can then be implemented. This is likely to have enormous impact on productivity and independence of elderly populations.

With high prevalence in the community-dwelling elderly population, hypertension may be an important factor influencing the development and progression of cognitive aging. Although hypertension is a major health problem that has been shown to affect cognition, very little is known about its effects on the molecular status, especially the epigenetic status, of brain regions critical to cognition. However, significant literature is accumulating describing effects of selective cognitive tasks on epigenetic mechanisms in rodents. The known effect of hypertension on cognition, and the accumulating literature on the epigenetic bases of selected cognitive capacities leads to the overarching hypothesis to be tested by this project: epigenetic changes induced in a rodent model of hypertension will mirror the known relationships between cognitive tasks and epigenetic mechanisms in rodents. We focus on epigenetic mechanisms since these are major factors in the regulation of gene expression. Specifically, this project is aimed at determining epigenetic changes induced by hypertension in subregions of the brain known to be important to cognition. We will study brains from 20 normotensive controls and 20 hypertensive behaviorally characterized rats, who have also received high resolution in vivo magnetic resonance imaging (MRl) scans of brain structure and white matter integrity. Male Fischer 344 rats have the cytochrome P450 promoter (Cyp1a1) inserted to up-regulate the expression of the mouse renin (Ren2) gene, Administration of 0,15% indole-3-carbinol {I3C) to the diet of these transgenic rats activates the Cyplal promoter to induce a gradual onset of hypertension. Dependent variables will be gene expression and the major epigenetic mechanisms, DNA methylation and histone modifications, as well as measures of cognitive performance and patterns of MRl gray and white matter integrity. Defined sub-regions, of the hippocampus will be isolated by laser capture microdissection. In order to obtain sufficient starting material successive sections from the same brain will be pooled. We will assess DNA methylation on a genome wide basis by bisulfite conversion, amplification and NimbleGen arrays. PCR will be used to assess DNA methylation of specific genes based on previous findings of others and array data produced by this project. We will assess selected histone modifications by chromatin immunoprecipitation followed by PCR of selected genes. The genes we select are those previously shown to be differentially expressed in brain regions, with an emphasis on hippocampus, in association with learning and memory. We will test our major hypothesis by statistical determination of hypertension-induced epigenetic changes in our model and then compare the resulting data with epigenetic changes associated with cognitive behaviors and MRl gray and white matter integrity. We will use multivariate analyses to estimate the extent to which epigenetic variables account for cognitive status in the experimental animals.

? DESCRIPTION (provided by applicant): Dramatic advances have been made in recent years in the theory of how information may be represented, stored and retrieved in neural networks and in the methodology for studying interactions among groups of neurons. Animal models of aging in rodents suggest that altered connectivity and plasticity mechanisms within the hippocampus contribute to altered network function associated with changes in spatial cognition. In addition to changes in temporal lobe circuits and episodic memory during aging, some of the earliest alterations detected in memory across the lifespan occur in frontal lobe-dependent tasks, including working memory and attention. Each of these cognitive functions, of course, is essential for effective interaction with our environment. Only humans spontaneously develop Alzheimer's disease. Thus other animals provide a good model of normative age changes. Even in humans, the proportion of people across the USA over 71 who are demented, from all causes, is 14%. This suggests that it is critical to understand normal cognitive aging processes in their own right, as this reflects 86% of aged individuals. The two Aims of this proposal are to better understand the underlying causes of two hallmarks of cognitive aging - behavioral slowing and multi-tasking deficits. Both of these cognitive operations are sensitive to prefrontal cortical function and different aspects of working memory. The systematic studies proposed in each Aim address each of these questions utilizing the animal model best suited to gaining insight into the origins of these two phenomena of cognitive aging in humans. Aim 1 examines working memory and the effect of age on speed of network dynamics in young and aged rats while performing the W-track continuous alternation task and recording simultaneously from the prefrontal cortex (PFC) and the hippocampus. The questions addressed in this Aim include how the aging brain adapts to the changed dynamics intrinsic to both hippocampus and PFC, and how these structures interact or compete during aging to find solutions to this spatial working memory problem. While rodent models have the advantage of the use of unrestrained behavior conditions and more complex recording configurations, there are cases in which there is significant evolutionary advantage to using nonhuman primates, particularly with respect to the prefrontal cortex and tasks that do not require free movement. Aim 2, therefore, examines working memory in young and aged bonnet macaques in an interference task that evaluates multi-tasking ability in the awake, behaving state while monitoring activity across populations of neurons recorded in PFC. In this Aim, the cellular correlates of multi-tasking are examined for the first time in an aging primate model, to assess how aging weakens the resilience of working memory circuits in the face of interference. For these different questions, each model has unique strengths and will allow us to begin to bridge the gap between principles learned from studying animal models, to those that underlie the neural basis of human cognitive aging.

? DESCRIPTION (provided by applicant): The postdoctoral training application entitled The Neurobiology of Aging and Alzheimer's Disease is submitted through the University of Arizona on behalf of the Arizona Alzheimer's Consortium. The training faculty chosen from the Consortium consists of highly interactive investigators whose scientific interests include aging and Alzheimer's disease, and spans fly, rodent, and nonhuman primate models of aging, as well as human studies in normal aging and in Alzheimer's disease. Six institutions within the Consortium contribute primary mentors to this Training Program, including: Arizona State University, the Barrow Neurological Institute, the Banner Sun Health Research Institute, the Translational Genomics Research Institute, the University of Arizona, and the Banner Alzheimer's Institute. Faculty have complementary strengths in brain imaging, computer science, genomics, molecular biology, basic systems, behavioral and cognitive neuroscience, neuropathology, and clinical research to promote the scientific understanding of the aging brain and early detection of Alzheimer's disease, as well as effective treatment and prevention therapies. Each Fellow will have a Professional Development Committee (PDC) that consists of 4 Mentors. A primary research Mentor in whose laboratory the Fellow will be based, who is chosen from the list of Training Faculty included within the tables of this grant, and a secondary research Mentor from this list, but from any other institution amongst the consortium. The secondary Mentor plays an active role in the research project undertaken by the Fellow, which goes beyond simply making facilities available. A tertiary Mentor may be drawn from the Training Faculty list, but can also be selected on the basis of experimental or pedagogical expertise, even if not among the Training Faculty. Additionally, a fourth member of the PDC will be chosen from amongst the 5 person leadership team of Director, co-Directors or Associate Directors, who will commit to provide an additional level of oversight. The Director chosen will be selected on the basis of suitability for assisting with the Fellow's research project. We believ that exposure to the unique Arizona tradition of cooperation and collaboration that knows no institutional boundaries will facilitate a significant enrichment of skills sets, professional development, and publication record of the Fellow during the training period. In addition to multi-laboratory research exposure, another unique programmatic aspect takes the form of three half or full day workshops per year. These include statewide events that Training Faculty attend (Annual Arizona Alzheimer's Consortium Retreat and Arizona Alzheimer's Consortium Annual Scientific Conference), as well as professional events that include broader national and international participation (Society for Neuroscience meeting). We believe the goal to give the Fellow the freedom to draw broadly from facilities and expertise within the Consortium will allow the best experiments to be designed to test important questions, and will instill an appreciation for collaboration and interdisciplinary interaction that will provide the trainee a solid foundatio for productive future work.

Abstract The effect of aging on the human brain shows wide individual variation ranging from early onset Alzheimer's disease (AD) to maintenance of cognitive clarity into the 10th decade. The challenge is to understand why aging can have such disparate outcomes, and why it contributes so profoundly to the risk of neurodegenerative disease. We have examined aging and AD from the perspective of molecular pathways that underlie memory consolidation and determined that a gene termed NPTX2 provides an important clue to human cognitive failure. NPTX2 is expressed by pyramidal neurons and secreted at their excitatory synapses on parvalbumin interneurons (PV) to control inhibitory circuit function. NPTX2 and markers of PV function are prominently down-regulated in the brain of humans with AD, and CSF levels of NPTX2 correlate with both disease state and cognitive performance. NPTX2 is not down-regulated in the brains of individuals who maintain cognitive clarity despite amyloid accumulation (asymptomatic AD). These and other findings support the hypothesis that NPTX2 is associated with brain resilience critical for cognition and fails in the shift from healthy to unhealthy aging. Aim 1 will identify signaling pathways associated with preserved or deteriorated NPTX2 expression across the spectrum from older individuals with exceptional cognition to those with AD. Studies use an approach of targeted proteomics combined with bulk and single nuclei RNAseq, and will specifically examine the hypothesis that NPTX2 loss-of-function is associated with changes in interneuron cell properties. Aim 2 extends the goals of Aim 1 to establish the cellular mechanism of NPTX2 down-regulation using isogenic human iPS neurons encoding familial mutations of APP and PS1. iPS neurons with fAD mutations show profound and specific reductions of NPTX2 expression and provide an extraordinary opportunity to isolate and validate critical disease pathways. Analyses will include TMT differential mass spectroscopy and RNAseq. Candidate pathways will be manipulated and tested using CRISPR and pharmacological approaches. Aim 3 will provide the first test of the hypothesis that NPTX2 loss of function (LOF) in the adult brain is causal for circuit dysfunction and cognitive decline in the context of AD pathogenesis. Experiments use a newly established rat genetic model for conditional deletion of NPTX2 in a transgenic APP/PS1 AD model (Tg344- AD). Analyses will include high density electrophysiological recordings in hippocampal subregions CA1 and CA3 together with behavior tests and histopathological assessments of AD markers. Single nuclei RNAseq performed in CA3 will define the signature of NPTX2 LOF in the context of amyloid pathology. These data will be cross-referenced with findings from Aims 1 and 2 as part of an integrated interspecies analysis of the cause and consequences of NPTX2 LOF. Combined studies will deepen our understanding mechanisms that can confer cognitive health or bias the brain towards disease.

SUMMARY/ABSTRACT: Overall Project The strategic vision of the Precision Aging Network (PAN) is to develop the essential scientific knowledge to understand the discrepancy that currently exists between cognitive healthspan and human lifespan. We must reveal the neural mechanisms that 1) account for optimal brain performance in old age resulting in healthy cognitive function, and 2) those that underlie decline in brain function leading to age-related cognitive impairment (ARCI), Alzheimer?s disease (AD), or Alzheimer?s disease-related dementias (ADRD). The ultimate goal of the PAN is to develop not only a strong scientific foundation for the essential knowledge needed to match cognitive healthspan with human lifespan, but also to leverage big data approaches that apply precision medicine concepts to prolong optimal brain function. To achieve this goal of sustaining optimal cognitive function in old age, and to extend quality of life for people across levels of risk for ARCI, AD, or ADRD, we maintain that methodologies such as those developed and implemented in the PAN will be required. Although ?chronological age? is consistently associated with increasing incidence of disability, including chronic brain disorders such as AD and ADRD, the exact mechanistic relationships between ?biological age? and decline in brain function is not known. The number of people now living with some form of dementia is estimated to be 50 million worldwide, which is expected to double every 20 years. Because of the enormous heterogeneity in brain and cognitive function among individuals in their 70s, 80s and 90s, the urgent challenge for science, medicine and healthcare providers is to discover interventions that are individually effective in delaying or preventing ARCI, AD, or ADRD. Untangling the complex relationship between age and cognitive performance requires a strategy that includes the study of very large, diverse, well-characterized and longitudinally sampled populations. This will require ?big data? but also the means to translate the massive amounts of information gathered into ?smart data? or ?knowledge?. This demands radically different conceptual models. Currently, no single approach adequately identifies the means to modify personal aging trajectories for improved brain health in individuals. The approach proposed in PAN is designed to overcome obstacles of earlier methods. The focus is on how to distinguish the various combinations of age, sex, genetics, race-ethnicity, health, lifestyle choices and environmental factors that influence brain drivers that increase susceptibility to dysfunction, as well as those factors that increase brain protection and resistance against dysfunction. The fundamental principle of the precision medicine approach is to ?individualize?. This will enable strong and specific predictions for each person to close the gap between cognitive healthspan and human lifespan. The root of this concept is in the teachings of Hippocrates, who said ? ?It is more important to know what sort of person has a disease than to know what sort of disease a person has.?