Alcino J. Silva - US grants

DESCRIPTION (provided by applicant): Previous studies suggested that the alpha-Calmodulin Kinase II (a-CaMKII) plays a critical role in hippocampal synaptic plasticity and in hippocampal-dependent learning and memory (L&M). Recent work from our laboratory also indicates that this kinase is required for memory consolidation in neocortical sites. These L&M studies, however, were limited by the fact that a-CaMKII was deleted in many brain regions. Using newly developed techniques, we have now derived mutant lines to generate sub-region (CA1, CA3 and dentate gyrus) and cell-type (excitatory neurons) restricted deletions of this kinase. Additionally, we have also used Cre -recombinase in Herpes Simplex Viral (HSV) vectors to specifically manipulate genes in a regional-specific manner. With these unique tools we plan to determine the role of this kinase in hippocampal pre- and post-synaptic plasticity, in learning and in memory consolidation. The specific aims of this proposal are: 1- To determine the role of a-CaMKII in either CAl, CA3 or dentate gyrus in learning and memory. Various models suggest specific roles for each hippocampal sub-region in L&M. We will use mice with post-natal and region restricted null mutations of a-CaMKII to test the role of this kinase in four forms of hippocampal-dependent learning: spatial learning in the Morris water maze, working memory in the 8-arm maze, contextual discrimination with fear conditioning, and social recognition. 2 - To test the role of pre- and post-synaptic a-CaMKII on the induction of long-term potentiation (LTP) in CAl, CA3 and dentate gymus. We will test the pre and post-synaptic role of this kinase in synaptic plasticity not only in CA1, but also in CA3 and dentate gyrus. These studies will also be essential to interpret the behavioral analysis of the mutant lines proposed in Specific Aim #1. 3 - To test the hypothesis that a-CaMKII-dependent plasticity in the hippocampus is critical for early stages of memory consolidation, but that later stages of consolidation require a-CaMKII-dependent plasticity in cortical sites. Recent findings in our laboratory suggest that a-CaMKII is critical for LTP and for memory consolidation in the neocortex. To directly test this hypothesis, we will use cortical and hippocampal transgenic and viral manipulations of a-CaMKII function. The studies proposed here will not only further our understanding of the role of a-CaMKII in synaptic plasticity and in L&M, but they will also be critical for insights into cognitive deficits, such as those associated with aging.

cAMP response element binding protein; long term memory; cyclic AMP receptors; electrophysiology; neurophysiology; neocortex; fear; conditioning; synapses; mutant; brain; cerebellum; hippocampus; neurons; phosphorylation; avoidance behavior; association learning; long term potentiation; neuropsychological tests; genetically modified animals; laboratory mouse; transfection; immunocytochemistry;

DESCRIPTION (Adapted from applicant's abstract): Specific learning disabilities are the most common neurological complication in children with neurofibromatosis type I (NF1), a neurological disorder that affects 1/4000 people worldwide. The inherent complexity of these cognitive deficits, and the complications of pursuing their study in patients, motivated us to study them in mice mutant for the neurofibromin gene (Nf1+/-). Additionally, uncovering the causes for the learning deficits in Nf1 mutant mice will reveal molecular, cellular, and neuroanatomical substrates of learning and memory. It is important to note that in both mice and humans the NF1 mutation is present throughout development. We have recently shown that the Nf1+/- mice are a model for the learning disabilities caused by the mutation of the NF1 gene in humans. Neurofibromin is a GTPase Activating Protein (GAP) thought to down regulate Ras function. Consistent with this, our laboratory recently found that the heterozygous N-ras mutation (N-ras+/-) "cures" the spatial learning deficits of the Nf1+/-mice, demonstrating that the modulation of Ras signaling is critical for learning and memory. The specific aims of this proposal are as follows. 1) To characterize the learning/behavioral impairments of the Nf1+/-mutant mice. I propose to expand the behavioral characterization of the Nf1+/-mice, and to determine whether either the N-, K-ras mutations, or increases in AMPA function (the Nf1 mice have decreased AMPA function) can rescue the behavioral impairments of the Nf1/-mutants. 2) To identify the cellular mechanisms responsible for the learning impairments of the Nf1+/- mutant mice. The experiments proposed will test the hypothesis that the learning impairments of the Nf1+/-mice are caused by increased RAS signaling which disrupts synaptic transmission. 3) To derive mice with neural specific and inducible mutations of Nf1. In both mice and humans the NF1 mutation is present throughout development, and therefore studying the causes for the learning deficits in the Nf1+/-mice will further our understanding of the learning impairments that can be associated with NF1 patients. However, to pinpoint where and when the loss of neurofibromin affects learning, we will derive neural specific and inducible mutations of Nf1. The studies proposed here will not only further our understanding of the function of neurofibromin in the brain, but they will also be crucial for finding treatments for the debilitating neurological impairments associated with Neurofibromatosis Type I.

Changes in learning and memory (L&M) occur with aging, but little is known about their underlying molecular and cellular causes. Mice show age-related social transmission of food preference (STFP), and contextual conditioning. Remarkably, a mull mutation of the K+ channel auxiliary subunit Kvbeta1.1 improves L&M in these tasks, specifically in aged mice. In contrast, this mutation either impaired or had no effect in the performance of young mice in these tasks. Consistent with the hypothesis that an increased slow after-hyperpolarization (sAHP) contributes to age- related L&M deficits, the sAHO of the aged Kvbeta1.1 mutants is comparable to that of young wild type (WT) mice, but significantly smaller than aged Wts. The specific aims of this proposal are: To further characterize the behavioral determinants underlying the rescue of L&M in aged Kvbeta1.1-/- mutant mice. To determine the time course for the L&M improvements triggered by the Kvbeta1.1.-/- mutation. To identify the electrophysiological mechanisms underlying the rescue of hippocampal-dependent L&M in the Kvbeta1.12.-/- mutants. To derive mice with neural specific and inducible mutations of Kvbeta1.1. The studies proposed here will not only further our understanding of the role of kvbeta1.1. in age-dependent L&M, but they will also be crucial for developing strategies to overcome these L&M impairments. Additionally, these studies will further our understanding of the possible connections between sAHP, LTP and L&M.

Recent developments demonstrate that mouse forward and reverse genetic approaches will play an important role in unraveling the molecular and cellular mechanisms underlying behavior. An important aspect of this effort will be the standardization and automation of many of the key behavioral tasks used in these studies. Results with standardized tests can be compared more easily between laboratories, and automation of these tasks will allow high-throughput testing of large numbers of subjects required for behavioral screens of mutant mice. Here, we propose to automate two key procedures that can be used to test a number of behaviors, including learning and memory. The first is fear conditioning, a Pavlovian task where animals learn to fear a conditioned stimulus (context, tone) previously associated with an unconditioned stimulus such as a foot shock. The second i8s social recognition, an ethologically-based task that takes advantage of an animal s ability to recognize and remember other conspecifics. In addition to automating these two tasks, and testing their usefulness with a number of mutant mice, and mouse inbred strains, we also propose to determine the involvement of specific brain regions in the various components of these two tests. The results from the neuroanatomical lesion experiments will provide valuable information to interpret the deficits of mutants revealed in these tasks. The studies proposed here will not only be useful in automating and standardizing two very powerful behavioral procedures, but they will also generate interesting new information about molecular and neuroanatomical substrates of memory formation.

Mental retardation is caused by a variety of factors including mutations that affect brain development and therefore impair cognitive function. However, some of the genes involved in brain development may also have roles in the adult CNS. Disruption of these adult functions could result in learning deficits independently of any developmental abnormalities. Importantly, these post-development impairments in function may be more easily reversible than changes established during development. For example, previous studies involved the Notch pathway in neurogenesis. Additionally, recent findings demonstrated the expression of Notch 1 in the post-natal brain suggesting that this pathway may also have a role in modulating function in the adult brain. Additionally, recent findings demonstrated the expression of Notch 1 in the post-natal brain suggesting that this pathway also have a role in modulating function in the adult brain. Interestingly, our laboratory found that Notch 1 heterozygous mice (Notch+/-) have specific spatial learning and memory deficits, while motor coordination and other behaviors required to test spatial learning seemed unaffected by this mutation. Since Notch 1 is expressed in the adult hippocampus, we propose to study the impact of the Notch+/- mutation in hippocampal physiology and in other hippocampal-dependent learning and memory. To determine whether disruption of the Notch pathway specifically in adult neurons can affect cognitive function, we will take advantage of the LBD-inducible strategy that we recently used successfully to regulate the transcription factor CREB in adult mice. We will manipulate Notch function by fusing the RBP-J(R218H) dominant-negative gene with a mutant estrogen receptor ligand-binding domain (LBD/G521R). Injection of tamoxifen (the inducer) should trigger the activation of the Notch 1 dominant-negative repressor. We will use both types of mutant mice to address the following questions: 1- Is the Notch pathway required for learning and memory? Is it required for any other behaviors? 2- Is the Notch pathway required for hippocampal synaptic function?

[unreadable] DESCRIPTION (provided by applicant): We are requesting partial support for the next Molecular and Cellular Cognition (MCC) Meeting. The Conference will be held in the San Diego Convention Center on October 21st and 22nd, 2004, right before the annual meeting of the Society for Neuroscience. The MCC meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc) and physiological (electrophysiology, optical physiology and other cellular approaches) to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive [unreadable] processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly. These meetings have been organized under the sponsorship and leadership of the Molecular and Cellular Cognition Society (MCCS), a new group whose main function is to organize meetings and promote interaction and collaborations among laboratories working in this general area. Although there are several learning and memory meetings in the USA and abroad, the Molecular and Cellular Cognition meeting is unique because it brings together individuals that integrate molecular, physiological and behavioral approaches in studies of memory and related disorders. Although the molecular and cellular cognition field is relatively new, it has already had a profound impact on neuroscience research. Importantly, in the last 5 years there has been an exponential increase in both the number of papers published, and the number of new groups that joined this area of research, which currently includes more than 100 laboratories. Currently, this is the only periodic meeting in the field, an invaluable opportunity to exchange information, and develop this young field. The 2002 and 2003 meetings were highly successful, attracting each year a diverse group of approximately 400 participants from North America, Europe, and Asia, and we have every reason to believe that the 2004 meeting will be equally successful. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Specific learning disabilities are the most common neurological complication in children with neurofibromatosis type I (NF1), a disorder affecting 1/4000 people world-wide. This genetic disease is caused by mutations in the NF1 gene which encodes neurofibromin, a Ras GTPase activating protein that is highly expressed in the brain. Our studies of mice mutant for the neurofibromin gene (Nf1+/- ) indicated that these mutants showed enhanced GABAA-mediated inhibition, deficits in long-term potentiation (LTP) and in spatial learning. In this application we propose to test the hypotheses that the spatial learning deficits of mice with an heterozygous-null germ-line Nf1 mutation (Nf1+/- mice), that model closely the human condition, are due to enhanced inhibition (either because of pre- or post-synaptic changes) that leads to deficits in LTP and subsequently to abnormalities in learning. We propose to pin-point both the cellular mechanism by which the Nf1+/- mutation affects inhibition, plasticity and learning and the brain region(s) affected by this mutation. To accomplish this we will use transgenic mice with cell-type (inhibitory or excitatory neurons) restricted deletions of Nf1, Adeno-Associated Virus type 2 Cre-recombinase (AAV-Cre) driven and region-specific (hippocampus, prefrontal cortex) deletions of Nf1, as well as a number of pharmacological, electrophysiological and behavioral tools. The specific aims of this proposal are: SPECIFIC AIM #1 - To determine whether deletions of Nf1 in either hippocampus or prefrontal cortex can account for the learning deficits of the Nf1 mutant mice. SPECIFIC AIM #2- To determine the critical cellular locus for neurofibromin's role in learning and memory. SPECIFIC AIM #3 - To determine how neurofibromin affects GABA-mediated inhibition and LTP. Although there is a great deal of data that implicate Ras/MAPK signaling in plasticity and learning, it is still unclear how this signaling pathway modulates these complex processes. The studies proposed here will further our understanding of the role of neurofibromin/Ras/MAPK signaling in the modulation of GABA-mediated inhibition, LTP and learning. Importantly, they will also be crucial for developing targeted treatments for the debilitating learning disabilities associated with Neurofibromatosis Type I.

Although in the last ten years there have been significant inroads into the molecular, cellular and systems mechanisms that mediate the early stages of contextual fear conditioning, a model of emotional memory, later stages of this process remain poorly understood. Recently our laboratory reported genetic, imaging and reversible lesion evidence that supports the idea that long-term memory for contextual conditioning depends on cortical regions, such as the anterior cingulate. Now, we propose to use a combination of genetics, transgenics, electrophysiology and 2-photon in vivo imaging to unravel the molecular and cellular mechanisms of cortical plasticity that underlie remote emotional memory. The specific aims of Project 1 are: 1- To identify genes specifically required for remote memory for contextual conditioning. In our Reverse Forward Genetic (RFG) pilot screen, out of 55 transgenics and KOs selected with a random number generator from the Jackson Laboratory collection, we found that 2 affect specifically 7-day memory (remote) for contextual conditioning, without disrupting 1-day memory (recent), general activity levels or shock reactivity. We now propose to extend this screen and test contextual fear conditioning in another 350 transgenic and KO mutants. These mutants will then be screened for somatosensory (Project 2) and visual (Project 3) plasticity deficits, as a preamble for mechanistic studies (see below) to unravel the molecular and physiological mechanisms underlying the later stages of cortical and behavioral plasticity. 2- To derive region and temporally specific mutations for genes that affect long-term memory for contextual conditioning. We propose to use the loxP/Cre recombinase system to control the cell types, brain regions and temporal expression of the mutations isolated in aim 1. The resulting mice will be tested for memory deficits and will also be studied in Projects 2 and 3. 3- To uncover cortical molecular mechanisms underlying the turnover and stability of spines in the anterior cingulate. Plasticity, including behavioral plasticity (i.e. remote memory), is thought to involve changes in neuronal structure required for consolidation and stability of stored information. We propose to use 2-photon scanning confocal in vivo imaging to examine whether mutations that affect remote memory also affect turnover and stability of spines in cortical regions required for remote memory (i.e. anterior cingulate) in trained and untrained (contextual conditioning) mutants and controls. These studies will parallel related imaging studies carried out in Projects 2 and 3. All together, the studies described here and related studies in Projects 2 and 3 will unravel fundamental molecular, cellular and structural mechanisms of how the neocortex encodes and stores information. These findings will have a key impact on how we study and treat disorders associated with emotional memory.

All three projects in the Center require mutant mice derived at UCLA. The Mouse Genetics Core will reside in[unreadable] a SPF facility at UCLA and will facilitate the back-cross, mating and dissemination of the mutant mice[unreadable] required by all three Projects in the center. The facility will maintain the lines, genotype them and send them[unreadable] when needed to the participating laboratories. An important role for the facility will be to transfer mutations[unreadable] into stable genetic backgrounds that could then be retested and sent to the laboratories in the Center. Some[unreadable] of the mice that will be tested in the genetic screen proposed in Project 1 are not in stable, balanced genetic[unreadable] backgrounds. The Mouse Genetics Core will use speed congenics and other techniques to transfer selected[unreadable] mutations into stable and defined genetic backgrounds. The facility will also be responsible for the[unreadable] distribution of mice with conditional mutations of genes identified in the plasticity screens proposed. The[unreadable] integration of studies in the Center is completely dependent on the uniformity of the genetic background of[unreadable] the mice tested. Since phenotypes, including brain phenotypes, are sensitive to genetic background, it would[unreadable] be impossible to integrate findings from different laboratories in the Center, if each laboratory used mice with[unreadable] varying genetic background. Thus, providing mice of uniform genetic background is a crucial and key[unreadable] function of the Mouse Genetics Core.

[unreadable] Description (Provided by Applicant): How is progress going to be made in understanding memory, including emotional memory and its disorders? We propose that it is important to start looking at phases of memory beyond immediate and short-term memory, beyond the initial involvement of the hippocampus. We need to alter the focus from these initial events to longer-term processes such as structural reorganization. Our refocus needs to switch from the molecular processes controlling receptor modification to those controlling synaptic and dendritic structure. Furthermore, our attention should also change from hippocampus to neocortex, as a wealth of evidence implicates neocortex in remote memory. The overall goal of the proposed Conte Center for Forward Genetic Approaches to Cortical Plasticity is to identify novel mechanisms of neural plasticity that operate in the neocortex and are critical for both remote memory and cortical plasticity. To accomplish this goal we propose to use state-of-the-art transgenic, electrophysiological and imaging approaches, including a novel genetic screen designed to identify mouse KOs and transgenics that affect remote, but not recent memory. The following is an outline of the framework of the Center that takes advantage of the long-standing (>10 years) collaborative relationship among its members: 1) The Silva laboratory will identify KOs and transgenics that have 7-, but no 1-day memory deficits for contextual conditioning, one of the most studied rodent models of emotional memory. Importantly, a pilot screen of 55 mutants already identified two with normal 1-day, but deficient 7-day memory. 2) The selected 7-day memory mutants will be screened for somatosensory (Fox Laboratory) and visual (Stryker Laboratory) cortical plasticity deficits. Out of the two memory mutants selected so far in our screen, one has been studied by the Fox group and found to have abnormal somatosensory plasticity! Our previous collaborative work also identified another mutation with the same properties (aCaMKII null heterozygous mutation). 3) Mutants that affect all three forms of plasticity will be studied collaboratively by all three laboratories with electrophysiology, in vivo imaging and behavioral tools. 4) Key genes identified in the screen will be floxed and the resulting conditional mutants will be studied in detail by the Center. The key idea is to leverage the wealth of tools and information available for somatosensory and visual cortical plasticity to understand the plasticity mechanisms underlying the harder-to- study process of remote memory storage in neocortical networks. The studies proposed here will not only shed light on poorly understood mechanisms of remote memory, they will also provide important insights for the development of treatments for memory disorders associated with psychiatric and neurologic conditions such as schizophrenia, depression and Alzheimer's disease. [unreadable]

The Administrative Core will direct, and coordinate the numerous fiscal, scientific, educational, and outreach functions of the Center. These will include a) the coordination of the scientific activities of the Center, including biennial meetings with the Board of Advisors, biannual meetings among the Pis and core facility directors of the Center, an annual meeting of the laboratory members of the Center before the Society for Neuroscience meeting, the exchange of students and post-docs among the laboratories involved in the Center, b) dissemination of key findings of the Center both to the scientific community (i.e. distribution of key papers to appropriate people in the field, organization of symposia on mechanisms of cortical plasticity, etc.) and general public (i.e., organization of public lectures and coordination with appropriate University Institutions of press releases describing key findings in the Center), oversight over the web site of the center, a key tool for information and sharing of data and c) financial administrative role in the yearly budget allocations and reports for the Projects and Cores of the Center

DESCRIPTION (provided by applicant): Neurofibromatosis Type I (NF1) and Noonan syndrome (NS), two related disorders with mental health phenotypes, are associated with cognitive deficits in nearly 1 in 2000 people worldwide. Compelling molecular and cognitive parallels between NF1 and NS suggest that their learning problems are caused by a common mechanism. Previous studies in our lab suggested the following mechanistic hypothesis for their cognitive deficits, that we propose to test here: 1) The NS and NF1 gene products regulate the activation of Ras/MAPK/ synapsin I signaling in hippocampal inhibitory terminals; 2) This signaling pathway modulates GABA release; 3) During learning NS and NF1 mutations result in the over activation of this signaling pathway, abnormally high increases in inhibition, disruptions in the activation of principal neurons, and deficits in the induction of long-term potentiation (LTP). Our specific aims are: 1. To test the hypotheses that learning triggers SHP-2/neurofibromin/Ras/MAPK-dependent increases in GABA-release, and that the NF1 and NS mouse mutations inappropriately increase these inhibition enhancements and therefore disrupt learning. This aim will focus on how learning changes inhibition. 2. To test the hypothesis that increased inhibition in Nf1 mice (and potentially NS mutants) prevents the activation of principal neurons in the hippocampus during learning, and consequently causes deficits in hippocampal-dependent learning. We propose to use transgenic fluorophores that track neuronal activity, as well as a novel two-photon imaging technique Micro Optic Probes (MOPs), to track longitudinally the activation patterns of hippocampal CA1 neurons during training in a spatial learning task. 3. To test the hypothesis that SHP-2 activating mutations in mouse hippocampal inhibitory neurons lead to Ras/MAPK/synapsin I-dependent increases in GABA release in the CA1 region, CA1 LTP deficits and hippocampal-dependent learning impairments. To test this hypothesis, we will use mouse mutants we already have and two inducible mutants that will restrict a SHP-2D61G NS mutation to either adult excitatory or inhibitory neurons. We will also use pharmacological inhibitors that target the mechanisms tested, including a FDA-approved drug. We will determine whether these drugs reverse the biochemical, physiological and behavioral deficits in animal models of NS. One of these pharmacological inhibitors (lovastatin), currently being tested in NF1 clinical trials, could also eventually be tested in NS clinical trials. PUBLIC HEALTH RELEVANCE: The animal model studies proposed here are designed to further the understanding of mechanisms of learning and memory, including those involved in the learning disabilities associated with two related genetic disorders: Neurofibromatosis type I (NF1) and Noonan syndrome (NS). Additionally, the drug studies proposed could be the preface for clinical trials of cognitive deficits associated with NS, and other mental health disorders, including learning disabilities, autism and schizophrenia.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

? DESCRIPTION (provided by applicant): Mutations in the Ras/ERK signaling pathway cause a class of complex neurodevelopmental disorders (Rasopathies) [1] associated with cognitive impairments and autism [2]. Our mouse model studies of NS showed that dominant-active PTPN11 gene mutations (principal cause of NS) enhance Ras/ERK activation at basal levels, which then increase the basal levels of synaptic AMPA receptors; Higher basal levels of AMPA receptors occlude CA1 long-term potentiation (LTP) and consequently disrupt hippocampal-dependent learning tasks, including object/place, spatial and contextual learning & memory. Studies in NS patients and our studies in mice showed that the phenotypes of the PTPN11D61G mutation are more severe than those of the PTPN11N308D mutation, a wonderful tool to address causation in NS studies, including those proposed here. Importantly, we showed that adult treatments, with drugs that decrease Ras/ERK signaling, reverse the molecular, physiological and behavioral impairments of the NS mutant mice; Additionally, we recreated these impairments with viral vector manipulations targeted to the hippocampus (HPC) in adult mice, thus directly involving this structure and confirming that disruption of adult mechanisms contribute to the NS-associated cognitive deficits. Here, we propose integrative multidisciplinary studies with state-of-the-art approaches, such as head-mounted fluorescent miniscopes in freely moving mice and in vivo 2-photon microscopy, to address three key hypotheses: 1) Since our previous studies were focused on the hippocampal CA region, we now propose that PTPN11 NS gene mutations also affect Ras/ERK signaling, AMPA receptor function and synaptic plasticity, in another brain region key for spatial learning, the retrosplenil cortex (RSC), 2) disruptions of spatial representations in HPC and RSC, two reciprocally connected areas critical for spatial processing, contribute to the spatial learning and memory impairments in NS mutant mice, and 3) RSC-dependent molecular, cellular, and behavioral deficits, as well as circuit deficits in RSC and HPC, can be reversed in adult NS mutants by an FDA approved drug (lovastatin) that we demonstrated reverses the NS-associated molecular, cellular and behavioral deficits in HPC. Beyond elucidating molecular, cellular and circuit mechanisms responsible for the spatial learning deficits associated with Rasopathies, which we hope will lead to treatments, the studies proposed here will also further our knowledge of the functional significance and interdependence of spatial representations in HPC and RSC, and they will be critical to understand other behavioral phenotypes associated with Rasopathies such as autism.

? DESCRIPTION (provided by applicant): One of the biggest challenges in neuroscience is to understand how neural circuits in the brain process, encode, store, and retrieve information. Meeting this challenge will require methods to record the activity of intact neural networks in freely behaving animals. Spectacular advances with the development of genetically encoded indicators of neural activity and optogenetic actuators now call for methods to image and manipulate the activity of large populations of identified neurons in freely moving mice over prolonged periods of time. Multi-channel imaging is needed to unequivocally identify individual neurons based on their unique gene expression profiles or projection patterns, and a flexible platform is needed so that the scopes can be easily adapted to address diverse neuroscience questions. Optogenetic capability is needed to draw causal connections between cellular activity patterns and behavior. Finally, currently available technology is limited because it requires the mice to be tethered by wires, limiting their range of behaviors and ability to interact with other animals or their environment. In addition, commercial miniaturized scopes are extremely expensive, and cannot be altered to meet individual end-user needs. Here, we seek to remedy shortfalls in existing technology by developing truly open source next-generation two-channel optogenetics-capable, wireless, miniaturized microscopes for imaging and tracking activity patterns of large neural-cell populations in freely moving mice. We will design, manufacture, optimize and test: a two-channel microscope for imaging two fluorophores (Aim 1), an optogenetics-capable microscope for imaging and optogenetic excitation (Aim 2), a wireless miniaturized microscope (Aim 3), and a microscope with combined two-channel, optogenetics, and wireless capability (Aim 4). All throughout, we will build an online environment for sharing our microscopes with the neuroscience community, lifting barriers for others to build, modify, and implant the microscopes and analyze neural activity data with our software. Our new wearable microscopes will have a transformative impact on neuroscience by permitting for the first time the imaging and manipulation of the activity of hundreds of identified neurons and other cells such as astrocytes in freely behaving animals.

? DESCRIPTION (provided by applicant): Mutations in the Ras/ERK signaling pathway cause a class of complex neurodevelopmental disorders (Rasopathies) [1] associated with cognitive impairments and autism [2]. Our mouse model studies of NS showed that dominant-active PTPN11 gene mutations (principal cause of NS) enhance Ras/ERK activation at basal levels, which then increase the basal levels of synaptic AMPA receptors; Higher basal levels of AMPA receptors occlude CA1 long-term potentiation (LTP) and consequently disrupt hippocampal-dependent learning tasks, including object/place, spatial and contextual learning & memory. Studies in NS patients and our studies in mice showed that the phenotypes of the PTPN11D61G mutation are more severe than those of the PTPN11N308D mutation, a wonderful tool to address causation in NS studies, including those proposed here. Importantly, we showed that adult treatments, with drugs that decrease Ras/ERK signaling, reverse the molecular, physiological and behavioral impairments of the NS mutant mice; Additionally, we recreated these impairments with viral vector manipulations targeted to the hippocampus (HPC) in adult mice, thus directly involving this structure and confirming that disruption of adult mechanisms contribute to the NS-associated cognitive deficits. Here, we propose integrative multidisciplinary studies with state-of-the-art approaches, such as head-mounted fluorescent miniscopes in freely moving mice and in vivo 2-photon microscopy, to address three key hypotheses: 1) Since our previous studies were focused on the hippocampal CA region, we now propose that PTPN11 NS gene mutations also affect Ras/ERK signaling, AMPA receptor function and synaptic plasticity, in another brain region key for spatial learning, the retrosplenil cortex (RSC), 2) disruptions of spatial representations in HPC and RSC, two reciprocally connected areas critical for spatial processing, contribute to the spatial learning and memory impairments in NS mutant mice, and 3) RSC-dependent molecular, cellular, and behavioral deficits, as well as circuit deficits in RSC and HPC, can be reversed in adult NS mutants by an FDA approved drug (lovastatin) that we demonstrated reverses the NS-associated molecular, cellular and behavioral deficits in HPC. Beyond elucidating molecular, cellular and circuit mechanisms responsible for the spatial learning deficits associated with Rasopathies, which we hope will lead to treatments, the studies proposed here will also further our knowledge of the functional significance and interdependence of spatial representations in HPC and RSC, and they will be critical to understand other behavioral phenotypes associated with Rasopathies such as autism.

PROJECT SUMMARY Age is the strongest risk factor for Alzheimer?s disease, and therefore, targeting factors that contribute to the onset of this disorder may be key for early prevention (1-3). Here, we propose that increases in neuroinflammatory CCL5/CCR5 chemokine signaling in middle-age(4-8) trigger the down-regulation of MAPK/CREB signaling(9-15), and that this contributes to the onset of memory deficits with aging and Alzheimer?s disease, including source and relational memory problems(16-20). Our memories depend not only on the ability to recall individual elements/items, but also on processes that link these elements/items (source/relational memory). Importantly, source/relational memory is more sensitive to age-related decline than item memory(16-20). Recently(21), our laboratory found that although in middle-aged(14-16 month old) mice contextual memory is still preserved, these mice show robust impairments in linking the memories for two contexts, a possible early sign of age-related memory deficits, including in Alzheimer?s disease. Two memories are linked when the recall of one triggers the recall of the other(21, 22). Accordingly, our studies showed that the CA1 neuronal ensembles activated by two linked contexts display significant overlap in young, but not in middle-aged mice(21). Thus, the deficit in ensemble overlap we found in middle-aged mice could underlie their memory linking deficits(21). Additionally, recent studies, including those carried out in the previous period of this project(23-34), are consistent with the hypothesis that learning triggers CREB activation and a subsequent temporary increase in neuronal excitability(35-37), that for a time biases the allocation of a subsequent memory to the neuronal ensemble encoding the first memory. We and others(21, 22) showed that this overlap between neuronal ensembles links these memories across time, such that the recall of one memory leads to the recall of the other. Our preliminary studies showed that a CCR5 null-mutation increases CREB signaling and rescues deficits in memory linking in middle-aged mice. Here, we propose to test whether increases in CCL5/CCR5-activation, and subsequent decreases in CREB-signaling, contribute to age-related decreases in CA1 excitability(Aim 1), whether this alters memory allocation in CA1(Aim 2), leading to the lack of overlap between CA1 neuronal ensembles encoding distinct contexts(Aim 2), and therefore, age-related impairments in memory linking(Aim 3). Importantly, we will test whether an FDA approved CCR5 inhibitor could be used to treat age-related memory and linking impairments. We will focus on CA1 because we showed that chemogenetically rescuing excitability in a subset of CA1 pyramidal neurons was sufficient to rescue contextual memory linking in middle-aged mice (21). Age-related increases in CCL5/CCR5 neuroinflammatory mechanisms(4-8) and their adverse effects on brain CREB signaling(38), could contribute to the onset of cognitive impairments in aging as well as in Alzheimer?s disease, since higher neuroinflammation(39) and beta- amyloid-dependent lower CREB levels, have been implicated in this disorder(40, 41).

PROJECT SUMMARY Molecular, cellular and circuit mechanisms for memory-linking deficits in psychiatric disorders Source and relational memory problems are commonly associated with a number of psychiatric conditions, including schizophrenia [1, 2], and major depression [3]. A key component of these complex cognitive problems is the inability to properly link information about items and events acquired at different times. Unfortunately, very little is known about how the brain routinely links and integrates information across time. Additionally, abnormal levels of Chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES), a ligand for the C-C chemokine receptor type 5 (CCR5), have been found in association with major depression [4], and schizophrenia[5], but it is unclear whether and how changes in this cytokine signaling system affects the complex cognitive phenotypes associated with these disorders. Recent studies in our laboratory revealed that CCR5 in neurons negatively regulates MAPK/CREB signaling, and that this modulates not only memory formation[6], but also how memories are linked across time, a mechanism that could contribute to source and relational memory problems in schizophrenia [1, 2], and major depression [3]. Results from recent studies, including those from our laboratory [7-18], suggest that learning triggers CREB activation and a subsequent temporary increase in neuronal excitability[19-21], that for a time biases the allocation of a subsequent memory to the neuronal ensemble encoding the first memory. We just reported that the resulting overlap between neuronal ensembles encoding both memories could link these memories across time, such that the recall of one memory leads to the recall of the other [22]. Here, we propose to use state-of-the-art tools, such as a new generation of head-mounted fluorescent microscopes developed in our lab, TetTag mice, a new optogenetic tool, and chemogenetics, to test the novel hypothesis that a) the opposing roles of CCL5/CCR5 and CREB signaling modulate neuronal excitability in CA1 (Aim 1), b) and consequently memory allocation in this structure (Aim 2), c) that memory allocation mechanisms determine the overlap between neuronal ensembles encoding distinct memories (Aim 2), and therefore, d) supports memory linking across time (Aim 3). Importantly, the highly mechanistic experiments proposed here will also include an FDA approved drug known to inhibit CCR5, and that could be used to help treat possible memory linking problems caused by deregulation of CCR5 in psychiatric conditions. These studies will focus on CA1 since this structure has been implicated in human relational memory[23], and the hippocampus is involved in cognitive deficits associated with schizophrenia [1, 24], and major depression [25]. Thus, the studies proposed will not only further our understanding of how memory allocation affects memory linking across time, a novel concept in memory research, they will address a possible neuroinflammatory mechanism[26] that could contribute to source and relational memory problems.

To understand how the brain processes information, creates and retrieves memories, and makes decisions it is necessary to record the activity of thousands of brain cells simultaneously. New small and light-weight microscopes have been developed that can be carried on the heads of laboratory mice and rats. These microscopes take advantage of new probes that sense calcium levels and flash bright when a brain cell becomes active. The Neuronex Neurotechnology Hub has built new miniature microscopes that not only sense light but can also directly record the electrical activity of the large numbers of cells deep in the brain. This combination of electrical and optical recordings gives scientists the new ability to read out how large groups of brain cells and brain regions work together as the brain senses, learns, plans and executes actions. The Neuronex Neurotechnology Hub will also create new computer systems that can analyze these activity patterns extremely quickly (within small fractions of a second). This rapid feedback system will allow investigators to rapidly probe how the activity of specific groups of brain cells is linked to each behavior. Finally, the Hub will build and test a new miniature microscope called a "light field miniature microscope". This version of the microscope will allow investigators to make 3-D movies of brain activity, greatly improving their view of the large network of brain cells. All these technologies will be openly shared with neuroscience community through a website (miniscope.org), such that each laboratory can build each of these devices themselves at very low cost. The Hub will hold workshops to teach scientists how to build and use the various devices. Finally the hub will reach out to the broader community by holding classes for K-12 and college students, and demonstrating how these devices can give us a view of brain function.

This Neurotechnology Hub will develop and share next-generation miniaturized in vivo sensing devices that integrate optical and electrophysiological recording from hundreds or thousands of neurons in behaving animals. These devices will be coupled with energy-efficient computing hardware for real-time signal processing and closed-loop feedback capabilities. The Hub will also create light field miniaturized microscopes that will allow three dimensional optical recordings of network activity in freely behaving animals. Last, the Hub will manufacture and distribute custom made, 3 dimensional silicon microprobes for large scale electrophysiological recordings. Making these devices widely available for neuroscience research and teaching will have significant broader impacts, by accelerating discovery and broadening outreach. The devices and techniques will be distributed widely to a large community of researchers, as previously done with the open-source miniaturized microscope developed by the PIs (the website at miniscope.org already has >2500 registered users and >250 labs using our microscope), as well as with silicon microprobes (>100 devices have been shared with users). Hence, the Hub will have a broad impact upon neuroscience research, facilitating many future advances in our understanding of the neural basis for emotion, cognition, and behavior, with a high potential to catalyze major new discoveries. The PIs will establish an outreach program through partnership with the Minority Access to Research Careers program at UCLA, as well as the UCLA Center for Excellence in Engineering and Diversity (CEED), to involve highly diversified high school and undergraduate students in this research. This NeuroTechnology Hub award is funded by the Division of Emerging Frontiers within the Directorate for Biological Sciences as part of the BRAIN Initiative and NSF's Understanding the Brain activities.

PROJECT SUMMARY Cognitive deficits are a significant clinical problem associated with HIV infection (HIV-associated neurocognitive disorders or HAND). We have recently[1] showed that an HIV coat peptide (HIV Gp120 V3 loop peptide or HIV-V3 peptide), known to bind and activate a receptor that mediates HIV cellular entry (CCR5), caused acute memory deficits[1], that could be prevented by a CCR5 knockout or viral vector-mediated knockdown of this receptor[1]. Additionally, while transgenic overexpression of CCR5 in neurons resulted in hippocampus-dependent learning and memory deficits[1], decreasing the function of CCR5 enhances hippocampus-dependent memory (e.g., spatial and contextual learning) by enhancing canonical memory mechanisms, including MAPK/CREB signaling leading to enhanced long-term potentiation (LTP). Knock-down of this receptor in retrosplenial cortex (RSC) also enhances spatial learning, and two-photon microscopy studies of spine turnover in this structure showed that CCR5 knock out increases spine turnover before learning and spine clustering after learning, two structural markers of enhanced learning and memory. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity and learning and memory, and demonstrate that while knockdown of CCR5 enhances plasticity and learning, over-activation of this receptor has the opposite effect[1], suggesting that CCR5 over-activation by viral proteins may contribute to HAND[1] [2]. Here, we propose integrative multidisciplinary studies with state-of-the-art approaches, such as head- mounted fluorescent microscopes (miniscopes) developed in our laboratory that can be used to track neuronal activation in freely moving mice [3]. These studies have two key goals: 1- To determine whether the GFAP- gp120 transgenic model [2, 4] or the HIV-V3 peptide specifically targeted to the adult RSC, disrupt MAPK and CREB signaling, LTP, spatial and contextual learning and memory, and whether this can be prevented by a CCR5 knockout, viral knockdown of this receptor, or by a FDA approved drug that inhibits CCR5 (Maraviroc) with or without combination antiretroviral therapy (cART); 2- To test whether spatial representations, measured with GCAMP6f and miniscopes, in the hippocampus and in the RSC are disrupted by the HIV-V3 peptide or in the GFAP-gp120 transgenic model, and whether this can be prevented by a CCR5 knockout, CCR5 viral knockdown, or by cART with or without Maraviroc. Beyond elucidating molecular, cellular and circuit mechanisms responsible for the learning and memory deficits caused by HIV proteins, the studies proposed here will also test specific treatments that could be potentially used to design clinical interventions targeted to the earlier stages of HIV infection. The relative lack of neuropathology attributed to HIV infection in cART-treated subjects [5], argues that the mechanisms responsible for HAND are likely due to functional alterations in neurons, such as the ones we propose to study here.