Leslie C. Griffith - US grants

9421360 Calcium/calmodulin-dependent protein kinase II (CaM kinase) has been shown to be involved in learning and memory in both mammals and fruit flies. This enzyme has properties which have suggested that it may be a "molecular switch", capable of initiating changes in cellular properties. Studies of CaM kinase in Drosophila melanogaster have shown that this enzyme is required for learning and memory in an associative behavior. In addition, it has been shown that the larval neuromuscular junction of flies expressing an inhibitor of CaM kinase produce abnormal repetitive firing to single stimuli, and have depressed plasticity. This model system is being used to investigate the molecular basis of these effects. A mutant lacking CaM kinase provides a template on which to test theories about the enzymatic properties of the kinase which are required for normal neuronal activity.

Calcium/calmodulin-dependent protein kinase II (CaM kinase) has been implicated in synaptic plasticity in both vertebrates and invertebrates and has properties which suggest that it may be a "molecular switch". Studies in Drosophila melanogaster and in mice have shown that CaM kinase is required for learning and memory. The objectives of the proposed study are threefold: 1) Identification of proteins that interact with CaM kinase. Using the yeast two-hybrid system, we will identify and characterize substrates, regulators and localizers of CaM kinase that are involved in neuronal function. The in vivo phosphorylation of Eag, a potassium channel subunit, will be studied. 2) Identification of genes that interact with CaM kinase. Mutations at the CaMK locus are lethal in the homozygous state, but viable as CaMK/+ heterozygotes. We will perform an enhancer screen to identify genes which, when mutant, enhance lethality of CaMK/+ heterozygotes. 3) Characterization of the larval NMJ phenotype of CaML/+ flies. We will assess the effects on identified ionic currents and neuronal plasticity at the NMJ in mutants lacking one copy of the CaMK gene. 4) Characterization of the membrance properties of CNS neurons which require CaM kinase for plastic behavior. We will study cultured CNS cells that have been shown to be involved in CaM kinase-dependent courtship conditioning. We will assess the effects of CaM kinase on identified currents in these cells. Cognitive functions such as learning and memory are impaired in many disease states. Understanding the biochemical basis of normal changes in neuronal properties is an important first step in understanding how pathological processes can disrupt brain function. The ability to genetically manipulate CaM kinase in DrosophiLa will allow us to understand not only its biochemical role, but its role in cellular and behavioral processes. The funding of this proposal would significantly enhance the ability of the PI to accomplish these goals by reducing her teaching and administrative duties. The interdisciplinary nature of this research program necessitates central involvement of the PI in training of students and postdocs in techniques and areas in which they are inexperienced. The PI will also be involved with the integration of electrophysiology into the research program as a new tool for studying the roles of CaM kinasein neuronal function.

Calcium/calmodulin-dependent protein kinase II (CaM kinase) has been implicated in synaptic plasticity in both vertebrates and invertebrates. This enzyme has properties which suggest that it may be a "molecular switch". Studies in Drosophila melanogaster have shown that CaM kinase is required for learning and memory in an associative behavior, courtship conditioning. The objectives of the proposed study are threefold: 1) Analysis of the biochemistry localization and in vivo function of the Drosophila calcium/calmodulin-dependent protein kinase II isoforms. This kinase, like its mammalian counterparts, has been shown to consist of multiple subunits that show sequence variation between the CaM-binding and association domains. Variations in CaM kinase activity of the isoforms may have functional consequences in the nervous system. The effects of the variable region on regulation of CaM-binding and substrate specificity will be investigated. To determine if differential localization plays a role in the function of the kinase, subcellular distribution of the isoforms will be studied. Finally, we will determine if individual isoforms can rescue viability and behavior on a CaM kinase null background. Understanding the regulation and function of this kinase will allow fuller understanding of its role in neuronal function. 2) Identification of proteins that interact with CaM kinase. We intend to identify and characterize substrates, regulators and localizers of CaM kinase that are involved in neuronal function. One such substrate, Eag, a potassium channel subunit, will be studied with respect to its in vivo phosphorylation. Additional substrates and proteins which interact with CaM kinase to localize and/or regulate it will be identified using the yeast two-hybrid system. Identification of such proteins will allow us to begin to assemble biochemical pathways of synaptic plasticity. 3) Identification of genes that interact with CaM kinase. Mutations at the CaMK locus are lethal in the homozygous state, but viable as CaMK/+ heterozygotes. We will perform an enhancer screen to identify genes which, when mutant, enhance lethality of CaMK/+ heterozygotes. The genes obtained in this screen will provide insight into the functions of CaM kinase and allow us genetic access to biochemical pathways that may be involved in synaptic plasticity. Cognitive functions such as learning and memory are impaired in many disease states. Understanding the biochemical basis of normal changes in neuronal properties is an important first step in understanding how pathological processes can disrupt brain function. CaM kinase has been proposed to play a role in many plastic processes, from long-term potentiation to whole animal behavior. The ability to genetically manipulate CaM kinase in Drosophila will allow us to understand not only its biochemical role, but its role in cellular and behavioral processes.

This research project is designed to take a CaM kinase-dependent plastic behavior and define the neuronal circuits that generate that behavior. We will manipulate kinase substrate activity genetically and monitor the effects of these manipulations on whole animal and synaptic behavior. These experiments will provide a well defined, genetically and physiologically accessible model for learning and memory, define biochemical pathways downstream of CaM kinase required for generation of this behavior and correlate specific changes in the properties of synapses with alterations in behavior. Specific aims include: 1. Identification of neuronal circuits requiring CaM kinase for plastic behavior. We will use GAL4/UAS expression of modulators of CaM kinase activity to define the neural substrates of courtship conditioning. We will investigate long-term memory in this paradigm. A neural network model will be generated to allow qualitative and quantitative predictions of the effects of manipulation of the circuit on behavior. 2. Characterization of plastic behavior in animals with altered CaM kinase-dependent biochemical pathways. We will investigate the role of the CaM kinase substrates Eag and Adf-1 in the production of plasticity in the courtship conditioning assay. Dominant mutant transgenic strains will be constructed to test the function of phosphorylation of these substrates in behavior. 3. Characterization of the effects of alterations in CaM kinase and its substrates on synaptic plasticity at the larval neuromuscular junction. We will use the third instar neuromuscular junction (NMJ) to investigate synaptic plasticity in animals with alterations in CaM kinase and its substrates. Genetic methods allowing expression of transgenes in either the pre- or postsynaptic cell will allow definitive assignment of aspects of CaM kinase function to one side of the synapse. Cognitive function such as learning and memory are impaired in many disease states. Understanding the biochemical basis of normal changes in neuronal properties is an important first step in understanding how pathological processes can disrupt brain function. CaM kinase has been proposed to play a role in many plastic process, from long-term potentiation to whole animal behavior. The proposed experiments are directed at allowing the first direct correlation between a biochemical activity, cell excitability and a whole animal behavioral output.

DESCRIPTION (provided by applicant): A number of signal transduction pathways have been implicated in behavioral, developmental and synaptic plasticity using pharmacological and genetic manipulations. Molecules identified as important in the generation of both short-term and long-term plasticity include CaMKII and PKA. The overlapping use of a few biochemical pathways to set up the circuitry for, and mediate, complex behaviors implies the existence of important temporal and/or spatial constraints on activity. This proposal is will use novel methods in Drosophila to detect and manipulate the spatial and temporal patterns of activation of protein kinases. Specific Aim #1 will probe the role of CaMKII autoregulation and localization in associative memory formation using temporally controlled expression of mutant kinase transgenes in cells known to be part of the memory circuit. Concurrent temporal and spatial control will be achieved by using either selectively inhibitable kinase transgenes or a tetracycline-controlled GAL4 enhancer trap strategy. The role of modulation of excitability as an important downstream consequence of CaMKII activation will be tested by manipulation of Eag potassium channel activity. Specific Aim #2 will identify the developmental window during which CaMKII acts to modify adult circuit formation. Once this window is established we will identify the sensitive cell groups. The morphological and gene expression consequences of early manipulation of CaMKII will be explored. Specific Aim #3 will develop and implement genetically based sensors to measure the activation of kinases in real time in intact behaving animals. The initial sensor will detect kinases such as PKA that activate gene expression via CREB and are also involved in short-term plasticity. These experiments will give us temporal and spatial information about the use of biochemical pathways during development and behavior. The high level of synteny in the biochemistry of neuronal function between mammals and flies leads to the expectation that these studies will have relevance for human learning disabilities.

DESCRIPTION (provided by the applicant): This grant requests funds for the purchase of a BIAcore 3000 surface plasmon resonance instrument for the analysis of biomolecular interactions in a variety of systems. The Biology and Biochemistry Departments of Brandeis do not currently possess any instruments with the capabilities of a BIAcore 3000 for the analysis of protein-protein, protein-membrane, protein-small molecule and protein-DNA interactions. The acquisition of a BIAcore instrument would significantly enhance the research capabilities of the life science group at Brandeis. The instrument would be housed in the Core Biomolecules Synthesis and Analysis Facility. This facility is staffed and instrument management mechanisms (financial, maintenance) are already in place and will be supplemented to accommodate the BIAcore. The BIAcore 3000 would be accessible to all NIH-supported investigators at Brandeis University as well as others on a time-available basis. The six investigators in the user group are interested in both traditional uses of the instrument (kinetics and equilibria of protein-protein interactions) and in development of new techniques. Projects include: regulation of CaM trapping by autophosphorylation of CaMKII isoforms; characterization of assembly of synaptic protein complexes; characterization of ligand/inhibitor binding and protein domain interactions of IMPPDH; regulation of exonuclease ssDNA binding by auxiliary subunits; characterization of splicing factor protein-protein interactions; measurement of Ab:Ag affinities during B cell differentiation; identification of ligands for orphan nuclear hormone receptors. Institutional commitment for acquisition and maintenance of a BIAcore 3000 is high. The salary of the Principal Operator, as well as space in the Facility, and funds for maintenance of the instrument are guaranteed into the foreseeable future.

DESCRIPTION (provided by applicant): CaMKII plays an important role in learning and memory formation in Drosophila as well as in other animals. We have recently found that CaMKII alters the excitability of identified neurons in the third instar larval nervous system of Drosophila. We will study the mechanisms of this modulation and explore its possible role in neuronal plasticity. Genetic access to these identified neurons will allow mechanistic studies of intrinsic property modulation that have not been possible in other systems. Our specific aims are: 1) Determine the effects of genetic and pharmacological manipulation of CaMKII on the excitability and behavior of central neurons. We will look at the effects of CaMKII on firing properties such as threshold, spike amplitude, spike waveform and firing frequency. We will define the voltage-dependent currents that are modulated by short-term and long-term alteration of kinase activity. 2) Determine the cellular mechanisms of activity-dependent modulation of intrinsic properties. We have shown that activity can induce a long-lasting increase in neuronal excitability in Drosophila motor neurons. We will investigate this phenomenon to determine the roles of firing pattern, calcium influx and activity of the postsynaptic cell in induction. We will determine if activity can change the response of the neuron to its normal presynaptic partners. 3) Determine the signal transduction mechanisms of activity-dependent modulation of intrinsic properties. We will define the conductances that are modulated by this process and investigate the role of CaMKII using transgenes and drugs. These studies will provide insight into a fundamental mechanism of plasticity and define novel roles for CaMKII in the regulation of short-term and long-term changes in excitability. Understanding these pathways will advance our knowledge of the basic processes that shape behavior.

CaMKII plays an important role in learning in Drosophila as well as in other animals. Localization of CaMKII allows this highly abundant enzyme to achieve specificity of action. Scaffolding interactions can also regulate the activity of CaMKII. We have characterized two such interactions: an interaction with the Eag potassium channel that renders the enzyme constitutively active without kinase autophosphorylation and an ATP-dependent interaction with Camguk (Cmg), a MAGUK protein that enhances inactivating autophosphorylation of the kinase. Our specific aims are: 1) Determine the role of alternative splicing in the subcellular localization of CaMKII. CaMKII localization is aberrant in a mutant that alters CaMKII isoform ratios and CaMKII can enter the nucleus in eag mutants. We will investigate the mechanisms of CaMKII subcellular localization and nuclear translocation. 2) Determine the role of the CaMKIhEag complex. We will define the molecular basis of the interaction and use this information to design specific inhibitors to use in vivo in electrophysiological and behavioral assays. 3) Determine the role of the CaMKIhCmg complex. We will define the molecular basis of the interaction and use inhibitors and overexpression to probe the role of Cmg in the regulation of CaMKII activity. Cmg promotes formation of a calcium-insensitive pool of CaMKII which may be important in modulating calcium signaling during plasticity. These studies will provide insight into the regulation of a protein that is fundamental to synaptic plasticity. Understanding these pathways will advance our knowledge of the basic processes that shape behavior.

DESCRIPTION (provided by applicant): Neurogenetic and molecular neurobiological investigations of sex-specific behavior in Drosophila are proposed. These studies will focus on two of the most salient, quantifiable, and behaviorally important features of the fixed action patterns that define these phenomena: the male's species-specific courtship song and separate components of mating itself. Among the experiments proposed are those involving manipulation of genes that function within the sex-determination hierarchy (SDH) of Drosophila melanogaster: fruitless (fru) and doublesex (dsx). The fru gene will be manipulated to delve into the neural substrates of courtship song, which cannot be performed by fruitless mutants lacking male-specific FRU protein in their ventral nerve cord (VNC); and of mating biology, two elements of which are dramatically perturbed in certain fru mutants. For the other SDH factor (dsx), similar transgene-based experiments are proposed to study the manner by which doublesex participates in programming VNC development and (potentially) regulating its ongoing function--both in the context of a sharply defined abnormality of singing behavior in dsx mutants. The doublesex experiments will be performed against a backdrop of an all-too-common view that sexual differentiation of Drosophila's nervous system is not influenced by this gene--which is belied by the behavioral phenomenon just referred to, along with a recent finding that DSX proteins are found in distinctive patterns within the metamorphosing and mature VNC. With respect to distal genic effectors of courtship actions, it is proposed to assess the effects of a fruitless-associated neurochemical factor (serotonin, or 5HT) that is strongly surmised to coordinate a key feature of mating, along with analyzing genes encoding 5HT-synthetic enzymes that are candidates to be controlled by the FRU transcription factor; and to manipuate neural expression of a song-controlling ion-channel gene (slowpoke). These SDH and effector-related experiments will involve applications of extant mutants (including those recently induced); genetic mosaics; and both spatial plus temporal manipulations of these genes' expressions, via utilization of existing transgenes and creation of novel molecularly engineered, reproductive-related factors, including those cloned from different Drosophila species and bioassayed in D. melanogaster

The context in which a sensory experience occurs can strongly influence whether it is perceived as pleasurable or aversive. In Drosophila, the volatile lipid pheromone cis-vaccenyl acetate (cVA) is produced by males and transferred to females during mating. When presented on eggs laid at food sources, it acts as an attractant. When presented on a mature female, it causes both instantaneous and long-term suppression of male courtship. In both of these situations the presence of another odor dictates the behavioral response to cVA. Our overall goal is to understand how olfactory context can change the immediate effects of a pheromone and lead to long-term changes in behavior. In this grant we will define the circuitry that is involved in sensing cVA and courtship-stimulating pheromones and identify the mechanisms that integrate pheromone signals to create appropriate modulation of courtship initiation. We will use genetically encoded probes of neuronal activity to assess the contribution of local antenna! lobe circuits to association of these cues. These experiments will provide insights into how odor information is processed and into howolfacotry memory is formed. Research in invertebrate model systems has arguably been the driver of the field of molecular memory in mammals. The high degree of structural and molecular conservation in the olfactory system and in mechanisms of learning makes work on olfactory plasticity in Drosophila germane to human health for the many developmental and acquired conditions that affect learning and memory. Specific aims are: 1. Define the olfactory neuron inputs that are required for sensing cVA and courtship-stimulating female pheromones. 2. Investigate the mechanisms of association between stimulatory pheromones and cVA in the antennal lobe. 3. Investigate the mechanisms of context integration in the antennal lobe. 4. Characterize the role of dTrpAl, a non-specific cation channel, in suppression of male-male courtship (collaboration with Garrity Lab). The olfactory system of Drosophila and the molecular basis of learning in this organism are highly homologus to that of humans. Understanding of the cellular mechnaism of olfactory learning in this organism will provide valuable information that can be used in designing therapeutics for human disorders of cognition such as Alzheimer's disease and mental retardation.

DESCRIPTION (provided by applicant): The public health and economic impact of circadian and sleep misalignment had grown enormously with the increasingly global and 24 hour nature of our society, and costs are in the billions. In spite of this, however, we know very little about the mechanisms by which sleep and the circadian clock influence memory circuits. Memory formation is bidirectionally sensitive to sleep levels. Sleep in the temporal window following training can enhance formation of memory, while sleep deprivation has negative effects on its formation. In Drosophila, we have a very detailed understanding both of the processes that underlie memory formation and of the circadian clock and sleep circuits. In this proposal we will leverage this knowledge to understand, for the first time at a cellular level, the direct links between memory and sleep/circadian systems. In Aim 1 we demonstrate that the fly circadian circuit and the fly memory circuits have direct anatomical connections. Further, we show that a circadian neuropeptide which has been associated with arousal is required for normal memory. We hypothesize that these connections provide the pro-arousal function of the circadian clock that helps maintain cognitive function over the course of the day. We will investigate the molecular function of the connections between these peptidergic clock cells and memory circuits and their behavioral role. Understanding connectivity of the clock and associative memory processes at this level will provide us with the ability to develop novel cognitive protection and enhancement strategies. In Aim 2 we demonstrate that the very process that underlies formation of stable associative memory, concurrent activation of neurons that drive memory consolidation, also drives sleep. This sleep is tightly linked to the learning process and we hypothesize that it is critical for consolidation. We will define the molecular and cellular processes that cause sleep and consolidation in this circuit and investigate the role of concurrently generated sleep in behavior. Understanding this linkage will provide important insight into how manipulation of sleep can be used to enhance memory formation.

The activity of individual neurons that are embedded in brain circuits has critical influence on system output. In this project, we will determine how the set point for neuronal firing rate is coded in different neuron types and how it can be homeostatically maintained in the face of time, environmental change and plasticity. We have shown that firing rate set points in Drosophila central neurons, like mammalian cortical neurons, involves the activity of CaM kinases. We will use genetic techniques coupled with central neuron electrophysiology to determine how these set points are structured in both excitatory glutamatergic neurons and inhibitory GABAergic neurons. We will use transcriptional profiling to determine the output programs that are engaged when firing rates diverge from their set point in these cell types. By comparison to similar gene sets extracted from mammalian glutamatergic and GABAergic neuron types, we will determine if these output programs represent an evolutionarily conserved genomic response to firing rate perturbation. Lastly, we will perform genetic screens in Drosophila to identify new components of the core firing rate sensor. The mammalian homologs of the genes identified in these screens will be tested to determine if they are part of the homeostatic firing rate sensor in the rodent nervous system. Crustacean homologs will similarly be tested in the stomatogastric ganglion. In aggregate, these studies will be central to formulating an understanding of the nature of homeostasis of firing rate. The fundamental and general nature of the problem suggests that the strategies used by vertebrate and invertebrate neurons will be similar, and that a multi-species approach will allow us to efficiently uncover the salient features of the system.

? DESCRIPTION (provided by applicant): Precise temporal and spatial regulation of gene expression is essential to many aspects of nervous system development, function and plasticity. Among several classes of gene regulatory factors, non-coding RNAs have emerged as a rich potential source of regulatory mechanism in the central nervous system. In particular, microRNAs (miRs) provide sequence-specific control over target mRNA translation and stability that can tune the levels of downstream proteins quite precisely thus improving the stability and robustness of molecular networks. However, comprehensive analysis of miR function within the intact nervous system has been very challenging, leaving open key questions such as: How complex is the miR regulatory landscape for neural circuits that mediate essential behaviors? Are these miRs acting mainly during neural development or are they reused to manage ongoing neural circuit activity and adaptation to stimuli? To what extent are miR mechanisms utilized in many parts of the brain, or do they regulate distinct sets of target genes in different cell types and/or developmental stages? In order to address these questions, we have assembled a team of accomplished investigators prepared to work in unison using multiple robust behavioral and cellular assays as part of an integrated program. Our team includes Drs. David Van Vactor (Harvard Medical School), Leslie Griffith (Brandeis University), Ronald Davis (Scripps Institute), and Dennis Wall (Stanford University), who will each assume responsibility for key components of this joint program. We will use Drosophila as a model organism that offers many sophisticated genetic tools complementary to the innovative tools we will develop. Drosophila has proven to be particularly effective for identification and dissection of cellular and molecular mechanisms underlying well conserved behaviors. This model is also accessible to a full range of techniques for determining the detailed cellular and physiological phenotypes of mutants in specific pathways, thus offering a system ideal for mapping out miR functions on a comprehensive scale followed by mechanistic dissection that will effectively leverage a wealth of tools and knowledge. Together, we will (i) build and apply new genetic tools, (ii) apply these tools to identify miRs required in multiple neural circuits, (iii) discover the mechanisms and regulatory strategies for miR function in each context, and then (iv) compare each model to distinguish general and specific strategies and examine their conservation. This will be the first analysis of its kind in the nervous system. Our preliminary findings already identify convergence between different circuits that will prioritize our detailed studies of several miRs: miR-13, miR-92, miR-190 and let-7. Preliminary analysis of miR-92 already points to a series of highly conserved downstream genes implicated in both neural circuit development and synaptic plasticity from insects to mammals, providing a set of specific mechanistic hypotheses that we will test in the three model circuits to define the regulatory logic for each validated target.

Project 2 Project Leader: Griffith, Leslie C. Project Summary / Abstract The public health and economic impact of circadian and sleep misalignment had grown enormously with the increasingly global and 24-hour nature of our society, and costs are in the billions. In spite of this, however, we know very little about the mechanisms by which sleep is regulated. This project will provide the first comprehensive look at the function of microRNAs (miRs) in sleep. The dominant model of how the amount of sleep is determined is that the major regulators of sleep are the circadian clock and homeostatic sleep drive. It has become clear in the last few years, however, that other physiological variables can also exert modulatory influences. We have recently completed a screen of a library of miR-sponges and identified specific miRs which appear to be essential for each of these levels of control and we propose to understand the roles of these miRs at the molecular and cellular levels. This project is also part of a more integrated assault on the conserved roles of miRs in plasticity. Sleep and synaptic plasticity have been posited to be intimately related for two reasons. The first is that sleep itself is a daily exercise in rewiring: sensory inputs and motor outputs are suppressed. The second is that one of the most compelling theories of why we sleep is that we need a daily ?resetting? of our circuitry to keep synaptic weights and circuit activity with a linear range to prevent saturation of our information storage capacity. Our work on sleep-regulating miRs will be analyzed in the context of experiments done by the Van Vactor lab on NMJ synaptic plasticity and by the Davis lab on associative learning to give us a glimpse into the more fundamental roles of miRs in regulation of synaptic function. Specific Aims: 1: Determine the complete set of microRNAs which control sleep. 2. Determine how miR190 regulates sleep plasticity. 3. Determine the role of miR92a/b in the regulation of sleep by the clock. 4. Determine the role of let-7 in the hormonal regulation of sleep.

? DESCRIPTION (provided by applicant): The ability to understand how brains generate behavior both in normal and pathological situations relies on our understanding of the neural computations carried out by behavioral circuits. The nature of chemical communication between neurons is determined by the specific neurotransmitters released onto postsynaptic targets and it was thought for many years that neurons released a single type of transmitter onto all their targets. In recent years, however, it has become clear that key neurons in vertebrate and invertebrate circuits involved in addiction, memory formation, feeding behavior and reproduction contain neurons that violate this rule, releasing multiple neurotransmitters. In this proposal we develop genetic tools to allow mapping of the distribution of specific neurotransmitter release sites in single neurons in multiple colors by modifying the endogenous genetic loci of vesicular transporters for neurotransmitter with fluorescent proteins. This allows both accurate and complete accounting of transmitter release sites since the marker's generation and turnover rely on processes that control the presence of the endogenous protein. Single cell resolution is obtained via an intersectional version of this strategy in which split fluorescent proteins become reconstituted only in specific cells. There is no current technique in any system which can provide this level of resolution. This technology will provide the ability to determine, in complex neurons releasing several chemical substances, the spatial distribution of each of the chemicals and its relationship to downstream targets of that neuron. It will also allow the mapping of temporal changes, either developmental or plasticity-induced, in neurotransmitter release. The technique is developed initially for use in Drosophila, a model organism which has been immensely important for our understanding of both the genetic and circuit basis of behavior, but as a general strategy can also be adapted for use in mammalian brain.

Project Summary/Abstract Over the 24 year tenure of this grant, Brandeis has built a multi-disciplinary program encompassing faculty in Biology, Biochemistry, Chemistry, Mathematics and Psychology. We educate students in the full range of Neuroscience topics from the molecular biology of neuronal non-coding RNAs to the cognitive effects of aging. Each member of our faculty collaborates with multiple others, and most projects involve several levels of analysis. Our students are part of an intellectually and spatially integrated neuroscience community. The resulting cohesion is reflected in every aspect of the program: coursework, rotations, thesis supervision, the shared availability of advanced instrumentation and the collegiality of interactions. The breadth of opportunity and interaction at Brandeis and close attention we pay to student progress allows each student to develop according to her/his individual needs, and results in a low attrition rate. While we are a relatively small program, over the last five years our program has graduated 18 students (3 of whom were members of underrepresented minority groups). Our students graduate with excellent credentials and go on to obtain positions in academia, health-care, government and industry that directly contribute to the NIH mandate to benefit human health. Our students enter Neuroscience from a remarkably wide variety of different backgrounds (Psychology, Biology, Biochemistry, Chemistry, Physics, Computer Science, Engineering, and Mathematics). A strong aspect of our program has always been its emphasis on quantitative thinking. Every area of Neuroscience is increasingly driven by large data sets and all Neuroscientists must be able to at least understand the principles of their analysis. In this renewal we rethink our approach to quantitative literacy and propose a curricular reform that will train our students in the fundamentals of quantitation rather than in specific high level methods. By giving them computational tools and by teaching them to code, all of our students will have a solid foundation for rigorous research at any level. The program of course work, rotations, multiple small-group colloquia, proposition exams, and participation in teaching necessarily consumes the bulk of a student's time during the first two years. They cannot (and we would not want them to) have their laboratory research as their sole focus prior to their third year at Brandeis. Therefore, they are not supported on research grants in these first critical years. This training grant provides crucial funding to support students while they develop a broad set of intellectual skills. There are 22 mentors in our program and we are requesting funds for 8 trainees.

Experiments show that interference with CaMKI I after LTP can erase LTP, a strong indication of the importance of CaMKll in the LTP maintenance process. CaMKll holoenzymes contain 12 catalytic subunits. In the ON state, each subunit is phosphorylated and therefore active. When a site becomes dephosphorylated, it can be refreshed (rephosphorylated) by a neighboring active subunit. Aim 1. Is refresh dependent on activity? The CaMKll refresh process requires low levels of Ca, but whether achievement of this level is dependent on spontaneous neural activity is not known. Therefore, a fundamental question of interest is whether the maintenance of LTP or memory requires neural activity. In Aim 1A, we will test this in acute hippocampal slices. In Aim 1B, we will explore whether activity is necessary for maintaining memory at the behavioral level using Drosophila. Although it is known that CaMKll is important for Drosophila memory, experiments have not yet tested whether CaMKll is important in memory maintenance. Given the importance of this issue for interpreting the effects of activity on memory, we will conduct the critical erasure test for determining whether CaMKll mediates memory storage in Drosophila. Aim 2. Does CaMKll subunit exchange occur in vivo: a potential mechanism for molecular refresh? According to theoretical models, switch stability could long outlive the lifetime of any subunit if CaMKll underwent protein turnover by subunit exchange: a newly inserted unphosphorylated subunit could be phosphorylated by a neighboring phosphorylated subunit, thereby providing a molecular refresh. We will make the first attempts to test whether subunit exchange occurs in living cells (Drosophila and hippocampus) and characterize its activity-dependence. Aim 3. Computational modelling: what kinds of neural activity are required to refresh CaMKll phosphorylation? The level of resting Ca2+, and that during spontaneous action potentials or mEPSPs can be estimate, as well as the rate of these reactions. We will use a verified computational model of CaMKll to determine whether these brief Ca2+ events are sufficient to refresh the phosphorylated state of CaMKll and thus ensure the stability of stored information. The results will bear importantly on the fundamental question of whether refresh reactions are mediated by spontaneous activity, or alternatively, are dependent on a network process that replays memories. RELEVANCE (See instructions): Understanding the processes that store memory at synapses will have major implications for several health problems. In particular, addiction has been demonstrated to involve persistent changes in CaMKll at synapses in the basal ganglia networks that are critical for addictive behaviors. The proposed work may provide ways to turn off CaMKll and thus reduce addictive behaviors. CaMKll has also been strongly implicated in memory disorders and stroke.

Project Summary/Abstract Activity-dependent synaptic modifications (LTP/LTD) are a major candidate for the mechanism of memory. LTP involves induction, maintenance, and expression processes. This proposal seeks to elucidate the molecular basis of the maintenance process, the process that underlies the engram. The critical test of any hypothesis regarding maintenance is the ?erasure test? in which an inhibitor is applied after LTP/memory is established. If this blocks LTP/memory and the effect persists after the inhibitor is removed, the inhibitor must have erased a maintenance process. We have conducted the erasure test using an inhibitor of CaMKII (CN-peptide). We found that application of this peptide after LTP induction produced erasure of saturated LTP. We now propose two experiments that test the role of CaMKII? in memory maintenance at the behavioral level. In the first, we ask whether a dominant-negative form of CaMKII? can erase conditioned place avoidance. We present strong preliminary evidence that it does. The second test is the ?occlusion test.? It has been shown that activated kinase (CaMKII?*) enhances synaptic transmission that occludes synaptically induced LTP. We will virally express CaMKII?* and test two predictions: that because this maximally increases all synaptic weights it should destroy memory function, and furthermore, that learning under these conditions should not be possible. Preliminary evidence supporting these predictions is presented. Other experiments in this proposal are aimed at understanding the nature of the CaMKII? complex that stores the engram. There are strong reasons to suspect that what maintains LTP is actually the complex of CaMKII? with NMDAR (and perhaps also densin- 180). Studies of the binding of proteins to CaMKII have relied on in vitro work, and there has been no previous method for studying the complexes formed during actual LTP induction. Thus, crucial information regarding the complex formation and persistence is lacking. We have developed and validated a novel optical method based on FLIM-FRET. Our preliminary evidence demonstrates that LTP induction produces complex of CaMKII? with GluN2B in spines and that the formation is synapse specific. We will determine the duration of the complex under conditions that either induce short-lasting LTP (early LTP) or produce both early and late LTP. We will also examine how the duration of the complex depends on factors that enhance (e.g., BDNF) or prevent (e.g., protein synthesis inhibitors) late LTP. This approach will be extended to study the interaction of CaMKII? with densin-180. Having identified properties of the complex that underlies LTP, in vivo experiments will be conducted to test whether disruption of protein interactions within the complex can disrupt maintenance of the engram.

Abstract Memories of salient events in our lives are part of what makes us individuals. CaMKII has been shown to be required in both vertebrates and invertebrates for short-term (STM) and long-term memory (LTM). CaMKII protein is enriched at synapses, and is synthesized locally in response to activity patterns that lead to LTM formation. Both of these features require specific sequences present in the distal untranslated part of the CaMKII mRNA. In spite of extensive work on local translation of CaMKII, several fundamental questions remain unanswered: Is there a requirement for somatic factors in local translation? We will disrupt the connection between the cell body and the synapse to test whether transport of somatic material to the synapse is required for activity-stimulated local synthesis of CaMKII. How is the information specifying local translation encoded in mRNA? Using transgenes encoding fluorescent reporters and real-time assays of new protein synthesis, we will determine what sequences are required for CaMKII synaptic localization and activity-dependent translation. What are the cellular components that read this information? We will do a bioinformatically-driven candidate gene screen in parallel with RNA affinity purification to identify proteins that regulate basal and plasticity-stimulated CaMKII accumulation. How does disruption of local translation of CaMKII affect LTM? We will use conditional genome editing to remove mRNA sequences that specify local translation or to replace them with specific mutants. We will determine which cells in the adult learning circuit use this information during LTM formation. This project utilizes cutting-edge genetic, cell biological and optical methods to address the molecular basis of a phylogenetically-conserved mechanism of plasticity in a way that will further our understanding of complex behaviors.