Prevention of obesity is an important scientific and clinical problem, because it causes metabolic syndrome. Leptin is a key molecule that controls energy balance. Leptin over-expressing transgenic mice have lean phenotype.However recombinant leptin administration fails to cure obesity because of leptin resistance. Overcoming leptin resistance is important in therapeutic approach. 2003-Several reports suggest a link between obesity and local inflammation in adipose tissue. Although leptin is an important regulator of energy balance and has potent effect on immune cells to produce pro-inflammatory cytokines, little is known about the metabolic aspects of leptin signaling in the immune system. I hypothesize that leptin signaling in the immune system may contribute to the control of metabolic status through pro-inflammatory cytokine secretion. Thus leptin sensitivity of macrophages may affect the inflammation of the organism, which would affect the metabolic status. I propose to address this possibility by analyzing the effect of leptin sensitivity of immune cells in mice. In this context, I will generate bone marrow chimera mice lacking leptin signaling in immune cells. And I perform in vivo analysis of leptin sensitivity. To gain insight into the effect of leptin on T cells, I will perform a microarray analysis of T cells treated with leptin in vitro, in order to characterize the effect of leptin on gene expression and to identify key gene products involved in coupling between the immune system and the metabolic status. If this hypothesis turns out to be correct, it would provide an important advance to our understanding of the links between inflammation and metabolism.

A new paradigm of gene regulation has emerged recently with the discovery of small regulatory RNAs and associated proteins that belong to the Argonaute family (Ago and Piwi proteins). microRNAs, a class of ~ 22 nucleotide RNAs are bound to Ago proteins and function by base paring with microRNA-recognition elements (MREs), found in their numerous mRNA targets, and direct either target mRNA endonucleolytic cleavage or translational repression. Piwi proteins in the testis associate with a novel class of 26 to 30 nucleotide small RNAs termed Piwi-associated RNAs (piRNAs). The functions of Piwi proteins and piRNAs are completely unknown. Whether piRNAs exist in other tissues where Piwi proteins are expressed is also unknown. Because of the similarity of domains of Piwi proteins with Ago proteins, I hypothesize that, as is the case of Ago/miRNA ribonucleoprotein, binding of a piRNA/Piwi ribonucleoprotein to a target RNA leads to silencing of its expression and plays an important role in meiosis in male germ cells. To unravel the functions of Piwi proteins and piRNAs, I propose to identify and validate human and mouse piRNA targets. I will use bioinformatics to predict piRNA-recognition elements (PREs). I will also utilize a biochemical approach based on crosslinks between target RNAs and piRNA/Piwi ribonucleoprotein followed by anti-Piwi immunoprecipitations to isolate RNAs that associate physically with piRNA/Piwi ribonucleoproteins. Moreover, I will investigate whether piRNAs are expressed in non-germ cells by performing immunoprecipitations with anti-Piwi antibody and identifying Piwi-associated RNAs. Finally I will validate the piRNA targets using mice as model system.

Meiosis is a specialized cell division process that produces haploid gametes through two rounds of chromosome segregation. The first meiotic chromosome segregation is different from that in mitosis, because homologous chromosome pairs, not sister chromatid pairs, segregate from each other. To allow this unique segregation, it is important that homologous chromosome biorientation is established until metaphase I, by which homologous kinetochore pairs face the opposite spindle poles whereas sister kinetochores face the same poles. Previous studies suggest that chromosome condensation and specific action of microtubules to the chromosome surface may contribute to formation of the bioriented structure of the homologous chromosomes. However, it has not been examined how spatiotemporal structural change and dynamic behavior of the chromosomes lead to establishment of homologous chromosome biorientation. Thus, I will visualize chromosome dynamics with high-resolution microscopy in live mammalian meiosis. To this end, I will use the mouse oocyte system, because induction of meiosis, expression of engineered proteins and perturbation of protein function are available in an in vitro culture. I will use advanced techniques of automated confocal microscopy and also selective plane illumination microscopy (SPIM) to establish quantitative high-resolution 4D imaging assays for chromosome biorientation. I will then use these assays to probe the molecular mechanisms for homologous chromosome biorientation, by functional perturbations of the cytoskeleton and candidate proteins such as condensins and Polo kinase by RNAi, specific drugs, or female germline specific conditional knockouts.

The hypothesis that cell division failure that result in genetically unstable tetraploid (4N) cells promotes tumorigenesis was proposed almost 100 years ago, and it has been supported by extensive in vitro and clinical correlations. The host laboratory recently provided direct experimental evidence in support of this hypothesis: tetraploidy, at least in combination with p53 loss, could promote the formation of aneuploid tumors. However, the mechanisms underlying transformation of 4N cells are poorly understood. To define the mechanisms, I propose experiments with the following aims. Aim 1: although numerous links have been demonstrated between p53 loss and tetraploidy, the role of p53 loss in transformation of 4N cells has not been determined. Therefore I will investigate whether p53 loss promotes tumorigenesis by 4N cells through aberrant DNA damage checkpoint response and/or suppression of apoptosis. This will be performed using mouse 4N embryonic stem (ES) cells that are genetically stable and wild type for p53. The p53 in this system will be depleted by lentivirus-mediated RNA interference. Aim 2: since mitotic 4N cells contain 4 centrosomes, they can form an abnormal multipolar spindle that results in catastrophic multipolar mitoses, leading to genome instability and tumorigenesis by 4N cells. However, this idea has never been tested yet, because the extra centrosomes in such cells cluster into two pairs. To test this idea, I will knock down a motor protein required for centrosome clustering (HSET) and force 4N ES cells to undergo multipolar mitoses. This experiment will determine whether multipolar mitoses promote DNA damage, genome instability, and tumorigenesis.

The goal of my proposal is to extend MADM system to study the logic of neural circuit organization and formation in the mouse brain. MADM (for Mosaic Analysis with Double Markers) is a recently developed molecular genetic method that can be used to label small populations of cells, to perform lineage analysis, to visualize wiring patterns, and to conditionally knockout genes only in labeled cells. The current MADM system has two major limitations for general phenotypic analysis that I plan to address: (i) MADM-based gene knockouts rely on the availability of a pair of the MADM cassettes between the gene of interest and the centromere. With a novel strategy that utilizes a random integration of MADM-transgene into ES cells, I plan to construct a “MADM mouse library” where all mouse euchromosomes are targeted by MADM cassettes. This library will enable researchers to make conditional mutant cell populations for nearly all genes in the mouse genome, and will be shared worldwide as a useful genetic resource. (ii) Spatial regulation of MADM is currently still limited. I will generate new Cre lines using BAC transgenic mice to provide spatial control of cell labeling and gene disruption. As a model, I will establish a spatially regulated MADM system in higher olfactory neurons using a precursor marker gene in the olfactory brain. I will analyze the spatial organization and conditional mutant phenotypes of olfactory cortical afferents. These experiments will be an essential first step in the broad application of MADM-based molecular genetics to other brain regions for the better understanding of the logic of mammalian neural circuit organization and formation.

Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable for the expression of most genes in eukaryotes. Splicing involves two transesterification steps within the spliceosomal complex, an elaborate molecular ensemble formed by the ordered interaction of four small nuclear ribonucleoproteins (snRNPs), U1, U2, U5, and U4/U6, and numerous splicing factors with the pre-mRNA. To date, six kinds of proteins are identified that are specific to U5 snRNPs Snu40p, Dib1p, Prp28p, Snu114p, Brr2p, and Prp8p in yeast. Notably, Prp8p (280 kDa) is highly conserved among species and essential for nuclear pre-mRNA splicing in vitro and in vivo. Prp8p can be crosslinked not only to loop1 of U5 snRNA which tethers the 5’ and 3’ exon during splicing reaction, but also to 5’ splice site (5’SS), branchpoint, and 3’ splice site (3’SS) of the pre-mRNA, perhaps acting as a cofactor in RNA catalysis. In addition, Prp8p binds strongly with the ATP-dependent RNA unwindase, Brr2p, and, EF-2 homologous GTPase / putative translocation inducer, Snu114p in yeast. Moreover, it is interesting that specific mutations near the C-terminus of Prp8p cause the retina disorder, Retinitis Pigmentosa. With this fellowship, I will do a dramatic shift from my previous studies first, I will purify this ternary complex by S.cerevisiae expression system and tri-(U4/U6)•U5 snRNP by tandem affinity purification (TAP) method, then I will determine the structure of Prp8p/Brr2p/Snu114p ternary complex by X-ray crystallography and the envelope of the tri-(U4/U6)•U5 complex by cryo-electron microscopy, which will provide important insights into the catalytic core of the spliceosome.

The control of translation initiation is an important mechanism for the regulation of gene expression in eukaryotes, and is known to be involved in cell growth, cancer, and stress responses such as starvation, hypoxia, ER-stress, and viral infection. At least 12 essential initiation factors (eIFs) play central roles in facilitating initiation as well as in translational regulation. Although distinct functions of each eIF are so far been characterized genetically and biochemically, it remains unclear how they interact with one another on ribosome to exert the integrated functions. As most eIFs form intermediate complexes with ribosome, initiator Met-tRNAi, or mRNA at specific stage of initiation, it is important to analyze the structures of the initiation intermediates to understand basis of functional association of eIFs. The purpose of the research proposed here is to investigate the initiation muti-factor complex assembly and its relation with individual functions. The study is also focused on structure and function of the largest and yet most uncharacterized initiation factor eIF3 consisting of 13 different subunits. The proposal will potentially shed new light into the comprehensive understanding of initiation factor-driven initiation and its regulation. The focus of my PhD work was to reveal the structure of archaeal homologs of translational apparatuses by X-ray crystallography. Studying the eukaryotic initiation complex extends my scope into the larger and complicated system as well as expanding my repertoire of techniques to preparation of multi-subunit factors from eukaryotic cells, and structural analysis with cryo-electron microscopy reconstitution.

Zinc is an essential micronutrient for organisms but is also toxic when present in excess. The yeast Zrt1, a zinc uptake transporter, is a member of ZIP family of transporters that are involved in metal uptake in bacteria, fungi, plants, and mammals. During the transition from zinc-limiting to zinc-replete growth conditions, the Zrt1 transporter is inactivated by endocytosis for preventing zinc accumulation to a toxic level. Zrt1 is ubiquitinated shortly after zinc treatment and prior to endocytosis. A region of Zrt1 required for ubiquitination and endocytosis in response to zinc was mapped and was designated as a potential ‘‘metal response domain’’ (MRD). The aim of this study is to understand how cells sense zinc concentration and transduce the signal to ubiquitinate Zrt1. I propose three hypotheses regarding the molecular mechanism: Zrt1 senses zinc as it’s being transported Zinc binds to MRD and its binding status controls ubiquitination An intra-or extra-cellular zinc binding protein (zinc sensor) transduces the signal directly or indirectly to MRD by protein-protein interaction. I will test these hypotheses by directed approaches and also by identification and characterization of novel proteins required for the mechanism. This study will contribute for understanding of the molecular mechanisms of substrate-dependent endocytosis of various transporters, important processes for nutrient homeostasis in various organisms.

The project aim is to investigate how the unconscious affects overt human behaviors. I will conduct fMRI and EEG in human subjects. They will first be primed by presentation of a variety of stimuli, including subliminal stimuli, followed by behavioral experiments to examine the consequences for overt behavior. Importantly, I will apply an independent component analysis (ICA) approach to extract individual signals from mixed signals of neuroimaging experiments. Using ICA, I will categorize the signal depending on the signal pattern and amplitude, and then relate them to behaviors. Furthermore I will use TMS to examine the functional importance of areas identified with fMRI and then use DWI and probabilistic tractography to examine whether co-activated areas are interconnected. This research field is entirely different from the themes of my previous molecular research work. I will now, however, attempt to investigate whether analytic approaches similar to those used in my previous research, focusing on Ca2+ patterning and cell function, are useful for understanding the factors that determine human behaviors. The focus on the identification of activation patterns is common in both researches. I expect that this project will help me broaden my knowledge and understanding of basic cognitive neuroscience and that it would also provide a good opportunity for training in functional imaging techniques. Importantly, I can learn to understand different points of view and interact with people from different scientific backgrounds. As a result, this project may potentially allow me to liaise between researchers in the fields of molecular biology and cognitive neuroscience.

Selective maturation of T cells in the thymus is critical for establishing one of the major limbs of the adaptive immune system. To produce a useful repertoire that lacks most potentially autoreactive clones, immature T cells undergo "positive" and "negative" selection steps. In this proposed study, I plan to investigate how regulation of ion fluxes in response to antigen receptor interaction with self peptide-presenting stromal cells in the thymus contributes to the positive and negative selection of double-positive (CD4+CD8+) immature T cells. To accomplish this specific aim, I will study the in situ behavior and intracellular Ca2+ response of double-positive T cells in freshly prepared thymic slices using two-photon laser scanning microscopy (established in the host laboratory) in combination with electrophysiological and biophysical techniques that I have acquired in my previous research on the physiological regulation of signaling in cells with excitable membranes, such as cardiomyocytes and neurons. The special focus of this proposed project is on the Ca2+ response and on ion channels. I will examine the potential roles of ion channel regulation in fine-tuning the Ca2+ response evoked by T cell receptor stimulation during selection Ca2+ events in the thymus. I believe that such interdisciplinary studies would enable me to make significant contributions in this field.

The specific aim of this proposal is the direct observation of the protein dynamics at the active site in the presence or absence of an intramolecular disulfide bond by use of vibrational echo methods. While the conformational changes of a protein are intimately coupled to its function, the conformational dynamics of a protein are difficult to discern. The Fayer lab at Stanford University specializes in physical and biophysical chemistry and performed the first ultrafast vibrational echo experiments approximately 10 years ago. On the other hand, I have used several molecular biological approaches to investigate the relationship between structure and function of proteins. I will combine physical chemistry and molecular biology to investigate the regulatory role of a disulfide bond. Neuroglobin (Ngb) is induced by neuronal hypoxia and protects neurons from hypoxia, which suggests that this protein may have a role in sensing or responding to neuronal hypoxia. Structural analysis has shown the presence in Ngb of a intramolecular disulfide bond which stresses the protein, and breaking the disulfide bond provides additional structural degrees of freedom of the protein, resulting in a release of O2. To understand the effects of the disulfide bond for the activity of Ngb, I will utilize vibrational echo methods for direct detection of the protein dynamics on time scales not accessible by other methods. The direct observation of the protein dynamics for Ngb in the presence or absence of the intramolecular disulfide bond will provide information on the redox-regulated activity of proteins.

Cryptochrome (CRY) is a blue/UV-A light receptor found in bacteria, plants, and animals including human. CRY phosphorylation is the only known light-dependent biochemical reaction of CRY. Although CRY contains no apparent sequence similarity to any protein kinase or ATP-binding structure, it has been demonstrated that CRY can autophosphorylate in vitro, which may represent a novel mechanism of protein phosphorylation in general. However, the underlying molecular mechanism and other important questions, such as whether CRY is phosphorylated by other kinases in vivo and whether CRY phosphorylates other proteins, remain to be elucidated. Here I propose to use a number of chemical-genetics approaches to address these questions, fully utilizing the information obtained from the recently solved crystal structure of the CRY N-terminal domain. As a graduate student, I studied structure-function relationship of plant red/far-red light receptor phytochrome using molecular genetics methods. Then I strongly felt the limitation of my previous approaches and how important the structural information of a protein to one’s understanding of its action mechanism. I proposed a chemical genetics study, not only because it is a new technique in itself, but also because I am lack of any experience in the structure-based study of protein function. The proposed research plan will allow me to gain expertise in this critical area of biological research and help me to reach my personal goal of becoming an independent investigator in photobiology.

Tight control of gene expression in response to the environmental signal is one of the most important characteristics of life. When cells are exposed to growth factors, the signal is transduced from cell surface receptors to the cascade of signal transduction, and leads activation of specific genes. Serum Response Factor (SRF) was identified as a DNA-binding transcription factor that is required for serum-induced gene expression. Many mammalian genes that are important for regulation of cell proliferation and differentiation, including SRF-target gene c-fos, are regulated at transcriptional elongation. In the absence of serum stimuli, RNA polymerase II (Pol II) is recruited to the c-fos gene promoter, but release from the promoter and elongation of transcription can be induced only after serum treatment, allowing rapid induction of serum-induced gene expression. However, the mechanisms that specifically connect growth factor signaling and stimulation of Pol II release and elongation have not been understood. Here I propose a research plan to analyze the relationship between signal-regulated transcription factors and post-Pol II recruitment steps of transcription, particularly, by focusing on post-translational modifications of Pol II that controls elongation and other transcription-associated events. This research experience is clearly different from my thesis research, in which I studied proteolysis in yeast, as I change the model to mammalian cells, and as I characterize the steps leading to protein synthesis. I would expect to obtain important insights for my future study, by learning about multiple aspects of gene expression as an interconnected biological system.

Over the past several years, genes have been identified that alter the lifespan of model organisms. Sir2 was originally found as the gene involved in gene silencing in yeast. Sir2 and its homologues are prominent among these. Sir2 encodes an enzyme that functions as NAD+-dependent deacetylase. In yeast, calorie restriction extends the lifespan of mother cells, and it is dependent on Sir2 activity for longevity. In rodents, calorie restriction also extends lifespan. However the molecular mechanism in rodents is not understood. In mammals, there exist seven Sir2 homologues, called sirtuins. SirT1 is the closest to yeast Sir2, and it also has NAD+-dependent deacetylase activity. SirT1 deacetylates not only histones but also other proteins such as p53 and FOXO. Though yeast Sir 2 only localizes in nuclei, mammal sirtuins localize in various compartments in cell. Surprisingly, SirT3, SirT4 and SirT5 are found in mitochondria. It is known that mitochondria play the most important role in energy production and metabolism in cells. Moreover the connection between aging and mitochondria has been strongly suggested for long time. Therefore I believe that Sirtuins in mitochondria also have an important role in metabolism regulation and lifespan extension during calorie restriction. Among the mitochondrial sirtuins, I will especially focus on SirT5 because the functional role of SirT5 is totally unknown. SirT5 possesses the NAD+-dependent protein deacetylace activity in vitro, but the functional targets are not identified yet. In this project, I will try to identify the functional targets of SirT5 and the SirT5 containing complexes, and elucidate the function of SirT5 in mitochondria.

The occurrence and advancement of metabolic syndromes resulting from obesity is closely associated with adipocyte differentiation and the extent of subsequent fat accumulation. To elucidate the mechanisms underlying adipocyte differentiation as well as the commitment of mesenchymal cells into adipocytes is important to understand the symptoms of obesity-related metabolic syndromes. My research interest is to investigate the mechanisms of mesenchymal cell commitment in detail and the relationships between the molecules that govern the commitment and obesity-related metabolic diseases. To examine them, first I will identify key genes that influence the commitment of adipocytes. Then I will examine their functions in adult using murine models. To identify the key genes, I will make use of two methods-namely gene silencing by RNA interference (RNAi) in mesenchymal cells and gene array analysis. To apply a large-scale library of RNAi-inducing shRNA expression retroviral vectors, I will newly develop a cell system to screen for the genes that are involved in the commitment and/or differentiation of mesenchymal stem cells into adipocytes. I will also make full use of the method of gene array analysis to identify the key genes. Following the identification stage, by generating the candidate gene-deficient mice, I will further investigate the effects of the genes on metabolic disorders from the standpoint of maintenance of homeostasis of energy through the regulation of mesenchymal cell commitment and/or differentiation. The study proposed in this application will greatly broaden my technical abilities and allow me to pursue scientific interests.

Exploring molecular mechanism for asymmetric cell division is important to understand not only the development of an organism but also the differentiation and proliferation of multi-potent stem cells. Imbalance of such cell division results in tumorigenesis caused by overproliferation. I previously had focused on the intracellular signaling events for establishing neuronal polarity in vitro by using biochemical and cell biological approaches. In this project, I will investigate how neural stem cells precisely and continuously generate differentiating and self-renewing daughter cells by asymmetric cell divisions in Drosophila nervous system during development. Recently, the host laboratory has found that a protein Brain tumor (Brat) localized asymmetrically in neuroblasts and inhibited cell growth in the daughter cells. Homozygous brat mutants result in the production of a tumor-like neoplasm in the larval brain, suggesting that asymmetric segregation of Brat in neuroblasts controls self-renewal versus differentiation. Specific aims of my research proposal are 1) to examine how Brat inhibits neuroblast self-renewal by systematically isolating Brat binding proteins, and 2) to search mutants causing overproliferation or underproliferation in the larval brain by genome-wide RNAi screening for systematic surveys of gene function in vivo. I would like to combine my biochemical and cell biological talents with developmental biology and Drosophila genetics in the host laboratory for my future career, which could allow rapid progress especially in a field analyzing complex mechanisms of living organisms.

Although it is widely accepted that neurons in the neocortex have wide variety of morphological and physiological characteristics, the mechanism underlying diversity of cortical neurons remains largely unknown. The host laboratory has developed a method for transcriptome analysis of specific neuronal type and constructed a database. The objective of this proposal is to reveal mechanisms of transcriptional regulation in maintenance of specific characteristics of cortical neurons. To achieve the goal, I will attempt to identify transcriptional factors (TFs) that are responsible for characteristic neuronal phenotypes. I will screen TFs which are differentially expressed among neuronal types from the database. To examine the roles of TFs in the maintenance of neuronal characters, I will silence or overexpress them and examine morphological and physiological phenotypes in specific type of neurons. And then I will look for downstream genes of TFs by transcriptome analysis and address what genes make actual characteristics. I expect this proposed project will provide first detailed mechanism for neuronal diversity, which are indispensable for understanding cortical microcircuits and analysis of mouse model of brain disorder. Through this project I will learn neurophysiology and genome informatics that I have never experienced in the previous field, molecular and cellular biology. I believe integrative approaches of physiology, molecular biology, and informatics will provide new insights in neuroscience in post-genomic era. This fellowship will give me a chance of acquiring those techniques, which will be very beneficial for establishment of my own research in near future.

(1) My proposed project: Oocyte maturation in Drosophila is supported by nurse cells, which supply the oocyte with nutrients through cytoplasmic bridges. Maternal components are sorted for transport in a temporal-specific manner, but the underlying mechanism is largely unknown. The objective of this project is to elucidate the molecular basis of intercellular transport between nurse cells and the oocyte. To achieve the goal, I will dissect the transport events by using several transport reporters available in my host lab. Imaging of cultured egg chambers, inhibitor experiments, and domain analysis allow us to examine transport dependence on cytoskeletal organization or novel sequence motifs. Moreover, I propose a genetic screen to identify mutants showing transport defects I will mutagenize flies carrying a GFP reporter transgene, and isolate mutants that have abnormal GFP localizations. After I identify responsible genes of those mutants, I will examine their functions and dissect the spatiotemporal regulation of the transport machinery. (2) How it differs from my previous research experience and how this will serve my professional growth: I have been studying planar cell polarity in wing. In the proposed research, I will study intercellular transport in oogenesis by using forward genetics, which is completely new to me. Besides, I will be working with germ-line cells that are substantially different from somatic cells. This training will provide me an opportunity to study new features of cells and signaling pathways. The genetic screen will form the foundation for my future when I establish my own research program and educate students as an independent investigator.

Normal aging is associated with structural and physiological changes in prefrontal systems. However the significance of these effects to behavior is not well understood. To study this question, I will focus on orbitofrontal cortex (OFC) and basolateral amygdala (ABL) as a model system and examine the effects of aging on this system using a Pavlovian reinforcer devaluation task. In the proposed experiments, I will compare task performance in young and aged rats in order to characterize the effects of aging on the orbitofrontal-dependent functions. I expect that a subset of aged rats will be impaired on this task. To identify the neural substrate of this deficit, I will then record neural activity in OFC and ABL during training in the same Pavlovian reinforcer devaluation task. I expect to find abnormal encoding in this circuit that accounts for the age-related behavioral deficit. I believe that these experiments will further our understanding of the normal operation of this circuit and also of how this operation is disrupted in normal aging. My host laboratory has substantial experiences applying methods of chronic neural recording in awake behaving rats powerful methods to examine the cognitive brain functions. Thus I will be able to learn these neurophysiological techniques and learn to analyze animal behavior and cognitive brain functions. The fellowship will enable me to advance my career from my previous research of sensory system to that of cognitive brain functions.

Permanent memory is believed to be stored in the neocortical areas in a distributed manner. Initially the hippocampus connects these neocortical areas, and repeated reactivation of acquired activity patterns in the hippocampus eventually establishes direct cortico-cortical connections which support memory independently of a functional hippocampus. This hypothesis is supported by the reports that the spiking patterns of the hippocampal and neocortical neurons during a task are reactivated in a coordinated fashion during subsequent rest period (memory trace reactivation) however, it has never been revealed whether cortico-cortical connections are actually established as a consequence of memory trace reactivation. To study this, I will combine neuronal ensemble recording, which is a novel technique for me, with pharmacological treatments, in which I have already developed expertise. I will record neural activity of the rat neocortical areas during sleep after retrieval of recently or remotely acquired spatial memory and investigate the effect on coordinated reactivation of these areas of acute hippocampal inactivation and/or cotico-cortical disconnections. And I will investigate chronic hippocampal inactivation or chronic blockade of neocortical NMDA receptors during consolidation on coordinated reactivation for remotely acquired memory. I expect that the outcome will provide important missing evidence in support of the trace-reactivation theory of memory consolidation. Besides, by using a task schedule in accord with previous behavioral studies, this project can bridge the gap between mnemonic processes revealed by behavioral study and the neuronal activity data.