Textbooks present motor memory as monolithic: once acquired, never forgotten. They dissociate it from declarative memory by suggesting that motor memory does not have a short-term form. However, recent theoretical and experimental results have overturned this view, suggesting that motor memory is supported by processes that have multiple timescales: fast processes that learn quickly but decay rapidly with time, and slow processes that are less sensitive to movement errors but show better retention. Indeed, it appears that formation of motor memory may be a gradual transformation from the fast to the slow processes, and this transformation may depend on time and the statistical properties of the learner’s performance errors. We propose to use this idea to shed new light on one of the fundamental problems in neuroscience: the distinct functions of the cortical motor structures vs. the cerebellum in learning and retention of skilled movements.

The unsolved problem is illustrated by two examples: whereas neurophysiological and neuroimaging experiments have found correlates of adaptation in the primary motor cortex (M1), functional disruption of this area by transcranial magnetic stimulation (TMS) appears to produce no effects on acquisition, yet produces post-adaptation amnesia, i.e., rapid forgetting. In contrast, while the cerebellum is crucial for adaptation, damage to this structure appears to spare some skills that were acquired before the damage. Our basic hypothesis is that the faster timescales of memory are dependent on the cerebellum, effectively learning to predict the consequences of motor commands and correcting the motor commands via internal feedback, and the slower timescales are dependent on the cerebral cortex, effectively learning to produce the motor commands appropriate for the specific conditions of the task.

Our project combines computational, psychophysical, neuropsychological, and neurophysiological approaches to investigate how time and statistics of performance. We will use common eye and arm movement paradigms in behavioral and neurophysiological experiments to explore how controlled changes in sensory and motor noise effect motor control and learning. This will enable us to directly relate high level behavior to neural mechanisms

Interstitial fluid flow strongly affects, in multiple ways, the structure and function of living tissues. Quantitative understanding of the effects of interstitial flow on cellular functions as well as on the interactions between cells and their environment during tissue development will improve the effectiveness of therapeutic treatment for native tissues, permit the efficient fighting off against persistent biofilm infections, and enable the successful engineering of functional artificial tissues. The proposed research project aims at the investigation of the effect of fluid flow on the spatiotemporal dynamics of bone cell proliferation and tissue development in 3D scaffolds confined within a microfluidic device. Visualization experiments will be performed under various flow regimes, such as transverse perfusion (which mimics the flow driven by the pressure difference between vessels in vivo) and parallel perfusion driven either by constant flow rate or by constant pressure drop. The response of bone cells to changes in the magnitude and/or the direction of the average fluid velocity through the scaffold will also be investigated. The experimental data will be analyzed and interpreted with the help of recently developed advanced theoretical models and computer simulation methods. In the context of this fellowship, I will have the opportunity to conduct research abroad on a very exciting and important scientific theme in one of the leading groups in tissue engineering, and to receive training on several experimental methods, including bone cell and tissue culture in 3D, confocal laser microscopy, and preparation of polymeric scaffolds.

The growth of animal organs is precisely controlled. When genes affecting growth, such as the Myc or Minute genes, are expressed unequally, competition is induced between adjacent cells. Cells that carry more copies of growth genes, divide faster than their neighbors within a tissue. Importantly, their growth advantages do not lead to an increase in tissue size because they trigger the death of neighboring cells in order to compensate for their own expansion. How cells communicate to achieve this is not known, however, regulators that limit tissue growth, such as members of the Hippo pathway, have been suggested to play a role. To identify components of the cell competition pathway, I propose to conduct a genome-wide RNAi screen in cultured Drosophila cells. To accomplish this, marked cell lines will be exposed to genetic knockdown and cultured with their wild-type counterparts. Assessment of relative overgrowth in one population, and death in the other, will identify novel candidates that induce or regulate cell competition. I will then test, in vivo, the genes identified from the screen in both developing tissues where competition has been documented, and ones where it has not. Importantly, candidates also will be investigated in tissues undergoing turnover in the adult to assess the role of cell competition in tissue homeostasis.

Plants make use of two-component systems for signal transduction, and these are involved in vital cellular processes such as the response to cytokinins, ethylene, red light, and osmosensing. Previous studies have been focused on functional characterization of two-component elements in plant signal transduction, in particular cytokinin signaling. To elucidate how two-component elements control various aspects of plant development, expanding analysis of the two-component pathway to other elements of the network is required.
My first aim is to identify novel transcription factors regulated by the two-component signaling pathway through genome-based screens. Based on previous results that the type-B response regulators (type-B ARRs) act as transcriptional regulators in the two-component pathway, I will isolate candidates showing altered expression in type-B arr mutant backgrounds. My second aim is to investigate the precise role of newly identified candidate transcription factors through physiological and molecular analysis. My third aim is to elucidate the connections between the two-component pathway and the other cellular networks by constructing functional/physical interaction maps among components. Through this study, I will contribute to an understanding of how the two-component gene network acts to alter plant growth and development..

It has been suggested that the large recognition diversity of cell surface proteins is required for complex neuronal wiring. Drosophila Down syndrome cell-adhesion molecule (Dscam) is a cell surface protein that belongs to immunoglobulin superfamily. Dscam can generate up to 38016 isoforms by alternative splicing and shows specific homophilic binding. Based on these features, Dscam is thought to be a very attractive molecule that could explain the mechanism of specific neuronal circuit formation. Dscam homophilic binding induces repulsion signaling and thereby contributes to dendritic self-avoidance and axon branching. However, the molecular mechanisms that translate adherent homophilic binding into repulsion are not understood. Here, I suggest to identify novel binding proteins of Dscam by biochemical method and to investigate their importance in vivo by genetic method. My host supervisor Dr. Schmucker has suggested that each Dscam isoform has non-redundant function during axon targeting of mechanosensory neurons. To examine this possibility, it is important to investigate expression pattern and localization of Dscam isoforms at protein level. To this end, I propose to use extracellular domain of Dscam isoform fused to a reporter protein such as protein phosphatase (AP). Next, I investigate whether intercellular Dscam-Dscam interactions could function as adhesion during axon/dendrite targeting and synapse development. To this end, I examine that whether same sets of isoforms are expressed at pre- and post-synaptic region and examine the role of Dscam in localization of neuronal transmitter receptor and synapse transmission.

Cell migration is driven by the polymerization of cytoskeletal proteins into filaments that push out the leading edge of the cell by a biochemically and biophysically complex process. The goal of our research is to provide an integrated picture of how the biochemical activities of cytoskeletal proteins affect individual filament mechanics and overall network properties to produce useful force and movement.

We will study two cytoskeletal protein machineries: actin of eukaryotic cells and Major Sperm Protein (MSP) of Caenorhabditis elegans sperm cells. Both machineries have very similar functionalities in terms of motile force and velocity, yet actin and MSP have different biochemical properties (sequence, structure, interaction partners) and thus likely different biophysical properties (mechanical stiffness and strength). By comparing these two very different cytoskeletal systems that have identical roles in cell movement, we hope to define the universal biochemical and physical properties governing cell motility.

To achieve this goal, we have assembled an interdisciplinary team that includes experts in actin and MSP biochemistry and in biopolymer mechanics on both the single filament and collective network scales. On the one hand, we will use biochemical manipulation and in vitro motility assays to test how biochemical regulation affects actin- and MSP-driven propulsion. The physical repercussions of biochemical tuning will be investigated by microrheology and scanning probe microscopy to measure the forces generated by polymerizing actin and MSP filaments and their elastic properties. For actin, we will focus on how certain domains and phosphorylation states of the Drosophila Enabled/Vasodilator-stimulated Phosphoprotein (Ena/VASP) family affect movement. The Ena/VASP proteins are key players in cell motility, but have been little studied from a mechanical point of view. For MSP, we will establish for the first time a purely in vitro motility assay and use this set-up to study MSP-associated factors that will be identified during the course of this project. This will be the first detailed biochemical/biophysical study of this propulsion system in the model organism C. elegans.

Ultimately protein chemistry and mechanical properties will be integrated into a complete description of cell movement in the context of two very different cytoskeletal systems.

Bacteria were long thought to take up nutrients, grow, and divide as individual unicellular organisms. However, this strict unicellular view has been challenged by the discovery of bacterial cell-to-cell communication systems and the observation of rudimentary multicellular behaviors. A promising model system is our model of choice, Bacillus subtilis. Undomesticated B. subtilis creates architecturally complex, sessile aggregates called biofilms which are robust in undomesticated strains. With-in the context of a biofilm, it was discovered that spore, motility and extracellular matrix production have a distinct spatial organization. Utilizing cell-type specific promoters and fluorescent reporter molecules coupled with fluorescence microscopy I will attempt to map out the spatial anatomy of a B. subtilis, I will try to study the mechanisms whereby the biofilm interacts with the environment and investigate differentiation of disturbed biofilms. Furthermore, I will ask how small molecules produced by different cell types and which participate in cell to cell communication alter the spatial organization of cells within a B. subtilis biofilm. The overall goal of the study is to identify, characterize, and understand new genetic and physiological components of extracellular matrix production and complex colony architecture in undomesticated B. subtilis

Synapses function as a central interface in neuronal circuits that determines cognitive processes such as learning and memory generation. Their function as minimal information storage units requires the spatiotemporal control of posttranscriptional gene expression within neuronal processes. In a complex network, a variety of RNA-binding proteins (RBPs) facilitate the localisation of specific transcripts to these processes where they are translated. In this proposal, I will develop and apply different biochemical and genetic strategies to gain insights into this mRNA localisation network. To determine the global profile of process-localised RNAs, microgrid colonisation that facilitates the isolation of neuronal processes will be followed by deep sequencing. To monitor the contribution of individual RBPs, this will be combined with overexpression and depletion of the respective proteins. As a complementary approach, I will map binding sites of the RBPs within the RNA population using UV cross-linking and immunoprecipitation (CLIP) that allows detection of protein/RNA interactions in vivo. In a comprehensive bioinformatic analysis, these protein/RNA interaction maps will be correlated with the RNA localisation profiles generated during depletion/overexpression experiments. This will allow to detect effects of binding site positioning as well as the concerted action of RBPs. Taken together, these achievements will contribute to our understanding of the global RNA localisation network in neurons.

Shaping the animal body by Hox genes is a leading topic in developmental biology. Insects, where Hox genes were discovered, provide excellent models for such research. The Hox gene Ultrabithorax (Ubx) changes Drosophila wings on the third thoracic segment (T3) into halteres. However, based on recent studies in other insects I hypothesise that the primary importance of Ubx is in the neighbouring first abdominal segment (A1), where Ubx transforms legs to glandular organs, the pleuropodia. Pleuropodia transiently appear in most insect embryos. Perhaps because they are absent in the Drosophila model, the pleuropodia have been neglected in genetic analyses and their function is unknown. Taking advantage of the genetically tractable insects, the beetle Tribolium castaneum and the cricket Gryllus bimaculatus, I aim to clarify for what purpose and how Ubx generates these glands. I will deduce pleuropodia function by analysing their specifically expressed genes isolated by EST sequencing. Transcript profiling and gene silencing by RNAi will be employed to unravel how Ubx modifies the A1 appendage and why could its effects in the neighbouring body segment be so different. In the springtail Folsomia candida I will test the hypothesis that insect pleuropodia and the glandular A1 appendages of springtails have common origin. If so, then the role of Ubx in A1 appendage specification is more ancient than the insects. The study will yield novel findings on insect development, physiology and evolution. Collaboration with the leading expert Prof. Akam will be essential for this project and for my successful entrance to the new field of Evolutionary Developmental Biology.

Multiple sclerosis (MS) is the major cause of neurological disability among young adults, for which thus far no cure exists. Its pathogenesis is hallmarked by demyelination and inflammation in the CNS. The current strategy in MS treatment development is immunomodulation. However, to stop progression of deficits, remyelination is needed. Recent insights have revealed that oligodendrocyte (OL) pathology is an important feature of MS pathogenesis. While remyelination can be observed, these myelinating cells seem incapable of this in the progressive stage of the lesion and the disease. This is probably caused by a depletion of OLs and an incapacity to recruit and differentiate sufficient numbers of OL progenitors (OPC). Another approach in MS therapy development is therefore reconstitution of the OL population through regenerative therapy.
In the current project I will explore the possible use of NSC in MS therapy. I will
1. Elucidate the intracellular pathways and downstream epigenetic mechanisms that are involved in the differentiation of adult NSC into OLs. In addition, I will explore the possibility to generate OLs from inducible pluripotent stem (iPS) cells, which can be obtained from skin fibroblasts.
2. Study the impact of inflammatory mediators on NSC fate. I will test the effect of factors implicated in MS pathogenesis and highly expressed in MS lesions on the survival and differentiation potential of NSC.
3. Explore the possibility of engraftment of NSC or OPCs in in vivo models of MS. In addition, I will investigate the impact of approved and potential new immunomodulatory therapies for MS on CNS regeneration in in vivo models of remyelination and neurogenesis.

Alterations in signal transduction cascades underlie the development of cancer. However, despite the identification of numerous signaling pathways aberrant in human tumors, very little data exists on the heterogeneity of cellular signaling in individual cancer cells. Importantly, there has been little examination of how changes in individual cancer cell signaling effect proliferation, apoptosis, and tumorigenesis. Thus, it would be valuable to monitor cancer cell signaling at the single-cell level in a way that maintains viability for functional analysis. Synthetic biology can allow one to make such measurements through the use of chimeric sensor molecules that can quantitatively reflect the activation status of a signaling pathway in live cells. I propose to apply synthetic biology approaches to characterize single-cell signaling in colon cancer. First, I will develop synthetic adaptor systems to detect activation of the PI3-kinase and MAPK signaling cascades in human colon cancer cells. Second, I will use these synthetic systems to assess signaling crosstalk in subpopulations with different degrees of pathway activation; these subpopulations will also be examined for effects on proliferation, apoptosis, and the production of tumor-initiating cells. Finally, I will characterize the effects of differential signaling populations on tumorigenesis in vivo. In total, these approaches should help to elucidate the role of different signaling pathways in colon cancer progression and enhance the therapeutic targeting of these pathways in the disease.

Understanding the molecular basis of mitochondrial function, and more specifically of mitochondrial DNA transcription, is a major challenge which raises the hope to pharmacologically cure mitochondria-related diseases such as neurodegenerative and metabolic disorders. Towards this goal, we propose to investigate the mechanisms regulating the activity of the mitochondrial transcription processes in response to specific cellular energetic demands. We will first focus on the elucidation of the molecular mechanisms regulating the mitochondrial transcription termination factor 3 (MTERF3) activity and beyond this, we aim, in the second part of this research work, to identify new factors that could, in addition to the basal mitochondrial transcription machinery, fine-tune mitochondrial transcription and ensure a coherent cellular energetic response. Since we have some evidence that MTERF3 protein levels are mainly regulated at the post-translational level, we will endeavour to identify putative partners and enzymes responsible for MTERF3 stability and/or activity, by using cellular and in vivo models challenged by physiological situations that require an adjustment of mitochondrial activity. In a second part, the identification of new factors impacting on the mitochondrial transcription will be addressed through a ChIP-western and protein-protein crosslinking approaches performed on cellular models of modulation of the mitochondrial activity.

Peroxisomes are ubiquitous eukaryotic organelles which serve in beta-oxidation of fatty acids, hydrogen peroxide-based respiration and oxidative stress defense. Peroxisome biogenesis disorders (PBDs) produce defective organelles which result in a neurological disease referred to as Zellweger’s syndrome. Recent reports in yeast suggest peroxisomes originate from the ER through membrane budding in a process that is dependent on the integral membrane protein Pex3. In pex3∆ mutant cells, peroxisomes are absent and the peroxisomal protein Pex15 is trapped in the ER membrane. Pex3-dependent peroxisome biogenesis suggests a new pathway from ER to peroxisomes.
Anterograde transport from ER to Golgi is a well known vesicular pathway. An in vitro system was used to study the molecular mechanism and led to the discovery of COPII vesicles. In this research plan, a novel ER to peroxisome transport reaction will be studied. Yeast microsome and crude extracts will be used to develop an in vitro system and generate ER-derived peroxisomal precursor vesicles. The precursor vesicles will be isolated by fractionation and immunoisolation. The vesicle protein content will be analyzed by mass spectrometry to examine protein sorting from ER resident and secretory proteins. The mechanism by which this sorting occurs will be pursued by fractionation of any cytosolic proteins required for the pre-peroxisome budding event.

The retinoblastoma tumor suppressor (Rb) is a key modifier of diverse cellular processes including cell cycle progression, DNA repair, DNA replication and differentiation. Loss or deregulation of Rb function can lead to cancer development. Rb interacts and forms multiprotein complexes with a variety of cellular proteins and these interactions are abolished by phosphorylation or the binding to viral proteins. How Rb masters the assembly of functional multiprotein complexes and how phosphorylation of Rb and protein binding lead to modulation of this activity is currently not clear. Existing experimental evidence suggests that intramolecular rearrangements induced by phosphorylation or protein binding play an important role. However accurate structural information as to the nature, mechanism and extend of such rearrangements has thus far not been produced. The goal of the proposed research is to structurally characterize multidomain fragments of Rb in order to define the conformational impact of protein binding and/or phosphorylation. Furthermore, this research aims to provide the structural characterization of the complexes formed by Rb and c-Jun oncoprotein or BRCA1 tumor suppressor, two binding partners involved in Rb activities distinct from the widely studied cell cycle regulatory function of Rb. Structural insights provided by the proposed work on Rb/c-Jun and Rb/BRCA1 complexes will help dissect the significance of Rb during differentiation and DNA repair and provide key information on the mechanism of Rb regulation with the view that such knowledge could lead to strategies that antagonize or reverse cancer development.

Modulations of the actin cytoskeleton coordinate tissue movements to shape organs, heal wounds, promote cell migration and split a mother cell into two daughter cells during cytokinesis. Recent work on fission yeast cytokinesis has shown that the cytokinetic contractile ring assembles via the coalescence of a broad band of about 65 precursor nodes. Each node contains a small number of formin molecules, which promote the assembly of actin filaments, as well as about 20 molecules of Myosin-II motor. A computer generated simulation based on careful observations of the movement of nodes marked by fluorescently tagged components proposed a model by which myosins in one node capture and exert force on actin filaments that grow from neighboring nodes thus pulling the nodes together into a ring. My proposed research focuses on three outstanding issues: 1) the ultra-structural features of nodes and associated actin filaments, 2) the dynamics and organization of actin filaments during node coalescence and 3) the contractile force generated by the actomyosin apparatus. To achieve my goals, I propose to use a combination of quantitative fluorescence microscopy and reconstitution of actin polymerization in live permeabilized cells, electron microscopy to elucidate the structure of nodes and microsurgery by two-photon laser emission to sever the actomyosin ring and measure its contractile force. This work will combine different microscopy techniques, protein biochemistry and mathematical modeling to further our knowledge of contractile structures.

Mitochondria are unique organelles that adapt cellular metabolism to changes in energy and nutrients. Indeed, mitochondria metabolize glucose-derived pyruvate, fatty acids or amino acids to produce ATP. Some molecular aspects of how mitochondrial metabolism is regulated during changes in nutrient availability and metabolic challenges are unknown. In yeast, Sir2 protein is activated by the dietary regimen calorie restriction. In mammals, Sirtuins (SIRT1-7), Sir2 homologs, are NAD-dependent deacetylases or NAD-dependent ADP-ribosylases. This NAD dependence links their activity with metabolism and makes sirtuins good candidates as metabolic sensors. SIRT4 is a member of the sirtuin family localized in mitochondria. The functions of this protein are largely unknown. However recently, it has been shown that SIRT4 ADP-ribosylates and down-regulates glutamate dehydrogenase (GDH) activity, impacting insulin secretion. GDH, an enzyme found in the mitochondrial matrix, regulates the usage of amino acids into energy production. These data establishes a link between SIRT4 activity and the regulation of cellular metabolism. By using in-vitro biochemical assays and SIRT4 knock-out mice, I will test the role of SIRT4 in the regulation of metabolic pathways during diet-induced obesity further defining its role during metabolic stress. Another goal of this project is to understand the impact of SIRT4 on signaling pathways that regulate cellular metabolism. These studies have great implications towards understanding regulation of metabolic programs by sirtuins and their link with mitochondrial bioenergetics.

Inositol has been exploited by evolution to generate a multitude of inositol polyphosphates structures (either soluble or lipid-bound species) that represents important class of signaling molecules controlling any aspects of cell physiology. The intrinsic inability of the inositol ring to be detected using standard spectrophotometric techniques means that, to date, it has not been successfully imaged (localized) within cells. Direct biochemical analysis (but not imaging) of inositides uses radioactive 3H-inositol labeling, followed by high performance liquid chromatographic separation of the 3H-inositol-containing species. Indirect imaging of lipid inositides species has been possible using specific fluorescent probes: the specificity of such probes, however, and their correct use is controversial and the literature harbors several examples of their misuse. We intend to use multi-isotope imaging mass spectrometry (MIMS) to analyze inositides signaling in cells. MIMS, which is the combination of a novel type of secondary ion mass spectrometer with tracer methods and intensive quantitative image analysis, allows the imaging and quantification of intracellular molecules labeled with stable isotopes. MIMS will allow high resolution direct quantitative imaging of inositol molecules once labeled with stable isotopes such 13C-inositol or 18O-inositol. This novel imaging approach should provide fundamental information about how cells handle inositol: the intracellular sites and concentration of its accumulation, the speed and location of lipid inositides synthesis. Additionally, further development of MIMS should allow the discrimination of the different inositol polyphosphates species. Consequently, multi-isotope imaging mass spectrometry may shed light on how the dynamic network of signaling inositides is maintained and regulated inside the cell.

Chromatin structural dynamics regulate diverse cellular functions that influence survival, growth, and proliferation. Disruption of chromatin homeostasis is thought to fundamentally impact the development and progression of cancers. One of the major mechanisms for regulating chromatin structure involves the reversible covalent post-translational modification of histone proteins by chemical moieties such as acetyl-, methyl- and phospho- groups. These chemical marks constitute an epigenetic code that can be maintained in dividing cells, and inherited across generations. The methylation of lysine residues on histones is performed by histone lysine methyltransferases (KMT). The NSD3 (Nuclear receptor binding SET Domain 3) protein is a candidate KMT that is amplified in breast cancer and involved in a recurrent translocation implicated in the pathogenesis of acute myeloid leukemia. Preliminary work in the Gozani lab indicates that NSD3 has a novel methylase activity on histone H3, however the role of this activity in regulation of chromatin structure and oncogenesis are currently uncharacterized. Two aims are proposed: In the first Aim, I plan to characterize the enzymatic activity of NSD3 and identify its substrates utilizing biochemical, chemical biological, and proteomic approaches. The goal of the second Aim is to elucidate the chromatin regulatory functions of NSD3 in normal cells and in cancer. The overall goal of this proposal is to develop comprehensive understanding of the role protein lysine methylation plays in chromatin biology and in modulating cancer regulatory signaling pathways.

A prominent manifestation of plant evolution is the diversity of vegetative and floral (inflorescence) branching systems. Branch number and pattern is determined by groups of stem cells at growing tips, called shoot apical meristems, which uniquely alter their branching potential through environmentally and intrinsically regulated processes called phase transitions. Beyond genetic frameworks, little is known about the molecular networks controlling phase transitions, especially in relation to distinct growth habits. For example, it is unclear to what extent vegetative to inflorescence and inflorescence to flower transitions are similar or different in plants such as Arabidopsis in which meristems grow continuously (monopodial), and plants such as tomato in which growth ends with the inflorescence and is renewed from lateral meristems (sympodial). The sympodial habit of tomato, which represents plants like trees, is a powerful system to study phase transitions, because it develops three distinct shoot systems, each of which undergoes a transition. We propose to use tomato as a model to study the molecular dynamics of sympodial phase transitions by (i) defining the temporal dynamics of gene expression as meristems mature from vegetative to reproductive using new quantitative RNA sequencing tools, and (ii) visualizing the expression dynamics of specific regulators in real time using live-imaging fluorescent microscopy. This project will enable the formulation and validation of pathways and networks underlying a widely deployed growth habit, which can ultimately be used to model meristem growth dynamics and provide new insights into mechanisms of natural variation.

This project aims to investigate how the embryonic brain develops its circuitry. We have evidence that electrical activity during development helps to define the way new neuronal circuits are formed, but we don’t know any of the detailed cellular mechanisms of the origin of these activities and of how they may control additional wiring patterns. The key problem is that standard electrode based measurements cannot provide data from multiple sites in the nervous system with cellular resolution. To solve this problem we will utilize new voltage sensitive dyes and non-linear optics to permit 4D (time plus 3 spatial dimensions) mapping of the system with resolutions that can permit the recording of activity down to the cellular and sub-cellular levels. The team of Drs. Sato and Momose-Sato has worked for many years to map electrical connectivity in the developing central nervous system. They have used optical recording as their primary method. But their previous instrumentation did not allow them to attain the necessary spatial resolution to understand the development of the circuitry at the cellular level. Dr. Pavone is an expert in biophotonics and has developed a unique non-linear optical random access microscope that will be ideal for this project. He will initially use this instrument to investigate the propagation of electrical activity at different stages of embryonic development. He will then enhance his non-linear optical microscope to permit its use with specialized voltage sensitive dyes in 1µm3 volumes within the tissue so as to investigate the cellular and subcellular mechanism electrical propagation. Dr. Loew has worked for many years to synthesize voltage sensitive dyes for optical recording of electrical activity. His laboratory will be responsible for the synthesis of the voltage sensitive dyes that will be required in this project. He will also characterize these dyes in vitro and in vivo. Additionally, the Loew lab pioneered the use of second harmonic generation (SHG) to image changes in membrane potential; Dr. Pavone’s microscope was designed explicitly to exploit the promise of SHG for 3D imaging of electrical activity in neuronal tissue. Importantly, the new science and the new methods developed in this proposed research will broadly impact the study of neuronal circuitry in both the developing and adult brain.