Extracellular vesicles (EVs) are found in all body fluids and play an important role in cell-cell communication by being able to fuse with target cells while carrying and transferring various signaling molecules. They are also known to be involved in several types of disease. Several studies suggest that the mechanical properties of particles play a major role in their cellular uptake. However, a systematic investigation of this effect is still lacking. Hence, I propose to test the hypothesis that mechanical properties of EVs affect the efficiency and kinetics of their uptake by cells. To this end, I will prepare synthetic lipid vesicles (SVs) with varying stiffness by incorporating a crosslinked internal polymeric shell and characterize their mechanical strength. Next, I will measure the forces involved in the adhesion and possibly internalization of the vesicles by cultured cells. I will do this using advanced nanomanipulation techniques such as Atomic Force Microscopy (AFM) and Acoustic Force Spectroscopy (AFS), combined with fluorescence imaging. I will test whether lipid mixing and content mixing between the vesicle bilayer and the cell membrane take place by double fluorescent labeling of the aqueous core and the lipid membrane. A better understanding of the link between SVs mechanical properties and uptake rate and mechanism will provide insight into fundamental biological processes mediated by vesicles, e.g the release of neurotransmitters in synapses, viral infections etc. In addition, my findings will facilitate the design of efficient synthetic exosomes for future therapeutic solutions.

During the last decades, signaling pathways responsible for the initiation of gastrulation in mammalian embryos have been identified. However, the physical rules governing the tissue spatial patterning and the extensive morphogenetic movements occurring during that process are still elusive. We have recently shown using micro-patterned adhesive substrates, that geometric confinement is sufficient to trigger self-organized patterning in hESCs and that this intrinsic tendency of ESCs to make patterns can be harnessed to provides a quantitative assay to study early developmental patterning and differentiation. By taking advantage the micro-fabrication and the micro-fluidics toolboxes, we propose here to study how the physical (stiffness of the substrate, size and shape of the tissue) and chemical (spatio-temporal profile of differentiating signals) properties of the cells micro-environment are affecting the final pattern, in order to shed light on the complex morphogenetic processes occurring during mammalian gastrulation.

Shelterin, a complex of proteins that recognizes repetitive DNA sequences present in the telomeres, efficiently acts as a suppressor of chromosomal fusions and in turn promote cellular genomic integrity. In addition to telomeres, regions of highly repetitive DNA sequences – often involving non-B DNA secondary structural conformations – can be found throughout the genome. These regions are often involved in various genomic rearrangements involving insertion, deletion or inter-chromosomal recombination. The mechanism by which such deleterious events occur is still poorly understood. We hypothesize that in addition to its estabilished role at telomeres, shelterin binds non-telomeric repetitive elements and blocks recombination initiating from these sites. The proposed project aims to analyze the distribution of key shelterin components, such as TRF2, in the repetitive-rich regions genome-wide as well as its specific accumulation at DNA damaged sites. Moreover, specialized and aberrant recombination events, which occur at the repetitive DNA regions in vivo will be assessed by utilizing TRF2-ablated mouse model systems. Finally, to understand the role of shelterin complex at the recombinogenic-prone sites, we will compare the TRF2-associated protein complex in telomere and other repetitive regions that are susceptible to genomic rearrangements. These approaches will enable us to learn ways in which genome stability is preserved. The completion of the project will provide a deeper understanding of diseases associated with trinucleotide repeat instability, as well as various lymphoma-derived cancers.

Many cortical areas cooperate to produce visually-guided behaviors but the way this cooperation happens is not well understood. Specifically, I will address the competing hypotheses of whether cortical areas interact in a “serial” versus a “distributed” fashion; that is, whether each area performs a distinct processing step in turn or whether some steps are accomplished jointly across areas. To distinguish between these hypotheses, I propose to use novel microelectrodes to record simultaneously from six key cortical areas in the pathway from sensation to action: primary and anteromedial visual cortex; posterior parietal cortex; cingulate cortex; primary and secondary motor cortex. Recordings will be made while awake, head-fixed mice perform a task in which they use their forepaws to turn a wheel left or right to indicate the location of the higher-contrast of two visual stimuli presented on a screen. I will analyze the responses of populations of neurons recorded from each area to determine whether and when the following pieces of information can be decoded on single trials: the onset and contrast of each visual stimulus; the integrated evidence available to the animal to solve the task, i.e. the difference in contrast between the two stimuli; the choice of the animal; the timing of the onset and the duration of the wheel-turn response. The temporal evolution of these signals on single trials will reveal whether there is a serial processing chain versus a computation distributed across cortical areas, thus answering a fundamental question about the way cortical areas cooperate to perform visually-guided behaviors.

Stem cell niches, composed of cells or cells together with extracellular matrix, produce secreted or cell surface factors, such as Notch, Wnt, FGF, and EGF, to control stem cell renewal, maintenance, or survival through the activation of various signaling pathways. Deciphering the relationships between these pathways is critical to our understanding of the niche function. The Drosophila adult midgut has emerged as an attractive system to study adult stem cells. Although many conserved signaling pathways regulating Drosophila intestinal stem cells (ISCs) have been characterized, major gaps in our understanding are (1) whether their activation are sequential or simultaneous, and (2) how stem cells integrate these signals to change the program of gene expression during regeneration.

I propose to characterize how stem cells are regulated during regeneration. In Aim 1, Dynamic fluorescent Pathway (DFP) reporters will be used to visualize where and when signaling pathways are activated. DFP reporters will provide quantitative read-outs of pathway activities and help dissect the logic of signaling events. In Aim 2, I will use the Targeted DamID (TADA) method to profile the transcriptional changes following the activation of signaling pathways to decipher their crosstalk and integration. In Aim 3, I will perform a genome-wide RNAi screen to identify transcription factors modulating stem cell proliferation. Together, these studies will provide an unprecedented systems-level understanding of the signaling pathways involved in stem cell-niche interactions during the regenerative process.

Over 100 post-transcriptional RNA modifications are known with N6-methyladenosine (m6A) being the most frequent. m6A has been reported to play a role in many human biological processes (e.g. obesity and cancer), but the detailed mechanisms remain unknown. Detailed study of the function of m6A requires the site-specific introduction of m6A into specific RNAs. However, this appears difficult using endogenous m6A modification enzymes because only a few such enzymes modify a wide range of RNAs. Here, I propose a methodology for introducing site-specific m6A modification in vivo by engineering of the C/D small nucleolar ribonucleoprotein (snoRNP). The C/D snoRNP catalyzes the 2’-O methylation of a substrate RNA and consists of four proteins and a C/D snoRNA, which recognizes the substrate RNA by base pairing. Therefore, the substrate specificity of C/D snoRNP can be easily changed by altering the C/D snoRNA sequence. In this research, I will engineer the catalytic protein of C/D snoRNP to catalyze m6A modification using rational design and directed evolution. First, the catalytic core of C/D snoRNP will be redesigned based on the structure of known m6A modification enzymes. Then, the engineered C/D snoRNP will be randomly mutated to construct a library to perform directed evolution. After directed evolution, the activity and substrate specificity of the engineered C/D snoRNP will be determined. Finally m6A modifications of target RNAs will be altered to assess the biological response. A similar approach could also be applied to other RNA modifications and this technique has the potential to be a fundamental tool to control and study various RNA modifications.

The molecular chaperone Hsp90, a dimeric ATP-driven molecular machine, is one of the most abundant proteins found in eukaryotic cells. This specialised chaperone is known to function as part of several complexes to assist in several essential cellular functions including the folding, maintenance and degradation of client proteins, although the exact mechanisms by which these functions occur is not yet known. In order to elucidate the function of this crucial molecule, the mechanisms and roles of the large conformational changes it undergoes and the forces involved must be studied. To achieve this, experimental techniques will be used that provide excellent force sensitivity and high spatial resolution. In combination with in vivo experiments, I will use custom-built optical trapping and fluorescence setups together with biochemical approaches and mutagenesis, which will provide me with a unique set of tools with which to delineate the specific domain orientations and forces that are required for the function of Hsp90. This will enable me to address the timely question of how the conformations and mechanics of Hsp90 are influenced by co-chaperones and client proteins.

Follicular helper T (Tfh) cells play a key role for inducing antigen-specific responses. Although substantial numbers of Tfh cells reside in the gut-associated lymphoid tissues, it is not fully understood how Tfh cells develop and whether Tfh cells enter the memory pool. To clarify the mechanisms for Tfh differentiation and maintenance in the gut, I will investigate commensal-specific CD4 T cells throughout the course of bacterial colonization. For this purpose, I will focus on segmented filamentous bacteria-(SFB) induced responses, because SFB preferentially induces both Th17 and Tfh cells. Since Th17 cells show plasticity into Tfh cells, I will examine the role of RORgt in Tfh cell development and memory T cell formation, using our established transgenic mice bearing SFB-specific T cell receptor. Next, to identify genes responsible for Tfh cell development, genome-wide analyses will be performed comparing gene expression profiles between wild type and RORgt-deficient Tfh cells. In addition, a proteomic approach will be used for identification of Bcl6 binding partners. For this analysis knock-in mice bearing tagged Bcl6 will be newly generated using the CRISPR-Cas9 system. In addition, based on these data, knockout-based screening will be done in primary CD4 T cells. Focusing on the genes most strongly associated with Tfh cell development, I will generate mutant mice using the CRISPR-Cas9 system. Using these mice, I will investigate molecular mechanisms by which the targeted gene regulates the Tfh program in coordination with Bcl6 in vivo. Through this proposed project I will elucidate how Tfh cells develop and are maintained in response to luminal bacteria.

A wide range of microbes can synthesize and secrete toxic compounds or protein-based virulence factors to inhibit the competing organisms by disrupting core cellular activities. To counteract this, eukaryotes have evolved elaborate mechanisms to surveil cellular and organismal integrity and defend against the xenobiotic attacks. Previous studies have revealed some xenobiotic-related surveillance and defense pathways in C. elegans. However, the potential memory of such responses in these pathways has not been studied. Since naturally an organism can be challenged by the same xenobiotic insults repeatedly within a specific habitat, and the xenobiotic threats to a parental generation stand a good chance of also threatening the next generation, it is reasonable to hypothesize that the surveillance and defense system could be sensitized by continuous exposures to particular toxins and for a short generation time organisms like C. elegans, perhaps even “remembered” across generations. To test this, I will establish appropriate systems to investigate whether the mitochondrial surveillance and defense pathways activated by xenobiotic attacks or disruption of mitochondrial genes can be sensitized both within one generation and between generations in C.elegans. Further, I will perform large-scale genetic screens to identify genes involved in sensitization processes and dissect the functions and underlying mechanisms of the identified genes. This research will contribute to the understanding of the eukaryotic innate immune system and provide potential new pharmaceutical strategies to treat mitochondrial dysfunction diseases and innate immunity disorders.

Traditionally, enzyme catalytic power has been assigned to active site residues that make and break chemical bonds and provide electrostatic interactions to stabilize a reaction’s transition state. However, simply adding these residues to water does not provide significant catalysis -these residues allow rate enhancements only in the context of the folded protein matrix -the enzyme scaffold. Furthermore, electrostatic interactions are not localized to the enzyme active site, as the enzyme scaffold has many charged and polar groups in the regions surrounding the active site. Despite the clear importance of the enzyme scaffold for catalysis, the general properties of the scaffold and how it contributes to enzymatic rate enhancements have been little studied and are not well understood. I propose to determine the properties of the enzyme scaffold and how they promote catalysis, with a focus on the well-behaved model enzyme Ketosteroid Isomerase (KSI). I will use a multidisciplinary approach, combining room temperature crystallography and solution NMR to characterize the dynamic and structural properties of the enzyme scaffold in complexes representative of the distinct species along the KSI catalytic cycle. Electrostatic properties of the scaffold will be assessed, and the range at which they have influence will be tested. The research proposed here subscribes to the overall goal of developing a predictive understanding of how specific properties of an enzyme scaffold contribute to catalysis and to the ambitious long-term goal of designing enzymes that rival natural enzymes in efficiency and specificity.

Autonomic innervation has been implicated as an important modulator of pancreas development and glucose homeostasis. Despite efforts to characterize the role of sympathetic/parasympathetic nerves, whether innervation directly controls endocrine function remains controversial. I propose to study the role of innervation in endocrine pancreas development and maintenance by using the zebrafish model. Pancreatic islet innervation has been observed in human, mouse and zebrafish with differences in the extent of innervation and specific cell types that are innervated. The zebrafish is well suited for the in vivo study of islet development with time-lapse imaging and has several advantages over other vertebrate models, including rapid embryogenesis, transparency during embryonic and early larval stages, high regenerative capacity and genome editing tools. Innervation studies in rodents and humans have been limited to specific snapshots in time and depth of the tissue, not allowing one to capture the temporal and three-dimensional spatial interactions that define the dynamic nature of innervation as the endocrine pancreas develops and matures. With reporters labelling specific neuronal and endocrine cells and tools for temporally controlled tissue specific ablation of neurons and knockdown of genes encoding specific neurotransmitter receptors, I plan to characterize innervation during pancreas development and elucidate the role of innervation on the maintenance of islet architecture, regeneration and function in response to metabolic demand. The results of this research can have a major impact on our understanding of pancreatic innervation dynamics, signalling and function.

An important aim of neuroscience is to decipher how neurons process environmentally and internally generated signals. In vivo, neocortical microcircuits are spontaneously active, providing an ongoing source of intrinsic excitatory and inhibitory network activity. This dynamic activity is integrated with sensory information and then transformed into an appropriate output. Neuronal computations have mostly been characterized in vitro, meaning our current knowledge of how neurons process information under realistic network conditions in vivo is incomplete. Recent theoretical studies indicate that in vivo network activity fundamentally alters the way in which neocortical excitatory pyramidal cells integrate synaptic input. The purpose of my proposal is to experimentally bridge this knowledge gap using an “all optical” method to non-invasively interrogate neuronal computations rapidly in 3D. This approach will be complemented by optogenetics, electrophysiology, and biologically detailed modeling. I will focus on the mouse visual and somatosensory cortical microcircuit formed by layer 5 pyramidal cells, the principal cortical output, and Martinotti interneurons, which provide strong feedback inhibition to the dendrites of pyramidal cells. L5 pyramidal cells express nonlinear mechanisms that are thought to affect spiking output during multimodal sensory integration. In contrast, virtually nothing is known about sensory computations performed by Martinotti interneurons or how they modulate L5 pyramidal cell activity. The potentially paradigm-shifting results derived from this research may transform our understanding of how neurons compute sensory information in vivo.

Humans and mice originate from pluripotent cells in the inner cell mass (ICM) of the embryo. This ICM is traditionally assumed to form after the 8-cell stage from a process termed ‘asymmetric’ cell division, which cleaves cells in parallel to the embryo surface and directly allocates one daughter cell inside. Further, it is believed that asymmetric division segregates polarity proteins and putative cell fate determinants (‘cell polarity’ hypothesis). Although asymmetric division is thought to be the primary morphological mechanism forming the ICM, it has not been directly demonstrated and it remains unclear whether biomechanical forces acting within the embryo would even allow it.

The Plachta lab has recently designed an in silico model demonstrating how adhesion and tension forces can generate virtual embryos with normal number of inner cells, faithfully recreating mammalian development. But unlike previous assumptions, these embryos feature very few asymmetric divisions. Instead, most inner cells originate from ‘symmetric’ divisions and subsequently internalize by apical constriction. Importantly, combining advanced imaging technologies of living mouse embryos and a novel custom membrane segmentation technology confirmed that asymmetric divisions are uncommon and unnecessary, and that apical constriction is the prime cellular mechanism allocating cells to the ICM in vivo.

These recent findings provide new understanding how the first embryonic structures form during mammalian embryogenesis. The goal of this proposal is to reveal how molecular mechanisms determine the polarity and fate of pluripotent cells coordinated by apical constriction-dependent ICM formation.

Non-coding RNAs that migrate between cells and organisms and act non-autonomously have recently attracted much attention. The inherent properties of RNA interference, i.e. sequence specificity, provide these “mobile RNAs” with the ability to affect essentially any gene in neighboring cells or tissues, while foreign RNA uptake can inform an organism about its habitat. In both plants and nematodes, mobile RNAs mediate cell signaling, antiviral defense, and facilitate transgenerational epigenetic inheritance. In mammals, extracellular miRNAs are highly abundant in many body fluids, making them attractive candidates for mediating trans-cellular communication. However, mammalian mobile RNAs have not yet been demonstrated definitively, and foreign RNA uptake through mammalian intestine is controversial. Major gaps in our understanding are the biochemical properties of mobile RNAs, and their mechanism of action, if any, in mammals. I propose to generate novel assays to investigate RNA uptake and mobility in mammalian “mosaic” organoid cultures. Using these assays, I will investigate how different cellular niches within the intestine respond to foreign RNA, whether small RNA can act non cell-autonomously, and what properties equip small RNAs with the ability to mediate trans-cellular communication. Ultimately, my studies are expected to generate new insights into small RNA based communication, and might resolve crucial issues in RNA-based drug delivery.

There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

The aim of this project is to understand how molecular forces ‘scale-up’ to generate biological outcomes. We focus on understanding the contractile actomyosin cell cortex, a sub-micron thick network that lies beneath the plasma membrane that supports cellular and tissue morphogenesis. Ultimately, functional outcomes and active mechanical behaviour at the larger scale must arise from the intrinsic biophysical properties of the force-generating molecules of the cortex. However, how these properties emerge is unclear. Our consortium will implement novel experimental approaches solutions to tackle this question. First, we will develop myosin mutants that allow us to modulate specific parameters of motor function (e.g. force, step-size, directionality, torque; Bryant) under control by light; we call this ‘Optomechanics’. Second, we will develop hydrodynamic active-gel theory for describing essential physical behaviors arising from molecular-scale force and torque generation in the cortex (Grill). We will apply these approaches to understand the contribution of the cortex to cellular morphogenesis during cytokinesis (Grill) and junctional tissue homeostasis (Yap). For this purpose, we bring expertise in myosin biophysics and molecular bioengineering (Bryant), cortical mechanics and theory (Grill), and cell junctions and morphogenesis (Yap). The bioengineering team will generate validated reagents that will be implemented in cells by the biological teams; results from the latter will reveal how modifications in myosin force generation at the single molecule level relate to in-vivo function. We will combine optical imaging, laser nanodissection with hydrodynamic theory to develop quantitative descriptions of cortical mechanics during cytokinesis in the C. elegans zygote. In parallel, we will define the impact of cadherin on cortical mechanics at mammalian epithelial cell-cell junctions, and how this influences mechanical coherence of populations. This combination of molecular manipulation with theory will allow us to describe how actomyosin mechanical activity at cell and tissue scales emerges from myosin motor function at the molecular scale, and how myosin molecular force drives cell- and tissue-level mechanics.

The crucial importance of secreted extracellular scaffolding proteins (ESPs), such as precerebellins, neuronal pentraxins, and C1q-like molecules, for the formation and function of neuronal synapses in vivo has only recently been discovered. These molecules share a modular structure, with domains that specifically interact with pre- and postsynaptic cell surface receptors to bridge neurons across the synaptic cleft. Guided by structural information of known ESPs, we aim to develop synthetic connector molecules that will expand the range and affinity of trans-synaptic interactions. These synthetic ESPs will likely become new and powerful tools to manipulate the number and properties of excitatory and inhibitory synapses, and to regulate synaptic communication and cognitive abilities in mouse models in vivo.
To achieve our goals, we will develop and implement a highly multi-disciplinary approach based on the diverse and complementary expertise and state-of-the-art techniques established in the applicants’ laboratories. Armed with structural and biophysical knowledge about physiological ESPs and their receptor interactions, we will design hybrid molecules that contain novel combinations of binding domains and thus promote a novel spectrum of molecular interactions across neuronal synapses. The synthetic ESPs will be further refined by mutagenesis or by replacing native domains with nanobodies of desired specificity. These novel neuronal connectors will be recombinantly produced, purified and delivered into subregions of the hippocampus and the cerebellum by acute or chronic injections using osmotic pumps. We will also express genes encoding the designer ESPs using viral vectors with neuron type-specific promoters. Electrophysiology, confocal microscopy, nanoscopy and behavioral analyses will be then employed to verify the targeting and functional efficacy of synthetic ESPs at the synaptic, neural network and organismic levels. Their effects on motor and spatial learning, reversal learning, pattern separation and completion will be studied in combination with recordings of neural activity oscillations and single-cell firing.
Considering the huge potential variety and versatility of these novel tools, we anticipate an immediate impact onto basic neuroscience and, in longer term, potential new avenues for the treatment of neuropsychiatric disorders.

Understanding how activity in large-scale brain networks causes behavior remains a fundamental challenge in neuroscience due to the inherent experimental limitations in the number of neurons that can be simultaneously recorded or manipulated. Unravelling complex biological systems to which we have limited access to measure or control the internal dynamics is notoriously difficult. Control engineering has solved this challenge for the control of complex man-made systems, such as aeroplanes and chemical plants, for which also only a small fraction of the state variables are accessible and controllable. Despite its clear potential, control engineering has not yet been adopted by neuroscience.
One mechanism by which information is thought to be integrated across multiple brain areas is by coherent oscillations of the local field potential at particular frequencies. Despite indirect evidence for such orchestration of cortical activity, a causal demonstration of oscillation-based information flow during behaviour has not yet been established. We will develop data-driven models of the interaction between cortical areas by using unscented Kalman filtering. These mathematical models then allow us to design control algorithms that apply optogenetic perturbations to control cortical interaction dynamics by manipulation of the temporal activity structure. We will develop mathematical models to determine the direction of information flow between brain areas by recording neural activity in auditory, visual and prefrontal cortex, during the performance of a demanding auditory-visual discrimination paradigm. Having identified the neural signatures associated with different behavioural states, we will perform closed-loop control of frequency-dependent information flow in the behaving animal to test the hypothesis that dynamic interactions between cortical areas play a causal role in perception and decision-making.
By combining control engineering and neurophysiology we will develop a new class of mathematical models which allow us to describe and manipulate neural activity, and therefore enable unique insights into brain function.

A new frontier in RNA biology has emerged in the last 5 years with the discovery that small RNAs (sRNAs) exist outside of cells and can be transferred from one cell to another in a functional form. This has been proposed as a mode of cell-to-cell communication and, in theory, could also enable communication between different organisms. Yet the basic mechanics of extracellular sRNAs are not understood, including how specificity in secretion is achieved and how sRNAs enter a functional RNA interference (RNAi) pathway in recipient cells. Without these details, it is difficult to predict the extent to which mobile sRNAs operate across biology, and to discern how to harness this mechanism in medicine. We approach these questions by examining sRNA transport in a host-pathogen system. We have found that an Argonaute protein (HpWAGO) and sRNAs, including microRNAs (miRNAs), are secreted by Heligomosoides polygyrus, a gastrointestinal nematode that infects mice. We will capitalize on combined expertise in nematode sRNA biology (Claycomb), host-pathogen interactions (Buck) and small RNA-target evolution (Abreu-Goodger) to examine the functional properties of HpWAGO and its sRNAs in both nematodes and mouse cells. Our approach integrates genetic and molecular techniques along with high-throughput proteomics and Next Generation sequencing. We exploit the nematode C. elegans for functional studies because H. polygyrus is genetically intractable and sRNA biology is well understood in C. elegans. The results from both systems will be computationally integrated to understand the mode of action, identify binding partners and their functional effects on gene expression, and explore the evolution of parasitic sRNA-related loci and their targets. Since there is no precedent for secretion of RNA or Argonaute proteins by nematodes, we will attempt to recapitulate a functional sRNA secretion system in C.elegans, which would bolster the nascent field of extracellular RNA communication and enable us to further dissect the secretion mechanism. Our overarching goal is to provide the first detailed characterization of an RNAi pathway that can mediate interactions between an extracellular pathogen and its host, which will enable us to predict the breadth of this mechanism across biology and lay a foundation for its functions in host-pathogen co-evolution.

Cortical pyramidal neurons (CPNs) are the basic computational units of the cortex. CPNs integrate local and distant inputs, and innervate various targets. Our knowledge about processing of these inputs by CPNs, and on the identity of CPN's targets, is limited due to the difficulty to record intracellularly from multiple spatial locations. We propose to apply a novel protein-based voltage imaging technology combined with optogenetic actuation ('optopatch') to record and manipulate CPNs in brain slices. Using optopatch we will study the details of voltage propagation in CPN dendrites following focal optogenetic stimulation in different compartments. Then we will describe voltage propagation and spiking output in response to synaptic inputs, by stimulating local and thalamic cells while imaging voltage propagation in single CPNs. This will provide a detailed map of inhibitory and excitatory local and distant connections into CPNs. Next, we will map the functional connections within a cortical column using simultaneous activation and recordings of multiple identified cells. Together, we will provide detailed description of computations performed by single cortical cells, and of the functional connections between them. Due to the high-throughput nature of these experiments, they could be readily repeated with other cell types, different cortical regions and various disease models. Success in implementing this approach in brain slices will be an important step toward in-vivo 'all-optical electrophysiology', a technological leap that may revolutionize neuroscience research.