Rafael Yuste - US grants

The neocortex constitutes the larger part of the brain in mammals and is the primary site of mental functions. No unitary theory of how the cortex works exists. Nevertheless, the basic structure of the cortex develops in stereotyped fashion, is similar in different parts of the cortex and in different mammals, and has not changed much in evolution since its appearance. Because of this, it is conceivable that a "canonical" cortical microcircuit may exist and implement a basic algorithm. Anatomical and physiological studies have suggested that the synaptic connectivity of the cortical microcircuitry is complex, but not random. In the previous cycle of the award we developed an optical method using calcium imaging of slices, to track excitatory circuits in neocortical slices. With this "optical probing" method we have reconstructed synaptic circuits in layer 5 from mouse primary visual cortex (V1) and have discovered extraordinary target specificity in several projections from layer 5 pyramidal neurons. These circuits were precise and identical in different animals suggesting that the neocortex is indeed built out of scores of precise circuits with dedicated functions. For this next cycle we propose a "frontal attack" on the cortical microcircuitry of mouse V1 using a large-scale optical probing effort with the goal to achieve a relatively complete reconstruction of the inter- and intralaminar excitatory circuitry. We will test whether the precision found in layer 5 applies to other neocorticat excitatory connections, as well as search for general rules in this circuit diagram. Our second goal is to test whether there are indeed canonical 'microcircuits, by reconstructing layer 5 circuits in mouse somatosensory cortex (S 1) and compare them with those in V1. In these experiments we will use transgenic mice strains that express GFP in subpopulations of neurons, a novel two-photon stimulation method that enables us to stimulate at will any neuron in the field of view, and exploratory microarray studies to find clusters of genes specifically expressed on subtypes of cortical cells. These basic studies will shed light on the structure of the functional units of the cortex and contribute to build bridges between system and cellular, molecular and biophysical level studies of visual cortex. In addition, they will help understand the central pathophysiological consequences of amblyopia and strabismus and improve analysis of visual evoked potentials and early diagnosis of visual pathologies.

DESCRIPTION: (from applicant's abstract) Epilepsy affects about 2 percent of the world population and is particularly frequent in children. Relatively little is known about how epileptic seizures propagate across and recruit apparently normal cortical circuits. Given the complexity of the neocortex, where dozens of classes of excitatory and inhibitory neurons are involved in different circuit functions, it is likely that the initiation and spread of epileptic discharges are differentially controlled by specific neuronal classes. Over the last decade, we have developed an optical approach using calcium imaging from population of neurons to study neocortical circuits and to image their activation in three dimensions with 2 photon excitations. Using this strategy, we can optically detect action potentials in the somata from dozens or hundreds of neurons, image epileptiform events with single cell resolution and detect which neurons participate in different types of epileptiform events. We propose a systematic effort to understand the role of different classes of neocortical neurons in the initiation and propagation of epilepsy. We will use calcium imaging of neuronal populations during pharmacological-induced epileptiform events in neocortical slices from juvenile (P9 - P20) rat somatosensory cortex, in order to better understand the circuit mechanisms responsible for juvenile epilepsy and at the same time image the transition from interictal to ictal events. The experiments will be carried out combining whole cell recordings and biocytin reconstructions with state-of-the-art imaging techniques, including two-photon microscopy, a photodiode array and a fast cooled CCD camera. Our first goal is to characterize morphologically and physiologically the neurons involved in spontaneous and evoked interictal and ictal epileptiform events. Our final goal is to apply a novel optical probing method to reconstruct the circuitry underlying epileptiform events by revealing the postsynaptic targets that are triggered by layer 5 IB neurons, or other potentially key cell classes. The answers to these questions could have therapeutic implications for targeting specific neurons or cell layers which play a critical role in epileptiform events. Also, our results will be particularly useful in identifying the cellular and circuit mechanism responsible for the lower seizure threshold of developing and juvenile neocortex and the transition from interictal to ictal events.

DESCRIPTION (Adapted from applicant's abstract): Dendritic spines are the major sites of synaptic input in the mammalian CNS and have been traditionally been considered stable structures. Nevertheless, as initially suggested by Francis Crick and confirmed recently by data from our group and by others, spines are motile in both dissociated cultures and in brain slices. Spine motility is action-based and appears to be intrinsic to the neuron. Because of the importance of spines in the cortical circuit, spine motility could have potentially, major consequences in the development and function of the cortex. In our previous work we discovered that spine motility in mouse cortex is down-regulated during the postnatal ages that herald the end of the critical period for monocular deprivation. Although the critical period in primary visual cortex has been studied extensively for many decades, it is still unclear what factors terminate it. Based on this correlation we hypothesized that the end of the critical period is due to the lack of motility of the spines. We want to examine this hypothesis in detail combining gene-gun GFP transfection, two-photon imaging, image deconvolution and electron microscopy of spines in brain slices from mouse primary visual cortex, as well as in vivo imaging, deprivation and pharmacological experiments. The first aim will focus in characterizing the motility in different cortical layers in mouser V1B and in reconstructing at the ultrastructural level the previously imaged spines. The ability of finding in serial reconstructions the same spines imaged in two-photon time-lapse movies will allow us to examine with unprecedented detail whether there are any correlations between the presence and type of motility and the presence and type of presynaptic terminal. The second aim will seek to identify the cellular mechanisms mediating the motility, with special emphasis on the downstream targets of the Rho family of small TGPases and in the examination of the role of synaptic activity in this process. The third aim will directly test if spine motility lays a causal role in the critical period, by examining whether it exists in vivo and by analyzing the consequences of blocking it in the monocular deprivation paradigm. These studies will shed light on the role of structural plasticity in the development of the visual cortex. In addition, they will help discern the cortical consequences of monocular deprivation, effects which may underlie amblyopia and strabismus, as well as help design therapeutic strategies aimed at compensating for these deficits. A more complete understanding of the development of visual cortex will also improve the measurement of acuity, contrast sensitivity and chromatic sensitivity of preverbal children and in early diagnosis of visual pathologies.

Progress in the biological sciences and medicine relies increasingly on methods, approaches, and strategies derived from synergistic interactions with the physical sciences and engineering. One notable example of this is the use of optical methods for biosensing and bioimaging. Furthermore, the tremendous nanoscale device fabrication capabilities built up in microelectronics and photonics furnish unparalleled opportunities for leveraging highly integrated platforms for on-chip biological sensor systems. By their nature, these applications cross through multiple disciplines and require a team with diverse expertise in the fundamental light/tissue interaction, complex optical instrumentation and imaging tools, and relevant biological systems. In this Integrative Graduate Education and Research Training (IGERT) program a new generation of scientists and engineers will be trained through a set of five research thrusts that cross three fundamental core competency areas: optics, photonics, and sensor electronics; biomolecular detection and cellular-level analysis; and applications to medicine and public health. Each IGERT trainee will be empowered to work at the boundaries between the disciplines and will be uniquely capable of contributing to advancements in this important emerging field. With 19 faculty members representing academic departments across Columbia University's School of Engineering and Applied Science, School of Arts and Sciences, Mailman School of Public Health, College of Physicians and Surgeons, and Teachers College, and incorporating strong interaction with City College, Queens College, and The Cooper Union in New York City, the IGERT trainees will experience a truly diverse community sharing in the integrated educational and research activities and will be exposed to a wide spectrum of cutting-edge applications. An external advisory board including industrial and government labs will provide additional connections between the IGERT and outside partners. Educationally, this IGERT program fulfills a compelling need to train a diverse workforce of U. S. scientists and engineers trained in an area of large and growing competitive importance to the United States. The proposed enrichment program provides IGERT fellows with enhanced training through experience in industry and government laboratories, seminars on professional development, career guidance, entrepreneurship, and discussion of ethical issues. Significant resources are committed to ensuring recruitment and retention of fellows from underrepresented groups. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

One of the greatest challenges in computational neuroscience is to reconstruct the connectivity of large, complex neuronal networks. The ability to decipher circuit connectivity would have a fundamental impact on our understanding of the dynamical properties and the functional organization of the nervous system. Knowledge of prevalent connectivity patterns will also shed light on the developmental constraints and learning rules under which the network might be operating.

Recent developments open new possibilities for collaborative efforts to tackle this basic problem. First, advances in two-photon imaging and photostimulation methods make it possible to observe the simultaneous activity of large ensembles of neurons, while stimulating neurons in arbitrary spatiotemporal patterns. Second, new statistical methods for extracting action potential timing information from calcium imaging data, and for modeling the response properties of small collections of neurons, are now efficient enough that they may be implemented on-line and scaled up to understand the function of large networks.

The investigators will combine these new experimental and analytical methods to estimate, for the first time, the connectivity diagram of large neocortical circuits, using two-photon calcium imaging of spontaneous and evoked activity in thalamocortical slices. A key novel step here is to directly verify the estimated circuit model with two-photon glutamate uncaging, which allows any neuron in the circuit to be activated (with single-cell resolution) while the evoked postsynaptic responses are monitored.

This interdisciplinary project has three complementary specific aims: (1) Develop statistically-optimal methods for real-time inference of spike timing from calcium imaging data. (2) Use these spike timing inference methods to estimate the network connectivity from large-scale multineuronal calcium-imaging of cortical slices. (3) Confirm the derived connectivity maps with glutamate uncaging and patch clamping, by photoactivating putative presynaptic neurons while recording intracellularly from postsynaptic cells. The proposed methods should also prove applicable to study other central and peripheral regions of the nervous system; data analysis software will be made publicly available online, to enhance the infrastructure for research and education in computational neuroscience.

IDBR: CMOS cameras for high-frame-rate time-correlated single-photon counting

Recent advances in biological imaging techniques, particularly those exploring molecular dynamics, are outpacing technological innovation. Fluorescence lifetime holds great potential as a biomarker that can reveal changes in a fluorophore's local chemical and physical environment, as well as the binding dynamics of single proteins through excited state interactions and Förster resonance energy transfer (FRET). Many of the latest active dyes, molecular probes and even transgenic labeling strategies exploit FRET to enable real-time observation of cellular processes both in-vitro and in-vivo. While FRET can be detected using intensity-only measurements, quantitation can be dramatically impaired by experimental factors such as photobleaching, whereas lifetime-based FRET measurements are significantly more robust. Nevertheless, adoption and widespread use of fluorescence lifetime imaging microscopy (FLIM) for biological research has been hindered by two major factors: the speed with which FLIM images can be acquired and the cost and complexity of the instrumentation required for FLIM. In this multidisciplinary proposal, a novel two-dimensional high-frame-rate complementary metal-oxide-semiconductor (CMOS) fluorescent lifetime camera chip based on single-photon avalanche diodes (SPADs) will be developed. This chip will be applied to both wide-field and laser-scanning-based microscopy techniques to enable several important advances in FLIM imaging. In widefield imaging, this will result in acquisition of images at a incident-photon-limited frame rate as high as 1 kHz.

Solid-state imagers are based primarily on two technologies, charged-coupled device (CCD) and CMOS. Both of these imaging technologies are based on converting photons to electrons and collecting many of these electrons to produce a measurable signal. These imagers are now employed in digital cameras of every type, from cell phone cameras to the high-end cameras employed in biological imaging. Since optical techniques are so pervasive in probing biological systems, cameras represent the fundamental interface between the biological world and the solid-state world. In this effort, an entirely new camera chip will be designed based on a device that, instead of collecting electrons produced by photons, counts them, one-by-one. This enables very high sensitivity for photon detection. At the same time it allows resolution of very short (and dim) optical events (on the order of 10's of ps). Such capabilities will enable new types of biological imaging applications. This project supports the multidisciplinary training of graduate and undergraduate students and a significant K-12 outreach effort.

DESCRIPTION (provided by applicant): Mental disease, including schizophrenia, depression and autism spectrum disorders, are still poorly understood, although it is clear that they mostly represent cortical disorders. The cortex is the primary site of higher mental functions, and despite extensive research, there is still no unified theory of how the cortex works. This is partl due to the fact that neuroscientists have traditionally relied on microelectrodes to record the activity of individual cells. However, cortical circuits are composed of millions of neurons and it is conceivable that single cell measurements alone will not be sufficient to unravel function of the brain. Optical imaging techniques tackle this emergent level of neuronal circuit activity and enable to image the activity of neuronal ensembles, in vitro and in vivo, while preserving single cell resolution, something that brain imaging techniques such as MRI or PET, cannot do. Moreover, the development of genetically encoded photosensitive proteins (optogenetics) and optochemical (caged) compounds offers the opportunity to not only image the activity of many neurons but also to optically control them. In spite of their potential, current optical imaging techniques suffer form the fact that they rely on lasers which have to be moved to each pixel to build an image, making the imaging slow. Moreover, common laser microscopy is performed in 2D. To supersede those problems, we have recently developed a novel form of microscopy that uses spatial light modulators (SLM), to split the laser beam into a holographic pattern that can be used to image (or photoactivate) neurons simultaneously in 3D. SLM microscopy has the potential of becoming the ideal method with which to explore the role of neural circuits in brain diseases. Boulder Nonlinear Systems and Columbia University propose to combine their expertise in building SLMs and in SLM microscopy in a two-phase project with the ultimate goal of making SLM microscopy a practical reality in neuroscience and clinical research. In the first phase we plan to build a compact, inexpensive, user- friendly system that enables fast, 3D imaging and photoactivation of neurons. The device will be self-aligning and integrated with appropriate software so that it can be used, out of the box, for applications in several neurobiological projects including imaging intact neural network activity, optical manipulation of neuronal firing, functional mapping of brain connectivity, investigating neurovascular coupling, and also be used for assaying neuronal activity in animal models of brain disease. In Phase II we will extend the design to support electrophysiological recording with two-photon excitation, allowing 3D imaging and photostimulation of cortical neurons in living animals, such as awake behaving rodent preparations. PUBLIC HEALTH RELEVANCE: Microscopy with spatial light modulators (SLMs) enables use of optical techniques to study neuronal circuit activity, to both monitor and manipulate the activity of neuronal ensembles, in vitro and in vivo. Boulder Nonlinear Systems and Columbia University propose the development of a compact, inexpensive, user-friendly SLM based microscope (Pocketscope) that enables fast, 3D imaging and photoactivation of neurons. The device will find widespread use in neuroscience research including imaging intact neural network activity, optical manipulation of neuronal firing, functional mapping of brain connectivity investigating neurovascular coupling, and also be used for assaying neuronal activity in animal models of brain disease.

DESCRIPTION (provided by applicant): Novel two-photon caged GABA compounds Understanding how GABAergic inhibition works is necessary to decipher the function of brain and the pathophysiological processes of diseases that affect it, including many epilepsy and addition syndromes. At the same time, there is no unified agreement as to how exactly GABAergic inputs function and even the basic question of whether they are inhibitory or excitatory is actively debated. Part of the reason for these controversies is the fact that inhibitry inputs target subregions of the postsynaptic cells, and there are not good tools to investigate these inputs with high spatial resolution. Local activation of receptors in living neurons can be achieved by two-photon photorelease of caged compounds. Indeed, two-photon uncaging of glutamate has revolutionized current understanding of excitatory transmission and integration in mammalian neurons. Unfortunately, opto-chemical tools to photorelease GABA are scant, even though they would be extremely useful to study the function of GABAergic inhibition. In this proposal we introduce a novel family of two-photon caged GABA compounds. Specifically, we will test and characterize the two-photon release of three different chemical generations of caged GABAs and use the best ones to generate high-resolution maps of GABAergic responses on living pyramidal neurons from mouse neocortical slices. Finally, we will confirm the accuracy of these maps using a novel 3D high- throughput electron microscope to identify symmetric synapses. The proposed work will expand the chemical toolbox of biological uncaging to include novel high-quality caged GABA compounds that can be photo-released with two-photon lasers. These new compounds will enable the detailed investigation of the functional effects of GABAergic transmission on selective subcellular compartments, something likely to have a major impact on our understanding of how inhibition alters normal and diseased brain function, since some of these compounds can be used to control epilepsy. Finally, our data will reveal, for the first time, the functional effect of GABAergic inputs onto dendritic spines. Since spines mediate most excitatory connections, these results could also alter our understanding of how excitatory inputs are integrated. PUBLIC HEALTH RELEVANCE: Novel two-photon caged GABA compounds Although GABAergic circuits mediate most of the inhibition in the brain, and are affected in many brain pathologies, their function is poorly understood, partly because they act with great spatial selectivity onto subregions of the neurons. We propose the development of novel opto-chemical tools that will allow to optically activate GABAergic inputs onto neurons with unprecedented spatial resolution, thus enabling the dissection of their functional properties. Our work could result in the generation of novel optical methods to control the activity of neurons in hyper-excitable pathological states such as in epilepsy. !

DESCRIPTION (provided by applicant): Astrocytic regulation of neuronal synchronization. The function that astrocytic glia play is poorly understood, and their traditional role, as supportive of neurons, captures only a small part of many functional outputs that astrocytes may have. Recent research on astrocytes has revealed important effects on synaptic transmission, but their role in the circuit as a whole has been less studied, partly due to lack of circuit-level assays of the interaction between astrocytes and neurons. Using two-photon imaging, we have recently discovered that, in neocortical brain slices, stimulation of a single astrocyte can lead t widespread neuronal synchronization, in the form of UP states. Conversely, blocking astrocytic signaling by the injection of BAPTA into individual astrocytes can reduce the number of spontaneous neuronal UP states. We propose to test in a series of direct experiments if astrocytes regulate UP states in vivo. We have developed new two-photon and genetic techniques that enable us to image and manipulate the activity of astrocytes in vivo and examine how it relates to neuronal UP states. Our central hypothesis is that the activity of astrocytes precedes, and is causally related, to the occurrence of UP states. Specifically, in a first aim we will perform fast two-photon calcium imaging, with SLMs, of neuronal and astrocytic activity during UP states in Brainbow mice where astrocytic territories are labeled, and examine whether there are spatio- temporal correlations between the activity of astrocytes and neurons. In a second aim we will optically stimulate astrocytes, using two-photon uncaging of RuBi- glutamate, IP3 or ChR2 photoactivation, and test whether this increases or alters spontaneous UP states. Our proposed work could establish a causal link between astrocytic function and neuronal synchronization in vivo, thus providing a novel circuit-level functional role for astrocytes. UP states are thought to underlie slow-wave sleep and resting state fMRI signals, so our research could directly involve astrocytes in the circuit mechanisms of those global brain states. Our group has a unique combination of optical and circuit neuroscience skills and this research could introduce novel approaches into the study of astroglia.

DESCRIPTION (provided by applicant): Astrocytes are not merely passive members of the circuit, but that they may also be directly involved with neuronal computation. Indeed, astrocytes are well suited to a potential widespread role in the circuit: they tile the CNS with near- complet coverage, are connected into an extensive syncytium via gap junctions, and processes from a single astrocyte can contact up to tens of thousands of synapses. In fact, we have recently shown how even the stimulation of single astrocyte can control circuit-wide neuronal synchronizations in the cortex. Tools to study astrocytes are significantly less developed than those available to investigate neuronal function and, consequently, most research on astrocytic information processing has been limited and incomplete. Astrocyte researchers need methods to selectively measure and manipulate the function of astrocytes in vivo. Two-photon excitation, which penetrates living tissue while affording single-cell resolution, combined with compatible optochemical or optogenetic probes, could be an ideal technical platform for future astrocyte research. We propose to develop novel two-photon and computational tools to provide subcellular and circuit-level analysis of astrocytic function in neural circuits in vivo, using a nvel astrocyte-specific Brainbow mouse line. These tools include two- photon optogenetic and caged compounds, methods to image groups of astrocytes in 3D, and computational algorithms to reveal interactions of astrocytes with neurons in the circuit. With this award, we will provide the astroglia community with a wide range of optical and computational tools that can be readily adapted for use in vivo. This work will enable the investigation of the roles of astrocytes in shaping neuronal activity in the cortical microcircuit and beyond, and thus could have a fundamental impact on how we view neuro-glia processing, as well as on solidifying the role of an overlooked circuit constituent-the astrocyte-in cortical synchrony and computation. Finally, this work could introduce a novel potential means to assay or control neuronal function for potential therapeutic purposes: through specific activation of astrocytes.

DESCRIPTION (provided by applicant): Novel caged dopamine compounds. The abnormal regulation of dopamine receptors has been postulated as a prominent pathophysiology of several mental disorders, such as schizophrenia, bipolar disorder and depression. However, how dopamine receptors modulate information flow in individual neurons and neuronal circuits in prefrontal cortex is still poorly understood. Local activation of receptors in living neurons cn be achieved by two-photon photorelease of caged compounds. Indeed, two-photon uncaging of glutamate has revolutionized current understanding of excitatory transmission and integration in mammalian neurons. Unfortunately, opto-chemical tools to photorelease dopamine are scant, even though they would be extremely useful to study the function of dopaminergic modulation. We propose to use a newly synthesized caged compound, RuBi-Dopa, which can be photoreleased with two-photon lasers, to optically activate dopamine receptors with high precision and map the distribution of functional dopamine responses in spines from prefrontal pyramidal neurons, studying how their activation alters glutamatergic transmission. Finally, we will test how dopamine affects the temporal patterns of multi-neuronal firing in prefrontal cortex, by uncaging RuBi-Dopa while performing two-photon calcium imaging in vivo in awake preparations. The proposed work will expand the chemical toolbox of biological uncaging to include novel high-quality caged dopamine compounds that can be photo- released with two-photon lasers. These new compounds will enable the detailed investigation of the functional effects of dopaminergic transmission on selective subcellular compartments, something likely to have a major impact on our understanding of how dopamine alters normal and diseased brain function. It is possible that some of these compounds could be used to develop optical therapies for mental disease. Finally, our data will reveal, for the first time, the functional efect of dopaminergic inputs onto dendritic spines. Since spines mediate most excitatory connections, these results could also alter our understanding of how excitatory inputs are integrated.

DESCRIPTION (provided by applicant): Functional Connectomics of the Neocortical Microcircuit The cortex constitutes the primary site of higher cognitive functions and mental disease. No unified theory of how the cortex works exists yet, due to our basic ignorance about its microcircuits (i.e. the detailed connectivity patterns of any cortical area), and also because it is likely that its function is based on an emergent level, determined by the states of activity of large neuronal ensembles. Two-photon calcium imaging and photo-activation techniques enable us to simultaneous record and optically manipulate the activity of larger neuronal populations, while maintaining single cell resolution. Using such techniques we have encountered signs of what could be a highly distributed and essentially random cortical microcircuit. Based on these results, we propose the idea that the cortex is a random circuit, meaning that each synaptic connection is chosen by chance, independently from others. These circuits, mathematically analogous to completely connected ones, would maximize the distribution of information and enable the appearance of emergent functional states. This model runs contrary to the traditional view of the cortex, one that arose from sampling individual neurons, as a very specific machine where the connectivity and function of each neuron is precisely determined. Using this award, I want to test the hypothesis that the cortex is a random network, applying novel two-photon methods in a large-scale and systematic study of the mouse cortical microcircuit. I propose a three-pronged approach: 1- Image the activity of an entire cortical module in a mouse, to detect all spikes from all cells. 2- Perform a ?Circuit Cracker? analysis to obtain the blueprint of connectivity of the module. 3- Optically manipulate the population activity to test whether it behaves as a random circuit. Experiments will be done in mouse cortex in vivo, with awake, head-restrained preparations, under sensory stimulation and rest. Transgenic strains will be used to selectively label identified subpopulations of cells, and several cortical areas will be examined to explore common modular features. The proposed work will provide, for the first time, a complete description of the activity of any neural circuit and the blueprint of the cortical circuit and will pioneer ?Functional Connectomics?, i.e., deciphering the connectivity of the circuit from its functional correlations. If the data confirm that the microcircuit is indeed random, our results could also usher in a novel model for cortical function, one based on the existence of emergent functional states. This model could replace the current paradigm, and enable a more efficient understanding of the pathophysiology of cortical diseases, such as epilepsy and schizophrenia.

This award supports an international meeting to coordinate the numerous independent large-scale brain projects that are now underway in many different countries. NSF has the congressional charge to lead the coordination of international Brain Projects, and this meeting will serve as a critical step toward the establishment of a Global Brain Initiative, replicating the success of the Human Genome Projects and other international worldwide scientific collaborations. The meeting will host over 400 US and international attendees, at Rockefeller University in association with the nearby United Nations General Assembly, and feature as speakers a group of international scientists and administrators from all the major brain projects around the world. The potential broader impacts of coordination among these projects for advancing neuroscience, understanding brain function, and improving treatment for neurological disease is immense.

The goal of the meeting is two-fold: 1) to write a platform paper supporting cooperation, to be signed by heads of brain projects worldwide, and 2) to create a committee with representatives from all brain projects to promote collaboration and cooperation. The two organizers led the teams of researchers that launched the US BRAIN initiative, have gathered wide private and public support for this meeting, and are based at two local universities. A diverse group of researchers will participate, and online access to the meeting will assure broad dissemination.

Dendritic spines mediate essentially all excitatory connections and are thus critical elements in the brain but their function is still poorly understood. In particular, a key question is whether or not they are electrical compartments. To explore this, researchers have used cable theory and Goldman-Hodgkin-Huxley-Katz models, which form a theoretical foundation responsible for many cornerstone advances in neuroscience. However, these theories break down when applied to small neuronal compartments, such as dendritic spines, because they assume spatial and ionic homogeneity. Taking advantage of advances in computational power, we will explore the application of a broader theory that incorporates the Poisson-Nernst-Planck (PNP) approximation and electrodiffusion to more accurately model the constraints that the nanostructure of the spines place on electrical current flow. Specifically, we will combine multiscale modeling, asymptotic and simulations of partial differential equations to extract features from data and experimental approaches to study how the geometry and composition of a dendritic spine affect the electrical and ionic fluxes and the coupling between the synapse and the dendrite. We will test the predictions of electrodiffusion combining cutting-edge voltage imaging methods with 2-photon glutamate uncaging in vitro and in vivo and nanopipette recordings of spines from mouse pyramidal neurons. Our broader theoretical analysis of could be instrumental to understand their physiological role in neuronal circuits. This work will explore an alternative theoretical formulation to the established cable theory, in order to understand quantitatively the biophysical properties of dendritic spines. We aim to generate the most rigorous mathematical model of spines to date and tackle key questions in neurobiology: how synaptic voltages in neuron are shaped at spines, how they propagate to dendrites and how this is regulated by ionic channels and dendritic and spine geometry. Finally, this proposal will help to link the form and the function of neurons, with a detail which has never been carried out before, something that could help interpret functionally many peculiar morphological characteristics of neuronal microstructures, in both normal and pathological processes.

One way to decipher a complex biological problem, such as understanding how the brain works, is by using a simpler system that enables greater experimental or computational access. Hydra is a small, transparent relative of the jellyfish, and represents the first animals to have evolved a nervous system. Correspondingly, the nervous system of Hydra is very simple, with a few hundred neurons forming a net which tiles the body of the animal, without ganglia or brain. In spite of this simplicity, Hydra's nerve net generates a rich range of nimble behaviors, including contracting, elongating, bending, searching and somersaulting. Recently, the investigators of this project developed genetically altered Hydra strains in which the activity of neurons and muscles causes them to generate a light signal. Thus, the investigators can directly observe the activation of every neuron and muscle cells in an animal while it is behaving. Because of this, they can use statistical methods to analyze how neural activity drives movements. They discover basic principles of how simple nervous systems control muscles to produce behaviors. Given that Hydra has no brain, this project may reveal how complex movement can be organized without any central coordination. Further, Hydra has an extraordinary ability to regrow: its cells are constantly being replaced, and a complete Hydra body can reform from even very small pieces of the animal. Understanding how the nerve net of Hydra continues to produce stable behavior in the face of rapid turnover may advance understanding of how nervous systems can repair themselves. The study of Hydra with an integrated imaging/computational approach serves as an appealing platform for outreach opportunities. The research introduces members of the general public to neuroimaging and essential biology and mathematical neuroscience. It also provides training opportunities for researchers at all levels. The Hydra system is deeply integrated into summer courses at the Marine Biological Laboratory and provides cross-cutting projects for students from diverse backgrounds.

This project aims to decipher the relation between the activity of a nervous system, the muscles it controls and the behavior the muscles generate using the cnidarian Hydra. The investigators focus on decoding the neural basis of a few elementary behaviors that can be rigorously identified and that are generated by the endodermal and ectodermal nerve nets. The investigators use calcium imaging of every neuron and every muscle cell in mounted Hydra preparations during contractile behaviors. To analyze the required data sets, the investigators develop algorithms to track cells in the moving, deforming animal and apply dimensionality reduction methods to discover spatiotemporal patterns of movement corresponding to muscle activation patterns. The end product is a quantitative model that explains how contractile behaviors are generated. As another deliverable, the techniques developed to track neurons and discover spatiotemporal patterns are made widely available in an open source platform and may be of use in other systems. This proposed work will help establish Hydra as a model neural system for which a complete accounting of neural activity and behavior may be rigorously approached.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Dendritic spines cover dendrites of most mammalian neurons and receive almost all excitatory connections in the cortex. Although their role in these circuits is therefore likely to be crucial, the function of spines is still poorly understood. Spines are chemical compartments, and this could provide the biochemical isolation necessary to implement input-specific synaptic plasticity. But recent experiments have suggested that, in addition, spines could compartmentalize voltage. This could have a major impact on excitatory synaptic potentials, altering them as they are injected into the dendrites. In fact, by regulating the spine neck dimensions, dendritic spines could rapidly control synaptic strength. While there is in vitro data supporting this hypothesis, there is currently no direct measurements of spine voltages in vivo. Our goal is to build tools to determine if spines indeed have an electrical function in vivo. We propose two types of optical tools to image and optically manipulate spines in mouse visual cortex in vivo. In the first aim we will build, calibrate and test two novel Genetically Encoded Voltage Indicators (GEVIs), which will be designed for optimal two-photon cross section and for targeting to dendritic spines. In the second aim, we will pilot the use of simultaneous two-photon imaging and optogenetics of individual spines in vivo and we will also synthesize and test a RuBi caged-TTX for two-photon photorelease in vivo. Our research will develop tools that could enable the systematic study of the function of dendritic spines and other neuronal nanocompartments. Testing the electrical function of spines could also help to better understand the pathophysiology of many mental retardation syndromes, characterized by abnormally long spines.

To understand the physical and biological principles underlying the self-assembly of the nervous system of the fresh-water polyp Hydra vulgaris, the PIs propose a collaboration between theoreticians and experimentalists that will focus on this problem. The PIs will image the activity of every neuron and muscle cell during re-aggregation of Hydra, and analyze it with novel imaging, statistical and mathematical tools, correlating the emergence of phase transitions in the neuronal and muscle activity with the appearance of specific behaviors. This work will provide a deep insight into algorithms and mechanisms of self-assembly, with important repercussions for control theory, neuroscience, soft-matter physics, robotics and network science. Due to Hydra's extreme regenerative ability, this work may enable synthetic biology of an organism with a nervous system; and the work will have future repercussions for the field of neuroregeneration. The techniques developed for tracking of neurons in a moving and distorting animal will be made widely available and will be of use in other systems. The study of Hydra with the proposed integrated imaging/computational approach will serve as an imaginatively appealing platform for a range of outreach opportunities introducing members of the general public to mathematical neuroscience, and training opportunities for students at all levels. In particular, the Hydra system can be deeply integrated into the full spectrum of courses at the Marine Biological Laboratory and provide cross-cutting projects for students from diverse backgrounds.

One of the most fascinating aspects of biological organisms is their self-assembly: bodies put themselves together without external directions, to yield robust, resilient and adaptive living systems. This is particular dramatic in the cnidarian Hydra vulgaris. This small, transparent polyp has unique regenerative properties, demonstrated in the ability to completely regenerate itself after its body has been dissociated into individual cells. This remarkable self assembly, regenerating a normal animal, is complete within a few days, occurs robustly in culture dishes under a microscope and has surprisingly not yet been studied systematically. This project goes to the heart of this problem and will apply the a state-of-the-art toolset of modern neuroscience and computational approaches to this fundamental problem.

This collaborative US/France project is supported by the US National Science Foundation and the French Agence Nationale de la Recherche, where NSF funds the US investigator and ANR funds the partners in France.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.