Mriganka Sur - US grants

I shall use the technique of injection physiologically identified axons intracellularly with horseradish peroxidase (HRP) to define the terminal morphology of retinal ganglion cell axons in normal and visually deprived adult cats and in normal kittens. The HRP completely stains an axon and permits detailed light microscopic visualization of terminal processes. Frist, the regions of termination of single, W-, X- and Y-cell axons within the thalamus and midbrain, along with morphologies of terminal zones, will be specified in normal adult cats. Second, axonal arborizations will be characterized in adult cats raised with monocular lid suture. Our initial observations indicate that monocular deprivation causes expansion of X-cell and reduction of Y-cell retinogeniculate terminal fields in the A-laminae of the lateral geniculate nucleus (LGNd). These results suggest competition between X-cell and Y-cell terminations in the LGNd during development, and also suggest retrograde effects of binocular competition as a cofactor. These mechanisms will be evaluated. Third, to directly identify developmental sequences, similar experiments will be initiated in normal kittens. Concurrently, retrogradely labelled retihal ganglion cell somata will be described in the normal and monocularly deprived adult cats and in kittens. Thus, the morphology of entire neurons from cell body to axonal terminations will be characterized. These studies, besides providing basic structure-function correlations of the anatomical organization of physiological pathways at the single-cell level, will provide information fundamental to understanding neural mechanisms of amblyopia. Experiments beyond this grant period will focus on longitudinal effects of visual deprivation on the development of the retinogeniculate pathway. Taken together, these experiments constitute a long-term study of how physiologically defined ganglion cell types distribute information centrally, how these pathways develop, and how the visual environment influences their development.

We propose experiments to examine the anatomical substrate and physiological consequences of a dramatic rerouting of retinal projections to the auditory thalamus and cortex in ferrets. The organization of the visual pathway in normal ferrets in very similar to that in cats. We have successfully induced, with appropriate surgery in neonatal ferrets, retinal projections into the medial geniculate nucleus (MGN), the principal auditory thalamic nucleus. The MGN with retinal input retains its projections to auditory cortex. We now propose to study visual responses from, and the extrinsic and intrinsic connectivity of, the MGN and primary auditory cortex in operated ferrets reared to adulthood. Specific electrophysiological experiments include mapping the projection of the visual field onto the MGN and primary auditory cortex, studying the types of retinal input and receptive fields of visual cells in the MGN, and the receptive field properties of visual cells in primary auditory cortex. Anatomical experiments include defining in detail the projection of the two eyes to the auditory thalamus and cortex, the cells of origin of retinal projections to the MGN, and the subcortical and cortical connectivity of auditory thalamus and cortex in operated animals. We shall initiate ultrastructural studies of the synaptic organization of MGN cells with retinal input. We shall compare these features with corresponding features of the lateral geniculate nucleus (LGN) and primary visual cortex, or the MGN and primary auditory cortex, of normal animals. The overall goal of our experiments is thus to address a fundamental issue in development: whether structure and function in a target is determined by intrinsic properties of the target, or by its inputs during development.

There is increasing evidence that synaptic transmission between retinal ganglion cell axons and neurons in the lateral geniculate nucleus (LGN) is mediated primarily by excitatory amino acids. The goal of this proposal is to examine the role of NMDA and non-NMDA excitatory amino acid receptors on neurons LGN in (a) retinogeniculate transmission through the adult LGN, and (b) the development of retinogeniculate projections. These experiments follow from the hypothesis that NMDA receptors an be used for two distinct tasks in the brain: for regulating the level of postsynaptic activity in normal adult function, and for regulating synaptic connections during development. Specific questions include: (1) Are there NMDA and non-NMDA excitatory amino acid receptors on LGN neurons in adult cats and ferrets? Are these receptors at retinogeniculate synapses or at corticogeniculate synapses? (2) How does blocking these receptor sub-types affect the visual responses of adult LGN neurons? (3) Can NMDA receptors at retinogeniculate synapses, in tandem with cortical inputs on LGN neurons, serve to gate transmission of visual information through the LGN to visual cortex? (4) Are there NMDA and non-NMDA receptors on LGN neurons at early stages of development of the visual pathway? How do these receptors contribute to the excitatory postsynaptic responses of developing LGN cells? (5) How does blockade of NMDA and non-NMDA receptor affect the development of retinogeniculate connections? In particular, what role do these postsynaptic receptors play in two key events in retinogeniculate development: formation of eye- specific layers, and, in ferrets, the formation of on and off sublayers? (6) How are NMDA receptors involved in synaptic potentiation, and hence in the consolidation and elimination of retinogeniculate synapses, during development? The first three questions relate to the adult LGN and will be addressed using (a) extracellular recording and microiontophoresis, and (b) intracellular recording in slices of LGN. The next three relate to the developing LGN and will be addressed using (a) minipump infusion of agonists and antagonists, and (b) intracellular recording in slices of LGN at different ages.

We propose experiments that would examine a fundamental issue in development: whether structure and function in a target structure is determined by intrinsic properties of the target or by its inputs during development. We nave induced, by appropriate surgery in neonatal ferrets, retinal projections into the medial geniculate nucleus (MGN), the principal auditory thalamic nucleus. The MGN with retinal input retains its projections to auditory cortex. We have shown that retinal input to the MGN in "rewired" animals arises from small retinal ganglion cells with slow conduction velocities, that the pathways for visual inputs to the primary auditory cortex (AI) is from the retina via the MGN, that neurons in the MGN and AI have visual receptive fields, and that a map of visual space exists in AI. In the next grant period, we shall examine: (1) the structure of individual retinal ganglion cells that project to the MGN in rewired animals and compare the cells with ganglion cells in the normal retina; (2) the structure of single retinal axon arbors within the MGN in rewired animals, and compare the arbors with those of retinal axons in normal targets; (3) the physiological properties of visual cells and the visual field map in the MGN; (4) the physiological properties of visual cells in AI, and compare their response features with cells in primary visual cortex (VI); (5) the arbors of single thalamocortical axons that project from the MGN to AI in rewired animals; (6) the mapping of thalamocortical fibers in AI after silencing cortical cells by infusion of muscimol; (7) the spatial distribution of thalamocortical excitation and intracortical inhibition in slices of rewired and normal AI; (8) the intracortical restriction of visual receptive fields in AI by microiontophoresis of bicuculline; and (9) whether rewired ferrets perceive visual stimuli presented to the rewired pathway as visual or as auditory. The experiments follow from, and extend, our work in the previous grant period. The studies have implications not only for mechanisms of normal development but also for mechanisms underlying sparing and recovery of function after early trauma to the brain.

There is increasing evidence that synaptic transmission between retinal ganglion cell axons and neurons in the lateral geniculate nucleus (LGN) is mediated primarily by excitatory amino acids. The goal of this proposal is to examine the role of NMDA and non-NMDA excitatory amino acid receptors on neurons LGN in (a) retinogeniculate transmission through the adult LGN, and (b) the development of retinogeniculate projections. These experiments follow from the hypothesis that NMDA receptors an be used for two distinct tasks in the brain: for regulating the level of postsynaptic activity in normal adult function, and for regulating synaptic connections during development. Specific questions include: (1) Are there NMDA and non-NMDA excitatory amino acid receptors on LGN neurons in adult cats and ferrets? Are these receptors at retinogeniculate synapses or at corticogeniculate synapses? (2) How does blocking these receptor sub-types affect the visual responses of adult LGN neurons? (3) Can NMDA receptors at retinogeniculate synapses, in tandem with cortical inputs on LGN neurons, serve to gate transmission of visual information through the LGN to visual cortex? (4) Are there NMDA and non-NMDA receptors on LGN neurons at early stages of development of the visual pathway? How do these receptors contribute to the excitatory postsynaptic responses of developing LGN cells? (5) How does blockade of NMDA and non-NMDA receptor affect the development of retinogeniculate connections? In particular, what role do these postsynaptic receptors play in two key events in retinogeniculate development: formation of eye- specific layers, and, in ferrets, the formation of on and off sublayers? (6) How are NMDA receptors involved in synaptic potentiation, and hence in the consolidation and elimination of retinogeniculate synapses, during development? The first three questions relate to the adult LGN and will be addressed using (a) extracellular recording and microiontophoresis, and (b) intracellular recording in slices of LGN. The next three relate to the developing LGN and will be addressed using (a) minipump infusion of agonists and antagonists, and (b) intracellular recording in slices of LGN at different ages.

We propose experiments that would examine a fundamental issue in development: whether structure and function in a target structure is determined by intrinsic properties of the target or by its inputs during development. We nave induced, by appropriate surgery in neonatal ferrets, retinal projections into the medial geniculate nucleus (MGN), the principal auditory thalamic nucleus. The MGN with retinal input retains its projections to auditory cortex. We have shown that retinal input to the MGN in "rewired" animals arises from small retinal ganglion cells with slow conduction velocities, that the pathways for visual inputs to the primary auditory cortex (AI) is from the retina via the MGN, that neurons in the MGN and AI have visual receptive fields, and that a map of visual space exists in AI. In the next grant period, we shall examine: (1) the structure of individual retinal ganglion cells that project to the MGN in rewired animals and compare the cells with ganglion cells in the normal retina; (2) the structure of single retinal axon arbors within the MGN in rewired animals, and compare the arbors with those of retinal axons in normal targets; (3) the physiological properties of visual cells and the visual field map in the MGN; (4) the physiological properties of visual cells in AI, and compare their response features with cells in primary visual cortex (VI); (5) the arbors of single thalamocortical axons that project from the MGN to AI in rewired animals; (6) the mapping of thalamocortical fibers in AI after silencing cortical cells by infusion of muscimol; (7) the spatial distribution of thalamocortical excitation and intracortical inhibition in slices of rewired and normal AI; (8) the intracortical restriction of visual receptive fields in AI by microiontophoresis of bicuculline; and (9) whether rewired ferrets perceive visual stimuli presented to the rewired pathway as visual or as auditory. The experiments follow from, and extend, our work in the previous grant period. The studies have implications not only for mechanisms of normal development but also for mechanisms underlying sparing and recovery of function after early trauma to the brain.

DESCRIPTION: Orientation specificity, or the selective response to bars of light of particular orientations, is a fundamental property of neurons in primary visual cortex (VI). The circuitry underlying the generation of orientation specificity and the map of orientation selective cells remains unsolved. New hypotheses, and recent advances in techniques for studying cortex, give promise of significant new insight into the problem. The investigator proposes the hypothesis that short-range intracortical excitatory synapses, which form the majority of synapses on every cell type in cortex, are critical for generating orientation selectivity. Complementarily, long- range inhibition is crucial for the organization of orientation selective cells into an orientation map. Specific questions he proposes to addresses are as follows. (1) Can thalamic imputs alone to VI neurons generate orientation tuning? (2) How does the development of orientation tuning relate to the development of synaptic transmission in cortex? (3) How does the amplitude and reliability of unitary synaptic transmission develop in VI? (4) How do long-range horizontal connections modulate the strength of excitation and inhibition on individual neurons and on neuronal populations in VI? The investigator proposes that the influence of horizontal connections can be facilitatory or suppressive depending on the relative drive between receptive field center and extraclassical surround. (5) How does the orientation map develop in relation to the development of lateral synaptic transmission and long-range, clustered, connections of neurons in the superficial layers. (6) What roles do specific postsynaptic and presynaptic mediators of activity-dependent development, NMDA receptors and nitric oxide (NO), play in the development of orientation tuning, orientation maps and horizontal connections? These experiments would employ intracellular recording from VI neurons using whole cell techniques in vivo and in slices in vitro, optical imaging of the activity of populations of neurons, and intracellular dye labeling of neuronal morphology. Different approaches would often be used in the same animal, so that cellular and integrative development can be directly correlated.

vision; biomedical facility; biomedical equipment development;

The long term objective of the proposed research is to understand how cortical neurons integrate sensory inputs in space and time and hence contribute to sensory perception. The goal of the present application is to elucidate the substrates and mechanisms of rapid, context‑dependent response modulation ("dynamics") in adult rat primary sornatosensory (SI) cortex. We propose to use whole cell recording of intracellular currents in vivo, simultaneous single unit recording from multiple neurons, and optical imaging of intrinsic signals from an expanse of cortex, to characterize the detailed electrophysiological changes that occur in individual SI neurons and in the SI map during stimulation of single whiskers and multiple whiskers. We propose the following specific aims: (1) Examine the spatial spread and temporal pattern of subthreshold input to SI neurons. The sum of the excitatory and inhibitory inputs to a neuron, its subthreshold receptive field, provides the spatial substrate for dynamic integration. We shall use intracellular recording to describe the extent of subthreshold receptive fields as well as the temporal characteristics of single‑whisker responses that are critical for multi-whisker interactions. (2) Exornine cortical integration during temporal modulation -of single whiskers. We have recently used optical imaging techniques to demonstrate a sharpening of the spread of cortical activity with increasing frequency of whisker stimulation. To explicate the changes underlying this paradigmatic case of dynamic integration, we shall record intraceBu1arly from SI neurons during stimulation of individual center and surround whiskers at increasing frequency. (3) Examine cortical integration during multi‑whisker stimulation, particularly as amplitude, frequency and direction of motion of central and surround inputs is varied. We shall carry out parametric studies of multi‑whisker responses, as well as examine specific hypotheses derived from a model of cortical dynamics postulating how interactions between center and surround whiskers sharpen the representation of salient inputs in cortex. Information from these experiments will be important for understanding the integration of multiple inputs in sensory cortex, and more generally for understanding cortical mechanisrns of object detection and discrimination.

DESCRIPTION (provided by applicant): The Department of Brain and Cognitive Sciences at the Massachusetts Institute of Technology requests renewal of their major training grant. The department is organized to promote interdisciplinary training and basic research in neuroscience and behavior, approached with the experimental power of modern cellular and molecular biology and of systems neuroscience and cognitive science, combined with the theoretical strength of computational methods coming from the field of artificial intelligence. Trainees begin laboratory work under one or more advisors through lab rotations in the first term and subsequently joining a laboratory, working on problems in learning and memory, neural development, vision, or motor control. Required coursework can be completed in two to three years, with a two-term sequence of core courses in the first year and a flexible array of graduate lecture courses, seminars, and guided reading. Oral and written reports to the faculty and at professional meetings and in journals mark the students' progress through completion of a thesis, which is usually based on the second major project, begun in the third year. Most students continue in research careers, armed with skills in more than one of the disciplines of cellular and molecular neuroscience, neuroanatomy, neurophysiology, neuropharmacology, neuropsychology, psychophysics, and computational neuroscience. Trainees will, in general, have strong backgrounds in the natural sciences (e.g., undergraduate majors in biology, chemistry, physics, or electrical engineering). Occasional trainees will already hold a graduate degree in another field. Candidates for the graduate program will be chosen for the department Graduate Committee constituted for the propose of overseeing this program and will be evaluated on the basis of interviews, talent for research as demonstrated by past performance, letters of recommendation, grades, and GRE scores.

DESCRIPTION (provided by applicant): The major goal of this project is to discover molecular mechanisms that regulate the connectivity of the visual cortex during development. Surprisingly little is known about the molecular substrates which underlie the formation of the highly specific sets of connections which both characterize and underlie the normal functioning of the visual system. This knowledge is critical not only for understanding visual system development, but to also enable us to genetically target specific populations of neurons to determine their function in visual processing or to deliver therapies following disease or injury to the nervous system. (1) We shall identify molecules that are important in regulating the initial sets of input and output connections of primary visual cortex. To do this, we will utilize high-density DNA microarrays to discover genes that are expressed in visual cortex at very early stages in development in the mouse. It is likely that the genes that regulate the initial connections will be differentially expressed between cortical areas at these times. We will prepare biotinylated cRNA samples from visual, somatosensory and auditory cortex from embryonic and neonatal mice. Data will be analyzed to find genes that are consistently enriched in visual cortex compared to other sensory neocortical regions. (2) We shall examine the spatial and temporal expression of promising candidate genes using in situ hybridization. We will determine which populations of neurons express the candidate genes by combining in situ hybridization with anatomical tracing experiments and immunohistochemistry. (3) We shall identify molecules that regulate the activity dependent refinement of connections which occurs during the critical period. We will therefore prepare samples from the visual cortex of mice in the mid-critical period for ocular dominance plasticity and from mature mice. We will analyze the data to find genes that show up- or down-regulation during the critical period compared to expression levels seen in neonates and mature animals. (4) We shall assess whether promising candidate genes identified in our third aim are regulated by visually-driven activity, by performing in situ hybridization on tissue from visually-deprived animals and comparing the signal strengths. In the longer term, the function of genes which show a spatial and/or temporal distribution pattern which suggests they play a role in the regulation of connectivity will be assessed using in vitro co-culture techniques and anatomical and physiological analysis of transgenic mouse models.

DESCRIPTION (provided by applicant): Understanding how brain pathways form, how the pattern of activity conveyed by them shapes processing networks, and how inputs, pathways and networks together mediate behavior, are central themes in understanding mammalian brain development and plasticity. We propose to examine mechanisms responsible for the specific targeting of projections from the retina to the thalamus, and utilize an induced miss targeting of projections to ask how patterned activity shapes the function of subsequent structures. Retinal projections to visual thalamic targets such as the lateral genicutate nucleus (LGN) require specific molecular cues, and these are altered when retinal projections are routed to the medial geniculate nuclus (MGN) of the auditory thalamus. Such rewiring then provides a means to examine how a very different pattern of activity, that driven by vision rather than by audition, influences the development, organization and function of pathways which normally mediate auditory functions and behaviors. Specific questions are: 1. What are the molecular determinants and mechanisms responsible for generating target specificity in retinothalamic projections? We hypothesize that: retinal projections to specific targets, are mediated by molecules such as the ephrins that also generate topographic order. We shall use wild type mice and mice lacking ephrin A2/A5 or Eph B2/B3 to examine whether retinal projections to the LGN and rewired MGN are similarly disrupted. Additional factors also operate during normal development to generate specificity of axon projections. We will use laser micro-dissection and DNA micro-array analyses to discover genes and signaling molecules that normally regulate containment of retinal ganglion cell axons to the LGN and that promote miss targeting of these axons to the MGN after rewiring. 2. How does the pattern of input activity influence visual feature processing networks in cortex? We hypothesize that a key role for patterned activity is to shape the cortical networks that generate and map multiple stimulus features according to rules of coverage and continuity. We will use optical imaging and single unit recording in ferret primary visual cortex (V1) and rewired primary auditory cortex (A1) to examine the relationships between maps of retinotopy, orientation, ocular dominance, spatial frequency, and direction. 3. How does visual input influence the hierarchical processing of cortical information? We hypothesize that visual activity shapes the serial processing of visual motion in cortex. We will examine the analysis of motion, including direction selectivity, in a hierarchy of areas in the visual cortex and rewired auditory cortex. 4. Can a behavior be specified by its inputs, measured as the influence of vision on fear conditioning? We hypothesize that visual inputs directed to the auditory thalamus instruct the function of subsequent projections and structures. We will use a fear conditioning paradigm, exploiting the slow rate of acquisition of visual compared to auditory cued fear, to examine whether visual inputs routed to the auditory pathway accelerate visual cued fear conditioning in rewired mice.

[unreadable] DESCRIPTION (provided by applicant): Fundamental to understanding how neuronal circuits are created in cortex is defining the mechanisms by which electrical activity is transduced into structural changes in neurons and connections. Primary visual cortex (V1) has been a proving ground for describing the phenomena and mechanisms of activity- dependent plasticity during development. Within V1, ocular dominance plasticity, particularly during an early, well-defined critical period, is a model for understanding functional and structural changes initiated by visual activity. We propose to define the structural correlates of rapid functional plasticity during the critical period; in so doing, we seek to understand the mechanisms that sequentially transduce functional drive into structural changes in dendrites and axon terminals. In particular, spines are sites of the vast majority of excitatory synapses in cortex, and how their structure relates to functional plasticity in the intact cortex remains virtually unknown. We will use the techniques of intrinsic signal optical imaging, high resolution two-photon laser scanning microscopy in vivo and in vitro, and viral expression of exogenous proteins, in ferrets and mice, to examine: (1) the time course of functional changes in the ferret visual cortex during the critical period for ocular dominance plasticity; (2) the structural correlates of rapid functional changes in the ferret visual cortex; (3) structural changes in spines with varying synaptic drive in ferret visual cortex; (4) functional and structural changes in the mouse visual cortex following short- and long-term term visual deprivation, including changes in different layers and specific cell classes; (5) specific molecular mechanisms, including the roles of CaMKII, actin and the extracellular matrix, involved in translating functional changes to structural reorganization in the visual cortex. Together, these experiments will examine in unprecedented detail the extent and time course of structural changes at single synapses in the visual cortex, and reveal mechanisms underlying their dynamic regulation by vision. Such information is critical for explaining pathologies of cortical development, and for suggesting strategies for treatment. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Technical Summary: The precision of vision is thought to be anchored by selective response features of neurons in the visual pathway, by detailed representations of these features, and by precise relationships between the representations. Neurons in primary visual cortex (V1) are selective for multiple features of visual stimuli, for example orientation and spatial frequency, and are arranged into maps with orderly progression of feature preferences. The cellular resolution at which multiple feature maps and their relationships exist is unclear. We propose to investigate the details of map organization at the level of individual neurons in V1, using two photon imaging of calcium indicators. The technique allows an unprecedented combination of spatial resolution and coverage density, which will enable us to describe the stimulus feature representations of all individual neurons in a cortical volume and examine specific proposed relationships between these representations. Furthermore, there is growing evidence that astrocytes, which constitute over half of the cells in the human brain, have unique functions that not only derive from neuronal responses and representations but may also shape them. In addition, astrocytes are suspected to have a key role in the translation of neuronal activity into vascular and metabolic changes, the readout of which is a crucial component of many imaging modalities in neuroscience and medicine. Novel approaches, including specific cellular markers, optical probes of cellular function and structure, viral expression of fluorescent proteins, and genetically engineered mice with optical reporters of activity-dependent genes, provide new ways to examine the cooperative roles of neurons and astrocytes in cortical organization and function. We propose to use these approaches in combination with in vivo two-photon calcium imaging of cells in V1, optical imaging of intrinsic signals, in vivo structural imaging of spines, pharmacological manipulations, and electrophysiological recording, to examine the visual response properties, feature selectivity and feature representation of astrocytes. We will dissect the influence of astrocytes on particular components of signals that enable intrinsic optical imaging (and fMRI), and examine mechanisms by which astrocytes influence neurons, assayed by the development, and adult plasticity, of orientation selectivity. Together, these experiments will provide an extraordinarily detailed view of the representation of visual features in the cortex, and the interactions between neurons and astrocytes that underlie these representations. From a broader perspective, astrocytes are strongly implicated in a number of pathological conditions in the brain, including epilepsy, ischemia/stroke and hepatic encephalopathy. Understanding the function of astrocytes within a well-defined cortical circuit will go a long way towards defining early signatures of malfunction, developing a mechanistic understanding of the role of astrocytes in these pathological states, and suggesting strategies for their treatment. Significance: Astrocytes, which make up over half the cells in the human brain, are known to be integral to the maintenance of neuronal metabolism through their interactions with the vasculature of the brain, and have been linked to several neuropathological conditions. In a major paradigm shift, they have recently been shown to interact cooperatively with neurons. The planned experiments will examine visual responses and representations of neurons and astrocytes in the visual cortex and clarify the role of astrocytes in shaping neuronal circuits, providing a new avenue for the development of therapeutic tools against a variety of diseases including stroke and epilepsy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Distinct cell types in the visual cortex contribute in unique ways to cortical circuits and function. Furthermore, the location of cortical cells within maps enables them to have specific functions. We propose to use state-of-the-art tools to examine the role of specific cell types in visual cortex circuits in vivo. First, we will examine how neurons in ferret visual cortex simultaneously map multiple response features at single cell resolution, using two-photon imaging of calcium signals. We hypothesize that neuronal representations maximize continuity and coverage by creating precise representations of response features that contain spatially offset regions of high and low rates of change. Second, we will examine the response characteristics and mappings of neuronal populations which project to specific cortical targets. We will use retrograde labeling of fluorescent tracers combined with two-photon imaging to compare response features and representations of neurons that project to area PSS, and hence to a putative motion-processing stream, with neurons that project to area 21, and hence to a putative form- processing stream. Third, we will compare the responses of inhibitory neuron subpopulations in ferret V1 recorded with high resolution imaging in vivo and subsequently identified by immunohistochemical markers ex vivo, and examine the hypothesis that specific inhibitory neuron subsets have distinct response features and tuning. We will examine the response features of inhibitory neuron subpopulations in mouse V1 marked by a genetic cre-lox system, and examine the hypothesis that parvalbumin- and calretinin-expressing interneurons have distinct response properties. In addition, we will examine the function of these interneuron types in mice with selective genetic deletion of one subpopulation or the other, in which we predict particular influences on response features of excitatory neurons. Fourth, we will examine whether individual neuronal dendrites show integrative responses, which are summed and thresholded at the soma to impart unique responses to neurons. We will examine response features of individual dendrites and dendritic compartments of single neurons in ferret V1 that have specific projections such as to area PSS and area 21. We will examine their dendritic responses by labeling with either (a) intracellular injection of calcium indicator dye, or (b) a novel genetically engineered CaMKII1 FRET probe, and compare responses to those at the soma recorded by visualized whole-cell patch recording. Together, we expect these studies to contribute significantly to an understanding of how specific cell types in visual cortex contribute to cortical function, and thus how cortical dysfunction might arise from diseases that target a particular type of cell. PUBLIC HEALTH RELEVANCE: Significance Distinct populations of neurons contribute to the development and function of neural circuits for vision; the precise organization of these cell populations and integration of their inputs within cortical area V1 is necessary for the establishment of coherent internal representations of visual stimuli. This project aims to explore the integrative properties of visual cortex on spatial scales ranging from individual dendrites to neural networks, identifying vectors of visual system dysfunction and targets for their intervention.

DESCRIPTION (provided by applicant): The exquisite alignment of projections from the two eyes in central visual structures is fundamental for a precise representation of the visual world. We have discovered that the transmembrane protein Ten_m3 is a critical regulator of this process. Mice that lack Ten_m3 show profound abnormalities in mapping of ipsilateral projections relative to contralateral projections, and marked deficits in visual behavior which are reversed by acute monocular inactivation. This indicates that altered interocular interactions act to suppress vision, and that the upstream cortical circuitry is sufficiently intact to mediate visual behavior in the absence of these interactions. These mice allow the opportunity to not only understand the functional consequences of a binocular mismatch, but to probe the mechanisms which underlie binocular vision itself. We will determine the mechanism by which Ten_m3 regulates axon guidance. The pronounced ipsilateral mistargeting predicts that inputs to V1 will be altered in Ten_m3 null mice. We will use anatomical and functional techniques to determine the distribution of ipsilateral and contralateral inputs to V1. We will test the intriguing prediction that functional ocular dominance columns will form in V1, and assess the role of visual experience in their generation. The data suggest there will be a functional misalignment between the ipsilateral and contralateral inputs to V1. The responses, receptive fields and maps of ipsilateral and contralateral inputs, and their interactions, will be examined using electrophysiological recording, intrinsic signal imaging, and two-photon imaging. We will explore the possibility that altered interocular interactions lead to an exaggerated form of interocular suppression and that V1 is an important locus of this effect. Based on its expression pattern, we propose that Ten_m3 regulates cortical arealization and connectivity and will determine its role in these processes. The intracellular signaling pathways which operate downstream of Ten_m3 are unknown. Our data shows that mice lacking zic4 display the inverse phenotype to Ten_m3 mutants. The nature of the interactions between Ten_m3 and zic4 will be determined and novel components of this signaling pathway identified. These findings will have important implications for understanding how abnormal binocular disparity leads to dysfunction in visual disorders such as strabismus and amblyopia, and for developing strategies for their treatment. PUBLIC HEALTH RELEVANCE: Ten_m3 has been shown to be instrumental in development of the visual system. Improper processing of visual inputs can result in amblyopia, strabismus, or blindness;in addition, deficits in sensory processing have been linked to autism and other disorders of cognitive and social development. The planned experiments promise to elucidate the mechanisms by which Ten_m3 expression affects the development of binocular vision, and thus to provide a novel basis for the design of therapies for visual dysfunction.

DESCRIPTION (provided by applicant): Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder and the leading known genetic cause of autism in girls. RTT is characterized by normal early development followed by cognitive, motor and language regression. Mutations in the X-linked MECP2 (methyl-CpG binding protein 2) gene account for 90% of RTT cases. The neurobiology of MECP2 is fundamental to understanding the mechanisms of RTT and to the identification of therapeutics for the disorder. Mutant mice that lack MeCP2 or express a truncated MeCP2 protein recapitulate many features of RTT. Recent evidence points to the hypothesis that the deficits of RTT arise from a recoverable failure of synaptic and circuit development in the brain, and molecular analyses of cortical development and plasticity point to mechanisms that suggest a novel therapeutic strategy for the disorder. We propose two specific aims. In aim 1, we will use a mouse model of RTT with a germline null mutation of MeCP2, to examine at multiple levels of analysis the hypothesis that a MeCP2 deficit causes synapses and circuits to remain in an immature state. First, we will quantify the brain expression of key synaptic maturation molecules that are downstream of Insulin-like Growth Factor 1 (IGF1) and Brain-derived Neurotrophic Factor (BDNF), that we hypothesize are downregulated in MeCP2 deficient mice. Second, we will use two-photon imaging of neurons and their dendrites across time in vivo to evaluate structural correlates of spine maturation. Third, we will measure functional synapse maturation and circuit plasticity through intracellular electrophysiology in vitro and optical imaging of visual cortex in vivo during experience-dependent plasticity. Fourth, we will assess the organismal physiology of the animals along metrics of maturation in central control systems, including locomotion, heart rate, respiration, and survival rates. Fifth, we will evaluate the mice on behavioral tests that characterize RTT, designed to quantify anxiety, learning and social interaction. Lastly, we will apply microarray and bioinformatics analyses to identify IGF1 related synapse maturation pathways specific to MeCP2. These measurements will provide detailed quantifications of the MeCP2 mutant phenotype and a concrete series of benchmarks for evaluating the effectiveness of the proposed treatment. In aim 2, we will apply recombinant human IGF1 systemically, across ranges of dose and duration, to MeCP2 mutant mice to test the hypothesis that treatment with IGF1 would ameliorate symptoms of the disorder by causing synapses and circuits to rapidly mature. Since IGF1 crosses the blood-brain barrier and is approved by the FDA for pediatric use for other indications, we expect that these hypotheses, if supported, will advance the use of recombinant human IGF1 for treating Rett Syndrome.

The cortex consists of two major cell types: neurons and glia. Most research in cortical function has focused primarily on the role of neurons in signal processing. Glial cells, including astrocytes, have been considered as secondary actors in brain function, providing physical and metabolic support to neurons. In primary visual cortex (V1), precise neuronal responses and representations are considered to anchor visual processing. However, astrocytes contact synapses as well as blood vessels, and recent evidence suggests that astrocytes receive synaptic inputs and influence neuronal as well as vascular responses. The accumulated results of the past decade have led to the ?tripartite synapse? concept, in which excitatory synapses in cortex are composed of a presynaptic, a postsynaptic, and an astrocytic element. An over-arching and novel theme of this proposal is that astrocytes partner with neurons in synaptic transmission and plasticity. Any complete framework for understanding and modeling the network basis of cortical responses must account for both astrocytic and neuronal contributions. The goal of this proposal is to combine experimental and computational modeling approaches to understand the role astrocytes play in the generation, development and plasticity of neuronal responses in visual cortex.

Many aspects of astrocyte biology and physiology have been described in vitro, but little is known about the role of astrocytes in the context of intact functional circuits. This project will utilize novel experimental approaches, including specific cellular markers, optical probes of cellular function, and genetically engineered mice with optical reporters, that provide new ways to examine the cooperative roles of neurons and astrocytes in visual cortex responses and representations. The US laboratory of Mriganka Sur has pioneered the use of these approaches, in combination with in vivo two-photon calcium imaging of cells, optical imaging of intrinsic signals, and electrophysiological recording, to study the influence of astrocytes on visual processing. The modeling portion of this project will develop the first network models of visual cortex to include astrocyte influences on synaptic transmission, in addition to neuronal excitation and inhibition. The German laboratory of Klaus Obermayer has made seminal contributions to a computational understanding of how visual cortex networks generate, develop and alter emergent responses. Previous joint efforts of the Sur and Obermayer groups have been influential in revealing operating regimes of visual cortex networks, the influence of map structure on cortical network function, and the dynamics of feature-selective responses. In each instance, computational models influenced experiments, and vice versa.

This project is jointly funded by Collaborative Research in Computational Neuroscience and the Office of International Science and Engineering. A companion project is being funded by the German Ministry of Education and Research (BMBF).

? DESCRIPTION (provided by applicant): Perceptual decision-making involves multiple cognitive components and diverse brain regions. To perform a perceptual decision, an individual must process an incoming sensory percept, retain this information in short- term memory, and choose an appropriate motor action. Research using delayed-response tasks in nonhuman primates has revealed that sensory and choice information is distributed across a hierarchy of cortical areas, with task-relevant information flowing from sensory to association to motor regions. However, a mechanistic understanding of how circuits in these regions transform and maintain information during such tasks is lacking, due to limited ability to identify and manipulat specific circuits in the primate brain. By developing a memory- guided task for head-fixed mice, we intend to leverage the genetic tractability of the mouse to address these questions. We have developed a perceptual decision task for mice that involves separate sensory, memory, and action epochs. Using large-scale population calcium imaging (Aim 1), we can simultaneously measure the activity of 1000+ neurons during the task, and across multiple brain regions (visual, parietal, and frontal motor cortex). This will allow us to record how neural activity in different cortical areas correlates with different epochs of the task. Our preliminary results indicate a diversity of different response types in each of the three areas studied, including delay-period activity in a large proportion of parietal and motor cortical neurons. These huge and complex data sets require us to employ new statistical methods (Aim 2) to analyze cell-type-specific and region-specific population activity patterns. In collaboration with Emery Brown, we will use state-space approaches to infer how single cells and cortical areas encode information about the task. To investigate the specific circuits and projection pathways underlying the task (Aim 3), we will use retrograde tracers such as rabies virus (in collaboration with Ian Wickersham) to label neurons that project to a particular brain region, or even to a single task-responsive neuron, and measure their functional role during the task. In collaboration with Kwanghun Chung, we will then use CLARITY for multiple-protein immunostaining of the entire brain. These techniques in combination will allow us to link the molecular identity and connectivity profile of each neuron with its functional role in the task. Finally, we plan to test the causal role of these brain regios and circuits using novel ontogenetic tools (Aim 4). Using transgenic mice that express ChR2 in inhibitory neurons, we will transiently inactivate each brain region during specific epochs of the task. This will allow us to determine the necessity and time course of involvement of each brain region. We will lastly manipulate the activity of anatomically-defined and computationally-identified subsets of neurons within each brain region, to determine whether specific subpopulations play a causal role in behavior. By integrating a wide range of cutting-edge experimental and computational tools, and assembling a collaborative team with multidisciplinary expertise, we hope to transform understanding of the neural substrates underlying memory-guided perceptual decisions.

This award is jointly made by two programs: Instrument Development for Biological Research program (IDBR), and Emerging Frontiers (EF), in the Directorate of Biological Sciences (BIO).

Short-term memory is a crucial component of cognitive function and pervades nearly all aspects of our mental lives. Previous research has shown that short-term memory involves multiple cognitive components and diverse brain regions. However, it is not mechanistically understood what regions are involved when, what neuronal subsets are recruited within these regions, or how they interact to represent information relevant to behavior. This proposal aims to elucidate the role of visual, association, and motor cortex in mice performing a visually-cued short-term memory task. This will be accomplished using massive-scale two-photon calcium imaging in behaving mice to measure activity of thousands of neurons simultaneously across these multiple brain regions. Subsequently, optogenetic manipulation of brain regions and of computationally identified neuronal assemblies will be used to determine their causal role in behavior. These technologies and results will have wide impact on understanding neural circuits underlying behavior and cognition. New approaches will be introduced for massive-scale mapping of single neuron activity in relation to a quantifiable behavior. New ways to determine circuit connectivity, and novel combination computational and optogenetic technologies to manipulate critical circuit components, will be introduced. These large data sets will be made widely and freely available, enabling other research groups to avail of these data for novel analyses.

The goal of this proposal is to develop novel tools and provide unprecedented information on neuronal activity patterns and circuits in order to understand the role of multiple cortical areas during short-term memory in mice. Classical electrophysiological recordings are limited to relatively small numbers of neurons with unknown identity. In addition, while microstimulation or pharmacological manipulations can be used to activate or inhibit all the neurons within a local area, it is not possible to selectively excite or inhibit specific neuronal subpopulations that are known to play a role in the behavior. The proposal addresses these issues by developing novel tools to study mice performing a visually-cued memory-guided discrimination task. First, methods for massive scale imaging (up to ten thousand neurons simultaneously) of multiple cortical regions spanning several millimeters in the mouse cortex will be developed. Second, mice will be trained on a visually cued short-term memory task with suitable behavioral richness, including separate sensory, memory and response epochs, so that activity in distributed cortical regions (such as visual, parietal, and frontal motor cortices) can be imaged and the role of individual areas in each epoch can be ascertained. Third, targeted inactivation of specific brain areas will be performed to determine their role in the behavior. Finally, computationally identified neuronal subsets in specific areas will be stimulated in order to determine if they are sufficient for altering behavior. Together, these will be the first studies in the field to link behavior, extremely large-scale multiple-area recordings, and causal manipulations of areas and identified neuronal assemblies. By introducing tools for a radically different approach from previous analyses of memory and memory-guided functions, it is expected that the project will have a significant impact on the field.

? DESCRIPTION (provided by applicant): Rett Syndrome (RTT) is a devastating neurodevelopmental disorder and the leading known genetic cause of autism in girls. Mutations in the X-linked gene MECP2 (methyl-CpG binding protein 2) account for the vast majority of RTT cases. The neurobiology of MECP2 is fundamental to understanding the mechanisms of RTT and identifying therapeutics for the disorder. MeCP2 is an epigenetic modulator of gene expression that has recently been shown to interact significantly with microRNA machinery; these interactions are at the core of MeCP2 mechanisms. Multiple lines of evidence point to a role for MeCP2 in successive stages of brain development, including prenatal neurogenesis, postnatal development of connections and function, and experience-dependent synaptic plasticity. We hypothesize that the pleiotropic effects of MeCP2 are mediated in prenatal development via a set of early regulated miRNAs that influence neurogenesis; during postnatal development through a different set of miRNAs that regulate Insulin-like growth factor 1 (IGF1) signaling; and in late development into adulthood via a third set of miRNAs that influence synaptic function and plasticity. The goal of this proposal is to employ cutting-edge miRNA methodologies, in combination with stem cell, behavioral, two-photon imaging, and targeted electrophysiological approaches, to reveal the function of MeCP2-related miRNAs at different developmental stages. In aim 1, we will examine the role of MeCP2 and downstream miRNA-mediated pathways in prenatal neurogenesis, using isogenic human RTT model cell lines (aim 1a), 3-D cerebral organoids (aim 1b), and mouse models (aim 1c). Our findings to date implicate miR-199 and -214 in the aberrant regulation of prenatal neurogenesis as a result of MeCP2 deficiency; we will analyze the functional mechanisms and molecular pathways downstream of these miRNAs. In aim 2, we will determine the influence of postnatal MeCP2-regulated miRNAs on IGF1 signaling, and their potential role in RTT therapeutics. We will examine the regulation of LIN28a and the let-7 family of miRNAs downstream of BDNF, and their ability to regulate IGF1 expression, in Mecp2 deficient mice (aim 2a). We will investigate whether normalizing the levels of molecular alterations using the ß2 adrenergic receptor agonist clenbuterol can positively impact survival and a range of phenotypes in Mecp2 deficient mice (aim 2b), along with synergistic interactions between clenbuterol and IGF1 as a potent mechanism-based combination therapeutic for RTT (aim 2c). In aim 3, we will examine the role of MeCP2 and late-expressed miRNAs such as miR-132 in regulating experience-dependent cortical plasticity. We will determine whether restoring expression of miR-132 in the visual cortex of Mecp2 mutant mice can restore normal age-dependent maturation of ocular dominance plasticity (aim 3a). We will also examine whether IGF1 (and subsequently, clenbuterol and the combination of clenbuterol and IGF1) upregulates miR-132 expression, and can act through its downstream mechanisms to influence plasticity (aim 3b).

? DESCRIPTION (provided by applicant): Inhibitory interneurons of the visual cortex powerfully shape the responses of their target cells. As our knowledge of cortical inhibitory neuron types has grown, it has become clear that they participate in inhibitory as well as disinhibitory circuit, and are influenced by bottom-up as well as top-down inputs. The individual and collective responses of neurons in successive stages of cortical processing are also shaped by contextual influences, and are crucially modulated by task-dependent behavior. The goal of this proposal is to reveal mechanisms of excitatory-inhibitory interactions and their role in contextual modulation in visual cortex, using a range of approaches that include unique mouse lines, cell-specific targeted recordings, high-density 2-photon calcium imaging of 3-dimensional volumes, novel optogenetic probes and paradigms, specific control of modulatory systems, and behavioral paradigms coupled with dissection of area-specific neuronal responses. We hypothesize that inhibitory circuits can dynamically regulate both the amplitude and the timing of cortical activity depending on behavioral context. To clarify the functional impact of different inhibitory neuron classes on the amplitude of responses in their target pyramidal cells, we will use a novel single-pulse optogenetic probe. Our hypothesis is that temporal coactivation of these neurons and their target cells dynamically dictates their function within intact circuits (Aim 1). Additionally, many inhibitory subtypes express receptors for acetylcholine (ACh), a neuromodulator known to strongly affect visual cortical activity during behavior, leading to changes in the timing of population activity: desynchronization in the local field potential and reduced correlation between neurons, which together improve coding of visual stimuli. We will use targeted in vivo and slice recordings in combination with optogenetic techniques in specific mouse lines to investigate both the effects of ACh on defined interneuron types, as well as the causal role of these cell types in mediating cholinergic effects on cortical processing (Aim 2). Changes in both response amplitude and timing have also been proposed to improve sensory processing during attention-demanding behavior. We will develop a visual discrimination task for mice and use large-scale (>1000 neurons simultaneously) 2-photon calcium imaging to measure neural responses during performance of the task (engaged) or during passive viewing of the same stimuli (passive) (Aim 3). Using specific mouse lines, we will measure responses from identified excitatory and inhibitory populations, from primary visual and posterior parietal cortex as well as from cholinergic axons in the visual cortex. We will investigate cell-specific changes in firing rate, between-neuron correlations, and between-trial reliability as a function of behavioral engagement. Together, these experiments should provide fundamental information on the circuits and pathways by which contextual influences shape cortical processing, and reveal mechanisms relevant to dysfunction in a wide range of brain disorders.

In primary visual cortex (V1), precise spatiotemporal neuronal responses are known to underlie visual processing. Though neuronal roles in visual processing have been well studied, the role of non-neuronal cells, particularly astrocytes, in cortical synapses and circuits remains poorly understood. Cortical astrocytes contact and ensheathe nearly all excitatory synapses, creating discrete functional units consisting of a presynaptic input, a postsynaptic spine and an astrocyte process. A crucial function of astrocytes is the active uptake of glutamate from the synaptic cleft via transporters, particularly GLT1. We propose that astrocytes contribute fundamentally to V1 circuits via GLT1 activity, actively shaping synaptic and neuronal response profiles. Focal Ca2+ transients potentially related to synaptic glutamate uptake have recently been demonstrated within astrocyte processes, and synaptic transmission shown to actively recruit astrocytic GLT1 to sites of synaptic activity. Novel high- resolution imaging techniques, together with cell-specific markers, new optical probes, and genetically engineered mice with specific temporal and spatial control of protein expression, enable us to analyze the crosstalk between astrocyte and neuronal activity at unprecedented resolution in awake mice in vivo. We aim to take advantage of the exquisite organization of visual inputs to V1 neurons to examine the interaction of Ca2+ microdomains, mitochondria and glutamate transporters in astrocyte processes, the functional contribution of astrocyte transporters to neuronal synapses and circuits during visual processing, and the impact of altered glutamate transport on the development and plasticity of V1 circuits. In Aim 1, we will examine astrocyte microdomain Ca2+ responses to visual stimuli, including orientation-specific gratings and complex natural images, their relationship to mitochondria, and how genetic or pharmacological reduction of GLT1 impacts the specificity and reliability of astrocyte and cell-specific neuronal responses. In Aim 2, we will determine the functional relationship between single dendritic spines and adjacent astrocytic processes using simultaneous dual-wavelength imaging of astrocytes and neurons during visual stimulation. We will also determine how GLT1 reduction affects astrocytic process and neuronal spine responses and structures. In Aim 3, we will determine the role of GLT1 in the development and plasticity of astrocyte responses and visual cortex circuits. We will examine how germline reduction of GLT1 alters neuronal and astrocyte microdomain responses during normal development and following monocular deprivation, along with the sharpening of orientation selectivity and the binocular matching of orientation preference. Our overarching goal is to critically examine the hypothesis that astrocytes and their transporters are integral functional partners with neurons in the function and development of cortical circuits. As such, an understanding of normal and abnormal function in a host of neurodevelopmental and neurodegenerative disorders will require incorporating the role of astrocytes.

Astrocytes are a major class of non-neuronal cells in the brain whose crosstalk with neurons at the synaptic and circuit levels remains poorly understood. While in vivo two-photon microscopy has revealed spatiotemporally diverse astrocytic signatures of intracellular Ca2+ transients, the scarcity of tools that manipulate the genetic makeup and physiological activity of astrocytes with spatial and temporal precision in vivo has restricted investigation of their physiological impact on neurons to predominantly correlational studies. Here, we propose developing three novel and mutually independent tools that target three crucial functions of astrocytes: gene expression, intracellular signal transduction, and glutamate uptake. In Aim 1, we will develop a CRISPR/Cas9- based platform to simultaneously knockout multiple genes selectively in astrocytes. Current mouse astrocytic gene ablation studies rely on a small number of Cre-LoxP recombinase transgenic lines, which target only a single gene and often lack temporal and spatial control. We propose creating a novel astrocyte-specific, temporally inducible, CRISPR/Cas9 conditional transgenic mouse model with an innovative viral platform for ablating multiple genes using a single virus Multi-gRNA, Cys4-mediated, Universal Targeting System (MRCUTS). We will apply this system in cultured astrocytes to target the Itpr2 and Adra1a/b genes (aim 1a), validate the tool and compare its efficacy to current Cre-LoxP methods (aim 1b), and probe a new functional role of astrocytes in arousal by using MRCUTS to simultaneously ablate two subtypes of noradrenergic receptors (Adra1a/b) (aim 1c). In Aim 2, we will develop a method for optogenetically activating G-protein signaling cascades in astrocytes. Current methods for modulating astrocyte signaling, such as DREADDs, lack temporal precision. We will develop and characterize the use of optogenetically activated G-protein receptors (opto-XR) in astrocytes to probe astrocyte signal transduction on physiologically-relevant timescales, first in vitro (aim 2a), then in vivo using 2-photon microscopy to measure astrocyte calcium dynamics (aim 2b), and subsequently explore the effects of astrocytic G-protein signal transduction on neuronal physiology using opto-XR in conjunction with astrocyte-neuron dual-calcium imaging (aim 2c). In Aim 3, we will develop an in vivo method for optogenetically disrupting glutamate uptake by astrocytes. Screening for mutations in ChR2, and combining four mutations, results in a light-gated ion channel, ChromeQ that possesses order-of-magnitude reductions in calcium and proton conductance while increasing sodium currents. We will record from astrocytes in acute brain slices to parameterize optogenetically activated sodium currents and determine effects on both astrocyte transporter currents and nearby neurons (aim 3a), examine how disrupting glutamate uptake via chromeQ affects astrocyte calcium dynamics and neuronal response properties in vivo (aim 3b), and explore the effects of ChromeQ on neuronal physiology and motor learning (aim 3c). The tools proposed here will enable a deeper understanding of astrocyte-neuron crosstalk in normal brain function and its disruption in brain disorders.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily affecting girls. In its classical form, RTT is predominantly caused by mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). MeCP2 is a multifunctional regulator of gene expression which regulates transcription through diverse mechanisms such as DNA-binding, interaction with transcription factor complexes, modulation of chromatin structure and regulation of miRNAs ? mechanisms that are engaged pleiotropically through different developmental stages. MeCP2 was considered to act predominantly through late development into adulthood, but recent clinical studies of RTT children point to very early signs of the disorder. The early developmental mechanisms of MeCP2 are poorly understood. We previously used RTT patient iPSCs to show that reduction of MeCP2 leads to overexpression of miRNA-199 and miRNA-214, an increase in neural progenitors, and reduction in neurogenesis and neuronal migration in cortical organoids. We now propose to analyze the migration deficits in detail, and examine the mechanisms underlying the deficits. The objective of this proposal is to develop a novel live-cell imaging platform merging 3D stem cell technologies, microfluidics and multiphoton microscopy, and combine it with state-of-the- art molecular approaches, including mass spectrometry proteomics and single cell RNA sequencing, to examine mechanisms of neuronal migration deficits associated with RTT-causing mutations in MECP2. In Aim 1, we propose to develop label-free third-harmonic generation three-photon microscopy and use it to characterize neuronal migration deficits in RTT organoids compared to isogenic controls. We will additionally develop a microfluidics-based live imaging platform where organoids can be stably imaged and neurons tracked for days. In Aim 2, we will examine the consequence of MECP2 mutations on downstream molecular pathways involved in neuronal differentiation and migration. We will examine mechanisms of anomalous overexpression of AKT in RTT organoids and neural progenitors, and use a proteomic and phospho-proteomic screen to define new proteins and pathways of neuronal migration dysregulated in RTT. We will exploit the transcriptomic profile of single cells to reveal cell types, populations and transcriptomic differences between RTT and control organoids. In Aim 3, we will use the technologies of Aim 1, and results of Aim 2, to examine the role of implicated signaling pathways in neuronal migration. We will interrogate the function of AKT and downstream signaling molecules, and that of new proteins, including modulators of cell adhesion and cytoskeleton organization identified from our screens, that are predicted as involved in migration. We will validate in vivo in mice the ability of specific pathways and focal adhesion proteins to rescue RTT neuronal migration deficits. Together, we expect that these results will advance our understanding of mechanisms involved in deficits of early cortical development in RTT, and suggest potential novel therapeutics targeting these stages.