Elly Nedivi - US grants

The goal of this proposal is to understand the cellular mechanisms of activity-dependent plasticity through molecular genetic studies of rat visual system development. We have previously cloned a large number of candidate plasticity-related genes (CPGs). Preliminary screening of these CPGs has identified six whose expression is modulated by light-driven neural activity in the visual cortex of the adult rat, and is also regulated during postnatal cortical development. In this proposal two of these CPGs, CPG2 and CPG15, will be used as molecular probes to test the following hypothesis: 1) CPG expression is regulated by physiological activity during rat visual system development. 2) CPGs regulated by physiological stimuli participate in activity- dependent developmental plasticity. To show that CPG2 and CPG15 developmental expression during the critical period for ocular dominance segregation. To further relate activity- dependent expression of CPG2 and CPG15 to critical period plasticity we will study how this expression is effected by monocular deprivation in rats where critical period plasticity is delayed by dark rearing. Based on sequence homologies, CPG2 and CPG15 have predictable cellular and subcellular distributions. To test these predictions, we propose to generate polyclonal antibodies against bacterially expressed CPG2 and CPG15. These antibodies will permit immunolocalization of the endogenous proteins. To test CPG2 and CPG15 participation in cellular events related to plasticity we will apply antisense oligonucleotides to primary neuronal cultures in order to abolish cellular expression of these proteins. As we expect CPG2 and CPG15 to be participating in synaptic restructuring associated with synaptic plasticity, the effects of antisense oligonucleotide application will be assayed by a morphological analysis of changes in dendritic structure and alterations in spine density.

Plasticity, the property of the brain that allows it to constantly adapt to change, is a prominent feature of brain development as well as learning and memory in the adult. Learning and memory is only a specific case of the brain's ability to modify connections in response to altered input. Connections between neurons (synapses) that are used often become stronger, while those that are unstimulated gradually dwindle away. How does activity modify a synapse to make it "strong"? Neuronal activity induces expression of specific plasticity genes whose protein products then bring about molecular changes in the neurons, strengthening their response to a given stimulus. One approach to understanding the cellular mechanisms of activity dependent synaptic plasticity is to identify and characterize participating genes and their proteins' functions.

This POWRE proposal addresses the functional characterization of one plasticity gene that was previously isolated by Dr. Nedivi: cpg15. Virally mediated gene transfer will be used to overexpress cpg15 in the cerebral cortex, and the effect on synaptic strength will be assayed several days later by electrophysiological recording. Manipulating gene expression in vivo and examining the ensuing changes that occur in a functional cortical circuit can help elucidate how a specific gene, like cpg15, may participate in the synaptic events that lead to cortical plasticity. The experiments in this POWRE project will add an electrophysiological dimension to Dr. Nedivi's study of synaptic plasticity, which until now only included molecular biology and imaging technologies. Such a cross-disciplinary approach is absolutely necessary for integrating information obtained by different disciplines of neurobiology into a unified view of the molecular mechanisms underlying brain plasticity.

DESCRIPTION (provided by applicant): Use-dependent selection of optimal connections is a key feature of neural circuit development and in the mature brain underlies functional adaptation of sensory maps as well as learning and memory. Patterned activity guides this circuit refinement through selective stabilization or elimination of specific neuronal branches and synapses. The thalamocortical circuit is central to mammalian brain function, yet the molecular signals that mediate activity-dependent synapse and arbor stabilization and maintenance in this circuit are unknown. cpg15, discovered in a screen for activity-regulated genes, is expressed in the visual system in response to light. Spatio-temporal cpg15 expression coincides with activity-dependent wiring of thalamocortical circuits. The CPG15 protein promotes dendritic and axonal arbor growth, and enhances synaptic maturation. Preliminary analysis of general and cortex-specific cpg15 KO mice shows that although cpg15 is present in both thalamus and cortex, it is thalamic CPG15 that may be critical for maturation of thalamocortical synapses and cortical neuronal arbors. These findings suggest a novel mechanism by which thalamocortical inputs can regulate maturation of cortical synapses through signaling by CPG15. We propose to test the hypothesis that CPG15 is required and sufficient for experience-dependent development of the visual system, specifically in thalamocortical circuits. Aim 1: To test whether cpg15 is required for synapse and dendrite stabilization and experience-dependent plasticity during visual cortex development, and whether the source of CPG15 is thalamic or cortical. Synapse, spine, and dendritic arbor development as well as ocular dominance (OD) plasticity will be assessed during the critical period for OD plasticity in mouse visual cortex. To dissect the role o input- versus target-derived CPG15 signaling in the development of thalamocortical circuits, analyses will be done first in cpg15 KO mice and then in mice with cortex or thalamus-specific deletion of cpg15. Functional synapse development will be assayed by whole-cell patch-clamp recording in visual cortex slices, monitoring spontaneous AMPAR- and NMDAR-mediated mEPSCs and evoked NMDAR/AMPAR EPSC ratios. During recording, cells will be filled with biocytin and later traced to assess for delayed or stunted dendritic arbor growth. Synapse and spine dynamics on pyramidal neurons in visual cortex will be visualized in vivo using newly developed tools for dual color two-photon microscopy, and posthoc immunohistochemistry will discriminate subcortical vs. cortical inputs onto in vivo imaged synapses. Optical intrinsic signal imaging will be used to measure eye-specific responses, as well as OD plasticity in response to MD. Aim 2: To determine whether cpg15 expression from a thalamic vs. cortical source is sufficient to rescue defects in synapse and dendritic arbor stabilization and experience-dependent plasticity. Lentivirus-mediated gene transfer will be used to re-introduce CPG15 into visual cortex or thalamus of cpg15 KO mice in vivo and assess if CPG15 rescues defects in synapse and arbor stabilization and functional maturation, as well as OD plasticity.

DESCRIPTION (provided by applicant): The goal of this proposal is to develop a system for chronic in vivo imaging of neuronal morphology in the intact rodent visual cortex. A system that can be used to directly test predictions regarding the site and extent of structural dynamics that occur in visual cortex on a day to day basis and in response to visual input. By combining this innovative imaging technology with state of the art mouse genetics we can begin to address the molecular basis of cortical structural plasticity. This integrative approach will not only revolutionize our ability to understand this fundamental aspect of brain function, but can identify molecules with therapeutic potential that can promote plasticity in visual cortex and may be used to compensate for insults at lower levels of the visual pathway. To monitor in vivo structural dynamics of neurons in the mammalian visual cortex, we will fabricate a custom designed two-photon microscope for imaging single neurons within a three-dimensional volume of the mouse visual cortex with high fidelity and at short time scales. Multi-photon microscopy allows for vital imaging deep into scattering tissue at sub-cellular resolution with minimal photodamage or phototoxicity. We will use this microscope to image in vivo and reconstruct entire dendritic trees and axon collaterals of EGFP (enhanced green fluorescent protein) expressing neurons in the visual cortex of transgenic mice. These mice express EGFP driven by the thy1 promoter in a random subset of cells sparsely distributed within the superficial cortical layers. The EGFP fills the cells so that they are brightly fluorescent all the way to the tips of their terminals, allowing visualization of their entire structure including filopodia and dendritic spines. We will image these neurons over time in anesthetized animals through a cranial window that allows repeated access to the same cells. To identify molecules that play a role in the structural remodeling that underlies visual cortical plasticity, we will cross mouse lines that are knockouts or transgenic for candidate plasticity genes (CPGs) with the thyl-EGFP transgenic line. This will create mice that are deficient or overexpressing a specific gene and have fluorescent cells in their cortex. Monitoring the structural dynamics of cortical neurons in these mice and comparing them to those of EGFP labeled cortical neurons from wild-type animals, will reveal to what extent the manipulated genes play a role in structural plasticity of the mammalian cortex. Genes could also be introduced into the cortex transiently by virally mediated gene transfer. Some of the first genes tested will be CPGs that we isolated in a screen for activity-regulated genes involved in synaptic plasticity.

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Characterizing the dynamic potential of cortical neurons will provide a baseline for future testing of molecules with therapeutic potential for promoting plasticity in the cerebral cortex. Such molecules may be used to compensate for insults or deterioration at multiple levels of the visual pathway. To investigate the mechanisms underlying structural plasticity in the mammalian brain we utilized a multi- photon microscope system for chronic in vivo imaging of neuronal morphology in the intact rodent cerebral cortex. Using this system we have imaged and reconstructed the dendritic trees of neurons in visual cortex of thy1-GFP transgenic mice. These mice express GFP in a random subset of neurons sparsely distributed within the superficial cortical layers that are optically accessible through surgically implanted cranial windows. We will chronically image neurons in the superficial layers of the neocortex in control thy1-GFP mice, or thy1-GFP mice before, during, and after visual perturbations, to address the following aims: Specific aim 1: To clarify cell type-specific rules that delimit structural plasticity in the adult cortex, we will conduct a survey of structural dynamics in a cross section of neurons that reflects the diversity of neocortical cell types within the superficial layers of visual cortex. A comparative analysis of visual, somatosensory, and pre-frontal cortex will allow us to address whether interneuron remodeling is a general phenomenon. Specific aim 2: To investigate the role of visual experience in structural dynamics of layer 2/3 cortical neurons, we will manipulate visual input using experimental protocols that produce ocular dominance (OD) plasticity in the adult rodent cortex: prolonged monocular lid suture, monocular lid suture preceded by a previous monocular deprivation (MD), or monocular lid suture preceded by dark adaptation. For comparison, we will also apply two additional manipulations, binocular deprivation (BD) and monocular blockade by intraocular TTX injection. By comparing dendritic arbor changes in the cortex of untreated adult thy1-GFP mice with those in mice after a long MD, after a brief MD primed by a previous deprivation or by dark adaptation, after BD, or intraocular TTX injection, we can test the hypothesis that only the forms of activity that produce OD shifts will also enhance structural plasticity in the imaged neurons. Principle component analysis and cluster analysis will be used to quantitatively classify and analyze the morphological and cellular characteristics of imaged neurons. Cluster analysis should provide insight as to whether there are interneuron cell types that are more structurally plastic than others, and whether subsets of interneurons exhibit structural plasticity correlated with OD plasticity. [unreadable] [unreadable] [unreadable]

The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Our previous findings show that while pyramidal neurons in layer 2/3 of adult visual cortex show little, if any, change in branch tip length over time, GABAergic non-pyramidal interneurons display significant dendritic branch tip remodelling driven by visual experience in an input and circuit-specific manner. The fact that structural plasticity of interneurons is continuous through adulthood raises the intriguing possibility that local remodelling of inhibitory connections may underlie adult cortical plasticity. Yet, how experience alters inhibitory circuitry is unclear, and how modifications to inhibitory and excitatory circuits are locally coordinated remains unaddressed. In the previous funding period we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical layer 2/3 pyramidal neurons in vivo during normal and altered visual experience. We found that the rearrangements of inhibitory synapses and dendritic spines are locally clustered, mainly within 10 ¿m of each other, and that this clustering is influenced by experience. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement a newly developed three-color labeling system to independently and simultaneously monitor the formation and disappearance of dendritic spines along with appearance or removal of the post-synaptic density in these spines, and the appearance and removal of inhibitory synapses along the same dendrites. Using spectrally resolved two-photon microscopy we will 1) monitor the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals; 2) monitor the effects of experience- dependent plasticity on coordination of inhibitory and excitatory synapse remodeling; 3) examine the specificity of afferent inputs to coordinated excitatory/inhibitory synaptic pairs. Further, 4) we will develop and implement spectrally resolved multifocal multiphoton microscopy to further enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.

? DESCRIPTION (provided by applicant): The goal of this proposal is to develop new methods for high speed monitoring of sensory-driven synaptic activity across all inputs to single living neurons in the context of the intact cerebral cortex. Although our focus is on understanding how synaptic inputs are integrated across a single neuron embedded in an intact circuit, the next generation random access imaging technology we propose is more broadly applicable for monitoring multi-cellular activity representing large intra-and inter areal neuronal networks. The approach improves on the speed and sensitivity of current random-access technology by nearly 2 orders of magnitude, enabling high- throughput interrogation of up to 104 independent locations within a fraction of a millisecond and compatible with imaging using next generation voltage sensitive indicators. In Aim 1 we propose to generate a comprehensive structural map that will allow random access scanning of all excitatory and inhibitory synapses on functionally defined pyramidal cell types expressing a genetically encoded Ca+2 indicator. The data generated in this Aim will be used to develop image segmentation algorithms to quickly convert structural images of the dendritic tree and the associated synapses into a 3D location map with grid coordinates for sparse sampling of activity patterns at known locations using a fast random access imaging approach described in Aim 2. In Aim 2 we will construct and develop an imaging system allowing high throughput, random addressing within 10-100 ms of approximately 10,000 locations corresponding to all excitatory synapses and other functionally relevant dendritic and somal sites on a single neuron. In Aim 3 we will test and validate the utility of our approach.

? DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the mechanisms of cortical structural plasticity by combining innovative in vivo imaging technology with classical visual manipulations. This integrative approach holds the potential to revolutionize our understanding of adaptive circuit modification, a fundamental aspect of brain function. Our previous findings show that while pyramidal neurons in layer 2/3 (L2/3) of the adult mouse visual cortex show little, if any, change in branch tip length over time, GABAergic non-pyramidal interneurons display significant dendritic branch tip remodelling driven by visual experience in an input and circuit-specific manner. The fact that structural plasticity of interneurons is continuous through adulthood raises the intriguing possibility that local remodelling of inhibitory connections may underlie adult cortical plasticity. Yet, how experience alters inhibitory circuitry is unclear, and how modifications to inhibitory and excitatory circuits are locally coordinated remains unaddressed. Recently, we developed a method for labeling inhibitory synapses in vivo and simultaneously monitored inhibitory synapse and dendritic spine remodeling across the entire dendritic arbor of cortical L2/3 pyramidal neurons in vivo during normal and altered visual experience. We found that the rearrangements of inhibitory synapses and dendritic spines are locally clustered, mainly within 10 µm of each other, the spatial range of local intracellular signaling mechanisms, and that this clustering is influenced by experience. However, previous imaging intervals were typically 4 days. Thus, the nature of the coordinated inhibitory and excitatory synaptic dynamics remained temporally unresolved in terms of whether the two events occur simultaneously or one of the two drives the change, while the other adjusts to it. It is also unclear whether synapses that behave in a coordinated manner are ones driven by specific afferent inputs, and how visual experience increases coordination between excitatory and inhibitory synaptic changes. In this proposal we seek to characterize with high temporal resolution the nature of the coordinated insertion and removal of excitatory synapses and neighboring inhibitory synapses in the neocortical circuit. To this purpose we will implement a newly developed three-color labeling system to independently and simultaneously monitor postsynaptic markers representing the full synaptic complement onto individual L2/3 pyramidal neurons in mouse visual cortex. Using spectrally resolved two-photon microscopy we will 1) monitor the temporal sequence of inhibitory and excitatory synapse remodeling in vivo across the full dendritic arbor of L2/3 pyramidal neurons at short time intervals; 2) monitor the effects f experience-dependent plasticity on coordination of inhibitory and excitatory synapse remodeling; 3) examine the specificity of afferent inputs to coordinated excitatory/inhibitory synaptic pairs. Further, 4) we will develop and implement spectrally resolved multifocal multiphoton microscopy to enhance imaging speed and allow interrogation of synaptic dynamics at even shorter time intervals.

? DESCRIPTION (provided by applicant): Spectrally-resolved imaging is ubiquitous in numerous biological studies ranging from mapping synapse dynamics, to monitoring of intracellular signaling, and studying protein-protein interactions. The ability to independently monitor the lifetime and dynamics of cellular structures, such as the synapse, the nucleus, protein trafficking vesicles, and various other multicomponent complexes is critical to revealing their cellular function as well as their assembly and disassembly. While spectrally resolved visualization of 3-4 different proteins in the same cell is quite routine using confocal microscopy in fixed brain sections or in cell culture, dynamic multi-protein imaging in vivo remains a challenge, yet many intra- and inter- cellular interactions are dependent on the context of an intact tissue. Our goal is to develop and implement spectrally resolved technologies that are compatible with high throughput multiphoton microscopy to allow large volume, in vivo imaging of multicomponent subcellular structures. In the first two aims we propose testing two novel spectrometric approaches for large volume, high-speed imaging, with respective strengths and weaknesses, that can be tailored to tackle different imaging needs. In the third aim, we will develop a highly efficient wavelet based Poisson denoised spectral un-mixing algorithm that can potentially enhance both approaches by allowing accurate analysis of images with far lower SNR. Aim 1: Design a dispersive spectrometer (DS) to enable hyperspectral imaging in a multifocal multiphoton microscope (MMM) system utilizing multianode PMTs (MAPMT). Aim 2: Design a Fourier transform spectrometer (FTS) to enable hyperspectral imaging in MMM and wide-field multiphoton microscopy (WFMM) systems. Aim 3: Develop a morphology-guided, Poisson-denoised maximum likelihood (MLE) spectral decomposition algorithm to reduce SNR requirement of raw images.

Many mutations resulting in neurodevelopmental and neuropsychiatric disorders target synaptic proteins. Synapse remodeling and loss precedes cell death in neurodegenerative diseases, such as Alzheimer's or Parkinson's, and addictive drugs are known to alter synaptic function. The convergence of many brain disorders at the synapse, indicate that its integrity is critical to normal brain function. Monitoring synapse lifetime and assembly/disassembly in vivo has been hampered by the difficulty of discretely labeling and simultaneously tracking the recruitment and assembly of its individual components. Technology for robust, real-time visualization of synapse formation and loss in vivo would enable the exploration of this fundamental feature of brain development and plasticity, and its dysfunction in brain disease. Our goal is to address this need by developing high-resolution, high throughput temporal focusing (TF) two-photon microscopy for large-volume imaging of synapse assembly-disassembly in the living mouse brain. We propose two aims: 1) Design and implement TF two-photon microscopy for imaging an entire neocortical neuron at synaptic resolution in vivo in less than one minute. Imaging small structures in vivo, especially within the context of the full dendritic arbor, imposes significant time demands due to the need for increased sampling and longer dwell times. Currently, imaging an entire neuron at synaptic resolution takes 60-90 minutes. It is impossible to track events on the order of hours or minutes with such long scan times, or to stably image in awake mice. We propose a novel parallelized approach, line scanning TF two-photon microscopy, to enable in vivo imaging with throughput at least two orders of magnitude higher than point scanning, but with comparable resolution and signal-to-noise ratio. We will test the feasibility of this approach for imaging synaptic structural dynamics in real time, in the awake mouse. 2) Incorporate multi-spectral capabilities into a TF imaging system to enable in vivo tracking of multiple synaptic labels across a single neuron. Visualizing multiple discrete subcellular structures in vivo requires methods for efficient spectrally resolved imaging in deep tissue. We have achieved simultaneous three-color imaging with a single focus scanning multiphoton microscope using Ti-Sapphire lasers and an optical parametric amplifier (OPA) as light sources. However, these devices do not provide light pulses with sufficient peak power for highly parallelized imaging. We will extend the capability of the high-throughput line-scan TF imaging system by implementing multi-color excitation using a regenerative amplifier delivering femtosecond pulses at 1040 nm combined with a tunable OPA providing additional pulses in the 650-1600 nm range. This will allow simultaneous excitation of a broad palette of fluorescent proteins, enabling the tracking of multiple synaptic components at once. The technology we propose will provide a new and powerful tool for dissecting the synaptic roots of many disorders that affect formation, stability, and plasticity of excitatory and inhibitory synapses.

Many brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Unfortunately, our ability to monitor synaptic or circuit failures as they occur has been hindered by the difficulty of visualizing synapses in vivo. Here we propose in vivo monitoring of the ?order of operations? in synapse formation and elimination, and identifying the steps and molecules controlling experience-dependent synapse selection. We focus on the visual system, where there is a well-characterized toolkit for manipulating experience. We hypothesize that the dynamics of a synapse's assembly and disassembly, and its propensity to remodel, are intimately linked to its connection identity and proteomic content. To test this, we propose the following aims: Aim1: To track the structural remodeling of inhibitory synapses and how it relates to their afferent input specificity and proteomic content. We will label Somatostatin and Parvalbumin inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to monocular deprivation, and analyze their proteomic content in relation to dynamic history and afferent identity. To this purpose, we will implement triple color two- photon microscopy to simultaneously track, in vivo, both pre- and postsynaptic elements of inhibitory synapses, followed by Magnified Analysis of Proteome (MAP), a combination of tissue clearing and expansion microscopy, for super resolution analysis of synaptic protein content across the entire neuron. Aim 2: To track the structural remodeling of excitatory synapses and how it relates to their afferent input specificity and proteomic content. Using a similar strategy as in Aim 1, we will discriminate general thalamic, LGN, and LP inputs to excitatory synapses across the arbor of L2/3 pyramidal neurons, track their daily dynamics and response to dark adaptation, and analyze their proteomic content in relation to dynamic history and afferent identity. Aim 3: To dissect, at a molecular level, experience-dependent selection and stabilization of excitatory synapses. CPG15/neuritin is an activity-regulated gene product critical for synapse stabilization and maturation. In vivo imaging in WT and CPG15 knockout mice revealed that while spine formation occurs normally in the absence of visual experience or CPG15, in both cases PSD95 recruitment to nascent spines is deficient. CPG15 expression in the absence of activity is sufficient to restore normal PSD95 recruitment and spine stabilization, suggesting it acts as an activity-dependent synapse selector. We ask how CPG15 loss impacts molecular events in synapse formation and maturation. Aim 4: To develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of four distinct genetically encoded fluorophores marking different cellular proteins. We will develop new labeling and two-photon microscope configurations for in vivo monitoring of up to four synaptic components at once, with options for addressing a variety of experimental questions.