Sacha B. Nelson - US grants

Information is passed from cell to cell in the brain by functional contacts called synapses. The rate, pattern and precise timing of those signals represent aspects of coding by the nerve cell that brings information to the synaptic junction, and so is called the pre-synaptic neuron. The transmission to the post-synaptic neuron then determines the pattern of activity of that neuron in turn. The synaptic transmission is not perfect, but has some factor of reliability, so that a sequence of pre-synaptic nerve impulses turnq into some sequence of post-synaptic responses that may not be identical. This project utilizes a preparation of an isolated slice of brain tissue from the visual cortex, as a model system where individual neurons can be visualized under the microscope while electrophysiologically recording their signals. Recordings are made simultaneously from connected pairs of neurons, identified by injected fluorescent tracers. Results will give estimates of the reliability of this important stage of visual cortical processing, and will be important to the current controversy about bursting and oscillating activity in areas of the cortex. These studies will have an impact not only on visual neuroscience, but also on general aspects of synaptic function, computational neuroscience, our understanding of cortical organization and perception, and even on understanding mechanisms of learning.

DESCRIPTION: The responses of neurons in primary visual cortex (V1) diminish with time during repeated visual stimulation. This response adaptation is believed to underlie perceptual effects such as contrast threshold elevation, and tilt and movement aftereffects. Unlike light adaptation, which occurs primarily in the retina, contrast adaptation is known to be cortical in origin, since it is absent from the visual responses of lateral geniculate neurons. Although contrast adaptation has been well characterized functionally, the cellular mechanisms responsible remain unknown. The proposed experiments will test the hypothesis that cortical response adaptation is a direct consequence of the modulation and temporal transfer properties of visual cortical synapses. The initial aim is to use whole cell recording and electrical stimulation in vitro, coupled with recently described computational techniques, to extract the temporal transfer characteristics of visual cortical synapses. Preliminary results indicate that excitatory inputs to V1 pyramidal neurons exhibit use-dependent synaptic depression whose magnitude and time course are sufficient to account for the kinetics and frequency-dependence of response adaptation in vitro.. The second and third aims will examine the relationship between synaptic depression and response adaptation directly, using whole cell and field potential recording in vivo. The key question is whether or not electrically-evoked synaptic depression and visually-evoked response adaptation occlude each other. Additional preliminary results reveal that excitatory synapses in V1 are potently depressed by adenosine, an endogenous neuromodulator which is released in an activity-dependent fashion by cortical neurons. The fourth aim is to determine whether a build up of adenosine contributes to more long lasting adaptation effects by measuring adaptation of extracellular responses during iontophoretic application of adenosine receptor agonists and antagonists. These experiments will address the mechanisms underlying a central feature of how cortical neurons respond to visual stimuli. Since adaptation can readily be measured psychophysically electrophysiologically in human subjects, understanding its cellular mechanism may provide an important link between perception and the physiology of cortical synapses.

DESCRIPTION (Adapted from Applicant's abstract): Visual cortical circuits exhibit experience-dependent plasticity within well defined critical periods. A popular hypothesis is that heightened plasticity during the critical period results from enhanced activation of cortical NMDA receptors. Biophysical properties of postsynaptic NMDA receptors and of presynaptic glutamate release at cortical synapses change dramatically during the critical period. Despite spectacular advances in our understanding of the molecular mechanisms underlying many of these changes, we know remarkably little about their functional consequences for how NMDA currents summate during physiologically relevant patterns of activity. Nevertheless, it is widely believed that a major factor that determines whether or not a given pattern of presynaptic activity will cause long-term synaptic change, and the sign of that change, is the time course and amplitude of the resulting NMDA current in the postsynaptic cell. Here we propose to measure the development of six aspects of excitatory synaptic transmission that are key determinants of the dynamics of NMDA receptor activation during patterned activity: 1) presynaptic short-term synaptic plasticity and the resulting dynamics of AMPA-mediated currents, 2) the ratio of NMDA and AMPA activation at individual cortical synapses, 3) the kinetics of unitary NMDA-mediated currents, 4) voltage-dependent block of NMDA receptors by magnesium ions, 5) saturation of NMDA receptors during repetitive activation, and 6) desensitization/deactivation of NMDA receptors in response to calcium influx. Single and dual whole cell recordings in slices will be used to study the development of glutamatergic transmission mediated by AMPA and NMDA receptors in layers 2/3 and 4 of rat visual cortex. We will then manipulate this development by rearing rats under conditions of monocular and binocular deprivation. An essential goal of our studies will be to develop a comprehensive quantitative description of how these factors interact to alter the dynamics of excitatory transmission during normal and altered visual development.

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this project is to quantitatively understand the rules that govern the induction and expression of long-term plasticity at neocortical synapses. Altering sensory experience, both during development and in the adult, can lead to potent changes in the organization of cortical receptive fields. A common theme of these plasticity experiments is that neurons representing parts of the periphery which are stimulated together become strengthened and linked, while neurons representing parts of the periphery which are lesioned or stimulated in an uncorrelated fashion become weakened and disconnected. Depending on the paradigm, many mechanisms, both within the cortex and more peripherally may contribute, but it is widely assumed that Hebbian plasticity at cortical synapses is a key component of experience-dependent changes in sensory representation. Despite the attractiveness of this idea, it has been difficult to formulate in a rigorous and quantitative way. A quantitative formulation requires that we know not merely that "neurons that fire together wire together," but exactly how much firing at what frequency and with what degree of correlation are required. To that end we will systematically vary the rate and timing with which pre- and postsynaptic neurons fire during paired recording in neocortical slices. Based on these recordings, we will develop a comprehensive and predictive picture of the rules governing the induction and expression of long term plasticity at synapses between thick-tufted layer 5 pyramidal neurons. In addition we will make whole cell recordings from neurons in the somatosensory cortex in vivo and determine whether or not the rules measured in slices also apply to synaptic responses evoked electrically or by stimulating the facial whiskers. The results of this basic research on the link between cortical synaptic plasticity and the reorganization of somatosensory representations may provide clues that will be useful in treating derangements of cortical plasticity during dementia or in promoting recovery of function following brain injury or stroke.

DESCRIPTION (provided by applicant): Despite a hundred years of anatomical work and nearly fifty years of electrophysiology, a full understanding of the neuronal cell types that comprise the visual cortex is lacking. There is widespread disagreement as to how cell types defined on the basis of morphology, axonal projections, cellular physiology, and neurochemistry relate to one another or to functional categories of neurons defined by their response properties in vivo. Despite the difficulty of the problem, progress is crucial if cellular and molecular work done in rodents is to be related to in vivo physiology performed in carnivores and primates, and to clinical observations in humans. Here we propose to combine anatomy, physiology and gene expression analysis to discover sets of markers that can be used to identify cortical cell classes. Subsets of pyramidal neurons that share laminar and sublaminar position, dendritic morphology and axonal targets have been identified on the basis of expression of fluorescent proteins in transgenic mice. Fluorescence activated cell sorting will be used to isolate these neurons and Affymetrix gene chips will be used to measure their patterns of gene expression. Multiple strains of mice will be used in which variant fluorescent proteins are expressed in different subsets of neurons to allow comparisons of gene expression across different cell types. Whole cell recording in slices will be used to characterize the intrinsic electrophysiology of each candidate neuronal cell class. Their morphological properties will be measured from intracellular fills; their synaptic connectivity will be assessed from dual whole cell recording, and their suspected long range projections will be confirmed using retrograde tracers. This work may serve as a foundation for identifying homologous cortical cell types across regions, across species and across development. In addition, the assays of gene expression in identified neuronal types may provide a more selective and sensitive assay of changes in gene expression induced by altered sensory experience or by neurological diseases.

[unreadable] DESCRIPTION (provided by applicant): The objective of this grant is to develop and teach a new course in the neurobiology of disease at Brandeis University. The course will be offered to predoctoral trainees in the Brandeis Neuroscience training program and to other members of the Brandeis neuroscience community. The course will bring together faculty from five Brandeis University departments as well as clinicians and clinician-scientists from Boston area hospitals and medical schools. It will consist of lectures from Brandeis and outside faculty, student discussion of primary biomedical literature, attendance at Grand Rounds Conferences at nearby hospitals, small group meetings with clinical mentors, and small group research projects aimed at elucidating potentially fruitful approaches to key remaining problems in the pathobiology of particular diseases or sets of diseases. These projects and the outside lectures will be made available to the broader community via a course web site. If our training mission is successful, it will contribute to the education of a new generation of basic neuroscientists more comfortable with the facts, concepts and terminology of neurological and psychiatric medicine and allied fields. Such training is likely to better equip tomorrow's researchers to quickly identify key clinically relevant basic scientific problems that require solution for improved diagnosis and treatment of neurological and psychiatric diseases such as schizophrenia, depression, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, mental retardation, autism, and epilepsy. Improved dialogue and communication between basic scientists and clinicians is likely to contribute to more rapidly moving advances at the bench back into the clinic. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A fundamental feature of cortical neurons is the way they transform input current into action potential output. Many cortical neurons adapt: the interval between successive action potentials increases in response to constant current injection. The ion channels mediating this behavior have been sought for more than two decades. We discovered that corticospinal neurons in motor cortex have the opposite behavior: the interval between successive action potentials decreases. The mechanisms of this firing rate acceleration are also not known. We have used microarrays to profile gene expression in distinct populations of cortical neurons. These experiments have identified potassium channels that may underlie adaptation and acceleration. Here we will test the role of these channels in endowing cortical cell types with distinct firing properties. We will determine how firing properties differ across anatomically and genetically defined cell types and how these properties vary across cortical regions. We will also study the emergence of cell type specific firing properties during development, and determine whether or not the development and maintenance of intrinsic firing properties is activity dependent. Significance: Cortical circuits malfunction in epilepsy, stroke and developmental disorders such as mental retardation and autism. Understanding the molecular and physiological properties that distinguish different classes of neurons that make up cortical circuits may illuminate the malfunction of these circuits during disease.

[unreadable] DESCRIPTION (provided by applicant): Rett Syndrome is a devastating developmental disorder due in most cases to mutation of the gene Mecp2. Affected individuals lose or fail to develop many normal language, motor and cognitive abilities. Mutant mice lacking part or all of the gene recapitulate many features of the human disease. However, the precise molecular and cellular interactions by which Mecp2 causes disease remain largely unknown. We found recently that mice lacking normal Mecp2 have reduced cortical activity due to a shift in the balance between excitation and inhibition. We also found that gene expression is altered in cortical neurons, but that different sets of genes are affected in different neuronal cell types. We will determine if this effect of loss of Mecp2 is more general by examining gene expression in several other cell types and will identify which changes occur earliest in development. Rett patients have severe learning disabilities and Mecp2 mutant mice show altered synaptic plasticity. The defects in synaptic plasticity could be primary effects of loss of Mecp2 function, or could be secondary to changes in brain circuitry. In order to decide between these possibilities we will examine long- term potentiation and depression at individual synaptic connections under conditions that minimize the potential impact of other changes in the circuit. We will also examine a homeostatic form of plasticity called synaptic scaling. This is a plasticity mechanism that normally keeps cortical networks stable in the face of changing activity levels. Disruption of scaling could contribute to altered activity levels following loss of Mecp2 function. Finally, we will determine whether or not the changes in gene expression and physiology observed in mice that lack Mecp2 altogether, also occur in heterozygous mice that lack only one copy of Mecp2. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Synapses are specialized sites of cell-cell contact that mediate communication between neurons in the nervous system. It is now widely believed that aberrant development or function of either excitatory or inhibitory synapses contributes to neurological impairments such as mental retardation, autism spectrum disorders and epilepsy. To understand how synapse dysfunction underlies these neurological disorders, it is paramount that we discover how synapses form and function in the non-perturbed state. The NIH-sponsored research of the five investigators in the user group is specifically focused on this research goal: understanding how synaptic connections are formed, modified, and maintained in the nervous system of a variety of experimental organisms. Projects include: mechanisms of synapse formation in the central and peripheral nervous system, neuromodulation, and the regulation of intrinsic excitability. Array tomography is a novel imaging modality that represents a new approach to high resolution imaging of synaptic structure in intact nervous systems. The Zeiss Axio Imager Z2 fluorescence imaging system that we propose to purchase will allow this user group to study synaptic structure and function at an unprecedented resolution using an innovative sectioning and reconstruction strategy devised by Dr. Stephen Smith and colleagues at Stanford University. Traditional immunohistochemsitry using antibodies against synaptic markers on brain tissue sections yields poor resolution of synaptic puncta due to antibody penetration problems in relatively thick tissue sections (e.g. 255m) and limited resolution along the Z axis during imaging, even when employing confocal microscopy. Array tomography circumvents these issues by antibody staining of ultrathin cryosections (e.g. 70nm) of nervous system tissue. There are numerous additional benefits to utilizing array tomography over traditional immunohistochemistry including the possibility of obtaining ultrastructural information from the tissue using scanning electron microscopy after immunofluorescence imaging and, perhaps most importantly for our purposes, the ability to perform multiple rounds of antibody staining of the same tissue section. As the position of the tissue is fixed on the slide, repeated staining with different antibodies against synaptic proteins will allow cataloging of which synaptic components are present or absent at all of the synapses in a single neuron. All five investigators in the user group have reached the limit of what can be achieved in assaying synapse morphology and function with traditional immunofluorescence methods. Thus, it is critical to furthering the research mission of these groups that the array tomography imaging technology be available on the Brandeis campus. PUBLIC HEALTH RELEVANCE: Numerous studies now point to defects in synapse formation as a possible cause for neurological disorders such as autism, mental retardation, and epilepsy. One approach to understanding how aberrant synapse formation contributes to these widespread neurological impairments is to first investigate how synapses are formed, maintained, and function in the non-pathological state using light microscopy. Acquisition of the Zeiss Axioimager Z2 System for Array Tomography will allow for unprecedented high-resolution imaging of synapses from the nervous system of a variety of experimental organisms.

DESCRIPTION (provided by applicant): Manipulating the activity of identified neurons in brain circuits is essential for studying how they are organized and how they produce behavior. Manipulating circuit activity can also be used to treat neurological and psychiatric diseases including Parkinson's Disease, depression and epilepsy. Optogenetic tools have revolutionized our ability to manipulate neuronal activity. We propose to create a set of complementary thermogenetic tools. These have the potential to reliably provide stronger levels of activation and may be activated with less invasive stimulus delivery systems. At present, such tools are used exclusively in flies. We propose to modify these tools to optimize them for use in mammalian systems.

DESCRIPTION (provided by applicant): Genetic manipulation of the mammalian visual system is currently limited by available tools for targeting gene knockout or overexpression to specific neuronal cell types, and is further hampered by an incomplete understanding of the normal gene expression in those cell types. This proposed studies will use a novel genetic strategy, lentiviral enhancer trapping, to generate new driver strains of mice expressing cre recombinase and tet activator in specific cell types. Recently developed cell type specific expression profiling will be used to map gene expression in these and other neurons within the central visual system. The genetic and genomic tools and resources developed will then be used to study the cellular and molecular mechanisms underlying experience dependent plasticity in layer 4 input neurons and layer 6 output neurons of the primary visual cortex. PUBLIC HEALTH RELEVANCE: Many features of the visual system visual system are conserved across mammals. This project will develop genetic tools for identifying and manipulating specific neuronal cell types within the mouse visual system and will map patterns of gene expression within these cell types. The genetic and genomic tools developed will be used to investigate mechanisms of experience dependent plasticity in the visual cortex.

DESCRIPTION (provided by applicant): Neuronal cell types are the building blocks of brain function and differences between cell types are critical to understanding nervous system disease. New genetic and genomic resources are increasingly available for identifying and manipulating specific cell types in the mouse. We will create a unified gateway to these reagents and a platform for integrating and sharing data obtained with them to enhance progress on the difficult problem of identifying the neuronal cell types of the mammalian brain. We will implement a searchable, freely shared open source database of resources and experimental results. We will annotate the anatomical location of targeted cell types within each of ~90 existing driver/reporter strains by aligning individual sections to the Allen Brain Referenc Atlas and will develop new strains using a novel lentiviral enhancer trap strategy in mice. For a subset of cell types we will perform genome-gene expression profiling to enhance insights from this unbiased metric of relationships between neuronal cell types. We will integrate the cell type database and improved atlas viewer with tools for viewing cell type-specific morphology, physiology and gene expression data, and will link these data with other available brain atlas and genomic databases. The ability to freely search and compare cell-type specific phenotypes and gene expression data will enable members of the scientific community to develop specific hypotheses about the molecular bases of specific cellular phenotypes and to improve schemes for classifying neuronal cell types. PUBLIC HEALTH RELEVANCE: Neuronal cell types are the functional building blocks of the brain. We will create a Database of Mammalian Neuronal Cell-types. To enhance the usefulness of this effort we will generate new strains of mice that allow genetic targeting of specific cell types and will profile the gene expression patterns that make cell types different from one another. The great majority of brain diseases are diseases that primarily affect specific cell types, and therefore integrating and improving our understanding of cell types in the mammalian brain may help study of these diseases.

PROJECT SUMMARY (See instructions): Homeostatic regulation of inhibition is a fundamental problem faced by many neural circuits. However, the rules governing homeostasis of inhibitoty neurons may differ from those operating in excitatory neurons, since the inhibitory neurons must he regulated, not only by their own firing, but by the overall level of actirity in the circuit. Altered homeostatic regulation of inhibition has been implicated in brain disorders including schizophrenia, autism and autism spectrum disorders and epilepsy. Furthermore, genes implicated in these disorders have potent actions on specific subtypes of cortical interneurons, but the relationships between these pathways and homeostatic regulation of actirity are incompletely understood. We vrill investigate the role of DNA methylation in regulating the setpoint or operating range of the largest subgroup of cortical interneurons, the parvalbumin positive fast-spiking (FS) neurons in mice. We vrill study the physiological, epigenetic, and transcriptional consequences of deleting enzymes that mediate methylation, selectively in these neurons . We vrill study the interaction between this regulation and regulation by actirity and other signaling pathways known to regulate these neurons including neuregulin 1 and its receptor ErbB4. In a second set of studies we investigate the role of micro RNA pathways in homeostatic plasticity of FS cell excitabUity, first by deleting the bios3nithetic enzyme dicer in FS neurons, and then by using novel profiling methods to identify actirity regulated microRNAs in these neurons. Finally, we ask which neuronal populations' actirity is the critical signal driring various forms of homeostatic regulation of inhibition. Using a combination of optogenetic activation and silencing strategies, we vrill determine which components of homeostatic plasticity are cell autonomous, and which depend on network actirity.

Array tomography is a new approach to high resolution imaging of protein localization in intact tissue devised by Stephen Smith's laboratory at Stanford. This core will provide project scientists with protocols, reagents, scientific advice, technical assistance, equipment and software needed to carry out Array Tomographic analyses of the localization of synaptic and cellular protein complexes. Array Tomography is quickly becoming the imaging technique of choice for probing synapse density and structure using immuno-fluorescence in mammalian brain sections. Major benefits over traditional immunohistochemistry include greater antibody penetration in ultrathin sections, the possibility of obtaining ultrastructural information from the tissue using electron microscopy after immunofluorescence imaging, and perhaps most importantly for our purposes, the hydrophilic LR White resin, coupled with the ultra-thin tissue sections, allows multiple rounds of antibody staining of the same tissue section. Repeated staining with different antibodies will allow much more detailed study of the co-localization and activation state of signaling molecules and complexes within the soma, nucleus, or synapses of individual neurons. Because relationships between these elements can be characterized within a single cell, non-linear relationships that would be lost upon averaging can be captured. Heterogeneity between cells, something which is likely highly biologically relevant, can be identified and quantified. Only array tomography gives us the technical ability to carry out these kinds of analysis. Pattern of Core usage: tissue preparation, embedding and staining will be performed by individual project scientists with advice and training as needed from experienced core users and/or core staff. Thin sectioning will be performed by a trained technician in the core facility, and automated imaging and image processing will be performed by project scientists working with core staff in the facility. This will maximize flexibility in terms of tissue and antibodies used while maximizing cost effectiveness and data quality by allowing project scientists to share a single microscope and ultramicrotome staffed by technical and scientific personnel dedicated to perfecting the histological, optical and computational aspects of the technique. Administrative structure of the Array Tomography Core A is described in the Administrative Core B.

? DESCRIPTION (provided by applicant): Recent genetic advances have driven significant progress in scientists' abilities to classify and map neuronal cell types within the brains of mode organisms like laboratory mice. To better delineate neuronal cell types in the human brain, however, it is critical to have a deeper understanding of the way that neuronal cell types evolve across mammals. As a first step toward achieving this understanding, corresponding neuronal cell types will be directly compared in two closely related mammalian species: mice and rats. By closely examining differences in the properties of these cells, including the genes they express, we hope to identify genomic elements that control the properties of neuronal cell types, and to infer properties of the corresponding cell types in the human brain. Improving the precision with which we can classify human neuronal cell types could have a transformative impact on our ability to understand pathological changes in neuropsychiatric disease.

DESCRIPTION (provided by applicant): Genetic manipulation of the mammalian visual system is currently limited by available tools for targeting gene knockout or overexpression to specific neuronal cell types, and is further hampered by an incomplete understanding of the normal gene expression in those cell types. This proposed studies will use a novel genetic strategy, lentiviral enhancer trapping, to generate new driver strains of mice expressing cre recombinase and tet activator in specific cell types. Recently developed cell type specific expression profiling will be used to map gene expression in these and other neurons within the central visual system. The genetic and genomic tools and resources developed will then be used to study the cellular and molecular mechanisms underlying experience dependent plasticity in layer 4 input neurons and layer 6 output neurons of the primary visual cortex.

Developmental disorders including Autism Spectrum Disorders and Intellectual Disability can lead to reduced cortical activity. Paradoxically, these same disorders also greatly increase the risk for developing seizures. Multiple homeostatic plasticity mechanisms can compensate for reduced activity by increasing excitatory synaptic transmission and cellular excitability, and/or by decreasing inhibitory synaptic transmission. But these normally beneficial mechanisms can have maladaptive effects, especially when reduced activity is prolonged and occurs early, during a critical period of circuit formation. For example, activity blockade in vivo in rat or mouse neocortex, induces seizures, but only if it occurs early and for a prolonged period. Here we explore the mechanisms underlying this Maladaptive Compensatory Plasticity (MCP) in cultured neocortical slices. Activity blockade produces a qualitative change in subsequent synchronized activity that persists following prolonged deprivation when activity is restored. This is accompanied by a dramatic shift in the balance between excitation and inhibition. Physiological and imaging studies are consistent with a dramatic change in synaptic connectivity. Aim 1 will identify the critical physiological features of MCP, that separate it from normal homeostatic plasticity. By blocking activity in single neurons, and by varying the timing and duration of activity blockade, we will distinguish cell autonomous from network effects, and determine which are critical for persistent effects of MCP. Using synapse imaging techniques and paired recording, we ask whether induction of MCP alters the number of functional excitatory and inhibitory synapses. Aims 2 develops the novel idea of push/pull transcriptional regulation of homeostatic plasticity. We identify a pair of closely related transcription factors (TFs) that are potently and progressively upregulated during blockade of activity. Intriguingly, these TFs are part of a pathway that opposes compensatory plasticity, since compensatory responses are exaggerated when they are knocked out. CRISPR-based manipulations will be used to alter TF expression selectively in specific cell types. RNAseq will be used to identify candidate targets, and chromatin assays will distinguish direct and indirect targets. Finally, we will initiate in vivo studies to more directly test the role of homeostatic plasticity and its transcriptional regulation in audiogenic seizures. Together these studies may identify new strategies for mitigating maladaptive consequences of normally beneficial plasticity mechanisms.