Bernardo L. Sabatini - US grants

DESCRIPTION (provided by applicant): The striatum is a key brain area that plays a central role in the initiation and execution of voluntary movements. Medium spiny neurons (MSNs) are the most common cell type in the striatum and they give rise to the inhibitory projection that striatum sends to other brain areas involved in motor coordination. Degeneration or perturbed function of MSNs occurs in several human movement disorders including Huntington's and Parkinson's as well as in certain psychiatric illnesses and drug addiction. As in most neurons, stimulus-evoked calcium entry into MSNs regulates many processes including neuronal excitability, circuit connectivity, and gene transcription. Recently, perturbed calcium handling has been identified in patients with Huntington's disease as well as in mouse models of the disease. Interestingly this defect is detectable before the appearance of any motor symptoms and may hint that altered calcium regulation directly leads to the neuronal dysfunction seen in later stages of the disease. Despite the existence of well-established techniques for the study of calcium handling in neurons, the properties of calcium signaling in dendrites of MSNs remain relatively unexplored. The proposed work will use a combination of 2-photon laser scanning microscopy, 2-photon laser uncaging, and whole-cell electrophysiology to analyze how MSNs integrate the activity of many synapses. Our study will examine how nonlinear interactions between the many types of voltage-gated ion channels found in MSNs and synaptic activity dictate both membrane depolarization and calcium signaling. A novel technique will be used to determine if the spatial arrangement of active synapses on the dendrite determines the net effect of synaptic activity on membrane depolarization. Furthermore we will examine whether these nonlinearities play a role in the induction of synaptic plasticity. Lastly, the effect of dopamine on synaptic integration and calcium handling will be examined. Ultimately, we hope to understand how the specialized electrophysiological and morphological properties of MSNs influence striatal function and how perturbation of these processes contributes to human disease.

The regulated production of proteins in neurons in response to extracellular stimuli has emerged as an important and common mechanism for the modification of synapses. In addition, ample evidence now supports that proteins can be made within neuronal subcompartments such as dendrites. However, the spatial scale over which protein translation is independently regulated and whether it is truly controlled locally in dendrites by focal synaptic activity are unknown. Furthermore, the pathways that link synaptic stimuli to the control of protein production are unclear. In many cell types, the regulation of protein translation in response to extracellular stimuli occurs through the mTOR pathway. An important role for the mTOR pathway is neurons is indicated by the association of mutations in the pathway with the human neurological disorders Tuberous Sclerosis Complex (TSC) and Lhermitte-Duclos Disease (LDD) and the finding that pharmacological inhibitors of mTOR interfere with several forms of long-term synaptic plasticity. The research proposed here will examine the regulation of the mTOR pathway by synaptic stimuli and its role controlling dendritic and synaptic growth. Traditional biochemcial approaches will be used to systematically map the sets of stimuli that activate or repress protein translation in neurons. 2-photon uncaging of ; glutamate will be used to focally activate synapses and to probe the spatial scale of regulation of protein translation in dendrites. The perturbation of dendritic spine growth and synaptic plasticity resulting from interfering with the mTOR pathway and from genetic mutations associated with TSC and LDD will be determined. Lastly, electrophysiogical analysis and calcium imaging will be used to uncover functional defects arising from interfering with the mTOR pathway.

DESCRIPTION (provided by applicant): The experience-dependent modification of synapses in the brain is thought to underlie the ability of animals to learn new behaviors and form memories. A great deal is known about the mechanisms that regulate synapses that signal via fast, classical neurotransmitters such as glutamate and GABA. However, the brain also contains a diverse and wide-spread system of synapses that signal through the release of short peptides called neuropeptides. Despite the importance of neuropeptides and neuropeptide receptors in modulating mammalian behavior and their known role in the pathogenesis of human disease, relatively little is known at a cellular level about the physiological consequences of peptidergic signaling. Similarly, neither the stimuli that trigger neuropeptide release nor its modulatory effects on classical synaptic transmission in the mammalian brain are well understood. We propose that neuropeptides rapidly regulate neurons and synapses with high temporal and spatial precision. We will examine this hypothesis using novel tools that allow the precisely-timed and spatially-delimited delivery of neuropeptides within brain tissue. We will use this approach in conjunction with optical and electrophysiological analysis of glutamatergic and GABAergic synapses to determine the modulatory effects of neuropeptides on cellular and synaptic physiology within the mammalian brain. We will initially focus on the actions of Tachykinin and Opioid family peptides and hope to eventually include Vasopressin, Oxytocin, and other neuropeptides. The action of these peptides will be examined in the hippocampus and basal ganglia, brain regions that have established relevance in human disease and behavior and with which the laboratory is familiar. In summary, we will use innovative approaches to study fundamental and relatively unexplored aspects of neurophysiology. These studies will advance our basic understanding of the mammalian brain and begin to lay the groundwork necessary to eventually understand the contribution of perturbed peptidergic signaling to neurological diseases. PUBLIC HEALTH RELEVANCE: Neurons in the mammalian brain use peptides to send signals and modulate brain function. Although perturbations of these "neuropeptide" signaling systems contribute to human neurological and psychiatric diseases, how they regulate neuron function is largely unknown. Here we propose to generate tools to rapidly activate neuropeptide signaling systems and to use these to understand how neuropeptides regulate neurons and synapses in the mammalian brain.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-NS-103: Breakthrough technologies for neuroscience under the ARRA of 2009, NIH Challenge Grants in Health and Science Research (RFA-OD-09- 003) under the National Institute of Neurological Disorders and Strokes (NINDS). Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescent imaging of neuronal structure and function deep within neural tissue. However, due to the near infrared (NIR) photons necessary for deep tissue penetration and excitation in 2PLSM, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here we propose to develop supraresolution 2PLSM fluorescence imaging to analyze small neural structures deep within brain tissue. Deep tissue supraresolution imaging beyond the classical Abbe diffraction limit is accomplished by using NIR lasers for 2-photon excitation and 1-photon stimulation emission depletion (STED). Preliminary data using a prototype microscope demonstrates 3-fold supraresolution imaging to depths of ~100 microns. With further refinements, combined 2PLSM/ STED supraresolution microscopy will achieve ~10 fold improvement in resolution over conventional 2PLSM, allowing nanoscale imaging within live tissue. This improved resolution permits monitoring of fine-scale synapse and neuronal structural plasticity in situ. Given that perturbations of neural structure are common in models of human neuropsychiatric diseases, better methods such as 2PLSM/STED must be developed to evaluate these defects in intact brain tissue. High resolution imaging deep within the brain is difficult due to the poor ability of light to propagate into this tissue. We propose to design and implement a supraresolution microscope that allows imaging within brain tissue at resolution beyond the diffraction limit.

To enable rapid, high-throughput fiuorescence microscopy for the automated analysis of dendrific architecture, cellular morphology, and synaptic connectivity

DESCRIPTION (provided by applicant): Medium and high-throughput assays (i.e., screens) have generally not been applied to mammalian neurons because of the difficulties in culturing them in large numbers and because of the low efficiency with which the genetic makeup of neurons can be altered. Furthermore, because many aspects of neuronal function can only be assayed with electrophysiological assays, follow-up analysis and validation of screening hits is difficult. We propose to use automated imaging approaches to analyze synapse number and neuronal structure in vitro in a scalable format. We have implemented tissue culture and immunostaining approaches to monitor the number and types of synapses formed onto neurons in multi-well plates. We will couple this analysis with lentivirus mediated introduction of short-hairpin RNAs to induce RNA interference against genes expressed in neurons. This will be performed in concert with transcriptional analysis of neurons to determine the key changes in gene expression that correlate with structural and synaptic changes. The proposal represents a significant collaboration between several groups with expertise in functional analysis of neurons, automated analysis of images, viral mediated manipulation of gene expression, and whole-genome transcriptional analysis. We hope that our work will lead, for the first time, to a turn-key and robust method of analysis of neuron and synapse structure suitable for scalable, whole-genome analysis. Such a system will permit the unbiased and systematic analysis of pathways involved in neuropsychiatric diseases including neurodegenerative diseases such as Alzheimer's and Parkinson's as well as neurodevelopmental disorders such as mental retardation and autism. PUBLIC HEALTH RELEVANCE: Massively parallel analysis of cells in many conditions has allowed the discovery of key pathways that control cell function. Unfortunately, these techniques have not been applied to neurons due to difficulties in handling, manipulating, and analyzing large numbers of brain cells. We propose to develop imaging-based techniques to analyze neurons in dishes at a high throughput in order to find pathways that control their development and susceptibility to disease.

Project Abstract The many classes of neurons in the mammalian brain use many different neurotransmitters to communicate. Nevertheless, it has generally been assumed that each neuron uses one principal neurotransmitter. This notion has dominated the analysis of the contributions of synaptic transmission to circuit function and behavior. However, we and others have found that many neurons in the mammalian brain actually release several neurotransmitters at the same time, often targeting each neurotransmitter to a specific and different postsynaptic cell class. We propose that the co-released neurotransmitters act in concert to have consistent and mutually reinforcing effects on their enclosing circuit. Here we propose to study the integration and coordination of peptidergic, GABAergic, and cholinergic signaling and reveal how these diverse signaling molecules act together to dictate the activity and plasticity state of cerebral cortex. The pathways that we have uncovered are potentially powerful means of regulating cortical function and may, in the future, be exploited to restore cognitive function in neurodegenerative disorders.

DESCRIPTION (provided by applicant): The mammalian brain, including the human brain, contains dozens of peptides that act as neurotransmitters between cells but whose function and modes of action are relatively unknown. This is particularly true in the basal ganglia in which the expression of peptides and of peptide receptors varies from cell to cell and nucleus to nucleus. The basal ganglia play a central role in controlling motor action selection - i.e. deciding what we do - and are at the heart of human disorders such as Parkinson's, Huntington's and drug addiction. We propose to study the function of peptides in the basal ganglia in order to understand how they signal and control the circuitry of this structure. We will make use of new technology developed in the laboratory that allows us to precisely release peptides in mammalian brain tissue using pulses of light. This will be used to analyze peptide function in tissue in which specific classes of neurons are marked or placed under optogenetic control. The proposed work will shed light on the basic biology of this conserved signaling system and will inform how its perturbation contributes to disease.

The major goals of this core were to establish a core facility for the high content analysis of neurons and neural networks. The aim was started with high content cellular imaging systems from BD biosciences to capture fluorescence images of cells in 96 and 384 well format as necessary for morphology based shRNA and drug screens. Since then we have significantly expanded the imaging resources with the addition of an Olympus VS120 slide scanning microscope, a Leica laser capture microdissection system, an Olympus Viva View environmental time lapse imaging microscope, and a home built selective plane imaging microscope (SPIM). With these technologies the core has the capabilities to analyze cells and circuits at a wide range of spatiotemporal resolutions in order to reveal their anatomical organization as well as the relationship between cellular content and function. The major goals have not changed but the capabilities have been expanded via the acquisition of the components described above.

? DESCRIPTION (provided by applicant): Behaviors are sequences of actions that are executed in the proper order and correct setting to achieve a goal. Action sequences and their association with the specific environmental contexts in which they are beneficial can be hardwired, as in the case of innate behaviors, or learned and flexible, as in the case of adaptive responses to changing surroundings. The basal ganglia, a complex set of phylogenetically ancient subcortical nuclei, collect sensorimotor information from across the cortical mantle and project via output nuclei to thalamic structures that regulate action; this circuit organization suggests that the basal ganglia may play key roles in modulating ongoing patterns of action. Consistent with this possibility, neurological and psychiatric diseases that disrupt basal ganglia function also disrupt action selection, sequencing and execution. Furthermore, neural correlates have been identified within the basal ganglia that predict, accompany and lag different features of behavior. However, three key questions remain open about the relationship between basal ganglia activity and behavior. First, it is unclear whether the basal ganglia primarily encode behavioral sequences, the action components of behavioral sequences, or both. Second, because of the temporal diversity of task-related activity observed in the basal ganglia, it is not clear whether activity in specific populations of neurons is causal for behavior. Finally, because most research into basal ganglia function involves overtraining in operant tasks, it is not clear what the core principles of action encoding are that govern basal ganglia function during spontaneously generated patterns of behavior like exploration. Here we propose to take advantage of a novel 3D machine vision technology uses Baysean inference to classify spontaneous behavior on fast (e.g. neural) timescales to probe the causal relationships between neural activity in the basal ganglia and action. We will focus our analysis of the main output nucleus of the basal ganglia; the substantia nigra pars reticulate (SNpr). We will first seek to identify predictive neural correlates within the SNpr for action components and behavioral sequences by combining our behavioral analysis methods with dense electrical recordings, both during normal exploration and during the execution of innate approach and avoidance behaviors triggered by odor cues from foods, conspecifics and predators. We will then test the causal relationship between activity in these SPnr neurons and specific features of behavior by using closed- loop optogenetics to subtly alter global patterns of activity within SPnr neurons themselves. This work will shed light on the mechanisms used by the brain to create self-generated patterns of action, and yield important clues about how the links between neural activity and action are altered during disease.

? DESCRIPTION (provided by applicant): The brains of mammals contain an extraordinarily large number of neurons whose activity and interconnections determine the function of circuits that monitor our sensory environment, dictate our motor choices, form memories, and guide all behavior. However we do not understand how the activity of these circuits governs brain activity. A fundamental limitation has been the inability to monitor and control the activity of a significan fraction of brain cells at any one time - thus typical studies of the neural underpinnings of behavior monitor at most ~100 cells simultaneously, or approximately one millionth of the total. In order to gain insight how circuit computations are carried out and subsequently control behavior, we will develop two novel technologies. The first is a radical new class of electrode with 50-100 times more recording sites than is typical and with on-board electronics, allowing unprecedented quality recordings of high number of neurons. The second is a novel way to deliver light into the brain in a controlled manner in order to be able to perturb the activity of neurons with high precision.

Project Summary The release of urine, known as micturition, is a tightly controlled process. Micturition is an important physiological function necessary to maintain water and salt balance, as well as to expel unwanted molecules from the blood. However, in many animals urine is also released in specific places to communicate with other members of the same species and its release is avoided in other places to avoid detection by predators. Descending projections from the ?pontine micturition center? (PMC) to the spinal cord are conserved in mice and humans and trigger urine release. The PMC receives inputs from many higher brain centers and integrates information from these to control micturition. For this reason, in many neuropsychiatric diseases the central control of micturition is compromised resulting in urine incontinence, urine retention, or the volitional release of urine at socially inappropriate times and places. Here we propose to map and study these pathways in mice to understand how circuits that underlie central control of micturition.

Abstract Modern neuroscience relies heavily on tools to visualize the structure, and at times the function, of the nervous system. This includes tools that examine structure at the level of few nanometers (electron microscopy) to those that examine whole human brain (MRI). Between these extremes are approaches to examine cellular sub-compartments and organelles, cellular morphology, small-scale circuit organization, and whole rodent or drosophila brains. Through the Institutional Center Core Grant to Support Neuroscience Research program, we established at Harvard Medical School and Boston Children's Hospital a Center Imaging Core that supports research in our community directed at understanding the structure and function of the nervous system in healthy animals and in models of human disease. The core has been operational for approximately 6 years, and has facilitated or directly made possible research projects throughout our community. The core operates both turnkey systems for daily use, specialized microscopes for chronic, high-content, and high-throughput imaging, as well as custom pipelines for imaging at the highest resolution in tissue and cells. The core has been successful, supporting over 35 NINDS projects across 27 laboratories. For the upcoming grant period, we have expanded the set of microscopes available and have reorganized and simplified the administrative and technical support structure to increase efficiency. Beyond its research mission, the Center will also continue to serve as a nexus for collaborative interactions across neuroscience communities and an educational hub for learning about imaging technologies and approaches. Through the sustained development of this Center, we hope to continue to provide cost-effective and productive services to NINDS researchers while further deepening ties between these two vibrant neuroscience communities.

Project Summary On a moment-by-moment basis each animal must consider its environment, past experience, and present goals to choose future actions. For example, presented with food a hungry animal will likely eat, although not if a predator is near or if the context in which the food appears was previously associated with an adverse output. The process of action selection depends on the proper function of the basal ganglia (BG). Perturbations of BG contribute to many diseases including Parkinson's, Huntington's, Tourette's, and obsessive-compulsive disorders as well as to drug addiction. How activity in the BG mediates action selection and reinforces the repetition of actions associated with positive outcomes is still unknown. Work from a variety of species, indicates that the entopeduncular (EP) and parafascicular (PF) nuclei are important for these processes and contribution to action outcome evaluation and action selection, respectively. Here we propose to carry out anatomical, functional, and behavioral studies in mice to understand the circuitry of these nuclei and how their activity contributes to action selection and evaluation. We make use of new tools and behavioral paradigms to address these unknowns and use anatomical studies to guide our in vivo analyses.

Project abstract Animals, including humans, interact with their environment via self-generated and continuous actions that enable them to explore and subsequently experience the positive and negative consequences of their actions. As a result of their interactions with the environment, animals alter their future behavior, typically in a manner that maximizes positive and minimizes negative outcomes. Furthermore, how an animal interacts with its environment and the actions that it chooses depend on its current environment, its past experience in that environment, as well as its internal state. Thus, the actions taken by an animal are dynamic and evolving, as necessary for behavioral adaptation. It is thought that both the execution of actions, in particular goal-oriented actions, and the modification of future behavior in response to the outcome of actions, depend on evolutionarily old parts of the brain called the basal ganglia. Within the basal ganglia, cells that produce dopamine have a profound influence on behavior, including human behavior, and their activity appears to encode for features of the environment and animal experience that are important for directing goal-oriented behavior. Here we bring together a team of experimental and computational neurobiologists to understand how these dopamine- producing cells modulate behavior and basal ganglia circuitry. We will use unifying theories and models to integrate information acquired over many classes of behavior. Completing the proposed work, including the technical advances and biological discoveries, will provide a platform for future analyses of related circuitry and behaviors in many species, including humans.

Project Summary We propose to acquire a machine that performs serial two-photon tomography of fluorescent cells in whole organs. The machine, a TissueCyte 1000 Imaging Platform manufactured by TissueVision, is an automated system that serially sections and images whole tissues, and will allow users to create anatomical datasets of brains and other organs automatically. The technology has been transformative for neurobiology research as it allows unbiased analysis of fluorescence throughout the entire brain. This enables comparisons of anatomical tracts across experiments, genotypes, and conditions. Furthermore, it greatly improves experimental rigor and systematic discovery as it eliminates the many user-dependent steps typical to other fluorescence imaging modalities. Lastly, it provides data in a format that has been extensively validated, for which automated analysis routines exist, and in which the hosting community has extensive experience. The P30 Neurobiology Imaging Facility at Harvard Medical School/Boston Children's Hospital (HMS/BCH; NINDS P30 NS072030) will house the equipment, provide technical expertise to the entire neuroscience community, and continue to create an opportunity for collaborations across laboratories and institutions. This technology will catalyze research at HMS/BCH focusing on nervous system disorders by facilitating a systematic comparison of three-dimensional anatomical tracts in animal models of nervous disease and interrogate the circuit basis for neurological diseases.

Project abstract The proposal brings together an interdisciplinary team of investigators from Harvard University, Harvard Medical School, Columbia, and UC Berkeley to uncover the function of dopaminergic neurons in controlling action timing, selection, and reinforcement. The Administrative Core will provide support for interactions among the team members as well as between the team, NIH, and external advisory board. In conjunction with the Team Leader and the Internal Advisory Committee, the Administrative Core will also set timelines and milestones, organize real and virtual meetings of the team members, monitor progress, and generate corresponding reports.