Karen Zito - US grants

One of the most remarkable events in development of the nervous system is the construction of the diverse and complicated array of neural circuits that underlie behavior, from the simple circuit of a motor reflex to the complex circuits that underlie learning and memory. The research objective of this project is to understand how neural circuits are initially established during development and how they are modified by sensory experiences. Specifically, the goal of this project is to identify the patterns of neural activity that act to stabilize newly formed circuit connections in the mammalian brain. Advanced imaging techniques and electrophysiology will be used to stimulate individual synaptic connections and to monitor their strength and stability, in order to identify those activity patterns that lead to stabilization of neural circuits. Neural activity patterns that lead to increase in the strength of individual synaptic connections are expected also to lead to stabilization of circuit connections. Results from these experiments will advance our understanding of the mechanisms by which neural circuits are established and stabilized during development of the nervous system. In addition, this research project will provide training opportunities for both undergraduate and graduate students in the laboratory. The educational objective of this project is to develop an outreach program at UC Davis in order to advance public awareness about the progress and benefits of brain research and to encourage grade school students to choose scientific career paths. This program will serve to increase participation in outreach activities from a large and diverse group of undergraduate and graduate students with the goal that these students will be encouraged to continue outreach activities throughout their scientific careers.

Abstract The growth and retraction of dendritic spines with synapse formation and elimination is thought to underlie experience-dependent changes in brain circuitry during development and in the adult brain, and also may play a role in neurodevelopmental disorders. While much focus has been given to spine growth and associated synapse formation as a key step in the development of brain circuits, the mechanisms of spine retraction and synapse disassembly are ill-defined. During late postnatal development, after initial connectivity has been established, dendritic spine densities decrease and half of all synapses are lost in some regions of the cortex. This period coincides with a period of intense learning, suggesting that spine and synapse elimination may have an integral role in learning and memory. Indeed, many neurological disorders that result in mental retardation have been associated with spine loss and spine morphology changes. The primary goal of the proposed studies is to determine the mechanisms that govern the retraction of dendritic spines and the disassembly of synapses during brain development, plasticity, and disease. We have recently shown that spine shrinkage is initiated by both input-specific and locally competitive activity patterns that lead to synaptic weakening and that spine retraction is tightly coupled to disassembly of the postsynaptic density. We currently have three aims. First, we will determine the molecular mechanisms that drive input-specific spine shrinkage and retraction and synaptic weakening. Second, we will define the cellular and molecular signaling mechanisms by which competition between neighboring synapses drives spine shrinkage and retraction, and what role these competitive mechanisms play during circuit plasticity in vivo. Finally, we will determine how synaptic interactions and molecular composition are regulated during input-specific and heterosynaptic spine shrinkage and retraction. To achieve these goals, we will use focal photolysis of caged glutamate to stimulate individual spines, combined with electrophysiology to measure spine synapse function, calcium and fluorescence lifetime imaging to measure real-time signaling in dendritic microdomains, and electron microscopy to monitor pre- and postsynaptic ultrastructural changes and molecular alterations during spine retraction. The combined use of advanced two-photon imaging techniques, electrophysiology, and electron microscopy at single synapses will provide an innovative and powerful way to identify the mechanisms that govern the retraction and disassembly of spine synapses. Results from our experiments will rigorously address the mechanisms of spine retraction and synapse disassembly, thereby filling major gaps in our current understanding of neural circuit refinement during development and experience-dependent plasticity. Ultimately, basic knowledge of the mechanisms of spine elimination has strong potential to facilitate the development of therapeutics for neurological diseases.

One of the primary challenges in neuroscience is to understand how sensory experiences drive the changes in brain circuits that underlie learning. Notably, the current lack of adequate tools to monitor the functional properties of individual synaptic connections has been holding back efforts to define how complex neuronal firing patterns drive changes in neuronal connectivity in the brain. This EAGER proposal is focused on the development and application of radically novel fluorescent probes to visualize integrated neural activity at individual synapses. If successful, these innovative probes will be transformative for the field; broad application of these probes by the neuroscience community will enable discovery of the rules that link sensory-driven neural activity to the structural changes in synaptic connectivity underlying learning. This knowledge would revolutionize current understanding of the dynamic changes in brain structure and function during learning. Furthermore, this project will foster close collaboration between groups of investigators with complementary expertise, and thus will create a vibrant environment for interdisciplinary training of the next generation of scientists.

This EAGER proposal addresses one of the major unsolved problems in neuroscience: how complex patterns of neural activity at multiple synapses interact to drive experience-dependent changes in circuit connectivity. The specific goal is to develop and apply novel fluorescent probes for visualizing the history of neural activity at individual synapses. These innovative probes will facilitate mapping the function and structure of the neural circuitry underlying a specific physiological process or behavioral task. To accomplish this goal, a multidisciplinary approach will be used that incorporates genetic strategies, computation-guided protein design, two-photon imaging, and electrophysiology. First, candidate sensors will be generated through a high-throughput, multi-step sensor screening process. Next, the sensitivity and kinetics of the sensors will be characterized in neurons in vitro, ex vivo, and in vivo. Finally, the sensors will be implemented in brain slices to probe the activity-dependent mechanisms that drive the formation and stabilization of synaptic connections. Ultimately, these sensors will be used to define the activity-dependent mechanisms that drive circuit changes in vivo during complex behavioral tasks. The proposed research would provide much needed imaging tools of synaptic activity that are compatible with a variety of advanced imaging techniques, such as wide-field, confocal and two-photon microscopy, and would dramatically enhance understanding of how the history of neural activity at individual synapses and their neighbors can influence long-term stability of neural circuit connections.

One of the major challenges in neuroscience is to understand the experience-dependent mechanisms that drive the changes in neural circuits that underlie complex behaviors, and how these mechanisms are altered in disease. Altered synaptic transmission has been implicated in a number of human neurological and psychiatric disorders, including epilepsy, schizophrenia, autism and addiction. Considerable recent evidence from our labs and others has demonstrated that specific patterns of neural activity at individual synapses can drive the growth, stabilization and elimination of synaptic connections. However, how complex patterns of neural activity at multiple synapses in vivo interact to drive changes in circuit connectivity remains poorly defined. Specifically, the relative role of neural activity at clustered versus distributed synaptic inputs, and that of integrated versus patterned neural activity, in driving synaptic and circuit plasticity has been difficult to determine, primarily due to the lack of adequate imaging probes to monitor the history of activity at individual synapses. New tools are needed. The overall objective of this research proposal is to develop novel glutamate sensors for large-scale monitoring of the activity of individual synapses in the living behaving animal. We will focus on two specific objectives: (1) developing glutamate integrators for visualizing the history of neural activity at individual synapses in large fields of view during short behavioral epochs and (2) developing glutamate highlighters with slower kinetics that will enable large-scale monitoring of transient responses at individual synapses in the living animal. Existing tools for monitoring glutamatergic signaling at individual synapses, such as the genetically- encoded glutamate sensor, iGluSnFR, are excellent for characterizing the activity of individual synapses in small fields of view with high temporal precision; however, due to the fast kinetics and transient nature of the glutamatergic responses at individual synapses, these sensors are not suitable for real-time monitoring of synapses in large fields of view. Here, we propose to develop glutamate sensors that permit monitoring activity at individual synapses over larger fields of view and also with the spatial and temporal resolution to mark activated synapses amongst the distributed circuitry. Indeed, our proposed glutamate integrators will transform the activity of transient synaptic inputs into permanent labels of active synapses, enabling access to information mapping neural activity to the structure of the neural circuitry underlying a specific physiological process or behavioral task. To accomplish these goals, we will use a multidisciplinary approach incorporating genetic strategies, computation-guided protein design, two-photon imaging, glutamate uncaging and electrophysiology. First, we will use high-throughput, multi- step sensor screening, photophysical characterization, and ligand-binding specificity measurements to generate candidate sensors. Next, we will generate synaptically targeted sensors, and characterize the sensitivity and kinetics of lead sensors in dissociated neuronal culture and in brain slices. Finally, we will characterize the expression, sensitivity and kinetics of lead sensors in vivo in zebrafish and in mice. If successful, the proposed research will provide much needed imaging tools of synaptic activity that are compatible with a variety of advanced imaging techniques, such as wide-field, confocal and two-photon microscopy, and would dramatically enhance our understanding of how the history of neural activity at individual synapses and their neighbors can influence long-term stability of neural circuit connections.