Haining Zhong - US grants

DESCRIPTION (Provided by the applicant) Abstract: Great advances in our knowledge are often the result of innovative technological advances. For example, combining light microscopy with Golgi staining led to the birth of modern neuroscience. My proposal aims to provide a major leap in the methodology of neuroscience research that holds the potential of transforming future studies of brain function and dysfunction. Brain function relies on tiny specialized structures at the synapse where protein molecules are arranged with nanometer precision. Small changes in synaptic machinery are viewed as early manifestations of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population ages. However, it has been challenging to examine synaptic protein organization at a sufficiently high level of resolution, especially in brain tissue. Surprisingly, this limitation does not lie in the microscopic methods. Fluorescence super-resolution microscopic methods such as the photoactivated localization microscopy (PALM) that can probe with 10-nm resolution have been developed. However, obstacles associated with protein labeling, sample preparation and data interpretation have prevented their application to brain tissue. Herein, I propose to develop innovative methodologies to label endogenous synaptic proteins and prepare brain samples for PALM. We will also establish a novel approach for visualizing the super-resolution protein organization within a cellular context to aid the interpretation of the data. We will do so by combining innovative developments across multiple disciplines, including molecular biology, biochemistry, genetics, super-resolution fluorescence microscopy, electron microscopy and computer programming. If established, our techniques will revolutionize the methodologies used in neuroscience research by providing unprecedented abilities to obtain fine details of synaptic architecture. These methods will be applicable to the study of other cellular proteins. The ability to identify previously undetectable subtle changes will also enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic and other cellular structures. Public Health Relevance: Subtle changes in protein organization in the brain are an early manifestation of many neurological disorders and neurodegenerative diseases, which are increasingly prevalent as the population continues to age. We aim to develop methods that can visualize these previously-undetectable changes to enable early diagnoses and an enhanced mechanistic understanding of neurological diseases affecting synaptic structures.

DESCRIPTION (provided by applicant): Brain function relies on the coordinated actions of trillions of tiny specialized structures called synapses, which serve as communication gateways between neurons. Small changes in these synaptic machineries are the early manifestation of many neurological disorders and neurodegenerative diseases, which become increasingly prevalent as the population ages. Proper synaptic function relies on the precise, yet dynamic, regulation of the abundance and subcellular localization of synaptic proteins. Monitoring the spatiotemporal organization of these proteins in live neurons under native conditions is an important step toward understanding their function. However, it remains challenging to visualize endogenous synaptic protein organization in neurons in their native habitat - in living brain tissue or in living animals. The majority of studies investigating protein dynamics rely on the overexpression of fluorescently tagged proteins of interest. Unfortunately, overexpression can alter protein stoichiometry, trafficking, subcellular localization, and signaling, ultimately affecing cellular function. Although 'knock-in' strategies can, in principle, be used to label the target molecule with a fluorescent protein and express it at endogenous levels, most knock-in approaches result in the global expression of the labeled protein. Global expression leads to high background fluorescence and a lack of cell-specific contrast in intact tissue, making high-resolution imaging studies of protein dynamics difficult. To address these problems we propose to develop an innovative mouse knock-in strategy called Conditional Labeling by Exon Duplication, or CLED, to fluorescently tag specific proteins and express them at endogenous levels in a sparse subset of neurons. By using an innovative approach that combines the Cre/LoxP site- specific recombination strategy with exon duplication, we will express the tagged proteins under the control of endogenous transcriptional and translational regulation while maintaining cell-specific, Golgi-staining-like high contrast. As a proof of principle, in Specific im 1, we will tag the critical synaptic protein PSD-95 with the yellow fluorescent protein mVenus, and, in Specific Aim 2, we will use the resulting transgenic mouse line to examine functionally significant dynamics of PSD-95 organization. Once established, our proposed strategy will complement current microscopy techniques and sample preparation methods to provide a previously unattainable, highly sensitive, dynamic view of the spatiotemporal organization of specific synaptic proteins in situ and in vivo. The same strategy can be applied to visualize proteins that carry out non-neuronal functions. The acquired knowledge and principles from these novel transgenic mouse lines will facilitate future studies of human brain function and disease.

? DESCRIPTION (provided by applicant): Two primary modes of chemical communication occur between neurons in the brain: fast synaptic transmission, such as that mediated by glutamate and GABA, which directly control the electrical activities of neurons, and slow synaptic transmission, such as that mediated by norepinephrine and dopamine, which regulate subcellular signaling events that cannot be measured directly from neuronal electrical activities. Slow synaptic transmission, which is also called neuromodulation, plays important modulatory roles in regulating excitability, synaptic plasticity and other aspects of neuronal function, and eventually imposes powerful control over the function of fast synaptic transmission. However, unlike fast synaptic transmission, which can be monitored directly via an increasing number of modern approaches such as multi-electrode recording, voltage imaging and calcium imaging methods, much less is known about the precise neuromodulatory events that occur in living animals because there has not been an established method to reliably record the relevant activities triggered by neuromodulation in individual neurons in vivo. To overcome this problem, we propose a novel approach for examining neuromodulatory activities with single-neuron resolution in vivo by imaging the activity of cyclic AMP (cAMP) and protein kinase A (PKA). The cAMP/PKA pathway is a common downstream signal transduction pathway for both dopamine and norepinephrine. Although genetically encoded cAMP/PKA sensors based on Förster resonance energy transfer (FRET) have been used for experiments in vitro, their application in vivo has been difficult due to lower signal-to-noise ratios under the more challenging in vivo imaging conditions. We propose a multipronged approach to eliminate several bottlenecks encountered with current FRET imaging approaches to maximize the signal-to-noise ratio. Our approach includes: 1) developing and improving cAMP/PKA sensors, 2) implementing a FRET imaging modality that is more effective than conventional FRET measures in light-scattering brain tissue, 3) correcting light aberrations associated with in vivo imaging conditions, and 4) developing novel mouse reagents for high-contrast, reproducible FRET imaging. We will validate the utility of this method for monitoring neuromodulatory activities by determining the spatiotemporal patterns of norepinephrine action in anesthetized mice using optogenetic approaches and in behaving mice using different stress stimulations. If successful, our efforts will provide a previously unattainable ability to conduct large- scale monitoring of neuromodulatory activities in the brain at the cellular and circuitry levels. This ability to quantitate neuromodulation will complement the measurements of fast synaptic transmission to enhance our understanding of brain function underlying animal behavior.

PROJECT SUMMARY The functions and connectivity of neurons are established and manifested by their constituent proteins. Monitoring the organization of individual proteins in specific neuronal subtypes in live brain tissue may therefore provide important readout of cellular and circuit properties underlying brain function. However, it remains challenging to visualize endogenous synaptic proteins in individual neurons in live tissue. Most studies rely on the overexpression of fluorescently tagged proteins of interest. However, protein overexpression can alter protein stoichiometry, trafficking, subcellular localization, and cell signaling, ultimately affecting cellular and circuit functions. Although `knock-in' strategies can in principle bypass protein overexpression, these strategies are rarely employed because typical knock-in approaches result in global expression of the labeled protein in all cells where the target protein is normally expressed. Global expression results in high fluorescence background and a lack of contrast in tissues, making high-resolution imaging difficult. To solve the above problems, we recently developed a novel genetic strategy called endogenous labeling via exon duplication (ENABLED). We have used this method to label the critical postsynaptic marker protein PSD-95 with the yellow fluorescent protein mVenus in all neurons, in a sparse subset of neurons, or in specific neuronal subtypes. Unlike the conventional approach to visualizing PSD-95, which involves PSD-95 overexpression, our strategy does not result in altered neuronal functions, and, for the first time, allows for the monitoring of PSD-95 at endogenous levels in individual neurons in living mice. Despite these advantages, the ENABLED strategy can be further optimized to broaden its applicability and enhance its sensitivity. Furthermore, to comprehensively examine neuronal functions and connectivity, multiple types of synaptic markers will need to be labeled. Here, we request funds to optimize the ENABLED strategy and use it to label the excitatory and inhibitory postsynaptic markers, PSD-95 and gephyrin, respectively, with 5-fold stronger signal in mice. We will also generate ENABLED mice in which PSD-95 can be labeled using different colors for dual-color analyses with other proteins. We will make the reagents available to the neuroscience community to provide fellow researchers with an unprecedented ability to monitor synaptic connectivity and plasticity under physiological conditions in brain slices and in behaving animals.

PROJECT SUMMARY The amygdala plays a central role in diverse learned behaviors. By integrating the sensory information with stress, punishment, and reward signals, the circuitry within the amygdala is thought to be modified during learning to mediate specific behavioral outcomes. However, the circuit principles governing what is changed and how different types of learning give rise to qualitatively distinct behaviors remains largely unknown. It has been recognized that an important step towards dissecting the circuitry mechanism underlying amygdala- dependent learning is to determine the activities of individual neurons within discrete amygdala circuits before, during, and after a learning task. However, this goal has been challenging to achieve for technical reasons. First, the amygdala is buried deep within the brain, making it difficult to access by imaging methods, such as calcium imaging, which has become a technique of choice for interrogating neuronal action potential activities with cellular resolution over large neuronal populations. Second, the stress and reward signals are in part encoded as neuromodulatory activities, which do not usually result in direct changes in neuronal electrical activities and cannot be measured by calcium imaging or voltage measurements. Measuring neuromodulation in vivo, especially during behavior, remains challenging. Adding to the difficulty, the identity of individual amygdala circuits, as well as where each circuit receives input and where it sends output, are only partially understood. We plan to meet these challenges by integrating the most recent, complementary technological advances from the three co-PIs. In defined behavioral paradigms we will image calcium as a proxy for neuronal firing in the amygdalae of behaving mice by performing two-photon imaging via a tiny GRIN lens (?~0.5 mm), which offers optical access to deep brain structures with relatively little damage. Simultaneously through the same GRIN lens, we will image the activity dynamics of the cAMP/protein kinase A (PKA) signaling pathway, which is a common downstream signaling pathway for many neuromodulators, including norepinephrine and dopamine, as readout for stress/reward-induced neuromodulatory signals by using two-photon fluorescence lifetime imaging microscopy. In conjunction, we will perform computation-based anatomical circuitry analyses to dissect novel functional subdivisions of the amygdala, and identify the input-output of each subdivision with cell-type specificity. Based on these techniques, we will systematically map circuits, including previously unknown circuits, within the amygdala and determine how neurons from each circuit are recruited by and contribute to the generation of specific behaviors.

PROJECT SUMMARY  A major goal of the BRAIN initiative is to understand neuronal connectivity and plasticity in the context of  animal behavior. The functions and connectivity of neurons are established and manifested by their constituent  proteins. Monitoring the organization of individual proteins in specific neuronal subtypes in behaving animals  may therefore provide an important readout of cellular and circuit properties underlying animal behavior.  However, it remains challenging to visualize endogenous synaptic protein organization in individual neurons in  living animals. Most studies rely on the overexpression of fluorescently tagged proteins of interest. Protein  overexpression can alter protein stoichiometry, trafficking, subcellular localization, and cell signaling, ultimately  affecting cellular and circuit functions. Although ?knock-­in? strategies can in principle bypass problems  associated with protein overexpression, they result in global expression of the labeled protein, leading to high  fluorescence background and a lack of cell-­specific contrast. Other alternative labeling methods for visualizing  endogenous proteins, such as the intracellular expression of fluorescently tagged intrabodies and CRISPR-­ mediated gene editing, also have their own limitations, including potential off-­target effects.   To solve the above problems, we recently developed a novel genetic strategy called endogenous labeling via  exon duplication (ENABLED). We have used this method to label the critical postsynaptic marker protein PSD-­ 95 with the yellow fluorescent protein mVenus in all neurons, in a sparse subset of neurons, or in specific  neuronal subtypes. Unlike the conventional approach to visualizing PSD-­95 via overexpression, our strategy  does not result in altered neuronal functions, and, for the first time, allows for the monitoring of PSD-­95 at  endogenous levels in individual neurons in living mice. Despite these advantages, the ENABLED strategy can  be further optimized to broaden its applicability and to enhance its sensitivity. Furthermore, to comprehensively  examine neuronal functions and connectivity, additional synaptic proteins will need to be labeled at both the  presynaptic and postsynaptic sides. Here, we request funds to optimize the ENABLED strategy and use it to  label 12 additional critical synaptic proteins in mice. We will also generate ENABLED mice in which the  synaptic proteins can be labeled using different colors for simultaneous imaging. The reagents we generate will  be made available to the neuroscience community to provide researchers with an unprecedented ability to  monitor synaptic connectivity and plasticity under physiological conditions in behaving animals.