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): 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 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.