Tianyi Mao - US grants

DESCRIPTION (provided by applicant): The basal ganglia play essential roles in behavior, including movement control and learning. The striatum is the primary input station of the basal ganglia where it processes and sorts information from the cortical areas and the thalamus into downstream pathways. Defects in striatal function are responsible for the cognitive and behavioral deficits observed in neuropsychiatric disorders, including Parkinson's disease, obsessive-compulsive disorder, and drug addiction. Great strides have been made toward understanding striatal function at two levels. First, at the behavioral level, much has been learned about the crucial roles of the striatum in action selection and execution. Second, at the single cell level, the molecular and physiological properties of individual striatal neurons as wel as their overall roles in behaviors have been examined extensively. However, our understanding of the circuit mechanisms that bridge striatal behavioral functions and the cellular properties of individual striatal neurons remains in its infancy. The neuronal circuitry in other brain regions i often organized around functional subdivisions (e.g., cortical columns) and cell types (e.g., layer 5A and 5B cortical pyramidal neurons). Although the striatum has been grossly divided into three divisions according to their functions and it is known to consist of at least five major neuronal subtypes, its functional subdivision-dependent and cell-type- specific microcircuits are not fully understood. Herein, we propose to fill this gap by examining the striatal subdivision-dependent and cell-type-specific microcircuits in mice, a genetically tractable system required for unambiguously defining cell types. We will do so by investigating the organization of the thalamostriatal projections, which consist of one of the two major excitatory inputs to the striatum, at both anatomical and functional levels. We will use an innovative combination of anatomical tracing, imaging, genetic, optogenetic, and physiological approaches. We expect that our study will provide a complete functional thalamostriatal wiring diagram and uncover the principles behind how information from the thalamus is segregated into the downstream cell-type-specific and functional subdivision-specific circuits. Our acquired knowledge will synergize with current knowledge regarding the striatum at the levels of behavior and single-neuron properties to advance our understanding of how the striatum functions and how the thalamus contributes to the function of the basal ganglia as a source of upstream input. This knowledge will also pave the way for future studies of striatal function and pathology by providing a benchmark for circuit connectivity under normal conditions.

? 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.

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. 

PROJECT ABSTRACT With the ongoing opioid crisis, there is a tremendous need for an in-depth understanding of opioid actions and the underlying mechanisms at the cellular and circuit levels. The striatum integrates excitatory inputs from the interconnected cortex and thalamus to form a triangular circuit that mediates critical brain functions, including motor control, affective pain, decision-making, and reward. Opioids impose strong modulation of this circuit, but their specific actions, such as ?where? and ?how? they act, are not fully understood. The overarching goal of our proposal is to comprehensively elucidate how individual elements in the thalamo-cortico-striatal triangular circuit are modulated by distinct opioid receptor agonists and how these modulations alter the function of the circuit. The thalamo-cortico-striatal circuit is organized based on specific subregions within the cortex, thalamus, and striatum. During the previous funding period, we established the first comprehensive thalamo-cortico-striatal circuit wiring diagram, which allowed us to identify and delineate subregion- specific connectivity. In our preliminary studies, we have identified the exact convergent sites of the anterior cingulate cortex (ACC) and the mediodorsal (MD) thalamus, both of which play critical roles in affective pain and reward, in the dorsomedial striatum (DMS). This MD-ACC-DMS circuit presumably drives pain and reward-associated executive functions. Different subtypes of opioid receptors are expressed in all three of these brain regions, making this circuit a likely substrate for opioids. However, the precise actions of agonists in the context of specific opioid receptor types, cell types, and brain subregions are poorly characterized in this circuit. In the current proposal, we will use cutting-edge tools to dissect subregion-specific, cell type-specific, opioid receptor type-specific, and synapse-specific modulation of the synapses in the MD-ACC-DMS circuit. Specifically, we will take advantage of our unique research strengths, including the novel connectomic information we acquired during the previous funding period, our novel imaging capability for directly visualizing subcellular cAMP/PKA signaling downstream of opioid receptors in living tissue, and our establishment of novel brain slice preparations for monitoring opioid modulation of multi-synaptic information propagation. Using these approaches, we will identify the action sites (Aim 1), the underlying intracellular signaling mechanisms (Aim 2), and the functional impacts (Aim 3) of distinct activated opioid receptors. Our proposed experiments will result in an in-depth, mechanistic understanding of the actions of opioid receptors in the MD-ACC-DMS circuit that may facilitate the development of strategies to more effectively address the role of opioids in analgesia and addiction.