Matthew R. Banghart, Ph.D. - US grants

Project Summary Neuromodulators play an important role in our life by dynamically adapting the brain to an ever-changing environment. By simultaneously activating multiple receptor classes that are distributed amongst distinct components of neural circuits, neuromodulators can effectively rewire the brain in a reversible manner. However, the involvement of specific neuromodulators in distinct cognitive processes has been difficult to establish. Neuropeptides, in particular, are a unique and mysterious class of neuromodulator that are released from neurons that also release classic neurotransmitters such as glutamate or GABA. The behavioral contexts and neural activity patterns that drive neuropeptide release have been difficult to establish experimentally due to a lack of spatiotemporally precise tools for monitoring neuropeptide levels in the brain. Our current understanding is based largely on studies that rely on microdialysis for detection and pharmacology to interfere with or mimic neuropeptide signaling in behaving animals, leaving the following fundamental questions unanswered: When and where are neuropeptides released in the brain during distinct behaviors? Once released, how far do they spread from release sites, and how long do they remain to influence neural activity? To address these and related questions, we propose to engineer Nanobody-Evolved Optical Neuropeptide Sensors (NEONS) that will report the presence of neuropeptide in the extracellular space. NEONS will readily interface with contemporary imaging technology to provide highly sensitive, spatially and temporally precise monitoring of neuropeptide release in the brain. Our approach relies on the ability to evolve nanobodies in the laboratory that bind to neuropeptides with high affinity. Uniquely, our strategy is to engineer two nanobodies that form a ternary complex in the presence of neuropeptide and to translate complex formation into an optical signal using fluorescent proteins. In Specific Aim 1 we will implement a novel, powerful method for directed evolution of nanobodies developed in the Chang Laboratory called GAIN recombination. In Specific Aim 2 we will engineer these dual-binding affinity elements into optical sensors by fusing them with fluorescent proteins that function as FRET pairs and by tethering the nanobodies to circularly permutated GFP. The resulting NEONS will be optimized and characterized in brain tissue in Specific Aim 3 using fluorescence microscopy. NEONS promise to enable large-scale, potentially whole brain imaging of neuropeptidergic transmission in behaving animals. Such unbiased experiments will reveal otherwise invisible roles for neuropeptides in specific brain regions and will motivate further studies that dissect the mechanisms by neuropeptides transform circuit function to regulate behavior.

DESCRIPTION (provided by applicant): The striatum integrates limbic and sensorimotor information to drive goal-directed behaviors. Opioid peptides and their receptors are abundant in the striatum, but a clear role for endogenous opioid signaling in action selection has not been established. This proposal aims to define key components of the striatal circuitry that process limbic information in mice and to understand how opioid peptides alter these circuits in the context of reward-guided behavior. A systematic anatomical and functional analysis of limbic projections to opioid-rich striatal microcircuits will be conducted using genetic tracing methods and ontogenetic tools in brain slices. New photochemical reagents will be employed in combination with ontogenetic to identify opioid-sensitive circuit components and determine how intercompartmental volume transmission shapes the spatiotemporal dynamics of opioid signaling. Genetically-targeted calcium imaging experiments will reveal how these actions translate into changes in network function. To provide evidence for endogenous opioid release in the context of goal-directed behaviors, activity patterns observed during reward-guided tasks will be driven in brain slices ontogenetically while electrophysiological monitoring opioid signaling in brain slices. To identify a functional role, opioid receptors will be blocked during reward-guided behavioral assays using a new photoactivatable antagonist that enables transient inhibition within discrete striatal sub-regions in vivo. To detect the release and spread of endogenous opioids with high sensitivity good spatiotemporal resolution, optical sensors will be developed by chemically modifying receptors so that they report the presence of endogenous agonists. A chemical- genetic strategy for cell-specific pharmacology will be developed to reveal which cellular targets of opioid signaling underlie behavioral responses to opioid peptides and opiate drugs. By targeting native receptors in genetically-identified cells, ths tool provides a powerful means to study opioidergic pathways in the brain. Collectively, these studies will uncover mechanisms by which opioids modulate a neural circuit involved in goal-oriented behavior. By quantifying signaling dynamics in new ways, the role of volume transmission will emerge. Although these novel techniques focus on opioids, the underlying principles are general, and should be applicable to signaling molecules beyond the nervous system. The anticipated findings should facilitate our understanding of disorders in which goal-directed actions are compromised such as Parkinson's and Huntington's diseases as well as attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), and addiction to substances of abuse.

The secretion of growth factors, peptide hormones, neuropeptides and biogenic amines from dense-core vesicles (DCVs) in neurons and endocrine cells is a tightly-regulated event that drives physiological processes such as feeding, digestion, energy storage, lactation, emotion and analgesia. Compromised DCV release is implicated in metabolic and neurological disorders such as diabetes, eating disorders, depression, drug addiction, and Huntington?s disease. Yet the molecular pathways that govern the release of DCVs, particularly in electrically excitable cells of the nervous and endocrine systems, remain largely undefined. The objective of this proposal is to uncover molecular mechanisms that regulate DCV secretion. Our central hypothesis is that the signaling pathways that govern DCV release vary between different classes of cells, and between different populations of DCVs within the same cell, according to their selective expression and trafficking of key, as of yet unidentified regulatory molecules. We further posit that, similar to small synaptic vesicles, DCV release is tightly controlled by neuromodulatory signaling through G protein-coupled receptors (GPCRs). Our innovative hypothesis challenges the existing paradigm that focuses exclusively on intracellular calcium as the primary molecular determinant of DCV release. The discovery of diverse release mechanisms will provide a new understanding for long-standing questions surrounding the challenges associated with evoking neuropeptide secretion. We will test our hypothesis by addressing the following key knowledge gaps: 1) an understanding of the neural activity patterns and wide range of intracellular calcium concentrations that drive DCV release in different neuron classes, 2) and understanding of how neuromodulatory biochemical signaling can adjust the activity and/or calcium requirements for release, 3) elucidation of endogenous GPCRs that can carry out this novel form of neuromodulatory cross-talk, 4) elucidation of the diverse protein machineries associated with DCVs containing different cargoes in different cell classes. The proposed research builds on 1) our recent establishment of several assays for monitoring the actions of tachykinin and opioid neuropeptides in the striatum, 2) our recent discovery of diverse conditions for driving endogenous tachykinin and opioid neuropeptide release, 3) our successful development of photoactivatable peptides for mimicking, and thus calibrating, spatiotemporal aspects of endogenous release, and 4) the recent development of optical sensors that report peptide release in brain tissue. Uncovering the general principles that govern DCV release will establish new connections between intercellular and intracellular signaling pathways and reveal how they are integrated at the molecular level in numerous biological systems that transmit information via DCV secretion. In the long term, we anticipate that the unique signaling pathways uncovered can be exploited to treat metabolic diseases, psychological disorders and neurodegenerative disease, and for chronic pain, latter of which is urgently needed to address the Opioid Crisis. By uncovering new connections between signaling pathways that are fundamental to human physiology in both health and disease, the findings of this work will likely impact numerous scientific fields, including cancer, cardiology, development, gastroenterology, and neuroscience. 1

Project summary The mammalian brain is remarkably dynamic and can quickly adjust its functional state in response to changes in the environment. For example, when a salient event occurs, the brain enters a mode that enhances memory formation. Such brain state changes occur too rapidly to be due to anatomical rewiring. Instead, they are thought to arise from the action of neuromodulators (NMs) and neuropeptides (NPs). Unlike small-molecule NMs, such as acetylcholine and monoamines, NPs are not generally released as the major neurotransmitter from specialized neurons and they are not recycled after release. Instead most neurons synthesize and release NPs in addition to fast transmitters such as glutamate and GABA, and peptide clearance is governed by diffusion and proteolysis. Although long utilized as anatomical markers, our understanding of NP signaling is only cursory. Insights into the cellular code of peptidergic communication are only now emerging from large- scale transcriptional profiling studies that reveal the distribution of peptides and their receptors across cell types. These have revealed a differentiated anatomic distribution of NP-receptor pairs across cell types that poise NPs as important mediators of trans-cellular communication in neural circuits. However, the functional significance of NP signaling is extremely difficult, if not impossible, to study using current tools, which do not reveal the timing and location of NP signaling in vivo, or the consequences of NP signaling on neural circuit activity. Thus, new technologies are needed to enable gain- and loss-of-function studies that precisely target the normal location and timing of NP activity in behaving animals. To overcome these technical barriers, we assembled a multi-disciplinary team to develop, validate, apply, and disseminate next-generation optical toolkits for functional analysis of the spatiotemporal dynamics of NP signaling during behavior. Our toolkits include: 1) photoactivatable agents to rapidly deliver NPs (or drugs that target NP receptors) to their sites of action with high spatiotemporal precision; 2) genetically-encoded NP sensors to report when NPs are released and over what temporal and spatial scales they act: 3) new optical and genetic approaches for cell- and region-specific recording and manipulation of NP action using these probes at multiple sites in the mammalian brain simultaneously. Combining these methods with functional studies in behaving animals, we aim to establish paradigms for determining the necessity and sufficiency of NP signaling for the modulation of circuits in vivo. We will determine the context and location of NP release, the ensuing spatiotemporal pattern of NP receptor activation, and the effects this has on neuronal physiology and behavior. We will actively disseminate these toolkits to the neuroscience community. Broad applications in various brain regions and species will reveal the dynamic contribution of NPs to the control of brain circuits and plasticity. This knowledge will provide building blocks and pave the ways to refine theory and develop novel therapeutics for neurological and neuropsychiatric disorders.

Project summary The mammalian brain is remarkably dynamic and can quickly adjust its functional state in response to changes in the environment. For example, when a salient event occurs, the brain enters a mode that enhances memory formation. Such brain state changes occur too rapidly to be due to anatomical rewiring. Instead, they are thought to arise from the action of neuromodulators (NMs) and neuropeptides (NPs). Unlike small-molecule NMs, such as acetylcholine and monoamines, NPs are not generally released as the major neurotransmitter from specialized neurons and they are not recycled after release. Instead most neurons synthesize and release NPs in addition to fast transmitters such as glutamate and GABA, and peptide clearance is governed by diffusion and proteolysis. Although long utilized as anatomical markers, our understanding of NP signaling is only cursory. Insights into the cellular code of peptidergic communication are only now emerging from large- scale transcriptional profiling studies that reveal the distribution of peptides and their receptors across cell types. These have revealed a differentiated anatomic distribution of NP-receptor pairs across cell types that poise NPs as important mediators of trans-cellular communication in neural circuits. However, the functional significance of NP signaling is extremely difficult, if not impossible, to study using current tools, which do not reveal the timing and location of NP signaling in vivo, or the consequences of NP signaling on neural circuit activity. Thus, new technologies are needed to enable gain- and loss-of-function studies that precisely target the normal location and timing of NP activity in behaving animals. To overcome these technical barriers, we assembled a multi-disciplinary team to develop, validate, apply, and disseminate next-generation optical toolkits for functional analysis of the spatiotemporal dynamics of NP signaling during behavior. Our toolkits include: 1) photoactivatable agents to rapidly deliver NPs (or drugs that target NP receptors) to their sites of action with high spatiotemporal precision; 2) genetically-encoded NP sensors to report when NPs are released and over what temporal and spatial scales they act: 3) new optical and genetic approaches for cell- and region-specific recording and manipulation of NP action using these probes at multiple sites in the mammalian brain simultaneously. Combining these methods with functional studies in behaving animals, we aim to establish paradigms for determining the necessity and sufficiency of NP signaling for the modulation of circuits in vivo. We aim to determine the context and location of NP release, the ensuing spatiotemporal pattern of NP receptor activation, and the effects this has on neuronal physiology and behavior. We will actively disseminate these toolkits to the neuroscience community. Broad applications in various brain regions and species will reveal the dynamic contribution of NPs to the control of brain circuits and plasticity. This knowledge will provide building blocks and pave the ways to refine theory and develop novel therapeutics for neurological and neuropsychiatric disorders.