Fan Wang - US grants

DESCRIPTION: (provided by the applicant) My lab is interested in studying the molecular mechanisms and neuronal circuitry that mediate distinct orofacial pain, using the mouse as a model system. In the craniofacial region, pain transmission is mediated by the trigeminal sensory neurons that innervate diverse tissue on the entire face. Interestingly, the perceived pain originated from different areas is different. For example, the pain of a headache is usually very different from dental pain. Furthermore, different craniofacial regions tend to be associated with or develop distinct chronic pains. We hypothesize that the three trigeminal divisions innervating different targets should express distinct molecular programs and engage in distinct neural circuits, thus enabling them to mediate different orofacial pain. In Specific Aim 1, we will identify the molecular differences among the separate divisions of trigeminal ganglion using genomic approaches. The goal is to generate comprehensive lists of molecular signatures for neurons innervating distinct head/face regions and answer the question: "How heterogeneous are nociceptive trigeminal neurons". In Specific Aim 2, we will use some of the molecular markers that we already identified to genetically map the neuronal projections from specific trigeminal populations and study the nociceptive sensory maps in normal mice and in mice with experimentally induced chronic pain. These studies should allow us to address two fundamental questions in pain research: (1) Do different trigeminal neurons form convergent projections at certain locations in the hindbrain? (2) Is abnormal axon sprouting the structural basis for chronic pain? Finally, in Specific Aim 3, we will test the functions of two of the maxillomandibular expressed transcription factors to prove, in principle, that genes specifically present in some but not all trigeminal divisions are important for either their specialized development requirements or their specific functions.

Rodents use whiskers as their primary tactile sensors. Individual whiskers are innervated by a group of touch sensory neurons located in the trigeminal ganglion, and are mapped onto the brain by the central axons from the same set of trigeminal neurons. The discovery of the whisker- related large modular structures (barrelettes in the hindbrain, barreloids in the thalamus and barrels in the cortex) in 1970s made the trigeminal-whisker system a popular prototypic model for studying somatosensory maps and touch sensory circuits. Yet to date, we still have limited knowledge of the precise neural circuits assembled inside each whisker-representing unit. The objective of this proposal is to dissect the micro-circuits formed by different classes of touch sensory neurons within individual barrelettes, and to begin to identify the molecular mechanisms controlling the precise assembly of these barrelette circuits. These studies will provide the much- needed knowledge for future understanding of somatosensory information coding and processing and ultimately sensory perception. The mechanisms uncovered in this study will also have broad implications for understanding neuropathological diseases associated with abnormal wiring in the somatosensory system such as Autism and neuropathic orofacial pain.

A major challenge in neuroscience research is to identify the precise functional neural circuits that are responsible for diverse nervous system processes such as sensory perception, learning and memory, as well as the aberrant circuits that underline the pathological conditions such as chronic pain, depression, addiction, and epilepsy. Previous work in this area has focused on designing molecular sensors that detect transient voltage change or calcium influx using optical methods. However, the use of these sensors for studying in vivo complex neural physiology, and pathophysiology processes of the mammalian nervous system have been extremely limited. Moreover, these sensors do not allow for anatomical re-construction of the activated circuits. In this EUREKA application, we propose to develop novel genetically encoded anatomical probes for visualizing neuronal activity and plasticity directly. These probes will label activated neurons and synapses (and those most likely undergo plasticity) with a marker (e.g. a fluorescent protein) for easy histological detection and anatomical tracing. The chief principle underlying this unconventional design of probes is to use activity (strong calcium influx) to induce the translation of an otherwise un-translated marker protein. Successful development of the activity-induced anatomical probes will greatly advance our understanding of the functions and dysfunctions of the nervous system.

DESCRIPTION (provided by applicant): Pre-motor Neural Circuits for Exploratory Movement Abstract Movements performed by animals in order to explore external objects are called exploratory movements. Humans use delicate and complex movements of fingers/fingertips to discern the texture, shape and other physical properties of objects, and to manipulate tools. Rodents explore their physical environment through rhythmic sweeping of their vibrissae (whisking), and thus serve as a major model for studying neural circuits controlling exploratory touch movements. The final common control of the tactile vibrissae is provided by motor neurons located in the lateral facial nucleus (vFMNs). The objective of this proposal is to discover and characterize the premotor circuitry that directly regulates the activities of vFMNs. We will identify the connectivity maps of premotor neurons that provide monosynaptic input for the different vFMNs controlling vibrissa protraction and retraction. We wil also determine the neurotransmitter phenotypes of identified premotor neurons, and characterize the functional inputs of different premotor neurons onto vFMNs using electrophysiological and optogenetic approaches. Furthermore, we will determine how developmental changes in the vFMN premotor circuitry enable the postnatal emergence of bilaterally coordinated and often synchronized exploratory whisking behavior. Identifying the structural and functional wiring diagram of these premotor neural circuits is a critical step for investigating the generation and voluntary control f exploratory movements. Results from this study will also provide new foundations for understanding motor control of hand and finger movements in humans, and thus can help lead to the design of superior neuroprosthetics devices to restore exploratory movements following paralysis or amputation.

DESCRIPTION (provided by applicant): It is hypothesized that animals' perceptual, emotional, or behavioral processes are governed/caused by specific patterns of brain activities, or firings of selected populations (ensembles) of neurons across multiple regions in the nervous system. Indeed recent advancements in optic imaging and multi-electrode recording technology have begun to reveal the inordinately complex dynamics of large populations of neurons in awake, behaving vertebrates such as larval zebrafish and rodents. However, these visualization/recording experiments could not definitively establish the causal relationships between the activities of neuronal ensembles and their functions. A toolkit that enables neuroscientists to be not only observers, but also actuators of the observed ensembles is critically needed for causal neuroscience. The difficulty to developing such a toolkit lies in th complexity of the mammalian brain, which contains billions of neurons and trillions of synapses. Thus, individual neurons are likely to participate in different active ensembles at different time points. Hence the ensembles associated with a given behavioral or perceptual process are emergent properties arising out of the complicated interactions among millions of neurons. Therefore, ensembles are unlikely to be genetically pre-determined, and molecule or cell-type based methods are not useful for labeling and manipulating them. To overcome this difficulty, we will develop a novel toolkit consisting of two key components: (1) a mouse line designed to express, transiently and selectively, a very unstable foreign receptor only in activated neurons, and (2) engineered non-toxic, pseudo-typed viruses that can only infect neurons expressing this foreign receptor. In this way, timed-injection of the pseudo-typed viruses will allow us to specifically and permanently capture the recently activated ensembles (which are the ones expressing the viral receptor). The engineered viruses can int

Tactile sensation is paramount to how we experience the physical world around us. Species like mice use their facial whiskers to navigate and assess their environment. Although there is a basic understanding of how brainstem trigeminal neurons, the first central relay for whisker-derived signals, respond to passive tactile stimuli, little is known about their activities and functions during active behaviors. Tactile exploration is an active sensing process, which adapts to behavioral purposes, and responses of trigeminal brainstem neurons are likely adjusted accordingly. Furthermore, brainstem projection neurons contribute to two main ascending pathways, lemniscal and paralemniscal, but the functions of these two distinct pathways remain unclear. Our goal for this high-risk exploratory grant application is to develop a chronic in vivo recording preparation, perform recordings from optogenetically identified lemniscal or paralemniscal projection neurons during active behaviors, and use causal manipulations to determine their functions. Recording and manipulating pathway-specific neurons in vivo, and making accurate behavioral measurements in freely moving animals are both challenging. We will use a combination of technological innovations to overcome these challenges. The outcome of this project will establish a platform for future in-depth dissection of how touch sensory information is integrated, transformed and delivered to different processing streams by trigeminal brainstem to generate perception, invoke emotion and/or inform decisions.

Project Summary An inability to form and maintain social bonds typifies a wide range of neuropsychiatric and neurodevelopmental disorders. These social deficits stem in large part from impaired expressive and receptive vocal communication skills. Surprisingly, exactly how vocal communication promotes social affiliation is not well understood, in part because the underlying neural circuits remain poorly described. Here we propose the use of a novel genetic approach to selectively tag neurons that are active during social encounters that elicit vocalizations. We will combine this innovative method with in vivo imaging, electrophysiology, chemical and optogenetic perturbations of neural activity, and behavioral measurements to identify neural circuits that facilitate expressive and receptive aspects of vocal communication in the service of social affiliation. In Aim 1, we will test the idea that a specific subpopulation of neurons in the midbrain periaqueductal gray (PAG) is required for male and female mice to produce vocalizations used during their social interactions. In Aim 2, we will manipulate the activity of these PAG neurons to suppress or augment vocalization, allowing us to test the idea that these vocalizations promote social affiliation. In Aim 3, we will test the idea that prefrontal cortical (PFC) neurons that provide input to PAG vocalization neurons are important in regulating vocalization as a function of social context. In Aim 4, we will either reversibly silence or image PFC neurons that provide input to the PAG to test the idea that they play a role in generating affiliative social responses in males and females listening to a vocalizing individual. These studies will identify the neurons and circuits that gate vocalization during social encounters and promote social affiliation in response to these acoustic signals. This research will also build the foundation for future studies that explore how these circuits are affected in mouse models of neuropsychiatric disorders characterized by social communication and affiliation deficits, such as autism spectrum disorder and schizophrenia.

Cortical Signature and Modulation of Pain Abstract/Project Summary Pain perception contains two main dimensions: the sensory-discriminative and the affective-cognitive aspects. In this proposal, we will focus on the cortical signature and modulations of the sensory aspects of pain using mouse models. Pain can be largely divided into inflammatory or neuropathic pain. A common condition in both types of pain is mechanical allodynia: externally applied innocuous gentle touch becomes painful. Paradoxically, pain elicits self-initiated recuperative behaviors such as rubbing and massaging of the painful regions; and the mechanical stimuli from such self-generated behaviors generally relieve pain. The neural circuit mechanisms underlying the opposite effects of external versus self-applied mechanical stimuli in pain conditions remain poorly understood. Based on previous research as well as our preliminary studies, we hypothesize that corticospinal projecting neurons from primary somatosensory (S1) cortex facilitate mechanical hypersensitivity in pain models, whereas specific motor cortex projection neurons play key roles in suppressing tactile allodynia in self-initiated recuperative behaviors (such as licking and wiping in mice). We further hypothesize that the cortical pain signature can be read out from activity patterns of large populations of individual S1 neurons by comparing their activities in the painful versus non-painful or pain- relieved conditions. We will use a combination of viral-genetic labeling of specific cortical neurons, in vivo calcium imaging and in vivo multi-electrode extracellular recording in freely behaving mice, optogenetics- assisted slice physiology, opto/chemicogenetic manipulations, and computational analyses to test our hypotheses.

ABSTRACT Chronic pain is a major health problem that afflicts one third of Americans. New research that can aid the development of new pain-relieving strategies is urgently needed. Our proposal focuses on identifying and validating a new central analgesic circuit. This project is based on a highly innovative hypothesis that the strong analgesic effect of general anesthesia (GA) is in part carried out by GA-mediated activation of the endogenous analgesic circuits. In preliminary studies, we discovered that a subset of GABAergic neurons located in the central amygdala (CeA) become strongly activated and express high level of the immediate early gene Fos under GA (hereafter referred to as CeAGA neurons). Excitingly, using our recently developed activity-dependent tagging system called CANE (Capturing Activated Neuronal Ensemble), we were able to capture CeAGA neurons and discovered that activating these neurons exerted profound pain-suppressing effects in an acute pain model and a chronic orofacial neuropathic pain model. Based on these exciting preliminary results, we propose to identify and validate CeAGA neurons? analgesic functions in multiple mouse pain models conducted in different labs (Wang and Ji, co-PI). In aim 1, we will systematically activate and silencing CeAGA analgesic neurons and test the consequences of such bi- directional manipulations on regulating the sensory-discriminative and emotional-affective aspects of pain processing in naïve mice and in several mouse models of acute and chronic pain models with cross validation between the two labs. In aim 2, using the state-of-the-art in vivo imaging technology, we will test the hypothesis that the spontaneous activities of CeAGA analgesic neurons are severely reduced in various chronic pain models compared to naïve conditions, leading to pain hypersensitivity in these models (due to the suppression of this endogenous analgesic circuit); and complimentary, we will use slice electrophysiology to examine changes in intrinsic and evoked properties of CeAGA analgesic neurons in normal and chronic pain conditions which may explain the altered in vivo activities. In aim 3, based on preliminary results showing extensive axonal projections of CeAGA to many brain areas, we will identify the critical subsets of CeAGA projection pathway(s) for their analgesic effect in different chronic pain models. We expect to identify shared common pathways that need to suppressed by specific subtypes of CeAGA analgesic neurons in all models, and such information will be critical for developing precise CeAGA-based therapies. In summary, this research is expected to identify and validate a novel central analgesic circuit whose power can be harnessed to treat chronic pain.