Timothy E. Holy, PhD - US grants

DESCRIPTION (provided by applicant): Inhibition plays a central role in the nervous system, but many of its features remain mysterious. For example, there are many types of inhibitory neurons, each with strikingly-different shapes, electrical properties, and connections. However, when examined in other ways for example in terms of their activity to natural stimuli in the intact circuit in many cases these diverse types appear surprisingly uniform. While one suspects there must be a reason that the brain contains so many types of inhibitory neurons, currently the major tasks attributed to inhibition could seemingly be accomplished with far fewer types. The olfactory system has long been one of the most attractive systems for studying the roles of inhibition, in part because it spotlights many of its puzzles. Because olfaction has many different processing streams (a multitude of receptor genes to detect a multitude of chemicals), the olfactory system represents an excellent opportunity to look for specificity in the patterns of inhibitory function. However, using conventional techniques, the sheer diversity of olfactory systems also makes this a daunting challenge. Within the olfactory system, we will focus on a particular stage of processing called the glomerular layer, which collectively encodes all the information the animal has available about olfactory stimuli. We will leverage a new imaging technology developed in our laboratory, Objective-Coupled Planar Illumination (OCPI) microscopy, to image the glomerular layer of the accessory olfactory bulb in its entirety. This wil allow us to exhaustively analyze the interactions between different glomeruli. Our experiments will study the consequences of inhibition at two stages within the glomerular layer roughly, its inputs and outputs and thereby serve to test several candidate mechanisms of inhibitory specificity. Collectively, these experiments will reveal the logic of a key stage of sensory processing in the nervous system.

DESCRIPTION (provided by applicant): Steroid hormones regulate many aspects of mammalian physiology. Steroids pass through membranes and bind to nuclear receptor/transcription factors, initiating a change in gene transcription. Particularly in the nervous system, steroids also exert effects that are too rapid to be attributed to this mechanism. Recently, my laboratory discovered a novel mode of steroid action: sulfated steroids are detected by neurons residing in the vomeronasal organ, which provide sensory input to the accessory olfactory system. These sensory neurons express several hundred different types of G protein-coupled receptors, and different neurons detect different sulfated steroids. Sulfated steroids are present in natural bodily secretions, such as urine, and represent a prime candidate for organizing mammalian social behavior. In mice, behavioral or physiological responses mediated by the accessory olfactory system frequently involve integration of multiple cues;in particular, there are several examples in which male-derived stimuli act in opposition with female-derived stimuli, and the integration of these cues leads to a single behavioral response. We hypothesize that these behaviors arise as a direct consequence of the neuronal circuitry that processes sensory data about sulfated steroids. To test this hypothesis, we propose both electrophysiological and behavioral experiments that will elucidate the functions of this circuitry in analyze complex steroidal blends. In aim 1, we will determine how mitral cells of the accessory olfactory bulb encode information about individual sulfated steroids, and test how circuit processing changes the representation inherited from the sensory neurons. In aim 2, we will examine the role of male and female steroidal cues in mixture suppression, a likely physiological correlate of male/female opponent behavioral processes. In aim 3, we will examine the role of olfactory-detected sulfated steroids in estrous cycle suppression, focusing in particular on the role of glucocorticoids. In aim 4, we will examine the role of sulfated steroids in protecting against pregnancy block triggered by the urine of strange males. These aims will illuminate the novel role of steroids in the olfactory regulation of mammalian reproductive behaviors. PUBLIC HEALTH RELEVANCE: A better understanding of steroid effects in the nervous system offers hope for treatment of stress, anxiety, and mood disorders, as well as to shed light on sex differences in neuronal development. Most experiments on steroid effects are performed in rodents, but interpretation of the results is complicated by differences between rodents and humans. Olfactory detection may represent a major, previously-unrecognized mechanism for steroid action in mice;understanding this phenomenon will provide critical new insights for interpreting studies that alter the steroid milieu.

DESCRIPTION (provided by applicant): In studies of neural plasticity, one of the outstanding challenges is to connect our growing understanding of cellular and synaptic mechanisms to their consequences for plasticity of whole neuronal circuits. A promising approach is to use optical microscopy to observe the changes in activity that accompany learning;however, conventional methods like multiphoton microscopy cannot monitor neuronal events over large regions at high speeds. A new technique, Objective-Coupled Planar Illumination (OCPI) microscopy, promises orders-of- magnitude increases in the sensitivity and speed of optical physiology. This proposal focuses on extending the spatiotemporal resolution of OCPI microscopy to observe neuronal activity and plasticity at subcellular resolution in thousands of neurons simultaneously. PUBLIC HEALTH RELEVANCE: The mechanisms of learning are among the great unsolved mysteries of the brain, and memory deficits are a major medical problem. To better understand the neuronal basis of learning, we will develop new instruments for observing the changes in the brain during memory formation.

DESCRIPTION Abstract Deciphering how the brain processes sensory information and makes behavioral decisions ultimately requires methods to record simultaneously from all the neurons in a local circuit. Of techniques used to measure neuronal activity, optical microscopy stands out as one of the few tools with the spatial resolution needed for dense ensemble recordings in thick tissue. Recently, significant progress has been made in developing probes and instrumentation for measuring neuronal activity using fluorescence. For studies of neural circuits, however, this strategy is fundamentally limited by the phototoxicity of the fluorophore: long-term, high-speed imaging needed to study circuits delivers a light dose that damages (and ultimately destroys) fluorescently-labeled cells. To overcome this problem, I propose to develop new approaches to measure spiking activity without fluorescence. The methods we will develop will perform at speeds sufficient to image large three-dimensional volumes of intact tissue thousands of times per second, sensitive enough to record each action potential, and sufficiently non-damaging to record from the same set of neurons for periods of hours. This technical advance will provide an unprecedented look at how neuronal activity generates the central computational functions of the brain. Public Health Relevance This project will develop a new non-invasive method for recording brain activity. One immediate application of the technology will be in neurosurgery, where it could be used for rapid local diagnostics during resections of tumors or epileptogenic tissue. THE FOLLOWING RESUME SECTIONS WERE PREPARED BY THE SCIENTIFIC REVIEW OFFICER TO SUMMARIZE THE OUTCOME OF DISCUSSIONS OF THE REVIEW COMMITTEE ON THE FOLLOWING ISSUES. VERTEBRATE ANIMAL (Resume): ACCEPTABLE COMMITTEE BUDGET RECOMMENDATIONS: The budget was recommended as requested. SCIENTIFIC REVIEW OFFICERS NOTES: Since the NIH Director's Pioneer Award applications are reviewed differentl

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Among mice, pheromones and other social odor cues convey information about sex, social status, and identity;however, the molecular nature of these cues is essentially unknown. To identify these cues, we screened chromatographic fractions of female mouse urine for their ability to cause reproducible firing rate increases in the pheromone-detecting vomeronasal sensory neurons (VSNs) using multielectrode array (MEA) recording. Active compounds were found to be remarkably homogenous in their basic properties, with most being of low molecular weight, moderate hydrophobicity, low volatility, and possessing a negative electric charge. Purification and structural analysis of active compounds revealed multiple sulfated steroids, of which two were identified as sulfated glucocorticoids, including corticosterone 21-sulfate. Sulfatase-treated urine extracts lost >80% of their activity, indicating that sulfated compounds are the predominant VSN ligands in female mouse urine. As measured by MEA recording, a collection of 31 synthetic sulfated steroids triggered responses 30-fold more frequently than did a similarly sized stimulus set containing the majority of all previously reported VSN ligands. Collectively, VSNs detected all major classes of sulfated steroids, but individual neurons were sensitive to small variations in chemical structure. VSNs from both males and females detected sulfated steroids, but knock-outs for the sensory transduction channel TRPC2 did not detect these compounds. Urine concentrations of the two sulfated glucocorticoids increased many fold in stressed animals, indicating that information about physiological status is encoded by the urine concentration of particular sulfated steroids. These results provide an unprecedented characterization of the signals available for chemical communication among mice.

DESCRIPTION (provided by applicant): A central goal of neuroscience is to understand how animal and human behaviors are generated by the circuits of the brain. Progress towards this goal has relied in part on the ability to record the activity of neuronal populations. To record densely from a local circuit, one of the most widely-used techniques is calcium imaging. Calcium imaging by two-photon microscopy currently allows investigators to record from hundreds of neurons simultaneously; while a major improvement over past approaches, it is nevertheless a small fraction of the total number of neurons in almost any local circuit. The difficulty of determining which neurons first compute a particular decision or pattern of activity is, in my view, the most important technical barrier to a comprehensive understanding of how neuronal circuits drive behavior. Recently, we developed a method, called Objective-Coupled Planar Illumination (OCPI) microscopy, to perform fast fluorescence imaging of whole tissue volumes. Instead of collecting data from one pixel at a time, it uses a thin sheet of light to collect entir images at once. OCPI microscopy has allowed us to record from approximately ten thousand neurons simultaneously at high speeds and signal-to-noise ratio. For our scientific work on the olfactory system, OCPI microscopy has revealed what the sensory periphery detects, how chemical stimuli are encoded, the spatial arrangement of circuits in the brain, and even how different individuals perceive the world. Here I propose to extend the domain of applicability of OCPI microscopy. In aim 1, we will develop new methods to see deeper into the living brain. In aim 2, we will develop procedures to tag individual neurons based on their physiological properties. In aim 3, we will develop and share algorithms to process the terabyte-sized datasets produced by OCPI microscopy. These aims will allow OCPI microscopy to address new questions and to be disseminated widely throughout the neuroscience community.

Abstract ?Social odors??sometimes called ?pheromones??are key regulators of infant feeding, reproduction, and aggression. Once detected by the sense of smell, these signals act to modulate the animal's hormonal status. Conversely, steroid metabolites in urine have emerged as one of the best-understood classes of cues detected by the vomeronasal organ, one of the principal sensory organs for pheromones. This tight integration between hormones and olfaction is believed to serve as an ?honest signal? in which information about physiological status is inferred from metabolites of the hormones that control it. In mammals, the identity of most pheromones remains mysterious. In mice, despite 40 years of investigation, the principal sex-specific vomeronasal cues in natural stimuli like urine were controversial or unknown. Recently, my laboratory developed new techniques to perform large scale unbiased screens for the ligands that activate vomeronasal sensory neurons. Using these tools and combining them with neurophysiology, endocrine manipulations, and analysis of behavior, we have identified the first female sex pheromones for the mouse. With support from the NIH, we propose to identify most or all of the male-specific urinary cues that activate the vomeronasal organ. Specifically, we propose to (1) purify male-specific ligands; (2) solve their chemical structures, and (3) identify their pattern of expression and contributions to two behaviors, the Vandenbergh Effect (puberty acceleration) and the Bruce Effect (pregnancy block). These aims promise to open new doors in our understanding of the relationship between sex, olfaction, hormones, and behavior.

This proposal, from the Neuroscience Program in Washington University?s Division of Biology and Biomedical Sciences (DBBS), is a new application to the Jointly Sponsored Ruth L. Kirschstein National Research Service Award Institutional Predoctoral Training Program in the Neurosciences. The overarching goals of our Neuroscience program are to equip our trainees with a firm foundation in nervous system function and dysfunction, the ability to identify problems and design strategies to address them critically and rigorously, and the skills required to perform, present, and mentor others in research. The strengths of our current training program include a strong and evolving curriculum to address critical areas of modern neuroscience and the skills necessary for success in any neuroscience career, a focus on improving diversity of students in neuroscience and retaining diverse students in the program, a collegial and collaborative atmosphere, broad institutional support, multiple neuroscience-related opportunities for community outreach and teaching and a supportive administrative structure that facilitates all aspects of the educational process, from recruitment of students to thesis defense and beyond. This proposal builds on these features with ongoing and future initiatives aimed at improving quantitative, experimental and statistical thinking, facilitating interdisciplinary and/or advanced training in areas relevant to a student?s research, modernizing curriculum delivery, providing evidence-based ethics training to address well-publicized problems of rigor and reproducibility, and assessing the impact of these initiatives and modifying their implementation as needed. We are requesting 11 slots for students in their 1st and 2nd years. Students will emerge from this program with a stronger foundation in experimental and statistical thinking, ethics and methods to improve rigor and reproducibility. Faculty in the program will also benefit from exposure to emerging methods and approaches in these areas.