Raphael Pinaud - US grants

DESCRIPTION (provided by applicant): Brain function depends on precisely organized neural networks. A central problem in contemporary neuroscience has been to determine how the specific organization of synaptic inputs is established in the brain during development. Uncovering the identity of key molecules involved in this process and elucidating how they are dynamically engaged as a result of development and experience is fundamental for a comprehensive understanding of the mechanisms underlying the establishment, maturation and functionality of the synaptic circuitry. Previous studies conducted in sensory systems have shown that both intrinsic and experiential factors are required for the adequate formation of synaptic circuitry and its performance. In particular, significant research into these questions has been carried out in the mammalian auditory brainstem, where a series of post-natal developmental changes underlie the adequate establishment of neuronal connectivity and, consequently, intact hearing. Much of these studies have focused in the medial nucleus of the trapezoid body (MNTB), a relay station of the ascending auditory pathways involved in the localization of sounds in the horizontal plane, and embedded in the superior olivary complex. Input into MNTB neurons occurs through a highly specialized synaptic terminal adapted for high-fidelity transmission of auditory information. This synaptic terminal is known as the Calyx of Held and consists of the largest known synaptic terminal in the mammalian central nervous system. Due to its size, known neurochemical properties and convenient anatomical localization, this synaptic contact on MNTB neurons has been extensively used to determine the biophysical and ultrastructural underpinnings of synaptic transmission supporting hearing. Despite a large body of literature on the biophysical changes that correlate with the development of the MNTB circuitry, the underlying molecular substrates responsible for such modifications are largely unknown. Furthermore, no systematic studies to date have attempted to identify and dissociate the molecular events are regulated by developmental stage (age) from those that are impacted by sensory experience. In this application, we aim to significantly fill this current gap in knowledge. We propose to use high-throughput quantitative proteomics and mass spectrometric approaches to uncover the cascade of protein regulatory events associated with the post-natal development of MNTB circuitry. More specifically, we intend to use two-dimensional differential in-gel expression (2D-DIGE)-based proteomics to identify age- and experience-regulated proteins in the MNTB, both before and after hearing onset. In addition, we propose to investigate how abnormal sensory experience impacts the dynamic regulation of the MNTB proteome throughout post-natal development. To achieve this goal, we intend to combine high-throughput quantitative proteomics and mass spectrometry to study mouse models of early- and late-onset deafness and, consequently, identify protein regulatory events that are altered in these conditions. We predict that the results of this research will significantly contribute to the emergence of a coherent picture of the molecular and cellular processes underlying the development of central auditory pathways. Moreover, this research is expected to provide open new avenues for the development of pharmacological and/or molecular strategies aimed at ameliorating or recovering hearing dysfunction, as in the case of deafness. We propose to systematically investigate how the coordinated activation of key molecules guide the maturation of neuronal circuits that control hearing function, and how interference with normal audition impacts these molecular programs. A thorough understanding of the molecular events that underlie the establishment and maturation of hearing-related circuits may open potential avenues for ameliorating or restoring dysfunctional audition, as in the case of deafness.

Recently, Dr. Pinaud's research group demonstrated that the female hormone estrogen controls how auditory signals are processed and interpreted by auditory neurons in the brain. These are exciting findings because they indicate that estrogen insufficiency may impact hearing function and manipulations directed at estrogen levels may ameliorate or recover hearing dysfunction triggered by hormonal deficits. Although these novel findings are promising, it is necessary to uncover the mechanisms by which estrogen impacts the functionality of auditory neurons and the functional relevance of this hormonal modulation. This project will address these gaps by using a combination of molecular biology, electrophysiology and high-throughput proteomics approaches to understand: 1) how estrogen controls the activation of auditory neurons by acoustic signals; 2) how estrogen shapes selectivity of neuronal responses to auditory signals; 3) the gene expression programs controlled by estrogen in auditory neurons. This work should significantly impact our understanding of how estrogen controls auditory processing and affects long-term gene expression activated by auditory signals in the brain. Consequently, these efforts are a critical first step in the path to exploring how hormonal manipulations may be used to shape how efficiently vertebrates extract acoustic cues for the environment. This project will also generate resources for the scientific community. Specifically, part of the results of this project will be posted on Dr. Pinaud's website (www.pinaudlab.org) during the progression of this project. In addition to the scientific impact of this project, two educational programs will provide 1) students of under-represented minorities with opportunities to participate in these studies, and 2) workshops to children at the Elementary School level, as well as their parents, on concepts in neuroscience and the importance of basic scientific research. The project will also include an exchange program with the Brazil to bring students of under-represented minorities to conduct research in this US laboratory. This program does not constitute a one-way exchange, but rather is an opportunity for young scientists from both cultures to participate in learning experiences that differ significantly from their own programs and backgrounds. This exchange could lay the foundation for future international collaborations that could significantly impact this research area.

DESCRIPTION (provided by applicant): Estrogens play a central role in the brain's regulation of reproductive behavior, sexual development and cognitive processes such as learning and memory formation. Recent evidence suggests that estradiol (E2) also impacts auditory processing and perception. Auditory event-related potentials correlate with the plasma E2 levels in humans during the menstrual cycle and auditory processing is more efficient in younger and older females relative to males. Hearing disorders and lower auditory processing efficiency also occur in women with Turner's syndrome, who are deficient in E2. Direct evidence for E2 as a regulator of hearing-driven neural activity and plasticity-associated gene expression was recently presented for central auditory circuits. Despite key advancements on the characterization of a novel sensory-neuroendocrine interaction, the intracellular mechanisms by which E2 regulates hearing-driven gene expression are unknown. It is also unclear how E2 modulates the tuning and effectiveness with which auditory signals are processed and encoded by central auditory neurons. Here we address this gap by studying the auditory system of songbirds, a unique model for the study of auditory processing of communication signals. We focus on the caudomedial nidopallium (NCM), a forebrain auditory area thought to be analogous to the mammalian auditory association cortex. NCM neurons are selective to species-specific auditory signals, are involved in the perceptual processing of songs, and the formation of auditory memories. NCM is also the site of a major overlap between the auditory and neuroendocrine systems. High levels of aromatase and estrogen receptors are found in NCM. E2 levels also rapidly increase in NCM with socially-relevant auditory experiences. Finally, locally-produced E2 regulates auditory-evoked activity and plasticity-related gene expression in NCM. The molecular bases of E2's effects on NCM neurons, and the functional outcomes of such modulation to NCM's physiology and auditory-based behaviors are unknown. This proposal has five objectives that, together, will address these gaps: First, in-situ hybridization will be used to determine if auditory stimulation activates neurons that either produce or are sensitive to E2 in NCM. Second, a combination of intracerebral pharmacology in awake birds and biochemical methods will determine the specific intracellular mechanisms by which E2 influences auditory-driven genomic responses in NCM neurons. Third, patch-clamp electrophysiological studies will determine how E2 exert rapid effects in synaptic transmission and plasticity within NCM circuitry. Fourth, in-vivo neurophysiology coupled to local pharmacology will determine how E2 impacts the spectrotemporal tuning, and the effectiveness with which NCM neurons encode auditory signals. Finally, local pharmacological manipulations in awake animals coupled to behavioral assays will determine if normal E2 modulation of NCM is required for auditory discrimination. Our long-term goal is to elucidate the mechanistic bases and functional roles of E2's modulation of NCM's physiology during the auditory and perceptual processing of songs. We also aim to uncover how manipulations of steroid hormone levels may be used to alter auditory processing towards recovery of abnormal auditory function. This proposal will yield significant information on how the vertebrate brain interprets behaviorally-relevant communication signals, and will shed light on the neural basis of experience- dependent changes that underlie critical auditory-based behavioral processes such as auditory discrimination. PUBLIC HEALTH RELEVANCE: We propose to thoroughly determine how a classic steroid hormone, 17[unreadable]-estradiol, impacts the cellular physiology and the processing of complex communication signals in the vertebrate brain. In addition, we aim to understand how this hormone engages sensory-regulated, plasticity-associated gene expression, and ultimately modulates higher-order behaviors such as auditory discrimination.