Dwight Bergles - US grants

DESCRIPTION (provided by applicant): Glutamatergic synaptic transmission supports the rapid transfer of information between neurons in the mammalian CNS and undergoes changes in efficacy that may underlie memory storage and learning. An essential step in glutamatergic transmission involves removal of glutamate from the extracellular space by Na+-dependent transporters. Immunocytochemical, electrophysiological, and molecular/transgenic studies indicate that glutamate uptake by astroglial cells is critical for preventing the accumulation of glutamate, maintaining the sensitivity of receptors, and preserving the specificity of synaptic transmission. The close proximity of astrocyte processes to synapses and the high density of transporters in these membranes suggest that they may also shape the activation of receptors during first few milliseconds after release. This involvement of glutamate transporters in excitatory transmission raises new questions about how the activity of these transporters is regulated. We hypothesize that mechanisms exist to rapidly adjust the capacity of uptake to changes in glutamate release. We have shown that the activity of glutamate transporters can be monitored in astroglial cells in acute brain slices using electrophysiological techniques, providing the means to study the properties and regulation of these transporters in their native membranes. Studies in our laboratory indicate that activation of metabotropic glutamate receptors (mGluR) on hippocampal astrocytes triggers a dramatic enhancement of glutamate transporter currents in these cells. We propose to determine the mGluRs and transporters responsible for this enhancement, the mechanisms involved, and the consequences of this enhancement for glutamate clearance and receptor occupancy during synaptic transmission. These studies of the basic mechanisms of glutamate transporter regulation may reveal how the action of glutamate is constrained despite rapid and maintained changes in the activity of excitatory afferents. The dual role of glutamate as an excitatory transmitter and excitotoxin, suggests that glutamate transporters could be effective therapeutic targets in treating disorders of cognitive impairment, and in preventing neuronal damage associated with stroke and neurodegenerative diseases. Understanding the endogenous mechanisms of transporter regulation may thus provide us with new approaches for treating neurological diseases.

[unreadable] DESCRIPTION (provided by applicant): Glial cells in the mammalian CMS are responsible for creating and maintaining an environment where neuronal activity can be sustained, and are capable of modulating this activity. This close interrelationship between neurons and non-neuronal cells suggests that mechanisms may exist to rapidly adjust glial cell behavior in response to changes in the needs of surrounding neurons. Our previous studies indicate that NG2 cells (also known as oligodendrocyte precursor cells, or OPCs), a class of progenitor cells found ubiquitously in both gray and white matter, express functional ionotropic receptors for glutamate and GABA in situ, suggesting that conventional neurotransmitters may have widespread roles in cell signaling. These low affinity receptors are activated in NG2 cells by the quantal release of transmitter from neurons, which result in transient depolarizations of the NG2 cell membrane. The existence of synaptic signaling between neurons and NG2 cells in the hippocampus raises many new questions about the role of this rapid communication in regulating the properties and development of these enigmatic cells. We hypothesize that neuron-NG2 cell synaptic signaling is a ubiquitous mechanism for regulating the proliferation and development of NG2 cells in the brain. The availability of transgenic mice in which the fluorescent protein DsRed is expressed in all NG2 cells provides us with an unprecedented opportunity to study the interaction between neurons and NG2 cells within intact slices of mammalian brain. We propose to use single cell electrophysiological methods, high resolution electron microscopy, and transgenic manipulation of glutamate receptors in NG2 cells, to define the properties of receptors expressed by NG2 cells in different brain regions, the mechanisms responsible for activation of these receptors, and the role of this signaling in regulating NG2 cell behavior. These studies will evaluate the specific hypothesis that Ca2+ influx through these AMPA receptors plays a central role in regulating the proliferation and differentiation NG2 cells. Because these cells serve as oligodendrocyte progenitors and have multipotent capability, a better understanding of the factors that regulate the NG2 cells behavior in situ may lead to new strategies for preventing myelin damage in pre-term infants, and replacing neurons and glia that have been injured as a result of ischemia or lost through disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Spontaneous activity in developing sensory systems has been shown to be important for the growth and survival of projection neurons as well as the refinement and stabilization of sensory maps in the brain. In the developing cochlea, bursts of action potentials occur in afferent spiral ganglion neurons prior to the onset of hearing, activity that has been traced to inner hair cells (IHCs). Although IHCs are capable of generating Ca2+ action potentials during this period, the depolarizing stimulus required to initiate these events has not been identified. Whole-cell recordings from IHCs and supporting cells located adjacent in IHCs in ex vivo cochleas from young rodents revealed the presence of spontaneous inward currents that were capable of inducing large depolarizations. This activity was coincident with changes in the optical properties of the tissue when visualized using IR/DIC imaging, indicating that these events can be monitored non- invasively. Spontaneous electrical and optical activity was blocked by P2 purinergic receptor antagonists and gap junction inhibitors, suggesting that ATP and gap junctions/hemichannels are involved in initiating these events. Remarkably, this activity is no longer observed after the onset of hearing. This discovery of spontaneous purinergic signaling in the developing organ of Corti raises many new questions about the mechanisms responsible for producing this activity, the role that this ATP-mediated signaling plays in driving afferent firing, and the cause of the disappearance of the activity after hearing onset. We hypothesize that these ATP driven depolarizations of IHCs are responsible for initiating activity in developing auditory pathways. The preservation of this activity in both acute and cultured cochleas in which appropriate cell- cell interactions are maintained provides us with an unprecedented opportunity to understand the mechanisms responsible for these robust phenomena. We propose to use IR/DIC and confocal fluorescence imaging, photolysis, and both whole cell and extracellular recording to investigate the mechanisms underlying spontaneous activity in supporting cells and hair cells in the developing organ of Corti. These studies will evaluate the specific hypothesis that spontaneous oscillations in [Ca2+]i within supporting cells triggers both inward currents and the release of ATP that depolarizes IHCs.Relevance The studies outlined in this proposal seek to understand the mechanisms responsible for initiating spontaneous activity in supporting cells, hair cells, and afferent dendrites in the developing cochlea. This activity has been shown to have a profound influence on survival of target neurons in brainstem nuclei, the physiological properties of these auditory neurons, and the pattern of synaptic connectivity in these regions. Most congenital forms of deafness result from mutations in connexin 26, a gap junction protein highly expressed by cochelar supporting cells. As our preliminary results suggest that connexin hemichannels may play a role in ATP release from supporting cells, the studies outlined here may help explain how these mutations lead to deafness. Furthermore, these studies may reveal one mechanism by which activity can be induced in afferent nerves in the absence of sound, which may have direct relevance to human conditions such as tinnitus.

[unreadable] DESCRIPTION (provided by applicant): Spontaneous activity in developing sensory systems has been shown to be important for the growth and survival of projection neurons and may help to refine and stabilize sensory maps in the brain. Recent in vivo studies indicate that bursts of action potentials occur in afferent spiral ganglion neurons prior to the onset of hearing; however the mechanisms responsible for generating this activity have not been determined. Our preliminary studies indicate that inner hair cells (IHCs) in the developing cochlea are periodically depolarized by ATP that is released spontaneously from neighboring supporting cells. These ATP-dependent depolarizations can trigger Ca2+ action potentials in IHCs, suggesting that extracellular ATP may be responsible for initiating activity in auditory nerve fibers. Here, we propose to test the hypothesis that `spontaneous' activity in primary afferent spiral ganglion neurons prior to the onset of hearing requires purinergic receptor activation. We will use cochlear explant cultures prepared from prehearing rats to characterize the patterns of spontaneous activity that occur in spiral ganglion neurons, and determine whether this activity is dependent on ATP receptors and requires gap junctions/hemichannels. In addition, we will examine the firing patterns of spiral ganglion neurons in vivo, by making extracellular recordings from these neurons in prehearing rats. To test whether the in vivo firing of spiral ganglion neurons is mediated by ATP, we will infuse P2 receptor antagonists into the cochlea while recording extracellular action potentials from these neurons. These experiments seek to define the patterns of activity carried by auditory nerve fibers during the prehearing period and determine if this activity is mediated by the release of ATP within the cochlea. Ultimately, the data gained from these mechanistic studies will enable us manipulate this activity in vivo to determine the specific role that activity plays in auditory system development. PUBLIC HEALTH RELEVANCE: This proposal seeks to determine the mechanisms responsible for initiating sensory-independent activity in auditory nerves before the onset of hearing. As our preliminary findings suggest that ATP release from supporting cells may play an important role in the initiation of this activity, these studies may yield new insight into how mutations in gap junctions and other supporting cell-associated genes result in deafness. Furthermore, our experiments may have direct relevance to human conditions where sound in perceived in the absence of sensory input, such as peripheral tinnitus. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The mammalian CNS contains an abundant, widely distributed population of glial progenitors known as NG2 cells (also termed oligodendrocyte precursor cells or polydendrocytes) that have the ability to develop into oligodendrocytes and undergo dramatic changes in response to injury and demyelination. Although these cells retain the capacity to generate oligodendrocytes in the adult brain and spinal cord, most NG2 cells in the adult CNS do not differentiate and remain in a progenitor state. NG2 cells are arranged in a grid-like manner in all gray and white matter regions, extend highly ramified processes into the surrounding neuropil, and form direct synapses with neurons, raising the possibility that they modulate the activity of neurons and the flow of information through neural circuits. Moreover, NG2 cells proliferate, increase expression of NG2, and contribute to the formation of glial scars in response to both acute and chronic injury, suggesting that they may help limit neurodegeneration and promote repair. Nevertheless, our knowledge about the roles of these abundant glial cells, and the consequences of their change in behavior following ischemic injury, is very limited. To define the functions of NG2 cells in the adult brain, we recently developed transgenic mice that allow selective ablation of NG2 cells in vivo. Using these mice, NG2 cells can be removed from the brain without inducing reactive changes in astrocytes or microglial cells, or causing paralysis or death, indicating that this approach can be used to help define the functions of these ubiquitous glial cells. In the proposed studies, we will selectively ablate NG2 cells from the CNS in vivo, and examine whether their absence results in alterations in neuronal activity, signaling at synapses, axonal conduction, or specific aspects of behavior, such as spatial memory, anxiety, or sensorimotor control. In addition, we will examine whether removal of NG2 cells alters the response of other glial cells to focal ischemia and the extent of neuronal injury. Together, these exploratory studies have the potential to reveal new roles for this enigmatic population of glial cells in the adult brain, and deepen our understanding of interactions that occur between neurons and glial cells in physiological and pathological conditions. If NG2 cells participate in neuromodulation and CNS repair, they would represent an additional therapeutic target with the potential to reduce abnormal neuronal activity, prevent neurodegeneration in chronic disease, and promote recovery and repair following stroke.

C1A. MULTIPHOTON IMAGING CORE C1 A l ¿ Establishment and setup of the Core: The Multiphoton Imaging (MPI) Core JHU became fully operational in November 2006 with support from this grant and funds provided by the institution. During the first year of funding, design and renovation of dedicated space suitable for imaging was completed, instrumentation was purchased and installed, and a fulltime technician was hired and trained. Dr. Bergles serves as the Director of this Core and is assisted by Dr. Pucak, who serves as Core Manager of the facility. The goals of this Core are to provide access to cuttingedge instrumentation for: 1) analyzing protein localization, protein dynamics, and protein-protein interactions with high resolution; 2) performing time-lapse imaging of multiple fluorophores in living cells and tissues; and 3) combining high-resolution imaging of fluorescently tagged proteins or ion indicator dyes with electrophysiological monitoring of electrical activity. The MPI Core was originally located on the 10th floor of the Wood Basic Science building in the Basic Science research cluster on the JHU medical campus. With institutional support, this facility was relocated in September 2009 down the hall to newly renovated space on the 10^ floor of the immediately adjacent Preclinical Teaching Building. This move increased the size of the facility 2-fold to ~500 sq. ft., which allowed reorganization of the microscope layout for more efficient use (necessitated after the installation of the second microscope), addition of a dedicated preparation area, and expansion of the image processing suite. The Core is currently divided into one large room (~350 sq. ft.) that houses a vibration isolation table, two microscopes and associated equipment, and an outer vestibule (~150 sq. ft.) that is used as an office by the senior research technician responsible for day-to-day operation of the facility and as a site for analysis of imaging and electrophysiology data. The original instrumentation for this Core (Zeiss LSM 510 NLO Meta) was provided by funds from the JHU SOM, and all space for the Core was provided by the Department of Neuroscience. Scheduling for this facility is aided by an interactive web-based calendar accessible through the JHU Neuroscience portal. This Core is located on the same floor as the laboratories of Core members Drs. Bergles, Ginty, Huganir, Kolodkin (PI) and Sockanathan, and it is readily accessible by the other investigators listed in this proposal who have laboratories on other floors in the same building or in adjacent buildings. The Preclinical Teaching Building is sen/ed by two elevators and is connected by a covered bridge to other buildings at JHU SOM, providing convenient access to the three Cores for all NINDS investigators in clinical and basic science departments.

DESCRIPTION (provided by applicant): The Neuroscience Graduate Program, which was begun in 1983, has its headquarters in the Department of Neuroscience at The Johns Hopkins University School of Medicine. Consisting of 64 faculties drawn from various departments across the University, it serves as the hub of a broad spectrum of efforts for the training of graduate students, encompassing molecular, cellular, developmental, systems, cognitive and computational neuroscience as well as neurobiology of disease. Each year, from a pool of-200 applicants, we typically matriculate 10-12 Ph.D. candidates as well as 1-4 candidates for combined M.D./Ph.D. degrees (who are admitted through a separate process). Students enter the program with diverse undergraduate backgrounds ranging from computer science to biochemistry. In the first year they are required to take a year-long integrative lecture course with lab entitled Neuroscience and Cognition as well as a seminar on Science, Ethics and Society. Research opportunities are presented to students through a Departmental Retreat, Lab Lunches (which feature work-in-progress) and a Mini-symposium series by Program Faculty specifically designed to help first-year students choose their research rotations. This information is used to help pick three 12-week lab rotations which are typically completed by the end of the first academic year, following which, a thesis lab is selected. By the end of the second year, students complete 6 additional elective courses, many of which are chosen from a list of 12 small seminar-style courses in Neuroscience specialties. Following completion of a Comprehensive Exam at the end of Year 2, students write and defend a Thesis Proposal which is written in the form of a Pre-doctoral NRSA. Each student is advised by two Prethesis Advisors in Years 1 -2 (at 3 month intervals) and an individualized Thesis Advisory Committee thereafter (at 6 month intervals). Thesis Advisory Committees make reports to the Graduate Program Steering Committee which carefully tracks the progress of each student in the program as well as setting overall program policy. At present, 85 students are enrolled in the Neuroscience Graduate program. The average time to complete the Ph.D. has been 5.3 years. Of the students who have graduated from our program greater than 93% have remained in academic biomedical research. Here, we request stipend support for five students during their first two years in the program. RELEVANCE: The mission of the Program is to train the next generation of neuroscientists to teach and perform research in both basic and clinical neurosciences. The Training Program also serves as a focal point for the faculty and for fostering interactions among students and other investigators doing research in neuroscience.

Astrocytes extend highly branched processes that ensheath excitatory synapses, providing a barrier to diffusion and the means to localize transporters near sites of release. This tripartite structure, consisting of presynaptic and postsynaptic elements and associated astrocyte processes, limits interactions between densely packed synapses, and allows astrocytes to modulate synaptic signaling through the release of neuroactive molecules (gliotransmitters) in response to a rise in intracellular Ca2-H. Despite the many in vitro studies that have implicated astrocytes in synaptic plasticity, our knowledge about their roles in synaptic modulation in vivo is limited, in part, due to difficulties associated with monitoring and manipulating astrocyte activity in the intact CNS. Due to its uniform structure and accessibility, the cerebellar cortex offers many advantages for analyzing neuron-astrocyte interactions. This proposal will use in vivo two photon imaging, in combination with newly developed transgenic mice that allow cell-specific expression of genetically encoded Ca2-H indicators, to monitor Bergmann glia activity in response to voluntary movement. The mechanisms responsible for initiating these events, such as activation of Ca2+ permeable AMPA receptors, will be evaluated by selective disruption of AMPA receptor signaling in Bergmann glia. A further goal of these studies is to investigate the involvement of the Ca2-H release-activated Ca2-H (CRAC) channel complex in generating these transients, by genetically deleting Orail and STIM1 from Bergmann glia. The effects of disruption of this robust form of signaling on motor coordination will be evaluated to assess the broader consequence of Ca2+ signaling in these glial cells. These studies will serve as a crucial template with which to understand the role of astrocytes in modulating excitatory synapses in other brain regions relevant for mental health.

PI: Li, Xingde
Proposal: 1430040
Title: MRI: Development of Ultralight Biophotonic Neuro-cellular Imaging Platform for Elucidating Brain Activity in Freely-behaving Animals

Significance
Development of this imaging platform will positively impact human lives, particularly in the areas of health care, safety, and human assistance, due to its portability, miniature probe design, and ability to operate in multiple modalities. The neuroscience research
will impact education, rehabilitation, and mental-health. The project involves people at many career stages, offering a rich multidisciplinary environment that integrates science and engineering. Detailed system designs and research results will be widely disseminated, accelerating the pace of neuroscience discovery. Exposure to this platform will allow scientists to extend their research into new physiological and pathological contexts. Moreover, the proposed imaging platform has significant clinical translation potential, as it enables minimally invasive visualization of cellular characteristics in vivo, in situ and in real time, without the need for tissue removal, which can be readily adapted for diagnostic analysis of other organ systems.

Technical Description:
This Major Research Instrument (MRI) project aims to develop an ultracompact, portable and head-mountable imaging platform, which will enable imaging of brain activity with subcellular resolution in freely behaving animals. The core of the platform is a novel fiber-optic, scanning two-photon fluorescence endomicroscope which is not commercially available. This ultra-microscope is capable of visualizing cellular activity with an imaging quality approaching that of large bench-top microscopy systems. In addition, a small footprint, tunable femtosecond Ti:Sapphire laser will also be developed and integrated to provide a portable platform for two-photon excitation. To facilitate identification of specific brain regions for high resolution imaging, a 2-mm fiber-optic confocal probe with a large field of view (FOV) will be developed and integrated into the platform; this confocal probe will be exchangeable with the two-photon ultra-microscope through the same head-mount base. The confocal probe also enables high-speed imaging of cellular dynamics, complementing the two-photon endomicroscope. The end result of this development effort will be a unique, highly adaptable imaging platform for shared-use, which can be transported to specific facilities/labs across JHU to address specific research questions. The imaging platform will be developed in close collaboration with four research labs at JHU that are engaged in studies of cellular and subcellular dynamics in the intact brain and whose studies have been limited by existing microscope-based imaging platforms.

? DESCRIPTION (provided by applicant): Approximately 5,596,000 people in the United States live with some form of paralysis; among these, 1,275,000 are paralyzed as the result of a spinal cord injury (SCI). The lack of treatment results in a constantly growing population. Each year, SCI cost to the health care system is roughly 40.5 billion. SCI triggers profound changes in the behavior of glial cells, which limit secondary injury and influence regeneration. However, the distinct roles of different glial cell types in the recovery from SCI are not well understood. NG2+ glial cells, a class of glial progenitors that generate oligodendrocytes in the developing and adul CNS, are rapidly mobilized following SCI; they increase their proliferation and migrate to the site of injury. The consequences of this recruitment of NG2+ cells are unknown. Although the behavior of these cells has been viewed primarily in the context of oligodendrocyte regeneration, recent studies suggest that they may play additional roles in the pathophysiology of SCI. In this exploratory research grant (R21) we propose to examine the fate of these highly dynamic cells and evaluate their contribution to functional recovery using in vivo genetic lineage tracing, in vivo two photon imaging, and in vivo selective cell ablation in a clinically relevant model of contusion-induced SCI. The knowledge gained from these studies may reveal new therapeutic strategies based on manipulation of this endogenous pool of progenitors as well as targeted manipulation of NG2+ cells (also termed oligodendrocyte precursor cells (OPCs) engineered for transplantation therapy.

? DESCRIPTION (provided by applicant): The Neuroscience Training Program (NTP) at The Johns Hopkins University was established in 1983 to provide students with advanced instruction and research training in the neurosciences. It now includes 85 training faculty in 18 different departments across the university, as well as several associated institutes where neuroscience research is performed. The program encompasses a broad array of research areas, including molecular, cellular, developmental, sensory, systems, cognitive and computational neuroscience, as well as neurobiology of disease, providing diverse training options and unique opportunities for collaboration for our students. We typically matriculate 10-12 PhD candidates each year, from a pool of ~300 applicants, and 1-4 additional candidates for combined MD/PhD degrees (who are admitted through a separate process). Students enter the program with diverse backgrounds ranging from computer science to biochemistry. To ensure that they learn the basic tenets of neuroscience, they are required to take a year-long integrative lecture and laboratory course, Neuroscience and Cognition, as well as statistics and a perspective/orientation course, Science, Ethics and Society. Students learn about research opportunities through a mini-symposium series led by Program Faculty (featuring short chalk talks), the Departmental Retreat, and Lab Lunches (which feature work-in-progress by NTP faculty). This information is used to help students arrange three 12-week laboratory rotations, which are typically completed by the end of the first academic year, and form the basis for selecting a thesis advisor. By the end of the second year, students have completed five elective courses, from 19 small seminar-style courses in different neuroscience specialties or relevant courses offered in other departments. Following completion of a comprehensive oral exam in the spring of Year 2, students write and defend a Thesis Proposal that is written in the form of a Predoctoral NRSA application. Each student is advised by two Pre-thesis Advisors in Years 1-2 (at 3 month intervals) and an individualized Thesis Advisory Committee thereafter (at 6 - 12 month intervals). Thesis Advisory Committees report student progress to the Graduate Program Steering Committee, which carefully tracks the advancement of each student in the program and establishes overall program policy. At present, 84 students are enrolled in the NTP. The average time to complete the PhD for the past ten years is 5.9 years. Of the students who have graduated from our program, 92% are pursuing careers in science or medicine. Here, we request stipend support for five students during their first two years in the program.

SUMMARY: Multiphoton Imaging Core The goal of the Multiphoton Imaging Core (MPI Core) is to provide NINDS-supported Primary Center Investigators, and other neuroscientists at JHU who perform research consistent with the mission of NINDS, access to advanced imaging technology and training to further their ongoing research programs. In particular, the MPI Core will help investigators analyze protein localization, protein dynamics, and protein-protein interactions with high temporal and spatial resolution in situ and in vivo; perform in vivo time-lapse imaging of multiple fluorophores to monitor cell dynamics; combine imaging of ion indicator dyes/proteins with physiological monitoring of electrical activity to assess cellular communication; obtain quantitative information about neuronal morphologies in situ in health and disease to define cell identity and cellular networks; and enable neuronal and glial activity to be monitored in freely moving rodents. This Center grant will provide funds necessary to support two full-time technicians who will manage day-to-day Core activities, provide training and assist in data collection and analysis. It will also support service contracts to maintain Core equipment, and funds to purchase supplies and additional microscope components (e.g. objectives, stages) to adapt existing instrumentation to the needs of its users. In the next funding period, we will substantially expand the capabilities of the MPI Core, by providing access to fiber optic imaging platforms that enable fluorescence imaging in awake, freely moving rodents, by adapting the custom two photon microscope for resonant scanning to enable rapid collection of 3D datasets and reduce motion artifacts, and by establishing a brain clearing and light sheet imaging service to define cell distribution and morphology in the intact CNS. These additional capabilities will allow Core users access to the most recent technology for observing cell structure and cellular activity to determine how these features influence normal behavior and neurological disease. The unique capabilities of the MPI Core will continue to serve as a crucial resource for NINDS-funded investigators at JHU by extending the research capabilities of individual investigators and by serving as a focal point for dissemination and practical training in advanced microscopic methods. Through these efforts, the MPI Core will help establish new strategies for studying brain function in health and disease that will benefit the broader neuroscience community.

? DESCRIPTION (provided by applicant): Auditory circuits in the mammalian CNS exhibit robust spontaneous activity before the onset of hearing. This activity originates within the developing cochlea and is dependent on excitation of inner hair cells (IHCs); however, little is known about the molecular mechanisms responsible for initiating spontaneous IHC activity or the consequences of this activity for development of auditory circuits in the brain. The studies outlined in this proposal will use in vivo genetic manipulations of key components of this pathway in combination with electrophysiological studies in isolated cochleae and in vivo imaging in unanesthetized mice to test the hypothesis that IHC activity is induced by the periodic release of ATP from inner supporting cells (ISCs). These studies will define the autoreceptor(s) responsible for detecting ATP and the mechanisms by which ISCs induce depolarization of nearby IHCs, events that ultimately trigger bursts of action potentials in spiral ganglion neurons (SGNs) and synchronous activity of neurons in central auditory circuits. The global activity patterns exhibited by auditory neurons before hearing onset will be defined in the inferior colliculus and primary auditory cortex (A1) during normal development and when sensory-independent activity from the cochlea is disrupted, providing fundamental new insight into the role of this cochlea-dependent activity in maturation of auditory circuits and the response of these circuits to deficits in activity during this crucial developmental stage. Changes in network activity in response to the loss of connexin 26, mutations of which are a major cause of non-syndromic deafness, will be examined to determine how disruption of gap junctional coupling among cochlear supporting cells alters the patterns of activity carried through nascent auditory circuits. In addition, the role of spontaneous activity in the refinement of dendritic and axonal projections of SGNs will be assessed through selective in vivo genetic manipulations of cells in the cochlea. These studies will provide greater insight into the fundamental mechanisms used to shape the circuits that process sound information.

Summary: To sustain neural signaling and enable plasticity throughout life, the nervous system relies on homeostatic interactions with a diverse population of glial cells, which create and modify the extracellular matrix, remove ions and neurotransmitters, provide metabolic support and promote functional reorganization of circuits in response to changing patterns of activity. Although most studies of glial cells have focused on the three main classes of glia ? astrocytes, oligodendrocytes and microglia ? the mammalian brain also contains an abundant, highly dynamic population of glial progenitors termed oligodendrocyte precursor cells (OPCs or NG2 glia). These lineage restricted progenitors play crucial roles in generating oligodendrocytes to enable production of new myelin sheaths during motor learning and replacement of myelin destroyed through disease, such as multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). OPCs remain widely distributed in gray and white matter throughout life, but exhibit profound changes in behavior with aging, including reduced proliferation and differentiation. Emerging evidence suggests that OPCs do more than serve as progenitors for oligodendrocytes, as they are found in regions where there is no myelin, and like microglia, OPCs migrate to sites of injury and contribute to scar formation, features unrelated to their role in oligodendrogenesis and myelin repair. OPCs share many other features with microglia ? they are present at a similar density, maintain a grid-like distribution, possess ramified, radially-oriented processes and are highly dynamic, continuously exploring their surrounding environment with motile filopodia. Moreover, recent evidence indicates that OPCs can transform into inflammatory OPCs (iOPCs) that engulf and present exogenous antigens through MHC class I and II when exposed to inflammatory cytokines, suggesting that they may modulate tissue inflammation. While microglia and astrocytes are known to undergo phenotypic changes in response to inflammation that profoundly influence the aging brain, much less is known about the role of OPCs in this context, despite their persistence in brain circuits. In part, this lack of knowledge stems from the limited molecular and physiological interrogation of OPCs that has been completed in vivo. OPCs are underrepresented in available single cell RNA-seq datasets and there have been no studies specifically designed to define how changes in their properties with aging are influenced by their prior behavior. We will leverage a diverse array of methodologies and our combined expertise in physiology, molecular and computational biology to define the causes and consequences of age-dependent changes in these ubiquitous glial cells. The new insight provided by these studies may lead to a deeper understanding of the homeostatic roles performed by these glial cells, and reveal new approaches for rejuvenating their regenerative potential to sustain brain function throughout life.