Anthony J. Ricci - US grants

Adaptation is a process by which hair cells extend their dynamic range, reducing saturation and preventing damage. A tonographic distribution in the adaptation rate of the mechano-electric transducer (MET) current has demonstrate in hair cells of the turtle auditory papilla leading to the suggestions that the rate of adaptation serves as a high pass filter to mechanical stimulation. The purpose of this proposal is to substantiate the filtering properties of adaptation and to elucidate the underling mechanisms involved in establishing this gradient. The possible mechanisms to be addressed include: (i) a distribution in the effective calcium buffering in the stereocilia of hair bundles along the papilla, (ii) a change in the Ca/2+ load per stereocilium, either by varying the number of MET channels per stereocilium or by a variation in the relative calcium permeability of the MET channel across the papilla, (ii) a change in the Ca2+ load per stereocilium, either by varying the number of MET channel across the papilla and, (iii) a change in the relative force exerted by the adaptation processes across the papilla. These possible mechanisms will be addressed by recording the MET currents from hair cells in a newly developed Intact turtle auditory papilla preparation using both whole cell and perforated patch techniques (i-iv). Both displacement-clamp and force-clamp stimuli will be employed. Single-channel measurements, calcium imaging experiments and flash photolysis experiments will be incorporated to better resolve the mechanisms involved in the regulation of calcium homeostasis and the adaptation process. Recently cyclic nucleotides have been reported to shift the set point of the displacement sensitivity of the hair bundle by some unknown mechanism. A fourth topic of study for this proposal will be to characterize the cyclic nucleotide effects and to determine the mechanisms of action. Understanding calcium dynamics in the hair bundle as well as the cyclic nucleotide effects on adaptation have broad implications into the physiologic function of auditory and vestibular hair cells and may underlie the large frequency range at which hair cells are capable of responding. Pathophysiologic implications of alterations in calcium homeostasis in the stereociliary bundle may be linked to both temporary and permanent threshold shifts and noise induced hearing loss.

DESCRIPTION (provided by applicant): The primary goal of this proposal is to develop a short-term culture of mature mouse cochlea. This is a new area of interest for the investigator in that the major model in the laboratory at present is the turtle auditory papilla. The development of this procedure will open avenues to the investigation of transgenic animals as well as allow for molecular manipulations of the tissue in the short term to investigate the physiology of hearing at the molecular level. The usefulness of this preparation is multifaceted. It can be used to explore mechanical, electrophysiological and synaptic properties of both sensory and nonsensory cells. By establishing a culture system that survives for several days gene expression studies using viral transfections or gene gun technology can be incorporated into physiological studies. In addition, the time course of processes leading to ototoxicity, either pharmacological or environmental (i.e. noise exposure) can also be investigated. Additionally, working with mouse cultures will allow for knockout/knockin mice to be investigated. This proposal attempts to lay the groundwork for these types of investigations by studying the mechanical and electrophysiological properties of mouse sensory hair cells maintained in an organotypic culture for up to four days. The specific aims are first to determine the most mature age that the organ of corti can be dissected from the mouse inner ear and viable electrophysiological recordings obtained at 35 degrees Celsius. Secondly, tissue from the age determined in specific aim 1 will be placed into culture for up to four days. Basic electrophysiological measurements including the complement of basolateral conductances and mechano- electric-transducer currents will be measured from cells at specific positions along the cochlea over time of culture to optimize conditions that maintain viability but do not promote differentiation of cell properties. This work qualifies as a feasibility study because mature mouse cochlea have not been successfully cultured, nor have electrophysiological measurements been made at physiological temperatures. Although several obstacles exist, if overcome, this preparation has great potential for exploring both the physiological and pathophysiological processing in the inner ear.

DESCRIPTION (provided by applicant): A mechanical tuning mechanism located in the sensory hair cell bundle and intimately associated with the mechano-electric transducer (MET) channels has been described. Termed fast adaptation, this calcium-dependent process has been postulated to underlie in part the cochlea active process, the mechanism responsible for the exquisite sensitivity of the auditory system. Perturbations of this system might result in elevated thresholds, temporary threshold shifts and tinnitus. Understanding the mechanisms responsible for the generation and regulation of adaptation of mechano-electric transduction is therefore critical if the long term goal is to design therapeutic treatments for these maladies. To this end, experiments are designed to quantitatively address several critical issues pertaining to the generation and regulation of the ton topic variations in fast adaptation. The first goal is to determine if intrinsic differences in MET channels exist between high and low frequency cells, specifically focusing on channel kinetics and single channel properties. An interaction between MET channels, probably through summation of intraciliary calcium has been postulated as a mechanism underlying the tontopic differences. Experiments are designed to directly test this hypothesis by coupling multiphoton imaging with electrophysiological recordings. A slower component of adaptation has been described that results in an increase in hair bundle compliance. This slow component may serve to prevent saturation and mechanical damage of the sensory hair bundle. Preliminary data suggests this component may be triggered by an intracellular release of stored calcium, and perhaps operate via a myosin motor, experiments are designed to characterize the mechanisms responsible for generating and the biochemical regulation of this slow form of adaptation. Hair cell calcium channels regulate membrane excitability and dictate transmitter release. Differential regulation of calcium channels based on which function the channels serves may be an important tool for signal processing. Characterization of the biophysical, pharmacological and biochemical properties of these channels is the fourth goal of this project and should yield some exciting new information regarding signal processing.

[unreadable] DESCRIPTION (provided by applicant): The hair cell afferent nerve synapse is critical for audition. Loss of function of this synapse leads to hearing loss and deafness. This synapse is highly sensitive to noise induced, age related hearing loss as well as ototoxic agents. Fundamental questions remain to be addressed regarding synaptic specializations and mechanisms of regulation of presynaptic release and post synaptic integration. These questions require that several technically challenging problems be addressed. The purpose of this proposal is to develop and implement these technologies. The first problem is that enzymatic treatments used in either isolating cells or removing tectorial or otolithic membranes alter synaptic properties and can in many instances eliminate receptors. Specific Aim (SA) 1 focusses on refining a nonenzymatic technique for removing tectorial membranes while maintaining functioning hair bundles. The technique involves using high divalent concentrations to soften the membrane and hair bundle connections to this membrane in order to eliminate mechanical perturbation of the hair bundle as the membrane is removed. The second issue is that present methods used to track membrane capacitance changes (as indicators of vesicular release) are limited in that they give only a steady-state response. Conductance changes, like calcium channel activation, compromise the capacitance measurement. SA 2 applies a dual sine wave technique, developed by Santos Sacchi, (2005) for measuring nonlinear capacitance in outer hair cells to estimate vesicle release. We will use the standard technology that we have mastered to compare the two techniques and determine the resolution and limitations of the dual sine wave method. This technique has the potential of allowing the kinetics of exocytosis to be determined directly and compared to the activation kinetics of the calcium current. The third obstacle is simultaneously recording from hair cells and primary afferent nerve. SA3 will develop this ability. We presently can record from each independently and maintain mechanical sensitivity of both the hair cell and synapse. We will then apply the capacitance techniques of SA2 with the recording additions of SA3 to directly measure EPSCs and correlate these amplitudes with the corresponding capacitance changes measured presynaptically. Together these technologies will allow us to determine the underlying mechanism responsible for multivesicular release. [unreadable] [unreadable] [unreadable]

Deflection of the sensory hair cell's hair bundle (mechanotransduction) results in an electrical response from the hair cell that is the primary signal for audition. Disruption of this pathway by noise, ototoxic agents or aging leads to maladies such as threshold shifts (both temporary and permanent), hearing loss, deafness at an extreme and may even underlie some forms of Tinnitus. Understanding the mechanisms involved in the mechanotransduction process should help identify sites for intervention and prevention of hearing loss and may also provide guidelines for development of replacement therapies such as hair cell regeneration. Although a great deal of knowledge regarding mechanotransduction has accrued over the past 25 years, many fundamental questions regarding mechanisms and functions remain to be elucidated. Experiments herein will use state of the art optical and electrical techniques coupled with tissue culture and pharmacological tools to probe several basic questions regarding hair cell mechanotransduction. Specific Aim (SA) 1 will determine whether the functional mechano electric transduction (MET) channels are located at the tops or along the sides of the stereocilia by taking advantage of the morphological arrangement of inner hair cell bundles. It will determine the number of channels per stereocilia by coupling calcium imaging of individual stereocilia with measurements of MET current. Finally, it will determine the role of membrane tension on channel gating which should give insight into whether the MET channel is directly tethered to the tip-link or cytoskeleton or whether it is activated via membrane stretch. SA2 will investigate the Ca2+ dependence of adaptation and activation by coupling Ca2+ imaging, Ca2+ uncaging and electrophysiological measurements of MET currents. These experiments will attempt to separate calcium-dependent responses from mechanical stimulation and should yield important new kinetic information regarding the calcium regulation of transduction and adaptation. Experiments will also explore the Ca2+ dependence of hair bundle mechanics. SA3 will investigate single channel MET properties to determine the mechanism by which Ca2+ regulates channel conductance and open time. SA4 will test the hypothesis that MET provides mechanical tuning to the hair cell.

The imaging core has four independent workstafions (Image Stafions 1-4), each incorporafing overlapping but independent imaging technologies. Three of four can be used for live cell imaging and two of four will have complementary electrophysiological recording stafions that allow the combinafion of powerful imaging and electrophysiological technologies. From an imaging perspective, the core facilities can accommodate multi-fluorophore immunocytochemical high-resolufion 3-dimensional imaging while also serving high speed physiological imaging. Addifionally, there are two dedicated computers with high end graphics boards, ample memory and processing power that can be used for image analysis offline from the imaging stafions. From conventional fluorescence and confocal imaging to high speed swept field confocal and two-photon imaging and uncaging, the core facility will serve to bring together ROI-funded invesfigators with a broad range of interests and expertise. The core will facilitate the use of this equipment so that translafional uses by the clinical faculty can be increased while at the same fime having the versafility to include users with common interests from mulfiple departments that share goals that are in agreement with the mission statement of the NIDCD. Clear examples of each of these uses exist already with the two-photon system being used to develop whole animal cochlea imaging that hopes to be ufilized in cochlear implant placements, while both the high speed swept field confocal system and the two photon system are presently being used to invesfigate synchronous firing in the cortex. This core grant proposal will allow us to expand the number of users and thus serve as a means to increase collaborafion across both basic science and clinical departments

Abstract: The peripheral auditory system communicates with the central nervous system through synapses between the sensory cells and the spiral ganglion neurons. This single synapse carries information regarding frequency, intensity and timing that is used by the CNS for to extract all information provided by the auditory modality. The synapse is specialized to operate across a broad frequency range, to respond to graded stimuli in a linear manner. The synapse maintains high rates of release and does not fatigue and is sensitive to receptor potentials often below 1 mV. The goal of this proposal is to delineate the mechanisms underlying these unusual synaptic properties. Both pre and postsynaptic mechanisms will be investigated. Specific Aim 1 addresses presynaptic issues and has three subaims; to identify the Ca2+ dependent components of vesicle release and trafficking, to determine the kinetics of release while identifying the rate limiting steps of trafficking and release, and to elucidate tonotopic differences in release properties, characterizing Ca2+ homeostatic mechanisms that regulate release and identifying phosphorylation dependent regulation of release and trafficking. Specific Aim 2 investigates postsynaptic processing with three goals; first to determine if there are tonotopic variations in afferent electrical properties that might serve to enhance frequency selectivity such as the kinetics and magnitude of voltage-dependent processes, second to characterize synaptic transmission at different locations to identify differences in receptor types and numbers and third to investigate mechanisms associated with multivesicular release. Structural, immunocytochemical, optical, electrophysiological and theoretical tools including several new technologies are included to better enable us to investigate these issues. Given that any deficits in synaptic transmission at the hair cell afferent fiber synapse results in auditory deficits, identifying novel mechanisms may provide unique avenues for intervention.

DESCRIPTION (provided by applicant): We are proposing to purchase a two photon mating system that is specialized for microendoscopy. The gradient refractive index (GRIN) lens microendoscope allows for imaging of previously inaccessible tissue. For our research core, this means imaging the cochlea, though this is not exclusive. Along with this system, we need to incorporate the ability to assess quality of hearing, which includes, ABR, distortion product otoacoustic emissions and tympanometry. Additionally, the ability to make electrophysiological measurements is required and so micromanipulators and an amplifier are requested. This device will expand our imaging core so that whole animal hearing experiments can be performed at the cellular and systems level. This technology will give us unprecedented access to living cochlea tissue, greatly expanding and enhancing ongoing research projects within this core. To date, most measurements of cochlea mechanics are single or dual point measurements at the level of the basilar membrane. This new technology allows imaging of multiple cells or cell parts, like sensory hair bundles, tectorial membrane, etc. Proof of principal experiments have been performed with a consultant and expert on GRIN lens (Mark Schnitzer) but are limited by being in a laboratory not equipped for acoustic isolation and on a system that is not dedicated to these experiments. This device has the potential to revolutionize auditory research in the whole animal and will expand the resources of our imaging core to incorporate imaging from the molecular to cellular to system. The technology is not limited to cochlear exploration and as demonstrated in the research projects section can be used to investigate heretofore inaccessible structures in living animals, such as deep brain regions and nasal mucosal cells. Together these projects incorporate 6 presently funded RO1s from 4 investigators representing five different departments. Also there are presently three junior investigators actively pursuing this technology. Part of the mandate of our core facility is to provide access and training to graduate students and residents and so the addition of this technology will greatly enhance training. In addition, this technology allows direct translational experiments to be performed in the whole animal. We have significant departmental support funding the service contract for the instrument, support personnel involved in the training and maintenance of the equipment and the setup costs for the equipment. PUBLIC HEALTH RELEVANCE: Hearing loss induced by noise exposure, aging or chemicals reduce quality of life and productivity of millions of people each year, costing our economy billions of dollars. The ability to directly assess the actions of chemicals or noise on the sensory tissue has long been hampered by the inability to access cochlear tissue, as it is surrounded by bone. The equipment requested, a two photon imaging system using microendoscopy allows minimally invasive surgery to result in our ability to directly monitor cochlea tissue in vivo and thus will expedite our understanding of the mechanisms associated with these forms of hearing loss.

Abstract: Aminoglycoside (AGs) antibiotics are used worldwide because of their potent antimicrobial activities and low cost. They are widely used despite the significant side effects of ototoxicity and nephrotoxicity. Recent evidence from independent laboratories demonstrated that AGs accumulate rapidly in hair cells because of their ability to enter these cells through the mechanoelectric transducer channel located near the tops of the stereocilia. Channel biophysical properties promote entry through the channel but limit exit through these same channels. Our data clearly show that ototoxicity can be prevented by blocking entry via these channels. The goal of this proposal is to develop novel non-ototoxic aminoglycosides. Our team has unique insights into the biophysical properties of the mechanically gated channels and can use this knowledge to design compounds that are sterically and/or electrically restricted from entering the channel and therefore the hair cell. Sites for modification are selected on the AG backbone so as not to interfere with antimicrobial activity. We have in hand the ability to investigate these compounds at the channel, cellular, end organ, system and whole animal level, using electrophysiological, optical, molecular and pharmacological means to monitor ototoxicity as well as antimicrobial activity. Complementary to the development of these compounds will be identifying the mechanism of entry of the AGs into the endolymph compartment. Upon identification of this pathway we will devise means to limit transport of existing AGs so that a co-treatment plan might prevent access of the AGs to the MET channel and thus limit entry into hair cells. The focus of this proposal is to design treatment regimes, either by creating novel AGs, or co-treatment plans, that will ameliorate ototoxicity due to AG administration. At the end of the proposed two-year research period, we will have applied our unique knowledge base and skill sets to gain insights into the effectiveness of either of these approaches in reducing ototoxicity caused AGs.

DESCRIPTION (provided by applicant): There is not yet a single unifying theory of cochlear amplification consistent with the organ of Corti cytoarchitecture, basilar membrane mechanics, and otoacoustic emissions (OAEs). Our central hypothesis is that the systematically organized Y-shaped structural elements between the reticular lamina and basilar membrane in the organ of Corti collectively form a mechanism for cochlear amplification in the best-frequency region of the basilar membrane, in which the angled outer hair cells (OHCs) provide an accumulating feed- forward force directed apically, and the oppositely angled phalangeal processes provide a feed-backward force directed basally. The feed-forward and feed-backward (FF/FB) amplification theory will be tested using anatomically realistic fluid-coupled 3D finite element computational models for the mouse and gerbil organs of Corti, constructed from two-photon and confocal microscopy images. After validation against previous physiological measurements and modeling results, the new models will be used to test the effects of FF/FB forces on cochlear amplification, as well as the effects of tectorial and basilar membrane mechanics. The hypothesis that the FF/FB amplifier concepts are compatible with theories and measurements of stimulus- frequency OAEs and distortion-product OAEs, and that selective modifications to the structure of the organ of Corti will produce predictable results, will be tested both in the model and experimentally using wild-type mice and alpha-tectorin protein mutated mice (TectaC1509G/+) that feature a shortened tectorial membrane. Cochlear models have typically assumed that the OHC force output is proportional to the stereociliary force input, with a gain ? assumed to be independent of cochlear location and frequency. We will improve upon this by creating a model for the OHC receptor potential that accounts for the basolateral conductances and cell wall capacitance, which we will then combine with an anatomically and physiologically realistic model for somatic motility in order to determine realistic values for ? as a function of location and frequency. The resulting ?(x,r,f) model will then be integrated into our FF/FB modeling frameworks as a further test of our central hypothesis. The scientific contributions stemming directly from this research are expected to be 1) a detailed 3D description of the Y- shaped elements in the organ of Corti across the different OHC rows, from base to apex; 2) an incorporation of this information into computational models for testing the FF/FB amplifier theories with realistic anatomy; 3) an improved understanding of how mechanisms of OAE generation and propagation relate to the FF/FB amplification theory; and 4) an enhanced understanding of the contributions of OHC basolateral conductances, receptor potential, and somatic motility to cochlear amplification. The resulting models will provide powerful new tools for future cochlear mechanics studies, including those involving normal, genetically modified, and regenerated mouse cochleae, and, due to homologs between humans and mice, the human cochlea as well.

DESCRIPTION (provided by applicant): Models of hair cell mechanotransduction have existed for more than 30 years based on groundbreaking work done in lower vertebrates. The models, largely based on physiological data, have shaped the thinking about hair cell mechanotransduction for a generation. Recent advances both challenge and support these basic tenets by providing new insights into the molecular machinery involved in mechanotransduction. Technological advancements now allow us to investigate mechanotransduction at the single cilia and molecular level. This is necessary to bring new data into perspective with existing, traditional theories. During the past funding period, we made use of the technological advancements and developed new methods that will allow us to directly probe mammalian mechanotransduction with unprecedented resolution. These methods include stimulation of hair bundles at high rates, imaging of bundle motion at high rates (>250kHz), imaging of fluorophores with a swept field confocal system at higher rates (500-2000 fps) and we have expanded our ability to do electrophysiology, and live hair bundle imaging followed by immunohistochemical or field emission SEM on these same bundles. With these tools we will determine the functional significance of tonotopic variations observed in the mechanotransduction process, testing the hypothesis that activation and adaptation kinetics provide tuning to outer hair cells. We will directly probe the function of USH1 syndrome proteins localized to the upper tip-link insertion point, near the tops of stereocilia. We will address new hypotheses regarding the function and molecular underpinnings of slow adaptation. We will directly couple morphological measurements with functional outcomes to determine how inner hair cell hair bundles are coupled together. Doing this is fundamental to understanding the complexity that is the mechanotransduction process.

? DESCRIPTION (provided by applicant): All auditory information received by the central nervous system is transferred across the inner hair cell - afferent fiber synapse. Dysfunction of this synapse due to aging, noise or genetic disorders reduces hearing dramatically. This remarkable synapse must transfer intensity and timing information faithfully for long periods of time. Ribbon synapses, like those in hair cells, are typically found in cells that use graded receptor potentials and need to have sustained release. The hair cell ribbon synapse is more robust than most, releasing at higher rates and sustaining release for longer periods of time. In order for synapses to operate in this manner they must have a robust means of recycling and replenishing vesicles to synaptic release sites. We have identified a nonlinear calcium dependent capacitance change that we hypothesize is a reflection of vesicle recruitment and replenishment. Our first major goal (Aim 1) is to determine whether this nonlinear change is in fact synaptically based and we will do that using paired recordings to identify a postsynaptic response, using a new glutamate sensor to determine if glutamate release mimics the capacitance response and by inactivating ribbons optically to see if we can ablate this nonlinear component. We have also identified a calcium induced calcium release process (CICR) associated with the nonlinear capacitance change. We are hypothesizing that vesicle trafficking is calcium dependent and that CICR is used as a means of ensuring continual vesicle supply while calcium at the synapse is tightly regulated. We will use optical, electrophysiological and immunocytochemical tools to investigate this possibility in aim 2. Otoferlin is a unique protein implicated to regulate vesicle fusion, trafficking and reuptake. We are postulating that biochemical modification of this protein via phosphorylation by CaM Kinase II or ubiquitination is responsible for targeting these different functions. We will investigate this hypothesis using electrophysiological, optical, molecular and immunocytochemical technologies.

? DESCRIPTION (provided by applicant): Aminoglycosides are a widely used broad spectrum antibiotic. A major side effect of these compounds is oto- (and vestibulo) and nephrotoxicity. Data obtained during an R21 award demonstrates that these compounds enter sensory hair cells of the inner ear via a specialized mechanosensitive ion channel in vitro. Using our unique knowledge of the biophysical properties of the mechanotransduction channel coupled with insights provided by crystallography data that identifies aminoglycoside interacting sites on the bacterial ribosome, we have developed novel compounds that greatly reduced oto- and nephrotoxicity in vivo, though at some cost to the breadth of antimicrobial activity. These proof-of-principal experiments serve as the basis for iteratively designing new compounds. The design component has three phases, first to use our initial screen as a starting point where we will alter the substituted group on sisomicin to try to better separate antimicrobial activity and the toxic side effects. Second we will synthesize and test a novel modification site based on new data arising from experiments on gentamicin derivatives. Finally, we will change the parent compound to better target specific microbes such as Pseudomonas organisms. We have developed the ability to test for purity and specificity of each compound. We can probe activity in terms of antimicrobial function as well as oto- and nephrotoxicity in in vivo and in vitro models. We can also test for sensitivity to the development of resistance and for vestibular pathologies. Together this iterative ability based on a novel hypothesis should yield a new class of nontoxic antibiotics.

Abstract: Age related hearing loss (ARHL) has a major impact on quality of life of more than 70% of those over the age of 70. It is a growing problem with significant socioeconomic ramifications. The cause of ARHL is complex and likely involves both environmental and genetic factors. Symptoms associated with ARHL, specifically, the initial loss of ability to discriminate sound in a noisy environment that progress to high frequency loss that can further progress to lower frequencies over time, suggest a common vulnerability that is independent of the initial trigger. We are postulating that common to ARHL is an initial loss of hair cell afferent fiber synapses. Much as with noise induced hearing loss, we hypothesize a selective loss of high threshold fibers. We will test these hypotheses using multiple genetic mouse models of ARHL. Each will be monitored using whole animal auditory testing including auditory brainstem responses and distortion product otoacoustic emissions to document the time course of onset of ARHL in each model. Specific Aim 2 will evaluate synaptic maintenance and proteins associated with pre and postsynaptic elements to further characterize the temporal windows associated with hearing loss. Specific Aim 3 will use electrophysiological and optical technologies to measure synaptic vesicle release and trafficking, monitor calcium homeostasis and record postsynaptic properties synaptic potentials as well as electrical properties. Each aim is well integrated with the goals and techniques available from each of the other investigators on this proposal and together we shall identify a global framework in which to address this important translational problem.

The Stanford Neuroscience Training Program remains the only doctoral degree granting entity for Neuroscience at Stanford. This interdisciplinary program consists of 91 students and 99 faculty from 28 departments (11 clinical, 17 basic scientists) and 4 schools. The breadth of departments illustrates the breadth of research areas which span molecular/cellular to systems and behavior, from human cognition to translational work. The training grant and its implementation is the central funding source, with 14 slots, and the foundation of the program. Our mission is to identify, recruit and train predoctoral PhD students to become the next generation of leaders in neuroscience at all societal levels. This training plan has four components: curriculum, research, mentoring and leadership, typically accomplished in under 6 years. The curriculum uses best practices in teaching to provide a foundation in neuroscience that allows for the rigorous identification of a scientific question, design, implementation and analysis of a research project culminating in an independent publication. A core module system challenges students to learn how different fields approach scientific problems. Students participate in journal clubs, a recurring responsible conduct and ethics course, and higher- level courses specifically governed by the interests and needs of each individual student. Research begins with rigorous and challenging rotations that allow students to explore new research areas and new technologies. From these rotations, students select a research laboratory to do their thesis work that is supervised by a faculty mentor and committee of experts. Training in experimental design, rigorous data collection and statistical analysis, is achieved through both didactic course work and direct application to their own work. Our mentoring approach is that it takes a village. From day 1 students have a First Year advisor and a senior student advisor. After selecting a laboratory students have a personal faculty mentor as well as a committee of advocates. They also have access to a senior advisory panel of faculty and both individual and group focused peer-mentoring groups. And finally, leadership includes involvement in directing all aspects of the program as student representatives on all committees as well as TAs for major courses, designing and teaching neuroscience courses and engaging in university and community outreach such as Brain Day and Stanford Summer Research Program. Within each component, we include elements for professional development like reading, critically evaluating and writing scientific papers, preparing and presenting scientific presentations to both professional and lay audiences, networking both academically and in industry as well as professional interactions related to biases such as gender, socioeconomic and race. The program's infrastructure combines faculty, student and administrative feedback on all major levels. There is a program committee, chaired by the director that oversees all infrastructure, an admissions and curriculum committee, used to oversee these two major undertakings and an informal advisory committee that includes other university leadership.

The Stanford Neuroscience Training Program remains the only doctoral degree granting entity for Neuroscience at Stanford. This interdisciplinary program consists of 91 students and 99 faculty from 28 departments (11 clinical, 17 basic scientists) and 4 schools. The breadth of departments illustrates the breadth of research areas which span molecular/cellular to systems and behavior, from human cognition to translational work. The training grant and its implementation is the central funding source, with 14 slots, and the foundation of the program. Our mission is to identify, recruit and train predoctoral PhD students to become the next generation of leaders in neuroscience at all societal levels. This training plan has four components: curriculum, research, mentoring and leadership, typically accomplished in under 6 years. The curriculum uses best practices in teaching to provide a foundation in neuroscience that allows for the rigorous identification of a scientific question, design, implementation and analysis of a research project culminating in an independent publication. A core module system challenges students to learn how different fields approach scientific problems. Students participate in journal clubs, a recurring responsible conduct and ethics course, and higher- level courses specifically governed by the interests and needs of each individual student. Research begins with rigorous and challenging rotations that allow students to explore new research areas and new technologies. From these rotations, students select a research laboratory to do their thesis work that is supervised by a faculty mentor and committee of experts. Training in experimental design, rigorous data collection and statistical analysis, is achieved through both didactic course work and direct application to their own work. Our mentoring approach is that it takes a village. From day 1 students have a First Year advisor and a senior student advisor. After selecting a laboratory students have a personal faculty mentor as well as a committee of advocates. They also have access to a senior advisory panel of faculty and both individual and group focused peer-mentoring groups. And finally, leadership includes involvement in directing all aspects of the program as student representatives on all committees as well as TAs for major courses, designing and teaching neuroscience courses and engaging in university and community outreach such as Brain Day and Stanford Summer Research Program. Within each component, we include elements for professional development like reading, critically evaluating and writing scientific papers, preparing and presenting scientific presentations to both professional and lay audiences, networking both academically and in industry as well as professional interactions related to biases such as gender, socioeconomic and race. The program's infrastructure combines faculty, student and administrative feedback on all major levels. There is a program committee, chaired by the director that oversees all infrastructure, an admissions and curriculum committee, used to oversee these two major undertakings and an informal advisory committee that includes other university leadership.