John A. White - US grants

The hippocampal formation, important for the process of laying down new memories, contains large amounts of zinc, located in synaptic vesicles and released with high-frequency stimulation. Furthermore, zinc exerts effects on many ion channels, some of which may be unique to the hippocampal region. Thus, it has been postulated, but not confirmed, that synaptic zinc and specialized zinc-sensitive ion channels act as a novel neuromodulatory system. As the next steps in testing this postulate, experiments are proposed here to test the following four hypotheses: Hypothesis A. The kinetics of zinc-induced block of voltage-dependent conductances are consistent with a neuromodulatory role. The kinetics of Zn2+-induced block of ion channels are crucial for determining whether this pharmacological effect has physiological significance. Using rapid solution-exchange and whole-cell patch-clamp techniques, these kinetics will be measured in dissociated neurons from the medial entorhinal cortex (MEC). Hypothesis B. Zinc-sensitive ionic conductances co-localize with Zn2+- positive nerve terminals in the hippocampal region. If specialized, Zn2+ -sensitive ion channels are the postsynaptic "receptors" of a novel neuromodulatory system, they are likely to be expressed in regions of the hippocampal formation other than the MEC. This hypothesis will be tested by recording from dissociated neurons from two additional Zn2+-rich regions: hippocampal region CA3 and the lateral entorhinal cortex (LEC). Hypothesis C. Zinc-sensitive Na+ channels of the MEC are structurally similar to cardiac Na+ channels. Demonstrating that Zn2+-sensitive ion channels are structurally related to cardiac Na+ channels, and hence different from known brain channels, would argue strongly that these channels are indeed "specialized." The structure of these channels will be probed using well-understood pharmacological manipulations in dissociated neurons. Hypothesis D. Endogenous, synaptically released Zn2+ modulates neuronal voltage-dependent conductances in the hippocampal region. To argue that Zn2+ modulates voltage-gated conductances in the MEC, LEC and region CA3, one must demonstrate that endogenous synaptically released Zn2+ reaches and affects these conductances. This demonstration will be attempted in recordings from hippocampal-entorhinal brain slices. The overall goal of this project - an understanding of the role zinc plays in the functioning of the hippocampal region - may prove crucial in understanding several devastating neurological disorders. Examples of brain disorders to which zinc metabolism in the hippocampal region has been linked include Alzheimer's disease, temporal lobe epilepsy, and stroke-related cell death.

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White
Introduction. Electrical activity in nerve and muscle cells is generated by populations of ion channels that "gate" (open or close) in response to changes in transmembrane voltage and/or the concentrations of crucial chemicals. In studies of the biophysical processes underlying electrical activity, scientists and engineers rely upon two basic recording configurations. In the first recording configuration, commonly referred to as current clamping, the researcher controls the amount of net transmembrane current (i.e., current across the cell membrane) and measures transmembrane voltage. In the second recording configuration, called voltage clamping, the researcher uses an electrical feedback circuit to control transmembrane voltage and measures transmembrane current. Current clamping is useful for characterizing the patterns of electrical activity generated by a given nerve or muscle cell; voltage clamping is useful for studying the biophysical mechanisms underlying a particular pattern of electrical activity.

More recently, a third very useful recording configuration has been developed, in which neither transmembrane current nor transmembrane voltage is the controlled variable. Instead, the researcher uses a sophisticated recording system to mimic in real time the electrical conductance associated with a given population of virtual ion channels. This recording mode, called dynamic clamping, allows the researcher to block native ion channels, and replace them with virtual analogs, the properties of which can be controlled precisely. Dynamic clamping and other real-time-computing-based protocols enable entirely new classes of experiments, in which (for example) the applied stimulus can mimic the behavior of a (blocked) dynamic component of the system, and thus be used to determine unequivocally the effects of the mimicked component of overall behavior.

Dynamic clamping and other real-time experimental techniques show enormous promise as important research tools. Dynamic clamping could be used, for example, to study the electrical effects of computer-designed pharmaceutical agents even before the agents are developed in the laboratory. So far, however, the impact of this technique has been educed by three interrelated difficulties. First, the technical complexities of its design are beyond the skills of most end-users, and no one has yet provided a flexible, powerful, turn-key system. Second, existing dynamic clamp systems do not account for the seemingly stochastic (i.e., probabilistic) nature of voltage-gated ion channels. Adding this capability would allow researchers to attack entirely new sets of exciting problems that are as yet unapproachable. Third, existing dynamic clamp systems cannot account for measured or assumed spatial distributions of ion channels.

Specific Aims. The specific aims of this proposal are (1) to complete construction of a stochastic dynamic clamp (SDC) system that can be used to study the actions of noisy virtual voltage- and ligand-gated ion channels in living cells; (2) to create a web-based system of support to help end-users adopt and use the SDC system for dynamic clamping and other real-time experimental applications; (3) to develop methods for representing virtual ion channels that are remote from the recording site in the cell body, and (4) to use the new SDC system to test specific hypotheses regarding the relative importance of noise from synaptic sources and noise from voltage-gated ion channels in limiting neuronal reliability.

The project will have educational impact at both the graduate and undergraduate levels, in the context of the classroom and research projects. Innovations from Specific Aims 1 and 3 will extend the dynamic clamp method to account for stochastic and spatially distributed channels. The investigators' support (Aim 2) will be a crucial step in developing a turn-key system. The system will be easily adaptable to apply techniques of real-time computation in many biomedical engineering applications. Field tests of the device (aim 4) will push the field of neurobiology in a more quantitative, information-oriented direction. In particular, they will advance the study of the biophysical underpinnings of neuronal reliability, which play a fundamental role in the understanding of coding strategies used by the nervous system. Such advances are not practically achievable without real-time computing technology.

The investigator and her colleagues collaborate in a group
project at the Center for BioDynamics (CBD) to provide
interdisciplinary education and training for graduate students
and postdoctoral-level investigators in the context of a vigorous
interdisciplinary research program that focuses on areas of
mutual interest in mathematics (especially dynamical systems),
biology, and engineering. Disciplines include mathematics,
biomedical engineering, aerospace/mechanical engineering,
biology, psychology, and physics. Training extends beyond the
usual classroom activities by engaging participants in a variety
of research projects as well. One of the major topics is dynamics
of the nervous system. The projects, which involve experiments,
modeling, and analysis, all deal with the variety of rhythms in
the nervous system and the potential functions of these rhythms
in key cognitive states and processes such as attention,
awareness, learning, and recall. A second major topic is
dynamics of gene expression. Progress in genomic research is
leading to maps of the building blocks of biology and fueling the
study of gene regulation, where proteins often regulate their own
production or that of other proteins in a complex web of
interactions. CBD projects focus on using techniques from
nonlinear dynamics, statistical physics, control theory, and
molecular biology to model, design, and construct synthetic gene
regulatory networks, and to probe naturally occurring gene
regulatory networks. The third major topic is the dynamics of
patterns and waves. Training activities include two weekly
working seminars, extra journal clubs and reading groups,
seminars to educate the CBD members in the research going on
within the Center, and a CBD-initiated team-taught course.
The Center for BioDynamics (CBD) helps to advance
understanding of difficult interdisciplinary problems at the
intersection of mathematics, biology, and engineering, and it
trains mathematicians, scientists, and engineers for the 21st
century workforce. It does this by combining traditional
classroom education with significant engagement of students and
postdocs in interdisciplinary teams working on current problems.
The disciplines involved are mathematics, biomedical engineering,
aerospace/mechanical engineering, biology, psychology, and
physics. One of the major topics is dynamics of the nervous
system. The projects in this topic seek to shed light on the
origin of the electrical activity in the brain, and how the brain
uses this activity to process sensory information, to think, and
to regulate movement. A second major topic is dynamics of gene
expression. The web of interactions among the proteins that are
produced by genes is complex; the projects associated with this
topic involve the design and construction of artificial gene
regulatory networks, and techniques to better understand
naturally occurring gene regulatory networks. The third major
topic is the dynamics of patterns and waves, occuring in a
variety of applications. Training activities include two weekly
working seminars, regular sessions to read scientific journals,
seminars to educate the CBD members in the research going on
within the Center, and a CBD-initiated team-taught course. The
project is supported by the Computational Mathematics, Applied
Mathematics, Computational Neuroscience, and Biological Databases
and Informatics programs and by the MPS Office of
Multidisciplinary Activities.

DESCRIPTION (provided by applicant): The long-term goal of the proposed work is to use coordinated electrophysiological, computational, and applied mathematical techniques to understand the biophysical underpinnings of synchronous activity in the brain. Because synchronous activity is linked to the behavioral state, seems important for learning and memory, and shows clear abnormalities in clinically important conditions like temporal lobe epilepsy, the results gained from this program should make important contributions to human health. The specific goal of the proposed research is to characterize neurons from brain slices of the hippocampal formation in terms of their abilities to generate coherent population activity via mutual excitation and/or inhibition. To this end, we will adapt and use "mapping" techniques from applied mathematics, along with custom-built "dynamic clamp" technology. Work will focus on the mechanisms underlying two EEG rhythms that appear together during active exploration and learning: the 4-12 Hz theta rhythm and 30-80 Hz gamma rhythm. The effects of known neuromodulatory agents (acetylcholine, norepinephrine, and serotonin), as well as those of a putative neuromodulator (zinc), will be assessed. This appraisal will focus on how the biophysical effects of neuromodulators may alter cellular synchronization properties and hence, change the population driving synchronous activity. Computational and experimental methods will be used to verify the validity of mapping measurements in predicting network behavior. Because the techniques to be used in the proposed work are generally applicable, with clear underlying assumptions, both the results and the newly developed techniques should be broadly pertinent for work in many brain structures that show coherent network activity. Work will be organized around two hypotheses: (A) The biophysical properties of excitatory neurons in excitation-dominated structures support synchronization by mutual excitation; the converse is true for inhibition-dominated structures. (B) Neuromodulators can change the functional architecture of the hippocampal formation by altering cellular properties that determine synchronization behavior.

DESCRIPTION(adapted from applicant's abstract): Single nerve cells are often unreliable: repeated presentations of identical stimuli can generate significantly different trains of action potentials. Because this response variability limits the accuracy of encoding by the nervous system, its biophysical underpinnings are of great interest. The immediate goal of this project is to understand how two major sources of neuronal noise - synaptic noise in the signal received from presynaptic cells and channel noise caused by the probabilistic gating of ion channels - contribute to and interact with the dynamics of excitatory neurons of the hippocampal formation, a brain region implicated in learning and memory. Excitatory neurons of the medial entorhinal cortex (MEC) and hippocarnpus provide a powerful test-bed for these experiments for several reasons. For example: some of these neurons exhibit prominent channel noise that may limit response reliability and shape network responses; different classes of these neurons have contrasting rhythmic properties that imply contrasting stimulus preferences under noisy conditions; and these neurons play a critical role in human memory in the healthy and compromised brain. Electrophysiological experiments will be conducted using standard methods and newly developed stochastic dynamic clamp technology. The latter approach allows direct exploration of the causal roles of specific biological noise sources in shaping neuronal electrical dynamics and reliability. Four hypotheses will be tested: A. Excitatory neurons in MEC and hippocampus exhibit significant levels of channel noise B. Properties of reliability differ significantly among principal cells of the hippocampal formation C. Channel and synaptic noise influence electrical dynamics and reliability in the MEC D. Biological noise influences the behavior of biologically-inspired network simulations The long-term goal of this project - to enhance our understanding of how molecular-level events contribute to excitability, rhythmicity, and encoding properties in nerve cells - is important for improving human health. A mechanistic understanding of this connection may lead to novel diagnoses and treatments for several debilitating neurological disorders that disrupt the information-processing capabilities of the hippocampal region, including temporal-lobe epilepsy and stroke-related cell death.

[unreadable] DESCRIPTION (provided by applicant): The 2nd annual GTReal workshop will be held in May or June 2006 at the campus of Boston University. The meeting will be attended by around 120 scientists at a range of levels of experience (students, postdoctoral fellows, faculty members). The meeting will include 3 invited speakers, 12-15 contributed talks, and 2-4 "breakout" sessions, in which attendees will discuss topics of mutual interest. There will also be a hands-on tutorials. Depending on the number of submissions, there may be posters as well. Following on the success of the first event, held in May 2005 at the Georgia Tech, GTReal will focus on the following topics: (1) Exchanging scientific results. Because of the technical hurdles involved, only a handful of investigators are using real-time "dynamic clamp" systems to mimic membrane conductances in excitable cells. These investigators have gone to this level of trouble because dynamic clamp techniques provide the best method to test specific, computationally based hypotheses in living systems. Invited and contributed talks will focus on exciting examples of dynamic clamp and other real-time applications in neurophysiology and other disciplines, as well as technical means used to obtain such results. (2) Building a community of developers and end-users. Over a decade after the seminal work on dynamic clamp, no turn-key system exists, but several groups have devised systems that seem promising. A major goal of the breakout sessions will be to continue to build a community of developers and end-users of real-time technology, based on the principles of open-source software development. The attendees of GTReal will constitute much of the core of this community. Tutorials and informal interactions will add to the cadre of skilled users and developers. (3) Extending the real-time approach to new types of experiments. Real-time techniques allow an unprecedented degree of quantitative hypothesis testing in biological experiments. A number of extensions of the technique would increase its impact dramatically. For example, within cellular neurophysiology, it should be possible to combine real-time technology and optical techniques for recording and stimulation. Real-time techniques also have the potential to revolutionize systems physiology and behavioral studies of sensory systems. [unreadable] [unreadable] [unreadable]

This project will advance the creation and support of a community of scholars, from undergraduate to faculty, working at the interfaces among dynamical systems and biological applications. The three main areas of focus are: 1. Analysis of systems with multiple length and time scales, including applications to pattern formation; 2. Mathematical neuroscience, including analytical methods for working with small networks and reduction of dimension techniques; 3. Gene regulatory networks, including the development of RNA switches, transcriptional bursting and programmable cells. These areas have major applications to issues concerning health and medicine. The project will build on the previous research and training experience of the Center for BioDynamics, co-directed by the Principal Investigator and one of the other senior faculty members. Trainees will be pre- and post-doctoral students who will take part in a wide variety of formal and informal activities, including special seminars, working groups, mini-symposia, laboratory work, journal clubs and social events, which will enable them to acquire the multiple scientific cultures needed to work in a trans-disciplinary manner. The pre-doctoral students will be from the departments of Mathematics or Biomedical Engineering; the postdoctoral associates will be drawn from a wide range of backgrounds, with a focus on applied math. In addition to their research activities, trainees will obtain experience teaching at different levels. Math department faculty and trainees will be involved in the construction of new interdisciplinary curricula for undergraduates in other departments, including Biology; the faculty will mentor the trainees in teaching the new curricula.

choice; stimulus /response

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15- NS-104: Early-Stage Therapy Development. The major goal of the proposed work is to develop novel technical and theoretical means to understand the mechanisms underlying temporal lobe epilepsy (TLE) and to assess new classes of possible treatments for this devastating disorder. TLE is often difficult to treat using currently available approaches and entails an economic cost of $12 billion dollars per year within the United States alone. The proposed work is transformative on several fronts. First, the work focuses on the putative role of glial support cells (specifically, astrocytes) in TLE. Although there is a convincing body of evidence that astrocytes are involved in epileptic dysfunction, this evidence has not yet gained wide acceptance, leaving approaches that focus on astrocytes underappreciated and underutilized. Second, the proposed approach depends on a novel imaging technique, called targeted path scanning (TPS), which allows recordings of neuronal and glial calcium transients in up to 100 cells simultaneously, with single-cell spatial resolution and excellent temporal resolution. The TPS approach allows the proposed research program to study mechanisms and putative treatments of TLE in interacting neuronal and glial networks, with spatiotemporal resolution that permits simultaneous analysis at both the cellular and network levels. The proposed study has two specific aims. Aim 1 addresses whether the properties of astrocytic population calcium transients are altered in brain slices derived from animals that have been subjected to the kainic acid (KA) model of TLE. These transients will be characterized, and compared with data from age-matched controls, in slices from animals during both the latent period (after induction of status epilepticus but before spontaneous seizures) and after spontaneous seizures have begun. Relevant properties to be studied include temporal frequency, magnitude, and spatial extent throughout the astrocytic network. Aim 2 focuses on interactions between astrocytic calcium transients and spike-driven calcium transients in nearby neurons. Specific questions to be addressed in this aim include: Are calcium transients in astrocytes and neurons spatially and/or temporally correlated? If so, which cell type in a given area leads the other? How do known anti-epileptic drugs affect calcium transients in astrocytes, calcium transients in neurons, and the potential interactions between the two cell types? Finally, can this ground-breaking technology be used as a network- based assay for the identification of novel anticonvulsant molecules for the treatment of pharmacoresistant epilepsy? The proposed project-a pioneering effort between a pharmacologist/epileptologist and a bioengineer-is translational in its focus and intent. The major goal of the proposed work is to develop new theories and approaches that could be invaluable in discovering new pharmacological treatments for the devastating seizure disorder, temporal lobe epilepsy. PUBLIC HEALTH RELEVANCE: Temporal lobe epilepsy is a seizure disorder with devastating effects, particularly in the large number of patients for whom current treatments are ineffective. The purpose of the proposed work is to use ground- breaking imaging technology to study this disease in interacting networks of both nerve cells and glial metabolic support cells. A likely outcome of the proposed work will be entirely new ways to assess potential pharmacological therapies for epilepsy.

DESCRIPTION (provided by applicant): To understand brain function mechanistically, and thus to take principled approaches in repairing damaged brains, biomedical scientists face the daunting task of bridging the gap between the electrophysiological properties of single cells and the emergent properties of neuronal networks. The proposed experiments will help bridge this gap for a problem of great relevance in cognition and learning and memory: the cellular bases of the coherent theta rhythm in the hippocampus. The central hypothesis is that a particular class of hippocampal inhibitory interneurons, called oriens lacunosum-moleculare (O-LM) cells, plays a crucial role in amplifying the theta rhythm in vivo and generating theta-rhythmic activity in vitro. Proposed brain-slice experiments rely upon a recently developed real-time dynamic clamp system to study the integrative properties of O-LM cells and to immerse living neurons in computer-simulated microcircuits. Building such hybrid microcircuits-small brain circuits containing biological and simulated neurons that interact in real time- allows one to test precise hypotheses of microcircuit function with unprecedented quantitative rigor. Additional proposed studies focus on the consequences of O-LM-cell projections to the distal dendrites of pyramidal cells, as well as the consequences of O-LM-cell loss for the theta rhythm in vivo and in vitro. The proposed research program has five aims: (1) To study the input-output properties of O-LM cells in response to artificial synaptic barrages that mimic the in vivo state. (2) To study how phase-locked, distal and proximal inhibitory inputs can lead to phase-locked sparse firing in excitatory pyramidal cells. (3) To study the effects of distal O-LM-based inhibition on phase-dependent selection of dendritic inputs to pyramidal neurons. (4) To study how input from oriens-lacunosum moleculare (O-LM) interneurons to pyramidal cells and fast- spiking interneurons contributes to self-organized theta and gamma rhythms in closed-loop networks. (5) To study the importance of synchronization of O-LM cells for rhythmic activity under manipulation of feedback input, artificial rhythmic drive from the septum, and other factors. The long-term goal of this research program is to understand, with quantitative and mechanistic rigor, the mechanisms by which both normal and abnormal rhythmic behaviors emerge in the hippocampus and other cortical regions. The work will be immediately relevant to understanding the theta and gamma rhythms. These two patterns of coherent activity seem crucial for normal cognition and learning and memory, and are disrupted in a broad range of conditions including epilepsy, schizophrenia, Parkinson's disease, and Alzheimer's disease. Because the proposed approach can show how specific membrane mechanisms contribute to network function, it is particularly useful for identifying new drug targets. An added bonus of the proposed approach is that the dynamic clamp technology developed for these studies may prove useful for therapeutic, feedback-controlled electrical stimulation of the brain.

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE), a devastating seizure disorder that is difficult to control with anticonvulsant drugs, often develops following an initia insult to the CNS. In order to better understand the process of epileptogenesis and to develop innovative therapeutic approaches for the management of TLE, animal models have been developed that exhibit some of the hallmarks of this seizure disorder: a period of status epilepticus (SE) which serves as the initial insult to the CNS, a variable latent period during which seizures do not occur, and the eventual development of recurrent, spontaneous seizures of temporal lobe origin. Recently we used the kainic acid (KA) model of TLE to investigate 'reactive' astrocytes in the hippocampus (HC), a brain region known to be involved in seizure generation. There is a significant increase in gap junction coupling between astrocytes following KA-induced status epilepticus (SE). Therefore, the astrocytic network architecture is altered in brain regions associated with seizure generation. We also discovered that astrocytes express kainate receptor (KAR) subunits following SE and hypothesize that activation of KARs can result in calcium (Ca2+) transients that induce the release of signaling molecules that modulate neuronal activity in the HC. The present application will use targeted path scanning 2-photon microscopy (TPS) to simultaneously evaluate rapid Ca2+ transients in large networks of astrocytes in brain slices obtained from animals treated with KA to induce SE. We employ in utero electroporation to target a genetically encoded Ca2+ indicating protein (Lck- GCaMP3) to the rat HC so that we can use brain slices obtained from adult animals to determine 1) if activation of KARs induces somatic Ca2+ signaling in networks of reactive astrocytes in the HC and 2) if KAR- induced and/or other agonist-induced Ca2+ signaling in the fine processes of reactive astrocytes induces the release of signaling molecules that directly influence network activity in HC brain slices obtained from KA- treated rats during both the latent period and chronic epilepsy. Finally, we will use electron microscopy to determine if there are ultrastructura changes in KAR expression, gap junction coupling, and dendritic ensheathment in the astrocyte compartment of the tripartite synapse of the CA1 and CA3 regions of the HC following KA-induced SE. The combined use of TPS with the stable expression of Lck-GCaMP3 in cells of the HC is a technical achievement that will contribute to our understanding of the functional role of KAR expression in astrocytes following status epilepticus (SE), both in the latent period and in chronic epilepsy. The proposed experiments will also determine how pathologic glial/neuronal interactions, both structural and functional, influence circuit activity during the development and persistence of epilepsy. Finally it is anticipated that the proposed experiments will lead to the identification of novel molecular targets for innovative therapeutic approaches for the treatment, prevention, and/or cure of this devastating seizure disorder.

Genetically-encoded reporters of neural activity are a transformative tool for understanding brain function because they allow for the simultaneous measurement of activity across many neurons defined by genetic and anatomical criteria. The current generation of such reporters use light to signal activity, which limits their ability to be used deep in brain tissue and across the full range of neuronal activity. The goals of the project are to overcome these limitations by developing reporter proteins that can be engineered to emit unique electrical or magnetic signals in response to neural activity. The project will also develop sensors that are optimized for detecting these signals from individual neurons in intact brain tissue in the freely-behaving animal. The proposed 'electrogenetic' toolbox will allow neural activity to be recorded with high fidelity from defined cell types across the entire physiological range of neuronal firing rates, from any location in the mammalian brain, and in the freely-behaving animal. This strategy leverages existing and widely available technology for recording electromagnetic signals in the brain, and thus has the potential to be rapidly adopted for a wide range of neuroscience applications.

This project will develop a new strategy for measuring neural activity from genetically-targeted neurons in the intact brain. An interdisciplinary team of investigators will first use gene therapy techniques to express candidate proteins in particular neurons, then will screen for electrical or magnetic signals using conventional electrodes or nanoscale magnetometer probes. Understanding how neurons of a particular type are activated in the behaving animal is crucial for understanding the neural basis of sensation, cognition and behavior. Indeed, the lack of tools for interrogating identified neurons while they are in action is a major impediment to understanding functional neural circuits in the brain. In addition to breaking this impasse in basic science, a potential broader impact of this project is that the tools to be developed in this proposal may provide information leading to improved diagnosis and treatment of nervous system disorders including mental illness, autism, addiction and epilepsy.

Funding is requested for an additional five years of support to continue and enhance an established training program entitled Quantitative Biology and Physiology (QBP). The core mission of this program continues to be to train PhD research scientists who demonstrate: (1) a quantitatively-based understanding of the principles underlying molecular biology, cell biology and physiology; (2) the ability to apply advanced techniques of computational modeling and quantitative measurement to understand biological and physiological systems; (3) the capacity to determine emergent properties biological systems or processes across length scales; and (4) the insight to apply their knowledge, in an academic or industrial setting, in order to improve human health. The QBP program includes the following crucial features (new features underlined): An extraordinarily talented set of the students who fit the multi-scale, highly quantitative approach of the program; a carefully curated list of 37 training faculty; a request to increase the number of slots to 12, and a shift to funding most students for their first two years, in order to strengthen the cultural identity of the program even further; an integrated structure of governance that includes elected student and faculty representatives; more detailed data collection, compatible with the new form structure imposed by NIH in Spring 2016; a mentoring plan for faculty without training experience; a well-considered curriculum that includes rigorous training in multi-scale biology, scale-independent analysis and modeling, and modern measurement techniques; multiple lab rotations that ensure exposure to problems at the molecular, cellular-tissue, and organ level biology and physiology; opportunities to conduct thesis research that is interdisciplinary, quantitative, integrative, and necessarily linked to experimental and/or clinical data; a requirement that all QBP trainees apply for independent fellowships, along with financial incentives for success in this effort; an exciting new course to introduce BME graduate students to relevant topics for research in industry; highly successful culture-building efforts, including a journal club and a yearly symposium, with increased formal faculty involvement and leadership; three dinners each year with clinical and academic thought leaders; ?grand rounds? events at which clinicians come to our campus and provide clinical context for engineering work; new efforts to increase the size of the pool and the yield of applicants from underrepresented groups. The BME department continues to attract high-caliber trainees who have organized to create an empowering identity. These trainees also sustain activities that foster the themes of the QBP program so as to enrich the entire institution. We have expanded our training mentor pool, improved the quality of the department as a whole, and revised our administrative approach based on experience and feedback. We look forward to the opportunity to continue this momentum during the next grant cycle.