Kendall H. Lee - US grants

DESCRIPTION (provided by applicant): This proposal describes a 5 year training program for the development of an academic career in neurosurgery. This program will promote the command of electrophysiology, electrochemistry, brain imaging and stereotactic and functional neurosurgery. Dr. Gary Sieck, Chair of Physiology and Biomedical Engineering, will serve as the primary mentor. A mentorship team of experts in brain imaging, electroanalytical chemistry, neuroengineering and neurosurgery will help the applicant elucidate the mechanisms by which Deep Brain Stimulation (DBS) treats Parkinson's disease. Our recent work demonstrates that subthalamic nucleus (STN) DBS may activate nerve endings to release neurotransmitters, including dopamine. The proposed experiments will entail electrophysiologic, electrochemical, and fluorescent microscopic recordings during DBS. The specific aims include the demonstration of 1) DBS results in changes in neuronal action potential firing and release of dopamine in the striatum. 2) The therapeutic benefit of STN DBS is mediated by dopamine release in the 6-OH DA lesioned rat model of Parkinson's disease. 3) STN DBS results in release of glutamate in the STN via astrocytic vesicular release mechanism. This study will be the first detailed analysis of the mechanism of DBS as it relates to DBS effect on astrocytic glutamate release and striatal dopamine release. Success in this project will open a new era in medical diagnosis and intervention, providing precise targeting of DBS electrodes and regulation of neurotransmitter that are critically involved in the therapeutic effectiveness of DBS. The applicant, Kendall Lee, M.D., Ph.D., is a new investigator and a neurosurgeon at the Mayo Clinic Rochester with a multidisciplinary background in neurology, neurosurgery, and neuroscience. He has demonstrated the ability to form unique and effective collaborations. To maximize the likelihood of success, the applicant has enlisted as advisors a highly skilled team of researchers: Charles Blaha, Ph.D. (expert in amperometry), Paul Garris, Ph.D. (expert in Parkinson's disease animal models and fast scan cyclic voltammetry), Eric Newman, Ph.D. (expert in astrocyte research), and Demetrius Maragonore, M.D. (Parkinson's disease Neurologist and researcher). In addition, Fredric Meyer, M.D. (Chair, Neurosurgery Mayo Clinic Rochester) will serve as career development advisor, to make certain a successful career in academic neurosurgery for the applicant.

DESCRIPTION (provided by applicant): Deep brain stimulation (DBS) within the basal ganglia complex is an effective neurosurgical approach for treating motoric symptoms of Parkinson's disease (PD). Elucidating DBS mechanisms for improving outcomes in PD and other targeted disorders has become a critical clinical goal in stereotactic and functional neurosurgery. We propose to address this issue by combining for the first time two powerful technologies, notably functional Magnetic Resonance Imaging (fMRI) and in vivo neurochemical monitoring to investigate DBS-mediated activation of basal ganglia network circuitry. For this purpose, we have developed an MRI-compatible wireless monitoring device to obtain chemically resolved neurotransmitter measurements at implanted microsensors in a large mammalian model (rhesus monkey). This device supports an array of electrochemical measurements that includes fast-scan cyclic voltammetry (FSCV) for real-time simultaneous in vivo monitoring of dopamine and adenosine release at carbon-fiber microelectrodes as well as fixed potential amperometry for monitoring of glutamate at enzyme-linked biosensors. Using electrophysiological targeting of the STN to implant appropriately scaled-down human DBS electrodes, our rhesus monkey model will enable us to employ fMRI to initially determine the major sites of activation in the basal ganglia during application of clinically-defined therapeutic (tDBS) versus non-therapeutic (nDBS) STN stimulation. We will then electrochemically monitor extracellular levels of glutamate, dopamine and adenosine release evoked by STN DBS in the brain areas identified by fMRI activation. Lastly, we propose to combine fMRI and FSCV recordings in the rhesus monkey to confirm a causal relationship between glutamate, dopamine and/or adenosine release and the fMRI-identified anatomical sites in the basal ganglia complex by determining the consequences of dopamine depletion and repletion mimicking advanced PD and pharmacological treatment of the disease, as well as adenosine depletion. The three Specific Aims are (1) identify using fMRI brain regions within the basal ganglia complex activated by STN DBS, (2) quantify glutamate, dopamine and adenosine release evoked by STN DBS of the brain region(s) identified by fMRI, and (3) correlate STN DBS-evoked glutamate, dopamine and adenosine release in the regions identified by fMRI with simultaneous fMRI before, during, and after pharmacological depletion and restoration of dopamine, and reductions in adenosine. We believe that the simultaneous combination of fMRI and electrochemistry offer a new and exciting approach that provides complementary anatomical mapping and neurochemical monitoring implicated in the therapeutic actions of STN DBS.

DESCRIPTION (provided by applicant): Deep brain stimulation (DBS) is an effective neurosurgical approach for a variety of neurological and psychiatric disorders including Parkinson's disease, essential tremor, epilepsy, and depression, among others. This research proposal is intended to advance DBS technology by developing a novel intraoperative monitoring approach based on electrochemical monitoring at the brain-electrode interface by use of carbon nanofiber (CNF) based electrode design. This approach utilizes technology developed at the Mayo Clinic, the Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS), an instrumentation system that combines digital telemetry with fast-scan cyclic voltammetry (FSCV) and amperometry, coupled to CNF based electrode technology developed at National Aeronautic and Space Administration (NASA) called WINCSnanotrode. Under Mayo IRB approved protocol, WINCS safety and feasibility has already been tested successfully in human patients undergoing DBS neurosurgery. Recently, CNF nanoelectrodes have been shown to be an excellent substrate for electrochemical detection demonstrating ultra high sensitivity, high signal to noise ratio, and rapid sampling, while at the same time providing an improved brain-electrode interface for more efficient stimulation, thereby conserving battery life. By virtue of its sub-second, chemically resolved recording, FSCV is recognized as state-of-the-art for measuring neurotransmitters, including dopamine, serotonin, norepinephrine, and adenosine, in laboratory animals. By correlating the release of neurotransmitter and therapeutic stimulation in real time, Mayo Clinic's WINCS coupled to NASA's WINCSnanotrode will provide a powerful new tool to assess the mechanism of DBS, guiding electrode placement, and testing accuracy and efficiency of stimulation. The three Specific Aims are (1) complete WINCS and WINCSnanotrode development to generate a device that is capable of use in humans for electrochemical sensing, (2) establish WINCSnanotrode approach for intraoperative neurochemical monitoring in a large-animal (pig) model of DBS neurosurgery, and (3) establish WINCS and WINCSnanotrode approach for intraoperative neurochemical monitoring in humans during DBS neurosurgery. We believe that WINCS and WINCSnanotrode technology engenders great potential to identify specific targets for DBS, to streamline the already long and difficult implantation procedure, to assess efficiency of stimulation parameter, and to improve the accuracy and efficiency of stimulating electrode.

? DESCRIPTION (provided by applicant): Determining the levels of neurotransmitters present in the living brain in real time is a matter of current scientific interest for research and clinicl reasons. Among these reasons is the need for understanding and mapping brain function and for improvement in the clinical application of deep brain stimulation (DBS). One analytical technique that holds potential promise in this application is fast-scan cyclic voltammetry (FSCV), however technical limitations have hindered its adoption for chronic in vivo use. Of particular difficulty has been the construction of a chronically-implantable FSCV electrode that possesses both the proper chemical properties for the monitoring of neurotransmitter levels as well as sufficient durability for chronic implantation in humans or animals. Carbon fiber has been used successfully under some circumstances, particularly at low voltage potentials, but at the higher voltages required for detection of neurotransmitters such as adenosine (e.g., up to +1.5V), its lifetime is very limited. Additionally, current FSCV methodology provides only a relative measure of neurotransmitter concentration or concentration change, but once an electrode is implanted chronically, recalibrating it on the bench becomes impossible. Due to buffer effects, proper bench calibration of a FSCV electrode intended for implantation is impossible in any case. Therefore, a means to extract absolute concentration data via FSCV with only in situ calibration would prove extremely valuable. Our work in these areas has been ongoing for several years, and initial results indicate that a coating of polycrystalline diamond film, when properly doped to provide electrical conductivity, yields electrodes that are both sufficiently sensitive and durable for chronic in vivo use. To construct these diamond-coated FSCV electrodes, our lab has already completed the construction of a chemical vapor deposition reactor to create polycrystalline diamond film-based electrodes. Initial results have been promising, but we propose to continue this development work. We have also determined that simple modifications to the FSCV procedure, combined with more sophisticated data analysis procedures, appear to allow for the determination of absolute analyte concentration. These two lines of work are mutually-reinforcing insofar as they are both focused on the goal of a long-term implantable FSCV neurotransmitter-monitoring device and, ultimately, a closed-loop DBS stimulator.

PROJECT SUMMARY Physician scientists (MD-PhDs) are uniquely positioned to address many of the challenges at the forefront of modern medicine. During the first 14 years of support, the Mayo Clinic MSTP has dedicated itself to training talented and passionate students to be critical, productive physician scientists. Our mission is to prepare our students for academic careers in basic, translational and clinical research, focused on studying fundamental questions and translating basic discoveries into medical advances. The program philosophy is that the skills required for this type of academic career are best developed in a basic research setting. However, the unique quality of the physician scientist is the ability to integrate basic studies with translational and clinical research to ultimately advance the practice of medicine, and the Mayo Clinic MSTP strives continually to improve the integration of clinical and basic research training. First awarded an MSTP grant in 2003 and renewed twice, our MSTP has continued to develop and mature while maintaining aspects that were previously praised by reviewers. The main strengths of our MSTP are: ? An enthusiastic training faculty of 71 mentors who provide extensive opportunities for cutting-edge interdisciplinary training in basic, translational, and clinical research; ? Outstanding current trainees who are passionate about the study of fundamental biological processes of relevance to human disease; ? A highly competitive applicant pool; ? An autonomous admissions process that enables selection of students based on their prior research experiences and excitement for biomedical research; ? An effective recruitment and retention plan to enhance diversity, with three URM students who completed training during the past five years and 12 URM or students with disabilities currently in training; ? Integration of medical and graduate school curricula, which allows students to complete three required graduate courses and two laboratory rotations during MS1 and MS2; ? Programmatic features, including the MSTP Selectives, Weekly MSTP Conferences, MSTP Annual Retreat, MSTP Clinical Experiences Program and MSTP Clinical Re-Entry Course, as well as individualized development support that respond to specific needs of MD-PhD trainees; ? Strong institutional support for education, which enables us to fund our MD-PhD students throughout their medical and graduate training, providing exceptional flexibility in choosing thesis laboratories; ? Exceptional research resources that enhance our students' educational experiences; ? A dedicated, interactive, and supportive team consisting of the Director, Associate Directors, administrators and student leadership.

PROJECT SUMMARY Subthalamic nucleus (STN) deep brain stimulation (DBS) is a common surgical treatment for Parkinson?s disease (PD). Despite over 20 years of clinical success, the therapeutic mechanisms of STN DBS remain elusive. However, it has become clear that DBS acts at the molecular, cellular, and systems levels in complex and sometimes contradictory ways. Current techniques such as electrophysiology, electrochemistry, and functional imaging commonly used to study pieces of this puzzle are limited in either resolution or behavioral paradigms these can be applied to, and have thus not provided all the information needed to parse out the complicated relationships between stimulation and evoked effects. Here, we propose the use of fluorescence calcium microscopy in GCaMP6f-expressing rats using a head-mounted miniature single photon system as a novel tool to bridge the gap between cellular and system level understanding of DBS in awake behaving animal models of PD. To this end, we will analyze neural activity changes in motor cortex evoked by stimulation of the STN during open field, stepping, cylinder tests, and apomorphine-induced rotations, all of which are stereotypical tests that have shown predictive validity for evaluation of movement and therapeutic efficacy in parkinsonian animals. The techniques proposed here provide a unique approach for answering questions about DBS mechanisms such as whether DBS-induced activation or pharmacologic inhibition of excitatory STN glutamatergic neuronal projections to the globus pallidus internus / substantia nigra reticulata produces detectable changes in motor cortex activity associated with changes in behavioral outcomes (e.g., open field, kinematic assessment of stepping, cylinder, and apomorphine-induced rotation tests).

PROJECT SUMMARY We propose to develop and optimize an advanced neurochemical recording technique that would be able to measure relatively rapid physiologically representative second-to-second changes in basal concentrations of specific neurochemicals, such as dopamine, in the brains of awake behaving animals. Microdialysis, a commonly used in vivo sampling technique, is able to measure changes that occur in basal levels. However, in practice the sampling timescale is significantly limited to minute-to-minute changes and it suffers from poor spatial resolution and induces significant tissue damage. As well, conventional in vivo electrochemical recording techniques, such as fast-scan cyclic voltammetry, are intrinsically limited to measuring phasic (stimulation-induced) changes in neurochemical concentrations and not changes in basal concentrations. The proposed electrochemical technique we call Multiple Cyclic Square Wave Voltammetry (M-CSWV) will enable second-to-second measurements of basal extracellular levels of neurochemicals with exceptional spatial resolution, sensitivity, specificity, and minimal tissue disturbance. This proposal leverages our unique expertise in neuroscience, electrochemistry, software development, and engineering to develop and validate this novel neurochemical recording technology for broad use in basic neuroscience research, clinical brain neuromodulation, and a variety of electrochemical applications. Our initial animal studies will guide and inform the application of our investigational technique for use by the general neuroscience and medical community. Our proposal seeks to (1) establish M-CSWV as a reliable research tool that is capable of identifying and quantifying basal dopamine extracellular levels in vivo with unsurpassed sensitivity and selectivity; and (2) validate the use of M-CSWV for in vivo chronic selective measurement of basal dopamine concentrations and application in an animal model of drug-induced neurochemical sensitization.