Xinyan Tracy Cui, Ph.D. - US grants

0729869
Cui
The main objective of this project is to implement an electroactively controlled conductive polymer drug release device. The system will utilize the unique properties of electroactive conducting polymers, which can be synthesized by electropolymerization with ionic drug molecules incorporated, allowing for flexible drug loading onto electrodes. Upon an electrical potential change, the polymer undergoes redox reactions that lead to the release of drug from the electrodes, with the release quantity controlled by the amplitude of electrical stimulation.
The specific aims of the project are: (1) in-vitro development and characterization of a conductin-based neurochemical system, and (2) in-vivo characterization of the neurochemical delivery system. The interdisciplinary research project includes elements of bioengineering, material science and neuroscience. This study will make important contributions to the field of neuro-electrode interfacing.

The research will support new course development, train graduate and undergraduate students and support programs for educating women and underrepresented minorities. The results will enhance scientific understanding in multi-disciplinary fields. This project will significantly impact localized drug delivery while electrically recording the neural response in real-time.

This Career award by the Biomaterials program in the Division of Materials Research to University of Pittsburgh is to study to facilitate, control and direct the growth, differentiation and functional integration of stem cells within a host tissue. This project seeks to develop a technology platform that can be used to study material-neural tissue interaction with a specific emphasis on the differentiation of neural stem/progenitor cells. That the stem cell fate can be influenced by the engineered substrate, specifically via the surface characteristics, controlled release of soluble factors and electrical stimulation is one of the most important questions in neural tissue engineering, tissue repair and regenerative medicine. Application of stimuli is expected to promote neural stem cells differentiation into functional neurons at desired locations and orientations, and is to promote functional tissue regeneration and repair. Electroactive conducting polymers such as polypyrrole and its derivatives will be used to build various surfaces for neural stem cells to grow and differentiate. The unique properties of these polymers will allow to systematically changing the surface characteristics, to release soluble factors at a high spatial and temporal resolution and to apply electrical stimuli. Conductive polymers will be electrodeposited on electrodes patterned on the bottom of cell culture wells for systematic and high-throughput cell culture studies to be performed with improved efficiency and precision.

This technology platform will create numerous multi-interdisciplinary training opportunities, including new courses and workshops to attract and educate graduate and undergraduate students, post-doctoral fellows and even medical doctors. The proposed research project may provide significant insight into methods of manipulating neural stem cells via their supporting substrates by engineered external stimuli, so that they can be of therapeutic value to neurological diseases or injuries, many of which are the most devastating of conditions. The educational plan will bring more impact to the rapidly growing field of neural engineering by promoting the engagement of scientists, engineers and clinicians, and attracting more young talent (graduate and undergraduate students, with extra effort on increasing participation of underrepresented groups) to the field and providing them with a wealth of multidisciplinary training and research experience.

DESCRIPTION (provided by applicant): Cortical implants possess tremendous research and therapeutic importance. Man-made devices are being developed to be placed in the brain to restore function, treat neurological disease or monitor the physiological environment. A common problem faced by devices used in these applications is the lack of brain tissue biocompatibility. Within the scope of this project, attention is focused on the development of neural recording electrode arrays used in brain-machine interface studies, in which motor signals recorded from these electrodes can be used to control an external machine, a technology that can potentially restore movement to the paralyzed. However, the current unsatisfactory chronic performance of these devices has greatly hindered their clinical translation. The implanted electrodes cause progressive brain tissue responses including neuronal loss and inflammatory gliosis, which may lead to signal loss. We hypothesize that promoting neuronal health around the implant and reducing chronic inflammation may improve the quality, reliability and longevity of chronic recording. To test these hypotheses we developed two unique biomaterial strategies. One is surface immobilization of brain-derived biomolecules on the implant to provide bioactive sites for host tissue to interact with in a less invasive manner. The other strategy is an electrically controlled drug release system that can release anti-inflammatory drugs directly from the electrode surface to modulate the inflammatory tissue response. First we will respectively characterize the effect of these two approaches on modifying the tissue responses around the implanted electrodes, and determine to what degree promoted neuronal health and reduced inflammation influence the chronic recording performance. After the assessment is completed, the surface modification and controlled drug release approaches will be systematically combined to investigate whether there is a synergistic effect on the improvement of tissue- implant interface and chronic recording. Our specific aims are 1) To determine the effect of promoted neuronal survival and health on chronic recording quality, longevity and reliability. 2) To investigate the role of inflammation on chronic recording quality, longevity and reliability. 3) To evaluate the effect of combined treatment of surface modification and drug release on chronic recording quality, longevity and reliability. As shown above, the proposed project has both basic research and applied objectives. Chronic recording improvement is our immediate application-driven goal. However, the fundamental understanding of brain tissue response to implants and how different biomaterial strategies may modulate these responses are critically important for all types of neural implants, including neural recording devices, stimulators, CNS drug delivery systems and biochemical sensors. PUBLIC HEALTH RELEVANCE: Man-made devices are being developed to be placed in the brain to restore function and treat neurological disease. The goal of this project is to investigate the novel approaches that can be taken to make the implanted device more brain friendly and thereby obtain improved performance longevity and reliability.

DESCRIPTION (provided by applicant): R01:Biomimetic Surface for Neural Implant PI: Tracy Cui Implantable microelectrode arrays for neural recording and stimulation have demonstrated tremendous research and clinical potential. Studies of brain tissue response to neural electrode arrays have revealed localized microglia activation, followed by astrocytic scarring and neural degeneration. These reactions are thought to contribute to the low yield and chronic failure of neural recording, although direct links have not been soundly established. Past studies characterizing the CNS response to implants have used postmortem histology at discrete time points. This approach suffers from a large degree of variability and fails to capture the dynamic molecular, cellular and vascular changes of the host. To address this issue, we have developed an experimental set-up to directly image the electrode-tissue interface in live animals using 2-photon microscopy in conjunction with electrical recording. Our previous work indicates that by coating the surface of neural probes with neural adhesion molecules, neuronal density around the device can be promoted while glial reaction attenuated. Meanwhile, neural recording quality is drastically improved. We hypothesize that promoting neuronal growth and health, and/or inhibiting microglia activation will lead to recording improvement. The specific objectives of thi project are to investigate the biological mechanisms of the coating's effect on recording and to evaluate the clinical potential of biomimetic coating in a brain machine interface (BMI) model. First, the acute neuronal and microglia responses to coated probes will be characterized in transgenic animals using two photon imaging and electrical recording for two weeks. Real time tissue characteristics (such as neuronal and neurite density, microglia density and morphology, vasculature change and BBB leakage) will be correlated to recording metrics(such as unit yield, SNR, amplitude of signal and noise as well as impedance). Several biomolecules that promote or inhibit neuronal growth or microglia activation will be immobilized on the Blackrock arrays to test our hypothesis. Secondly, the long-term benefit of the coatings on recording will be determine by testing the optimum coating conditions in rats for 6 months. Explants will be taken monthly to examine the coating longevity, while immunohistochemistry and microarray analysis of the tissue at the interface will be performed to characterize the cellular and molecular change over time. Thirdly, to assess the potential of biomimetic coating for clinical application, coated electrodes will be tested in rhesus monkeys in a brain-machine-interface (BMI) model. Recording metrics such as SNR, signal amplitude, unit yield and stability will be quantified over 2 years and compared to uncoated arrays. A novel functional metric will be developed to assess functionality of the recorded signals. BMI performance will be evaluated based on speed and accuracy. This proposal combines the cutting edge real-time imaging, effective biomaterial strategies and state of the art brain machine interface technology to understand the interactions between neural implants and host tissue. The findings will guide the development of seamless neural interface devices for BMI, visual and auditory prosthesis, deep brain stimulation for Parkinson's disease, depression and epilepsy, to name a few. The knowledge will also benefit other brain implants from biochemical sensing and therapeutic delivery to scaffold and stem cell transplant for treating neurological disorders.

? DESCRIPTION (provided by applicant): Inhibition of Neural Electrode-mediated Inflammation and Neuronal Cell Death A growing number of implantable neural electrode devices are being developed to map brain circuit or restore function and treat diseases. The performance of these devices hinges on the quality and stability of the electrode-neural tissue interface. Undesirable brain tissue responses, including persistent microglia activation and blood brain barrier breach, glial scarring, neuronal loss and degeneration, have been consistently reported in animal studies. For electrode devices that require intimate contact with host neurons, their performance functionality may be compromised by these responses. As an example, single unit neural recording via microelectrode arrays experiences deterioration in yield and quality over time, which is a major barrier to applications of this technology in long-term neuroscience research and clinical translation. There are many molecules and pathways involved in inflammation and neuronal death. We began our study by focusing on caspase-1, as caspase-1 is a key mediator of both inflammation and programmed cell death. Activation of caspase-1 is the earliest detectable event in neuronal apoptosis in vitro and in brains with ischemic, injury and neurodegenerative conditions. Furthermore, caspase-1 activates interleukin-1 ß (IL-1ß), a pro-inflammatory cytokine highly expressed in the tissue surrounding implanted electrodes, especially those that showed poor electrophysiological outcome. IL-1ß triggers inflammatory gliosis and exacerbates BBB breach; both are hypothesized causes of chronic recording failure. Therefore, we hypothesize that caspase-1 mediates the neuronal death and inflammation around neural implants and inhibiting caspase-1 may improve neuronal survival, reduce inflammation and lead to improved electrode performance. We have performed a preliminary study comparing the neural recording performance of microelectrode arrays implanted in caspase- 1 knockout (KO) vs. wild-type (WT) mice. The single unit yield and signal quality are significantly greater in the knockout animals over the 6 month time period, strongly supporting the critical role for caspase-1 in maintaining the quality of the electrode-tissue interface. However, closer examination of the recording over time revealed dynamic changes that cannot be interpreted with end-point histology. To better understand the mechanism(s) by which caspase-1-mediated pathways affect recording, we propose to use 2-photon live animal imaging to characterize the cellular and vascular responses to implanted neural probes in conjunction with neural recording and comprehensive tissue and biochemical analyses. Therapeutics targeting caspase-1 or the inflammation/cell death in general will be evaluated in an effort to improve the chronic neural interface. The drugs to be tested are caspase 1 specific inhibitor VX765, melatonin and minocycline. This proposal uses a multidisciplinary approach to uncover the molecular and cellular mechanism contributing to neural recording performance. The findings will increase our scientific understanding of neural implant pathology, and guide the development of therapeutic and/or biomaterial strategy for stable and reliable neural interface. Data and technology developed in this project may also contribute to the study of neuronal degeneration and inflammation in traumatic brain injury, stroke and neural degenerative diseases.

Cocaine is a highly addictive psychostimulant that exhibits region-specific activity throughout the brain. It is widely accepted that adolescents present a higher vulnerability to cocaine addiction than adults. Recent evidence has suggested that this increased vulnerability is biological in origin, thus raising the question of whether this age effect is due to differences in neural circuitry or local cocaine concentration in the brain. In order to investigate this and other important neuroscience questions, it is unequivocally necessary to develop cocaine sensing technology capable of directly measuring real-time transient events at multiple discrete regions throughout the brain. Current conventions for in vivo cocaine quantification (microdialysis, homogenized tissue composition, etc.) lack the necessary spatial and temporal resolution. We have recently developed an electrochemical aptamer-based in vivo cocaine sensor on a silicon based microelectrode array (MEA) platform capable of directly measuring cocaine from discrete brain locations. The sensor exhibits a detection limit of 1 µM with excellent spatial and temporal resolution and can maintain a reproducible detection over the course of 3 hours. After 3 hours, performance degradation was observed likely due to biofouling and aptamer detachment. We propose to develop and apply advanced dual polymer coating strategy to improve the sensitivity and stability of the sensor. The coatings include non-conductive and conductive zwitterionic polymers that are highly resistant to biofouling. To improve the aptamer binding efficiency and stability, a novel electrically conducting polymer will be developed capable of bio-conjugation with thiolated aptamers. We hypothesize that the incorporation of these polymer coatings will improve cocaine sensor performance over long-term implantation. The specific objectives of this project are to develop the methodology to pattern these polymer coatings on MEAs for the best sensing capability and fouling resistance and then test the ability of the polymer- modifed cocaine sensor to directly measure in vivo cocaine concentration reproducibly over a period of 72 hours. The local brain concentration of cocaine upon repeated IV injection will be compared between adult and adolescent rats to determine the origin of the age effect. The proposed sensor will serve as the first ever technology capable of measuring in vivo cocaine concentration over multiple hours and days. This technology has the potential to revolutionize our understanding of cocaine abuse and addiction. Additionally, the modified microelectrodes are also able to recording neurophysiological signals. Implantable sensors with dual functionality will have a broad impact on neuroscience research. Finally, the aptamer based electrochemical sensing platform can be generalized to a broad range of important analytes, while the highly functionalizable and fouling resistant coatings can be applied to other implantable biosensors throughout a broad range of biological research fields and medical diagnosis.

Microstimulation has been an invaluable tool for neuroscience researchers to infer functional connections between brain structures or causal links between structure and behavior. In recent years, therapeutic microstimulation is gaining interest for the restoration of visual, auditory and somatosensory functions as well as emerging applications in bioelectronic medicine. Current neural stimulation parameters and safety limits need to be revised for microelectrodes using more systematic and advanced methodologies. Stimulations via microelectrodes often require high charge injection for effective modulation of neural tissue without exceeding the threshold to harm the tissue or the electrodes. Therefore, advanced electrode materials with high charge injection capability and stability are highly desired. We have developed several types of stimulation materials based on conducting polymer PEDOT and nanomaterial composites. These materials present different charge transfer and electrochemical properties as well as biocompatibility, and the effects of these properties on microstimulation have yet to be comprehensively characterized. This proposal aims to establish new in vitro and in vivo models to examine the efficiency and safety of stimulation via multiple electrode materials, ranging from the clinically approved Pt and Iridium Oxide (IrOx) to the emerging PEDOT nanocomposites. Another challenge with micro-stimulation is its sensitivity to host tissue responses. Implantation of electrodes causes electrode fouling, progressive neuronal loss and inflammatory gliosis immediately surrounding the implants. Loss of nearby neurons and axons leads to decreased stimulation efficacy, while electrode fouling and gliosis increase impedance. Additionally, stimulation itself may further exacerbate host tissue responses if above the safety limit, which has yet to be defined for microelectrodes and emerging electrode materials. Using in vivo imaging in fluorescently labeled mice, we will examine the acute and chronic effects of microstimulation on neurons, microglia and vasculature, while monitoring the electrode material and electrochemical products. We will use an in vitro multielectrode arrays (MEA) system to study the effects of electrical stimulation on material and cells, in order to pinpoint the mechanisms of material and tissue damage. The first aime is to assess the efficiency and safety limit of neural stimulation via different electrode materials in vivo in acute experiments. For efficiency testing, we will implant the electrodes in the cortices of GCaMP mice and use 2-photon microscopy to image the calcium signal in order to determine stimulation threshold and optimum stimulation parameter for each electrode material. as a function of stimulation parameters. Stimulation threshold and efficiency for different pulse width, interphase period, bias potential and frequency from each electrode material type will be determined. For safety testing, we will use Syn-RCaMP/Cx3Cr1-GFP mice to visualize both neuronal and microglia cells and determine the damage threshold. The second aim is to examine the effects of stimulation on electrode materials and cultured cells in vitro. Using a high-throughput in vitro MEA system in which the six microelectrode materials can be deposited, we will stimulate at safe and unsafe parameters (identified in vivo from Aim 1) for up to 12 weeks. We will assess electrode material stability and analyze the stimulated media to identify electrochemical and degradation products. The toxicity of stimulated media will be tested in cultures of neuron, microglia, endothelial cells and neuron-microglia co-culture at varying doses to determine the detrimental effects of electrochemical and degradation products on these cells. Finally, we will directly stimulate the cells cultured on MEAs and characterize cell behavior using quantitative RNA and protein analysis, neural recording/stimulation and immunohistochemistry. The third aim is to characterize the chronic safety and stability of microstimulation in vivo from different electrode materials. Stimulation will be applied one hour per day to microelectrode arrays chronically implanted in Syn-RCaMP/Cx3Cr1-GFP animals for 12 weeks. In each weekly imaging session, we will measure the in vivo impedance, CV, charge injection limit, and stimulation threshold. The neuronal response (activity, health, density), microglia (morphology, coverage and motility) and BBB integrity will be recorded, and compared over time points between material types, and to the non-stimulated sites. In addition, we will closely track the electrode health with electrochemical interrogation, imaging and explant analysis.

This project develops novel devices and methods to record the electrical activity of large numbers of neurons while simultaneously identifying their specific cell types. Specific cell types have precise computational roles in neural information processing systems, but in most cases cell types are not identifiable from electrical activity alone. In cortical regions responsible for decision making, the difficulties posed by intermingled cell types are further complicated by layers, recurrent connections, and the multitude of interneuron types. In order to understand how neural information processing systems mediate decision making, it is necessary to (1) record simultaneously from many neurons to quantify high-dimensional activity, (2) identify and ascertain the precise computational roles of the cell types within those ensembles, and (3) chemically perturb neural ensembles to determine causal functionality. This project will for the first time enable all 3 of these capabilities simultaneously. The proposed neural interface will incorporate recording electrodes, neurochemical stimulators, and flat optical imager waveguides all in the slim form factor of an implantable micro-needle. This device will be used to study the detailed circuit-level functionality of specific cell types involved in the population activity of neurons. The collaboration between a team of engineers and biologists provides a unique interdisciplinary environment for training graduate and undergraduate students working on this project. The PIs will also design a new course on neurotechnology to teach students about the needs in neuroscience research and opportunities in engineering to design next generation neural interfaces.

This project incorporates an integrative approach based on innovations in technology (nanotechnology, photonics, and neurotechnology) as well as advancements in fundamental neurobiology and transcriptional profiling of cells based on optical tagging to shed light on the role of specific cell types on collective actions of neurons during behavior. Building on a recently developed polymer-based optical waveguide platform with embedded micromirror ports, the investigators will design a novel flat imager that can be monolithically integrated with micro-electrodes to optically image the cell identities, while simultaneously recording their electrophysiology activity. The proposed neural interface (i) is compact and flexible, (ii) combines high-density electrical recording with chemical stimulation, (iii) contains electrically actuated nanocomposite polymers, and (iv) enables on-shank fluorescent imaging using a novel micro-imager array based on parylene polymer photonic waveguides. The utility of this technology platform will be demonstrated for studying cell types involved in encoding sensory sensations in rats during whisker stimulation. The developed multimodal probes will also be disseminated to different neurobiology labs to be used in other experimental contexts to amplify the impact of the proposed project. The outcome of this cross-field research will be (i) a new technology platform that can be used to test various neuroscience hypotheses on the role of specific cell types in encoding and transforming information in brain and (ii) a valuable dataset that can enhance existing mathematical models of neuronal population activity by adding new dimensions to the existing large-scale data based solely on electrophysiology, and will enable an entirely new class of neurobiology experiments.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The ability to quantify spatially discrete dopamine (DA) concentration over a chronic, multi-week timescale is paramount to unlocking the mechanisms underlying healthy and disease state behavior. DA signaling throughout the brain occurs over multiple timescales. Phasic signaling results from high frequency burst firing, whereas tonic DA release is maintained by low frequency ?pacemaker firing?. For decades, fast scan cyclic voltammetry (FSCV) at carbon fiber microelectrodes (CFEs) has been used to record sub second phasic DA transmission, but measuring resting level, tonic DA concentrations has been a technical challenge. We have recently shown that conductive polymer nanocomposite coating consisting of poly(3,4-ethylenedioxythiophene) and acid functionalized carbon nanotubes (PEDOT/CNT) significantly increases electrode sensitivity and selectivity for DA, and when combined with a novel square wave voltammetry (SWV) protocol, is capable of measuring resting DA concentrations in vivo with high selectivity. The same coating can also record sub second DA release using FSCV. Microfabricated multielectrode arrays (MEAs) have been developed to monitor neurophysiological signals simultaneously from multiple recording sites with high spatial resolution. However, the poor sensitivity and selectivity of conventional metal electrodes have limited the use of MEAs for neurochemical sensing. By applying PEDOT/fCNT coating onto MEAs, we can increase both the sensitivity and selectivity of neurochemical detection from MEA. Furthermore, implantation of stiff MEAs inevitably causes neuronal damage and inflammatory glial response, both of which compromise sensor performance, especially for long-term applications. Recent advancement in MEA technology has revealed that flexible and subcellular sized implants significantly mitigate the foreign body response resulting in seamless integration within neural tissue. Here, we hypothesize that chronic multisite DA measurement can be enabled through combining the highly sensitive PEDOT/CNT coating with ultra-small, flexible neural recording probe technology. The first specific aim is to fabricate PEDOT/fCNT functionalized flexible MEA capable of detecting DA with sensitivities and LODs in the physiologically relevant concentration range. PEDOT/CNT coating conditions will be optimized for electrode sites on 16-channel, flexible SU-8 MEAs. DA sensing performance will be investigated using SWV and FSCV in the presence of interferents. Coating stability will be assessed via mechanical bending and agar insertion experiments. The second specific aim is to determine the efficacy of PEDOT/fCNT functionalized flexible MEAs for acute and chronic in vivo DA sensing. In the acute validation experiments, sensors will be implanted into the DS of anesthetized rats. SWV (measure basal DA) and FSCV (measure electrically evoked sub second DA) measurements will be recorded from 16 individual electrode sites spanning the entire sagittal length of the DS (4 mm) before and after the acute administration of either nomifensine (NOM, increase DA) or ?-methyl-DL-tyrosine (?MPT, decrease DA). In the chronic experiments, sensors will be used to record spatially discrete tonic and electrically evoked phasic DA over 28 days in 6-OH-DA lesioned rats over a period of 28 days post lesion. Post-mortem immunohistology will be conducted after 7, 14 and 28 days of probe implantation to assess inflammatory host tissue response and to monitor lesion formation. Observation of any spatially correlated changes in resting and electrically evoked DA relating to 6-OH-DA lesion formation will reveal the effectiveness of the chronic DA sensor and provide valuable physiological insight into Parkinson's Disease. This proposal has the potential to revolutionize the state of the art of neurochemical sensing by enabling high fidelity chronic measurement of tonic and phasic DA release from multiple discrete neuron groupings. The proposed in vivo experiments could shed light on the mechanisms of Parkinsonian DA compensation. On a broader sense, this technology will find a wide spread use throughout a range of basic neuroscience and clinical research areas with the ultimate goal of understanding healthy and diseased brain as well as developing effective therapies.

Project Summary The real-time measurement of neurotransmitters in vivo in living brain is of utmost importance for understanding brain functions in normal and pathological conditions and to improve diagnosis and treatments of neurological and neuropsychiatric diseases. High surface area carbon (HSAC), or nanocarbon, has been considered the ideal material for electrochemical detection of neurotransmitters, due to its outstanding electrochemical properties and chemical inertness. However, HSAC microelectrode arrays (MEAs) are difficult to fabricate, and the extreme environments needed for the nanocarbon synthesis limit the choice of substrate to rigid materials that can withstand high temperatures. Moreover, chemical doping to improve electrochemical sensing also requires high-temperature post-synthesis processing. Thus, there is an unmet need for fabricating implantable HSAC MEAs on flexible substrates with tunability of morphology and chemistry, for multisite measurements of neurotransmitters at different temporal resolutions (ms to min), within and across brain regions (µm to mm). To fill this gap, this project introduces a new laser-induced nanocarbon (LINC) fabrication technique, capable of patterning customizable types of HSAC on-demand directly on flexible polymers. LINC is a new direct-write process with the unprecedented ability for bottom-up growth of nanocarbons on polymers that act as the carbon source upon laser irradiation. Our inventive approach enables for the first time, a fast, low-cost, batch-fabrication of HSAC MEAs in a highly reproducible way, without the need of high-temperature carbon synthesis, or multistep microfabrication processes. Importantly, LINC allows in situ precise control of the nanocarbon atomic structure, nanoscale morphology, and surface chemistry. Thus, our HSAC MEAs will be tailored for high-sensitivity electrochemical detection of different neurotransmitters using two different electrochemical technique: fast scan cyclic voltammetry (FSCV), for capturing of fast phasic dynamics, and square wave voltammetry (SVW) for detecting tonic levels. Following a meticulous in vitro optimization, we will determine the effectiveness of the proposed HSAC MEA in performing electrochemical sensing of electroactive neurotransmitters for acute in vivo detection of 1) tonic (via SWV) and 2) electrically evoked (via FSCV) dopamine and serotonin release in the rat dorsal striatum and in the hippocampus (CA2 region) of rat brain, respectively or simultaneously. The successful completion of this project will provide 1) a cutting-edge technology with the potential to revolutionize the state- of-the-art of nanocarbon-based MEA fabrication for neurochemical applications, and 2) will provide the scientific community with a platform for unprecedented studies of neurotransmitters and their interactions in normal and pathological brain conditions.

Project Summary The ability to monitor activity of ensembles of neurons at single-cell resolution, chronically, over long time periods is greatly desired by neuroscientists. A variety of multi-electrode arrays (MEAs) have been developed for in vivo studies. These arrays are capable of revealing the activity of neuronal ensembles. Unfortunately, none of the devices on the market is fully capable of obtaining recordings that are simultaneously high-yield and high-quality, as well as stable and useful over months to years. This well-known challenge has greatly limited our ability to track the activity of populations of single neurons over a sufficient period of time to fully investigate circuit change during learning and memory, development and aging, or disease progression and wound healing. Additionally, the clinical use of brain machine interface (BMI), which utilize recorded neural activities to decode movement intent for controlling machine, has been hindered by the unstable and unreliable recording. We have developed a biomimetic coating composed of a brain-derived L1-cell adhesion molecule that mitigate the inflammatory host tissue reaction. In rodents, L1 coated NeuroNexas probes maintained high quality neural recording over the period of 16 weeks with significant higher single unit yield and signal to noise ratio than the uncoated control probes. Meanwhile, recordings in non-human primates (NHPs) with L1-coated Blackrock MEAs also demonstrated high quality performance in single unit yield and signal amplitude for at least 6 months. MEA manufacturers and users expressed strong interest in utilizing this technology. However, the coating made of biological protein is fragile and may lose bioactivity during the harsh environment of shipping, storage and sterilization. In order to make the L1 coating a technology that can be widely adopted by the neuroscience community, we propose to optimize the coating stability and develop practical protocols for coating preservation, storage, packaging, delivery and sterilization. The bioactivity of the coating prepared with different protocols will first be tested with cell cultures. Promising procedures will then be tested with implantation and recording in rodents at the University of Pittsburgh. Once optimum coating and procedures are determined, coated arrays will be delivered to users to evaluate the coating performance. Dr. Buzsaki (NYU) will test the L1 coated NeuroNexas arrays in freely moving rats. Dr. Schwartz (U. Pitt) and Dr. Chestek (U. Michigan) will test the L1 coated Blackrock arrays in NHPs for BMI studies. Users will work closely with us to define specific performance criteria in their recording applications, compare performance of coated and uncoated arrays, and provide user input for us to improve the packaging and delivery. Throughout the project, representatives from two MEA manufacturers, Blackrock Microsystems and NeuroNexus Technology, will serve as consultants to ensure compatibility of our procedures with their devices and guide us on the path to dissemination. The project will produce a coating technology that is both easy to adopt and generalizable to all types of state- of-art and emerging MEAs. Solving the practical issues of sterilization, packaging and delivery is a critical step toward commercial and clinical translation of the technology. High quality and stable of neural recording will greatly improve our ability to map brain activity in long-term experiments, and benefit brain-computer interfaces and other types of neural prostheses. In a broader sense, the protocols developed here for preserving immobilized protein during storage, delivery and sterilization should be applicable to other medical implants containing bioactive proteins, immunoassays, protein arrays, enzyme-based biosensors or any micro/nano devices that incorporate biological components.