Dendritic calcium signals in rhesus macaque motor cortex drive an optical brain-computer interface

Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging

Calcium imaging for stem cell grafts in mouse neocortex: continuous tracking and assessment of functional integration

Neural stem cells have the capacity to self-renew and differentiate into multiple specialized neural phenotypes, providing a promising therapeutic strategy to replace damaged or lost neurons in neurological diseases. Preliminary clinical trials have demonstrated that grafting neural stem cells in brain tissue could achieve some therapeutic effects. However, the outcomes are highly variable with various adverse effects. Unfortunately, an understanding of which factors underlie success or failure remains elusive.  To facilitate the development of safe and effective clinical therapies using grafted neural stem cells, it will be informative to improve our understanding of the environmental factors and means of controlling the process of integration. It is currently difficult to observe the characteristics of neural stem cell integration into host tissue continuously; the integration process is thus often deduced from snapshots of post-mortem tissue requiring sacrifice of transplanted animals at distinct time points, which is often inefficient and impractical to carry out. This dissertation addresses the problem by describing the development and use of a reliable experimental platform that enables continuous observation of stem cells grafted in mouse cortex throughout the course of integration. Current attempts to image neocortical regions on the surface of mouse brain typically use a small glass disc attached to the cranial surface. This approach, however, is often challenged by progressive deterioration in optical quality and permits limited tissue access after its initial implantation. I describe a design and demonstrate a two-stage cranial implant device developed with a remarkably versatile material, polydimethylsiloxane, which facilitates longitudinal imaging experiments in mouse cortex. The system was designed considering biocompatibility and optical performance. This enabled us to achieve sustained periods of optical quality, extending beyond a year in some mice, and allows imaging at high spatiotemporal resolution using wide-field microscopy. Additionally, the two-part system, consisting of a fixed headplate with integrated neural access chamber and optical insert, allowed flexible access to the underlying tissue. Finally, I demonstrate the technical feasibility of rapid adaptation of the system to accommodate varying applications requiring long-term ability to visualize and access neural tissue. Utilizing the two-part cranial window system, two distinct sources of neural stem cells dissected from distinct anatomical regions within mouse embryo and labeled with genetically encoded calcium indicators were transplanted into an adult mouse cortex. The cellular dynamics across hundreds of transplants were acquired periodically across several months using wide-field epifluorescence microscopy. This allowed longitudinal comparisons of cell and network activity from each animal. Immediately after transplantation, in both cell populations, the spontaneous network activity was dominated by a highly recurrent pattern of synchronous bursts, similar to the characteristic activity observed during early development of endogenous cells. Gradually, the network activity diversified and matured into complex activation patterns — network states with better information processing capacities. In an attempt to quantify functional integration of grafted cell-derived neurons with host neural network, several strategies were employed to capture the evolution in dynamic patterns of network activation, including cross-correlation, entropy, and information carrying capacity. Future work using such approach to analyze environmental factors on impacting neural stem cell integration in the native context will contribute to advanced stem cell therapy for neurological disorders.2020-09-28T00:00:00

Tools for interfacing, extracting, and analyzing neural signals using wide-field fluorescence imaging and optogenetics in awake behaving mice

Imaging of multiple cells has rapidly multiplied the rate of data acquisition as well as our knowledge of the complex dynamics within the mammalian brain. The process of data acquisition has been dramatically enhanced with highly affordable, sensitive image sensors enable high-throughput detection of neural activity in intact animals. Genetically encoded calcium sensors deliver a substantial boost in signal strength and in combination with equally critical advances in the size, speed, and sensitivity of image sensors available in scientific cameras enables high-throughput detection of neural activity in behaving animals using traditional wide-field fluorescence microscopy. However, the tremendous increase in data flow presents challenges to processing, analysis, and storage of captured video, and prompts a reexamination of traditional routines used to process data in neuroscience and now demand improvements in both our hardware and software applications for processing, analyzing, and storing captured video. This project demonstrates the ease with which a dependable and affordable wide-field fluorescence imaging system can be assembled and integrated with behavior control and monitoring system such as found in a typical neuroscience laboratory. An Open-source MATLAB toolbox is employed to efficiently analyze and visualize large imaging data sets in a manner that is both interactive and fully automated. This software package provides a library of image pre-processing routines optimized for batch-processing of continuous functional fluorescence video, and additionally automates a fast unsupervised ROI detection and signal extraction routine. Further, an extension of this toolbox that uses GPU programming to process streaming video, enabling the identification, segmentation and extraction of neural activity signals on-line is described in which specific algorithms improve signal specificity and image quality at the single cell level in a behaving animal. This project describes the strategic ingredients for transforming a large bulk flow of raw continuous video into proportionally informative images and knowledge

Finding Common Ground: The Common Marmoset as a Model to Accessing and Providing Insight into the Social Brain

Utilizing the immense strengths of the common marmoset (Callithrix jacchus) as a model organism, we executed three efforts with the common goal of revealing key insights into dissecting the social brain. First, we examined the hypothesis that there is a neural, functional architecture underlying face processing. Faces form a unique category of stimuli that bridge visual perception and social cognition. They are processed in dedicated areas of cortex, face patches, which are organized into an interconnected network. While insight into neurons within face patches has been explored through extracellular electrophysiology, the functional architecture of local ensembles of cells has remained elusive. Recently discovered face patches in the lissencephalic common marmoset brain provide the cortical access necessary in order to employ optical techniques to resolve both the functional properties as well as the spatial organization of these neural ensembles. Unique to marmoset face patches, the cortical boundaries of these patches overlap with areas usually attributed to early stage visual processing. In particular, the occipitally located face patch “O” overlaps with V2, an area usually thought to be composed of neurons with tuning properties to low-level visual stimuli such as, but not limited to, orientation and direction. Here, in the anesthetized marmoset, we demonstrated the areal parcelization of function along the dimensions of low-level visual stimuli and high-level visual stimuli including faces, objects, and bodies using two-photon microscopy. We found that the functional architecture revealed best supports naturalistic stimuli processing more so than face processing. This suggests the interpretation that face patch “O” is a selective measure of recurrent activity or feedback activity into natural scene processing of faces rather than an area of pure face perception. As an area of natural scene processing, there may be cell-type specific populations supporting such segregation and suppression of low-level feature responses. We developed a novel approach to characterize the entire region using iterative antibody staining with volumetric immunohistochemistry. From a genetic and circuits approach, social cognition has been implicated in numerous circuits across brain structures. One such circuit includes pyramidal neurons projecting from layer V of the medial prefrontal cortex (mPFC) to the nucleus accumbens (NAc), given its involvement in social-reward behaviors and depression in rodents and primates. This circuit presents an opportunity not only to study the genetic blueprint of a socially relevant circuit, but also to determine how it is conserved across rodents and non-human primates. Here we established retrograde viral Translating Ribosome Affinity Purification (retro-vTRAP), using techniques previously implemented only in rodents. retro-vTRAP enables sequencing of mRNA bound to EGFP-tagged polyribosomes from chosen projection neurons that have been virally labeled with an EGFP-L10a transgene. retro-vTRAP facilitates the study of gene expression patterns in specific neural cell types to be studied in the complex, heterogeneous tissue of the cortex, shedding new light on transcriptional differences in cognitively relevant cells across species. We implemented retro-vTRAP in marmosets, macaques, rats, and mice and found a single conserved, enriched gene ontology set across marmosets and the rodents. This gene set is involved in negative regulation of endoplasmic reticulum stress induced apoptosis and is implicated in depression and mechanisms of treatment. This enrichment specific to this projection may be part of a genetic signature of this projection and provide functional modulation in times of social stress. Lastly, from a developmental approach, we detailed a critical embryonic period of protracted growth resulting in a developmental delay in the marmoset compared to other species, relative to gestation length. This was revealed through use of highresolution, serial ultrasound scans of developing marmosets. We demonstrated that this delay period occurs during gastrulation and before neural tube closure. During this protracted period, the amnion undergoes massive restructuring and growth, creating a unique opportunity to utilize this window to introduce genetic manipulations creating a proxy of a transgenic animal without conventional transgenic methodologies. This may be transformative to building more accurate models of disorders, particularly cognitive disorders that cannot be fully replicated in rodent models

Carbon Fiber Electrode Arrays for Cortical and Peripheral Neural Interfaces

Neural interfaces create a connection between neural structures in the body and external electronic devices. Brain-machine interfaces and bioelectric medicine therapies rely on the seamless integration of neural interfaces with the brain, nerves, or spinal cord. However, conventional neural interfaces cannot meet the demands of high channel count, signal fidelity, and signal longevity that these applications require. I investigated the damage resulting from conventional Utah arrays after multiple years of implantation in the cortex of a non-human primate as a possible explanation for these limitations. The neuron density around the electrode shanks was compared to the neuron density of nearby healthy tissue, finding a 73% loss in density around the electrodes. The explanted arrays were imaged and characterized for degradation. Coating cracks, tip breakage, and parylene cracks were the most common degradation type. A significantly higher number of tip breakage and coating crack occurrences were found on the edges of the arrays as compared to the middle. In this work, I made clear the need for a minimally damaging alternative to the Utah electrode array. Neural interfaces composed of carbon fiber electrodes, with a diameter of 6.8 microns, could enable a seamless integration with the body. Previous work resulted in an array of individuated carbon fiber electrodes that reliably recorded high signal-to-noise ratio neural signals from the brain for months. However, the carbon fiber arrays were limited by only 30% of the electrodes recording neural signals, despite inducing minimal inflammation. Additionally, it was relatively unknown if carbon fibers would make suitable long-term peripheral neural interfaces. Here, I illustrate the potential of carbon fiber electrodes to meet the needs of a variety of neural applications. First, I optimized state-of-the-art carbon fiber electrodes to reliably record single unit electrophysiology from the brain. By analyzing the previous manufacturing process, the cause of the low recording yield of the carbon fiber arrays was identified as the consistency of the electrode tip. A novel laser cutting technique was developed to produce a consistent carbon fiber tip geometry, resulting in a near tripling of recording yield of high amplitude chronic neural signals. The longevity of the carbon fiber arrays was also addressed. The conventional polymer coating was compared against platinum iridium coating and an oxygen plasma treatment, both of which outperformed the polymer coating. In this work, I customized carbon fiber electrodes for reliable, long-term neural recording. Secondly, I translated the carbon fiber technology from the brain to the periphery in an architecture appropriate for chronic implantation. The insertion of carbon fibers into the stiffer structures in the periphery is enabled by sharpening the carbon fibers. The sharpening process combines a butane flame to sharpen the fibers with a water bath to protect the base of the array. Sharpened carbon fiber arrays recorded electrophysiology from the rat vagus nerve and feline dorsal root ganglia, both structures being important targets for bioelectric medicine therapies. The durability of carbon fibers was also displayed when partially embedded carbon fibers in medical-grade silicone withstood thousands of repeated bends without fracture. This work showed that carbon fibers have the electrical and structural properties necessary for chronic application. Overall, this work highlights the vast potential of carbon fiber electrodes. Through this thesis, future brain-machine interfaces and bioelectric medicine therapies may utilize arrays of sub-cellular electrodes such as carbon fibers in medical applications.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169982/1/elissajw_1.pd

Brain Computer Interfaces: Challenges to Clinical Viability Addressed in the Laboratory

Paralysis following spinal cord injuries, amyotrophic lateral sclerosis, stroke, and other disorders can intervene with signal transduction from the brain to the motor periphery, and eliminate the ability to perform volitional movements. Brain computer interfaces (BCI) directly measure brain activity associated with the user’s intent and translate the recorded brain activity into control signals for BCI applications, such as moving a computer cursor or a robot arm. While BCI technology has become an active and exciting field of research, much of the field’s development and achievements to date have taken place in the laboratory. The translation of BCI technology to the clinical setting is still not a reality. My thesis research has been dedicated to the objective of facilitating the translation of BCI systems from the primate lab to a clinical setting. That guiding objective has led me to work on several projects including: a technique that vastly improves the longevity of surgical implants in primate studies; a task that pushes the limits of sensorimotor performance – improving our knowledge of the function of primary motor cortex during realistic reaches and allowing us to quantify feedback effectiveness; characterizing the long-term tissue response to chronically implanted electrodes, and investigating how to optimally select parameters for neural information extraction. Each of these contributions will help bring BCI systems one step closer to clinical reality

Doctor of Philosophy

dissertationNeural recording devices are a therapeutic and diagnostic option for central nervous system (CNS) diseases and a vital component of neuroscience research. However, poor functional longevity is a major hurdle facing this broad class of devices. Decreases in functionality are associated, in part, with the foreign body response (FBR) surrounding chronically implanted recording devices; which includes chronic inflammation, astrogliosis, blood-brain barrier (BBB) leakiness, and neuronal cell death. Two potential areas for intervention were explored including the initial hemorrhage that results from device insertion and the neuroinflammatory sequela. Researchers have shown that cellular interactions with extracellular matrix (ECM) are able to affect both of these aspects of the FBR. The central hypothesis driving this work is that ECM coatings which target the initial hemorrhage, should decrease the FBR. This was investigated by coating silicon microelectrode arrays (MEAs) with ECM and implanting them into motor cortex of rats. Two ECM coatings were investigated, including the xenogeneic clinically-used Avitene Microfibrillar Collagen Hemostat and allogeneic astrocyte-derived ECM. Results show that the allogeneic astrocyte-derived ECM decreased astrogliosis within the recording zone at the 8-week time point. This decrease in astrogliosis may improve device functionality, as indicated by previous studies that correlated recording metrics to histology. Interestingly, the xenogeneic Avitene coating increased IgG within the recording zone at the 8-week time point. Collectively, these results show that ECM coatings with different genetic backgrounds and compositions are able to differentially affect specific aspects of the FBR. To broaden the knowledge on the FBR to neural recording devices, the FBR of headstage components used to anchor CNS devices to the skull was analyzed. Results showed that the FBR to fixation screws and fixation anchoring adhesive illicit a chronic FBR that has all of the hallmarks described for MEAs implanted in brain tissue. Moreover, results show evidence of persistent neuroinflammation below a variety of fixation screws including chronic macrophage activation, demyelination, and neural tissue loss. Understanding the FBR of fixation techniques, which is common to a wide variety of CNS devices, may improve the biocompatibility of existing devices and provide a reference for future biologically-informed device designs

Application of MRI Connectivity in Stereotactic Functional Neurosurgery

This thesis examines potential applications of advanced MRI-connectivity studies in stereotactic functional neurosurgery. Several new analysis methodologies are employed to: (1) build predictive models of DBS surgery outcome; (2) refine the surgical target and (3) help build a better understanding of the pathogenesis of the treated conditions and the mechanism of action of DBS therapy. The experimental component is divided into three main parts focusing on the following pathologies: (1) Parkinson’s disease (PD), (2) tremor and (3) trigeminal autonomic cephalalgias (TAC). Section I: In the first experiment (chapter 3), resting state fMRI was used to find radiological biomarkers predictive of response to L-DOPA in 19 patients undergoing subthalamic nucleus (STN) DBS for PD. A greater improvement in UPDRS-III scores following L-DOPA administration was characterized by higher resting state functional connectivity (fcMRI) between the prefrontal cortex and the striatum (p=0.001) and lower fcMRI between the pallidum (p=0.001), subthalamic nucleus (p=0.003) and the paracentral lobule. In the second experiment (chapter 4), structural (diffusion) connectivity was used to map out the influence of the hyperdirect pathways on outcome and identify the therapeutic ‘sweet spots’ in twenty PD patients undergoing STN-DBS. Clusters corresponding to maximum improvement in symptoms were in the posterior, superior and lateral portion of the STN. Greater connectivity to the primary motor area, supplementary motor area and prefrontal cortex was predictive of higher improvement in tremor, bradykinesia and rigidity, and rigidity respectively. The third experiment (chapter 5) examined pyramidal tract (PT) activation in 20 PD patients with STN-DBS. Volume of tissue activation (VTA) around DBS contacts were modelled in relation to the PT. VTA/ PT overlap predicted EMG activation thresholds. Sections II: Pilot data suggest that probabilistic tractography techniques can be used to segment the ventrolateral (VL) and ventroposterior (VP) thalamus based on cortical and cerebellar connectivity in nine patients who underwent thalamic DBS for tremor (chapter 6). The thalamic area, best representing the ventrointermedialis nucleus (VIM), was connected to the contralateral dentate cerebellar nucleus. Streamlines corresponding to the dentato-rubro-thalamic tract (DRT) connected M1 to the contralateral dentate nucleus via the dentato-thalamic area. Good response was seen when the active contact’s VTA was in the thalamic area with the highest connectivity to the contralateral dentate nucleus. Section III: The efficacy and safety of DBS in the ventral tegmental area (VTa) in the treatment of chronic cluster headache (CH) and short lasting unilateral neuralgiform headache attacks (SUNA) were examined (chapters 7 and 8). The optimum stimulation site within the VTa that best controls symptoms was explored (chapter 9). The average responders’ deep brain stimulation activation volume lay on the trigemino-hypothalamic tract, connecting the trigeminal system and other nociceptive brainstem nuclei, with the hypothalamus, and the prefrontal and mesial temporal areas

Cell Scaffold- and Drug-based Strategies for Improving the Integration of Neural Prostheses into Brain Tissue.

Neuroprosthetic devices record extracellular cortical signals which may be used to place exterior devices under a patient’s direct control. Therefore, these systems may restore function to individuals immobilized by paralysis or neurodegenerative disease. For neuroprosthetics to be useful in clinical and research settings, long-term, stable recordings must be achieved. However, these devices are plagued by recording instability, and the reactive tissue response that occurs after insertion into the brain is a likely cause. Specifically, neuronal density is reduced surrounding devices, and encapsulation (composed of microglia and astrocytes) isolates neuroprostheses from their neuronal signal sources. The research presented describes the development and evaluation of two strategies to improve the tissue response to neuroprostheses: (1) a neural stem cell (NSC)-seeded scaffold and (2) a cell cycle-inhibiting drug. NSCs were hypothesized to secrete factors, such as neurotrophins, which would improve device-tissue integration. The cells were encapsulated in an alginate hydrogel and seeded into a well on the devices. In the first study, in vitro testing identified the optimal alginate composition for NSC encapsulation. The second study characterized the relationship between alginate composition, degradation, and biocompatibility in vivo. The third study evaluated the effects of the NSC scaffold on the tissue response to implanted probes in vivo. The scaffold mitigated the early tissue response, but exacerbated it by six weeks post-implantation. Based on research showing a link between central nervous system injury and cell-cycle re-entry, the final study of the dissertation investigated the role of this phenomenon in the tissue response to neural prostheses. Specifically, the effects of a cell cycle-inhibiting drug (flavopiridol) on electrophysiology and tissue response metrics were explored. Flavopiridol reduced expression of a cell cycle protein in microglia surrounding probes three days after implantation and decreased impedance over the 28 day study period. Additionally, the data revealed novel, significant correlations between recording quality, impedance, and endpoint histology measurements. In conclusion, the studies demonstrate significant effects of two intervention strategies on tissue response and electrophysiology measurements, characterize alginate stability and its use as a NSC scaffold, and add insight into the relationship between the tissue-device interface and recording quality.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61573/1/erinfred_1.pd