Fabrication of a Multilayer Implantable Cortical Microelectrode Probe to Improve Recording Potential.

Intracortical neural probes are a key enabling technology for acquiring high fidelity neural signals within the cortex. They are viewed as a crucial component of brain-computer interfaces (BCIs) in order to record electrical activities from neurons within the brain. Smaller, more flexible, polymer-based probes have been investigated for their potential to limit the acute and chronic neural tissue response. Conventional methods of patterning electrodes and connecting traces on a single supporting layer can limit the number of recording sites which can be defined, particularly when designing narrower probes. We present a novel strategy of increasing the number of recording sites without proportionally increasing the size of the probe by using a multilayer fabrication process to vertically layer recording traces on multiple Parylene support layers, allowing more recording traces to be defined on a smaller probe width. Using this approach, we are able to define 16 electrodes on 4 supporting layers (4 electrodes per layer), each with a 30 µm diameter recording window and 5 µm wide connecting trace defined by conventional LWUV lithography, on an 80 µm wide by 9 µm thick microprobe. Prior to in vitro and in vivo validation, the multilayer probes are electrically characterized via impedance spectroscopy and evaluating crosstalk between adjacent layers. Demonstration of acute in vitro recordings in a cerebral organoid model and in vivo recordings in a murine model indicate the probe's capability for single unit recordings. This work demonstrates the ability to fabricate smaller, more compliant neural probes without sacrificing electrode density.

Neural engagement with online educational videos predicts learning performance for individual students.

Online educational materials are largely disseminated through videos, and yet there is little understanding of how these videos engage students and fuel academic success. We hypothesized that components of the electroencephalogram (EEG), previously shown to reflect video engagement, would be predictive of academic performance in the context of educational videos. Two groups of subjects watched educational videos in either an intentional learning paradigm, in which they were aware of an upcoming test, or in an incidental learning paradigm, in which they were unaware that they would be tested. "Neural engagement" was quantified by the inter-subject correlation (ISC) of the EEG that was evoked by the videos. In both groups, students with higher neural engagement retained more information. Neural engagement also discriminated between attentive and inattentive video viewing. These results suggest that this EEG metric is a marker of the stimulus-related attentional mechanisms necessary to retain information. In the future, EEG may be used as a tool to design and assess online educational content.

A Cerebral Organoid Connectivity Apparatus to Model Neuronal Tract Circuitry.

Mental disorders have high prevalence, but the efficacy of existing therapeutics is limited, in part, because the pathogenic mechanisms remain enigmatic. Current models of neural circuitry include animal models and post-mortem brain tissue, which have allowed enormous progress in understanding the pathophysiology of mental disorders. However, these models limit the ability to assess the functional alterations in short-range and long-range network connectivity between brain regions that are implicated in many mental disorders, e.g., schizophrenia and autism spectrum disorders. This work addresses these limitations by developing an in vitro model of the human brain that models the in vivo cerebral tract environment. In this study, microfabrication and stem cell differentiation techniques were combined to develop an in vitro cerebral tract model that anchors human induced pluripotent stem cell-derived cerebral organoids (COs) and provides a scaffold to promote the formation of a functional connecting neuronal tract. Two designs of a Cerebral Organoid Connectivity Apparatus (COCA) were fabricated using SU-8 photoresist. The first design contains a series of spikes which anchor the CO to the COCA (spiked design), whereas the second design contains flat supporting structures with open holes in a grid pattern to anchor the organoids (grid design); both designs allow effective media exchange. Morphological and functional analyses reveal the expression of key neuronal markers as well as functional activity and signal propagation along cerebral tracts connecting CO pairs. The reported in vitro models enable the investigation of critical neural circuitry involved in neurodevelopmental processes and has the potential to help devise personalized and targeted therapeutic strategies.

Neuronal substrates alter the migratory responses of nonmyelinating Schwann cells to controlled brain-derived neurotrophic factor gradients.

Neurodegeneration and dysfunction cause mobility impairment and/or paralysis in millions of adults, worldwide. Motor deficit and recovery in adults depend upon the plasticity of the neuromuscular junction (NMJ), a tripartite, biochemical synapse that transduces electrical impulses from the brain into voluntary contraction of skeletal muscle. Nonmyelinating Schwann cells (nmSCs) of the NMJ have been increasingly recognized as active synaptic partners with motor neurons and muscle and have become recent therapeutic targets for regeneration. nmSC synaptic transmission, plasticity, and growth are strongly modulated by brain-derived neurotrophic factor (BDNF), whose regenerative abilities have been explored through emerging biomaterials and tissue-engineered systems, as well as via clinical trials. Experimental models engineered to investigate integrated NMJ response(s) to local gradients of BDNF will both advance our understanding of key modulators of synaptic activity, postinjury, and aid in the development of NMJ-targeted, regenerative therapies to restore mobility. The current study examined the ability of nmSCs to respond to microfluidically controlled BDNF signaling upon different haptotactic substrates of motor neurons (MNs) and laminin adhesion coating. Tests seeding nmSCs sequentially with MNs illustrated that sequential seeding reported a fivefold increase in levels of tropomyosin receptor kinase B expression in response to BDNF signaling and a nearly fivefold increase in migration distance along BDNF gradients. By contrast, concurrent seeding of MNs and nmSCs upon laminin adhesion coating illustrated a difference in migration distance of less than one third-fold over control. Our findings are among the first to examine migratory responses of nmSCs for regenerative strategies and highlight the potential to restabilize NMJ synaptic activity by affecting nmSC behaviors through therapeutic BDNF and seeding with MNs.

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

A Milled Microdevice to Advance Glia-Mediated Therapies in the Adult Nervous System.

Neurodegenerative disorders affect millions of adults worldwide. Neuroglia have become recent therapeutic targets due to their reparative abilities in the recycling of exogenous neurotoxins and production of endogenous growth factors for proper functioning of the adult nervous system (NS). Since neuroglia respond effectively to stimuli within in vivo environments on the micron scale, adult glial physiology has remarkable synergy with microscale systems. While clinical studies have begun to explore the reparative action of Müller glia (MG) of the visual system and Schwann Cells (ShC) of the peripheral NS after neural insult, few platforms enable the study of intrinsic neuroglia responses to changes in the local microenvironment. This project developed a low-cost, benchtop-friendly microfluidic system called the glia line system, or gLL, to advance the cellular study needed for emerging glial-based therapies. The gLL was fabricated using elastomeric kits coupled with a metal mold milled via conventional computer numerical controlled (CNC) machines. Experiments used the gLL to measure the viability, adhesion, proliferation, and migration of MG and ShC within scales similar to their respective in vivo microenvironments. Results illustrate differences in neuroglia adhesion patterns and chemotactic behavior significant to advances in regenerative medicine using implants and biomaterials, as well as cell transplantation. Data showed highest survival and proliferation of MG and ShC upon laminin and illustrated a four-fold and two-fold increase of MG migration to dosage-dependent signaling from vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), respectively, as well as a 20-fold increase of ShC migration toward exogenous brain-derived neurotrophic factor (BDNF), compared to media control. The ability to quantify these biological parameters within the gLL offers an effective and reliable alternative to photolithography study neuroglia and their local ranges on the tens to hundreds of microns, using a low-cost and easily fabricated system.