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.

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.

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.