Yevgeny Berdichevsky, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The overall aim of this project is to develop an interface between organotypic brain neural circuits and electrophysiological recording equipment that is capable of long-term (weeks), highly parallel stimulation and recording of electrical activity at the level of individual cells and their processes. Our understanding of the role of individual neurons in the neural circuits of the central nervous system will benefit from the introduction of tools that allow the researcher to manipulate the circuit connectivity and monitor the electrical activity in vitro, over long term, and in a system that contains a basic functional unit of the in vivo circuit such as a cortical column or a slice of the hippocampus. We propose to develop an in vitro platform that combines organotypic brain slice cultures with a microdevice that is capable of geometric confinement of the axonal tracts connecting various parts of the circuit. The device will consist of polymer microchannels for axon guidance and an integrated microelectrode array for stimulation and recording of the signals in the neural circuit. The confinement of axons in insulated microchannels will enable parallel and independent stimulation of many axons in a pathway under study, mimicking the functionality of axonal pathways in vivo. We will focus on two model pathways, the thalamocortical pathway that can be recreated in vitro by coculturing slices from the thalamic nuclei and the primary cortices, and the perforant path, recreated by coculture of hippocampus and entorhinal cortex slices. These pathways, when recreated in vitro in a device that enables the researcher to apply stimulation selectively (to individual axons) and record from multiple cells in the recepient neural network over long term. The specific aims of this proposal are: (1) Fabrication of the interface microdevice with polymer channels for axon confinement and integrated multiple electrode array for long-term recording, (2) Organotypic cultures of perforant and thalamocortical pathways on microfabricated devices, and (3) Stimulation/recording of neural activity from model pathways. The platform developed in the course of proposed research will enable detailed investigation of the role timing-dependent synaptic plasticity plays in the development of neural circuitry, and of Hebbian learning mechanisms, contributing to understanding of the causes of developmental and learning disorders. The organotypic culture-electrode array platform also has an important application in medical research as an in vitro model for evaluation of the effects drugs, electrical stimulation, and/or cellular therapies (stem cells, etc) have on the neural circuitry, and on the growth rate, myelination, and signal conduction in the axons of the re-created pathway. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Currently available antiepileptic drugs (AEDs) are anticonvulsants that require continuous administration for suppression of seizures, and that do not prevent development of epilepsy. Furthermore, approximately 30% of patients develop drug refractory epilepsy. Therefore, discovery of drugs that can prevent or cure epilepsy, defined as antiepileptogenic or disease-modifying drugs, respectively, has been identified as a major epilepsy research goal. Development of these classes of drugs is complicated by the long time course of epileptogenesis and epilepsy progression. Variability in the duration of latent period and seizure intervals and frequency necessitate the use of continuous week-to-month long electrical recordings for sensitive, quantitative assessment of antiepileptogenic or disease-modifying effects. This proposal aims to increase the rate of drug development by significantly improving scalability of long-term electrical monitoring of epileptic activity in vitro. Hybrid microfluidic-multiple electrode array (Upsilon flow-MEA) chips will be developed for use with organotypic hippocampal culture model of epilepsy. Cultures will be placed in microwells, and maintained via perfusion of culture medium through inlet and outlet microchannels. This method will significantly reduce the area required for each culture. Each chip will be capable of monitoring 12-18 cultures simultaneously to quantitatively assess development and progression of epilepsy. Microchannel network will connect cultures into experimental groups to facilitate application of drugs, inhibitors, or other molecules. This platform will then be scaled up to conduct a pilot screen with a small molecule inhibitor library. The goal of the screen is to demonstrate an increase in scalability and experimental throughput achieved with Upsilon flow-MEA platform, and to discover new targets for antiepileptogenic or disease-modifying drugs.

? DESCRIPTION There is a great interest in imaging neuronal activity based on changes in fast intrinsic optical signals (e.g. changes in light scattering and phase) that occur on a millisecond timescale. Fast intrinsic optical signals are related to alteration in the complex refractive index and small volume changes near the neuron membrane, in response to the rapid osmotic changes associated with ion fluxes during action potentials. Optical coherence tomography (OCT) is an emerging biomedical imaging technology that provides label-free and depth-resolved images with micron-scale spatial resolution and sub-millisecond temporal resolution. OCT relies on detection of intrinsic optical contrast, eliminating the need for potentially toxic exogenous contrast agents or genetically- encoded indicators. OCT achieves over 100 dB sensitivity, enabling it to detect weak scattering changes associated with neuronal activity. In addition, OCT has extremely good phase sensitivity (

Abstract The NINDS Anticonvulsant Screening Program (ASP) has identified most of the anticonvulsants in clinical use today. However, one third of epileptic patients do not respond to these drugs. The ASP protocols are based on seizures induced by subjecting normal animals to acute convulsant conditions. We have developed a complimentary system of novel in vitro and in vivo assays of spontaneous seizures in chronically epileptic preparations. This two-stage screening system provides a unique focus on recurrent spontaneous seizures in chronic epilepsy models. The first stage is an in vitro assay comprised of the organotypic hippocampal slice culture, which develops electrographic seizure activity and corresponding biochemical biomarkers over the first week in vitro. The second stage is an in vivo assay comprised of the kainate model of epilepsy in which spontaneous seizures are monitored using continuous telemetry and supervised, blinded, computerized seizure detection. We used the rapid in vitro assay to screen over 400 compound-concentration combinations from the NINDS Custom Compound Collection. We found a lead compound, celecoxib, and then verified this lead by the second-stage testing in a randomized double blind in vivo crossover trial. Celecoxib had no effect on seizures induced by acute application of convulsants to normal brain tissue, suggesting that its anticonvulsant properties are unique to chronic epilepsy, and raising the possibility that its spectrum of action will be distinct from anticonvulsants discovered by the ASP protocols. The next step in development is medicinal chemistry to optimize celecoxib?s anticonvulsant efficacy. This is most feasibly accomplished through the UH2 / UH3 Blueprint Neurotherapeutics Network. As our discussions with BPN program officers clarified, to efficiently utilize the BPN medicinal chemistry program we must further develop the in vitro and in vivo assays and acquire additional data on our lead compound. The UH2/3 mechanism was considered the most appropriate funding mechanism by the NINDS program officer. In the R21 phase of this proposal, we will extend the in vitro assay?s concentration-response for celecoxib and 2,5 dimethyl celecoxib, a derivative that does not inhibit COX2 but has equal anticonvulsant efficacy in vitro. We will then characterize the assay?s reproducibility and Z factor. We will also establish the dose-response of the in vivo assays for celecoxib, and increase the number of in vitro and in vivo sites to two each in order to improve robustness and throughput, as well as engage outstanding younger investigators in this effort. In the R33 phase of the proposal, we will further characterize the lead compound by determining whether COX2 inhibition is necessary for anticonvulsant activity in the in vitro and in vivo assays.

Recent developments in optogenetics, patterned optical stimulation, and high-speed optical detection enable simultaneous stimulation and recording of thousands of living neurons. Connected biological living neurons naturally exhibit the ability to perform computations and to learn. The proposed project will engineer living neural network to compute for learning task. Experimental testbed will be built to allow optical stimulation and detection. Algorithms will be developed to train the living neuron networks. The proposed testbed can be used by neuroscientists to verify network-level hypotheses. Insights learned from the proposed research can inspire other neuromorphic architectures based on solid state devices. Throughout this project, graduate students will be trained in computer engineering, bioengineering, and signal processing. Students will have the opportunity to work on interdisciplinary research in these fields. New courses based on the results from the proposed work will be introduced and new modules will be added to existing curriculum. The proposed outreach activities aim to attract interest to computer engineering and neural engineering.

The goal of this project is to use optogenetic in vitro neural network to run learning applications. Living neural networks have spontaneous activities, which can interfere with precise modification of synaptic strength. This research will study how to stabilize the living neural network such that a Spike Time Dependent Plasticity (STDP)-based programming protocol can imprint the desired synaptic strengths onto a living neural network. This research will also investigate how to strategically design and apply an STDP-based protocol to maximize programming throughput and optimize convergence rate of the network states. On the algorithm side, the proposed research will study data representation and training algorithms that consider various constraints of the proposed wetware system. Learning algorithms will be designed to work on random neural networks of unknown topology. Observable details of neuron activities will be used to improve accuracy of learning tasks.

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.