Multidimensional large-scale, high-density in vitro recording facility for the investigation of neural systems function

Neural function in our central nervous system arises from complex interactions between different neuronal types within vast neural networks.

To understand how these networks operate, simultaneous recordings from large neuronal populations are essential. This can be achieved ex vivo (for example in brain slices) using either imaging (to visualise fluorescent markers expressed in various cell types), or arrays of electrodes (multielectrode arrays; MEAs) that record electrical activity simultaneously from hundreds to thousands of neurons that are functionally connected to each other (or combining both approaches).

When using brain slices, surface cells are damaged because of the slicing process that disrupts the integrity of their cellular processes. For that reason, these recordings are normally done in the depth of the tissue with penetrating electrodes or with optical recordings.

Traditional MEAs consist of planar electrodes that can record only from the surface of the tissue. They are useful to record from cells in dissociated cultures, growing on the electrodes, or from the isolated retina, where the output cells form a single superficial layer. But these planar MEAs are not amenable to record from brain slices. Most MEA systems currently on the market with penetrating electrodes are too large for delicate isolated neural tissue, and they are limited to having no more than 100 electrodes, which is insufficient to analyse the network dynamics that we aim to study in the related projects we propose..

With this proposal, we aim to establish a facility around a unique next-generation MEA system consisting of 4,096 densely packed electrodes, allowing recordings spaced at similar distance to that between adjacent neurones in neural networks. These electrodes consist of pillars that penetrate the tissue and can therefore record neural activity in depth, where cells are not damaged and neural networks are intact. We will combine this cutting-edge new recording system with a fluorescent microscope that will allow us to perform concurrent imaging of various physiological parameters while neurons signal to each other.

This novel platform will allow us to study neural function in health and disease at macroscopic scale in health and disease using brain slices, retinas (from rodents and human tissue) and human stem-cell-derived artificial organs (retina, inner-ear).

We expect that this new facility will attract exciting new collaborations and technology developments in Newcastle.

We seek funding to establish a unique, next-generation, multimodal platform for the in vitro study of brain function. The platform combines a large-scale, high-resolution multielectrode array (MEA) system with thousands of 3-dimensional (3D) electrodes with epifluorescence imaging to allow the study of neural network function at multiple scales in health and disease.

A significant limitation of current high density MEAs for in vitro neurophysiology is that they have arrays of planar electrodes. Such arrays can only record cells located superficially within the tissue, or in dissociated neuronal cultures, that come into direct physical contact with the electrodes. This is a serious disadvantage since cells in brain slices close to the cut surface may be damaged.

The MEA system (BioCAM DupleX from 3Brain) consists of 4,096 pillar electrodes capable of penetrating the tissue, thus allowing sampling of in vitro networks that is unparalleled in its extent and resolution. The pillars are 65 micrometer high, on 15 micrometer pedestals separated by microfluidic channels, allowing very efficient perfusion with physiological solutions under the tissue, which significantly improves its viability. The electrode pitch is 42 micrometers, close to cellular resolution in neural networks.

The MEA system will be combined with a high-speed widefield imaging system (Zeiss AxioExaminer Z1), an upright, fully motorized, fixed-stage microscope adapted for electrophysiology. The BioCAM DupleX consists of a small enclosure that can easily be positioned under the microscope. The microscope will be equipped with an environmental chamber, allowing us to perform prolonged recordings with organotypic cultures or organoids.

This novel, state-of-the-art experimental platform will allow us to investigate neural networks at multiple scales and unprecedented spatiotemporal resolution, shedding new light on neural network function in health and disease.

To understand brain function, it is imperative to know how neural networks function both at subcellular/cellular and systems levels. The cutting-edge experimental platform we seek to build to investigate neural networks' function will have impact at several levels.

The immediate impact will be on the scientists directly involved in the project, as outlined in the Case for Support, and their groups. Indeed, the new experimental platform will provide invaluable tools, allowing these groups to reach much more insightful knowledge about the intricacy of neural function. Once familiar with the new instrumentation, the various groups will begin to generate and analyse new data and they will be able to train new users (new group members, short- or long-term project students, technicians). The project will bring great benefit to the scientific officer for capacity building in this group of highly skilled professionals who are important to keeping the UK at the forefront of technological development.

The named collaborators in this proposal will also be able to enhance their research output by using the platform, and hopefully will be able to replicate it in their own universities. Group members will present the results at local seminars, workshops, national and international conferences and will publish them in appropriate scientific journals. This will raise the awareness of the broader scientific community, leading to wider recognition and new collaborations, raising the external impact. Producing new, impactful results will facilitate future grant applications, allowing important future scientific investigations, for the benefit of science in the UK and abroad.

The platform will also foster important collaborations with technology developers, more specifically people involved in lab-on-chip technologies. These include academic collaborators (e.g. Dr Zagnoni, Strathclyde University) and industrial partners (e.g. 3Brain). With these collaborators, we hope to be able to contribute to the development of better recording devices. With time, we expect our circle of technology developers to grow.

In terms of industrial partners, in addition to chip technology developers, we also expect to produce important developments in stem cell science with NewCells, one of our project partners, and with time, we anticipate to form new collaborations with other companies interested in tissue engineering, neurotoxicology/pharmacology and stem cell approaches.

We expect the scientific output generated with the new platform to have important impact on 3R's driven scientific research and policies. Indeed, it will showcase how reduced, in vitro preparations such as brain slices or organoids (human organs in the dish) can produce meaningful results for systems biology, providing extremely rich and insightful datasets that traditionally would have been possible only by performing in vivo experiments, at a much larger scale.

We also expect that the scientific output generated with our new experimental platform will have societal impact by providing novel approaches to understand how various neural systems function in health and disease. This will be communicated to the wider public during science festivals, the Brain Awareness Week, patient interest groups etc.