The beginning of the project was dedicated to the establishment of the procedure of plating E17 cortical cells and maintaining them on top of MEA chips. Along with biology fellow researchers, we have consolidated the optimal protocol to plate neurons on top of the glass MEA chip and maintaining them until a sufficient age (more than 30 days in-vitro), including the culture infection with a viral vector for expressing ChR2 protein (for light sensitivity). In parallel, the MEA hardware and software (including different accessories) were installed in the lab, after which custom software tools for data processing and analysis were developed. During this period, the ER went to the University of Genoa (Italy) for training in topics related to neuro-engineering, including MEA measurements and data analysis.
Following these procedures, we started to perform basic electrophysiological recordings with the MEA system, where we tested basic neuronal activity starting from spontaneous activity and followed by evoked response to electrical stimuli and long-term potentiation (LTP) protocols by electrical stimulation.
In the next step we installed the optical setup for optogenetic stimulation of ChR2 infected cultures. The digital light processor (DLP) system, including the optical instrumentation were installed in the laboratory. The DLP is a programable light projecting device based on digital micromirror device (DMD), which can create custom illumination patterns. The patterns are then projected on top of the sample and constitute a stimulation trigger for the genetically-modified neurons. Later we integrated the DLP and MEA to a joint operation. We then tested and characterized the response of neurons to light stimulation. Then in further experiments we tackled the question of memory formation in neuronal culture following light stimulation.
The last part of the project was devoted to developing advanced analytical tools with the aim of building a processing and control unit that reads the electrophysiological signals from MEA recordings and creates a corresponding map of the tested network, which further can control the stimulation location and pattern. An AI-based computational model was developed. This model obtains a simplified network (or a graph model), using the data acquired in MEA measurements as training dataset in order to decode the spatio-temporal signal patterns of the neuronal network and to extract its structure. The model is based on Reservoir computer network (RCN) approach, as it uses the fact that around each electrode of MEA, a complex neuronal circuit is embedded. The complexity of these circuits cannot be easily understood from the standard measurement analysis, and hence they are modeled as nonlinear networks with inner random connections. With this model we are able to extract a macroscopic graph representing the structure of the culture under test, where each node of this network represents a neural circuit (population of neurons); and the connections (edges) between them represent the weighted interaction between the populations.
The results of the ISLAND project will allow further applications and studies in-vitro and ex-vivo. The integrated optogenetic setup that has been developed in the project is already in use for further research of epileptic seizure suppression by light at NanoLab. The AI-model that has been developed during the project is able to retrieve functional connectivity of the tested neuronal network with high accuracy and hence various applications might arise from this work, such as pharmacological studies, brain mapping and more.
Dissemination:
1. 11th Optoelectronics and Photonics Summer School: NMP2021 – Neuromorphic Photonics, 20-26 June 2021, Monte Bondone, Trento, Italy. A talk introducing MSCA individual fellowship program and the ISLAND project was given (title: “Between biological and artificial neurons and MSCA-IF ISLAND project”).
2. International School on Bio hybrid interfaces, organic bio electronics and bio photonics Lake Como School of Advanced Studies, 5-9 September 2022, Como, Italy. A poster of the recent work in the ISLAND project was presented (title: “An integrated setup for in-vitro optogenetic experiments using AI to localize stimulation with a feedback of electrophysiological signals”).
3. SPIE Photonics West, 28 January - 3 February 2023, San Francisco, CA, USA. A poster about the ISLAND project was presented (title: “An integrated setup for in-vitro optogenetic experiments using AI to localize stimulation”).
4. (SUBMITED PAPER) 20th IEEE Conference on Computational Intelligence in Bioinformatics and Computational Biology (29-31 August 2023 – Eindhoven, The Netherlands)- a talk about the developed AI model is planed.
