Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative <em>In Vitro</em> Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the ...

Fondazione Istituto Italiano di Tecnologia (IIT)

In this study we report for the first time the localization of a photoreceptor pigment in the cilia of the colored heterotrich ciliates Blepharisma japonicum red and blue form, Fabrea salina, and Stentor coeruleus, as result of a confocal microscopy investigation. Optical sectioning confocal microscopy has been used for studying the spatial distribution of the pigment in the cell body, surprisingly showing that, besides its expected presence in the cortical region immediately below the cell membrane, it is located in the cilia too. In order to ascertain possible differences in the pigment fluorescence properties along the cell body, we have measured emission spectra from different parts of it (anterior, posterior, and cilia). Our results clearly indicate that in all cases the spectra are the same, within experimental errors. Finally, we have evaluated the pigment relative fluorescence efficiency of these ciliates. In an ordered scale from lower to greater efficiency, we have S. coeruleus, B. japonicum blue, B. japonicum red, and F. salina. The possible implications of our findings for the process of photosensory transduction are discussed.

Evidence for ciliary pigment localization in colored ciliates and implications for their photosensory transduction chain: a confocal microscopy study.

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion.

The formation of actin waves during regeneration after axonal lesion is enhanced by BDNF.

Au cours du d&eacute;veloppement, les axones des neurones dans le syst&egrave;me nerveux central des mammif&egrave;res perdent leur capacit&eacute; &agrave; se r&eacute;g&eacute;n&eacute;rer. Afin d&#39;&eacute;tudier le processus de r&eacute;g&eacute;n&eacute;ration, les axones des neurones de l&#39;hippocampe souris ont &eacute;t&eacute; partiellement endommag&eacute;s par un syst&egrave;me de dissecteur laser UVA. La possibilit&eacute; de livrer de tr&egrave;s faible puissance moyenne de l&#39;&eacute;chantillon r&eacute;duit les dommages collat&eacute;raux thermique et autoris&eacute;s &agrave; &eacute;tudier la r&eacute;g&eacute;n&eacute;ration des neurones de souris au cours des premiers jours in vitro. Mesures de spectroscopie de force ont &eacute;t&eacute; effectu&eacute;es pendant et apr&egrave;s l&#39;axone ablation avec un filet attach&eacute; &agrave; la membrane axonale et qui s&#39;est tenue dans un pi&egrave;ge optique. Avec cette approche, nous avons quantifi&eacute; l&#39;adh&eacute;rence de l&#39;axone au substrat et les propri&eacute;t&eacute;s visco-&eacute;lastiques de la membrane pendant la r&eacute;g&eacute;n&eacute;ration. La r&eacute;organisation et la r&eacute;g&eacute;n&eacute;ration de l&#39;axone est document&eacute;e par l&#39;imagerie live &agrave; long terme. Nous d&eacute;montrons que le BDNF r&eacute;gule l&#39;adh&eacute;sion neuronale et favorise la formation de vagues d&#39;actine pendant la r&eacute;g&eacute;n&eacute;ration apr&egrave;s une l&eacute;sion axonale.

La formation de vagues d&#39;actine pendant la r&eacute;g&eacute;n&eacute;ration apr&egrave;s une l&eacute;sion axonale est renforc&eacute;e par le BDNF.

Durante lo sviluppo, assoni di neuroni del sistema nervoso centrale dei mammiferi perdono la loro capacit&agrave; di rigenerare. Per studiare il processo di rigenerazione, assoni di neuroni hippocampal del mouse sono stati parzialmente danneggiati da un sistema dissettore laser a raggi UVA. La possibilit&agrave; di erogare potenza media molto bassa al campione ridotto il danno termico collaterale e ha permesso di studiare la rigenerazione assonale dei neuroni del mouse durante i primi giorni in vitro. Forza spettroscopia misurazioni sono state eseguite durante e dopo l&#39;assone ablazione con un tallone attaccato alla membrana assonale e tenuto in una trappola ottica. Con questo approccio, abbiamo quantificato l&#39;adesione dell&#39;assone al substrato e le propriet&agrave; viscoelastiche della membrana durante la rigenerazione. La riorganizzazione e la rigenerazione dell&#39;assone &egrave; stato documentato da immagini dal vivo a lungo termine. Qui ci dimostrano che il BDNF regola adesione neuronale e favorisce la formazione di onde di actina durante la rigenerazione dopo lesione assonale.

La formazione di onde di actina durante la rigenerazione dopo lesione assonale si arricchisce di BDNF.

A long-term live-imaging workstation to follow the development of cultured neurons during the first few days in vitro (DIV) is developed. In order to monitor neuronal polarization and axonal growth by live imaging, we built a micro-incubator system that provides stable temperature, pH, and osmolarity in the culture dish under the microscope, while preserving environment sterility. We are able to image living neurons at 2 DIVs for 48 h with a temporal resolution of one frame for every 2 min. The main features of this system are its ability to adapt to every cell-culture support, to integrate in any optical microscope, because of the relatively small dimensions (9.5×6.5×2.5  cm) and low weight of the system (<200  g), and to monitor the physiological parameters in situ. Moreover, we developed an image-analysis algorithm to quantify the cell motility, in order to characterize its complex temporal-spatial pattern. The algorithm applies morphological image processing operations on the temporal variations occurring in the inspected region of interest. Here, it is used to automatically detect cellular motility in three distinct morphological regions of the neurons: around the soma, along the neurites, and in the growth cone.

Motility flow and growth-cone navigation analysis during in vitro neuronal development by long-term bright-field imaging.

Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems.

Mattia Pesce

Fondazione Istituto Italiano di Tecnologia (IIT)

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model