Advancement of neural interfaces to probe network electrophysiology

To control the nervous system is to control the human body. The information needed to modulate physiological function flows through the intricate workings of neurons and neuronal networks. There is a severe lack of research tools to systematically access the nervous system and acquire reproducible, reliable, and physiologically relevant data. Microelectrode arrays (MEAs) and other electronic devices that interface with neural tissue have been a massive advance toward tapping into neural communication pathways; however, optimization of these systems is far from completion. In addition, well-defined activity patterns and characteristics of neural circuits remain difficult to characterize in vivo. In vitro neuroscience research follows a bottom-up strategy of integrating MEAs with neuronal cultures of controlled variables to methodically study how information is encoded, processed, and transmitted within networks. In this thesis, we build and establish a collection of valuable tools to tackle missing components of neuroelectronic interface optimization for in vitro research. First, we addressed the difficultly of extracting reproducible spatiotemporal data from randomly connected neurons in a culture dish and provide a means to engineer networks of defined structural and functional connectivity using polydimethylsiloxane (PDMS) guidance microstructures. Through clever structural design and integration with MEAs, we could record and stimulate the activity of directional, node-based networks with high reproducibility. Secondly, we increased the dimensional relevance of structured networks through the development and characterization of a paper-based scaffold substrate for 3D neuronal cultures. Paper-based substrates could be physically structured at high resolution with a laser cutter, produced at high sample number, and transferred to a MEA at multiple time points for high throughput data collection. Thirdly, we adapted the paper-based technology to create a simple, inexpensive, and versatile method of culturing astrocytes on the 3D substrate to act as supportive co-cultures for our low-density neuronal networks. Fourthly, we optimized the fabrication of a soft, stretchable MEA with embedded nanowires in PDMS and integrated the MEA with a custom, all-in-one microscope stage insert capable of mechanical and electrical stimulation and simultaneous electrical and optical readout. We validated the tool with a novel application to study the elusive mechanosensitive properties of dorsal root ganglion (DRG) sensory neurons after mechanical injury. Lastly, we tested the effect of a drug-eluting polymer coating on an in vitro model of the inflammatory tissue reaction to a foreign body that degrades the long-term function of neural implants. The anti-proliferative, anti-inflammatory, and neuroprotective compound rapamycin promoted a reduction of astrocytes around wire electrodes. Collectively, the presented cell-based assays, materials, and techniques expand the toolbox for probing neural electrophysiology with neuroelectronic interfaces

A novel postsynaptic role for DDC in synaptic plasticity in the adult mammalian hippocampus

While it is well known that netrin-1 and its receptor Deleted in Colorectal Cancer (DCC) are essential for normal neural development, it remains unclear why their expression persists in the adult nervous system. We have recently demonstrated that DCC is expressed at synapses in the mature mammalian brain and that it regulates synapse function and plasticity. The precise pre- and post-synaptic functions of DCC at established synapses in the adult is not yet understood. To study the specific post-synaptic role of DCC, we generated R4ag11::Cre/DCC floxed mice in which DCC expression is selectively eliminated from CA1 hippocampal pyramidal neurons once early development has been completed, while DCC expression in the pre-synaptic neurons of the adjacent CA3 region remained intact. This conditional loss of DCC from the CA3-CA1 post-synaptic neuron resulted in abnormal dendritic spine morphology in CA1 neurons in aging mice despite maintaining overall organization and spine density. Post-synaptic DCC deficiency in these transgenic animals also resulted in deficits in spatial memory and novel place recognition tasks but failed to disrupt novel object recognition memory, suggesting that selective deletion of DCC in CA1 neurons results in disturbances in the neural circuits underlying contextual spatial memory. Together, these findings provide evidence for a critical post-synaptic role for DCC in adult mice and that DCC contributes to memory consolidation during spatial navigation.S'il est bien connu que la nétrine-1 et son récepteur deleted in colorectal cancer (DCC) sont essentiels pour le développement normal du système nerveux, on ne sait pas pourquoi leur expression persiste dans le système nerveux adulte. Nous avons récemment démontré que DCC est exprimé au niveau des synapses dans le cerveau de mammifères mature et qu'il régule la fonction des synapses et leur plasticité. Les fonctions pré- et post-synaptiques précises de DCC au niveau des synapses établies chez l'adulte ne sont pas encore comprises. Pour étudier le rôle spécifique de DCC post-synaptique, nous avons généré des souris R4ag11 :: Cre / DCC floxé chez lesquelles l'expression de DCC est éliminée de manière sélective dans les neurones pyramidaux du CA1 de l'hippocampe une fois le début du développement achevé, tandis que l'expression de DCC dans les neurones pré-synaptiques de la région CA3 adjacente est restée intacte. Cette perte conditionnelle de DCC dans les neurones post-synaptiques du CA3-CA1 entrainent une morphologie dendritique anormale de la colonne vertébrale dans les neurones CA1 de souris âgées malgré le maintien global de l'organisation topographique et de la densitée dendritique de la colonne vertébrale. Le manque de DCC post-synaptique dans ces animaux transgéniques a également entraîné des déficits dans les tâches de reconnaissance de la mémoire spatiale et de nouveau lieu mais n'a pas réussi à perturber la mémoire de reconnaissance de nouveaux objets, ce qui suggère que la suppression sélective de DCC dans les neurones de CA1 entraîne des perturbations dans les circuits neuronaux sous-jacents de la mémoire spatiale contextuelle. Ensemble, ces résultats fournissent la preuve d'un rôle post-synaptique critique pour DCC dans des souris adultes et que DCC contribue à la consolidation de la mémoire lors de la navigation spatiale

“Brains on a chip”: Towards engineered neural networks

The fundamental mechanisms of complex neural computation remain largely unknown, especially in respect to the characteristics of distinct neural circuits within the mammalian brain. The bottom-up approach of building well-defined neural networks with controlled topology has immense promise for improved reproducibility and increased target selectivity and response of drug action, along with hopes to unravel the relationships between functional connectivity and its imprinted physiological and pathological functions. In this review, we summarize the different approaches available for engineering neural networks treated analogously to a mathematical graph consisting of cell bodies and axons as nodes and edges, respectively. After discussing the advances and limitations of the current techniques in terms of cell placement to the nodes and guiding the growth of axons to connect them, the basic properties of patterned networks are analyzed in respect to cell survival and activity dynamics, and compared to that of in vivo and random in vitro cultures. Besides the fundamental scientific interest and relevance to drug and toxicology tests, we also visualize the possible applications of such engineered networks. The review concludes by comparing the possibilities and limitations of the different methods for realizing in vitro engineered neural networks in 2D

Paper-based patterned 3D neural cultures as a tool to study network activity on multielectrode arrays

Cells in vitro behave differently if cultured in a 2D or 3D environment. In spite of the continuous progress over the recent years, methods available for realizing 3D cultures of primary neurons are still fairly complex, limited in throughput and especially limited in compatibility with other techniques like multielectrode arrays (MEAs) for recording and stimulating the network activity with high temporal precision. In this manuscript, a paper-based approach is presented using cellulose filter paper as a mobile substrate for 3D cultures of primary rat hippocampal and cortical neurons. Acting as 3D scaffolds for network development, filter membranes with different surface treatments were prepared to control network homogeneity and laser cut to change the network topology through spatial confinement. The viability of the prepared cultures was comparable to that of reference 2D cultures for over 4 weeks, and the mechanical stability of the paper substrates made it possible to transfer the cultures to MEA chips in an on-demand manner. Once the cultures were successfully transduced with a gene-encoded calcium indicator and transferred to a MEA chip, the optical and electrical signals of neuronal activity were simultaneously recorded and combined to study the different activity patterns with high spatiotemporal resolution. The high-throughput nature of the presented approach makes it a valuable tool for investigating the intimate relationship between topology and function, by studying the intrinsic parameters influencing network synchronization and signal propagation through the different activity patterns of 3D neural cultures with arbitrary topology. The developed platform provides a robust and simple alternative to existing 3D culturing technologies for neurons.ISSN:2046-206

Soft hydrogels featuring in-depth surface density gradients for the simple establishment of 3D tissue models for screening applications

Three-dimensional (3D) cell culture models are gaining increasing interest for use in drug development pipelines due to their closer resemblance to human tissues. Hydrogels are the first-choice class of materials to recreate in vitro the 3D extra-cellular matrix (ECM) environment, important in studying cell-ECM interactions and 3D cellular organization and leading to physiologically relevant in vitro tissue models. Here we propose a novel hydrogel platform consisting of a 96-well plate containing pre-cast synthetic PEG-based hydrogels for the simple establishment of 3D (co-)culture systems without the need for the standard encapsulation method. The in-depth density gradient at the surface of the hydrogel promotes the infiltration of cells deposited on top of it. The ability to decouple hydrogel production and cell seeding is intended to simplify the use of hydrogel-based platforms and thus increase their accessibility. Using this platform, we established 3D cultures relevant for studying stem cell differentiation, angiogenesis, and neural and cancer models

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development

Soft hydrogels featuring in-depth surface density gradients for the simple establishment of 3D tissue models for screening applications

Three-dimensional (3D) cell culture models are gaining increasing interest for use in drug development pipelines due to their closer resemblance to human tissues. Hydrogels are the first-choice class of materials to recreate in vitro the 3D extra-cellular matrix (ECM) environment, important in studying cell-ECM interactions and 3D cellular organization and leading to physiologically relevant in vitro tissue models. Here we propose a novel hydrogel platform consisting of a 96-well plate containing pre-cast synthetic PEG-based hydrogels for the simple establishment of 3D (co-)culture systems without the need for the standard encapsulation method. The in-depth density gradient at the surface of the hydrogel promotes the infiltration of cells deposited on top of it. The ability to decouple hydrogel production and cell seeding is intended to simplify the use of hydrogel-based platforms and thus increase their accessibility. Using this platform, we established 3D cultures relevant for studying stem cell differentiation, angiogenesis, and neural and cancer models

Modular microstructure design to build neuronal networks of defined functional connectivity

Theoretical and in vivo neuroscience research suggests that functional information transfer within neuronal networks is influenced by circuit architecture. Due to the dynamic complexities of the brain, it remains a challenge to test the correlation between structure and function of a defined network. Engineering controlled neuronal networks in vitro offers a way to test structural motifs; however, no method has achieved small, multi-node networks with stable, unidirectional connections. Here, we screened ten different microchannel architectures within polydimethylsiloxane (PDMS) devices to test their potential for axonal guidance. The most successful design had a 92% probability of achieving strictly unidirectional connections between nodes. Networks built from this design were cultured on multielectrode arrays and recorded on days in vitro 9, 12, 15 and 18 to investigate spontaneous and evoked bursting activity. Transfer entropy between subsequent nodes showed up to 100 times more directional flow of information compared to the control. Additionally, directed networks produced a greater amount of information flow, reinforcing the importance of directional connections in the brain being critical for reliable communication. By controlling the parameters of network formation, we minimized response variability and achieved functional, directional networks. The technique provides us with a tool to probe the spatio-temporal effects of different network motifs