Engineering an in vitro retinothalamic nerve model.

Understanding the retinogeniculate pathway in vitro can offer insights into its development and potential for future therapeutic applications. This study presents a Polydimethylsiloxane-based two-chamber system with axon guidance channels, designed to replicate unidirectional retinogeniculate signal transmission in vitro. Using embryonic rat retinas, we developed a model where retinal spheroids innervate thalamic targets through up to 6 mm long microfluidic channels. Using a combination of electrical stimulation and functional calcium imaging we assessed how channel length and electrical stimulation frequency affects thalamic target response. In the presented model we integrated up to 20 identical functional retinothalamic neural networks aligned on a single transparent microelectrode array, enhancing the robustness and quality of recorded functional data. We found that network integrity depends on channel length, with 0.5-2 mm channels maintaining over 90% morphological and 50% functional integrity. A reduced network integrity was recorded in longer channels. The results indicate a notable reduction in forward spike propagation in channels longer than 4 mm. Additionally, spike conduction fidelity decreased with increasing channel length. Yet, stimulation-induced thalamic target activity remained unaffected by channel length. Finally, the study found that a sustained thalamic calcium response could be elicited with stimulation frequencies up to 31 Hz, with higher frequencies leading to transient responses. In conclusion, this study presents a high-throughput platform that demonstrates how channel length affects retina to brain network formation and signal transmission in vitro.

Investigation of the input-output relationship of engineered neural networks using high-density microelectrode arrays.

Bottom-up neuroscience utilizes small, engineered biological neural networks to study neuronal activity in systems of reduced complexity. We present a platform that establishes up to six independent networks formed by primary rat neurons on planar complementary metal-oxide-semiconductor (CMOS) microelectrode arrays (MEAs). We introduce an approach that allows repetitive stimulation and recording of network activity at any of the over 700 electrodes underlying a network. We demonstrate that the continuous application of a repetitive super-threshold stimulus yields a reproducible network answer within a 15 ms post-stimulus window. This response can be tracked with high spatiotemporal resolution across the whole extent of the network. Moreover, we show that the location of the stimulation plays a significant role in the networks' early response to the stimulus. By applying a stimulation pattern to all network-underlying electrodes in sequence, the sensitivity of the whole network to the stimulus can be visualized. We demonstrate that microchannels reduce the voltage stimulation threshold and induce the strongest network response. By varying the stimulation amplitude and frequency we reveal discrete network transition points. Finally, we introduce vector fields to follow stimulation-induced spike propagation pathways within the network. Overall we show that our defined neural networks on CMOS MEAs enable us to elicit highly reproducible activity patterns that can be precisely modulated by stimulation amplitude, stimulation frequency and the site of stimulation.

Engineering circuits of human iPSC-derived neurons and rat primary glia.

Novel in vitro platforms based on human neurons are needed to improve early drug testing and address the stalling drug discovery in neurological disorders. Topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons have the potential to become such a testing system. In this work, we build in vitro co-cultured circuits of human iPSC-derived neurons and rat primary glial cells using microfabricated polydimethylsiloxane (PDMS) structures on microelectrode arrays (MEAs). Our PDMS microstructures are designed in the shape of a stomach, which guides axons in one direction and thereby facilitates the unidirectional flow of information. Such circuits are created by seeding either dissociated cells or pre-aggregated spheroids at different neuron-to-glia ratios. Furthermore, an antifouling coating is developed to prevent axonal overgrowth in undesired locations of the microstructure. We assess the electrophysiological properties of different types of circuits over more than 50 days, including their stimulation-induced neural activity. Finally, we demonstrate the inhibitory effect of magnesium chloride on the electrical activity of our iPSC circuits as a proof-of-concept for screening of neuroactive compounds.

Topologically controlled circuits of human iPSC-derived neurons for electrophysiology recordings.

Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with well-defined connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs), suggesting the formation of functional connections between the neurons of a circuit. Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.

Engineered Biological Neural Networks on High Density CMOS Microelectrode Arrays.

In bottom-up neuroscience, questions on neural information processing are addressed by engineering small but reproducible biological neural networks of defined network topology in vitro. The network topology can be controlled by culturing neurons within polydimethylsiloxane (PDMS) microstructures that are combined with microelectrode arrays (MEAs) for electric access to the network. However, currently used glass MEAs are limited to 256 electrodes and pose a limitation to the spatial resolution as well as the design of more complex microstructures. The use of high density complementary metal-oxide-semiconductor (CMOS) MEAs greatly increases the spatial resolution, enabling sub-cellular readout and stimulation of neurons in defined neural networks. Unfortunately, the non-planar surface of CMOS MEAs complicates the attachment of PDMS microstructures. To overcome the problem of axons escaping the microstructures through the ridges of the CMOS MEA, we stamp-transferred a thin film of hexane-diluted PDMS onto the array such that the PDMS filled the ridges at the contact surface of the microstructures without clogging the axon guidance channels. This method resulted in 23 % of structurally fully connected but sealed networks on the CMOS MEA of which about 45 % showed spiking activity in all channels. Moreover, we provide an impedance-based method to visualize the exact location of the microstructures on the MEA and show that our method can confine axonal growth within the PDMS microstructures. Finally, the high spatial resolution of the CMOS MEA enabled us to show that action potentials follow the unidirectional topology of our circular multi-node microstructure.

An experimental paradigm to investigate stimulation dependent activity in topologically constrained neuronal networks.

We present a stimulate and record paradigm to examine the behavior of multiple neuronal networks with controlled topology in vitro. Our approach enabled us to electrically induce and record neuronal activity from 60 independent networks in parallel over multiple weeks. We investigated the network performance of neuronal networks of primary hippocampal neurons until 29 days in vitro. We introduced a systematic stimulate and record protocol during which well-defined 4-node neural networks were stimulated electrically 4 times per second (4Hz) and their response was recorded over many days. We found that the network response pattern to a stimulus remained fairly stable for at least 12 h. Moreover, continuous stimulation over multiple weeks did not cause a significant change in the stimulation-induced mean spiking frequency of a circuit. We investigated the effect of stimulation amplitude and stimulation timing on the detailed network response. Finally, we could show that our setup can apply complex stimulation protocols with 125 different stimulation patterns. We used these patterns to perform basic addition tasks with the network, revealing the highly non-linear nature of biological networks' responses to complex stimuli.

FLRT3 Marks Direction-Selective Retinal Ganglion Cells That Project to the Medial Terminal Nucleus.

The mammalian retina extracts a multitude of diverse features from the visual scene such as color, contrast, and direction of motion. These features are transmitted separately to the brain by more than 40 different retinal ganglion cell (RGC) subtypes. However, so far only a few genetic markers exist to fully characterize the different RGC subtypes. Here, we present a novel genetic Flrt3-CreERT2 knock-in mouse that labels a small subpopulation of RGCs. Using single-cell injection of fluorescent dyes in Flrt3 positive RGCs, we distinguished four morphological RGC subtypes. Anterograde tracings using a fluorescent Cre-dependent Adeno-associated virus (AAV) revealed that a subgroup of Flrt3 positive RGCs specifically project to the medial terminal nucleus (MTN), which is part of the accessory optic system (AOS) and is essential in driving reflex eye movements for retinal image stabilization. Functional characterization using ex vivo patch-clamp recordings showed that the MTN-projecting Flrt3 RGCs preferentially respond to downward motion in an ON-fashion. These neurons distribute in a regular pattern and most of them are bistratified at the level of the ON and OFF bands of cholinergic starburst amacrine cells where they express the known ON-OFF direction-selective RGC marker CART. Together, our results indicate that MTN-projecting Flrt3 RGCs represent a new functionally homogeneous AOS projecting direction-selective RGC subpopulation.

Two septal-entorhinal GABAergic projections differentially control coding properties of spatially tuned neurons in the medial entorhinal cortex.

Septal parvalbumin-expressing (PV+) and calbindin-expressing (CB+) projections inhibit low-threshold and fast-spiking interneurons, respectively, in the medial entorhinal cortex (MEC). We investigate how the two inputs control neuronal activity in the MEC in freely moving mice. Stimulation of PV+ and CB+ terminals causes disinhibition of spatially tuned MEC neurons, but exerts differential effects on temporal coding and burst firing. Thus, recruitment of PV+ projections disrupts theta-rhythmic firing of MEC neurons, while stimulation of CB+ projections increases burst firing of grid cells and enhances phase precession in a cell-type-specific manner. Inactivation of septal PV+ or CB+ neurons differentially affects context, reference, and working memory. Together, our results reveal how specific connectivity of septal GABAergic projections with MEC interneurons translates into differential modulation of MEC neuronal coding.

Soft Electronics Based on Stretchable and Conductive Nanocomposites for Biomedical Applications.

Research on the field of implantable electronic devices that can be directly applied in the body with various functionalities is increasingly intensifying due to its great potential for various therapeutic applications. While conventional implantable electronics generally include rigid and hard conductive materials, their surrounding biological objects are soft and dynamic. The mechanical mismatch between implanted devices and biological environments induces damages in the body especially for long-term applications. Stretchable electronics with outstanding mechanical compliance with biological objects effectively improve such limitations of existing rigid implantable electronics. In this article, the recent progress of implantable soft electronics based on various conductive nanocomposites is systematically described. In particular, representative fabrication approaches of conductive and stretchable nanocomposites for implantable soft electronics and various in vivo applications of implantable soft electronics are focused on. To conclude, challenges and perspectives of current implantable soft electronics that should be considered for further advances are discussed.

Structural Basis of Teneurin-Latrophilin Interaction in Repulsive Guidance of Migrating Neurons.

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.

Regulation of Cerebral Cortex Folding by Controlling Neuronal Migration via FLRT Adhesion Molecules.

The folding of the mammalian cerebral cortex into sulci and gyri is thought to be favored by the amplification of basal progenitor cells and their tangential migration. Here, we provide a molecular mechanism for the role of migration in this process by showing that changes in intercellular adhesion of migrating cortical neurons result in cortical folding. Mice with deletions of FLRT1 and FLRT3 adhesion molecules develop macroscopic sulci with preserved layered organization and radial glial morphology. Cortex folding in these mutants does not require progenitor cell amplification but is dependent on changes in neuron migration. Analyses and simulations suggest that sulcus formation in the absence of FLRT1/3 results from reduced intercellular adhesion, increased neuron migration, and clustering in the cortical plate. Notably, FLRT1/3 expression is low in the human cortex and in future sulcus areas of ferrets, suggesting that intercellular adhesion is a key regulator of cortical folding across species.

FLRT structure: balancing repulsion and cell adhesion in cortical and vascular development.

FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Oxide nanotubes on Ti-Ru alloys: strongly enhanced and stable photoelectrochemical activity for water splitting.

The present work shows a significant enhancement of the photoelectrochemical water-splitting performance of anodic TiO(2) nanotube layers grown on low concentration (0.01-0.2 at% Ru) Ti-Ru alloys. Under optimized preparation conditions (0.05 at% Ru, 450 °C annealing) the water splitting rate of the oxide tubes could be 6-fold increased. Moreover, the beneficial effect is very stable with illumination time; this is in contrast to other typical doping approaches of TiO(2).