Background: Multielectrode arrays are widely used to analyze the effects of potentially toxic compounds, as well as to evaluate neuroprotective agents upon the activity of neural networks in short- and long-term cultures. Multielectrode arrays provide a way of non-destructive analysis of spontaneous and evoked neuronal activity, allowing to model neurodegenerative diseases in vitro. Here, we provide an overview on how these devices are currently used in research on the amyloid-β peptide and its role in Alzheimer’s disease, the most common neurodegenerative disorder. Main body: Most of the studies analysed here indicate fast responses of neuronal cultures towards aggregated forms of amyloid-β, leading to increases of spike frequency and impairments of long-term potentiation. This in turn suggests that this peptide might play a crucial role in causing the typical neuronal dysfunction observed in patients with Alzheimer’s disease. Conclusions: Although the number of studies using multielectrode arrays to examine the effect of the amyloid-β peptide onto neural cultures or whole compartments is currently limited, they still show how this technique can be used to not only investigate the interneuronal communication in neural networks, but also making it possible to examine the effects onto synaptic currents. This makes multielectrode arrays a powerful tool in future research on neurodegenerative diseases.

In this study, we devised a novel approach for culturing and electrophysiological recording of the enteric nervous system using custom-made glass substrates with spacers. This allows to turn cultures of isolated myenteric plexus upside down, enhancing the use of the microelectrode array technique by allowing recording of multiple cultures consecutively using only one chip. In addition, upside-down culture led to significant improvements in the culture conditions, resulting in a more in vivo-like growth.

Microelectrode surfaces covered with nanostructures derived from components of extracellular matrix, such as collagen fibers, have shown immense beneficial effects in promoting neuronal growth and cellular signaling. Synthetic nanostructures mimicking the features of biological nanostructures with durable conductive materials could promote the cell adhesion on microelectrode surfaces by providing topographical cues and simultaneously improve the charge transfer properties by reducing its global impedance. Therefore, an advanced nanostructuring method mimicking the structural and organizational features of natural collagen fibers onto metallic microelectrode surfaces has been presented here, which is adapted from previous technological achievements of the group and is based on nanoimprint lithography and gold electroplating. Surface characterization methods reveal an increase in surface area between 20% and 68% on the microelectrodes fabricated with two different nanostructure heights. Impedance spectroscopy measurements reveal reduction in impedance magnitude (at 1 kHz) between 22% and 41%, depending upon the nanostructure height and density on the microelectrode, which should subsequently modulate its charge transfer properties for biosensing application. Cell adhesion analysis performed with seal impedance measurements reveals a tighter coupling of enteric neurons on the nanostructured microelectrodes. Finally, extracellular recordings from enteric neurons exhibit a significant improvement in spike detection properties of the nanostructured microelectrodes.

Bacterial extracellular vesicles (EVs) have emerged as a relevant mode of communication between host and resident microbiota. Evidence is limited regarding the ability of Gram-positive EVs to interact with primary colonic epithelial cells. We propose a murine organoid-based in vitro model to study the impact of EVs from the lactic acid bacteria species Limosilactobacillus reuteri (LR) and Lactiplantibacillus plantarum (LP) on primary colonic epithelium. Previous EV characterization revealed significant differences in zeta-potential (LR: −0.0185 V vs. LP: −0.0103 V, p 0.0125) and particle concentration (LR: 3.38 × 10⁸ particles/mL vs. LP: 8.32 × 10⁸ particles/mL, p 0.0029) but not in lipoteichoic acid (LTA) content and long-chain fatty acid composition. Applied to colonic organoids, uptake efficiency differed between LR and LP EVs, with LR exhibiting higher internalization rates than LP (5 µg EV, 46.56% vs. 20.03%, p < 0.0001) at the expense of cellular viability. Internalized EVs partially co-localized with endo-lysosomal compartments and, depending on the bacterial species of origin, induced a proteomic stress response mimicking molecular features of neurodegenerative diseases. Proteomic profiling of EVs and parental bacteria revealed the existence of lipoproteins predicted to be embedded in the cytoplasmic membrane, potentially acting as adhesion factors to the epithelium. Pre-emptive blocking with LTA reduced EV uptake into the epithelium (1 µg/mL LTA: 3.77 × 10⁵ A.U. vs. control: 1.02 × 10⁶ A.U., p 0.0231). Efficiency of EV internalization differs between LR and LP and may be associated with LTA decoration. Studies using LTA-mutant Lactobacillus species are warranted to clarify the role of vesicle-bound LTA in the EV uptake machinery.

The functionality of the intestine is strongly affected by the microbiome, food, drugs and toxins. This influence can lead to a variety of diseases, which in turn can affect the entire organism¹. For studying the electrophysiological processes of the intestine in health and disease in vitro, a novel microelectrode system is developed and then characterized by electrochemical impedance spectroscopy (EIS). Additionally, EIS will be used to investigate the impedance behavior of different electrode-electrolyte and electrode-cell interfaces. In contrast to conventional planar microelectrode arrays, this novel system utilizes a three-dimensional spiral-like design. This enables to spatially detect the electrophysiological processes of complex tissue-like cell structures (see Figure 1). To ensure flexibility and biocompatibility, a polyimide-based insulating structure with an embedded titanium-gold-titanium metal stack is used. While titanium serves as an adhesion-promoting layer between gold and polyimide, the gold layer itself is used as the electrode material. The sensor is fabricated in a planar configuration on a glass wafer (see schematic representation of the process flow in Figure 2 and the fabricated sensor in Figure 3). First, a 50 nm adhesion-promoting aluminum layer is sputtered on the glass substrate and pre-structured by lithography and wet-etching. An 11 µm thick polyimide layer is then spin-coated and subsequently imidized by a curing process. Then, a metal stack of 20 nm titanium, 200 nm gold and 20 nm titanium is deposited by e-beam evaporation. The conductor tracks of the spiral-shaped electrodes are structured by lithography and wet-etching. An 2 µm thick polyimide layer serves to insulate the electrodes, which is again imidized by curing. The electrode and external contact pad areas are opened by reactive ion etching. For this purpose, a 150 nm sputtered and subsequently structured aluminum layer is used as a hard mask. A combination of SF₆ and O₂ mixture serves as the etching gas. An identical process sequence, which differs only in the mask of the lithography step is used to structure the polyimide frame and thus the contour of the system. Finally, the aluminum sacrificial layer is etched, after which the 3D system can be characterized electrochemically. For all electrode-characterizing electrochemical methods, a potentiostatic three-electrode setup with a 3 M KCl saturated Ag/AgCl reference electrode and a 2 mm gold disc counter electrode in a 0.1 M NaCl electrolyte is used. First, the devices are treated in an oxygen plasma to remove impurities from the openings of the electrodes. Cyclic voltammetry measurements are carried out to receive a stable open circuit potential (OCP), which is then later used as a DC-bias operating point for the EIS measurement. For this, 10 consecutive scans are performed at switching voltages between + 0.1 V and + 0.5 V against Ag/AgCl (3 M KCl) at a scan rate of 0.5 V/s. To obtain reproducible measurement results, the electrolyte is regenerated and the immersion time of the microelectrodes in the electrolyte is kept constant. The EIS measurements are then performed with a sinusoidal AC voltage of 10 mV (amplitude) and a DC operating point of 0 V against OCP in a frequency range from 100 kHz to 1 Hz for three measurement cycles. The electrolyte and the voltage are selected in such a way that faradaic reactions are suppressed and thus the main contribution of the current pertains to charging and discharging of the electric double-layer of the electrode-electrolyte interface. The measurement data is further analyzed in a frequency range where the electric double-layer capacitance is predominant and then fitted to an electrical equivalent circuit using complex non-linear least squares method (see Figure 4). The circuit describes a resistance, which is composed of solution and routing resistance, in series with a parallel circuit consisting of double-layer capacitance and charge-transfer resistance. Due to non-ideal behavior, the double-layer capacitance is modelled by a constant phase element, which is then converted into an average double-layer capacitance using an equation described by Brug et al.². Further EIS measurements will be carried out and modelled using more complex electrical equivalent circuits. For example, more application-based electrolytes such as cell culture medium and hydrogels with and without cells will be used. This serves not to characterize the electrode itself, but to investigate the impedance behavior of the electrode-electrolyte or the electrode-cell interface. Ultimately, electrophysiological recordings of enteric neurons will be spatially evaluated by the 3D microelectrode system. References B. Niesler et al., Nature reviews. Gastroenterology & hepatology, 18(6), 393–410 (2021). G. J. Brug et al., Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 176(1-2), 275–295 (1984). Figure 1

The enteric nervous system (ENS) regulates critical gastrointestinal (GI) processes such as motility, absorption, and secretion. Like any high-energy demanding tissue, the ENS relies on a well-coordinated vascular network for structural integrity and proper function. Disruptions in the neurovascular interplay are hypothesized to contribute to severe dysmotility conditions, including chronic intestinal pseudo-obstruction (CIPO). Thymidine Phosphorylase (TP), predominantly expressed in endothelial cells, plays a role in angiogenesis and vascular homeostasis, and reduced TP levels correlate with enteric neuronal loss in both genetic and idiopathic CIPO. To test whether reduced endothelial TP is sufficient to perturb enteric neurons, we exposed primary ENS-derived cells to conditioned medium from TP-silenced endothelial cells. This treatment was associated with time-dependent neurite network remodeling and changes in non-neuronal cell populations, including a trend toward glial expansion and increased fibroblasts number, while neuronal counts were not affected. However, neurons displayed morphological and molecular alterations, including neuronal swelling and increased neuronal Nitric Oxide Synthase expression. Microelectrode array recordings showed a biphasic functional response, with acute hyperexcitability followed by marked activity loss and reduced responsiveness to acetylcholine stimulation. A preliminary cytokine array analysis of conditioned medium further revealed a modest pro-inflammatory shift in TP-silenced endothelial cells, and a broader pro-inflammatory mediator profile in ENS-derived cells exposed to TP-silenced conditioned medium, compared to respective controls. Overall, these findings suggest that endothelial TP depletion is associated with altered ENS network organization and function in vitro, supporting further investigation of vascular-derived TP levels in ENS homeostasis. Graphical Abstract Conditioned medium from TP-silenced endothelial cells induces structural, cellular, and functional alterations in primary ENS-derived cultures, supporting a possible role for endothelial TP in enteric neuro-glia homeostasis. Image created with Biorender.com.