Harald Dermutz

Electrochemistry on a Localized Surface Plasmon Resonance Sensor

The optical signal of a localized surface plasmon resonance (LSPR)-based sensor combined with electrochemistry was investigated. Gold nanoparticles were immobilized on an indium tin oxide (ITO) substrate, which functioned as working electrode. Using cyclic voltammetry synchronized with LSPR sensing, surface reactions on gold were detected both electrically and optically. In the capacitive charging regime, optical signals linear to the applied potential were detected. Gold was found to be dissolved above the oxidation potential and partially redeposited during the reduction, which changed size and conformation of the gold nanoparticles. In kinetic measurements, slower potential establishment was observed at lower salt concentrations. Simulations by multiple multipole program (MMP) suggested the formation of a lossy layer by combination of charge depletion of gold and negative ion adsorption even below the reaction potential. We consider the results presented here of importance for any future sensors based on combined plasmonics and electrochemistry.

Local polymer replacement for neuron patterning and in situ neurite guidance.

By locally dispensing poly-L-lysine (PLL) molecules with a FluidFM onto a protein and cell resistant poly-L-lysine-graft-polyethylene glycol (PLL-g-PEG) coated substrate, the antifouling layer can be replaced under the tip aperture by the cell adhesive PLL. We used this approach for guiding the adhesion and axonal outgrowth of embryonic hippocampal neurons in situ. Cultures of hippocampal neurons were chosen because they mostly contain pyramidal neurons. The hippocampus is known to be involved in memory formation, and the stages of network development are well characterized, which is an asset to fundamental research. After fabricating diffuse PLL spots with 10-250 μm diameter, seeded hippocampal cells stick preferentially onto the spots migrating toward the spot center along the PLL gradient. Cell clusters were formed depending on the lateral size of the PLL dots and the density of seeded cells. In a second step of this protocol, the FluidFM is used to connect in situ the obtained clusters. The outgrowth of neurites, which are known to grow preferentially on adhesive substrates, is tailored by writing PLL lines. Antibody staining confirms that the outgrowing neurites are mostly axons, while the activity of the neurons is assessed by a calcium indicator, proving cell viability. The calcium signal intensity of two actively interconnected clusters showed to be correlated, corroborating the formation of vectored and polarized interconnections.

Ultrasoft Alginate Hydrogels Support Long-Term Three-Dimensional Functional Neuronal Networks.

Neuron development and function are exquisitely sensitive to the mechanical properties of their surroundings. Three-dimensional (3D) cultures are therefore being explored as they better mimic the features of the native extracellular matrix. Limitations of existing 3D culture models include poorly defined composition, rapid degradation, and suboptimal biocompatibility. Here we show that ionically cross-linked ultrasoft hydrogels made from unmodified alginate can potently promote neuritogenesis. Alginate hydrogels were characterized mechanically and a remarkable range of stiffness (10-4000 Pa) could be produced by varying the macromer content (0.1-0.4% w/v) and CaCl2 concentration. Dissociated rat embryonic cortical neurons encapsulated within the softest of the hydrogels (0.1% w/v, 10 mM CaCl2) showed excellent viability, extensive formation of axons and dendrites, and long-term activity as determined by calcium imaging. In conclusion, alginate is an off-the-shelf, easy to handle, and inexpensive material, which can be used to make ultrasoft hydrogels for the formation of stable and functional 3D neuronal networks. This 3D culture system could have important applications in neuropharmacology, toxicology, and regenerative medicine.

Simultaneous Scanning Ion Conductance Microscopy and Atomic Force Microscopy with Microchanneled Cantilevers.

We combined scanning ion conductance microscopy (SICM) and atomic force microscopy (AFM) into a single tool using AFM cantilevers with an embedded microchannel flowing into the nanosized aperture at the apex of the hollow pyramid. An electrode was positioned in the AFM fluidic circuit connected to a second electrode in the bath. We could thus simultaneously measure the ionic current and the cantilever bending (in optical beam deflection mode). First, we quantitatively compared the SICM and AFM contact points on the approach curves. Second, we estimated where the probe in SICM mode touches the sample during scanning on a calibration grid and applied the finding to image a network of neurites on a Petri dish. Finally, we assessed the feasibility of a double controller using both the ionic current and the deflection as input signals of the piezofeedback. The experimental data were rationalized in the framework of finite elements simulations.

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.

Local Chemical Stimulation of Neurons with the Fluidic Force Microscope (FluidFM)

Physiological communication between neurons is dependent on the exchange of neurotransmitters at the synapses. Although this chemical signal transmission targets specific receptors and allows for subtle adaptation of the action potential, in vitro neuroscience typically relies on electrical currents and potentials to stimulate neurons. The electric stimulus is unspecific and the confinement of the stimuli within the media is technically difficult to control and introduces large artifacts in electric recordings of the activity. Here, we present a local chemical stimulation platform that resembles in vivo physiological conditions and can be used to target specific receptors of synapses. Neurotransmitters were dispensed using the force-controlled fluidic force microscope (FluidFM) nanopipette, which provides exact positioning and precise liquid delivery. We show that controlled release of the excitatory neurotransmitter glutamate induces spiking activity in primary rat hippocampal neurons, as measured by concurrent electrical and optical recordings using a microelectrode array and a calcium-sensitive dye, respectively. Furthermore, we characterized the glutamate dose response of neurons by applying stimulation pulses of glutamate with concentrations from 0 to 0.5 mm. This new stimulation approach, which combines FluidFM for gentle and precise positioning with a microelectrode array read-out, makes it possible to modulate the activity of individual neurons chemically and simultaneously record their induced activity across the entire neuronal network. The presented platform not only offers a more physiological alternative compared with electrical stimulation, but also provides the possibility to study the effects of the local application of neuromodulators and other drugs.

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.