Low density cell culture of locust neurons in closed-channel microfluidic devices.

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.

The brain can eat: establishing the existence of a central pattern generator for feeding in third instar larvae of Drosophila virilis and Drosophila melanogaster.

To establish the existence of a central pattern generator for feeding in the larval central nervous system of two Drosophila species, the gross anatomy of feeding related muscles and their innervation is described, the motor units of the muscles identified and rhythmic motor output recorded from the isolated CNS. The cibarial dilator muscles that mediate food ingestion are innervated by the frontal nerve. Their motor pathway projects from the brain through the antennal nerves, the frontal connectives and the frontal nerve junction. The mouth hook elevator and depressor system is innervated by side branches of the maxillary nerve. The motor units of the two muscle groups differ in amplitude: the elevator is always activated by a small unit, the depressor by a large one. The dorsal protractors span the cephalopharyngeal skeleton and the body wall hence mediating an extension of the CPS. These muscles are innervated by the prothoracic accessory nerve. Rhythmic motor output produced by the isolated central nervous system can simultaneously be recorded from all three nerves. The temporal pattern of the identified motor units resembles the sequence of muscle contractions deduced from natural feeding behavior and is therefore considered as fictive feeding. Phase diagrams show an almost identical fictive feeding pattern is in both species.

Neuronal cell growth on iridium oxide.

Iridium oxide is an attractive material for the development of novel multi electrode array (MEA) systems that provide electrodes for stimulation as well as recording single neurons. In this study the biocompatibility of pure iridium and different iridium oxides that differ characteristically in their surface roughness was investigated using two different biological test systems, insect and vertebrate neurons. Iridium oxide surfaces were coated with Concanavalin A and poly-(D)-lysine. In detailed investigations (R(a) value determination, contact angle measurement, marker enzyme assay) the surface characteristics of non-modified and coated iridium oxide films were analysed, demonstrating that the materials can be successfully coated. Furthermore, we show that locust neurons grow well on all substrates tested, while chicken neurons need coated surfaces for proper adhesion. Increasing the roughness of iridium oxide films, which in principle could improve cell adhesion, did not improve the neurocompatibility. These results show that in future applications iridium oxide films can be used with surface morphologies previously shown to be optimal for stimulation purposes (cauliflower-like surface structure).

Iridium oxide microelectrode arrays for in vitro stimulation of individual rat neurons from dissociated cultures.

We present the first in vitro extracellular stimulation of individual neurons from dissociated cultures with iridium oxide (IrO(x)) electrodes. Microelectrode arrays with sputtered IrO(x) films (SIROF) were developed for electrophysiological investigations with electrogenic cells. The microelectrodes were characterized with scanning electron and atomic force microscopy, revealing rough and porous electrodes with enlarged surface areas. As shown by cyclic voltammetry and electrochemical impedance spectroscopy, the large surface area in combination with the good electrochemical properties of SIROF resulted in high charge storage capacity and low electrode impedance. Thus, we could transfer the good properties of IrO(x) as material for in vivo stimulation electrodes to multi-electrode arrays with electrode diameters as small as 10 mum for in vitro applications. Single rat cortical neurons from dissociated cultures were successfully stimulated to fire action potentials using single or trains of biphasic rectangular voltage-controlled stimulation pulses. The stimulated cell's membrane potential was simultaneously monitored using whole-cell current-clamp recordings. This experimental configuration allowed direct evaluation of the influence of pulse phase sequence, amplitude, and number on the stimulation success ratio and action potential latency. Negative phase first pulses were more effective for extracellular stimulation and caused reduced latency in comparison to positive phase first pulses. Increasing the pulse amplitude also improved stimulation reliability. However, in order to prevent cell or electrode damage, the pulse amplitude is limited to voltages below the threshold for irreversible electrochemical reactions at the electrode. As an alternative to increasing the amplitude, a higher number of stimulation pulses was also shown to increase stimulation success.

Pulse-clamp technique for single neuron stimulation electrode characterization.

Miniaturized electrodes, structures and devices are necessary to achieve high target selectivity during stimulation in single neuron networks, while significant charge transfer is still demanded. A reliable test method is required to evaluate charge injection capability for high resolution neural stimulation applications that demand both a large amount of charge injection and a small electrode size. A circuit designed for the pulse-clamp technique was employed to characterize the electrode charge-storage capability of microelectrodes of sizes smaller than 300 microm in diameter. The circuit allows different electrodes and surface modifications to be quickly and accurately compared. Pulse-clamp measurements are performed on planar microelectrodes in 154 mM phosphate buffered saline (PBS) solution with 400 micros long pulses at charges up to 40 nC. The pulse-clamp and cyclic voltammetry results of sputtered iridium oxide film (SIROF) electrodes of different sizes show charge losses of less than 3% and a superior reversible charge injection capability compared to platinum microelectrodes of the same size, even at higher charge density levels.

Prediction of visual evoked potentials at any surface location from a set of three recording electrodes.

Purpose of this study was to introduce a mathematical model which allows the calculation of a source dipole as the origin of the evoked activity based on the data of three simultaneously recorded VEPs from different locations at the scalp surface to predict field potentials at any neighboring location and to validate this model by comparison with actual recordings. In 10 healthy subjects (25-38, mean 29 years) continuous VEPs were recorded via 96 channels. On the base of the recordings at the positions POz', O1' and O2', a source dipole vector was calculated for each time point of the recordings and VEP responses were back projected for any of the 96 electrode positions. Differences between the calculated and the actually recorded responses were quantified by coefficients of variation (CV). The prediction precision and response size depended on the distance between the electrode of the predicted response and the recording electrodes. After compensating this relationship using a polynomial function, the CV of the mean difference between calculated and recorded responses of the 10 subjects was 2.8 +/- 1.2%. In conclusion, the "Mini-Brainmapping" model can provide precise topographical information with minimal additional recording efforts with good reliability. The implementation of this method in a routine diagnostic setting as an "easy-to-do" procedure would allow to examine a large number of patients and normal subjects in a short time, and thus, a solid data base could be created to correlate well defined pathologies with topographical VEP changes.

Iridium sputtered at varying pressures and target-substrate-distances evaluated for use as stimulation electrode material.

Iridium was sputtered as the top layer of stimulation electrodes. The coatings were varied in their morphology by adjusting the total deposition pressure and the working distance (WD) of target and substrate. It is shown that the resulting different kinds of morphologies have a strong influence on stimulation characteristics. The combination of high working gas pressure and small WD as well as the combination of medium working gas pressure and larger WD yield the best characteristics on macro-sized test electrodes.