Using Chronopotentiometry to Better Characterize the Charge Injection Mechanisms of Platinum Electrodes Used in Bionic Devices

The safe charge injection capacity and charge density of neural stimulating electrodes is based on empirical evidence obtained from stimulating feline cortices. Stimulation induced tissue damage may be caused by electrochemical or biological mechanisms. Separating these mechanisms requires greater understanding of charge transfer at the electrode-tissue interface. Clinical devices typically use a biphasic waveform with controlled current. Therefore, the charge injection mechanism and charge injection capacity of platinum was assessed on a commercial potentiostat by chronopotentiometry (controlled current stimulation). Platinum is a non-ideal electrode, charge injection by chronopotentiometry can be passed via capacitive and Faradaic mechanisms. Electrodes were tested under a variety of conditions to assess the impact on charge injection capacity. The change in electrode potential (charge injection capacity) was affected by applied charge density, pulse length, pulse polarity, electrode size, polishing method, electrolyte composition, and oxygen concentration. The safe charge injection capacity and charge density could be increased by changing the electrode-solution composition and stimulation parameters. However, certain conditions (e.g., acid polished electrodes) allowed the electrode to exceed the water electrolysis potential despite the stimulation protocol being deemed safe according to the Shannon plot. Multiple current pulses led to a shift or ratcheting in electrode potential due to changes in the electrode-solution composition. An accurate measure of safe charge injection capacity and charge density of an implantable electrode can only be obtained from suitable conditions (an appropriately degassed electrolyte and clinically relevant electrode structure). Cyclic voltammetric measurement of charge storage capacity can be performed on implantable electrodes, but will not provide information on electrode stability to multiple chronopotentiometric pulses. In contrast, chronopotentiometry will provide details on electrode stability, but the minimum time resolution of typical commercial potentiostats (ms range) is greater than used in a clinical stimulator (μs range) so that extrapolation to short stimulation pulses is required. Finally, an impedance test is typically used to assess clinical electrode performance. The impedance test is also based on a biphasic chronopotentiometic waveform where the measured potential is used to calculate an impedance value. Here it is shown that the measured potential is a function of many parameters (solution composition, electrode area, and surface composition). Subsequently, impedance test results allow electrode comparison and to indicate electrode failure, but use of Ohm’s law to calculate an impedance value is not valid

Alternative 3′ UTR Selection Controls PAR-5 Homeostasis and Cell Polarity in C. elegans Embryos

Cell polarity in one-cell C. elegans embryos guides asymmetric cell division and cell-fate specification. Shortly after fertilization, embryos establish two antagonistic cortical domains of PAR proteins. Here, we find that the conserved polarity factor PAR-5 regulates PAR domain size in a dose-dependent manner. Using quantitative imaging and controlled genetic manipulation, we find that PAR-5 protein levels reflect the cumulative output of three mRNA isoforms with different translational efficiencies mediated by their 3′ UTRs. 3′ UTR selection is regulated, influencing PAR-5 protein abundance. Alternative splicing underlies the selection of par-5 3′ UTR isoforms. 3′ UTR splicing is enhanced by the SR protein kinase SPK-1, and accordingly, SPK-1 is required for wild-type PAR-5 levels and PAR domain size. Precise regulation of par-5 isoform selection is essential for polarization when the posterior PAR network is compromised. Together, strict control of PAR-5 protein levels and feedback from polarity to par-5 3′ UTR selection confer robustness to embryo polarization

Centrosomes Can Initiate a Polarity Axis from Any Position within One-Cell C. elegans Embryos

SummaryThe stereotyped asymmetry of one-cell C. elegans embryos has proven to be an important model for identifying molecular determinants of cell polarity [1]. How polarity is initiated is less well understood. Polarity establishment depends on centrosomes [2–4], which use two molecularly distinct pathways to break symmetry [5–8]. In both, the centrosome's position adjacent to the cell cortex is thought to determine where polarization starts. Defects in centrosome-cortex juxtaposition correlate with defects in polarity establishment in several mutants [4, 9, 10], suggesting that these processes may be linked, but there is no direct test of this. Here we assess how centrosome position relative to the cortex affects polarity establishment. We find that centrosomes can initiate polarity from any position within the embryo volume, but centrosome-cortex proximity decreases the time required to initiate polarity. Polarization itself brings about close centrosome-cortex proximity. Prior to polarization, cytoplasmic microtubules constrain centrosome movement near the cortex, expanding the controversial role of microtubules during polarity establishment [4–6, 11–13]. The ability of centrosomes to induce a single polarity axis from any position within the egg emphasizes the flexible, self-organizing properties of polarization in C. elegans embryos and contrasts the common view of C. elegans development as invariant

Insights into the Electron Transfer Kinetics, Capacitance and Resistance Effects of Implantable Electrodes Using Fourier Transform AC Voltammetry on Platinum

The charge transfer mechanism at the electrode-solution interface was assessed by Fourier transform AC voltammetry (FTACV). The faradaic reactions that occur within the safe potential window on platinum had slow electron transfer kinetics. The charge transfer mechanisms during short chronopotentiometric stimulation of cells, is most likely dominated by capacitance. Impedance was modelled with a single time constant. FTACV was fit with a 2-component equivalent circuit comprising a series capacitor and resistor. Capacitance and resistance varied with electrode potential, area, topography, surface functionality and solution composition. Capacitance correlated with charge storage capacity measured by voltammetry. Increased capacitance reduced the change in potential during chronopotentiometry. Increased resistance resulted in uncompensated resistance, and a larger change in potential during chronopotentiometry. Uncompensated resistance in tissue may lead to the measured potential of an electrode being considerably higher than its true potential, leading to a conservative estimate of the safe operating potential window. An impedance test is used to assess electrode performance in vivo. The impedance test is a function of capacitance, faradaic charge and resistance. Impedance test results allow electrode comparison, indicating changes in electrode-tissue interface, electrode failure and power usage, however use of Ohm\u27s law to calculate an impedance value is not valid

Centrosomes direct cell polarity independently of microtubule assembly in C. elegans embryos.

Polarity establishment requires a symmetry-breaking event, resulting in an axis along which determinants are segregated. In Caenorhabditis elegans, oocytes are apolar and are triggered to polarize rapidly along one axis after fertilization. The establishment of this first polarity axis is revealed by the asymmetric distribution of PAR proteins and cortical activity in the one-celled embryo. Current evidence suggests that the centrosome-pronucleus complex contributed by the sperm is involved in defining the polarization axis. Here we directly assess the contribution of the centrosome to polarity establishment by laser ablating the centrosome before and during polarization. We find that the centrosome is required to initiate polarity but not to maintain it. Initiation of polarity coincides with the proximity of the centrosome to the cortex and the assembly of pericentriolar material on the immature sperm centrosome. Depletion of microtubules or the microtubule nucleator gamma-tubulin did not affect polarity establishment. These results demonstrate that the centrosome provides an initiating signal that polarizes C. elegans embryos and indicate that this signalling event might be independent of the role of the centrosome as a microtubule nucleator

Charge Injection from Chronoamperometry of Platinum Electrodes for Bionic Devices

The chronoamperometric response of platinum under biologically relevant conditions was investigated to understand how charge transfer across the electrode-tissue interface occurs during potential pulsing. Platinum behaves as a non-ideal electrode, passing capacitance and faradaic charge. The faradaic reactions are associated with oxide formation and removal, hydrogen and anion adsorption. The capacitance charge decayed within μs while the faradaic charge decay occurred over longer times. The total charge and the ratio of faradaic to capacitance charge was seen to vary with time, potential, electrode size, oxygen concentration, electrolyte and surface cleaning method. The charge transfer mechanisms result in an accumulation of charge during multiple potential pulses, mostly reductive charge under the conditions presented here. This modifies the composition of the electrode/solution interface. An accurate understanding of charge transfer at the electrode/tissue interface must subsequently be obtained under biologically relevant conditions (an artificial perilymph with low oxygen concentration for cochlear implants electrodes and artificial cerebrospinal fluid for neural implants) and with appropriate clinical electrodes

Using chronopotentiometry to better characterize the charge injection mechanisms of platinum electrodes used in bionic devices

The safe charge injection capacity and charge density of neural stimulating electrodes is based on empirical evidence obtained from stimulating feline cortices. Stimulation induced tissue damage may be caused by electrochemical or biological mechanisms. Separating these mechanisms requires greater understanding of charge transfer at the electrode-tissue interface. Clinical devices typically use a biphasic waveform with controlled current. Therefore, the charge injection mechanism and charge injection capacity of platinum was assessed on a commercial potentiostat by chronopotentiometry (controlled current stimulation). Platinum is a non-ideal electrode, charge injection by chronopotentiometry can be passed via capacitive and Faradaic mechanisms. Electrodes were tested under a variety of conditions to assess the impact on charge injection capacity. The change in electrode potential (charge injection capacity) was affected by applied charge density, pulse length, pulse polarity, electrode size, polishing method, electrolyte composition, and oxygen concentration. The safe charge injection capacity and charge density could be increased by changing the electrode-solution composition and stimulation parameters. However, certain conditions (e.g., acid polished electrodes) allowed the electrode to exceed the water electrolysis potential despite the stimulation protocol being deemed safe according to the Shannon plot. Multiple current pulses led to a shift or ratcheting in electrode potential due to changes in the electrode-solution composition. An accurate measure of safe charge injection capacity and charge density of an implantable electrode can only be obtained from suitable conditions (an appropriately degassed electrolyte and clinically relevant electrode structure). Cyclic voltammetric measurement of charge storage capacity can be performed on implantable electrodes, but will not provide information on electrode stability to multiple chronopotentiometric pulses. In contrast, chronopotentiometry will provide details on electrode stability, but the minimum time resolution of typical commercial potentiostats (ms range) is greater than used in a clinical stimulator (μs range) so that extrapolation to short stimulation pulses is required. Finally, an impedance test is typically used to assess clinical electrode performance. The impedance test is also based on a biphasic chronopotentiometic waveform where the measured potential is used to calculate an impedance value. Here it is shown that the measured potential is a function of many parameters (solution composition, electrode area, and surface composition). Subsequently, impedance test results allow electrode comparison and to indicate electrode failure, but use of Ohm\u27s law to calculate an impedance value is not valid

Directed Motion of Telomeres in the Formation of the Meiotic Bouquet Revealed by Time Course and Simulation Analysis

Chromosome movement is critical for homologous chromosome pairing during meiosis. A prominent and nearly universal meiotic chromosome reorganization is the formation of the bouquet, characterized by the close clustering of chromosome ends at the nuclear envelope. We have used a novel method of in vitro culture of rye anthers combined with fluorescent in situ hybridization (FISH) detection of telomeres to quantitatively study bouquet formation. The three-dimensional distribution of telomeres over time was used to obtain a quantitative profile of bouquet formation intermediates. The bouquet formed through a gradual, continuous tightening of telomeres over ∼6 h. To determine whether the motion of chromosomes was random or directed, we developed a computer simulation of bouquet formation to compare with our observations. We varied the diffusion rate of telomeres and the amount of directional bias in telomere movement. In our models, the bouquet was formed in a manner comparable to what we observed in cultured meiocytes only when the movement of telomeres was actively directed toward the bouquet site, whereas a wide range of diffusion rates were permitted. Directed motion, as opposed to random diffusion, was required to reproduce our observations, implying that an active process moves chromosomes to cause telomere clustering

Comparison of the In Vitro and In Vivo Electrochemical Performance of Bionic Electrodes

The electrochemical performance of platinum electrodes was assessed in vitro and in vivo to determine the impact of electrode implantation and the relevance of in vitro testing in predicting in vivo behaviour. A significant change in electrochemical response was seen after electrode polarisation. As a result, initial in vitro measurements were poor predictors of subsequent measurements performed in vitro or in vivo. Charge storage capacity and charge density measurements from initial voltammetric measurements were not correlated with subsequent measurements. Electrode implantation also affected the electrochemical impedance. The typically reported impedance at 1 kHz was a very poor predictor of electrode performance. Lower frequencies were significantly more dependent on electrode properties, while higher frequencies were dependent on solution properties. Stronger correlations in impedance at low frequencies were seen between in vitro and in vivo measurements after electrode activation had occurred. Implanting the electrode increased the resistance of the electrochemical circuit, with bone having a higher resistivity than soft tissue. In contrast, protein fouling and fibrous tissue formation had a minimal impact on electrochemical response. In vivo electrochemical measurements also typically use a quasi-reference electrode, may operate in a 2-electrode system, and suffer from uncompensated resistance. The impact of these experimental conditions on electrochemical performance and the relevance of in vitro electrode assessment is discussed. Recommended in vitro testing protocols for assessing bionic electrodes are presented