Donald R. Cantrell

Incorporation of the electrode-electrolyte interface into finite-element models of metal microelectrodes

An accurate description of the electrode-electrolyte interfacial impedance is critical to the development of computational models of neural recording and stimulation that aim to improve understanding of neuro-electric interfaces and to expedite electrode design. This work examines the effect that the electrode-electrolyte interfacial impedance has upon the solutions generated from time-harmonic finite-element models of cone- and disk-shaped platinum microelectrodes submerged in physiological saline. A thin-layer approximation is utilized to incorporate a platinum-saline interfacial impedance into the finite-element models. This approximation is easy to implement and is not computationally costly. Using an iterative nonlinear solver, solutions were obtained for systems in which the electrode was driven at ac potentials with amplitudes from 10 mV to 500 mV and frequencies from 100 Hz to 100 kHz. The results of these simulations indicate that, under certain conditions, incorporation of the interface may strongly affect the solutions obtained. This effect, however, is dependent upon the amplitude of the driving potential and, to a lesser extent, its frequency. The solutions are most strongly affected at low amplitudes where the impedance of the interface is large. Here, the current density distribution that is calculated from models incorporating the interface is much more uniform than the current density distribution generated by models that neglect the interface. At higher potential amplitudes, however, the impedance of the interface decreases, and its effect on the solutions obtained is attenuated.

Generation, identification and functional characterization of the nob4 mutation of Grm6 in the mouse

We performed genome-wide chemical mutagenesis of C57BL/6J mice using N-ethyl-N-nitrosourea (ENU). Electroretinographic screening of the third generation offspring revealed two G3 individuals from one G1 family with a normal a-wave but lacking the b-wave that we named nob4. The mutation was transmitted with a recessive mode of inheritance and mapped to chromosome 11 in a region containing the Grm6 gene, which encodes a metabotropic glutamate receptor protein, mGluR6. Sequencing confirmed a single nucleotide substitution from T to C in the Grm6 gene. The mutation is predicted to result in substitution of Pro for Ser at position 185 within the extracellular, ligand-binding domain and oocytes expressing the homologous mutation in mGluR6 did not display robust glutamate-induced currents. Retinal mRNA levels for Grm6 were not significantly reduced, but no immunoreactivity for mGluR6 protein was found. Histological and fundus evaluations of nob4 showed normal retinal morphology. In contrast, the mutation has severe consequences for visual function. In nob4 mice, fewer retinal ganglion cells (RGCs) responded to the onset (ON) of a bright full field stimulus. When ON responses could be evoked, their onset was significantly delayed. Visual acuity and contrast sensitivity, measured with optomotor responses, were reduced under both photopic and scotopic conditions. This mutant will be useful because its phenotype is similar to that of human patients with congenital stationary night blindness and will provide a tool for understanding retinal circuitry and the role of ganglion cell encoding of visual information.

Allelic variance between GRM6 mutants, Grm6nob3 and Grm6nob4 results in differences in retinal ganglion cell visual responses.

An electroretinogram (ERG) screen identified a mouse with a normal a‐wave but lacking a b‐wave, and as such it was designated no b‐wave3 (nob3). The nob3 phenotype mapped to chromosome 11 in a region containing the metabotropic glutamate receptor 6 gene (Grm6). Sequence analyses of cDNA identified a splicing error in Grm6, introducing an insertion and an early stop codon into the mRNA of affected mice (designated Grm6nob3). Immunohistochemistry of the Grm6nob3 retina showed that GRM6 was absent. The ERG and visual behaviour abnormalities of Grm6nob3 mice are similar to Grm6nob4 animals, and similar deficits were seen in compound heterozygotes (Grm6nob4/nob3), indicating that Grm6nob3 is allelic to Grm6nob4. Visual responses of Grm6nob3 retinal ganglion cells (RGCs) to light onset were abnormal. Grm6nob3 ON RGCs were rarely recorded, but when they were, had ill‐defined receptive field (RF) centres and delayed onset latencies. When Grm6nob3 OFF‐centre RGC responses were evoked by full‐field stimulation, significantly fewer converted that response to OFF/ON compared to Grm6nob4 RGCs. Grm6nob4/nob3 RGC responses verified the conclusion that the two mutants are allelic. We propose that Grm6nob3 is a new model of human autosomal recessive congenital stationary night blindness. However, an allelic difference between Grm6nob3 and Grm6nob4 creates a disparity in inner retinal processing. Because the localization of GRM6 is limited to bipolar cells in the On pathway, the observed difference between RGCs in these mutants is likely to arise from differences in their inputs.

Non-Centered Spike-Triggered Covariance Analysis Reveals Neurotrophin-3 as a Developmental Regulator of Receptive Field Properties of ON-OFF Retinal Ganglion Cells

The functional separation of ON and OFF pathways, one of the fundamental features of the visual system, starts in the retina. During postnatal development, some retinal ganglion cells (RGCs) whose dendrites arborize in both ON and OFF sublaminae of the inner plexiform layer transform into RGCs with dendrites that monostratify in either the ON or OFF sublamina, acquiring final dendritic morphology in a subtype-dependent manner. Little is known about how the receptive field (RF) properties of ON, OFF, and ON-OFF RGCs mature during this time because of the lack of a reliable and efficient method to classify RGCs into these subtypes. To address this deficiency, we developed an innovative variant of Spike Triggered Covariance (STC) analysis, which we term Spike Triggered Covariance – Non-Centered (STC-NC) analysis. Using a multi-electrode array (MEA), we recorded the responses of a large population of mouse RGCs to a Gaussian white noise stimulus. As expected, the Spike-Triggered Average (STA) fails to identify responses driven by symmetric static nonlinearities such as those that underlie ON-OFF center RGC behavior. The STC-NC technique, in contrast, provides an efficient means to identify ON-OFF responses and quantify their RF center sizes accurately. Using this new tool, we find that RGCs gradually develop sensitivity to focal stimulation after eye opening, that the percentage of ON-OFF center cells decreases with age, and that RF centers of ON and ON-OFF cells become smaller. Importantly, we demonstrate for the first time that neurotrophin-3 (NT-3) regulates the development of physiological properties of ON-OFF center RGCs. Overexpression of NT-3 leads to the precocious maturation of RGC responsiveness and accelerates the developmental decrease of RF center size in ON-OFF cells. In summary, our study introduces STC-NC analysis which successfully identifies subtype RGCs and demonstrates how RF development relates to a neurotrophic driver in the retina.

Fabrication of nanoelectrodes for neurophysiology: cathodic electrophoretic paint insulation and focused ion beam milling.

The fabrication and characterization of tungsten nanoelectrodes insulated with cathodic electrophoretic paint is described together with their application within the field of neurophysiology. The tip of a 127 mum diameter tungsten wire was etched down to less than 100 nm and then insulated with cathodic electrophoretic paint. Focused ion beam (FIB) polishing was employed to remove the insulation at the electrodes apex, leaving a nanoscale sized conductive tip of 100-1000 nm. The nanoelectrodes were examined by scanning electron microscopy (SEM) and their electrochemical properties characterized by steady state linear sweep voltammetry. Electrode impedance at 1 kHz was measured too. The ability of a 700 nm tipped electrode to record well-isolated action potentials extracellularly from single visual neurons in vivo was demonstrated. Such electrodes have the potential to open new populations of neurons to study.

Extracellular stimulation of mouse retinal ganglion cells with non-rectangular voltage-controlled waveforms

Neural prostheses rely upon electric stimulation to control neural activity. However, electrode corrosion and tissue damage may result from the injection of high charge densities. During electrical stimulation with traditional voltage-controlled square-wave pulses, the current density distribution on the surface of the stimulating electrode is highly nonuniform, with the highest current densities located at the edge of disk-shaped electrodes. Current density is implicated in tissue damage and electrode corrosion because it determines the charge density distribution. Through recent computer modeling work, we have found that Gaussian and sinusoidal stimulus waveforms produce a current density distribution that is significantly more uniform than the one produced by square-wave pulses. In this manner, these non-rectangular waveforms reduce the peak current densities without decreasing the efficacy of the neural stimulus. In the present work, we utilize an in vitro mouse retinal preparation to compare the same set of alternative stimulus waveforms. The -1V amplitude voltage-controlled stimuli were delivered through 20 µm diameter titanium nitride electrodes. Importantly, when normalized for the amount of injected charge, the data demonstrate that each waveform is similarly effective at eliciting a neural response. Also, the suprathreshold Gaussian and sinusoidal waveforms possessed much lower peaks in current. For this reason, these non-rectangular waveforms may be useful in reducing electrode corrosion and tissue damage.

A time domain finite element model of extracellular neural stimulation predicts that non-rectangular stimulus waveforms may offer safety benefits

Efforts to understand and model the process of extracellular neural electric stimulation have been driven by the desire to intelligently design neural prostheses in order to minimize tissue damage and to maximize the success for stimulating targeted neural structures. Tissue damage and electrode corrosion have been associated with high charge density, which is the integral of the current density passing through the electrode surface over the duration of the stimulus. Importantly, the current density distribution on the surface of stimulating electrodes can be extremely nonuniform, especially when high voltages or frequencies cause a decrease in the electrode-electrolyte impedance. Large current densities are found locally in regions of high curvature, such as the edge of a disk electrode or the tip of a conical electrode. We use a time domain finite element model of a platinum disk electrode and a simplified retinal ganglion cell to explore the potential for Gaussian and sinusoidal voltage-controlled stimulus waveforms to reduce the nonuniformity of the current densities on the electrode surface while maintaining stimulation efficacy. We model an overpotential-dependent electrode-electrolyte interfacial impedance consistent with the platinum-saline interface. An excitable cell membrane is incorporated using the Fohlmeister-Coleman-Miller model of a retinal ganglion cell. Both the electrode-electrolyte interface and the cell membrane were incorporated into the finite element model using a thin layer approximation. All simulations were performed in the COMSOL Multiphysics modeling environment. Rectangular stimulus waveforms were compared to waveforms of Gaussian and sinusoidal shapes. The results suggest that Gaussian and Sinusoidal waveforms may significantly decrease the nonuniformity of the current density distribution while retaining stimulation efficacy.

Modeling the electrode-electrolyte interface for recording and stimulating electrodes

The design of metal microelectrodes that produce minimal damage to tissue and can successfully record from and stimulate targeted neural structures necessitates a thorough understanding of the electrical phenomena generated in the tissue surrounding the electrodes. Computational modeling has been a primary strategy used to study these phenomena, and the Finite Element Method has proven to be a powerful approach. Much research has been directed toward the development of models for electrode recording and stimulation, but very few models reported in the literature thus far incorporate the effects of the electrode-electrolyte interface, which can be a source of very high impedance, and thus likely a key component of the system. To explore the effects that the electrode-electrolyte interface has upon the electric potential and current density surrounding metal microelectrodes, simulations of electrode-saline systems in which the electrodes were driven at AC potentials ranging from 10 mV to 500 mV and frequencies of 100 Hz to 10 kHz have been performed using the Finite Element Method. Solutions obtained using the thin layer approximation for the electrode-electrolyte interface was compared with those generated using a thin uniform layer, a representation that has previously appeared in the literature. Solutions using these two methods were similar in the linear regime of the interface however, the thin layer approximation has important advantages over its competitor including ease of application and low computational cost

A Novel Way to Go Whole Cell in Patch-Clamp Experiments

With a conventional patch-clamp electrode, an Ag/AgCl wire sits stationary inside the pipette. To move from the gigaseal cell-attached configuration to whole-cell recording, suction is applied inside the pipette. We have designed and developed a novel “Pushpen patch-clamp electrode,” in which a W wire insulated and wound with Ag/AgCl wire can move linearly inside the pipette. The W wire has a conical tip, which can protrude from the pipette tip like a push pen, a procedure we call the “Pushpen Operation.” We use the Pushpen operation to impale the cell membrane in cell-attached configuration to go whole cell without disruption of the gigaseal. We successfully recorded whole-cell currents from Chinese hamster ovarian cells expressing influenza A virus protein A/M2, after obtaining whole-cell configuration with the Pushpen operation. This novel method of achieving whole-cell configuration may have a higher success rate than the conventional patch-clamp technique.