Samsoon Inayat

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.

Patch clamp technique: Review of the current state of the art and potential contributions from nanoengineering

The patch clamp technique permits high-resolution recording of the ionic currents flowing through a cells plasma membrane. In different configurations, this technique has allowed experimenters to record and manipulate the currents that flow either through single ion channels or those that flow across the whole plasma membrane. Unfortunately, the conventional patch clamp method is laborious, requiring the careful fabrication of electrodes, skillful manipulation of the patch pipette towards a cell, and the clever design of electronics and apparatus to allow low-noise recordings. Advances in microfabrication offer promising technologies for high-throughput patch clamp recordings, particularly suitable for drug screening. This paper provides a review of the advances that have been made in the patch clamp technique over the years and considers where application of nanotechnology might provide significant contributions in the future.

Solid-supported membrane technology for the investigation of the influenza A virus M2 channel activity.

Influenza A virus encodes an integral membrane protein, A/M2, that forms a pH-gated proton channel that is essential for viral replication. The A/M2 channel is a target for the anti-influenza drug amantadine, although the effectiveness of this drug has been diminished by the appearance of naturally occurring point mutations in the channel pore. Thus, there is a great need to discover novel anti-influenza therapeutics, and, since the A/M2 channel is a proven target, approaches are needed to screen for new classes of inhibitors for the A/M2 channel. Prior in-depth studies of the activity and drug sensitivity of A/M2 channels have employed labor-intensive electrophysiology techniques. In this study, we tested the validity of electrophysiological measurements with solid-supported membranes (SSM) as a less labor-intensive alternative technique for the investigation of A/M2 ion channel properties and for drug screening. By comparing the SSM-based measurements of the activity and drug sensitivity of A/M2 wild-type and mutant channels with measurements made with conventional electrophysiology methods, we show that SSM-based electrophysiology is an efficient and reliable tool for functional studies of the A/M2 channel protein and for screening compounds for inhibitory activity against the channel.

Chemical stimulation of rat retinal neurons: Feasibility of an epiretinal neurotransmitter-based prosthesis

OBJECTIVE No cure currently exists for photoreceptor degenerative diseases, which cause partial or total blindness in millions of people worldwide. Electrical retinal prostheses have been developed by several groups with the goal of restoring vision lost to these diseases, but electrical stimulation has limitations. It excites both somas and axons, activating retinal pathways nonphysiologically, and limits spatial resolution because of current spread. Chemical stimulation of retinal ganglion cells (RGCs) using the neurotransmitter glutamate has been suggested as an alternative to electrical stimulation with some significant advantages. However, sufficient scientific data to support developing a chemical-based retinal prosthesis is lacking. The goal of this study was to investigate the feasibility of a neurotransmitter-based retinal prosthesis and determine therapeutic stimulation parameters. APPROACH We injected controlled amounts of glutamate into rat retinas from the epiretinal side ex vivo via micropipettes using a pressure injection system and recorded RGC responses with a multielectrode array. Responsive units were identified using a spike rate threshold of 3 Hz. MAIN RESULTS We recorded both somal and axonal units and demonstrated successful glutamatergic stimulation across different RGC subtypes. Analyses show that exogenous glutamate acts on RGC synapses similar to endogenous glutamate and, unlike electrical prostheses, stimulates only RGC somata. The spatial spread of glutamate stimulation was ≈ 290 μm from the injection site, comparable to current electrical prostheses. Further, the glutamate injections produced spatially differential responses in OFF, ON, and ON-OFF RGC subtypes, suggesting that differential stimulation of the OFF and ON systems may be possible. A temporal resolution of 3.2 Hz was obtained, which is a rate suitable for spatial vision. SIGNIFICANCE We provide strong support for the feasibility of an epiretinal neurotransmitter-based retinal prosthesis. Our findings suggest that chemical stimulation of RGCs is a viable alternative to electrical stimulation and could offer distinct advantages such as the selective stimulation of RGC somata.

Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation.

Subretinal stimulation of the retina with neurotransmitters, the normal means of conveying visual information, is a potentially better alternative to electrical stimulation widely used in current retinal prostheses for treating blindness from photoreceptor degenerative diseases. Yet, no subretinal electrical or chemical stimulation study has stimulated the OFF and ON pathways differentially through inner retinal activation. Here, we demonstrate the feasibility of differentially stimulating retinal ganglion cells (RGCs) through the inner nuclear layer of the retina with glutamate, a primary neurotransmitter chemical, in a biomimetic way. We show that controlled pulsatile delivery of glutamate into the subsurface of explanted wild-type rat retinas elicits highly localized simultaneous inhibitory and excitatory spike rate responses in OFF and ON RGCs. We also present the spatiotemporal characteristics of RGC responses to subretinally injected glutamate and the therapeutic stimulation parameters. Our findings could pave the way for future development of a neurotransmitter-based subretinal prosthesis offering more naturalistic vision and better visual acuity than electrical prostheses.

Development of a chemical retinal prosthesis: Stimulation of rat retina with glutamate

Retinal degenerative diseases cause partial or total blindness and affect millions of people worldwide, yet currently have no treatment. Retinal prostheses using electrical stimulation are being developed but face significant problems moving forward. Here we propose using chemical stimulation, via the neurotransmitter glutamate, to modulate retinal ganglion cell (RGC) spike rates. Our results demonstrate that it is feasible to stimulate RGCs in an explanted retina using focal ejections of glutamate from either subretinal or epiretinal sides. Preliminary evidence suggests we are primarily activating RGCs as opposed to bipolar cells. This is an important first step in the development of a chemical retinal prosthesis based on microelectromechanical systems (MEMS) technology.

Impedance characterization and modelling of an improved patch clamp device

The gold standard for studying the properties of ion channels is the patch clamp, a technique that measures pA currents passing through ion channels in an isolated patch of cell membrane. The authors sought to design an improved device for whole-cell patch clamp recordings using nanoscale pipettes which more readily form seals with the cell membrane and a movable metal electrode to decrease the access resistance, increase the signal bandwidth, and clear debris from the pipette tip. Electrical characterization of the instrument by impedance spectroscopy showed that advancing the metal electrode towards the pipette tip decreased the impedance distinctly in different frequency bands, the greatest decrease occurring between 1 and 100 kHz, and the smallest at 100 kHz. Finite element simulations of the electrode, incorporating the electrode/electrolyte interface, suggest that optimal signal transmission can be achieved with athick-walled pipette, nearly filled with alarge Ag/AgCl electrode, whose impedance can be reduced through surface modification.

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.