Matthew R. Behrend

An In Vitro Model of a Retinal Prosthesis

Epiretinal prostheses are being developed to bypass a degenerated photoreceptor layer and excite surviving ganglion and inner retinal cells. We used custom microfabricated multielectrode arrays with 200-mum-diameter stimulating electrodes and 10-mum-diameter recording electrodes to stimulate and record neural responses in isolated tiger salamander retina. Pharmacological agents were used to isolate direct excitation of ganglion cells from excitation of other inner retinal cells. Strength-duration data suggest that, if amplitude will be used for the coding of brightness or gray level in retinal prostheses, shorter pulses (200 mus) will allow for a smaller region in the area of the electrode to be excited over a larger dynamic range compared with longer pulses (1 ms). Both electrophysiological results and electrostatic finite-element modeling show that electrode-electrode interactions can lead to increased thresholds for sites half way between simultaneously stimulated electrodes (29.4 plusmn 6.6 nC) compared with monopolar stimulation (13.3 plusmn 1.7 nC, < 0.02). Presynaptic stimulation of the same ganglion cell with both 200- and 10- m-diameter electrodes yielded threshold charge densities of 12 plusmn 6 and 7.66 plusmn 1.30 nC/cm2, respectively, while the required charge was 12.5 plusmn 6.2 and 19 plusmn 3.3 nC.

Pulse generators for pulsed electric field exposure of biological cells and tissues

This paper describes three pulse generators: a spark gap switched coaxial cable, a spark gap switched Blumlein, and a solid state modulator, developed for applying ultrashort electrical pulses to biological materials in culture. Research has shown that ultrashort pulsed electric fields can induce apoptosis in biological cells, and that pulses as short as 10 ns with field amplitude greater than 1 W/m cause membrane phospholipid rearrangement and activation of the effector enzymes of apoptosis. Pulses of very short duration use only tens of mJ per mL per pulse to induce apoptosis and other intracellular effects without causing thermal trauma. The pulse generators discussed here, each of a different topology, deliver ns pulsed electric fields (nsPEF) to cells in liquid suspension, and can be modified to drive electrodes for external, surgical, or endoscopic treatment of tissues in situ.

Resolution of the Epiretinal Prosthesis is not Limited by Electrode Size

Epiretinal prostheses for the blind bypass diseased photosensitive cells in the retina, directly stimulating retinal neurons electrically and evoking signals that are relayed to the brain. Current clinical implants have few electrodes and provide limited visual acuity. Acuity may be improved by identifying electrode array design features and operational details that enhance or interfere with visual percept formation. We labeled all retinal ganglion cells in whole mount retina with a calcium reporter and then measured the number and pattern of cells responding, over a range of electrode diameters and stimulus durations. Span of the response scaled with electrode diameter for electrodes 60 μm and larger. Short stimulation pulse widths selectively activated cells nearest the electrode. Our measurements in the salamander retina suggest that the spatial resolution is 150 μm, which on a human retina is equivalent to 0.55° of human visual field and corresponding Snellen acuity of 20/660. Reading large print could be possible with such a prosthesis.

Nanosecond pulse Generator using fast recovery diodes for cell electromanipulation

Design and operation of a fast recovery diode based pulse generator is presented. The generator produces 3.5-ns-wide, 1200 V amplitude unipolar pulses or +/-600-V bipolar pulses into 50-/spl Omega/ load at the maximum repetition rate of 100 kHz. Pulses shorter than 10 ns are essential for the studies of biological cell response to high electric fields while avoiding ordinary electroporation effects dominant at long pulses. Bipolar pulses are used for the studies of biological cell response to high electric fields when the net transfer of charge is undesirable. The bipolar pulse is produced from a unipolar pulse with the help of a shorted transmission line. This transmission line delays and inverts the initial pulse, so the output is the sum of the initial and the inverted and delayed pulses. The use of mass-produced fast recovery surface-mount rectifier diodes in this circuit substantially simplifies the generator and results in low cost and very small footprint. Similar diode switched pulse generators have been described in the literature using mostly custom fabricated snap-recovery diodes. Here we give an example of an ordinary low-cost diode performing similarly to the custom fabricated counterpart. The diode switched circuit relaxes the requirement on the speed of the main closing switch; in our case, a low-cost power metal-oxide semiconductor field-effect transistor (MOSFET)-saturable core transformer combination.

Selective labeling of retinal ganglion cells with calcium indicators by retrograde loading in vitro

Here we present a retrograde loading technique that makes it possible for the first time to rapidly load a calcium indicator in the majority of retinal ganglion cells (RGCs) in salamander retina, and then to observe physiological activity of these dye-loaded cells. Dextran-conjugated calcium indicator, dissolved in water, was applied to the optic nerve stump. Following dye loading, the isolated retina was mounted on a microelectrode array to demonstrate that electrical activity and calcium activity were preserved, as the retina responded to electrical stimuli.

The Argus™ II retinal prosthesis: Factors affecting patient selection for implantation

The Argus II epiretinal prosthesis has been developed to provide partial restoration of vision to subjects blinded from outer retinal degenerative disease. To date, the device has been implanted in multiple subjects with profound retinitis pigmentosa as part of a worldwide clinical feasibility study (clinicaltrials.gov ID: NCT00407602). The Argus II is intended to provide partial restoration of functional vision. Most subjects showed an improvement in tasks assessing orientation & mobility, spatial-motor localization, and ability of discerning the direction of motion of moving stimuli. Roughly one third of subjects experienced measurable improvement in visual acuity with the implant. Some subjects identified words with high accuracy, a result that has also been reported by the leading subretinal implant group. Perceptual threshold was correlated with electrode-retina distance, electrode-fovea distance, and light sensitivity, either as single variables or in bivariate linear regression. Taken together these three variables may be used to inform patient selection and develop algorithms for the fitting of higher-electrode count systems. Visual acuity for future generations of the Argus implant may not hit theoretical limitations until arrays hold an excess of several hundreds of electrodes. Nevertheless, preliminary safety and efficacy data are supportive of the development of higher-resolution systems that target macular placement from implant design and surgical perspectives.

Imaging the response of the retina to electrical stimulation with genetically encoded calcium indicators

Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.

The Dependence of Spectral Impedance on Disc Microelectrode Radius

As microelectrodes gain widespread use for electrochemical sensing, biopotential recording, and neural stimulation, it becomes important to understand the dependence of electrochemical impedance on microelectrode size. It has been shown mathematically that a disc electrode, coplanar in an insulating substrate and exposed to a conducting media, exhibits an inhomogeneous current distribution when a potential step is applied. This distribution is known as the primary distribution, and its derivation also yielded an analytic solution for electrical resistance of the conducting media (Rs), between the disc surface and a distant ground, which is inversely proportional to disk radius [Rs = 1/(4Kr), where k is media conductivity and r is disk radius]. The dependence of spectral impedance on microelectrode radius, however, has not been explored. We verify the analytical solution for resistance using high-frequency (100 kHz) electrochemical impedance data from microelectrodes of varying radius (11-325 mum). For all disc radii, as we approach a lower frequency (rarr 10 Hz), we observe a transition from radial to area dependence (e.g., 1/r rarr 1/r2). We hypothesize that this transition is driven by the fact that the derivation of the primary distribution ignores concentration gradients, but that these gradients cannot be ignored at lower frequencies.

Fluorescence microscopy imaging of electroperturbation in mammalian cells.

We report the design, integration, and validation of a fluorescence microscopy system for imaging of electroperturbation--the effects of nanosecond, megavolt-per-meter pulsed electric fields on biological cells and tissues. Such effects have potential applications in cancer therapy, gene regulation, and biophysical research by noninvasively disrupting intracellular compartments and inducing apoptosis in malignant cells. As the primary observing platform, an epifluorescence microscope integrating a nanosecond high-voltage pulser and a micrometer electrode chamber enable in situ imaging of the intracellular processes triggered by high electric fields. Using specific fluorescence molecular probes, the dynamic biological responses of Jurkat T lymphocytes to nanosecond electric pulses (nanoelectropulses) are studied with this system, including calcium bursts, the polarized translocation of phosphatidylserine (PS), and nuclear enlargement and chromatin/DNA structural changes.

Ultrashort pulse electroporation: applications of high pulsed electric fields to induced caspase activation of human lymphocytes

Presents evidence for apoptosis, or programmed cell death, and up-regulation of a subset of important genes induced by intense electric fields of short duration (10s nsec). The fields are observed to perturb mitochondrial membranes and the compartmentalized intracellular environment of Jurkat T lymphocytes, and within hours caspase activation occurs. Intense, but low energy, fields can penetrate a biological cell and invoke mechanisms associated with apoptosis.