High-speed interferometric imaging reveals dynamics of neuronal deformation during the action potential.

Neurons undergo nanometer-scale deformations during action potentials, and the underlying mechanism has been actively debated for decades. Previous observations were limited to a single spot or the cell boundary, while movement across the entire neuron during the action potential remained unclear. Here we report full-field imaging of cellular deformations accompanying the action potential in mammalian neuron somas (-1.8 to 1.4 nm) and neurites (-0.7 to 0.9 nm), using high-speed quantitative phase imaging with a temporal resolution of 0.1 ms and an optical path length sensitivity of <4 pm per pixel. The spike-triggered average, synchronized to electrical recording, demonstrates that the time course of the optical phase changes closely matches the dynamics of the electrical signal. Utilizing the spatial and temporal correlations of the phase signals across the cell, we enhance the detection and segmentation of spiking cells compared to the shot-noise-limited performance of single pixels. Using three-dimensional (3D) cellular morphology extracted via confocal microscopy, we demonstrate that the voltage-dependent changes in the membrane tension induced by ionic repulsion can explain the magnitude, time course, and spatial features of the phase imaging. Our full-field observations of the spike-induced deformations shed light upon the electromechanical coupling mechanism in electrogenic cells and open the door to noninvasive label-free imaging of neural signaling.

Cortical Interactions between Prosthetic and Natural Vision.

Outer retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are among the leading causes of incurable blindness in the Western world [1]. Retinal prostheses have been shown to restore some useful vision by electrically stimulating the remaining retinal neurons [2]. In contrast to inherited retinal degenerative diseases (e.g., RP), typically leading to a complete loss of the visual field, in AMD patients the disease is localized to the macula, leaving the peripheral vision intact. Implanting a retinal prosthesis in the central macula in AMD patients [3, 4] leads to an intriguing situation where the patient's central retina is stimulated electrically, whereas the peripheral healthy retina responds to natural light stimulation. An important question is whether the visual cortex responds to these two concurrent stimuli similarly to the interaction between two adjacent natural light stimuli projected onto healthy retina. Here, we investigated the cortical interactions between prosthetic and natural vision based on visually evoked potentials (VEPs) recorded in rats implanted with photovoltaic subretinal implants. Using this model, where prosthetic and natural vision information are combined in the visual cortex, we observed striking similarities in the interactions of natural and prosthetic vision, including similar effect of background illumination, linear summation of non-patterned stimuli, and lateral inhibition with spatial patterns [5], which increased with target contrast. These results support the idea of combined prosthetic and natural vision in restoration of sight for AMD patients.

Honeycomb-shaped electro-neural interface enables cellular-scale pixels in subretinal prosthesis.

High-resolution visual prostheses require small, densely packed pixels, but limited penetration depth of the electric field formed by a planar electrode array constrains such miniaturization. We present a novel honeycomb configuration of an electrode array with vertically separated active and return electrodes designed to leverage migration of retinal cells into voids in the subretinal space. Insulating walls surrounding each pixel decouple the field penetration depth from the pixel width by aligning the electric field vertically, enabling a decrease of the pixel size down to cellular dimensions. We demonstrate that inner retinal cells migrate into the 25 µm deep honeycomb wells as narrow as 18 µm, resulting in more than half of these cells residing within the electrode cavities. Immune response to honeycombs is comparable to that with planar arrays. Modeled stimulation threshold current density with honeycombs does not increase substantially with reduced pixel size, unlike quadratic increase with planar arrays. This 3-D electrode configuration may enable functional restoration of central vision with acuity better than 20/100 for millions of patients suffering from age-related macular degeneration.

Characteristics of prosthetic vision in rats with subretinal flat and pillar electrode arrays.

OBJECTIVE: Retinal prostheses aim to restore sight by electrically stimulating the surviving retinal neurons. In clinical trials of the current retinal implants, prosthetic visual acuity does not exceed 20/550. However, to provide meaningful restoration of central vision in patients blinded by age-related macular degeneration (AMD), prosthetic acuity should be at least 20/200, necessitating a pixel pitch of about 50 µm or lower. With such small pixels, stimulation thresholds are high due to limited penetration of electric field into tissue. Here, we address this challenge with our latest photovoltaic arrays and evaluate their performance in vivo. APPROACH: We fabricated photovoltaic arrays with 55 and 40 µm pixels (a) in flat geometry, and (b) with active electrodes on 10 µm tall pillars. The arrays were implanted subretinally into rats with degenerate retina. Stimulation thresholds and grating acuity were evaluated using measurements of the visually evoked potentials (VEP). MAIN RESULTS: With 55 µm pixels, we measured grating acuity of 48 ± 11 µm, which matches the linear pixel pitch of the hexagonal array. This geometrically corresponds to a visual acuity of 20/192 in a human eye, matching the threshold of legal blindness in the US (20/200). With pillar electrodes, the irradiance threshold was nearly halved, and duration threshold reduced by more than three-fold, compared to flat pixels. With 40 µm pixels, VEP was too low for reliable measurements of the grating acuity, even with pillar electrodes. SIGNIFICANCE: While being helpful for treating a complete loss of sight, current prosthetic technologies are insufficient for addressing the leading cause of untreatable visual impairment-AMD. Subretinal photovoltaic arrays may provide sufficient visual acuity for restoration of central vision in patients blinded by AMD.

Tumor treating fields increases membrane permeability in glioblastoma cells.

Glioblastoma is the most common yet most lethal of primary brain cancers with a one-year post-diagnosis survival rate of 65% and a five-year survival rate of barely 5%. Recently the U.S. Food and Drug Administration approved a novel fourth approach (in addition to surgery, radiation therapy, and chemotherapy) to treating glioblastoma; namely, tumor treating fields (TTFields). TTFields involves the delivery of alternating electric fields to the tumor but its mechanisms of action are not fully understood. Current theories involve TTFields disrupting mitosis due to interference with proper mitotic spindle assembly. We show that TTFields also alters cellular membrane structure thus rendering it more permeant to chemotherapeutics. Increased membrane permeability through the imposition of TTFields was shown by several approaches. For example, increased permeability was indicated through increased bioluminescence with TTFields exposure or with the increased binding and ingress of membrane-associating reagents such as Dextran-FITC or ethidium D or with the demonstration by scanning electron microscopy of augmented number and sizes of holes on the cellular membrane. Further investigations showed that increases in bioluminescence and membrane hole production with TTFields exposure disappeared by 24 h after cessation of alternating electric fields thus demonstrating that this phenomenom is reversible. Preliminary investigations showed that TTFields did not induce membrane holes in normal human fibroblasts thus suggesting that the phenomenom was specific to cancer cells. With TTFields, we present evidence showing augmented membrane accessibility by compounds such as 5-aminolevulinic acid, a reagent used intraoperatively to delineate tumor from normal tissue in glioblastoma patients. In addition, this mechanism helps to explain previous reports of additive and synergistic effects between TTFields and other chemotherapies. These findings have implications for the design of combination therapies in glioblastoma and other cancers and may significantly alter standard of care strategies for these diseases.

Optimization of pillar electrodes in subretinal prosthesis for enhanced proximity to target neurons.

OBJECTIVE: High-resolution prosthetic vision requires dense stimulating arrays with small electrodes. However, such miniaturization reduces electrode capacitance and penetration of electric field into tissue. We evaluate potential solutions to these problems with subretinal implants based on utilization of pillar electrodes. APPROACH: To study integration of three-dimensional (3D) implants with retinal tissue, we fabricated arrays with varying pillar diameter, pitch, and height, and implanted beneath the degenerate retina in rats (Royal College of Surgeons, RCS). Tissue integration was evaluated six weeks post-op using histology and whole-mount confocal fluorescence imaging. The electric field generated by various electrode configurations was calculated in COMSOL, and stimulation thresholds assessed using a model of network-mediated retinal response. MAIN RESULTS: Retinal tissue migrated into the space between pillars with no visible gliosis in 90% of implanted arrays. Pillars with 10 µm height reached the middle of the inner nuclear layer (INL), while 22 µm pillars reached the upper portion of the INL. Electroplated pillars with dome-shaped caps increase the active electrode surface area. Selective deposition of sputtered iridium oxide onto the cap ensures localization of the current injection to the pillar top, obviating the need to insulate the pillar sidewall. According to computational model, pillars having a cathodic return electrode above the INL and active anodic ring electrode at the surface of the implant would enable six times lower stimulation threshold, compared to planar arrays with circumferential return, but suffer from greater cross-talk between the neighboring pixels. SIGNIFICANCE: 3D electrodes in subretinal prostheses help reduce electrode-tissue separation and decrease stimulation thresholds to enable smaller pixels, and thereby improve visual acuity of prosthetic vision.

Optimization of return electrodes in neurostimulating arrays.

OBJECTIVE: High resolution visual prostheses require dense stimulating arrays with localized inputs of individual electrodes. We study the electric field produced by multielectrode arrays in electrolyte to determine an optimal configuration of return electrodes and activation sequence. APPROACH: To determine the boundary conditions for computation of the electric field in electrolyte, we assessed current dynamics using an equivalent circuit of a multielectrode array with interleaved return electrodes. The electric field modeled with two different boundary conditions derived from the equivalent circuit was then compared to measurements of electric potential in electrolyte. To assess the effect of return electrode configuration on retinal stimulation, we transformed the computed electric fields into retinal response using a model of neural network-mediated stimulation. MAIN RESULTS: Electric currents at the capacitive electrode-electrolyte interface redistribute over time, so that boundary conditions transition from equipotential surfaces at the beginning of the pulse to uniform current density in steady state. Experimental measurements confirmed that, in steady state, the boundary condition corresponds to a uniform current density on electrode surfaces. Arrays with local return electrodes exhibit improved field confinement and can elicit stronger network-mediated retinal response compared to those with a common remote return. Connecting local return electrodes enhances the field penetration depth and allows reducing the return electrode area. Sequential activation of the pixels in large monopolar arrays reduces electrical cross-talk and improves the contrast in pattern stimulation. SIGNIFICANCE: Accurate modeling of multielectrode arrays helps optimize the electrode configuration to maximize the spatial resolution, contrast and dynamic range of retinal prostheses.

Validation of the Multidimensional Acculturative Stress Inventory on adolescents of Mexican origin.

The Multidimensional Acculturative Stress Inventory (MASI), a 36-item measure that assesses acculturative stress among people of Mexican origin living in the United States, was tested on 331 adolescent (14-20 years of age) high school students (204 female, 127 male) of Mexican origin. Exploratory factor analyses yielded 4 factors: bicultural practices conflict (9 items), Spanish competency pressures (8 items), English competency pressures (8 items), and bicultural self-consciousness (2 items). These factors accounted for 59.5% of the variance and correlated in the expected directions with criterion measures of acculturation and the Psychological General Well-Being Schedule. Bicultural practices conflict and bicultural self-consciousness emerged as the first and fourth factors for adolescents, which differed from the last 2 factors observed in a previous study of adults by Rodriguez, Myers, Mira, Flores, and Garcia-Hernandez (2002)--pressure to acculturate and pressure against acculturation. Comparisons of the MASI factor structures between adolescents and adults also revealed that English competency pressures and Spanish competency pressures played a prominent role for both adolescents in this study and adults in the study by Rodriguez et al. (2002). The congruence and difference in factor structure of the MASI between adolescents and adults indicates that both groups experience acculturative stress because of English- and Spanish-language competency pressures, but adolescents differentially experience difficulties in negotiating between American and Latino practices and identities. The results highlight the importance of assessing acculturative stress from both Latino and American culture and recognizing the varying levels of these sources of acculturative stress by generation.

Development of the Multidimensional Acculturative Stress Inventory for adults of Mexican origin.

The Multidimensional Acculturative Stress Inventory (MASI), a 36-item stress measure that was developed to assess acculturative stress among persons of Mexican origin living in the United States, was tested on a community sample of 174 adults (117 women, 57 men). Principal-components analyses yielded 4 stable and internally consistent factors: Spanish Competency Pressures (7 items), English Competency Pressures (7 items), Pressure to Acculturate (7 items), and Pressure Against Acculturation (4 items). These 4 factors accounted for 64.4% of the variance and correlated in the expected directions with criterion measures of acculturation and/or psychological adjustment. Further reliability and validity testing of the MASI is discussed as well as the utility of this measure in assessing acculturative stress among adults of Mexican origin.