Short-Term Effects of Gamma Stimulation on Neuroinflammation at the Tissue-Electrode Interface in Motor Cortex.

OBJECTIVES: The reliability of long-term neural recordings as therapeutic interventions for motor and sensory disorders is hampered by the brain tissue response. Previous work showed that flickering light at gamma frequencies (ie, 20-50 Hz) causes enhanced microglial recruitment in the visual cortex. The effects of gamma stimulation on glial cells surrounding implanted neural electrodes are not well understood. We hypothesized that invasive stimulation in the gamma frequency band increases microglial recruitment in the short term and reduces astrogliosis at the tissue-electrode interface. MATERIALS AND METHODS: Male Long Evans rats were implanted with dual-shank silicon microelectrode arrays into the motor cortex. After implantation, rats received one hour of 40-Hz stimulation at a constant current of 10 µA using charge-balanced, biphasic pulses on one shank, and the other shank served as the nonstimulated control. Postmortem, tissue sections were stained with ectodermal dysplasia 1 (ED1) for activated microglia, glial fibrillary acidic protein (GFAP) for astrocytes, and 4',6-diamidino-2-phenylindole (DAPI) for nonspecific nuclei. Fluorescent intensity and cell number as a function of distance from the tissue-electrode interface were used to quantify all stained sections. RESULTS: Fluorescent intensity for ED1 was nearly 40% lower for control than for stimulated sites (0-500 µm away from the implant), indicating increased microglial recruitment to the stimulated site (p < 0.05). Fluorescent intensity for GFAP was >67% higher for control than for stimulated sites (0-500 µm away from the implant), indicating reduced astrogliosis at the stimulated site (p < 0.05). No differences were observed in DAPI-stained sections between conditions. CONCLUSIONS: These results suggest that short-term gamma stimulation modulates glial recruitment in the immediate vicinity of the microelectrode. Future studies will investigate the long-term effects of gamma stimulation on glial recruitment at the tissue-electrode interface as a strategy to improve long-term recording reliability.

Charge Injection Enhancement Comparisons of Iridium Oxide Microelectrodes In Vitro and In Vivo Using a Portable Neurostimulator.

Microelectrodes are desired to deliver more charges to neural tissues while under electrochemical safety limits. Applying anodic bias potential during neurostimulation is a known technique for charge enhancement. Here, we investigated the levels of charge enhancement with anodic bias potential in vitro and in vivo using a custom-designed portable neurostimulator. We immersed our custom microelectrode probe in saline and measured voltage transients in response to constant current stimulation with and without a 500 mV anodic bias potential. We then inserted the same microelectrode probe into the primary motor cortex of the rat brain and measured voltage transients with the same electronics. Results showed that the charge injection capacity of the activated iridium oxide microelectrode site (with 2000 µm2 geometric surface areas (GSAs)) increased by the use of the anodic bias potentials in both in vitro and in vivo: from 10 nC/phase to 32 nC/phase for 200 µs pulse widths, and from 2 nC/phase to 8 nC/phase, respectively. Thus, the order of charge injection capacities of the four cases tested in this study is as follows (from the lowest to the highest): in vivo without anodic bias, in vivo with anodic bias, in vitro without anodic bias, and in vitro with anodic bias. This work also validated in vivo use of our new portable neurostimulator which received stimulation waveforms wirelessly.

A portable neurostimulator circuit with anodic bias enhances stimulation injection capacity.

Objective.Electrochemically safe and efficient charge injection for neural stimulation necessitates monitoring of polarization and enhanced charge injection capacity of the stimulating electrodes. In this work, we present improved microstimulation capability by developing a custom-designed multichannel portable neurostimulator with a fully programmable anodic bias circuitry and voltage transient monitoring feature.Approach.We developed a 16-channel multichannel neurostimulator system, compared charge injection capacities as a function of anodic bias potentials, and demonstrated convenient control of the system by a custom-designed user interface allowing bidirectional wireless data transmission of stimulation parameters and recorded voltage transients. Charge injections were conducted in phosphate-buffered saline with silicon-based iridium oxide microelectrodes.Main results.Under charge-balanced 200µs cathodic first pulsing, the charge injection capacities increased proportionally to the level of anodic bias applied, reaching a maximum of ten-fold increase in current intensity from 10µA (100µC cm-2) to 100µA (1000µC cm-2) with a 600 mV anodic bias. Our custom-designed and completely portable 16-channel neurostimulator enabled a significant increase in charge injection capacityin vitro. Significance.Limited charge injection capacity has been a bottleneck in neural stimulation applications, and our system may enable efficacious behavioral animal study involving chronic microstimulation while ensuring electrochemical safety.

A Simple Table-Top Technique for Multi-Signal Pseudo-Extracellular Recording.

Validation of neural probe performance often includes implantation in live animals, to assess ability to detect and distinguish signals generated by individual neurons. While this method is informative, an effective in vitro alternative would streamline device development and improve ethical considerations by reducing the use of animals in the validation of neural recording devices. Here, we describe a simple system using ball electrodes to apply multiple neural waveforms to phosphate buffered saline, which are simultaneously recorded by a microelectrode probe. Using this technique, our neural probe was able to detect and distinguish spikes from multiple units of roughly physiological amplitudes (~100 microvolts peak to peak), indicating promise as an in vitro alternative to animal testing for initial validation of neural recording devices.

Maximizing Charge Injection Limits of Iridium Oxide Electrodes with a Programmable Anodic Bias Circuit.

Efficacious stimulation of neural tissues requires high charge injection capacity while minimizing electrode polarization. Applying anodic bias on certain electrode materials is a way to enhance charge injection both in vitro and in vivo. We developed an embedded neurostimulator system that enabled a digital control of user-defined bias levels, without requiring a potentiometer or external voltage source. Comparison of charge injection with and without anodic-bias, as well as at different bias potentials were conducted in phosphate-buffered saline with Blackrock iridium oxide microelectrodes. Results showed that a nine-fold increase in current intensity and charge injection capacity, was achieved with a 0.7 V anodic bias and within electrochemically safe limits.

A Diagnostic Circuit for Crosstalk Detection in Microelectrode Arrays.

Current leakage between channels in microelectrode arrays is a sign of device failure and can lead to shorting of neural signals. The purpose of this project is to detect crosstalk between 32 channels of electrodes. We designed an embedded crosstalk detection system that can stimulate each electrode individually with a constant-current pulse and record voltage transients of the stimulated and adjacent electrodes to generate a matrix of crosstalk values. Charge injection in a phosphate buffered saline solution was used to check the condition of each electrode. A semi-wet condition was then used to determine the percent crosstalk between the channels. The analysis showed that there was minimal crosstalk between the electrodes, except for a known physical defect on the probe. The measurement technique enabled by the electronics circuit has the potential to be used in functional testing and screening of implantable devices.

Configuring intracortical microelectrode arrays and stimulus parameters to minimize neuron loss during prolonged intracortical electrical stimulation.

BACKGROUND: Previous studies have shown that neurons of the cerebral cortex can be injured by implantation of, and stimulation with, implanted microelectrodes. OBJECTIVES: Objective 1 was to determine parameters of microstimulation delivered through multisite intracortical microelectrode arrays that will activate neurons of the feline cerebral cortex without causing loss of neurons. OBJECTIVE: 2 was to determine if the stimulus parameters that induced loss of cortical neurons differed for all cortical neurons vs. the subset of inhibitory neurons expressing parvalbumin. METHODS: The intracortical microstimulation was applied for 7 h/day for 20 days (140 h). Microelectrode site areas were 2000 and 4000 µm2, Q was 2-8 nanocoulombs (nC) at 50 Hz, and QD was 50-400 µcoulombs/cm2. RESULTS: Neuron loss due to stimulation was minimal at Q = 2 Ncp, but at 8 Ncp, 20%-50% of neurons within 250 µm of the stimulated microelectrodes were lost, compared to unstimulated microelectrodes. Loss was greatest in tissue facing electrode sites. Stimulation-induced loss was similar for neurons labeled for NeuN and for inhibitory neurons expressing parvalbumin. Correlation between neuron loss and QD was not significant. Electrodes in the medullary pyramidal tract recorded neuronal activity evoked by stimulation in the cerebral cortex. The pyramidal neurons were activated by intracortical stimulation of 2 nC/phase. 140 h of microstimulation at 2 nC/phase and 50 Hz induced minimal neuron loss.

Unprotected sidewalls of implantable silicon-based neural probes and conformal coating as a solution.

Silicon-based implantable neural devices have great translational potential as a means to deliver various treatments for neurological disorders. However, they are currently held back by uncertain longevity following chronic exposure to body fluids. Conventional deposition techniques cover only the horizontal surfaces which contain active electronics, electrode sites, and conducting traces. As a result, a vast majority of today's silicon devices leave their vertical sidewalls exposed without protection. In this work, we investigated two batch-process silicon dioxide deposition methods separately and in combination: atomic layer deposition and inductively-coupled plasma chemical vapor deposition. We then utilized a rapid soak test involving potassium hydroxide to evaluate the coverage quality of each protection strategy. Focused ion beam cross sectioning, scanning electron microscopy, and 3D extrapolation enabled us to characterize and quantify the effectiveness of the deposition methods. Results showed that bare silicon sidewalls suffered the most dissolution whereas ALD silicon dioxide provided the best protection, demonstrating its effectiveness as a promising batch process technique to mitigate silicon sidewall corrosion in chronic applications.

Optical clearing reveals TNBS-induced morphological changes of VGLUT2-positive nerve fibers in mouse colorectum.

Colorectal hypersensitivity and sensitization of both mechanosensitive and mechanically insensitive afferents develop after intracolonic instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) in the mouse, a model of postinfectious irritable bowel syndrome. In mice in which ∼80% of extrinsic colorectal afferents were labeled genetically using the promotor for vesicular glutamate transporter type 2 (VGLUT2), we systematically quantified the morphology of VGLUT2-positive axons in mouse colorectum 7-28 days following intracolonic TNBS treatment. After removal, the colorectum was distended (20 mmHg), fixed with paraformaldehyde, and optically cleared to image VGLUT2-positive axons throughout the colorectal wall thickness. We conducted vector path tracing of individual axons to allow systematic quantification of nerve fiber density and shape. Abundant VGLUT2-positive nerve fibers were present in most layers of the colorectum, except the serosal and longitudinal muscular layers. A small percentage of VGLUT2-positive myenteric plexus neurons was also detected. Intracolonic TNBS treatment significantly reduced the number of VGLUT2-positive nerve fibers in submucosal, myenteric plexus, and mucosal layers at day 7 post-TNBS, which mostly recovered by day 28. We also found that almost all fibers in the submucosa were meandering and curvy, with ∼10% showing pronounced curviness (quantified by the linearity index). TNBS treatment resulted in a significant reduction of the proportions of pronounced curvy fibers in the rectal region at 28 days post-TNBS. Altogether, the present morphological study reveals profound changes in the distribution of VGLUT2-positive fibers in mouse colorectum undergoing TNBS-induced colitis and draws attention to curvy fibers in the submucosa with potential roles in visceral nociception.NEW & NOTEWORTHY We conducted genetic labeling and optical clearing to visualize extrinsic sensory nerve fibers in whole-mount colorectum, which revealed widespread presence of axons in the submucosal layer. Remarkably, axons in the submucosa were meandering and curvy, in contrast to axons in other layers generally aligned with the basal tissues. Intracolonic TNBS treatment led to pronounced changes of nerve fiber density and curviness, suggesting nerve fiber morphologies as potentially contributing factors to sensory sensitization.

Intraspinal stimulation with a silicon-based 3D chronic microelectrode array for bladder voiding in cats.

Objective.Bladder dysfunction is a significant and largely unaddressed problem for people living with spinal cord injury (SCI). Intermittent catheterization does not provide volitional control of micturition and has numerous side effects. Targeted electrical microstimulation of the spinal cord has been previously explored for restoring such volitional control in the animal model of experimental SCI. Here, we continue the development of the intraspinal microstimulation array technology to evaluate its ability to provide more focused and reliable bladder control in the feline animal model.Approach.For the first time, a mechanically robust intraspinal multisite silicon array was built using novel microfabrication processes to provide custom-designed tip geometry and 3D electrode distribution. Long-term implantation was performed in eight spinally intact animals for a period up to 6 months, targeting the dorsal gray commissure area in the S2 sacral cord that is known to be involved in the coordination between the bladder detrusor and the external urethral sphincter.Main results.About one third of the electrode sites in the that area produced micturition-related responses. The effectiveness of stimulation was further evaluated in one of eight animals after spinal cord transection (SCT). We observed increased bladder responsiveness to stimulation starting at 1 month post-transection, possibly due to supraspinal disinhibition of the spinal circuitry and/or hypertrophy and hyperexcitability of the spinal bladder afferents.Significance. 3D intraspinal microstimulation arrays can be chronically implanted and provide a beneficial effect on the bladder voiding in the intact spinal cord and after SCT. However, further studies are required to assess longer-term reliability and safety of the developed intraspinal microstimulation array prior to eventual human translation.

Gelatin embedding and LED autofluorescence reduction for rodent spinal cord histology.

We present two innovations in histological technique for rodent spinal cord: gelatin embedding and LED photobleaching. Gelatin embedding uses liquid gelatin solution to permeate delicate biological structures then solidify to provide mechanical support throughout dissection, vibratome sectioning, and staining. LED photobleaching uses high-intensity visible light during blocking and primary incubations to reduce autofluorescence in tissue sections before fluorescent secondaries are added. We found gelatin embedding improved mechanical stability without interfering with immunohistochemical staining. Gelatin embedding also preserved some spinal roots and provided an opportunity for dye-less and cut-less tracking of left/right orientation during free-floating staining, which is valuable for tissue samples that have no spare areas that can be marked. LED photobleaching greatly reduced autofluorescence and added essentially no extra time or labor to the process. Descriptions of the techniques and characterization data are provided.

Fabrication and modeling of recessed traces for silicon-based neural microelectrodes.

OBJECTIVE: Chronically-implanted neural microelectrodes are powerful tools for neuroscience research and emerging clinical applications, but their usefulness is limited by their tendency to fail after months in vivo. One failure mode is the degradation of insulation materials that protect the conductive traces from the saline environment. APPROACH: Studies have shown that material degradation is accelerated by mechanical stresses, which tend to concentrate on raised topographies such as conducting traces. Therefore, to avoid raised topographies, we developed a fabrication technique that recesses (buries) the traces in dry-etched, self-aligned trenches. MAIN RESULTS: The fabrication technique produced flatness within approximately 15 nm. Finite element modeling showed that the recessed geometry would be expected to reduce intrinsic stress concentrations in the insulation layers. Finally, in vitro electrochemical tests confirmed that recessed traces had robust recording and stimulation capabilities that were comparable to an established non-recessed device design. SIGNIFICANCE: Our recessed trace fabrication technique requires no extra masks, is easy to integrate with existing processes, and is likely to improve the long-term performance of implantable neural devices.

Extracellular single-unit recordings from peripheral nerve axons in vitro by a novel multichannel microelectrode array.

The peripheral nervous system (PNS) is an attractive target for modulation of afferent input (e.g., nociceptive input signaling tissue damage) to the central nervous system. To advance mechanistic understanding of PNS neural encoding and modulation requires single-unit recordings from individual peripheral neurons or axons. This is challenged by multiple connective tissue layers surrounding peripheral nerve fibers that prevent electrical recordings by existing electrodes or electrode arrays. In this study, we developed a novel microelectrode array (MEA) via silicon-based microfabrication that consists of 5 parallel hydrophilic gold electrodes surrounded by silanized hydrophobic surfaces. This novel hydrophilic/hydrophobic surface pattern guides the peripheral nerve filaments to self-align towards the hydrophilic electrodes, which dramatically reduces the technical challenges in conducting single-unit recordings. We validated our MEA by recording simultaneous single-unit action potentials from individual axons in mouse sciatic nerves, including both myelinated A-fibers and unmyelinated C-fibers. We confirmed that our recordings were single units from individual axons by increasing nerve trunk electrical stimulus intensity, which did not alter the spike shape or amplitude. By reducing the technical challenges, our novel MEA will likely allow peripheral single-unit recordings to be adopted by a larger research community and thus expedite our mechanistic understanding of peripheral neural encoding and modulation.

3D Reconstruction of the Intracortical Volume Around a Hybrid Microelectrode Array.

Extensive research using penetrating electrodes implanted in the central and peripheral nervous systems has been performed for many decades with significant advances made in recent years. While penetrating devices provide proximity to individual neurons in vivo, they suffer from declining performance over the course of months and often fail within a year. 2D histology studies using serial tissue sections have been extremely insightful in identifying and quantifying factors such as astroglial scar formation and neuronal death around the implant sites that may be contributing to failures. However, 2D histology has limitations in providing a holistic picture of the problems occurring at the electrode-tissue interface and struggles to analyze tissue below the electrode tips where the electrode tracks are no longer visible. In this study, we present 3D reconstruction of serial sections to overcome the limitations of 2D histological analysis. We used a cohort of software: XuvStitch, AutoAligner, and Imaris coupled with custom MATLAB programming to correct warping effects. Once the 3D image volume was reconstructed, we were able to use Imaris to quantify neuronal densities around the electrode tips of a hybrid microelectrode array incorporating Blackrock, Microprobes, and NeuroNexus electrodes in the same implant. This paper presents proof-of-concept and detailed methodological description of a technique which can be used to quantify neuronal densities in future studies of implanted electrodes.

Recessed Traces for Planarized Passivation of Chronic Neural Microelectrodes.

Implantable microfabricated neural electrodes have numerous neuroscientific research and clinical applications. However, these devices are prone to failure after several months in vivo. One mechanism is failure of passivation layers followed by corrosion of metal traces in the saline environment. It has been suggested that mechanical stress accelerates passivation layer failure and that stress is concentrated whenever passivation layers have a non-planar topography. Therefore, we developed a simple process for recessing metal traces within the substrate so that overlying passivation layers are planar. The process requires no extra masks and no post-passivation planarization steps.

A Wireless Neurostimulator System with an Embedded ARM™ Microprocessor.

In this study, a novel multi-layer printed circuit board (PCB)-based neurostimulator system with an embedded microprocessor is presented for applications in neuroprosthesis. The system integrates rechargeable batteries, a power management block, adjustable constant-current waveforms, voltage transient monitoring, and evoked neural response recording. The system can be configured to select channels among the 16 stimulation channels via Bluetooth communication wirelessly. Bench top measurements demonstrated that the system generated biphasic current waveforms with various stimulation parameters with approximately 407 mW of power consumption. Additional testing and validation with microelectrodes are underway.

Responses of neurons in the feline inferior colliculus to modulated electrical stimuli applied on and within the ventral cochlear nucleus; Implications for an advanced auditory brainstem implant.

Auditory brainstem implants (ABIs) can restore useful hearing to persons with deafness who cannot benefit from cochlear implants. However, the quality of hearing restored by ABIs rarely is comparable to that provided by cochlear implants in persons for whom those are appropriate. In an animal model, we evaluated elements of a prototype of an ABI in which the functions of macroelectrodes on the surface of the dorsal cochlear nucleus would be integrated with the function of multiple penetrating microelectrodes implanted into the ventral cochlear nucleus. The surface electrodes would convey most of the range of loudness percepts while the intranuclear microelectrodes would sharpen and focus pitch percepts. In the present study, stimulating electrodes were implanted chronically on the surface of the animal's dorsal cochlear nucleus (DCN) and also within their ventral cochlear nucleus (VCN). Recording microelectrodes were implanted into the central nucleus of the inferior colliculus (ICC). The electrical stimuli were sinusoidally modulated stimulus pulse trains applied on the DCN and within the VCN. Temporal encoding of neuronal responses was quantified as vector strength (VS) and as full-cycle rate of neuronal activity in the ICC. VS and full-cycle AP rate were measured for 4 stimulation modes; continuous and transient amplitude modulation of the stimulus pulse trains, each delivered via the macroelectrode on the surface of the DCN and then by the intranuclear penetrating microelectrodes. In the proposed clinical device the functions of the surface and intranuclear microelectrodes could best be integrated if there is minimal variation in the neuronal responses across the range of modulation depth, modulation frequencies, and across the four stimulation modes. In this study VS did vary as much as 34% across modulation frequency and modulation depth within a stimulation mode, and up to 40% between modulation modes. However, these intra- and inter-mode variances differed for different stimulation rates, and at 500 Hz the inter-mode differences in VS and across the range of modulation frequencies and modulation depths was = 24% and the intra-modal differences were = 15%. The findings were generally similar for rate encoding of modulation depth, although the depth of transient amplitude modulation delivered by the surface electrode was weakly encoded as full-cycle rate. Overall, our findings support the concept of a clinical ABI that employs surface stimulation and intranuclear microstimulation in an integrated manner.

A multispectral LED array for the reduction of background autofluorescence in brain tissue.

The presence of fixative-induced and cellular-derived artifactual autofluorescences (AAFs) presents a challenge in histological analysis involving immunofluorescence. We have established a simple and highly effective method for the reduction of AAFs that are ubiquitous in fixed mammalian brain and other tissues. A compact AAF-quenching photo-irradiation device was constructed using a commercially available light emitting diode (LED) array, cooling unit, and supporting components. The LED panel is comprised of an array of multispectral high intensity LEDs which serve as the illumination source for the photo-irradiation process. Rabbit and cat brain specimens of 5 µm- and 40 µm-thicknesses, respectively, were photo-irradiated for various durations. The AAFs were reduced to near tissue background levels after 24h of treatment for both deparaffinized and paraffinized rabbit brain specimens, and for the free-floating cat brain specimens. Subsequent immunofluorescence staining using primary antibodies against GFAP, NeuN, Iba-1, and MAP-2, and the corresponding Qdot(®) and Alexafluor(®) fluoroconjugates confirmed that the LED photo-irradiation treatment did not compromise the efficiency of the immunofluorescence labeling. The use of the device is not labor intensive, and only requires minimal tissue processing and experimental set-up time, with very low maintenance and operating costs. Finally, multiple specimens, in both slide and well-plate format, can be simultaneously photo-irradiated, thus, allowing for scalable batch processing.

Encoding of the amplitude modulation of pulsatile electrical stimulation in the feline cochlear nucleus by neurons in the inferior colliculus; effects of stimulus pulse rate.

OBJECTIVES: Persons without a functional auditory nerve cannot benefit from cochlear implants, but some hearing can be restored by an auditory brainstem implant (ABI) with stimulating electrodes implanted on the surface of the cochlear nucleus (CN). Most users benefit from their ABI, but speech recognition tends to be poorer than for users of cochlear implants. Psychophysical studies suggest that poor modulation detection may contribute to the limited performance of ABI users. In a cat model, we determined how the pulse rate of the electrical stimulus applied within or on the CN affects temporal and rate encoding of amplitude modulation (AM) by neurons in the central nucleus of the inferior colliculus (ICC). APPROACH: Stimulating microelectrodes were implanted chronically in and on the cats' CN, and multi-site recording microelectrodes were implanted chronically into the ICC. Encoding of AM pulse trains by neurons in the ICC was characterized as vector strength (VS), the synchrony of neural activity with the AM, and as the mean rate of neuronal action potentials (neuronal spike rate (NSR)). MAIN RESULTS: For intranuclear microstimulation, encoding of AM as VS was up to 3 dB greater when stimulus pulse rate was increased from 250 to 500 pps, but only for neuronal units with low best acoustic frequencies, and when the electrical stimulation was modulated at low frequencies (10-20 Hz). For stimulation on the surface of the CN, VS was similar at 250 and 500 pps, and the dynamic range of the VS was reduced for pulse rates greater than 250 pps. Modulation depth was encoded strongly as VS when the maximum stimulus amplitude was held constant across a range of modulation depth. This 'constant maximum' protocol allows enhancement of modulation depth while preserving overall dynamic range. However, modulation depth was not encoded as strongly as NSR. SIGNIFICANCE: The findings have implications for improved sound processors for present and future ABIs. The performance of ABIs may benefit from using pulse rates greater than those presently used in most ABIs, and by sound processing strategies that enhance the modulation depth of the electrical stimulus while preserving dynamic range.