Soft, Conductive, Brain-Like, Coatings at Tips of Microelectrodes Improve Electrical Stability under Chronic, In Vivo Conditions.

Several recent studies have reported improved histological and electrophysiological outcomes with soft neural interfaces that have elastic moduli ranging from 10 s of kPa to hundreds of MPa. However, many of these soft interfaces use custom fabrication processes. We test the hypothesis that a readily adoptable fabrication process for only coating the tips of microelectrodes with soft brain-like (elastic modulus of ~5 kPa) material improves the long-term electrical performance of neural interfaces. Conventional tungsten microelectrodes (n = 9 with soft coatings and n = 6 uncoated controls) and Pt/Ir microelectrodes (n = 16 with soft coatings) were implanted in six animals for durations ranging from 5 weeks to over 1 year in a subset of rats. Electrochemical impedance spectroscopy was used to assess the quality of the brain tissue-electrode interface under chronic conditions. Neural recordings were assessed for unit activity and signal quality. Electrodes with soft, silicone coatings showed relatively stable electrical impedance characteristics over 6 weeks to >1 year compared to the uncoated control electrodes. Single unit activity recorded by coated electrodes showed larger peak-to-peak amplitudes and increased number of detectable neurons compared to uncoated controls over 6-7 weeks. We demonstrate the feasibility of using a readily translatable process to create brain-like soft interfaces that can potentially overcome variable performance associated with chronic rigid neural interfaces.

Biomechanical micromotion at the neural interface modulates intracellular membrane potentialsin vivo.

Objective. Respiration and vascular pulsation cause relative micromotion of brain tissue against stationary implants resulting in repetitive displacements of 2-4µm (due to vascular pulsation) and 10-30µm (due to breathing) in rats. However, the direct functional impact of such tissue micromotion on the cells at the neural interface remains unknown. This study aims to test the hypothesis that micromotion in brain tissue causes changes in membrane potentials (MPs) through the activation of mechanosensitive ion channels.Approach. Intracellular MPs were recorded from Aplysia ganglion cells (n= 8) and cortical cells (n= 15)in vivoinn= 7 adult rats. Cyclic stresses between 0.2 and 4 kPa repeated at 1 Hz were tested in Aplysia ganglion cells. For thein vivoexperiments, 30µM of gadolinium chloride (Gd3+), a non-selective blocker of mechanosensitive ion channels, was used to assess the role of such ion channels.Main results. In Aplysia ganglion cells, there were no MP changes for <1.5 kPa, and action potentials were observed at >3.1 kPa. Drug studies utilizing 5-HT showed an 80% reduction in firing frequency from controls. Inin vivoexperiments, periodic pulsations (1-10 mV) were observed in the MPs of cells that corresponded to breathing and heart-rate. In response to the addition of 30µM Gd3+, we observed a significant reduction (0.5-3 mV) in the periodic pulsations in MP in all cortical cells across four different rats, suggesting the role of mechanosensitive ion channels in mediating MP fluctuations due to tissue micromotion at the neural interface.Significance.Under chronic conditions, the tissue at the interface stiffens due to scar tissue formation, which is expected to increase the likelihood of recruiting stretch-receptors due to tissue micromotion. It is speculated that such chronic sub-threshold pulsations in MPs might trigger the immune response at the neural interface.

Optogenetic modulation of cortical neurons using organic light emitting diodes (OLEDs).

OBJECTIVE: There is a need for low power, scalable photoelectronic devices and systems for emerging optogenetic needs in neuromodulation. Conventional light emitting diodes (LEDs) are constrained by power and lead-counts necessary for scalability. Organic LEDs (OLEDs) offer an exciting approach to decrease power and lead-counts while achieving high channel counts on thin, flexible substrates that conform to brain surfaces or peripheral neuronal fibers. In this study, we investigate the potential for using OLEDs to modulate neuronal networks cultured in vitro on a transparent microelectrode array (MEA) and subsequently validate neurostimulation in vivo in a transgenic mouse model. APPROACH: Cultured mouse cortical neurons were transfected with light-sensitive opsins such as blue-light sensitive channel-rhodopsin (ChR2) and green-light sensitive chimeric channel-rhodopsin (C1V1tt) and stimulated using blue and green OLEDs (with 455 and 520 nm peak emission spectra respectively) at a power of ~1 mW mm-2 under pulsed conditions. MAIN RESULTS: We demonstrate neuromodulation and optostimulus-locked, single unit-neuronal activity in neurons expressing stimulating opsins (34 units on n = 4 MEAs, each with 16 recordable channels). We also validated the optostimulus-locked response in preliminary experiments in a channel-rhodopsin expressing transgenic mouse model, where at least three isolatable single neuronal cortical units respond to OLED stimulation. SIGNIFICANCE: The above results indicate the feasibility of generating sufficient luminance from OLEDs to perform neuromodulation both in vitro and in vivo. This opens up the possibility of developing thin, flexible OLED films with multiple stimulation sites that can conform to the shape of the neuronal targets in the brain or the peripheral nervous system. However, stability of these OLEDs under chronic conditions still needs to be carefully assessed with appropriate packaging approaches.

Remote Stimulation of Sciatic Nerve Using Cuff Electrodes and Implanted Diodes.

We demonstrate a method of neurostimulation using implanted, free-floating, inter-neural diodes. They are activated by volume-conducted, high frequency, alternating current (AC) fields and address the issue of instability caused by interconnect wires in chronic nerve stimulation. The aim of this study is to optimize the set of AC electrical parameters and the diode features to achieve wireless neurostimulation. Three different packaged Schottky diodes (1.5 mm, 500 µm and 220 µm feature sizes) were tested in vivo (n = 17 rats). A careful assessment of sciatic nerve activation as a function of diode⁻dipole lengths and relative position of the diode was conducted. Subsequently, free-floating Schottky microdiodes were implanted in the nerve (n = 3 rats) and stimulated wirelessly. Thresholds for muscle twitch responses increased non-linearly with frequency. Currents through implanted diodes within the nerve suffer large attenuations (~100 fold) requiring 1⁻2 mA drive currents for thresholds at 17 µA. The muscle recruitment response using electromyograms (EMGs) is intrinsically steep for subepineurial implants and becomes steeper as diode is implanted at increasing depths away from external AC stimulating electrodes. The study demonstrates the feasibility of activating remote, untethered, implanted microscale diodes using external AC fields and achieving neurostimulation.

Compliant intracortical implants reduce strains and strain rates in brain tissue in vivo.

OBJECTIVE: The objective of this research is to characterize the mechanical interactions of (1) soft, compliant and (2) non-compliant implants with the surrounding brain tissue in a rodent brain. Understanding such interactions will enable the engineering of novel materials that will improve stability and reliability of brain implants. APPROACH: Acute force measurements were made using a load cell in n = 3 live rats, each with 4 craniotomies. Using an indentation method, brain tissue was tested for changes in force using established protocols. A total of 4 non-compliant, bare silicon microshanks, 3 non-compliant polyvinyl acetate (PVAc)-coated silicon microshanks, and 6 compliant, nanocomposite microshanks were tested. Stress values were calculated by dividing the force by surface area and strain was estimated using a linear stress-strain relationship. Micromotion effects from breathing and vascular pulsatility on tissue stress were estimated from a 5 s interval of steady-state measurements. Viscoelastic properties were estimated using a second-order Prony series expansion of stress-displacement curves for each shank. MAIN RESULTS: The distribution of strain values imposed on brain tissue for both compliant nanocomposite microshanks and PVAc-coated, non-compliant silicon microshanks were significantly lower compared to non-compliant bare silicon shanks. Interestingly, step-indentation experiments also showed that compliant, nanocomposite materials significantly decreased stress relaxation rates in the brain tissue at the interface (p < 0.05) compared to non-compliant silicon and PVAc-coated silicon materials. Furthermore, both PVAc-coated non-compliant silicon and compliant nanocomposite shanks showed significantly reduced (by 4-5 fold) stresses due to tissue micromotion at the interface. SIGNIFICANCE: The results of this study showed that soft, adaptive materials reduce strains and strain rates and micromotion induced stresses in the surrounding brain tissue. Understanding the material behavior at the site of tissue contact will help to improve neural implant design.

Long-term changes in the material properties of brain tissue at the implant-tissue interface.

OBJECTIVE: Brain tissue undergoes dramatic molecular and cellular remodeling at the implant-tissue interface that evolves over a period of weeks after implantation. The biomechanical impact of such remodeling on the interface remains unknown. In this study, we aim to assess the changes in the mechanical properties of the brain-electrode interface after chronic implantation of a microelectrode. APPROACH: Microelectrodes were implanted in the rodent cortex at a depth of 1 mm for different durations-1 day (n = 4), 10-14 days (n = 4), 4 weeks (n = 4) and 6-8 weeks (n = 7). After the initial duration of implantation, the microelectrodes were moved an additional 1 mm downward at a constant speed of 10 µm s(-1). Forces experienced by the microelectrode were measured during movement and after termination of movement. The biomechanical properties of the interfacial brain tissue were assessed from measured force-displacement curves using two separate models-a two-parameter Mooney-Rivlin hyperelastic model and a viscoelastic model with a second-order Prony series. MAIN RESULTS: Estimated shear moduli using a second-order viscoelastic model increased from 0.5-2.6 kPa (day 1 of implantation) to 25.7-59.3 kPa (after 4 weeks of implantation) and subsequently decreased to 0.8-7.9 kPa after 6-8 weeks of implantation in 6 of the 7 animals. The estimated elastic modulus increased from 4.1-7.8 kPa on the day of implantation to 24-44.9 kPa after 4 weeks. The elastic modulus was estimated to be 6.8-33.3 kPa in 6 of the 7 animals after 6-8 weeks of implantation. The above estimates suggest that the brain tissue surrounding the microelectrode evolves from a stiff matrix with maximal shear and elastic modulus after 4 weeks of implantation into a composite of two different layers with different mechanical properties-a stiff compact inner layer surrounded by softer brain tissue that is biomechanically similar to brain tissue-during the first week of implantation. Tissue micromotion-induced stresses on the microelectrode constituted 12-55% of the steady-state stresses on the microelectrode on the day of implantation (n = 4), 2-21% of the steady-state stresses after 4 weeks of implantation (n = 4), and 4-10% of the steady-state stresses after 6-8 weeks of implantation (n = 7). SIGNIFICANCE: Understanding biomechanical behavior at the brain-microelectrode interface is necessary for the long-term success of implantable neuroprosthetics and microelectrode arrays. Such quantitative physical characterization of the dynamic changes in the electrode-tissue interface will (a) drive the design and development of more mechanically optimal, chronic brain implants, and (b) lead to new insights into key cellular and molecular events such as neuronal adhesion, migration and function in the immediate vicinity of the brain implant.

Voltage Preconditioning Allows Modulated Gene Expression in Neurons Using PEI-complexed siRNA.

We present here a high efficiency, high viability siRNA-delivery method using a voltage-controlled chemical transfection strategy to achieve modulated delivery of polyethylenimine (PEI) complexed with siRNA in an in vitro culture of neuro2A cells and neurons. Low voltage pulses were applied to adherent cells before the administration of PEI-siRNA complexes. Live assays of neuro2a cells transfected with fluorescently tagged siRNA showed an increase in transfection efficiency from 62 ± 14% to 98 ± 3.8% (after -1 V). In primary hippocampal neurons, transfection efficiencies were increased from 30 ± 18% to 76 ± 18% (after -1 V). Negligible or low-level transfection was observed after preconditioning at higher voltages, suggesting an inverse relationship with applied voltage. Experiments with propidium iodide ruled out the role of electroporation in the transfection of siRNAs suggesting an alternate electro-endocytotic mechanism. In addition, image analysis of preconditioned and transfected cells demonstrates siRNA uptake and loading that is tuned to preconditioning voltage levels. There is approximately a fourfold increase in siRNA loading after preconditioning at -1 V compared with the same at ±2-3 V. Modulated gene expression is demonstrated in a functional knockdown of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in neuro2A cells using siRNA. Cell density and dendritic morphological changes are also demonstrated in modulated knockdown of brain derived neurotrophic factor (BDNF) in primary hippocampal neurons. The method reported here has potential applications in the development of high-throughput screening systems for large libraries of siRNA molecules involving difficult-to-transfect cells like neurons.Molecular Therapy-Nucleic Acids (2013) 2, e82; doi:10.1038/mtna.2013.10; published online 26 March 2013.

Packaging and Non-Hermetic Encapsulation Technology for Flip Chip on Implantable MEMS Devices.

We report here a successful demonstration of a flip-chip packaging approach for a microelectromechanical systems (MEMS) device with in-plane movable microelectrodes implanted in a rodent brain. The flip-chip processes were carried out using a custom-made apparatus that was capable of the following: 1) creating Ag epoxy microbumps for first-level interconnect; 2) aligning the die and the glass substrate; and 3) creating non-hermetic encapsulation (NHE). The completed flip-chip package had an assembled weight of only 0.5 g significantly less than the previously designed wire-bonded package of 4.5 g. The resistance of the Ag bumps was found to be negligible. The MEMS micro-electrodes were successfully tested for its mechanical movement with microactuators generating forces of 450 µN with a displacement resolution of 8.8 µm/step. An NHE on the front edge of the package was created by patterns of hydrophobic silicone microstructures to prevent contamination from cerebrospinal fluid while simultaneously allowing the microelectrodes to move in and out of the package boundary. The breakdown pressure of the NHE was found to be 80 cm of water, which is significantly (4.5-11 times) larger than normal human intracranial pressures. Bench top tests and in vivo tests of the MEMS flip-chip packages for up to 75 days showed reliable NHE for potential long-term implantation.

Adaptive movable neural interfaces for monitoring single neurons in the brain.

Implantable microelectrodes that are currently used to monitor neuronal activity in the brain in vivo have serious limitations both in acute and chronic experiments. Movable microelectrodes that adapt their position in the brain to maximize the quality of neuronal recording have been suggested and tried as a potential solution to overcome the challenges with the current fixed implantable microelectrodes. While the results so far suggest that movable microelectrodes improve the quality and stability of neuronal recordings from the brain in vivo, the bulky nature of the technologies involved in making these movable microelectrodes limits the throughput (number of neurons that can be recorded from at any given time) of these implantable devices. Emerging technologies involving the use of microscale motors and electrodes promise to overcome this limitation. This review summarizes some of the most recent efforts in developing movable neural interfaces using microscale technologies that adapt their position in response to changes in the quality of the neuronal recordings. Key gaps in our understanding of the brain-electrode interface are highlighted. Emerging discoveries in these areas will lead to success in the development of a reliable and stable interface with single neurons that will impact basic neurophysiological studies and emerging cortical prosthetic technologies.

Long-Term Neural Recordings Using MEMS Based Movable Microelectrodes in the Brain.

One of the critical requirements of the emerging class of neural prosthetic devices is to maintain good quality neural recordings over long time periods. We report here a novel MEMS (Micro Electro Mechanical Systems) based technology that can move microelectrodes in the event of deterioration in neural signal to sample a new set of neurons. Microscale electro-thermal actuators are used to controllably move microelectrodes post-implantation in steps of approximately 9 mum. In this study, a total of 12 movable microelectrode chips were individually implanted in adult rats. Two of the twelve movable microelectrode chips were not moved over a period of 3 weeks and were treated as control experiments. During the first 3 weeks of implantation, moving the microelectrodes led to an improvement in the average signal to noise ratio (SNR) from 14.61 +/- 5.21 dB before movement to 18.13 +/- 4.99 dB after movement across all microelectrodes and all days. However, the average root-mean-square values of noise amplitudes were similar at 2.98 +/- 1.22 muV and 3.01 +/- 1.16 muV before and after microelectrode movement. Beyond 3 weeks, the primary observed failure mode was biological rejection of the PMMA (dental cement) based skull mount resulting in the device loosening and eventually falling from the skull. Additionally, the average SNR for functioning devices beyond 3 weeks was 11.88 +/- 2.02 dB before microelectrode movement and was significantly different (p < 0.01) from the average SNR of 13.34 +/- 0.919 dB after movement. The results of this study demonstrate that MEMS based technologies can move microelectrodes in rodent brains in long-term experiments resulting in improvements in signal quality. Further improvements in packaging and surgical techniques will potentially enable movable microelectrodes to record cortical neuronal activity in chronic experiments.

Optoelectronic energy transfer at novel biohybrid interfaces using light harvesting complexes from Chloroflexus aurantiacus.

In nature, nanoscale supramolecular light harvesting complexes initiate the photosynthetic energy collection process at high quantum efficiencies. In this study, the distinctive antenna structure from Chloroflexus aurantiacusthe chlorosomeis assessed for potential exploitation in novel biohybrid optoelectronic devices. Electrochemical characterization of bacterial fragments containing intact chlorosomes with the photosynthetic apparatus show an increase in the charge storage density near the working electrode upon light stimulation and suggest that chlorosomes contribute approximately one-third of the overall photocurrent. Further, isolated chlorosomes (without additional photosynthetic components, e.g., reaction centers, biochemical mediators) produce a photocurrent (approximately 8-10 nA) under light saturation conditions. Correlative experiments indicate that the main chlorosome pigment, bacteriochlorophyll-c, contributes to the photocurrent via an oxidative mechanism. The results reported herein are the first to demonstrate that isolated chlorosomes (lipid-enclosed sacs of pigments) directly transduce light energy in an electrochemical manner, laying an alternative, biomimetic approach for designing photosensitized interfaces in biofuel cells and biomedical devices, such as bioenhanced retinal prosthetics.

Immobilization of functional light antenna structures derived from the filamentous green bacterium Chloroflexus aurantiacus.

The integration of highly efficient, natural photosynthetic light antenna structures into engineered systems while their biophotonic capabilities are maintained has been an elusive goal in the design of biohybrid photonic devices. In this study, we report a novel technique to covalently immobilize nanoscaled bacterial light antenna structures known as chlorosomes from Chloroflexus aurantiacus on both conductive and nonconductive glass while their energy transducing functionality was maintained. Chlorosomes without their reaction centers (RCs) were covalently immobilized on 3-aminoproyltriethoxysilane (APTES) treated surfaces using a glutaraldehyde linker. AFM techniques verified that the chlorosomes maintained their native ellipsoidal ultrastructure upon immobilization. Results from absorbance and fluorescence spectral analysis (where the Stokes shift to 808/810 nm was observed upon 470 nm blue light excitation) in conjunction with confocal microscopy confirm that the functional integrity of immobilized chlorosomes was also preserved. In addition, experiments with electrochemical impedance spectroscopy (EIS) suggested that the presence of chlorosomes in the electrical double layer of the electrode enhanced the electron transfer capacity of the electrochemical cell. Further, chronoamperometric studies suggested that the reduced form of the Bchl- c pigments found within the chlorosome modulate the conduction properties of the electrochemical cell, where the oxidized form of Bchl- c pigments impeded any current transduction at a bias of 0.4 V within the electrochemical cell. The results therefore demonstrate that the intact chlorosomes can be successfully immobilized while their biophotonic transduction capabilities are preserved through the immobilization process. These findings indicate that it is feasible to design biophotonic devices incorporating fully functional light antenna structures, which may offer significant performance enhancements to current silicon-based photonic devices for diverse technological applications ranging from CCD devices used in retinal implants to terrestrial and space fuel cell applications.