Significantly reduced inflammatory foreign-body-response to neuroimplants and improved recording performance in young compared to adult rats.

The multicellular inflammatory encapsulation of implanted intracortical multielectrode arrays (MEA) is associated with severe deterioration of their field potentials' (FP) recording performance, which thus limits the use of brain implants in basic research and clinical applications. Therefore, extensive efforts have been made to identify the conditions in which the inflammatory foreign body response (FBR) is alleviated, or to develop methods to mitigate the formation of the inflammatory barrier. Here, for the first time, we show that (1) in young rats (74±8 gr, 4 weeks old at the onset of the experiments), cortical tissue recovery following MEA implantation proceeds with ameliorated inflammatory scar as compared to adult rats (242 ± 18 gr, 9 weeks old at the experimental onset); (2) in contrast to adult rats in which the Colony Stimulating factor 1 Receptor (CSF1R) antagonist chow eliminated ∼95% of the cortical microglia but not microglia adhering to the implant surfaces, in young rats the microglia adhering to the implant were eliminated along with the parenchymal microglia population. The removal of microglia adhering to the implant surfaces was correlated with improved recording performance by in-house fabricated Perforated Polyimide MEA Platforms (PPMP). These results support the hypothesis that microglia adhering to the surface of the electrodes, rather than the multicellular inflammatory scar, is the major underlying mechanism that deteriorates implant recording performance, and that young rats provide an advantageous model to study months-long, multisite electrophysiology in freely behaving rats. STATEMENT OF SIGNIFICANCE: Multisite electrophysiological recordings and stimulation devices play central roles in basic brain research and medical applications. The insertion of multielectrode-array platforms into the brain's parenchyma unavoidably injures the tissue, and initiates a multicellular inflammatory cascade culminating in the formation of an encapsulating scar tissue (the foreign body response-FBR). The dominant view, which directs most current research efforts to mitigate the FBR, holds that the FBR is the major hurdle to effective electrophysiological use of neuroprobes. By contrast, this report demonstrates that microglia adhering to the surface of a neuroimplants, rather than the multicellular FBR, underlie the performance deterioration of neuroimplants. These findings pave the way to the development of novel and focused strategies to overcome the functional deterioration of neuroimplants.

Recording of cellular physiological histories along optically readable self-assembling protein chains.

Observing cellular physiological histories is key to understanding normal and disease-related processes. Here we describe expression recording islands-a fully genetically encoded approach that enables both continual digital recording of biological information within cells and subsequent high-throughput readout in fixed cells. The information is stored in growing intracellular protein chains made of self-assembling subunits, human-designed filament-forming proteins bearing different epitope tags that each correspond to a different cellular state or function (for example, gene expression downstream of neural activity or pharmacological exposure), allowing the physiological history to be read out along the ordered subunits of protein chains with conventional optical microscopy. We use expression recording islands to record gene expression timecourse downstream of specific pharmacological and physiological stimuli in cultured neurons and in living mouse brain, with a time resolution of a fraction of a day, over periods of days to weeks.

Ultrastructural Analysis of Neuroimplant-Parenchyma Interfaces Uncover Remarkable Neuroregeneration Along-With Barriers That Limit the Implant Electrophysiological Functions.

Despite increasing use of in vivo multielectrode array (MEA) implants for basic research and medical applications, the critical structural interfaces formed between the implants and the brain parenchyma, remain elusive. Prevailing view assumes that formation of multicellular inflammatory encapsulating-scar around the implants [the foreign body response (FBR)] degrades the implant electrophysiological functions. Using gold mushroom shaped microelectrodes (gMµEs) based perforated polyimide MEA platforms (PPMPs) that in contrast to standard probes can be thin sectioned along with the interfacing parenchyma; we examined here for the first time the interfaces formed between brains parenchyma and implanted 3D vertical microelectrode platforms at the ultrastructural level. Our study demonstrates remarkable regenerative processes including neuritogenesis, axon myelination, synapse formation and capillaries regrowth in contact and around the implant. In parallel, we document that individual microglia adhere tightly and engulf the gMµEs. Modeling of the formed microglia-electrode junctions suggest that this configuration suffice to account for the low and deteriorating recording qualities of in vivo MEA implants. These observations help define the anticipated hurdles to adapting the advantageous 3D in vitro vertical-electrode technologies to in vivo settings, and suggest that improving the recording qualities and durability of planar or 3D in vivo electrode implants will require developing approaches to eliminate the insulating microglia junctions.

Inflammatory Foreign Body Response Induced by Neuro-Implants in Rat Cortices Depleted of Resident Microglia by a CSF1R Inhibitor and Its Implications.

Inflammatory encapsulation of implanted cortical-neuro-probes [the foreign body response (FBR)] severely limits their use in basic brain research and in clinical applications. A better understanding of the inflammatory FBR is needed to effectively mitigate these critical limitations. Combining the use of the brain permeant colony stimulating factor 1 receptor inhibitor PLX5622 and a perforated polyimide-based multielectrode array platform (PPMP) that can be sectioned along with the surrounding tissue, we examined the contribution of microglia to the formation of inflammatory FBR. To that end, we imaged the inflammatory processes induced by PPMP implantations after eliminating 89-94% of the cortical microglia by PLX5622 treatment. The observations showed that: (I) inflammatory encapsulation of implanted PPMPs proceeds by astrocytes in microglia-free cortices. The activated astrocytes adhered to the PPMP's surfaces. This suggests that the roles of microglia in the FBR might be redundant. (II) PPMP implantation into control or continuously PLX5622-treated rats triggered a localized surge of microglia mitosis. The daughter cells that formed a "cloud" of short-lived (T 1 / 2 ≤ 14 days) microglia around and in contact with the implant surfaces were PLX5622 insensitive. (III) Neuron degeneration by PPMP implantation and the ensuing recovery in time, space, and density progressed in a similar manner in the cortices following 89-94% depletion of microglia. This implies that microglia do not serve a protective role with respect to the neurons. (IV) Although the overall cell composition and dimensions of the encapsulating scar in PLX5622-treated rats differed from the controls, the recorded field potential (FP) qualities and yield were undistinguishable. This is accounted for by assuming that the FP amplitudes in the control and PLX5622-treated rats were related to the seal resistance formed at the interface between the adhering microglia and/or astrocytes and the PPMP platform rather than across the scar tissue. These observations suggest that the prevention of both astrocytes and microglia adhesion to the electrodes is required to improve FP recording quality and yield.

Immunohistological and Ultrastructural Study of the Inflammatory Response to Perforated Polyimide Cortical Implants: Mechanisms Underlying Deterioration of Electrophysiological Recording Quality.

The deterioration of field potential (FP) recording quality and yield by in vivo multielectrode arrays (MEA) within days to weeks of implantation severely limits progress in basic and applied brain research. The prevailing hypothesis is that implantation of MEA platforms initiate and perpetuate inflammatory processes which culminate in the formation of scar tissue (the foreign body response, FBR) around the implant. The FBR leads to progressive degradation of the recording qualities by displacing neurons away from the electrode surfaces, increasing the resistance between neurons (current source) and the sensing pads and by reducing the neurons' excitable membrane properties and functional synaptic connectivity through the release of pro-inflammatory cytokines. Meticulous attempts to causally relate the cellular composition, cell density, and electrical properties of the FBR have failed to unequivocally correlate the deterioration of recording quality with the histological severity of the FBR. Based on confocal and electron microscope analysis of thin sections of polyimide based MEA implants along with the surrounding brain tissue at different points in time after implantation, we propose that abrupt FP amplitude attenuation occurs at the implant/brain-parenchyma junction as a result of high seal resistance insulation formed by adhering microglia to the implant surfaces. In contrast to the prevailing hypothesis, that FP decrease occurs across the encapsulating scar of the implanted MEA, this mechanism potentially explains why no correlations have been found between the dimensions and density of the FBR and the recording quality. Recognizing that the seal resistance formed by adhering-microglia to the implant constitutes a downstream element undermining extracellular FP recordings, suggests that approaches to mitigate the formation of the insulating glial could lead to improved recording quality and yield.

Multisite Intracellular Recordings by MEA.

The enormous advances made over the last 50 years in materials science, microelectronics, and nanoelectronics, together with the acknowledgment that substrate-integrated planar multielectrode arrays (MEA) are limited to recording of extracellular field potentials (FPs) rather than the entire electrophysiological signaling repertoire of the brain, have prompted a number of laboratories to merge the advantages of planar MEA technologies (non-damaging and durable) with those of the classical sharp and patch electrodes for intracellular recordings. Unlike extracellular planar electrode-based MEAs, the new generation of three-dimensional (3D) vertical nanoelectrodes are designed to functionally penetrate the plasma membrane of cultured cells and operate in a similar manner to classical intracellular microelectrodes. Although only approximately 10 years has elapsed since the development of the first vertical 3D nanostructure-based MEAs, this technology has progressed to enable recordings of attenuated intracellular action potentials (APs) and synaptic potentials from individual neurons, cardiomyocytes, and striated myotubes. Furthermore, recently the scaling advantages of nanochip/microchip fabrication technologies enabled simultaneously intracellular recordings of APs from hundreds of cultured cardiomyocytes, thus heralding a new milestone in MEA technology.In this chapter we present the earliest and today's cutting-edge achievements of this "young vertical nano-sensors MEA technology" at the single-cell and network levels, explain the biophysical principles and the various configurations used to form functional nanoelectrode/cell hybrids, and describe the quality and characteristic features of the recorded intracellular APs and subthreshold synaptic potentials by the vertical nanoelectrode-based MEA. Basic cell-biological mechanisms that curtail the length of time intracellular access by the nanoelectrodes are discussed, and approaches to overcome this problem are offered.Recent development of biotechnologies that use induced human pluripotent stem cells taken from healthy subjects and patients, and in vitro drug screening for the development of personalized medicine as well as basic brain research will benefit tremendously from the use of MEAs that record the entire brain electrophysiological signaling repertoire from individual cells within an operational network rather than only extracellular FPs.

Multisite Attenuated Intracellular Recordings by Extracellular Multielectrode Arrays, a Perspective.

Multielectrode arrays (MEA) are used extensively for basic and applied electrophysiological research of neuronal- and cardiomyocyte-networks. Whereas immense progress has been made in realizing sophisticated MEA platforms of thousands of addressable, high-density, small diameter, low impedance sensors, the quality of the interfaces formed between excitable cells and classical planar sensor has not improved. As a consequence in vitro and in vivo MEA are "blind" to the rich and important "landscape" of sub-threshold synaptic potentials generated by individual neurons. Disregarding this essential fraction of network signaling repertoire has become the standard and almost the "scientific ideology" of MEA users. To overcome the inherent limitations of substrate integrated planar MEA platforms that only record extracellular field potentials, a number of laboratories have developed in vitro MEA for intracellular recordings. Most of these novel devices use vertical nano-rods or nano-wires that penetrate the plasma membrane of cultured cells and record the electrophysiological signaling in a manner similar to sharp intracellular microelectrodes. In parallel, our laboratory began to develop a bioinspired approach in-which cell biological energy resources are harnessed to self-force a cell into intimate contact with extracellular gold mushroom-shaped microelectrodes to record attenuated synaptic- and action-potentials with characteristic features of intracellular recordings. Here we describe some of the experiments that helped evolve the approach and elaborate on the biophysical principles that make it possible to record intracellular potentials by an array of extracellular electrode. We illustrate the qualities and limitations of the method and discuss prospects for further improvement of this technology.

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes.

In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMµE) based MEA tightly engulf the gMµEs, forming a high seal resistance between the myotubes and the gMµEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5-0.9 V voltage pulse through the gMµEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10-30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMµE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine.

Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.

Substrate integrated planar microelectrode arrays is the "gold standard" method for millisecond-resolution, long-term, large-scale, cell-noninvasive electrophysiological recordings from mammalian neuronal networks. Nevertheless, these devices suffer from drawbacks that are solved by spike-detecting, spike-sorting and signal-averaging techniques which rely on estimated parameters that require user supervision to correct errors, merge clusters and remove outliers. Here we show that primary rat hippocampal neurons grown on micrometer sized gold mushroom-shaped microelectrodes (gMµE) functionalized simply by poly-ethylene-imine/laminin undergo self-assembly processes to form loose patch-like hybrid structures. More than 90% of the hybrids formed in this way record monophasic positive action potentials (APs). Of these, 34.5% record APs with amplitudes above 300 µV and up to 5,085 µV. This self-assembled neuron-gMµE configuration improves the recording quality as compared to planar MEA. This study characterizes and analyzes the electrophysiological signaling repertoire generated by the neurons-gMµE configuration, and discusses prospects to further improve the technology.

A feasibility study of multi-site,intracellular recordings from mammalian neurons by extracellular gold mushroom-shaped microelectrodes.

The development of multi-electrode array platforms for large scale recording of neurons is at the forefront of neuro-engineering research efforts. Recently we demonstrated, at the proof-of-concept level, a breakthrough neuron-microelectrode interface in which cultured Aplysia neurons tightly engulf gold mushroom-shaped microelectrodes (gMµEs). While maintaining their extracellular position, the gMµEs record synaptic- and action-potentials with characteristic features of intracellular recordings. Here we examined the feasibility of using gMµEs for intracellular recordings from mammalian neurons. To that end we experimentally examined the innate size limits of cultured rat hippocampal neurons to engulf gMµEs and measured the width of the "extracellular" cleft formed between the neurons and the gold surface. Using the experimental results we next analyzed the expected range of gMµEs-neuron electrical coupling coefficients. We estimated that sufficient electrical coupling levels to record attenuated synaptic- and action-potentials can be reached using the gMµE-neuron configuration. The definition of the engulfment limits of the gMµEs caps diameter at ≤2-2.5 µm and the estimated electrical coupling coefficients from the simulations pave the way for rational development and application of the gMµE based concept for in-cell recordings from mammalian neurons.