Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.
ABSTRACT: 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 (gM?E) 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??V and up to 5,085??V. This self-assembled neuron-gM?E configuration improves the recording quality as compared to planar MEA. This study characterizes and analyzes the electrophysiological signaling repertoire generated by the neurons-gM?E configuration, and discusses prospects to further improve the technology.
Project description: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 (gM?Es). While maintaining their extracellular position, the gM?Es record synaptic- and action-potentials with characteristic features of intracellular recordings. Here we examined the feasibility of using gM?Es for intracellular recordings from mammalian neurons. To that end we experimentally examined the innate size limits of cultured rat hippocampal neurons to engulf gM?Es 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 gM?Es-neuron electrical coupling coefficients. We estimated that sufficient electrical coupling levels to record attenuated synaptic- and action-potentials can be reached using the gM?E-neuron configuration. The definition of the engulfment limits of the gM?Es caps diameter at ?2-2.5??m and the estimated electrical coupling coefficients from the simulations pave the way for rational development and application of the gM?E based concept for in-cell recordings from mammalian neurons.
Project description:As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 microm thick and spaced 10 microm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electroporation, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.
Project description: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 (gM?E) based MEA tightly engulf the gM?Es, forming a high seal resistance between the myotubes and the gM?Es. 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 gM?Es 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 gM?E-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.
Project description:In this paper, a fine gold nanostructure synthesized on selective planar microelectrodes in micro-chip is realized by using an advanced hybrid fabrication approach incorporating growth of nanorods (NRs) with gold electroplating. By this developed nanostructure, integration of in-situ surface-enhanced Raman spectroscopy (SERS) detection with electrochemical impedance spectroscopy (EIS) measurement for label-free, nondestructive, real-time and rapid monitoring on a single cell has been achieved. Moreover, parameters of Au nanostructures such as size of nanoholes/nanogaps can be controllably adjusted in the fabrication. We have demonstrated a SERS enhancement factor of up to ~2.24 × 10(6) and double-layer impedance decrease ratio of 90% ~ 95% at low frequency range below 200 kHz by using nanostructured microelectrodes. SERS detection and in-situ EIS measurement of a trapped single cell by using planar microelectrodes are realized to demonstrate the compatibility, multi-functions, high-sensitivity and simplicity of the micro-chip system. This dual function platform integrating SERS and EIS is of great significance in biological, biochemical and biomedical applications.
Project description:OBJECTIVES:In this paper, we aim to detail the setup of a high spatio-temporal resolution, electrical recording system utilising planar microelectrode arrays with simultaneous optical imaging suitable for evaluating microelectrode performance with a proposed 'performance factor' metric. METHODS:Techniques that would facilitate low noise electrical recordings were coupled with voltage sensitive dyes and neuronal activity was recorded both electrically via a customised amplification system and optically via a high speed CMOS camera. This technique was applied to characterise microelectrode recording performance of gold and poly(3,4-ethylenedioxythiophene)/polystyrene sulfonate (PEDOT/PSS) coated electrodes through traditional signal to noise (SNR) calculations as well as the proposed performance factor. RESULTS:Neuronal activity was simultaneously recorded using both electrical and optical techniques and this activity was confirmed via tetrodotoxin application to inhibit action potential firing. PEDOT/PSS outperformed gold using both measurements, however, the performance factor metric estimated a 3 fold improvement in signal transduction when compared to gold, whereas SNR estimated an 8 fold improvement when compared to gold. CONCLUSION:The design and functionality of a system to record from neurons both electrically, through microelectrode arrays, and optically via voltage sensitive dyes was successfully achieved. SIGNIFICANCE:The high spatiotemporal resolution of both electrical and optical methods will allow for an array of applications such as improved detection of subthreshold synaptic events, validation of spike sorting algorithms and a provides a robust evaluation of extracellular microelectrode performance.
Project description:Glycosylphosphatidylinositols (GPIs) are lipid anchors allowing the exposure of proteins at the outer layer of the plasma membrane. In fungi, a number of GPI-anchored proteins (GPI-APs) are involved in the remodeling of the cell wall polymers. GPIs follow a specific biosynthetic pathway in the endoplasmic reticulum. After the transfer of the protein onto the GPI-anchor, a lipid remodeling occurs to substitute the diacylglycerol moiety by a ceramide. In addition to GPI-APs, A. fumigatus produces a GPI-anchored polysaccharide, the galactomannan (GM), that remains unique in the fungal kingdom. To investigate the role of the GPI pathway in the biosynthesis of the GM and cell wall organization, the deletion of PER1-coding for a phospholipase required for the first step of the GPI lipid remodeling-was undertaken. Biochemical characterization of the GPI-anchor isolated from GPI-APs showed that the PER1 deficient mutant produced a lipid anchor with a diacylglycerol. The absence of a ceramide on GPI-anchors in the ?per1 mutant led to a mislocation of GPI-APs and to an alteration of the composition of the cell wall alkali-insoluble fraction. On the other hand, the GM isolated from the ?per1 mutant membranes possesses a ceramide moiety as the parental strain, showing that GPI anchor of the GM follow a distinct unknown biosynthetic pathway.
Project description:Neural stem and progenitor cells (NSPCs) are heterogeneous populations of self-renewing stem cells and more committed progenitors that differentiate into neurons, astrocytes, and oligodendrocytes. Accurately identifying and characterizing the different progenitor cells in this lineage has continued to be a challenge for the field. We found previously that populations of NSPCs with more neurogenic progenitors (NPs) can be distinguished from those with more astrogenic progenitors (APs) by their inherent biophysical properties, specifically the electrophysiological property of whole cell membrane capacitance, which we characterized with dielectrophoresis (DEP). Here, we hypothesize that inherent electrophysiological properties are sufficient to define NPs and APs and test this by determining whether isolation of cells solely by these properties specifically separates NPs and APs. We found NPs and APs are enriched in distinct fractions after separation by electrophysiological properties using DEP. A single round of DEP isolation provided greater NP enrichment than sorting with PSA-NCAM, which is considered an NP marker. Additionally, cell surface N-linked glycosylation was found to significantly affect cell fate-specific electrophysiological properties, providing a molecular basis for the cell membrane characteristics. Inherent plasma membrane biophysical properties are thus sufficient to define progenitor cells of differing fate potential in the neural lineage, can be used to specifically isolate these cells, and are linked to patterns of glycosylation on the cell surface.
Project description:Electrophysiological variability in cardiomyocytes derived from pluripotent stem cells continues to be an impediment for their scientific and translational applications. We studied the variability of action potentials (APs) recorded from clusters of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using high-resolution optical mapping. Over 23,000 APs were analyzed through four parameters: APD30, APD80, triangulation and fractional repolarization. Although measures were taken to reduce variability due to cell culture conditions and rate-dependency of APs, we still observed significant variability in APs among and within the clusters. However, similar APs were found in spatial locations with close proximity, and in some clusters formed distinct regions having different AP characteristics that were reflected as separate peaks in the AP parameter distributions, suggesting multiple electrophysiological phenotypes. Using a recently developed automated method to group cells based on their entire AP shape, we identified distinct regions of different phenotypes within single clusters and common phenotypes across different clusters when separating APs into 2 or 3 subpopulations. The systematic analysis of the heterogeneity and potential phenotypes of large populations of hESC-CMs can be used to evaluate strategies to improve the quality of pluripotent stem cell-derived cardiomyocytes for use in diagnostic and therapeutic applications and in drug screening.
Project description:Patients with manifest atrioventricular accessory pathways (mAPs) have a greater tendency to develop atrial fibrillation (AF) compared with patients with concealed atrioventricular accessory pathways (cAPs). However, the risk factors of developing AF in patients with various atrioventricular accessory pathways (APs) are not clear.This retrospective study included 460 symptomatic patients with either cAPs (n = 246) or mAPs (n = 214) who underwent electrophysiological study and successful radiofrequency catheter ablation of APs. Clinical and electrophysiological characteristics were compared between cAPs and mAPs and between AF and non-AF groups with cAPs or mAPs. Independent risk factors of AF were analyzed using multivariate logistic regression.AF was more frequent in mAPs group than in cAPs group (23.4% vs 9.8%, p < 0.01). Clinical features were similar between cAPs and mAPs. Anterograde conduction properties served as the major electrophysiological feature of mAPs. Multivariate analysis indicated that mAPs, hypertension, post-ablation P wave dispersion (Pd), N-terminal proB-type natriuretic peptide (NT-proBNP) and creatinine were independent risk factors of AF in the complete cohort. Hypertension, post-ablation Pd and high-sensitivity C-reactive protein (hsCRP) were independent risk factors of AF in cAPs group. Post-ablation Pd, NT-proBNP, creatinine and shorter effective refractory period of anterograde accessory pathways (AAP ERP) were independent risk factors of AF in mAPs group.Results from this study demonstrate that the risk factors of AF are not homogenous between concealed and manifest APs, which might suggest heterogeneous pathogenesis of AF in these two types of APs.
Project description:BACKGROUND:In this study, a novel pelvic phantom was developed and used to assess the visibility and presence of artefacts from different types of commercial fiducial markers (FMs) on multi-modality imaging relevant to prostate cancer. METHODS AND MATERIALS:The phantom was designed with 3D printed hollow cubes in the centre. These cubes were filled with gel to mimic the prostate gland and two parallel PVC rods were used to mimic bones in the pelvic region. Each cube was filled with gelatine and three unique FMs were positioned with a clinically-relevant spatial distribution. The FMs investigated were; Gold Marker (GM) CIVCO, GM RiverPoint, GM Gold Anchor (GA) line and ball shape, and polymer marker (PM) from CIVCO. The phantom was scanned using several imaging modalities typically used to image prostate cancer patients; MRI, CT, CBCT, planar kV-pair, ExacTrac, 6MV, 2.5MV and integrated EPID imaging. The visibility of the markers and any observed artefacts in the phantom were compared to in-vivo scans of prostate cancer patients with FMs. RESULTS:All GMs were visible in volumetric scans, however, they also had the most visible artefacts on CT and CBCT scans, with the magnitude of artefacts increasing with FM size. PM FMs had the least visible artefacts in volumetric scans but they were not visible on portal images and had poor visibility on lateral kV images. The smallest diameter GMs (GA) were the most difficult GMs to identify on lateral kV images. CONCLUSION:The choice between different FMs is also dependent on the adopted IGRT strategy. PM was found to be superior to investigated gold markers in the most commonly used modalities in the management of prostate cancer; CT, CBCT and MRI imaging.