Biphasic recording In this lab you will study the response of the sciatic nerve of the frog to electrical stimulation using two pairs of stainless steel wire electrodes that are both in contact with the nerve, therefore recording the potential difference between two points on the nerve.
There are 12 such recording electrodes in the nerve bath, but only two can be connected to the differential amplifier at any one time. Modified from ADinstruments. All Rights Reserved. Delivering a sufficiently large stimulus to the nerve will result in an action potential that is quite a bit larger than a single intracellular action potential but looks remarkably similar.
This compound action potential CAP is the algebraic summation of all the action potentials produced by all the fibres that were fired by that stimulus. The nerve is made of thousands of axons whose size, myelination and position with respect to the stimulating and recording electrodes all affect the size of their contribution to the compound action potential.
Both the classic intracellular action potential and the compound action potential are biphasic. In other words, they have both positive and negative deflections, but for different reasons.
The negative phase of the intracellular action potential is attributed to the mechanism of after-hyperpolarization.
The negative phase of the CAP is due to the manner in which it is recorded, which will be explained below. There are two wire recording electrodes R1 and R2 touching the nerve, each connected to one input of the differential amplifier. The animation below illustrates how the shape of the CAP depends on the position of the two electrodes with respect to the travelling CAP.
For an in-depth explanation, please read on below. Before the stimulus is delivered, both wires should be measuring basically the same voltage. As the CAP passes the second electrode R2 , a deflection of the same size but opposite sign will be recorded.
When two out of the four nanoelectrodes are used to porate the HL-1 cell, the signals acquire a hybrid shape that contains extra- and intracellular potentials center region of the recording. When all four nanoelectrodes are intracellularly coupled, the signals acquire the more typical shape of the intracellular action potential.
Red lines are actual recordings, and blue lines are simulations. The analysis of the spike shape during subsequent poration of single 3D nanoelectrodes also permits the study of all of the specific contributions to the resulting recorded spike: the intracellular 3D nanoelectrodes that have been used for optoporation and penetrated the membrane , the extracellular 3D nanoelectrodes that have not been used for optoporation and remained outside the cell , and the extracellular planar electrode.
In Figure 5 b we show the proposed equivalent circuit for an HL-1 cell lying on four 3D nanoelectrodes that are used progressively for cell poration. Figure 5 c—f show the recorded and simulated spikes according to how many 3D nanoelectrodes penetrated the cell membrane. The 3D nanoelectrodes not used for opto-porating the cells remain extracellularly coupled to the cell and are taken into account in all simulations; their contribution to the recorded signal remains unchanged.
In addition, since we used MEA electrodes with size up to 30 um, in several cases some of the 3D nanoelectrodes were not covered by cell bodies or processes and were therefore directly exposed to the cell culture media. This experiment clearly presents the unique capability of plasmonic optoporation to selectively porate individual regions of a cell lying on one electrode and to record the extracellular and intracellular-like components.
In section S1 of the SI , we present the circuital analysis used to obtain the simulated spikes in Figure 5. We demonstrated the performance of 3D plasmonic nanostructures on multielectrode arrays for long-term and stable recordings of both intracellular and extracellular electrical spiking activity in primary mammalian neurons and cardiac derived HL-1 cells. Remarkably, the efficacy of the proposed method was demonstrated with the recording of spontaneous and unperturbed electrical activity with high SNR.
The key point enabling these results is the combination of vertical nanoelectrodes structured on planar microelectrodes with plasmonic optoporation. The former promotes a tight seal with the cell membrane that is essential for achieving a high signal-to-noise ratio in extracellular recordings. The latter enables an extremely local membrane poration process to gently penetrate the intracellular compartment with only the tip of the nanoelectrode, without affecting the tight seal.
Because the optical excitation used to induce the plasmonic cell-membrane poration is completely decoupled from the electrical recording circuit, hybrid MEA electrodes could be designed to optimize both extracellular and intracellular signals, and continuous recordings could be made throughout the poration event, without needing to adjust recording parameters such as amplifier gain.
This complete decoupling of plasmonic optoporation from electrical recording also allows recording electrical activity instantaneously after poration, revealing cellular events related to cell—electrode coupling when the membrane is locally porated.
Interestingly, in the presence of external forces, the 3D nanoelectrodes tend to bend rather than break or detach from the substrate; this flexibility might be an important factor to improve cellular adhesion to the nanoelectrodes and, consequently, the recording performance.
Although further optimizations will be needed to promote plasmonic optoporation toward the signal quality of a standard intracellular recording technique for primary neurons, our results demonstrate the vast potential of our approach and several appealing features to advance the quality of multisite electrophysiological recording technologies. The poration process can be scaled up to thousands of electrodes per minute, and both the fabrication process and the poration mechanism are compatible with high-density CMOS-MEAs see the SI.
This technology can be used for the selective and controlled intracellular delivery of nonmembrane-permeable molecules, 9 thus potentially enabling radical new experiments in which biomolecules are selectively delivered into neurons while the intracellular and the extracellular electrical activity are monitored on the large scale. Finally, for in vivo implantable probes, this approach might be combined with the recent advancements in integrated optical probes, 26 allowing the recording of intracellular signals from specific neurons and of the extracellular spikes and low-frequency signals of surrounding neuronal populations while minimizing the total number of individually routed electrodes.
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Gabriele C. The authors declare no competing financial interest. Nature nanotechnology , 7 3 , ISSN:. The ability to make electrical measurements inside cells has led to many important advances in electrophysiology.
The patch clamp technique, in which a glass micropipette filled with electrolyte is inserted into a cell, offers both high signal-to-noise ratio and temporal resolution. Ideally, the micropipette should be as small as possible to increase the spatial resolution and reduce the invasiveness of the measurement, but the overall performance of the technique depends on the impedance of the interface between the micropipette and the cell interior, which limits how small the micropipette can be.
Techniques that involve inserting metal or carbon microelectrodes into cells are subject to similar constraints. Field-effect transistors FETs can also record electric potentials inside cells, and because their performance does not depend on impedance, they can be made much smaller than micropipettes and microelectrodes.
Moreover, FET arrays are better suited for multiplexed measurements. Previously, we have demonstrated FET-based intracellular recording with kinked nanowire structures, but the kink configuration and device design places limits on the probe size and the potential for multiplexing. Here, we report a new approach in which a SiO2 nanotube is synthetically integrated on top of a nanoscale FET. This nanotube penetrates the cell membrane, bringing the cell cytosol into contact with the FET, which is then able to record the intracellular transmembrane potential.
Simulations show that the bandwidth of this branched intracellular nanotube FET BIT-FET is high enough for it to record fast action potentials even when the nanotube diameter is decreased to 3 nm, a length scale well below that accessible with other methods. Studies of cardiomyocyte cells demonstrate that when phospholipid-modified BIT-FETs are brought close to cells, the nanotubes can spontaneously penetrate the cell membrane to allow the full-amplitude intracellular action potential to be recorded, thus showing that a stable and tight seal forms between the nanotube and cell membrane.
We also show that multiple BIT-FETs can record multiplexed intracellular signals from both single cells and networks of cells. Nature Publishing Group. Action potentials have a central role in the nervous system and in many cellular processes, notably those involving ion channels. The accurate measurement of action potentials requires efficient coupling between the cell membrane and the measuring electrodes. Intracellular recording methods such as patch clamping involve measuring the voltage or current across the cell membrane by accessing the cell interior with an electrode, allowing both the amplitude and shape of the action potentials to be recorded faithfully with high signal-to-noise ratios.
However, the invasive nature of intracellular methods usually limits the recording time to a few hours, and their complexity makes it difficult to simultaneously record more than a few cells. Extracellular recording methods, such as multielectrode arrays and multitransistor arrays, are noninvasive and allow long-term and multiplexed measurements. However, extracellular recording sacrifices the one-to-one correspondence between the cells and electrodes, and also suffers from significantly reduced signal strength and quality.
Extracellular techniques are not, therefore, able to record action potentials with the accuracy needed to explore the properties of ion channels. As a result, the pharmacol.
The use of nanowire transistors, nanotube-coupled transistors and micro gold-spine and related electrodes can significantly improve the signal strength of recorded action potentials. Here, the authors show that vertical nanopillar electrodes can record both the extracellular and intracellular action potentials of cultured cardiomyocytes over a long period of time with excellent signal strength and quality.
Moreover, it is possible to repeatedly switch between extracellular and intracellular recording by nanoscale electroporation and resealing processes. Furthermore, vertical nanopillar electrodes can detect subtle changes in action potentials induced by drugs that target ion channels. Nature communications , 5 , ISSN:. Intracellular recording of action potentials is important to understand electrically-excitable cells. Recently, vertical nanoelectrodes have been developed to achieve highly sensitive, minimally invasive and large-scale intracellular recording.
It has been demonstrated that the vertical geometry is crucial for the enhanced signal detection. Here we develop nanoelectrodes of a new geometry, namely nanotubes of iridium oxide. When cardiomyocytes are cultured upon those nanotubes, the cell membrane not only wraps around the vertical tubes but also protrudes deep into the hollow centre.
We show that this nanotube geometry enhances cell-electrode coupling and results in larger signals than solid nanoelectrodes. The nanotube electrodes also afford much longer intracellular access and are minimally invasive, making it possible to achieve stable recording up to an hour in a single session and more than 8 days of consecutive daily recording.
This study suggests that the nanoelectrode performance can be significantly improved by optimizing the electrode geometry. Robinson, Jacob T. Deciphering the neuronal code - the rules by which neuronal circuits store and process information - is a major scientific challenge. Currently, these efforts are impeded by a lack of exptl. Here, the authors report a scalable intracellular electrode platform based on vertical nanowires that allows parallel elec.
Specifically, the authors show that their vertical nanowire electrode arrays can intracellularly record and stimulate neuronal activity in dissocd. The scalability of this platform, combined with its compatibility with silicon nanofabrication techniques, provides a clear path towards simultaneous, high-fidelity interfacing with hundreds of individual neurons.
A review. At present, the prime methodol. Although this methodol. On the other hand, intracellular recordings of the full electrophysiol. These, however, are limited to single cells at a time and for short durations. Recently a no. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users - with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.
Substrate integrated planar microelectrode arrays is the "gold std. Nevertheless, these devices suffer from drawbacks that are solved by spike-detecting, spike-sorting and signal-averaging techniques which rely on estd.
Of these, This study characterizes and analyzes the electrophysiol. Rabieh, Noha; Ojovan, Silviya M. In contrast to the extensive use of microelectrode array MEA technol. Here we demonstrate an empowering MEA technol. 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. 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. Journal of computational neuroscience , 32 1 , ISSN:. The spatial variation of the extracellular action potentials EAP of a single neuron contains information about the size and location of the dominant current source of its action potential generator, which is typically in the vicinity of the soma.
We used a dipole for the model source because there is extensive evidence it accurately captures the spatial roll-off of the EAP amplitude, and because, as we show, dipole localization, beyond a minimum cell-probe distance, is a more accurate alternative to approaches based on monopole source models.
Dipole characterization is separable into a linear dipole moment optimization where the dipole location is fixed, and a second, nonlinear, global optimization of the source location. The global source location was optimized by means of Tikhonov regularization that jointly minimizes model error and dipole size.
The particular strategy chosen reflects the fact that the dipole model is used in the near field, in contrast to the typical prior applications of dipole models to EKG and EEG source analysis. We applied dipole localization to data collected with stepped tetrodes whose detailed geometry was measured via scanning electron microscopy. Among various model error contributions to the residual, we address especially the error in probe geometry, and the extent to which it biases estimates of dipole parameters.
This dipole characterization method can be applied to any recording technique that has the capabilities of taking multiple independent measurements of the same single units. Neural Circuits , 6 , 80 DOI: Frontiers in neural circuits , 6 , 80 ISSN:.
Multielectrode arrays MEAs are extensively used for electrophysiological studies on brain slices, but the spatial resolution and field of recording of conventional arrays are limited by the low number of electrodes available. Here, we present a large-scale array recording simultaneously from electrodes used to study propagating spontaneous and evoked network activity in acute murine cortico-hippocampal brain slices at unprecedented spatial and temporal resolution.
We demonstrate that multiple chemically induced epileptiform episodes in the mouse cortex and hippocampus can be classified according to their spatio-temporal dynamics. Additionally, the large-scale and high-density features of our recording system enable the topological localization and quantification of the effects of antiepileptic drugs in local neuronal microcircuits, based on the distinct field potential propagation patterns. This novel high-resolution approach paves the way to detailed electrophysiological studies in brain circuits spanning spatial scales from single neurons up to the entire slice network.
The development of three-dimensional 3D synthetic biomaterials as structural and bioactive scaffolds is central to fields ranging from cellular biophysics to regenerative medicine. As of yet, these scaffolds cannot elec. Here, we address this challenge using macroporous, flexible and free-standing nanowire nanoelectronic scaffolds nanoES , and their hybrids with synthetic or natural biomaterials.
NanoES exhibited robust electronic properties and have been used alone or combined with other biomaterials as biocompatible extracellular scaffolds for 3D culture of neurons, cardiomyocytes and smooth muscle cells.
Furthermore, we show the integrated sensory capability of the nanoES by real-time monitoring of the local elec. Arrays of electrodes for recording and stimulating the brain are used throughout clin. To overcome this constraint, the authors developed new devices that integrate ultrathin and flexible silicon nanomembrane transistors into the electrode array, enabling new dense arrays of thousands of amplified and multiplexed sensors that are connected using fewer wires.
This system was used to record spatial properties of cat brain activity in vivo, including sleep spindles, single-trial visual evoked responses and electrog. It was found that seizures may manifest as recurrent spiral waves that propagate in the neocortex. The developments reported here herald a new generation of diagnostic and therapeutic brain-machine interface devices. Nano Lett. American Chemical Society. We present an advanced and robust technol. The presented architectures offer new and unconventional properties such as the realization of 3D plasmonic hollow nanocavities with high elec.
The 3D nature of the devices can overcome intrinsic difficulties related to conventional architectures in a wide range of multidisciplinary applications. Nanoscale , 7 , — DOI: Small , 11 , — DOI: Light Sci.
We present a theor. In this approach, electrons are accelerated in water by ponderomotive forces up to energies capable of exciting or ionizing water mols. This ability is enabled by the nanoelectrode structure extruding out of a metal baseplate , which allows for the prodn.
The electron injection is exptl. An understanding of the complex physics involved is obtained via a numerical approach that explicitly models the electromagnetic hot spot generation, electron-by-electron injection via multiphoton absorption, acceleration by ponderomotive forces and electron-water interaction through random elastic and inelastic scattering.
The model predicts a crit. Because of their high kinetic energy and large redn. Nanotechnology , 27 , DOI: BioMed Central Ltd. Background Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix mols.
With recent advances in nanotechnol. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diams.
Results We first checked the viability of cells grown on nanopillars with diams. It was obsd. We then calcd. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes.
However, when the size of nanopillars decreased to nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diam. Conclusions We have developed a simple approach to systematically study cell-substrate interactions on phys. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. Sakmann B, Neher E Patch clamp techniques for studying ionic channels in excitable membranes.
Gjerstad J, Valen EC, Trotier D, Doving K Photolysis of caged inositol 1,4,5-trisphosphate induces action potentials in frog vomeronasal microvillar receptor neurones. Pusch M, Neher E Rates of diffusional exchange between small cells and a measuring patch pipette.
Byerly L, Moody WJ Intracellular calcium ions and calcium currents in perfused neurones of the snail, Lymnaea stagnalis. Kostyuk PG Calcium ionic channels in electrically excitable membrane. Maconochie DJ, Knight DE A method for making solution changes in the sub-millisecond range at the tip of a patch pipette. Trautmann A, Siegelbaum S The influence of membrane patch isolation on single acetylcholine-channel current in rat myotubes. Hamill OP, Sakmann B Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells.
Patch clamp techniques for single channel and whole-cell recording. Molleman A Basic theoretical principles. Patch clamping. Wiley, Chichester, pp 5—42 Google Scholar. Windhorst U, Johansson H Modern techniques in neuroscience research: 33 tables. Marty A Ca-dependent K channels with large unitary conductance in chromaffin cell membranes.
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