Name: voltage biasing, cyclic voltammetry, &amp; electrical impedance spectroscopy for neural interfaces
Uploaded: 2012-02-24T13:00:00
Duration: 7 min 51 s
Description: Watch this Scientific Journal Video about Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces at JoVE.com

This research was supported by the National Institutes of Health (R03DC009339-02, NIDCD) and by the Defense Advanced Research Projects Agency (DARPA) Microsystems Technology Office (MTO), under the auspices of Dr. Jack W. Judy (jack.judy@darpa.mil) as part of the Reliable Neural Technology Program, through the Space and Naval Warfare Systems Command (SPAWAR) Systems Center (SSC) Pacific grant No. N66001-11-1-4013.

1. Set up the Electrochemistry Instrument
<ol>
 <li>Electrochemistry instrumentation such as a Methrohm Autolab PGSTAT (Utrecht, NL) is needed for EIS, CV and Rejuvenation. The FRA2 add-on enables EIS, and the channel multiplexer (MUX) add-on is useful for testing multi-channel electrodes. </li>
 <li>Build a headstage adapter to connect the channel MUX to the headstage. </li>
 <li>Make the connections. Connect the working and sensing electrodes to the channel MUX, and connect the reference and counter electrodes to the part of the headstage adapter connected to the current return path, typically an implanted stainless-steel or titanium bone screw. </li>
</ol>
2. Electrical Impedance Spectroscopy
<ol>
 <li>Start the Frequency Response Analyzer (FRA) software, and verify the Procedure file settings. The procedure should be set to test two multi-sine waveforms each consisting of 15 sines simultaneously ranging from 10 Hz to 30 kHz. The applied voltage should be 25 mV or less (see supplementary methods).</li>
 <li> Open and edit the Project file. The project uses the Procedure file, loops through each channel, and saves the result (see supplementary methods). </li>
 <li>Connect animal subject with a passive (no amplifiers) headstage. Active headstages will not pass input signals. </li>
 <li>Execute the Project file. Each channel takes tens of seconds depending on settings. </li>
 <li>Display and interpret result. Parse the output text files with MATLAB (Natick, MA), and make a Nyquist plot. A semicircle at higher frequencies indicates a tissue response. </li>
</ol>
3. Cyclic Voltammetry
<ol>
 <li>Start the General Purpose Electrochemistry System (GPES) software, and verify the Procedure file settings. The procedure should be set to sweep the voltage at 50 mV/s within the limits of hydrolysis which is between +0.8 and -0.6 V for typical neural electrode materials (Pt, Ir, IrOx). At least three scans should be run for the system to reach equilibrium. The results from the final scan are saved (see supplementary methods). The scan rate can be increased to 1 V/s to reduce measurement time; however, the shape of the I-V curve will likely change if the scan rate is faster than the charge transfer reactions occurring at the electrode-tissue interface. </li>
 <li>Open and edit the Project file. The project uses the Procedure file, loops through each channel, and saves the result (see supplementary methods). </li>
 <li> Connect animal subject with a passive headstage. </li>
 <li>Execute the Project file. Each channel takes about three minutes depending on settings. Increasing the scan rate to 1 V/s reduces measurement time to approximately ten seconds per channel. </li>
 <li>Display and interpret result. Parse the output text files with MATLAB, and plot the I-V relationship. The charge carrying capacity is quantified by integrating the area of the cathodic current within the CV. </li>
</ol>
4. Rejuvenation
<ol>
 <li>Start the General Purpose Electrochemistry System (GPES) software, and verify the Procedure file settings. Using the Steps and Sweeps method, the procedure should be set to step the voltage to 1.5 V for a duration of 4 seconds (see supplementary methods).</li>
 <li> Open and edit the Project file. The project uses the Procedure file, loops through each channel, and saves the result (see supplementary methods). </li>
 <li>Connect animal subject with a passive headstage. </li>
 <li> Execute the Project file. Each channel takes about ten seconds.
 4.5) Collect EIS and CV data and interpret results. </li>
</ol>
5. Representative Results
A typical workflow, including recordings, EIS, CV and rejuvenation, is shown in figure 1. Recordings and EIS are collected most frequently (daily or weekly) across all channels, while CV and rejuvenation can be used if spiking activity is no longer detectable.
EIS changes over the course of days to weeks after an electrode is implanted. When EIS data is displayed as a Nyquist plot, a semicircle at higher frequencies (near the origin) is indicative of the tissue response at the electrode site (Fig 2). 
CV produces a current-voltage (I-V) curve showing some hysteresis. The most relevant CV statistic is the charge carrying capacity, the area inside the I-V curve normalized by the electrode site area (Fig 3 a). Electrodes with large charge capacity are preferred for micro-stimulation. 
During rejuvenation a voltage pulse is applied that usually results in increased charge capacity and decreased impedance magnitudes (Fig 3a &amp; b). Spiking can also be restored in channels that previously had spikes (Figure 4a). While rejuvenation has only short-term effects on impedance and signal-to-noise ratio (SNR), this technique can be applied daily. Figure 4b &amp; c shows daily pre- and post-rejuvenation 1 kHz impedance magnitude and SNR data for a 16 channel array implanted in guinea pig cortex. Rejuvenation has a robust effect on lowering the 1 kHz impedance magnitude by an order of magnitude after each application. As a result of recovered signals and lower impedance, SNR increases after each rejuvenation session. Ultimately, all signals were lost following 160 days after implantation and rejuvenation was no longer effective. 
<img src="/files/ftp_upload/3566/3566fig1.jpg" alt="Figure 1" /> 
Figure 1. EIS is measured after each recording session. If no spikes are recorded on a channel that previously had spikes, and EIS shows a large tissue component that has increased over time, then CV and rejuvenation are tried on this channel. EIS and recordings are then used to determine if the treatment was successful.
<img src="/files/ftp_upload/3566/3566fig2.jpg" alt="Figure 2" /> 
Figure 2. EIS data displayed in a Nyquist plot of an electrode site immediately after implantation (blue), and 4 months later (green). Each point on the Nyquist plot represents the real and imaginary impedance at a single frequency. A partial semicircle due to the tissue around the site is evident at higher frequencies.
<img src="/files/ftp_upload/3566/3566fig3.jpg" alt="Figure 3" /> 
Figure 3. CV and EIS changes of an implanted iridium oxide electrode pre- and post-rejuvenation. (A) Rejuvenation increases the area of the I-V curve corresponding to an increased charge carrying capacity. (B) A significant shift in the impedance specra to lower impedance levels is generally observed after rejuvenation.
<img src="/files/ftp_upload/3566/3566fig4.jpg" alt="Figure 4" /> 
Figure 4. Effects of voltage biasing on recordings and impedance. (A) Pre- and post-rejuvenation recordings show spikes can be recovered on channels that had previously been silent. Daily pre- and post-rejuvenation results in a robust (B) drop in 1 kHz impedance magnitude and (C) increase in SNR for approximately 150 days after surgery. Errorbars represent standard error from data collected from a 16-channel array implanted in guinea pig cortex.

<ol>
	<li>Szarowski, D. H., Andersen, M. D., Retterer, S., Spence, A. J., Isaacson, M., Craighead, H. G., Turner, J. N., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+responses+to+micro&#45;machined+silicon+devices.">Brain responses to micro&#45;machined silicon devices.</a> Brain Res. 983, 23-35 (2003).</li><li>Vetter, R. J., Williams, J. C., Hetke, J. F., Nunamaker, E. A., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+neural+recording+using+silicon&#45;substrate+microelectrode+arrays+implanted+in+cerebral+cortex.">Chronic neural recording using silicon&#45;substrate microelectrode arrays implanted in cerebral cortex.</a> IEEE Trans. Biomed. Eng. 51, 896-904 (2004).</li><li>Williams, J. C., Hippensteel, J. A., Dilgen, J., Shain, W., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+impedance+spectroscopy+for+monitoring+tissue+responses+to+inserted+neural+implants.">Complex impedance spectroscopy for monitoring tissue responses to inserted neural implants.</a> J. Neural Eng. 4, 410-423 (2007).</li><li>Johnson, M. D., Otto, K. J., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+voltage+biasing+improves+unit+recordings+by+reducing+resistive+tissue+impedances.">Repeated voltage biasing improves unit recordings by reducing resistive tissue impedances.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 13, 160-165 (2005).</li><li>Otto, K. J., Johnson, M. D., Kipke, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+pulses+change+neural+interface+properties+and+improve+unit+recordings+with+chronically+implanted+microelectrodes.">Voltage pulses change neural interface properties and improve unit recordings with chronically implanted microelectrodes.</a> IEEE Trans. Biomed. Eng. 53, 333-340 (2006).</li><li>Pickup, P. G., Birss, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anodic+hydrous+oxide&#45;growth+at+iridium.">A model for anodic hydrous oxide&#45;growth at iridium.</a> J. Electroanal. Chem. 220, 83-100 (1987).</li></ol>

No conflicts of interest declared.

Neural recording prosthetic systems display a limited functional lifetime as recording capability decreases with time post-implantation. The likely contributor to diminishing performance is the reactive tissue response to the implanted device as a compact glial sheath functionally isolates the foreign object from healthy tissue1. Along with neural recording, electrochemical measurements (EIS and CV) are typically used for longitudinal monitoring of the electrode-tissue interface2,3. EIS is practically useful in assessing the recording capability of the interface. The impedance quickly increases with time post-implantation suggesting the reactive tissue response alters the electrical properties of the interface3. Additionally, EIS data can be used to model the cellular composition adjacent to the implanted electrode3-5. Cyclic voltammetry can be used to further investigate changes in recordings and EIS. The electrode material and roughness as well as the electrochemical reactions and the surrounding tissue influence the shape of the I-V curve. Large charge carrying capacity, determined from the area of the I-V curve, is usually preferred, especially for electrical micro-stimulation. Low charge capacity is often associated with increased EIS. The potential applied during CV may itself alter charge capacity and EIS, especially if the voltage range is large enough to drive redox reactions. 
The application of voltage biasing, or rejuvenation, can be used with the purpose of increasing charge carrying capacity, decreasing impedance, and increasing the number channels with recorded spikes5. Oxidation is likely occurring at the electrode interface during rejuvenation, and with iridium materials, a hydrous oxide monolayer forms at anodic potentials of 1.2 V6. It has been suggested that the formation of this monolayer may remove cellular and acellular material attached to the electrode resulting in lower impedance at the interface5. While rejuvenation can recover lost neural signals, it is most effective if used on channels that previously had spikes within a few days prior. Recordings, EIS, CV, and rejuvenation can best be used as complementary tools in monitoring the neural interface and improving the long-term functionality of implanted devices.

<table border="1">
<tbody>
 <tr>
 <td>Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Electrochemistry Instrument</td>
 <td>Metrohm Autolab</td>
 <td>PGSTAT128N</td>
 <td>add-ons: FRA2, channel MUX</td>
 </tr>
 <tr>
 <td>Passive Headstage</td>
 <td>Tucker-Davis Technologies</td>
 <td>&nbsp; </td>
 <td>model depends on connector and channel count</td>
 </tr>
 <tr>
 <td>26-pin female connector</td>
 <td>AMP</td>
 <td>5749069-2</td>
 <td>Headstage Adapter Or substitute appropriate connector for your headstage</td>
 </tr>
 <tr>
 <td>Banana Jacks</td>
 <td>Digikey</td>
 <td>J151-ND</td>
 <td>Headstage Adapter The Autolab channel MUX has banana plugs</td>
 </tr>
 </tbody>
</table>

voltage biasing, cyclic voltammetry, &amp; electrical impedance spectroscopy for neural interfaces

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive procedures, and can be used to monitor electrode performance over the long term. EIS measures electrical impedance at multiple frequencies, and increases in impedance indicate increased glial scar formation around the device, while cyclic voltammetry measures the charge carrying capacity of the electrode, and indicates how charge is transferred at different voltage levels. As implanted electrodes age, EIS and CV data change, and electrode sites that previously recorded spiking neurons often exhibit significantly lower efficacy for neural recording. The application of a brief voltage pulse to implanted electrode arrays, known as rejuvenation, can bring back spiking activity on otherwise silent electrode sites for a period of time. Rejuvenation alters EIS and CV, and can be monitored by these complementary methods. Typically, EIS is measured daily as an indication of the tissue response at the electrode site. If spikes are absent in a channel that previously had spikes, then CV is used to determine the charge carrying capacity of the electrode site, and rejuvenation can be applied to improve the interface efficacy. CV and EIS are then repeated to check the changes at the electrode-tissue interface, and neural recordings are collected. The overall goal of rejuvenation is to extend the functional lifetime of implanted arrays.

Watch this Scientific Journal Video about Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces at JoVE.com

Voltage Biasing, Cyclic Voltammetry, &amp; Electrical Impedance Spectroscopy for Neural Interfaces

The electrode-tissue interface of neural recording electrodes can be characterized with electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Application of voltage biasing changes the electrochemical properties of the electrode-tissue interface and can improve recording capability. Voltage biasing, EIS, CV, and neural recordings are complementary.

Electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measure properties of the electrode-tissue interface without additional invasive ...

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Presynaptic Dopamine Dynamics in Striatal Brain Slices with Fast-scan Cyclic Voltammetry

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Using fast-scan cyclic voltammetry to measure electrically evoked presynaptic dopamine dynamics in striatal brain slices.

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Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

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Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

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Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

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In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Laser Nanosurgery of Cerebellar Axons In Vivo

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This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

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Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

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Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Modeling Fast-scan Cyclic Voltammetry Data from Electrically Stimulated Dopamine Neurotransmission Data Using QNsim1.0

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is implicated in diseases and disorders including attention deficit hyperactivity disorder, Parkinson's disease, and traumatic brain injury. Thus, DA neurotransmission and the methods to study it are of intense scientific interest. In vivo fast-scan cyclic voltammetry (FSCV) is a method that allows for selectively monitoring DA concentration changes with fine temporal and spatial resolution. This technique is commonly used in conjunction with electrical stimulations of ascending DAergic pathways to control the impulse flow of dopamine neurotransmission. Although the stimulated DA neurotransmission paradigm can produce robust DA responses with clear morphologies, making them amenable for kinetic analysis, there is still much debate on how to interpret the responses in terms of their DA release and clearance components. To address this concern, a quantitative neurobiological (QN) framework of stimulated DA neurotransmission was recently developed to realistically model the dynamics of DA release and reuptake over the course of a stimulated DA response. The foundations of this model are based on experimental data from stimulated DA neurotransmission and on principles of neurotransmission adopted from various lines of research. The QN model implements 12 parameters related to stimulated DA release and reuptake dynamics to model DA responses. This work describes how to simulate DA responses using QNsim1.0 and also details principles that have been implemented to systematically discern alterations in the stimulated dopamine release and reuptake dynamics.

Fast-scan cyclic voltammetry can monitor in vivo dopamine neurotransmission in the context of drugs, disease, and other experimental manipulations. This work describes the implementation of QNsim1.0, a software to model electrically stimulated dopamine responses according to the quantitative neurobiological model to quantify estimates of dopamine release and reuptake dynamics.

Central dopaminergic (DAergic) pathways have an important role in a wide range of functions, such as attention, motivation, and movement. Dopamine (DA) is ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

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Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

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Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

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Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

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Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

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We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...