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 ...

voltage-biasing-cyclic-voltammetry-electrical-impedance-spectroscopy

Research

Education

JoVE Journal

JoVE Core

Electrical Engineering

Neuroscience

Unit 1

Sampling

Basics of Electric Circuits

SCIENTISTS IN ACTION

24.4K Views.  Purdue University. 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.

Video: Voltage Biasing, Cyclic Voltammetry, & Electrical Impedance Spectroscopy for Neural Interfaces

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

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and amphetamine), the role it plays in psychiatric illnesses (e.g. schizophrenia and Attention Deficit Hyperactivity Disorder), and its involvement in degenerative disorders like Parkinson's and Huntington's disease. Under normal physiological conditions, dopamine is known to regulate locomotor activity, cognition, learning, emotional affect, and neuroendocrine hormone secretion. One of the largest densities of dopamine neurons is within the striatum, which can be divided in two distinct neuroanatomical regions known as the nucleus accumbens and the caudate-putamen. The objective is to illustrate a general protocol for slice fast-scan cyclic voltammetry (FSCV) within the mouse striatum. FSCV is a well-defined electrochemical technique providing the opportunity to measure dopamine release and uptake in real time in discrete brain regions. Carbon fiber microelectrodes (diameter of ~ 7 &mu;m) are used in FSCV to detect dopamine oxidation. The analytical advantage of using FSCV to detect dopamine is its enhanced temporal resolution of 100 milliseconds and spatial resolution of less than ten microns, providing complementary information to in vivo microdialysis.

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

Extensive research has focused on the neurotransmitter dopamine because of its importance in the mechanism of action of drugs of abuse (e.g. cocaine and ...

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

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

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.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

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

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

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

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
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.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

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

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

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.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

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

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

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.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

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

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

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 ...