Work supported by National Institutes of Health Grants R01DC10146/R01DC010397, NIDCD P30 DC006276 Research Core, and HEI. Its content is solely responsibility of the authors and do not necessarily represent the official views of NIH or HEI. The authors declare no existing or potential conflict of interest.

1. Isolation of OHCs 
<ol>
<li>Begin this procedure by harvesting temporal bones from guinea pigs, mice or your mammalian animal model. </li>
<li>Next, open the temporal bones using a malleus nipper in order to expose the cochlea, and immerse them in Leibovitz L-15. Remove bone excess carefully, keeping the bony shell intact. Whereas this is a general procedure applicable to temporal bones of any mammalian species, minor changes to the technique may be necessary when dealing with temporal bones from very small animals. In old animals the bulla is usually calcified, introducing an additional complication to the procedure. </li>
<li>Under microscopical observation, open the apical region of the cochlea and remove stria vascularis and spiral ligament using the tip of a #11 scalpel blade, a micro point pick and a fine tweezer. </li>
<li>Take the Organ of Corti off from the cochlear modiolus using the tweezer, and put it in 1mg/ml collagenase in L-15 at room temperature for 5 min. </li>
<li>If OHCs from basal turns of the cochlea are needed, remove the bony shell covering the base of the cochlea with the pick, and separate the spiral from the temporal bone using the scalpel blade before removing the Organ of Corti. </li>
<li>Transfer the Organ of Corti to the recording chamber using a 50 &mu;L Hamilton syringe. Afterward, dissociate cells by reflux through the needle.</li> 
</ol>
2. Experimental Setup
<ol>
<li>Diagram of the External Alternating Electrical Field (EAEF) generator and their links with the image capture system (Fig. 1). The control circuitry was specially implemented at the Engineering Core of the House Ear Institute.</li>
<li>The experimental setup used in our experiments consists of an Axiovert 135TV inverted microscope (Zeiss, Thornwood, NY) with an alternative LED-based illumination system (High Power LED System-36AD3500, Lightspeed Technologies, Campbell, CA), two electronic micromanipulators (Eppendorf "Patchman", Germany), a PC-controlled ultra-high speed Photron Fastcam X 1024 PCI camera (Photron USA Inc.) in the Keller port and an additional regular CCD camera in the trinocular port. The Fastcam camera is able to capture images at high frequencies (up to 100,000 fps) and high resolution (e.g., 1024 x 1024 pixels at 1,000 fps, 512 x 128 at 10,000 fps, 384 x 96 at 18,000 fps, and so on). The images provided by the high-speed camera are directly observed in the PC monitor, whereas the CCD camera is connected to a different monitor. The LED-based illumination system works in two different modes: low-power analog and high-power digital. All the preliminary procedures (electrodes positioning, cell positioning, focus, etc) are performed using the low-power analog mode. The high-power illumination is switched on by the aperture of the camera shutter and then switched off by shutter closing, facilitating heat dissipation. Homemade software, also developed at the House Ear Institute Engineering Core, running on the same PC controls the trigger of the high-speed camera, the LED-based illumination system, and the EAEF. A conventional digital photo camera in the front port allows for still frames as needed. (Fig. 2 A). </li>
<li>Electrodes (two 0.25 mm-diameter Ag wires with tip distance of 0.8 mm) are driven to position using one of the electronic micromanipulators. Electrodes' position is monitored visually and through the microscopic image; a change in the focal plane of the image indicates that the electrodes touched the bottom of the experimental chamber. Initially, the electrical field is calibrated using an external electrode. This electrode measures the electric potential at different points, generating a "map" of the electrical field. If a single isolated outer hair cell is placed between the tips of the electrodes with its longitudinal axis parallel to the applied EAEF, it will move elongating and shortening at the same frequency of the electric field. If the cell is placed perpendicular to the field a different type of OHC response (bending) can be observed and investigated. (Fig. 2 B)</li>
</ol>
3. EAEF stimulation and image capture
<ol>
<li>Four different stimulation protocols are selectable (Fig. 3):
<ol>
	<li>continuous single frequency (Fig. 3 A). Note that stimulus mode, frequency, amplitude and wave type can be selected using the home-made control software (red circles). </li>
 <li>bursted single frequency (Fig. 3 B). The length of the bursts and gaps between burst are also selectable.</li>
 <li> linear sweep (Fig. 3 C). The initial and final frequencies are selectable.</li>
		<li> multi-stimulation (Fig. 3 D). Single frequency and linear sweeps can be combined in a single experiment. After selecting the corresponding parameters, the control software configures the system and allows the operator to initiate light-synchronized video recording and cell stimulation by clicking a single button in the computer screen.</li>
</ol>
</li>
<li>Images are captured in AVI format for further analysis at high frequencies. </li>
</ol>
4. Representative Results
<ol>
<li>In this movie, two isolated outer hair cells show changes in the length or curvature when they are being stimulated with an External Alternating Electrical Field that is parallel or transversal, respectively, to their longitudinal axis. (Movie #2).</li>
<li>OHCs motile responses are analyzed off-line using ProAnalyst software (Xcitex Inc., Cambridge, MA). "Feature Tracking" function in this software provides the distance between two points frame-by-frame (Fig. 4). During cell shortening the distance between points selected at the apex (Cuticular plate; red color) and the base of the cell (Basal pole; green color) is smaller, and increases with cell elongation. The movie shows the software analyzing frame-by-frame the changes in length. The panel at the bottom of the image shows the trace of the movement. In this example, the total change in length is about 6.5 pixels.</li>
<li>"Contour Tracking" function in ProAnalyst software can detect cell edge and automatically measure the area of the optical section (Fig. 5).</li>
<li>Polystyrene microspheres added to the bath solution randomly and firmly attach to the plasma membrane (Fig. 6 A). Different microspheres can be selected simultaneously, and the software can automatically track all of them frame by frame. In this way, the cells can be divided in sections and the motility of each section evaluated independently. (Fig. 6 A)</li>
<li>By selecting microspheres located on the lateral edges of the cell image, changes in length of each segment and changes in angle of one segment respect to other (bending) can also be independently evaluated. (Fig. 6B)</li>
</ol>
<img src="/files/ftp_upload/2965/2965fig1.jpg" alt="Figure 1"/> 
Figure 1. Diagram of the EAEF generator and their links with the image capture system.
<img src="/files/ftp_upload/2965/2965fig2.jpg" alt="Figure 2"/> 
Figure 2. A) Picture of the experimental setup. B) Detail of the microscope stage, with cartoons depicting the electrodes and a single OHC placed between them with its longitudinal axis parallel to the electrical field.
<img src="/files/ftp_upload/2965/2965fig3.jpg" alt="Figure 3"/> 
Figure 3. A) User interface of the homemade control software configured for single-frequency stimulation. The selected parameters are circled in red. B) User interface of the home-made control software configured for burst single-frequency stimulation. C) User interface of the home-made control software configured for Linear sweep stimulation. D) User interface of the homemade control software configured for multi-stimulation.
<img src="/files/ftp_upload/2965/2965fig4.jpg" alt="Figure 4"/> 
Figure 4. Single frame of an OHC with two points selected at the base (green) and the apex (red) of the cell, respectively, using the "Feature tracking" function of the ProAnalyst software. The curve below the cell shows the periodic changes in distance between the selected points associated with electrical stimulation. Moving the vertical bar different frames can be selected for individual analysis. 
<img src="/files/ftp_upload/2965/2965fig5.jpg" alt="Figure 5"/> 
Figure 5. The "Contour Tracking" function in ProAnalyst detects the cell edge and automatically measure the area of the optical section.
<img src="/files/ftp_upload/2965/2965fig6.jpg" alt="Figure 6"/> 
Figure 6. A) Captured image of an isolated OHC decorated with polystyrene microspheres (top), and the same image with five microspheres individually selected (bottom). A different color was assigned to each microsphere, and their displacements can be individually and automatically tracked frame by frame, analyzed and compared. B) Segments of arbitrary length can be defined by selecting polystyrene microspheres located on the edges of the cell, and changes in length of these segments as well as changes in orientation of one segment respect to others (cell bending) can be automatically evaluated frame by frame with the image analysis software.
Movie 1. Isolation of guinea pig OHCs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2965/2965_Movie1_web.mov">Click here to watch video</a>
Movie 2. OHCs parallel and perpendicular to the EAEF showing typical electromotility and bending responses. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/2965/2965_Movie2_web.mov">Click here to watch video</a>

<ol>
	<li>Frolenkov, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+insights+into+the+morphogenesis+of+inner+ear+hair+cells.">Genetic insights into the morphogenesis of inner ear hair cells.</a> Nat Rev Genet. 5, 489-498 (2004).</li><li>Ashmore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+outer+hair+cell+motility.">Cochlear outer hair cell motility.</a> Physiol Rev. 88, 173-210 (2008).</li><li>Dallos, P., Fakler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin,+a+new+type+of+motor+protein.">Prestin, a new type of motor protein.</a> Nature Rev. Mol. Cell Biol. 3, 104-111 (2002).</li><li>Frolenkov, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlear+outer+hair+cell+bending+in+an+external+electrical+field.">Cochlear outer hair cell bending in an external electrical field.</a> Biophys. J. 73, 1665-1672 (1997).</li><li>Matsumoto, N., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+Prestin-Dependent+and+Prestin-Independent+Components+from+Complex+Motile+Responses+in+Guinea+Pig+Outer+Hair+Cells.">Extraction of Prestin-Dependent and Prestin-Independent Components from Complex Motile Responses in Guinea Pig Outer Hair Cells.</a> Biophys J. 89, 4343-4351 (2005).</li><li>Matsumoto, N., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prestin-dependent+and+prestin-independent+motility+of+guinea+pig+outer+hair+cells.">Prestin-dependent and prestin-independent motility of guinea pig outer hair cells.</a> Hear Res. 208, 1-12 (2005).</li><li>Ashmore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable+cochlear+amplifier.">The remarkable cochlear amplifier.</a> Hear Res. 266, 1-17 (2010).</li><li>Kitani, R., Kakehata, S., Kalinec, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motile+responses+of+cochlear+outer+hair+cells+stimulated+with+an+alternating+electrical+field.">Motile responses of cochlear outer hair cells stimulated with an alternating electrical field.</a> Hearing Research. , (2011).</li><li>Dallos, P., Evans, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+outer+hair+cell+motility:+corrections+and+addendum.">High-frequency outer hair cell motility: corrections and addendum.</a> Science. 268, 1420-1421 (1995).</li><li>Frank, G., Hemmert, W., Gummer, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+dynamics+of+high-frequency+electromechanical+transduction+in+outer+hair+cells.">Limiting dynamics of high-frequency electromechanical transduction in outer hair cells.</a> Proc. Natl. Acad. Sci. 96, 4420-4425 (1999).</li><li>Santos-Sacchi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+limit+and+phase+of+outer+hair+cell+motility:+effects+of+the+membrane+filter.">On the frequency limit and phase of outer hair cell motility: effects of the membrane filter.</a> J. Neurosci. 12, 1906-1916 (1992).</li><li>Inou&#233;, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Video Microscopy. , (1986).</li></ol>

No conflicts of interest declared.

The experimental method presented here enables estimating OHC motile responses in the kHz range without any restriction to the cell's movement. Different stimulation protocols, additional markers (microspheres), as well changes in the orientation of the cell with respect to the electric field, make it possible to investigate new aspects of OHC motility with a level of detail previously inaccessible. Other methods, e.g. those using photodiodes 9 or laser Doppler vibrometry 10, require a tight control of the position of the cell. Here, in contrast, all the measurements are performed between points belonging to the same cell, and every displacement is only associated with changes in cell shape and not with their movement with respect to an external frame of reference. Reliable measurements of cross-sectional OHC area are also easily obtained, which allows for an estimation of fast changes in OHC volume. In addition, the continuous development of faster and more sensitive cameras and better image analysis software, guarantee a continuous improvement in the quality of the method. A downside of the technique, no control on the electric potential across the plasma membrane, is a limitation shared with all the current methods used to evaluate OHC motility in the kHz range. 
Thus, the method described here might be an important tool for hearing research, capable of providing new and important clues about the cellular and molecular mechanisms underlying OHCs' motile responses

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Leibovitz's L-15 </td>
 <td>Gibco</td>
 <td>21083</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase (Type 4)</td>
 <td>Sigma-Aldrich</td>
 <td>C5138</td>
 <td>1mg/mL in L-15</td>
 </tr>
 </tbody>
</table>

investigating outer hair cell motility with a combination of external alternating electrical field stimulation and high-speed image analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

Watch this Scientific Journal Video about Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis at JoVE.com

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

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13.4K Views.  House Ear Institute. A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

Video: Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

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, ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific frequency and in turn modulate ongoing cortical oscillatory activity. This neurotool allows the establishment of a causal link between endogenous oscillatory activity and behavior. Most of the tACS studies have shown online effects of tACS. However, little is known about the underlying action mechanisms of this technique because of the AC-induced artifacts on Electroencephalography (EEG) signals. Here we show a unique approach to investigate online physiological frequency-specific effects of tACS of the primary motor cortex (M1) by using single pulse Transcranial Magnetic Stimulation (TMS) to probe cortical excitability changes. In our setup, the TMS coil is placed over the tACS electrode while Motor Evoked Potentials (MEPs) are collected to test the effects of the ongoing M1-tACS. So far, this approach has mainly been used to study the visual and motor systems. However, the current tACS-TMS setup can pave the way for future investigations of cognitive functions. Therefore, we provide a step-by-step manual and video guidelines for the procedure.

Transcranial Alternating Current Stimulation (tACS) allows the modulation of cortical excitability in a frequency-specific fashion. Here we show a unique approach which combines online tACS with single pulse Transcranial Magnetic Stimulation (TMS) in order to "probe" cortical excitability by means of Motor Evoked Potentials.

Transcranial Alternating Current Stimulation (tACS) is a neuromodulatory technique able to act through sinusoidal electrical waveforms in a specific ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

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Effects of Transcranial Alternating Current Stimulation on the Primary Motor Cortex by Online Combined Approach with Transcranial Magnetic Stimulation

Transcranial Electrical Brain Stimulation in Alert Rodents

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats