This work was supported by National Institute on Deafness and Other Communication Disorders Grants R21DC013915 and R01DC012502, the Natural Sciences and Engineering Research Council of Canada and the European Research Council under the European Community&#8217;s Seventh Framework Programme (FP7/2007-2013 Grant Agreement no. 339152).

The current experimental protocol follows the guidelines of ethical conduct according to the Yale University Human Investigation Committee.
1. Electroenchephalopgaphy (EEG) Preparation
<ol>
	<li>Measure head size to determine the appropriate EEG cap.</li>
	<li>Identify the location of the vertex by finding the mid-point between nasion and inion with a measuring tape.</li>
	<li>Place the EEG cap on the head using the pre-determined vertex as Cz. Examine Cz again after placing the cap by using a measuring tape as done in 1.2. Note that the EEG cap is equipped with electrode holders and the placement of the 64 electrodes (or holders) is based on a modified 10-20 system with pre-specified coordinates system based on Cz11. 
	Note: This representative application uses a 64 electrode configuration to assess scalp distribution changes and for source analysis. For simpler applications (event-related potential changes in amplitude and latency) using fewer electrodes are possible. There are two additional electrodes for ground in the EEG system used here. Those electrode holders are also included in the cap.</li>
	<li>Apply electrode gel in the electrode holders using a disposable syringe.</li>
	<li>Attach EEG electrodes (including ground electrodes) into the electrodes holders matching the labels of the electrodes and to the electrode holders on the electrode cap.</li>
	<li>Clean the skin surface with alcohol pads. 
	Note: For electrodes for detecting eye motion (electro-oculography), the skin locations are above and below the right eye (vertical eye motion), and lateral to the outer canthus of the both eyes (horizontal eye motion); for somatosensory stimulation the skin lateral to the oral angle is cleaned.</li>
	<li>Fill the four electro-oculography electrodes with the electrode gel and secure the electrodes with double-sided tape to the sites noted in 1.6.</li>
	<li>Secure all electrode cables using a Velcro strap. If required, tape the cables to participant&#39;s body or the other locations that do not introduce any additional electrical or mechanical noise.</li>
	<li>Position the participant in front of the monitor and the robot for somatosensory stimulation. Secure all electrode cables again as in 1.8.</li>
	<li>Connect the EEG and electro-oculography electrodes (including the ground electrodes) into the appropriate connecters (matching label and connecter shape) on the amplifier box of the EEG system.</li>
	<li>Check to see that the EEG signals are artifact free and that the offset value is in an acceptable range (&#60;50 &#181;V or smaller). If noisy signals or large offsets that are usually indicative of high impedance are found, correct those electrode signals by adding additional EEG gel and/or repositioning hair that is directly under the electrode.</li>
	<li>Insert the EEG-compatible earphones and confirm that the sound level is in a comfortable range based on subject report.</li>
</ol>
2. Somatosensory Stimulation
Note: The current protocol applies facial skin stretch for the purpose of somatosensory stimulation. The experimental setup with the EEG system is represented in Figure 1. The details of the somatosensory stimulation device have been described in the previous studies1,7,12-14. Briefly, two small plastic tabs (2 cm wide and 3 cm height) are attached with double-sided tape to the facial skin. The tabs are connected to the robotic device using string. The robot generates systematic skin stretch loads according to experimental designs. The setup protocol for ERP recording is as follows:
<ol>
	<li>Place the participant&#39;s head in the headrest in order to minimize head motion during stimulation. Remove carefully the electrode cables between the participant&#39;s head and headrest.</li>
	<li>Ask the participant to hold the safety switch for the robot.</li>
	<li>Attach plastic tabs to the target skin location using double-sided tape for somatosensory stimulation. For the representative results12,13, in which the target is the skin lateral to the oral angle, place the center of the tabs on the modiolus, a few mm lateral to the oral angle with the center of the tabs at approximately the same height of the oral angle.</li>
	<li>Adjust the configuration of the string, string supports and the robot in order to avoid EEG electrodes and cables.</li>
	<li>Apply a few facial skin stretches (one cycle sinusoid at 3 Hz with a maximum force of 4 N) to check for artifacts due to the stimulation (usually observed as relatively large amplitude and lower frequency compared with the electrophysiological response). If artifacts are observed in the EEG signals, go back to 2.4.</li>
</ol>
3. ERP Recording
<ol>
	<li>Explain the experimental task to the subject and provide practice trials (one block = 10 trials or less) to confirm if the subject understands the task clearly. 
	Note: The experimental task and stimulus presentation for ERP recording are preprogramed in software for stimulus presentation.
	<ol>
		<li>In the representative test with combined somatosensory and auditory stimulation12, apply the somatosensory stimulation associated with skin deformation to the skin lateral to the oral angle. The pattern of stretch is a one cycle sinusoid (3 Hz) with a maximum force of 4 N. A single synthesized speech utterance that is midway in a 10-step sound continuum between &#34;head&#34; and &#34;had&#34; is used for auditory stimulation.</li>
		<li>Present both stimulations separately or in combination. In the combined stimulation, test three onset timings (90 msec lead and lag, and simultaneous in somatosensory and auditory onsets: see Figure 3A).</li>
		<li>Randomize the presentation of five stimulations (somatosensory alone, auditory alone and three combined: lead, simult. and lag). Vary the inter-trial interval between 1,000 and 2,000 msec in order to avoid anticipation and habituation. The experimental task is to identify whether the presented speech sound, which is the sound that is acoustically intermediate between &#34;head&#34; and &#34;had&#39;, was &#34;head&#34; by pressing a key on a keyboard. In the somatosensory alone condition, in which there is no auditory stimulation, the participants are instructed to answer not &#34;head&#34;.</li>
		<li>Record participant judgments and the reaction time from the stimulus onset to the key press using the software for stimulus presentation. Ask the participant to gaze a fixation point on the display screen in order to reduce artifacts due to eye-movement.</li>
		<li>Remove the fixation point every 10 stimulations for a short break. (See also other example of task and stimulus presentation12,13)</li>
	</ol>
	</li>
	<li>Start the software for ERP recording at 512 Hz sampling, which also records the onset time of stimulation in the timeline of ERP data. Note that the time stamps of the stimulation, which also includes the information about the type of the stimulation, are sent for every stimulus from the software for stimulus presentation. The two programs (for ERP recording and for the stimulus presentation) are running on two separate PCs that are connected through a parallel port.</li>
	<li>Set the software for the somatosensory stimulation to the trigger-waiting mode and then start stimulus presentation by activating the software for stimulus presentation. Note that the software for the somatosensory stimulation is also running on a separate PC from the other two PCs. Record 100 ERPs per condition. 
	Note: A trigger signal for the somatosensory stimulation is received through an analog input device that is connected to a digital output device in the PC for sensory stimulation. Single somatosensory stimulation is produced per one trigger.</li>
</ol>

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The authors have nothing to disclose.

The studies reported here provide evidence that precisely controlled somatosensory stimulation that is produced by facial skin deformation induces cortical ERPs. Cutaneous afferents are known as a rich source of kinesthetic information3,4 in human limb movement5,6 and speech movement7,8,21. Stretching the facial skin in a manner that reflects the actual movement direction during speaking induces a kinesthetic sense similar to the corresponding movement. The current method combining precis...

Speech production is dependent on both auditory and somatosensory information. The auditory and somatosensory feedback occur in combination from the earliest vocalizations produced by an infant and both are involved in speech motor learning. Recent results suggest that somatosensory processes contribute to perception as well as production. For example, the identification of speech sounds is altered when a robotic device stretches the facial skin as participants listen to auditory stimuli1. Air puffs to the cheek that coincide with auditory speech stimuli alter participants&#39; perceptual judgments2.
These somatosensory effects involve the activation of cutaneous mechanoreceptors in response to skin deformation. The skin is deformed in various ways during movement, and cutaneous mechanoreceptors are known to contribute to kinesthetic sense3,4. The kinesthetic role of cutaneous mechanoreceptors is demonstrated by recent findings5-7 that the movement-related skin strains are appropriately perceived as flexion or extension motion depending on the pattern of skin stretch6. Over the course of speech motor training, which is the repetition of specific speech utterance with concomitant facial skin stretch speech, articulatory patterns change in an adaptive manner7. These studies indicate that modulating skin stretch during action provides a method for assessing the contribution of cutaneous afferents to the kinesthetic function of the sensorimotor system.
The kinesthetic function of orofacial cutaneous mechanoreceptors has been studied mostly using psychophysiological methods7,8 and microelectrode recoding from sensory nerves9,10. Here, the current protocol focuses on the combination of orofacial somatosensory stimulation associated with facial skin deformation and event related potential (ERP) recording. This procedure has precise experimental control over the direction and timing of facial skin deformation using a computer-controlled robotic device. This allows us to test specific hypotheses about the somatosensory contribution to speech production and perception by selectively and precisely deforming facial skin in a wide range of orientations during both speech motor learning and directly in speech production and perception. ERP recording are used to noninvasively evaluate the temporal pattern and timing of the influence of somatosensory stimulation on orofacial behaviors. The current protocol then can evaluate the neural correlates of kinesthetic function and assess the contribution of the somatosensory system to both speech processing, speech production and speech perception.
To show the utility of the application of skin stretch stimulation to ERP recording, the following protocol focuses on the interaction of somatosensory and auditory input in speech perception. The results highlight a potential method to assess somatosensory-auditory interaction in speech.

<table border="0" cellpadding="0" cellspacing="0" width="673">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EEG recording system</td>
			<td style="width:163px;">Biosemi</td>
			<td style="width:147px;">ActiveTwo</td>
			<td style="width:147px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Robotic decice for skin stretch</td>
			<td style="width:163px;">Geomagic</td>
			<td style="width:147px;">Phantom Premium 1.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EEG-compatible earphones</td>
			<td style="width:163px;">Etymotic research</td>
			<td style="width:147px;">ER3A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Software for visual and auditory stimulation</td>
			<td style="width:163px;">Neurobehavioral Systems</td>
			<td style="width:147px;">Presentation</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electrode gel</td>
			<td style="width:163px;">Parker Laboratories, INC</td>
			<td style="width:147px;">Signa gel</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double sided tape</td>
			<td style="width:163px;">3M</td>
			<td style="width:147px;">1522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable syringe</td>
			<td style="width:163px;">Monoject</td>
			<td style="width:147px;">412 Curved Tip</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analog input device</td>
			<td style="width:163px;">National Instuments&#160;</td>
			<td style="width:147px;">PCI-6036E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Degital output device</td>
			<td style="width:163px;">Measurement computing</td>
			<td style="width:147px;">USB-1208FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

somatosensory event-related potentials from orofacial skin stretch stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.

This section presents representative event-related potentials in response to somatosensory stimulation resulting from facial skin deformation. The experimental setup is represented in Figure 1. Sinusoidal stimulation was applied to the facial skin lateral to the oral angle (See Figure 3A as reference). One hundred stretch trials were recorded for each participant with 12 participants tested in total. After removing the trials with blinks and eye movement ...

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Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

13.1K Views.  Haskins Laboratories. This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

Video: Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

High Density Event-related Potential  Data Acquisition in Cognitive Neuroscience

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part because fMRI data acquisition and analysis techniques are readily available. Because fMRI has excellent spatial resolution but poor temporal resolution, this method can only be used to identify the spatial location of brain activity associated with a given cognitive process (and reveals virtually nothing about the time course of brain activity). By contrast, event-related potential (ERP) recording, a method that is used much less frequently than fMRI, has excellent temporal resolution and thus can track rapid temporal modulations in neural activity. Unfortunately, ERPs are under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and low density ERP recording has poor spatial resolution. In an effort to foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition. Critically, high density ERPs offer the promise of excellent temporal resolution and good spatial resolution (or excellent spatial resolution if coupled with fMRI), which is necessary to capture the spatial-temporal dynamics of human brain function.

Event-related potential (ERP) recording is under utilized in Cognitive Neuroscience because data acquisition techniques are not readily available and this method often has poor spatial resolution. To foster the increased use of ERPs in Cognitive Neuroscience, the present article details key techniques involved in high density ERP data acquisition.

Functional magnetic resonance imaging (fMRI) is currently the standard method of evaluating brain function in the field of Cognitive Neuroscience, in part ...

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 ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Electrode Positioning and Montage in Transcranial Direct Current Stimulation

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a non-invasive and safe alternative to change cortical excitability2. The effects of one session of tDCS can last for several minutes, and its effects depend on polarity of stimulation, such as that cathodal stimulation induces a decrease in cortical excitability, and anodal stimulation induces an increase in cortical excitability that may last beyond the duration of stimulation6. These effects have been explored in cognitive neuroscience and also clinically in a variety of neuropsychiatric disorders &#x2013; especially when applied over several consecutive sessions4. One area that has been attracting attention of neuroscientists and clinicians is the use of tDCS for modulation of pain-related neural networks3,5. Modulation of two main cortical areas in pain research has been explored: primary motor cortex and dorsolateral prefrontal cortex7. Due to the critical role of electrode montage, in this article, we show different alternatives for electrode placement for tDCS clinical trials on pain; discussing advantages and disadvantages of each method of stimulation.

Transcranial direct current stimulation (tDCS) is an established technique to modulate cortical excitability1,2. It has been used as an investigative tool in neuroscience due to its effects on cortical plasticity, easy operation, and safe profile. One area that tDCS has been showing encouraging results is pain alleviation 3-5.

Transcranial direct current stimulation (tDCS) is a technique that has been intensively investigated in the past decade as this method offers a ...

Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain signal. Typically, researchers represent the brain's response as the mean across repeated experimental trials and disregard signal fluctuations over time as "noise". However, it is becoming clear that brain signal variability conveys meaningful functional information about neural network dynamics. This article describes the novel method of multiscale entropy (MSE) for quantifying brain signal variability. MSE may be particularly informative of neural network dynamics because it shows timescale dependence and sensitivity to linear and nonlinear dynamics in the data.

Neuroimaging researchers typically consider the brain's response as the mean activity across repeated experimental trials and disregard signal variability over time as "noise". However, it is becoming clear that there is signal in that noise. This article describes the novel method of multiscale entropy for quantifying brain signal variability in the time domain.

When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Extracting Visual Evoked Potentials from EEG Data Recorded During fMRI-guided Transcranial Magnetic Stimulation

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological effects. Specifically, by creating a transient interference with the normal activity of a target region and measuring changes in an electrophysiological signal, we can establish a causal link between the stimulated brain area or network and the electrophysiological signal that we record. If target brain areas are functionally defined with prior fMRI scan, TMS could be used to link the fMRI activations with evoked potentials recorded. However, conducting such experiments presents significant technical challenges given the high amplitude artifacts introduced into the EEG signal by the magnetic pulse, and the difficulty to successfully target areas that were functionally defined by fMRI. Here we describe a methodology for combining these three common tools: TMS, EEG, and fMRI. We explain how to guide the stimulator&#39;s coil to the desired target area using anatomical or functional MRI data, how to record EEG during concurrent TMS, how to design an ERP study suitable for EEG-TMS combination and how to extract reliable ERP from the recorded data. We will provide representative results from a previously published study, in which fMRI-guided TMS was used concurrently with EEG to show that the face-selective N1 and the body-selective N1 component of the ERP are associated with distinct neural networks in extrastriate cortex. This method allows us to combine the high spatial resolution of fMRI with the high temporal resolution of TMS and EEG and therefore obtain a comprehensive understanding of the neural basis of various cognitive processes.

This paper describes a method for collecting and analyzing electroencephalography (EEG) data during concurrent transcranial magnetic stimulation (TMS) guided by activations revealed with functional magnetic resonance imaging (fMRI). A method for TMS artifact removal and extraction of event related potentials is described as well as considerations in paradigm design and experimental setup.

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

How to Find Effects of Stimulus Processing on Event Related Brain Potentials of Close Others when Hyperscanning Partners

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen in one of two adjacent rooms. These rooms are separated by a glass window through which participants communicate to maintain feelings of togetherness while being fitted with the EEG cap. After checking for adequate EEG signals, the glass is covered by a curtain to prevent visual communication. Then, partners must be silent but are instructed to try to feel in the presence of their partner during the entire experiment. Just before it starts, participants are told that each of them will be presented with one image at a time and that these images will occur at the same time for both of them on their own screen. They are also instructed that, for each trial, the simultaneous images will always be different. However, unbeknownst to them, trials are randomized: only half of them are consistent with this instruction and actually include two different images. These trials form the DSC, that is, the different-stimuli condition. The other half of the trials are inconsistent with the instruction. They include two identical images and form the ISC (identical-stimuli condition). After the experiment, participants are sorted into two groups: those who reported that they felt in the presence of their partner during the majority of the trials and those who reported they did not. The impact of the stimulus processing of the partner is found by subtracting the mean voltages of the ERPs of the ISC (inconsistent with the instructions) from the ERPs of the DSC (consistent with the instructions) in at least two time windows (TWs): firstly, in the 75 to 150 ms TW, where the absolute values of these subtractions are greater, especially at right frontal sites, in those who felt in the presence of their partner than in those who did not; secondly, in the LPP time window (i.e., from 650 to 950 ms post onset), where ERPs are significantly less positive in the DSC than in the ISC in those in whom the raw results of the early (75-150ms) subtractions are negative.

This protocol describes key steps involved in assessing the sensitivity of the brain of one person to the stimulus processing of a close other by selecting pairs of partners, recording their electroencephalogram (EEG) simultaneously and computing their event-related brain potentials (ERPs).

The partners of each pair must be able to pass the McGill Friendship Questionnaire without communicating. Each partner is then seated in front of a screen ...