Name: non-restraining eeg radiotelemetry: epidural and deep intracerebral stereotaxic eeg electrode placement
Uploaded: 2016-06-25T15:00:00
Description: Watch this Scientific Journal Video about Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement at JoVE.com

The authors would like to thank Dr. Christina Ginkel (German Center for Neurodegenerative Diseases, DZNE), Dr. Michaela M&#246;hring (DZNE) and Dr. Robert Stark (DZNE) for assistance in animal breeding and animal health care. This work was financially supported by the Federal Institute for Drugs and Medical Devices (Bundesinstitut f&#252;r Arzneimittel und Medizinprodukte, BfArM) Bonn, Germany.

&#160;Ethics Statement: All animal experimentation was performed according to the guidelines of the local and institutional Council on Animal Care (University of Bonn, BfArM, LANUV, Germany). In addition, all animal experimentation was carried out in accordance with superior legislation, e.g., the European Communities Council Directive of 24 November 1986 (86/609/EEC) or individual regional or national legislation. Specific effort is made to minimize the number of animals used and their suffering.
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Implantable EEG radiotelemetry is of central relevance as it is a non-restraining technique allowing experimental animals to perform their full repertoire of behavior1,3. This is of major interest as the telemetric approach enables not only spontaneous EEG recordings but also recordings under cognitive tasks and circadian analytical setups, such as T-maze, radial maze, water maze, sleep deprivation tasks or whenever an EEG recording is necessary or helpful during complex cognitive or motor activity.
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Radiotelemetry is a most valuable methodological approach for measuring a variety of behavioral and physiological parameters in conscious, unrestrained animals of various sizes, particularly in the context of EEG, ECG, EMG, blood pressure, body core temperature or activity measurements 1-7. Theoretically, any species can be analyzed using implantable EEG radiotelemetry from laboratory rodents such as mice and rats to cats, dogs, pigs and primates 3,8. Even fish, reptiles and amphibians are subject to radiotelemetric investigation 9. Over the last two decades, implantable EEG radiotelemetry has proven to be valuable in the characterization of various transgenic animal models of human diseases, such as epilepsies, sleep disorders, neurodegenerative and neuropsychiatric disorders 7,10-12. In the past, numerous methodological approaches collecting physiological data including biopotentials from mice and rats have been described. Worn in jacket recorder systems, physical restraint methods, non-implanted radiotransmitters and tethered systems have received the main attention in the past 13,14. Nowadays, various systems for radiotelemetric implantation are commercially available. However, a literature screen also revealed 29 publications that describe the development of self-made radiotelemetric systems 15-40. Whereas home-made systems are likely to be less expensive and more user adapted, commercially available systems are straight forward, relatively easy to install and can be setup quickly.
Implantable EEG radiotelemetry has a number of advantages compared to competing techniques such as physical restraint methods, worn in jacket systems or tethered approaches. The latter are restraining by definition, i.e., the animal is unable to move or its normal behavior is impaired. It might even be necessary to anesthetize the animal for acquisition of reliable data. Modern tethered systems however are likely to be less restraining, but this needs to be scientifically validated. Radiotelemetry on the other hand allows animals to exhibit their full repertoire of behavior without spatiotemporal restrictions and thus, is thought to be superior to restraining approaches and be more predictive of the results that could be acquired in humans 1,3. It is known for quite a while that restraining approaches can dramatically alter fundamental physiological parameters, e.g., food intake, body core temperature, blood pressure and heart rate and physical activity for example 3. Tethered systems represent one still widely used classical restraining approach 13,14. The electrodes which are either epidural or deep electrodes are generally connected to a miniature socket which is anchored to the skull. The socket itself is exposed for attachment of a cable that allows relatively free movement of the animal. Although nowadays tethered systems have become extremely filigree and highly flexible, one of its major disadvantages is, that it is still semi-restraining. Besides, there might be a risk of infection at the electrode implantation site as the animals tend to manipulate any external devices originating from their body (head). Although wireless radiotelemetry technology in various species has already been described in the late 60s and has thus existed for decades, it has only recently become affordable, reliable, and relatively easy-to-use 10,41,42, particularly in small laboratory rodents such as mice and rats. Small, miniature implantable EEG transmitters are now commercially available and can be implanted in mice greater than 20 g (~10 weeks). Thus, the electrophysiological characterization of transgenic mouse models in particular has become a predominant field of application of implantable EEG radiotelemetry these days. Animal size is no longer an absolute experimental restriction whereas the life-span of the transmitters&#39; battery indeed is. Despite its limited life-time, implantable transmitter systems are capable of minimizing most disadvantages related to potential recording-associated stress by restraining systems. Rodents can present their complete armamentarium of physiological behavior including resting, locomotor activity (exploration) and sleep (REM, slow-wave sleep) 43,44. Importantly, implantable radiotelemetry can strongly reduce animal use 3. Currently, there is an intense discussion on how to limit the number of experimental animals in science and reduce their suffering. Clearly, animal experimentation and animal models of human and animal diseases are essential for our understanding of the bottom-line pathophysiology and subsequent progress in therapy. Furthermore, animal experiments are critical in drug research and development. They do substantially contribute to preclinical/toxicological studies in drug licensing thus committing to both human and animal care. It&#39;s noteworthy, that currently no alternatives are yet available to animal research to understand the complex pathophysiological mechanisms which would be otherwise impossible to be elicited. At the same time, the 3R, i.e., replacement, reduction and refinement strategy in the EU and the USA strongly encourages research into complementary and alternative methods. Radiotelemetry is an important example of a successful 3R strategy as it can reduce the number of experimental animals and their suffering compared to other techniques.
Here we provide a detailed and contiguous step-by-step approach to perform a subcutaneous pouch implantation of a radiofrequency transmitter in both mice and rats. This first sequence is followed by a description of stereotaxic epidural and deep intracerebral EEG electrode positioning. Special attention is paid to housing conditions, anesthesia, peri- and postoperative pain management and possible anti-infective treatment. The focus is on the computerized 3D stereotaxic approach to reliably target epidural and deep intracerebral structures. We also comment on frequent experimental pitfalls in EEG electrode implantation and strategies for reduction of trauma and optimization of pain management during postoperative recovery. Finally, examples of surface and deep EEG recordings are presented.

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non-restraining eeg radiotelemetry: epidural and deep intracerebral stereotaxic eeg electrode placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

This section illustrates examples obtained from surface and deep, intracerebral EEG recordings. Initially it should be stated that baseline recordings under physiological conditions are mandatory prior to subsequent recordings following e.g., pharmacological treatment. Such baseline recordings may provide valuable information about functional interdependence of brain rhythmicity with different behavioral states or sleep / circadian rhythmicity. Here, we show examples of recorded ...

Watch this Scientific Journal Video about Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement at JoVE.com

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

The overall goal of this experimental procedure is to record long-term electroencephalograms with stereotaxic epidural, and intracerebral electrode placement in different brain regions of freely moving rodents using non-restraining EEG radiotelemetry. This method can have answered key questions in the systemic integrative and translation in neuro-science field, such as dysrhythmia and neurological and neuropsychiatric diseases, as well as epilepsy. The main advantage of this technique is that it allows for epidural and deep intracerebral EEG recordings from freely moving rodents that serve as animal models of human diseases.Julien Soos, a graduate student from our laboratory will be demonstrating the technique. To prepare the epidural electrode, pull out of the silicone isolation, the tip of the transmitter lead with the forceps so it will be bent to a 90 degree angle. Shorten the tip of the lead.To prepare the deep electrode expose the stainless steel helix of the transmitter lead by pulling it out of the silicone isolation. Mechanically insert the deep electrode into the lead of the transmitter. Rewire the transmitter lead to the deep brain electrode to ensure a stable connection.Then bend the electrode to a 90 degree angle and shorten it to the required length. To begin this procedure, shave the head of a fully anesthetized mouse. Then disinfect the shaved area using 70%ethanol and an iodin based scrub.Place the animal in a prone position on a heating blanket to maintain it's body temperature during anesthesia. Using a scalpel, make a mid-line incision on the scalp from the forehead to the neck. Then open a subcutaneous pouch starting from the nuchal incision site along the lateral flank of the animal by blunt dissection with surgical scissors.Subsequently, inject one milliliter of 0.9%sodium chloride in the subcutaneous pouch. Afterward, place a transmitter with the sensing leads oriented crainially into the subcutaneous pocket at the flank close to the ventral abdominal region. In this procedure, place the anesthetized animal on the stereotaxic frame.Carefully position it's head with ear bars and a nose clamp so that the brahma and lambda cranium metric landmarks of the skull are at the same level. Next, clean the periosteum with cotton tips without damaging the temporal and occipital muscles. After that, apply hydrogen peroxide to the skull to exemplify the cranial suture, brahma and lambda.Prepare the 3D stereotaxic system for target coordinates. Drill the target holes at maximum velocity without applying pressure. Place the recording electrode on the motor cortex surface for recording EEG.Then fix the electrodes with dental cement to provide strong adhesion to the underlining neurocranium and leave the cement to dry for five minutes. Place the epidural reference electrode on the cerebellar cortex as it is an electroencephalographically silent region and fix it with cement as well. After having drilled the holes at the coordinates of interest, using the stereotaxic system attach the deep electrode to the vertical arm of the stereotaxic device.Place the deep electrode in the CA1 region. Subsequently, fix the electrode with dental cement to provide strong adhesion to the underlying neurocranium. Place the epidural reference electrode on the cerebellar cortex and fix it with dental cement as well.Leave the cement to dry for five minutes before closing the incision with sutures. This schematic shows that one epidural differential electrode is placed on the motor cortex. An additional intrahippocampal differential electrode is placed in the CA1 region of the hippocampus.Both pseudo reference electrodes are localized on the cerebellum. This coronal section illustrates the localization of the deep intracranial electrode for recording the electrohipppocampagram. And shown here, is the close up of the deep EEG electrode, the sensing lead of the radio frequency transmitter and their arrangement on top of the mirroring skull.These intrahippocampal CA1 recordings are taken from mice following acute treatment with kainate acid at day one, three, and five post-injection. This figure illustrates the examples of simultaneous epidural and deep EEGs in the pilocarpine model of mesial temporal lobe epilepsy in rats. Once mastered, this technique can be done within 30 to 45 minutes, depending on the number of electrodes to be implanted and the target regions if it is performed properly.While attempting this procedure, special attention has to be paid to the vital signs of the anesthetized animals. As transgenic and pharmacological mouse and rat models of human diseases can exhibit differences in anesthesia susceptibilities. Following this procedure, other behavioral, cognitive and electrophysiological masses, such as, T maze, regular maze or evoke potential recordings can be performed in order to address additional questions like, how rhythmicity depends on different functional stages or changes upon repetitive stimulus exposure.After watching this video, you should have a good understanding of how to perform accurate surface and deep EEG electrode placement in mice and rats using a three dimensional stereotaxic device and implantable EEG radiotelemetry.

non-restraining-eeg-radiotelemetry-epidural-deep-intracerebral

Research

JoVE Journal

Neuroscience

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Preterm EEG: A Multimodal Neurophysiological Protocol

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. 
In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked.
In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Electroencephalographic (EEG) Electrode Implantation on Mouse Brain

To begin, use an intraperitoneal anesthetized mouse and expose the skull Using standard surgical procedures. Attach the stainless steel rod mounted to the 2D electrode array on the backside of the flexible substrate to a manipulator. Then place the flexible substrate on the exposed skull.Adjust the location of channels 3 and 14 on the array to fit within the inferior colliculus. Draw four small circles at the locations of channels 3, 8, 9, and 14 on the skull with a permanent marker to serve as targeting landmarks. Next, dry the skull surface to improve the adherence of the dental cement and to electrically isolate the 2D electrode array on the flexible substrate from the mouse skull.Apply a layer of dental cement to the skull surface approximately one millimeter thick and allow the cement to cure for approximately 30 minutes. Align the flexible substrate in line with the small circular marks on the surface of the skull. Then position the tip of a dental drill on each electrode pad hole on the flexible substrate and carefully drill into the skull.Using a screwdriver, secure the miniature screw electrodes into the drill holes of the skull. Crimp the head of the screw electrode and the electrode pad firmly. Finally, measure the conductance between each screw electrode and the connector with testing equipment, such as an LCR meter to confirm electrical conductivity.

Electroencephalographic (EEG) Signal Recording System and Procedure

Electromyography (EMG) Electrode Placement

To begin, instruct the participant to sit in the experimental chair with their elbows resting on the arms of the chair and bent to allow the wrist or hand to rest comfortably on the desk workplace. Adjust the height of the chair and desk workplace as needed. Clean the skin over the abductor pollicis brevis or APB, first dorsal interosseous, or FDI and the ulnar styloid process, using a mildly abrasive cream placed on a round cotton pad.Wipe away any residue using an alcohol prep pad. For each muscle, place one disposable silver silver chloride adhesive electrode over the muscle belly. Place a second electrode on a nearby bony landmark as a reference.Finally, place one additional silver silver chloride adhesive electrode on the ulnar styloid process to serve as a ground. Connect each pair of electrodes and the ground to the electromyography or EMG amplifier and data acquisition system. Use channel one for the first dorsal interosseous and channel two for the abductor pollicis brevis.