Name: electrophysiological measurements and analysis of nociception in human infants
Uploaded: 2011-12-20T12:00:00
Duration: 9 min 18 s
Description: Watch this Scientific Journal Video about Electrophysiological Measurements and Analysis of Nociception in Human Infants at JoVE.com

This work was funded by the Wellcome Trust.

Before any studies can be conducted on infants' ethical approval and informed written parental consent needs to be sought.
In this study ethical approval was obtained from the University College Hospital Ethics Committee and informed written parental consent was obtained before each procedure. This study conformed to the standards set by the Declaration of Helsinki and Good Clinical Practice guidelines.
1. Data collection - setup
<ol>
 <li>Place a minimum of 16 individual disposable EEG Ag/AgCl cup electrodes on the infant's head after skin prepping according to the 10-20 electrode placement system (Figure 1A). More comprehensive coverage can be achieved using an EEG cap with embedded electrodes. Use of an EEG cap makes the process faster and less disruptive, particularly as electrodes are prepared with inductive gel before placing the cap on the head. The application of disposable EEG electrodes requires more time and skill, but generally results in a better recording. Consider reducing the number of electrodes if access to the infant is limited, but always use midline electrodes (Cz, CPz and FCz). Use FCz as the reference electrode for the recording.</li>
 <li>Use EEG conductive paste to optimise electrode/skin electric coupling.</li>
 <li>Place an elastic net over the electrodes to hold them in place.</li>
 <li>Tie the electrode leads together to minimise electrical interference.</li>
 <li>Place a ground electrode onto the chest or head.</li>
 <li>Place EMG electrodes on the biceps femoris of both legs after skin prepping (Figure 1B).</li>
 <li>To record ECG activity place lead 1 ECG electrodes onto the body after skin prepping (one electrode on the left side of the chest, one on the right and use the same ground electrode as for the EEG).</li>
 <li>Place a movement transducer on the abdomen to measure respiration.</li>
 <li>Place a pulse oximeter probe on the foot to measure oxygen saturation and heart rate. Make sure that the probe is secured in place and that a continuous signal is recorded without dropouts. The oximeter probe needs to be placed on the foot that is contralateral to the foot which you intend to stimulate.</li>
 <li>Set up a tripod-mounted camcorder to frame the face of the infant so that changes in facial expression can be recorded.</li>
 <li>Place a light-emitting diode (LED) in the camera frame. The LED is linked to the timing circuit so that it will flash when stimulation is presented to synchronise the EEG, EMG and video recording.</li>
</ol>
2. Data collection - recording
<ol>
 <li>Start video recording.</li>
 <li>Start pulse oximetry recording.</li>
 <li>Start EEG/EMG recording.</li>
 <li>Wait until the infant is settled.</li>
 <li>Hold the foot as if performing a heel lance and manually event mark the EEG and EMG recordings. This epoch will be used to identify a section of control background EEG and EMG.</li>
 <li>Apply touch stimulation by lightly tapping a rubber bung that is attached to the arm of a tendon hammer against the surface of the heel. Stimulate the foot that is not attached to the pulse oximeter. When the infant is stimulated the EEG/EMG and video recording must be event-marked in order to identify the time in the recording when the stimulation took place. The touch stimulation can be event-marked by attaching an impedance head to the tendon hammer which electronically links the stimulator to the recording equipment. The video recording is event-marked by the LED flash. Repeated touches may be applied and the stimulus can be applied to different regions of the body, i.e. the shoulder</li>
 <li>Apply a non-noxious control stimulation by rotating the lancet by 90 degrees and placing it against the foot so that when the spring-loaded blade is released it does not contact the skin. This event can be time-locked using an accelerometer attached to the top surface of the lance. The accelerometer detects the vibration that occurs when the blade is released.</li>
 <li>Perform the clinically-essential heel lance in accordance with clinical practice in the neonatal unit. Wait until the EEG activity is settled before doing the heel lance. Time-locking of the heel lance can be performed in the same way as for the control stimulation. Following the heel lance, do not squeeze the foot for at least 30 seconds to ensure that the recorded responses are solely due to the lance.</li>
 <li>Ensure that the required quantity of blood is collected and prepare the samples for clinical analysis.</li>
 <li>Save the data and stop all the recordings.</li>
 <li>Record the infant's demographic information and the experimental details and input them into a database for safe storage and future reference.</li>
 <li>Repeat this procedure in the required sample of infants. In this example the number of infants = 23.</li>
</ol>
3. EEG data analysis
<ol>
 <li>Create EEG epochs of 1.7 seconds that correspond to each touch, control and lance stimulation and background EEG. Epochs should start 0.6 seconds before each event. The number of epochs corresponding to each modality should be the same.</li>
 <li>Baseline correct the epochs by subtracting the mean baseline signal and high-pass filter them at 0.1 Hz.</li>
 <li>Consider the epochs recorded at CPz or Cz for further analysis and exclude epochs which were contaminated by movement artefact. Movement artefact is defined as a change in amplitude greater than 50&mu;V in less than 50ms.</li>
 <li>Align the traces to correct for latency jitter between 50 and 300 ms post stimulation and conduct Principal Component Analysis (PCA) in this time interval to identify the tactile potential (i.e. EEG activity related to tactile stimulation). Consider the epochs to be the variables and the time points the observations. PCA decomposes the EEG epochs into basic waveforms, termed principal components (PCs) and represent systematic variation in the amplitude of the signal across time points.</li>
 <li>Run one-way analysis of variance (ANOVA) on the weights of each of the first 2 PCs to determine which PC represents the tactile potential. This will be the PC whose weights were significantly larger following tactile stimulation compared to background EEG.</li>
 <li>Align the traces to correct for latency jitter between 300 and 700 ms post stimulation and conduct PCA in this time interval.</li>
 <li>Run one-way ANOVA on the weights of each of the first 2 PCs to determine which PC represents the nociceptive-specific potential. This will be the PC whose weights were significantly larger following noxious stimulation compared to tactile stimulation and background EEG.</li>
</ol>
4. EMG data analysis
<ol>
 <li>Calculate the root-mean-square (RMS) of the EMG signal in the first 1000 ms post stimulation for the control and lance stimuli.</li>
 <li>Perform a t-test on the RMS values to determine the nociceptive-specific spinal reflex withdrawal.</li>
</ol>











5. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3118/3118fig1.jpg" /> 
Figure 1. (A) (i) Diagram and (ii) photo of the electrode placements for EEG recordings (modified international 10/20 electrode placement system); (B) Diagram of the electrode placements for EMG recordings on the biceps femoris.
<img alt="Figure 2" src="/files/ftp_upload/3118/3118fig2.jpg" /> 
 Figure 2. (A) Examples of the sensory potential at Cz evoked by touch in 3 infants; (B) examples of the nociceptive-specific potential at Cz evoked by noxious lance in 3 infants. 
<img alt="Figure 3" src="/files/ftp_upload/3118/3118fig3.jpg" /> 
Figure 3. Dependence of the PC weights on stimulus modality at Cz (mean &plusmn; SEM). The PC obtained between 50-300 ms after the stimulus onset represents a tactile potential and the PC obtained between 300-700 ms after the stimulus onset represents a nociceptive-specific potential. The PCs (bold lines) are overlaid on the grand averages obtained across all stimulation types (background EEG, touch, noxious lance) after the individual traces were aligned in the stated time interval.
<img alt="Figure 4" src="/files/ftp_upload/3118/3118fig4.jpg" /> 
 Figure 4. (A) Example of EMG activity in one infant after (i) noxious heel lance and (ii) touch of the heel. (B) Mean (&plusmn;SE) EMG root-mean-square EMG activity in infants after the noxious heel and non-noxious touch stimuli.

<ol>
	<li>Fitzgerald, M., Walker, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infant+pain+management:+a+developmental+neurobiological+approach.">Infant pain management: a developmental neurobiological approach.</a> Nat. Clin. Pract. Neurol. 5 (1), 35-35 (2009).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+sucrose+as+an+analgesic+drug+for+procedural+pain+in+newborn+infants:+a+randomised+controlled+trial.">Oral sucrose as an analgesic drug for procedural pain in newborn infants: a randomised controlled trial.</a> Lancet. 376 (9748), 1225-1225 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evoked+potentials+generated+by+noxious+stimulation+in+the+human+infant+brain.">Evoked potentials generated by noxious stimulation in the human infant brain.</a> Eur. J. Pain. 14 (3), 321-321 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+infants+display+increased+noxious-evoked+neuronal+activity+in+the+brain+compared+to+healthy+age-matched+term-born+infants.">Premature infants display increased noxious-evoked neuronal activity in the brain compared to healthy age-matched term-born infants.</a> Neuroimage. 52 (2), 583-583 (2010).</li></ol>

The authors declare that they have nothing to disclose.

This video shows how electrophysiological responses, evoked by tactile and noxious stimulation, can be characterised in the human infant using EEG and EMG recordings. Studies of this type will help to understand the development and plasticity of human pain processing, and lead to improved clinical assessment and treatment of infant pain 1, 2.
The success of these experiments requires the close collaboration of a multidisciplinary team. Expertise is required in the field of neonatology, clinical neurophysiology, bioengineering and developmental neuroscience. 
It is essential that the infant's care take highest priority when these experiments are undertaken. The research nurse or doctor who performs the heel lance has responsibility for the infant's wellbeing and needs to ensure that the experiment is done in accordance with clinical practice. Approval from the hospital ethical committee and written parental consent are necessary to undertake these studies.
The heel lance is a clinically-required event and cannot be repeated for the purpose of the study. It is therefore essential that the recording is collected in a reliable and robust way that does not interfere with clinical practice 3, 4. In particular, it is crucial that the method used to time-lock the stimulation is reliable and does not impede the clinical blood collection.
All the measures of infant pain are necessarily indirect, as such it is important to ascertain that the recorded activity is specifically evoked by the noxious event and not other aspects of the infant's care 2. This can be achieved via careful technical design and post-processing of the data.

electrophysiological measurements and analysis of nociception in human infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

Watch this Scientific Journal Video about Electrophysiological Measurements and Analysis of Nociception in Human Infants at JoVE.com

Electrophysiological Measurements and Analysis of Nociception in Human Infants

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

electrophysiological-measurements-analysis-nociception-human

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Neuroscience

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...