There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.
24 Related JoVE Articles!
The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia
Institutions: Stanford University School of Medicine.
Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1
and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2
and to test potential therapeutic interventions 3,4
. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5
, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6
. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.
Neuroscience, Issue 21, Hypoxic-ischemic encephalopathy (HIE), 2 3 5-triphenyltetrazolium chloride (TTC), brain infarct
Forebrain Electrophysiological Recording in Larval Zebrafish
Institutions: University of California, San Francisco .
Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3
that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 μl during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo
screening of test compounds4
. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7
. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e.
, seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.
Developmental Biology, Issue 71, Neuroscience, Anatomy, Physiology, Neurobiology, Cellular Biology, Molecular Biology, Surgery, Seizure, development, telencephalon, electrographic, extracellular, field recording, in vivo, electrophysiology, neuron, activity, microsurgery, micropipette, epilepsy, Danio rerio, zebrafish, zebrafish larvae
P50 Sensory Gating in Infants
Institutions: University of Colorado School of Medicine, Colorado State University.
Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating.
Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e.
the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits.
Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.
Behavior, Issue 82, Child Development, Psychophysiology, Attention Deficit and Disruptive Behavior Disorders, Evoked Potentials, Auditory, auditory evoked potential, sensory gating, infant, attention, electrophysiology, infants, sensory gating, endophenotype, attention, P50
A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila
Institutions: Franciscan University of Steubenville, Franciscan University of Steubenville.
Video tracking systems have been used widely to analyze Drosophila melanogaster
movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster
. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster
mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless
) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.
Neuroscience, Issue 84, Drosophila melanogaster, movement tracking, seizures, video analysis, locomotion, metformin, behavior, seizure-like activity
Cortical Source Analysis of High-Density EEG Recordings in Children
Institutions: UCL Institute of Child Health, University College London.
EEG is traditionally described as a neuroimaging technique with high temporal and low spatial resolution. Recent advances in biophysical modelling and signal processing make it possible to exploit information from other imaging modalities like structural MRI that provide high spatial resolution to overcome this constraint1
. This is especially useful for investigations that require high resolution in the temporal as well as spatial domain. In addition, due to the easy application and low cost of EEG recordings, EEG is often the method of choice when working with populations, such as young children, that do not tolerate functional MRI scans well. However, in order to investigate which neural substrates are involved, anatomical information from structural MRI is still needed. Most EEG analysis packages work with standard head models that are based on adult anatomy. The accuracy of these models when used for children is limited2
, because the composition and spatial configuration of head tissues changes dramatically over development3
In the present paper, we provide an overview of our recent work in utilizing head models based on individual structural MRI scans or age specific head models to reconstruct the cortical generators of high density EEG. This article describes how EEG recordings are acquired, processed, and analyzed with pediatric populations at the London Baby Lab, including laboratory setup, task design, EEG preprocessing, MRI processing, and EEG channel level and source analysis.
Behavior, Issue 88, EEG, electroencephalogram, development, source analysis, pediatric, minimum-norm estimation, cognitive neuroscience, event-related potentials
Simultaneous EEG Monitoring During Transcranial Direct Current Stimulation
Institutions: Universidade Federal do Rio Grande do Sul, Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Harvard Medical School, De Montfort University.
Transcranial direct current stimulation (tDCS) is a technique that delivers weak electric currents through the scalp. This constant electric current induces shifts in neuronal membrane excitability, resulting in secondary changes in cortical activity. Although tDCS has most of its neuromodulatory effects on the underlying cortex, tDCS effects can also be observed in distant neural networks. Therefore, concomitant EEG monitoring of the effects of tDCS can provide valuable information on the mechanisms of tDCS. In addition, EEG findings can be an important surrogate marker for the effects of tDCS and thus can be used to optimize its parameters. This combined EEG-tDCS system can also be used for preventive treatment of neurological conditions characterized by abnormal peaks of cortical excitability, such as seizures. Such a system would be the basis of a non-invasive closed-loop device. In this article, we present a novel device that is capable of utilizing tDCS and EEG simultaneously. For that, we describe in a step-by-step fashion the main procedures of the application of this device using schematic figures, tables and video demonstrations. Additionally, we provide a literature review on clinical uses of tDCS and its cortical effects measured by EEG techniques.
Behavior, Issue 76, Medicine, Neuroscience, Neurobiology, Anatomy, Physiology, Biomedical Engineering, Psychology, electroencephalography, electroencephalogram, EEG, transcranial direct current stimulation, tDCS, noninvasive brain stimulation, neuromodulation, closed-loop system, brain, imaging, clinical techniques
Preterm EEG: A Multimodal Neurophysiological Protocol
Institutions: University of Helsinki , University of Helsinki , University of Helsinki , University of Helsinki .
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.
Neuroscience, Issue 60, neurophysiology, preterm baby, neonatal, EEG, evoked response, high density EEG, FbEEG, sensory evoked response, neonatal intensive care unit
EEG Mu Rhythm in Typical and Atypical Development
Institutions: University of Washington, University of Washington.
Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal resolution, EEG can be used to examine the neural response related to specific behaviors, states, or external stimuli. An example of this utility is the assessment of the mirror neuron system (MNS) in humans through the examination of the EEG mu rhythm. The EEG mu rhythm, oscillatory activity in the 8-12 Hz frequency range recorded from centrally located electrodes, is suppressed when an individual executes, or simply observes, goal directed actions. As such, it has been proposed to reflect activity of the MNS. It has been theorized that dysfunction in the mirror neuron system (MNS) plays a contributing role in the social deficits of autism spectrum disorder (ASD). The MNS can then be noninvasively examined in clinical populations by using EEG mu rhythm attenuation as an index for its activity. The described protocol provides an avenue to examine social cognitive functions theoretically linked to the MNS in individuals with typical and atypical development, such as ASD.
Medicine, Issue 86, Electroencephalography (EEG), mu rhythm, imitation, autism spectrum disorder, social cognition, mirror neuron system
A Neuroscientific Approach to the Examination of Concussions in Student-Athletes
Institutions: Elon University, Elon University, Duquesne University, Elon University.
Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement “may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an ‘impulsive’ force transmitted to the head.” Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual’s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.
Medicine, Issue 94, Concussions, Student-Athletes, Mild Traumatic Brain Injury, Genetics, Cognitive Function, Balance, Gait, Somatosensory
Simultaneous Scalp Electroencephalography (EEG), Electromyography (EMG), and Whole-body Segmental Inertial Recording for Multi-modal Neural Decoding
Institutions: National Institutes of Health, University of Houston, University of Houston, University of Houston, University of Houston.
Recent studies support the involvement of supraspinal networks in control of bipedal human walking. Part of this evidence encompasses studies, including our previous work, demonstrating that gait kinematics and limb coordination during treadmill walking can be inferred from the scalp electroencephalogram (EEG) with reasonably high decoding accuracies. These results provide impetus for development of non-invasive brain-machine-interface (BMI) systems for use in restoration and/or augmentation of gait- a primary goal of rehabilitation research. To date, studies examining EEG decoding of activity during gait have been limited to treadmill walking in a controlled environment. However, to be practically viable a BMI system must be applicable for use in everyday locomotor tasks such as over ground walking and turning. Here, we present a novel protocol for non-invasive collection of brain activity (EEG), muscle activity (electromyography (EMG)), and whole-body kinematic data (head, torso, and limb trajectories) during both treadmill and over ground walking tasks. By collecting these data in the uncontrolled environment insight can be gained regarding the feasibility of decoding unconstrained gait and surface EMG from scalp EEG.
Behavior, Issue 77, Neuroscience, Neurobiology, Medicine, Anatomy, Physiology, Biomedical Engineering, Molecular Biology, Electroencephalography, EEG, Electromyography, EMG, electroencephalograph, gait, brain-computer interface, brain machine interface, neural decoding, over-ground walking, robotic gait, brain, imaging, clinical techniques
Mouse Models of Periventricular Leukomalacia
Institutions: University of California, Davis.
We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.
JoVE Neuroscience, Issue 39, brain, mouse, white matter injury, oligodendrocyte, periventricular leukomalacia
Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex
Institutions: Washington State University.
Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1
. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4
. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6
. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7
. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8
; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.
Neuroscience, Issue 70, Physiology, Anatomy, Medicine, Pharmacology, Surgery, Sleep, rapid eye movement, glucose, glycolysis, pyramidal neurons, channelrhodopsin, optogenetics, optogenetic stimulation, electroencephalogram, EEG, EMG, brain, animal model
Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants
Institutions: Massachusetts General Hospital, Harvard Medical School, Université de Caen Basse-Normandie, Boston Children's Hospital, Harvard Medical School, ISS, INC..
Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2
). Thus, measures of CMRO2
are reflective of neuronal viability and provide critical diagnostic information, making CMRO2
an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2
) as a surrogate for cerebral oxygen consumption. However, SO2
is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2
are not sensitive enough to detect brain injury hours after the insult 1,2
, because oxygen consumption and delivery reach equilibrium after acute transients 3
. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2
with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi
) to yield an index of CMRO2
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.
Medicine, Issue 73, Developmental Biology, Neurobiology, Neuroscience, Biomedical Engineering, Anatomy, Physiology, Near infrared spectroscopy, diffuse correlation spectroscopy, cerebral hemodynamic, cerebral metabolism, brain injury screening, brain health, brain development, newborns, neonates, imaging, clinical techniques
Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue
Institutions: CNRS UMR 7241, INSERM U1050, Collège de France, Paris Descartes University, Sorbonne Paris Cité, CEA, Paris Descartes University, Paris Descartes University, La Pitié-Salpêtrière Hospital, AP-HP, Sorbonne and Pierre and Marie Curie University.
Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo
Medicine, Issue 92, electrophysiology, multi-electrode array, human tissue, slice, epilepsy, neocortex
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1
, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2
(Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3
. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5
. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8
. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9
, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8
. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Performing Behavioral Tasks in Subjects with Intracranial Electrodes
Institutions: Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Johns Hopkins University.
Patients having stereo-electroencephalography (SEEG) electrode, subdural grid or depth electrode implants have a multitude of electrodes implanted in different areas of their brain for the localization of their seizure focus and eloquent areas. After implantation, the patient must remain in the hospital until the pathological area of brain is found and possibly resected. During this time, these patients offer a unique opportunity to the research community because any number of behavioral paradigms can be performed to uncover the neural correlates that guide behavior. Here we present a method for recording brain activity from intracranial implants as subjects perform a behavioral task designed to assess decision-making and reward encoding. All electrophysiological data from the intracranial electrodes are recorded during the behavioral task, allowing for the examination of the many brain areas involved in a single function at time scales relevant to behavior. Moreover, and unlike animal studies, human patients can learn a wide variety of behavioral tasks quickly, allowing for the ability to perform more than one task in the same subject or for performing controls. Despite the many advantages of this technique for understanding human brain function, there are also methodological limitations that we discuss, including environmental factors, analgesic effects, time constraints and recordings from diseased tissue. This method may be easily implemented by any institution that performs intracranial assessments; providing the opportunity to directly examine human brain function during behavior.
Behavior, Issue 92, Cognitive neuroscience, Epilepsy, Stereo-electroencephalography, Subdural grids, Behavioral method, Electrophysiology
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool
Institutions: Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation.
Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability. The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed. The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing. The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient’s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing. This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research.
Medicine, Issue 88, Multiple Sclerosis, Multiple Sclerosis Functional Composite, computer-based testing, 25-foot walk test, 9-hole peg test, Symbol Digit Modalities Test, Low Contrast Visual Acuity, Clinical Outcome Measure
Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Institutions: University of New Mexico Health Sciences Center.
Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, model
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila
Institutions: University of California, Berkeley, University of California, Berkeley.
is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.
Neuroscience, Issue 26, elecrophysiology, Drosophila, seizures, epilepsy, giant fiber
In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy
Institutions: University of California, Berkeley.
Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37°C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.
Immunology, Issue 11, 2-photon microscopy, neonatal chimera, adoptive transfer, thymus
Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus
Institutions: University of Utah.
Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4
. In animal models, it is valuable to compare
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5
, following exercise 7
and exposure to air pollutants 8
, as well as determination of the antiarrhythmic efficacy of pharmacological
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min
, and results in mortality in 20% of cases 13
. Many individuals survive the SE, but die within 30 days 14,15
The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these
. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature
sudden and unexpected death associated with epilepsy (SUDEP) 18
. As with SE, the precise mechanisms mediating SUDEP are not known.
It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22
To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is
necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders
. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical
activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden
Medicine, Issue 50, cardiac, seizure disorders, QTc, QTd, cardiac arrhythmias, rat