Play fighting in the rat involves attack and defense of the nape of the neck, which if contacted, is gently nuzzled with the snout. Because the movements of one animal are countered by the actions of its partner, play fighting is a complex, dynamic interaction. This dynamic complexity raises methodological problems about what to score for experimental studies. We present a scoring schema that is sensitive to the correlated nature of the actions performed. The frequency of play fighting can be measured by counting the number of playful nape attacks occurring per unit time. However, playful defense, as it can only occur in response to attack, is necessarily a contingent measure that is best measured as a percentage (#attacks defended/total # attacks X 100%). How a particular attack is defended against can involve one of several tactics, and these are contingent on defense having taken place; consequently, the type of defense is also best expressed contingently as a percentage. Two experiments illustrate how these measurements can be used to detect the effect of brain damage on play fighting even when there is no effect on overall playfulness. That is, the schema presented here is designed to detect and evaluate changes in the content of play following an experimental treatment.
23 Related JoVE Articles!
Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children
Institutions: Swiss Tropical and Public Health Institute, Basel, Switzerland, University of Basel, Basel, Switzerland.
Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2
. Ascaris lumbricoides
, Trichuris trichiura
, and the two hookworm species, Ancylostoma duodenale
and Necator americanus
, are the most prevalent STHs3
. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4
. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis
, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6
. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8
, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis
, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis
larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9
. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10
. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12
. This line of scientific inquiry gained new momentum recently13,14,15
. The 20-meter (m) shuttle run test was developed and validated by Léger et al.16
and is used worldwide to measure the aerobic fitness of children17
. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13
. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.
Infection, Issue 66, Immunology, Medicine, Infectious Diseases, Soil-transmitted helminths, physical fitness, Kato-Katz technique, Baermann technique, 20-meter shuttle run test, children
Guidelines for Elective Pediatric Fiberoptic Intubation
Institutions: St. Jude Children's Research Hospital, Children's Hospital of Michigan, Children's Hospital of Michigan.
Fiberoptic intubation in pediatric patients is often required especially in difficult airways of syndromic patients i.e. Pierre Robin Syndrome. Small babies will desaturate very quickly if ventilation is interrupted mainly to high metabolic rate. We describe guidelines to perform a safe fiberoptic intubation while maintaining spontaneous breathing throughout the procedure. Steps requiring the use of propofol pump, fentanyl, glycopyrrolate, red rubber catheter, metal insuflation hook, afrin, lubricant and lidocaine spray are shown.
Medicine, Issue 47, Fiberoptic, Intubation, Pediatric, elective
A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome
Institutions: Université Claude Bernard Lyon, CNRS UMR 5534.
Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans
is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans
genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (≥75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation ≤60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (≥80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.
Basic Protocol, Issue 82, Caenorhabditis elegans, UV-mediated transgene integration, transgenic worms, irradiation, extrachromosomal, fluorescent
The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy
Institutions: University of Southampton.
Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.
Chemistry, Issue 85, gas chromatography, fatty acid, pregnancy, cholesteryl ester, solid phase extraction, polyunsaturated fatty acids
Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer
Institutions: Stanford University .
In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1
Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5
Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.
Bioengineering, Issue 69, CyTOF, mass cytometry, PBMCs, ICP-MS, multiparametric
Using Microwave and Macroscopic Samples of Dielectric Solids to Study the Photonic Properties of Disordered Photonic Bandgap Materials
Institutions: San Francisco State University.
Recently, disordered photonic materials have been suggested as an alternative to periodic crystals for the formation of a complete photonic bandgap (PBG). In this article we will describe the methods for constructing and characterizing macroscopic disordered photonic structures using microwaves. The microwave regime offers the most convenient experimental sample size to build and test PBG media. Easily manipulated dielectric lattice components extend flexibility in building various 2D structures on top of pre-printed plastic templates. Once built, the structures could be quickly modified with point and line defects to make freeform waveguides and filters. Testing is done using a widely available Vector Network Analyzer and pairs of microwave horn antennas. Due to the scale invariance property of electromagnetic fields, the results we obtained in the microwave region can be directly applied to infrared and optical regions. Our approach is simple but delivers exciting new insight into the nature of light and disordered matter interaction.
Our representative results include the first experimental demonstration of the existence of a complete and isotropic PBG in a two-dimensional (2D) hyperuniform disordered dielectric structure. Additionally we demonstrate experimentally the ability of this novel photonic structure to guide electromagnetic waves (EM) through freeform waveguides of arbitrary shape.
Physics, Issue 91, optics and photonics, photonic crystals, photonic bandgap, hyperuniform, disordered media, waveguides
Paired Whole Cell Recordings in Organotypic Hippocampal Slices
Institutions: University of Auckland, Stanford University.
Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.
Neuroscience, Issue 91, hippocampus, paired recording, whole cell recording, organotypic slice, synapse, synaptic transmission, synaptic plasticity
Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread
Institutions: Montana State University, Princeton University.
Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.
Virology, Issue 78, Infection, Immunology, Medicine, Molecular Biology, Cellular Biology, Microbiology, Genetics, Microscopy, Fluorescence, Neurobiology, Herpes virus, fluorescent protein, epifluorescent microscopy, neuronal culture, axon, virion, video microscopy, virus, live cell, imaging
Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation
Institutions: Department of Medicine, Cardiovascular Division, Cornell University.
In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.1
On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.2
Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.3
To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.
Biophysics, Issue 55, nuclear envelope, nuclear stiffness, nucleo-cytoskeletal coupling, lamin, nesprin, cytoskeleton, biomechanics, nuclear deformation, force transmission
Transmitting Plant Viruses Using Whiteflies
Institutions: University of Florida .
Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci
, a complex of morphologically indistinquishable species5
, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus
) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16
). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17
). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13
, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14
, new cultural approaches1,4,19
, or the selection and development of resistant cultivars7,8,18
, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7
. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.
Plant Biology, Issue 81, Virology, Molecular Biology, Botany, Pathology, Infection, Plant viruses, Bemisia tabaci, Whiteflies, whitefly, insect transmission, Begomovirus, Carlavirus, Crinivirus, Ipomovirus, host pathogen interaction, virus, insect, plant
Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG
Institutions: Macquarie University.
Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity . The electrophysiological activity of the brain generates electric fields - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 °C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.
Neuroscience, Issue 36, Magnetoencephalography, Pediatrics, Brain Mapping, Language, Brain Development, Cognitive Neuroscience, Language Acquisition, Linguistics
Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism
Institutions: University of Calgary Faculty of Medicine, Trinity College.
Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T+ Itpr3tf
/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as "inchworming," was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.
Behavior, Issue 89, mice, inbred C57BL, social behavior, animal models, autism, BTBR, motor stereotypy, repetitive
Portable Intermodal Preferential Looking (IPL): Investigating Language Comprehension in Typically Developing Toddlers and Young Children with Autism
Institutions: University of Connecticut.
One of the defining characteristics of autism spectrum disorder (ASD) is difficulty with language and communication.1
Children with ASD's onset of speaking is usually delayed, and many children with ASD consistently produce language less frequently and of lower lexical and grammatical complexity than their typically developing (TD) peers.6,8,12,23
However, children with ASD also exhibit a significant social deficit, and researchers and clinicians continue to debate the extent to which the deficits in social interaction account for or contribute to the deficits in language production.5,14,19,25
Standardized assessments of language in children with ASD usually do include a comprehension component; however, many such comprehension tasks assess just one aspect of language (e.g.
or include a significant motor component (e.g.
, pointing, act-out), and/or require children to deliberately choose between a number of alternatives. These last two behaviors are known to also be challenging to children with ASD.7,12,13,16
We present a method which can assess the language comprehension of young typically developing children (9-36 months) and children with autism.2,4,9,11,22
This method, Portable Intermodal Preferential Looking (P-IPL), projects side-by-side video images from a laptop onto a portable screen. The video images are paired first with a 'baseline' (nondirecting) audio, and then presented again paired with a 'test' linguistic audio that matches only one of the video images. Children's eye movements while watching the video are filmed and later coded. Children who understand the linguistic audio will look more quickly to, and longer at, the video that matches the linguistic audio.2,4,11,18,22,26
This paradigm includes a number of components that have recently been miniaturized (projector, camcorder, digitizer) to enable portability and easy setup in children's homes. This is a crucial point for assessing young children with ASD, who are frequently uncomfortable in new (e.g.
, laboratory) settings. Videos can be created to assess a wide range of specific components of linguistic knowledge, such as Subject-Verb-Object word order, wh-questions, and tense/aspect suffixes on verbs; videos can also assess principles of word learning such as a noun bias, a shape bias, and syntactic bootstrapping.10,14,17,21,24
Videos include characters and speech that are visually and acoustically salient and well tolerated by children with ASD.
Medicine, Issue 70, Neuroscience, Psychology, Behavior, Intermodal preferential looking, language comprehension, children with autism, child development, autism
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
Measuring Attentional Biases for Threat in Children and Adults
Institutions: Rutgers University.
Investigators have long been interested in the human propensity for the rapid detection of threatening stimuli. However, until recently, research in this domain has focused almost exclusively on adult participants, completely ignoring the topic of threat detection over the course of development. One of the biggest reasons for the lack of developmental work in this area is likely the absence of a reliable paradigm that can measure perceptual biases for threat in children. To address this issue, we recently designed a modified visual search paradigm similar to the standard adult paradigm that is appropriate for studying threat detection in preschool-aged participants. Here we describe this new procedure. In the general paradigm, we present participants with matrices of color photographs, and ask them to find and touch a target on the screen. Latency to touch the target is recorded. Using a touch-screen monitor makes the procedure simple and easy, allowing us to collect data in participants ranging from 3 years of age to adults. Thus far, the paradigm has consistently shown that both adults and children detect threatening stimuli (e.g.,
snakes, spiders, angry/fearful faces) more quickly than neutral stimuli (e.g.,
flowers, mushrooms, happy/neutral faces). Altogether, this procedure provides an important new tool for researchers interested in studying the development of attentional biases for threat.
Behavior, Issue 92, Detection, threat, attention, attentional bias, anxiety, visual search
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking
Institutions: Cornell University, University of Chicago, Manesar, India.
Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor & Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer & Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al.
, 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed & Clark, 2002).
Neuroscience, Issue 46, High-density EEG, ERP, ICA, gaze tracking, computer game, ecological validity
Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR
Institutions: Spedali Civili di Brescia.
T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106
cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
Immunology, Issue 94, B lymphocytes, primary immunodeficiency, real-time PCR, immune recovery, T-cell homeostasis, T lymphocytes, thymic output, bone marrow output
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
Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases
Institutions: Children's National Medical Center, George Washington University School of Medicine and Health Sciences.
The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing locomotive impairment in animal models of neuromuscular disease and efficacy of therapeutic drugs that may improve locomotion and/or muscle function. The open field activity measurement provides a different measure than muscle strength, which is commonly assessed by grip strength measurements. It can also show how drugs may affect other body systems as well when used with additional outcome measures. In addition, measures such as total distance traveled mirror the 6 min walk test, a clinical trial outcome measure. However, open field activity monitoring is also associated with significant challenges: Open field activity measurements vary according to animal strain, age, sex, and circadian rhythm. In addition, room temperature, humidity, lighting, noise, and even odor can affect assessment outcomes. Overall, this manuscript provides a well-tested and standardized open field activity SOP for preclinical trials in animal models of neuromuscular diseases. We provide a discussion of important considerations, typical results, data analysis, and detail the strengths and weaknesses of open field testing. In addition, we provide recommendations for optimal study design when using open field activity in a preclinical trial.
Behavior, Issue 91, open field activity, functional testing, behavioral testing, skeletal muscle, congenital muscular dystrophy, muscular dystrophy
A Method for Investigating Age-related Differences in the Functional Connectivity of Cognitive Control Networks Associated with Dimensional Change Card Sort Performance
Institutions: University of Western Ontario.
The ability to adjust behavior to sudden changes in the environment develops gradually in childhood and adolescence. For example, in the Dimensional Change Card Sort task, participants switch from sorting cards one way, such as shape, to sorting them a different way, such as color. Adjusting behavior in this way exacts a small performance cost, or switch cost, such that responses are typically slower and more error-prone on switch trials in which the sorting rule changes as compared to repeat trials in which the sorting rule remains the same. The ability to flexibly adjust behavior is often said to develop gradually, in part because behavioral costs such as switch costs typically decrease with increasing age. Why aspects of higher-order cognition, such as behavioral flexibility, develop so gradually remains an open question. One hypothesis is that these changes occur in association with functional changes in broad-scale cognitive control networks. On this view, complex mental operations, such as switching, involve rapid interactions between several distributed brain regions, including those that update and maintain task rules, re-orient attention, and select behaviors. With development, functional connections between these regions strengthen, leading to faster and more efficient switching operations. The current video describes a method of testing this hypothesis through the collection and multivariate analysis of fMRI data from participants of different ages.
Behavior, Issue 87, Neurosciences, fMRI, Cognitive Control, Development, Functional Connectivity
Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making
Institutions: University of Zurich, Royal Holloway, University of London.
Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1
and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2
. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4
. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-β-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.
Neuroscience, Issue 49, behavioral endocrinology, testosterone, social status, decision making
Making MR Imaging Child's Play - Pediatric Neuroimaging Protocol, Guidelines and Procedure
Institutions: Children’s Hospital Boston, University of Zurich, Harvard, Harvard Medical School.
Within the last decade there has been an increase in the use of structural and functional magnetic resonance imaging (fMRI) to investigate the neural basis of human perception, cognition and behavior 1, 2
. Moreover, this non-invasive imaging method has grown into a tool for clinicians and researchers to explore typical and atypical brain development. Although advances in neuroimaging tools and techniques are apparent, (f)MRI in young pediatric populations remains relatively infrequent 2
. Practical as well as technical challenges when imaging children present clinicians and research teams with a unique set of problems 3, 2
. To name just a few, the child participants are challenged by a need for motivation, alertness and cooperation. Anxiety may be an additional factor to be addressed. Researchers or clinicians need to consider time constraints, movement restriction, scanner background noise and unfamiliarity with the MR scanner environment2,4-10
. A progressive use of functional and structural neuroimaging in younger age groups, however, could further add to our understanding of brain development. As an example, several research groups are currently working towards early detection of developmental disorders, potentially even before children present associated behavioral characteristics e.g.11
. Various strategies and techniques have been reported as a means to ensure comfort and cooperation of young children during neuroimaging sessions. Play therapy 12
, behavioral approaches 13, 14,15, 16-18
and simulation 19
, the use of mock scanner areas 20,21
, basic relaxation 22
and a combination of these techniques 23
have all been shown to improve the participant's compliance and thus MRI data quality. Even more importantly, these strategies have proven to increase the comfort of families and children involved 12
. One of the main advances of such techniques for the clinical practice is the possibility of avoiding sedation or general anesthesia (GA) as a way to manage children's compliance during MR imaging sessions 19,20
. In the current video report, we present a pediatric neuroimaging protocol with guidelines and procedures that have proven to be successful to date in young children.
Neuroscience, Issue 29, fMRI, imaging, development, children, pediatric neuroimaging, cognitive development, magnetic resonance imaging, pediatric imaging protocol, patient preparation, mock scanner