High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning. Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.
24 Related JoVE Articles!
Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition
Institutions: California Institute of Technology, Johns Hopkins University School of Medicine, Johns Hopkins University School of Medicine, University Of Washington Medical Center, Johns Hopkins University School of Medicine.
The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for C
omputer tomography-guided f
radiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.
Neuroscience, Issue 81, Neural Stem Cells (NSCs), Body Weight, Radiotherapy, Image-Guided, Metabolism, Energy Metabolism, Neurogenesis, Cell Proliferation, Neurosciences, Irradiation, Radiological treatment, Computer-tomography (CT) imaging, Hypothalamus, Hypothalamic Proliferative Zone (HPZ), Median Eminence (ME), Small Animal Radiation Research Platform (SARRP)
Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia
Institutions: University of Colorado Anschutz Medical Campus, University Hospital of Essen.
Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo
using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo
pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo
using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo
bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro
and in vivo
and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.
Medicine, Issue 79, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters
Institutions: University of California, San Diego.
For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9
. However in vitro,
genesis of insulin producing cells from human fetal ICCs is low10
; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro
genesis of insulin producing cells from hESC is much less robust11-17
. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22
, far fewer exist for ICCs10,23,24
. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6
. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.
Medicine, Issue 87, human fetal pancreas, islet cell cluster (ICC), transplantation, immunofluorescence, endocrine cell proliferation, differentiation, C-peptide
Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis
Institutions: University of Texas Medical Branch.
Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3
H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.
Immunology, Issue 78, Virology, Chemistry, Infectious Diseases, Biochemistry, Genetics, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Arboviruses, Bunyaviridae, RNA, Nuclear, Transcription, Genetic, Rift Valley fever virus, NSs, transcription, click chemistry, MP-12, fluorescence microscopy, flow cytometry, virus, proteins, immunostaining, assay
Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer
Institutions: Colorado State University.
Reeves' muntjac deer (Muntiacus reevesi
) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7
, including several species of farmed cervids8-19
, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.
Medicine, Issue 83, Ultrasound, Reeves' muntjac deer, Muntiacus reevesi, fetal development, fetal growth, captive cervids
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Institutions: University of Houston.
Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.
Medicine, Issue 84, breast cancer, microRNA, estrogen, estrogen receptor, microarray, qPCR
Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Institutions: University of Alabama at Birmingham, INRA UR1067, INRA UR1037.
Due to the inherent difficulty and time involved with studying the myogenic program in vivo
, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata,
however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.
teleost fish) and full regeneration following appendage loss (i.e.
urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio
), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae
. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum
) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4
Basic Protocol, Issue 86, myogenesis, zebrafish, myoblast, cell culture, giant danio, moustached danio, myotubes, proliferation, differentiation, Danioninae, axolotl
The 5-Choice Serial Reaction Time Task: A Task of Attention and Impulse Control for Rodents
Institutions: Oberlin College.
This protocol describes the 5-choice serial reaction time task, which is an operant based task used to study attention and impulse control in rodents. Test day challenges, modifications to the standard task, can be used to systematically tax the neural systems controlling either attention or impulse control. Importantly, these challenges have consistent effects on behavior across laboratories in intact animals and can reveal either enhancements or deficits in cognitive function that are not apparent when rats are only tested on the standard task. The variety of behavioral measures that are collected can be used to determine if other factors (i.e
., sedation, motivation deficits, locomotor impairments) are contributing to changes in performance. The versatility of the 5CSRTT is further enhanced because it is amenable to combination with pharmacological, molecular, and genetic techniques.
Neuroscience, Issue 90, attention, impulse control, neuroscience, cognition, rodent
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
Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model
Institutions: University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine, University of Kentucky College of Medicine.
Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs
-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf
transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf
animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1
. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf
fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.
Medicine, Issue 79, Skin, Inflammation, Photometry, Ultraviolet Rays, Skin Pigmentation, melanocortin 1 receptor, Mc1r, forskolin, cAMP, mean erythematous dose, skin pigmentation, melanocyte, melanin, sunburn, UV, inflammation
Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation
Institutions: University of Heidelberg , Institut de recherches cliniques de Montreal.
Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero
with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e.
the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.
Neuroscience, Issue 77, Neurobiology, Genetics, Cellular Biology, Molecular Biology, Biomedical Engineering, Developmental Biology, Anatomy, Physiology, Embryo, Mammalian, Brain, Diencephalon, Hypothalamus, Genetic Techniques, Transfection, anesthesia, development, electrodes, electroporation, in utero, mammillary body, mouse, animal model
Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction
Institutions: University Hospitals Leuven, Monash University, Victoria, Australia, Katholieke Universiteit Leuven, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER).
Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero
, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero
cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.
Medicine, Issue 76, Developmental Biology, Biomedical Engineering, Molecular Biology, Anatomy, Physiology, Cardiology, Fetal Therapies, Obstetric Surgical Procedures, Fetal Development, Surgical Procedures, Operative, intrauterine growth restriction, fetal echocardiography, Doppler ultrasound, fetal hemodynamics, animal model, clinical techniques
A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation
Institutions: Children’s Hospital of Michigan, St. Jude Children’s Research Hospital.
We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which works especially well with poor mask fit.
Can not intubate, can not ventilate" (CICV) is a potentially life threatening situation. In this video we present a simulation of the technique we used in a case of CICV where oxygenation and ventilation were maintained by inserting an endotracheal tube (ETT) nasally down to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.
A 13 year old patient was taken to the operating room for incision and drainage of a neck abcess and direct laryngobronchoscopy. After preoxygenation, anesthesia was induced intravenously. Mask ventilation was found to be extremely difficult because of the swelling of the soft tissue. The face mask could not fit properly on the face due to significant facial swelling as well. A direct laryngoscopy was attempted with no visualization of the larynx. Oxygen saturation was difficult to maintain, with saturations falling to 80%. In order to oxygenate and ventilate the patient, an endotracheal tube was then inserted nasally after nasal spray with nasal decongestant and lubricant. The tube was pushed gently and blindly into the hypopharynx. The mouth and nose of the patient were sealed by hand and positive pressure ventilation was possible with 100% O2
with good oxygen saturation during that period of time. Once the patient was stable and well sedated, a rigid bronchoscope was introduced by the otolaryngologist showing extensive subglottic and epiglottic edema, and a mass effect from the abscess, contributing to the airway compromise. The airway was secured with an ETT tube by the otolaryngologist.This video will show a simulation of the technique on a patient undergoing general anesthesia for dental restorations.
Medicine, Issue 47, difficult ventilation, difficult intubation, nasal, saturation
Making Sense of Listening: The IMAP Test Battery
Institutions: MRC Institute of Hearing Research, National Biomedical Research Unit in Hearing.
The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.
Neuroscience, Issue 44, Listening skills, auditory processing, auditory psychophysics, clinical assessment, child-friendly testing
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
Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale
Institutions: Brown University, Women & Infants Hospital of Rhode Island, University of Massachusetts, Boston.
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.
Behavior, Issue 90, NICU Network Neurobehavioral Scale, NNNS, High risk infant, Assessment, Evaluation, Prediction, Long term outcome
Eye Tracking Young Children with Autism
Institutions: University of Texas at Dallas, University of North Carolina at Chapel Hill.
The rise of accessible commercial eye-tracking systems has fueled a rapid increase in their use in psychological and psychiatric research. By providing a direct, detailed and objective measure of gaze behavior, eye-tracking has become a valuable tool for examining abnormal perceptual strategies in clinical populations and has been used to identify disorder-specific characteristics1
, promote early identification2
, and inform treatment3
. In particular, investigators of autism spectrum disorders (ASD) have benefited from integrating eye-tracking into their research paradigms4-7
. Eye-tracking has largely been used in these studies to reveal mechanisms underlying impaired task performance8
and abnormal brain functioning9
, particularly during the processing of social information1,10-11
. While older children and adults with ASD comprise the preponderance of research in this area, eye-tracking may be especially useful for studying young children with the disorder as it offers a non-invasive tool for assessing and quantifying early-emerging developmental abnormalities2,12-13
. Implementing eye-tracking with young children with ASD, however, is associated with a number of unique challenges, including issues with compliant behavior resulting from specific task demands and disorder-related psychosocial considerations. In this protocol, we detail methodological considerations for optimizing research design, data acquisition and psychometric analysis while eye-tracking young children with ASD. The provided recommendations are also designed to be more broadly applicable for eye-tracking children with other developmental disabilities. By offering guidelines for best practices in these areas based upon lessons derived from our own work, we hope to help other investigators make sound research design and analysis choices while avoiding common pitfalls that can compromise data acquisition while eye-tracking young children with ASD or other developmental difficulties.
Medicine, Issue 61, eye tracking, autism, neurodevelopmental disorders, toddlers, perception, attention, social cognition
Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure
Institutions: Children's Memorial Research Center, Northwestern University.
Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2
. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8
. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.
Medicine, Issue 61, Zebrafish, fetal alcohol exposure, Danio rerio, development, mRNA expression, morpholino, ethanol exposure
The Measurement and Treatment of Suppression in Amblyopia
Institutions: University of Auckland, McGill University , McGill University .
Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3
, the majority of cases being monocular2
. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5
. Amblyopia is characterized by both monocular and binocular deficits6,7
which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8
. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12
. The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16
. This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15
Medicine, Issue 70, Ophthalmology, Neuroscience, Anatomy, Physiology, Amblyopia, suppression, visual cortex, binocular vision, plasticity, strabismus, anisometropia
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
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
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
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