Fear of certain threat and anxiety about uncertain threat are distinct emotions with unique behavioral, cognitive-attentional, and neuroanatomical components. Both anxiety and fear can be studied in the laboratory by measuring the potentiation of the startle reflex. The startle reflex is a defensive reflex that is potentiated when an organism is threatened and the need for defense is high. The startle reflex is assessed via electromyography (EMG) in the orbicularis oculi muscle elicited by brief, intense, bursts of acoustic white noise (i.e., “startle probes”). Startle potentiation is calculated as the increase in startle response magnitude during presentation of sets of visual threat cues that signal delivery of mild electric shock relative to sets of matched cues that signal the absence of shock (no-threat cues). In the Threat Probability Task, fear is measured via startle potentiation to high probability (100% cue-contingent shock; certain) threat cues whereas anxiety is measured via startle potentiation to low probability (20% cue-contingent shock; uncertain) threat cues. Measurement of startle potentiation during the Threat Probability Task provides an objective and easily implemented alternative to assessment of negative affect via self-report or other methods (e.g., neuroimaging) that may be inappropriate or impractical for some researchers. Startle potentiation has been studied rigorously in both animals (e.g., rodents, non-human primates) and humans which facilitates animal-to-human translational research. Startle potentiation during certain and uncertain threat provides an objective measure of negative affective and distinct emotional states (fear, anxiety) to use in research on psychopathology, substance use/abuse and broadly in affective science. As such, it has been used extensively by clinical scientists interested in psychopathology etiology and by affective scientists interested in individual differences in emotion.
22 Related JoVE Articles!
The Trier Social Stress Test Protocol for Inducing Psychological Stress
Institutions: Northern Arizona University.
This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1
Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2
In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3
These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3
Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3
Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4
In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1
Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1
Medicine, Issue 56, Stress, anxiety, laboratory stressor, cortisol, physiological response, psychological stressor
Psychophysiological Stress Assessment Using Biofeedback
Institutions: Cambridge Health Alliance, Harvard Medical School.
In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.
Neuroscience, Issue 29, Stress, biofeedback, psychophysiological, assessment
Training Synesthetic Letter-color Associations by Reading in Color
Institutions: University of Amsterdam.
Synesthesia is a rare condition in which a stimulus from one modality automatically and consistently triggers unusual sensations in the same and/or other modalities. A relatively common and well-studied type is grapheme-color synesthesia, defined as the consistent experience of color when viewing, hearing and thinking about letters, words and numbers. We describe our method for investigating to what extent synesthetic associations between letters and colors can be learned by reading in color in nonsynesthetes. Reading in color is a special method for training associations in the sense that the associations are learned implicitly while the reader reads text as he or she normally would and it does not require explicit computer-directed training methods. In this protocol, participants are given specially prepared books to read in which four high-frequency letters are paired with four high-frequency colors. Participants receive unique sets of letter-color pairs based on their pre-existing preferences for colored letters. A modified Stroop task is administered before and after reading in order to test for learned letter-color associations and changes in brain activation. In addition to objective testing, a reading experience questionnaire is administered that is designed to probe for differences in subjective experience. A subset of questions may predict how well an individual learned the associations from reading in color. Importantly, we are not claiming that this method will cause each individual to develop grapheme-color synesthesia, only that it is possible for certain individuals to form letter-color associations by reading in color and these associations are similar in some aspects to those seen in developmental grapheme-color synesthetes. The method is quite flexible and can be used to investigate different aspects and outcomes of training synesthetic associations, including learning-induced changes in brain function and structure.
Behavior, Issue 84, synesthesia, training, learning, reading, vision, memory, cognition
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols.
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Irrelevant Stimuli and Action Control: Analyzing the Influence of Ignored Stimuli via the Distractor-Response Binding Paradigm
Institutions: Trier University, Trier University.
Selection tasks in which simple stimuli (e.g.
letters) are presented and a target stimulus has to be selected against one or more distractor stimuli are frequently used in the research on human action control. One important question in these settings is how distractor stimuli, competing with the target stimulus for a response, influence actions. The distractor-response binding paradigm can be used to investigate this influence. It is particular useful to separately analyze response retrieval and distractor inhibition effects. Computer-based experiments are used to collect the data (reaction times and error rates). In a number of sequentially presented pairs of stimulus arrays (prime-probe design), participants respond to targets while ignoring distractor stimuli. Importantly, the factors response relation in the arrays of each pair (repetition vs. change) and distractor relation (repetition vs. change) are varied orthogonally. The repetition of the same distractor then has a different effect depending on response relation (repetition vs. change) between arrays. This result pattern can be explained by response retrieval due to distractor repetition. In addition, distractor inhibition effects are indicated by a general advantage due to distractor repetition. The described paradigm has proven useful to determine relevant parameters for response retrieval effects on human action.
Behavior, Issue 87, stimulus-response binding, distractor-response binding, response retrieval, distractor inhibition, event file, action control, selection task
Developing Neuroimaging Phenotypes of the Default Mode Network in PTSD: Integrating the Resting State, Working Memory, and Structural Connectivity
Institutions: Alpert Medical School, Brown University, University of Georgia.
Complementary structural and functional neuroimaging techniques used to examine the Default Mode Network (DMN) could potentially improve assessments of psychiatric illness severity and provide added validity to the clinical diagnostic process. Recent neuroimaging research suggests that DMN processes may be disrupted in a number of stress-related psychiatric illnesses, such as posttraumatic stress disorder (PTSD).
Although specific DMN functions remain under investigation, it is generally thought to be involved in introspection and self-processing. In healthy individuals it exhibits greatest activity during periods of rest, with less activity, observed as deactivation, during cognitive tasks, e.g.
, working memory. This network consists of the medial prefrontal cortex, posterior cingulate cortex/precuneus, lateral parietal cortices and medial temporal regions.
Multiple functional and structural imaging approaches have been developed to study the DMN. These have unprecedented potential to further the understanding of the function and dysfunction of this network. Functional approaches, such as the evaluation of resting state connectivity and task-induced deactivation, have excellent potential to identify targeted neurocognitive and neuroaffective (functional) diagnostic markers and may indicate illness severity and prognosis with increased accuracy or specificity. Structural approaches, such as evaluation of morphometry and connectivity, may provide unique markers of etiology and long-term outcomes. Combined, functional and structural methods provide strong multimodal, complementary and synergistic approaches to develop valid DMN-based imaging phenotypes in stress-related psychiatric conditions. This protocol aims to integrate these methods to investigate DMN structure and function in PTSD, relating findings to illness severity and relevant clinical factors.
Medicine, Issue 89, default mode network, neuroimaging, functional magnetic resonance imaging, diffusion tensor imaging, structural connectivity, functional connectivity, posttraumatic stress disorder
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
Assessment of Murine Exercise Endurance Without the Use of a Shock Grid: An Alternative to Forced Exercise
Institutions: VA Puget Sound Health Care System, Seattle Institute for Biomedical and Clinical Research, University of Washington, VA Puget Sound Health Care System.
Using laboratory mouse models, the molecular pathways responsible for the metabolic benefits of endurance exercise are beginning to be defined. The most common method for assessing exercise endurance in mice utilizes forced running on a motorized treadmill equipped with a shock grid. Animals who quit running are pushed by the moving treadmill belt onto a grid that delivers an electric foot shock; to escape the negative stimulus, the mice return to running on the belt. However, avoidance behavior and psychological stress due to use of a shock apparatus can interfere with quantitation of running endurance, as well as confound measurements of postexercise serum hormone and cytokine levels. Here, we demonstrate and validate a refined method to measure running endurance in naïve C57BL/6 laboratory mice on a motorized treadmill without utilizing a shock grid. When mice are preacclimated to the treadmill, they run voluntarily with gait speeds specific to each mouse. Use of the shock grid is replaced by gentle encouragement by a human operator using a tongue depressor, coupled with sensitivity to the voluntary willingness to run on the part of the mouse. Clear endpoints for quantifying running time-to-exhaustion for each mouse are defined and reflected in behavioral signs of exhaustion such as splayed posture and labored breathing. This method is a humane refinement which also decreases the confounding effects of stress on experimental parameters.
Behavior, Issue 90, Exercise, Mouse, Treadmill, Endurance, Refinement
An Alternative to the Traditional Cold Pressor Test: The Cold Pressor Arm Wrap
Institutions: Marquette University.
Recently research on the relationship between stress and cognition, emotion, and behavior has greatly increased. These advances have yielded insights into important questions ranging from the nature of stress' influence on addiction1
to the role of stress in neural changes associated with alterations in decision-making2,3
. As topics being examined by the field evolve, however, so too must the methodologies involved. In this article a practical and effective alternative to a classic stress induction technique, the cold pressor test (CPT), is presented: the cold pressor arm wrap (CPAW). CPT typically involves immersion of a participant's dominant hand in ice-cold water for a period of time4
. The technique is associated with robust activation of the sympatho-adrenomedullary (SAM) axis (and release of catecholamines; e.g.
adrenaline and noradrenaline) and mild-to-moderate activation of the hypothalamic-pituitary-adrenal (HPA) axis with associated glucocorticoid (e.g.
cortisol) release. While CPT has been used in a wide range of studies, it can be impractical to apply in some research environments. For example use of water during, rather than prior to, magnetic resonance imaging (MRI) has the potential to damage sensitive and expensive equipment or interfere with acquisition of MRI signal. The CPAW is a practical and effective alternative to the traditional CPT. Composed of a versatile list of inexpensive and easily acquired components, CPAW makes use of MRI-safe gelpacs cooled to a temperature similar to CPT rather than actual water. Importantly CPAW is associated with levels of SAM and HPA activation comparable to CPT, and can easily be applied in a variety of research contexts. While it is important to maintain specific safety protocols when using the technique, these are easy to implement if planned for. Creation and use of the CPAW will be discussed.
Behavior, Issue 83, Sympathetic Nervous System, Glucocorticoids, Magnetic Resonance Imaging (MRI), Neuroimaging, Functional Neuroimaging, Cognitive Science, Stress, Neurosciences, cold pressor, hypothalamic-pituitary-adrenal axis, cortisol, sympatho-adrenomedullary axis, skin conductance
Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers
Institutions: Johns Hopkins University School of Nursing, Arizona State University, Johns Hopkins University School of Nursing, Johns Hopkins University Bloomberg School of Public Health.
Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.
Medicine, Issue 82, Saliva, Dementia, Behavioral Research, Aging, Stress, saliva, cortisol, alpha amylase, dementia, caregiving, stress
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter
Institutions: Yale University, Virginia Tech, The Hebrew University of Jerusalem.
The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2
. Plant litter comprises the majority of detritus3
, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5
. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6
. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9
because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6
. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7
. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10
, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6
. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira
), a dominant grasshopper herbivore (Melanoplus femurrubrum
),and a variety of grass and forb plants9
Environmental Sciences, Issue 73, Microbiology, Plant Biology, Entomology, Organisms, Investigative Techniques, Biological Phenomena, Chemical Phenomena, Metabolic Phenomena, Microbiological Phenomena, Earth Resources and Remote Sensing, Life Sciences (General), Litter Decomposition, Ecological Stoichiometry, Physiological Stress and Ecosystem Function, Predation Risk, Soil Respiration, Carbon Sequestration, Soil Science, respiration, spider, grasshoper, model system
Telomere Length and Telomerase Activity; A Yin and Yang of Cell Senescence
Institutions: Albert Einstein College of Medicine , Albert Einstein College of Medicine , Albert Einstein College of Medicine .
Telomeres are repeating DNA sequences at the tip ends of the chromosomes that are diverse in length and in humans can reach a length of 15,000 base pairs. The telomere serves as a bioprotective mechanism of chromosome attrition at each cell division. At a certain length, telomeres become too short to allow replication, a process that may lead to chromosome instability or cell death. Telomere length is regulated by two opposing mechanisms: attrition and elongation. Attrition occurs as each cell divides. In contrast, elongation is partially modulated by the enzyme telomerase, which adds repeating sequences to the ends of the chromosomes. In this way, telomerase could possibly reverse an aging mechanism and rejuvenates cell viability. These are crucial elements in maintaining cell life and are used to assess cellular aging. In this manuscript we will describe an accurate, short, sophisticated and cheap method to assess telomere length in multiple tissues and species. This method takes advantage of two key elements, the tandem repeat of the telomere sequence and the sensitivity of the qRT-PCR to detect differential copy numbers of tested samples. In addition, we will describe a simple assay to assess telomerase activity as a complementary backbone test for telomere length.
Genetics, Issue 75, Molecular Biology, Cellular Biology, Medicine, Biomedical Engineering, Genomics, Telomere length, telomerase activity, telomerase, telomeres, telomere, DNA, PCR, polymerase chain reaction, qRT-PCR, sequencing, aging, telomerase assay
Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents
Institutions: Tufts University Cummings School of Veterinary Medicine, Manchester Metropolitan University.
Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.
Behavior, Issue 76, Neuroscience, Neurobiology, Physiology, Anatomy, Medicine, Biomedical Engineering, Neurobehavioral Manifestations, Mental Health, Mood Disorders, Depressive Disorder, Anxiety Disorders, behavioral sciences, Behavior and Behavior Mechanisms, Mental Disorders, Stress, Depression, Anxiety, Postpartum, Maternal Behavior, Nursing, Growth, Transgenerational, animal model
Creating Dynamic Images of Short-lived Dopamine Fluctuations with lp-ntPET: Dopamine Movies of Cigarette Smoking
Institutions: Yale University, Yale University, Yale University, Yale University, Massachusetts General Hospital, University of California, Irvine.
We describe experimental and statistical steps for creating dopamine movies of the brain from dynamic PET data. The movies represent minute-to-minute fluctuations of dopamine induced by smoking a cigarette. The smoker is imaged during a natural smoking experience while other possible confounding effects (such as head motion, expectation, novelty, or aversion to smoking repeatedly) are minimized.
We present the details of our unique analysis. Conventional methods for PET analysis estimate time-invariant kinetic model parameters which cannot capture short-term fluctuations in neurotransmitter release. Our analysis - yielding a dopamine movie - is based on our work with kinetic models and other decomposition techniques that allow for time-varying parameters 1-7
. This aspect of the analysis - temporal-variation - is key to our work. Because our model is also linear in parameters, it is practical, computationally, to apply at the voxel level. The analysis technique is comprised of five main steps: pre-processing, modeling, statistical comparison, masking and visualization. Preprocessing is applied to the PET data with a unique 'HYPR' spatial filter 8
that reduces spatial noise but preserves critical temporal information. Modeling identifies the time-varying function that best describes the dopamine effect on 11
C-raclopride uptake. The statistical step compares the fit of our (lp-ntPET) model 7
to a conventional model 9
. Masking restricts treatment to those voxels best described by the new model. Visualization maps the dopamine function at each voxel to a color scale and produces a dopamine movie. Interim results and sample dopamine movies of cigarette smoking are presented.
Behavior, Issue 78, Neuroscience, Neurobiology, Molecular Biology, Biomedical Engineering, Medicine, Anatomy, Physiology, Image Processing, Computer-Assisted, Receptors, Dopamine, Dopamine, Functional Neuroimaging, Binding, Competitive, mathematical modeling (systems analysis), Neurotransmission, transient, dopamine release, PET, modeling, linear, time-invariant, smoking, F-test, ventral-striatum, clinical techniques
Setting Limits on Supersymmetry Using Simplified Models
Institutions: University College London, CERN, Lawrence Berkeley National Laboratories.
Experimental limits on supersymmetry and similar theories are difficult to set because of the enormous available parameter space and difficult to generalize because of the complexity of single points. Therefore, more phenomenological, simplified models are becoming popular for setting experimental limits, as they have clearer physical interpretations. The use of these simplified model limits to set a real limit on a concrete theory has not, however, been demonstrated. This paper recasts simplified model limits into limits on a specific and complete supersymmetry model, minimal supergravity. Limits obtained under various physical assumptions are comparable to those produced by directed searches. A prescription is provided for calculating conservative and aggressive limits on additional theories. Using acceptance and efficiency tables along with the expected and observed numbers of events in various signal regions, LHC experimental results can be recast in this manner into almost any theoretical framework, including nonsupersymmetric theories with supersymmetry-like signatures.
Physics, Issue 81, high energy physics, particle physics, Supersymmetry, LHC, ATLAS, CMS, New Physics Limits, Simplified Models
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Ultrasound Assessment of Endothelial-Dependent Flow-Mediated Vasodilation of the Brachial Artery in Clinical Research
Institutions: University of California, San Francisco, Veterans Affairs Medical Center, San Francisco, Veterans Affairs Medical Center, San Francisco.
The vascular endothelium is a monolayer of cells that cover the interior of blood vessels and provide both structural and functional roles. The endothelium acts as a barrier, preventing leukocyte adhesion and aggregation, as well as controlling permeability to plasma components. Functionally, the endothelium affects vessel tone.
Endothelial dysfunction is an imbalance between the chemical species which regulate vessel tone, thombroresistance, cellular proliferation and mitosis. It is the first step in atherosclerosis and is associated with coronary artery disease, peripheral artery disease, heart failure, hypertension, and hyperlipidemia.
The first demonstration of endothelial dysfunction involved direct infusion of acetylcholine and quantitative coronary angiography. Acetylcholine binds to muscarinic receptors on the endothelial cell surface, leading to an increase of intracellular calcium and increased nitric oxide (NO) production. In subjects with an intact endothelium, vasodilation was observed while subjects with endothelial damage experienced paradoxical vasoconstriction.
There exists a non-invasive, in vivo
method for measuring endothelial function in peripheral arteries using high-resolution B-mode ultrasound. The endothelial function of peripheral arteries is closely related to coronary artery function. This technique measures the percent diameter change in the brachial artery during a period of reactive hyperemia following limb ischemia.
This technique, known as endothelium-dependent, flow-mediated vasodilation (FMD) has value in clinical research settings. However, a number of physiological and technical issues can affect the accuracy of the results and appropriate guidelines for the technique have been published. Despite the guidelines, FMD remains heavily operator dependent and presents a steep learning curve. This article presents a standardized method for measuring FMD in the brachial artery on the upper arm and offers suggestions to reduce intra-operator variability.
Medicine, Issue 92, endothelial function, endothelial dysfunction, brachial artery, peripheral artery disease, ultrasound, vascular, endothelium, cardiovascular disease.
The Resident-intruder Paradigm: A Standardized Test for Aggression, Violence and Social Stress
Institutions: University Groningen, Radboud University Nijmegen.
This video publication explains in detail the experimental protocol of the resident-intruder paradigm in rats. This test is a standardized method to measure offensive aggression and defensive behavior in a semi natural setting. The most important behavioral elements performed by the resident and the intruder are demonstrated in the video and illustrated using artistic drawings. The use of the resident intruder paradigm for acute and chronic social stress experiments is explained as well. Finally, some brief tests and criteria are presented to distinguish aggression from its more violent and pathological forms.
Behavior, Issue 77, Neuroscience, Medicine, Anatomy, Physiology, Genetics, Basic Protocols, Psychology, offensive aggression, defensive behavior, aggressive behavior, pathological, violence, social stress, rat, Wistar rat, animal model
Methods for ECG Evaluation of Indicators of Cardiac Risk, and Susceptibility to Aconitine-induced Arrhythmias in Rats Following Status Epilepticus
Institutions: University of Utah.
Lethal cardiac arrhythmias contribute to mortality in a number of pathological conditions. Several parameters obtained from a
non-invasive, easily obtained electrocardiogram (ECG) are established, well-validated prognostic indicators of cardiac risk in patients suffering
from a number of cardiomyopathies. Increased heart rate, decreased heart rate variability (HRV), and increased duration and variability of cardiac
ventricular electrical activity (QT interval) are all indicative of enhanced cardiac risk 1-4
. In animal models, it is valuable to compare
these ECG-derived variables and susceptibility to experimentally induced arrhythmias. Intravenous infusion of the arrhythmogenic agent aconitine has
been widely used to evaluate susceptibility to arrhythmias in a range of experimental conditions, including animal models of depression 5
, following exercise 7
and exposure to air pollutants 8
, as well as determination of the antiarrhythmic efficacy of pharmacological
It should be noted that QT dispersion in humans is a measure of QT interval variation across the full set of leads from a
standard 12-lead ECG. Consequently, the measure of QT dispersion from the 2-lead ECG in the rat described in this protocol is different than that
calculated from human ECG records. This represents a limitation in the translation of the data obtained from rodents to human clinical medicine.
Status epilepticus (SE) is a single seizure or series of continuously recurring seizures lasting more than 30 min
, and results in mortality in 20% of cases 13
. Many individuals survive the SE, but die within 30 days 14,15
The mechanism(s) of this delayed mortality is not fully understood. It has been suggested that lethal ventricular arrhythmias contribute to many of these
. In addition to SE, patients experiencing spontaneously recurring seizures, i.e. epilepsy, are at risk of premature
sudden and unexpected death associated with epilepsy (SUDEP) 18
. As with SE, the precise mechanisms mediating SUDEP are not known.
It has been proposed that ventricular abnormalities and resulting arrhythmias make a significant contribution 18-22
To investigate the mechanisms of seizure-related cardiac death, and the efficacy of cardioprotective therapies, it is
necessary to obtain both ECG-derived indicators of risk and evaluate susceptibility to cardiac arrhythmias in animal models of seizure disorders
. Here we describe methods for implanting ECG electrodes in the Sprague-Dawley laboratory rat (Rattus norvegicus), following SE,
collection and analysis of ECG recordings, and induction of arrhythmias during iv infusion of aconitine.
These procedures can be used to directly determine the relationships between ECG-derived measures of cardiac electrical
activity and susceptibility to ventricular arrhythmias in rat models of seizure disorders, or any pathology associated with increased risk of sudden
Medicine, Issue 50, cardiac, seizure disorders, QTc, QTd, cardiac arrhythmias, rat