Behavior, in its broadest definition, can be defined as the motor manifestation of physiologic processes. As such, all behaviors manifest through the motor system. In the fields of neuroscience and orthopedics, locomotion is a commonly evaluated behavior for a variety of disease models. For example, locomotor recovery after traumatic injury to the nervous system is one of the most commonly evaluated behaviors 1-3. Though locomotion can be evaluated using a variety of endpoint measurements (e.g. time taken to complete a locomotor task, etc), semiquantitative kinematic measures (e.g. ordinal rating scales (e.g. Basso Beattie and Bresnahan locomotor (BBB) rating scale, etc)) and surrogate measures of behaviour (e.g. muscle force, nerve conduction velocity, etc), only kinetics (force measurements) and kinematics (measurements of body segments in space) provide a detailed description of the strategy by which an animal is able to locomote 1. Though not new, kinematic and kinetic measurements of locomoting rodents is now more readily accessible due to the availability of commercially available equipment designed for this purpose. Importantly, however, experimenters need to be very familiar with theory of biomechanical analyses and understand the benefits and limitations of these forms of analyses prior to embarking on what will become a relatively labor-intensive study. The present paper aims to describe a method for collecting kinematic and ground reaction force data using commercially available equipment. Details of equipment and apparatus set-up, pre-training of animals, inclusion and exclusion criteria of acceptable runs, and methods for collecting the data are described. We illustrate the utility of this behavioral analysis technique by describing the kinematics and kinetics of strain-matched young adult, middle-aged, and geriatric rats.
24 Related JoVE Articles!
One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure
Institutions: Saint Louis University School of Medicine.
Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.
Biochemistry, Issue 40, metabolomics, gas chromatography/mass spectrometry, GC/MS, inborn errors, vitamin deficiency, BNA analyses, carbohydrate, amino acid, organic acid, urease
Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis
Institutions: House Ear Institute.
OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1
. OHCs are able to change shape —elongate, shorten and bend— in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3
. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4
. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6
. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8
. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11
. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12
. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies.
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.
Neuroscience, Issue 53, Outer Hair Cell, Electromotility, Slow Motility, External Alternating Electrical Field, High-speed Imaging Analysis, Cochlea
Measurement of Bioelectric Current with a Vibrating Probe
Institutions: University of California, Davis.
Electric fields, generated by active transport of ions, are present in many biological systems and often serve important functions in tissues and organs. For example, they play an important role in directing cell migration during wound healing. Here we describe the manufacture and use of ultrasensitive vibrating probes for measuring extracellular electric currents. The probe is an insulated, sharpened metal wire with a small platinum-black tip (30-35 μm), which can detect ionic currents in the μA/cm2
range in physiological saline. The probe is vibrated at about 200 Hz by a piezoelectric bender. In the presence of an ionic current, the probe detects a voltage difference between the extremes of its movement. A lock-in amplifier filters out extraneous noise by locking on to the probe's frequency of vibration. Data are recorded onto computer. The probe is calibrated at the start and end of experiments in appropriate saline, using a chamber which applies a current of exactly 1.5 μA/cm2
. We describe how to make the probes, set up the system and calibrate. We also demonstrate the technique of cornea measurement, and show some representative results from different specimens (cornea, skin, brain).
Bioengineering, Issue 47, electric, field, current, vibrating, probe
Construction of a Preclinical Multimodality Phantom Using Tissue-mimicking Materials for Quality Assurance in Tumor Size Measurement
Institutions: University of Kansas School of Medicine, University of Texas Health Science Center at San Antonio.
World Health Organization (WHO) and the Response Evaluation Criteria in Solid Tumors (RECIST) working groups advocated standardized criteria for radiologic assessment of solid tumors in response to anti-tumor drug therapy in the 1980s and 1990s, respectively. WHO criteria measure solid tumors in two-dimensions, whereas RECIST measurements use only one-dimension which is considered to be more reproducible 1, 2, 3,4,5
. These criteria have been widely used as the only imaging biomarker approved by the United States Food and Drug Administration (FDA) 6
. In order to measure tumor response to anti-tumor drugs on images with accuracy, therefore, a robust quality assurance (QA) procedures and corresponding QA phantom are needed.
To address this need, the authors constructed a preclinical multimodality (for ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI)) phantom using tissue-mimicking (TM) materials based on the limited number of target lesions required by RECIST by revising a Gammex US commercial phantom 7
. The Appendix in Lee et al.
demonstrates the procedures of phantom fabrication 7
. In this article, all protocols are introduced in a step-by-step fashion beginning with procedures for preparing the silicone molds for casting tumor-simulating test objects in the phantom, followed by preparation of TM materials for multimodality imaging, and finally construction of the preclinical multimodality QA phantom. The primary purpose of this paper is to provide the protocols to allow anyone interested in independently constructing a phantom for their own projects. QA procedures for tumor size measurement, and RECIST, WHO and volume measurement results of test objects made at multiple institutions using this QA phantom are shown in detail in Lee et al. 8
Biomedical Engineering, Issue 77, Bioengineering, Medicine, Anatomy, Physiology, Cancer Biology, Molecular Biology, Genetics, Therapeutics, Chemistry and Materials (General), Composite Materials, Quality Assurance and Reliability, Physics (General), Tissue-mimicking materials, Preclinical, Multimodality, Quality assurance, Phantom, Tumor size measurement, Cancer, Imaging
From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope
Institutions: Scuola Normale Superiore, Instituto Italiano di Tecnologia, University of California, Irvine.
It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn’t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn
-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on µm-sized membrane regions in the micro-to-milli-second time range.
Bioengineering, Issue 92, fluorescence, protein dynamics, lipid dynamics, membrane heterogeneity, transient confinement, single molecule, GFP
Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers
Institutions: University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, University of Wisconsin-Madison, USDA-ARS Dairy Forage Research Center, USDA-ARS Pasture Systems Watershed Management Research Unit.
Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.
Environmental Sciences, Issue 90, greenhouse gas, trace gas, gas flux, static chamber, soil, field, agriculture, climate
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
Nanomanipulation of Single RNA Molecules by Optical Tweezers
Institutions: University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York, University at Albany, State University of New York.
A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.
Bioengineering, Issue 90, RNA folding, single-molecule, optical tweezers, nanomanipulation, RNA secondary structure, RNA tertiary structure
Identification of Olfactory Volatiles using Gas Chromatography-Multi-unit Recordings (GCMR) in the Insect Antennal Lobe
Institutions: University of Washington.
All organisms inhabit a world full of sensory stimuli that determine their behavioral and physiological response to their environment. Olfaction is especially important in insects, which use their olfactory systems to respond to, and discriminate amongst, complex odor stimuli. These odors elicit behaviors that mediate processes such as reproduction and habitat selection1-3
. Additionally, chemical sensing by insects mediates behaviors that are highly significant for agriculture and human health, including pollination4-6
, herbivory of food crops7
, and transmission of disease8,9
. Identification of olfactory signals and their role in insect behavior is thus important for understanding both ecological processes and human food resources and well-being.
To date, the identification of volatiles that drive insect behavior has been difficult and often tedious. Current techniques include gas chromatography-coupled electroantennogram recording (GC-EAG), and gas chromatography-coupled single sensillum recordings (GC-SSR)10-12
. These techniques proved to be vital in the identification of bioactive compounds. We have developed a method that uses gas chromatography coupled to multi-channel electrophysiological recordings (termed 'GCMR') from neurons in the antennal lobe (AL; the insect's primary olfactory center)13,14
. This state-of-the-art technique allows us to probe how odor information is represented in the insect brain. Moreover, because neural responses to odors at this level of olfactory processing are highly sensitive owing to the degree of convergence of the antenna's receptor neurons into AL neurons, AL recordings will allow the detection of active constituents of natural odors efficiently and with high sensitivity. Here we describe GCMR and give an example of its use.
Several general steps are involved in the detection of bioactive volatiles and insect response. Volatiles first need to be collected from sources of interest (in this example we use flowers from the genus Mimulus
(Phyrmaceae)) and characterized as needed using standard GC-MS techniques14-16
. Insects are prepared for study using minimal dissection, after which a recording electrode is inserted into the antennal lobe and multi-channel neural recording begins. Post-processing of the neural data then reveals which particular odorants cause significant neural responses by the insect nervous system.
Although the example we present here is specific to pollination studies, GCMR can be expanded to a wide range of study organisms and volatile sources. For instance, this method can be used in the identification of odorants attracting or repelling vector insects and crop pests. Moreover, GCMR can also be used to identify attractants for beneficial insects, such as pollinators. The technique may be expanded to non-insect subjects as well.
Neuroscience, Issue 72, Neurobiology, Physiology, Biochemistry, Chemistry, Entomlogy, Behavior, electrophysiology, olfaction, olfactory system, insect, multi-channel recording, gas chromatography, pollination, bees, Bombus impatiens, antennae, brain, animal model
Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis
Institutions: Centre for Vision Research, York University, Centre for Vision Research, York University.
The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1
and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2
. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6
, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7
which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.
Neuroscience, Issue 64, fMRI, eyetracking, BOLD, attention, inhibition, Magnetic Resonance Imaging, MRI
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
Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival
Institutions: Swedish University of Agricultural Sciences, Uppsala University.
Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida
KT2440 and A. chlorophenolicus
A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1
Microbiology, Issue 78, Cellular Biology, Molecular Biology, Biochemistry, Biophysics, Basic Protocols, Cell survival, sucrose, polysaccharides, cellulose, Ficoll, freeze-drying, Pseudomonas putida, Arthrobacter chlorophenolicus, cells, cell culture
Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility
Institutions: Institute for Cardiovascular Research, VU University Medical Center, Institute for Cardiovascular Research, VU University Medical Center.
Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro
impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.
Bioengineering, Issue 85, ECIS, Impedance Spectroscopy, Resistance, TEER, Endothelial Barrier, Cell Adhesions, Focal Adhesions, Proliferation, Migration, Motility, Wound Healing
Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
Institutions: University of Ottawa, University of Ottawa, University of Ottawa.
Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals' access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal's body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal's body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.
Neuroscience, Issue 85, animal tracking, weakly electric fish, electric organ discharge, underwater infrared imaging, automated image tracking, sensory isolation chamber, exploratory behavior
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
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice
Institutions: Fujita Health University, Core Research for Evolutionary Science and Technology (CREST), National Institutes of Natural Sciences.
The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at https://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.
Behavior, Issue 85, Fear, Learning, Memory, ImageFZ program, Mouse, contextual fear, cued fear
Getting to Compliance in Forced Exercise in Rodents: A Critical Standard to Evaluate Exercise Impact in Aging-related Disorders and Disease
Institutions: Louisiana State University Health Sciences Center.
There is a major increase in the awareness of the positive impact of exercise on improving several disease states with neurobiological basis; these include improving cognitive function and physical performance. As a result, there is an increase in the number of animal studies employing exercise. It is argued that one intrinsic value of forced exercise is that the investigator has control over the factors that can influence the impact of exercise on behavioral outcomes, notably exercise frequency, duration, and intensity of the exercise regimen. However, compliance in forced exercise regimens may be an issue, particularly if potential confounds of employing foot-shock are to be avoided. It is also important to consider that since most cognitive and locomotor impairments strike in the aged individual, determining impact of exercise on these impairments should consider using aged rodents with a highest possible level of compliance to ensure minimal need for test subjects. Here, the pertinent steps and considerations necessary to achieve nearly 100% compliance to treadmill exercise in an aged rodent model will be presented and discussed. Notwithstanding the particular exercise regimen being employed by the investigator, our protocol should be of use to investigators that are particularly interested in the potential impact of forced exercise on aging-related impairments, including aging-related Parkinsonism and Parkinson’s disease.
Behavior, Issue 90, Exercise, locomotor, Parkinson’s disease, aging, treadmill, bradykinesia, Parkinsonism
Conducting Miller-Urey Experiments
Institutions: Georgia Institute of Technology, Tokyo Institute of Technology, Institute for Advanced Study, NASA Johnson Space Center, NASA Goddard Space Flight Center, University of California at San Diego.
In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth's atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2
, 200 mmHg of CH4
, and 200 mmHg of NH3
into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.
Chemistry, Issue 83, Geosciences (General), Exobiology, Miller-Urey, Prebiotic chemistry, amino acids, spark discharge
In Situ Neutron Powder Diffraction Using Custom-made Lithium-ion Batteries
Institutions: University of Sydney, University of Wollongong, Australian Synchrotron, Australian Nuclear Science and Technology Organisation, University of Wollongong, University of New South Wales.
Li-ion batteries are widely used in portable electronic devices and are considered as promising candidates for higher-energy applications such as electric vehicles.1,2
However, many challenges, such as energy density and battery lifetimes, need to be overcome before this particular battery technology can be widely implemented in such applications.3
This research is challenging, and we outline a method to address these challenges using in situ
NPD to probe the crystal structure of electrodes undergoing electrochemical cycling (charge/discharge) in a battery. NPD data help determine the underlying structural mechanism responsible for a range of electrode properties, and this information can direct the development of better electrodes and batteries.
We briefly review six types of battery designs custom-made for NPD experiments and detail the method to construct the ‘roll-over’ cell that we have successfully used on the high-intensity NPD instrument, WOMBAT, at the Australian Nuclear Science and Technology Organisation (ANSTO). The design considerations and materials used for cell construction are discussed in conjunction with aspects of the actual in situ
NPD experiment and initial directions are presented on how to analyze such complex in situ
Physics, Issue 93, In operando, structure-property relationships, electrochemical cycling, electrochemical cells, crystallography, battery performance
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Convergent Polishing: A Simple, Rapid, Full Aperture Polishing Process of High Quality Optical Flats & Spheres
Institutions: Lawrence Livermore National Laboratory.
Convergent Polishing is a novel polishing system and method for finishing flat and spherical glass optics in which a workpiece, independent of its initial shape (i.e.
, surface figure), will converge to final surface figure with excellent surface quality under a fixed, unchanging set of polishing parameters in a single polishing iteration. In contrast, conventional full aperture polishing methods require multiple, often long, iterative cycles involving polishing, metrology and process changes to achieve the desired surface figure. The Convergent Polishing process is based on the concept of workpiece-lap height mismatch resulting in pressure differential that decreases with removal and results in the workpiece converging to the shape of the lap. The successful implementation of the Convergent Polishing process is a result of the combination of a number of technologies to remove all sources of non-uniform spatial material removal (except for workpiece-lap mismatch) for surface figure convergence and to reduce the number of rogue particles in the system for low scratch densities and low roughness. The Convergent Polishing process has been demonstrated for the fabrication of both flats and spheres of various shapes, sizes, and aspect ratios on various glass materials. The practical impact is that high quality optical components can be fabricated more rapidly, more repeatedly, with less metrology, and with less labor, resulting in lower unit costs. In this study, the Convergent Polishing protocol is specifically described for fabricating 26.5 cm square fused silica flats from a fine ground surface to a polished ~λ/2 surface figure after polishing 4 hr per surface on a 81 cm diameter polisher.
Physics, Issue 94, optical fabrication, pad polishing, fused silica glass, optical flats, optical spheres, ceria slurry, pitch button blocking, HF etching, scratches
Interview: Protein Folding and Studies of Neurodegenerative Diseases
Institutions: MIT - Massachusetts Institute of Technology.
In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.
Neuroscience, issue 17, protein folding, brain, neuron, prion, neurodegenerative disease, yeast, screen, Translational Research
A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia
Institutions: Universite de Montreal, Universite de Montreal, Universite de Montreal.
There are several lines of evidence supporting the role of de novo
mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo
mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo
mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1
and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo
mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2
. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo
mutations. This is the case for autism and schizophrenia3
. The male-to-female ratio of mutation rate is estimated at about 4–6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo
mutations would more frequently come from males, particularly older males4
. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo
mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo
mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.
Medicine, Issue 52, de novo mutation, complex diseases, schizophrenia, autism, rare variations, DNA sequencing