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Dolphins adjust species-specific frequency parameters to compensate for increasing background noise.
PUBLISHED: 04-09-2015
An increase in ocean noise levels could interfere with acoustic communication of marine mammals. In this study we explored the effects of anthropogenic and natural noise on the acoustic properties of a dolphin communication signal, the whistle. A towed array with four elements was used to record environmental background noise and whistles of short-beaked common-, Atlantic spotted- and striped-dolphins in the Canaries archipelago. Four frequency parameters were measured from each whistle, while Sound Pressure Levels (SPL) of the background noise were measured at the central frequencies of seven one-third octave bands, from 5 to 20 kHz. Results show that dolphins increase the whistles' frequency parameters with lower variability in the presence of anthropogenic noise, and increase the end frequency of their whistles when confronted with increasing natural noise. This study provides the first evidence that the synergy among SPLs has a role in shaping the whistles' structure of these three species, with respect to both natural and anthropogenic noise.
Authors: Wen-ju Pan, Garth Thompson, Matthew Magnuson, Waqas Majeed, Dieter Jaeger, Shella Keilholz.
Published: 08-19-2010
To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.
23 Related JoVE Articles!
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A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes
Authors: Alexander R. Harris, Simeon J. Morgan, Gordon G. Wallace, Antonio G. Paolini.
Institutions: La Trobe University, University of Wollongong, ARC Centre of Excellence for Electromaterials Science, RMIT University.
New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics. This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters. This article develops a protocol for in vitro and in vivo testing of neural recording electrodes. Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed. This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models. The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio. This protocol can easily be adapted to test other electrode materials and designs. The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators. The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.
Neuroscience, Issue 85, Electrochemistry, Electrophysiology, Neural Recording, Neural Implant, Electrode Coating, Bionics
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Construction and Characterization of External Cavity Diode Lasers for Atomic Physics
Authors: Kyle S. Hardman, Shayne Bennetts, John E. Debs, Carlos C. N. Kuhn, Gordon D. McDonald, Nick Robins.
Institutions: The Australian National University.
Since their development in the late 1980s, cheap, reliable external cavity diode lasers (ECDLs) have replaced complex and expensive traditional dye and Titanium Sapphire lasers as the workhorse laser of atomic physics labs1,2. Their versatility and prolific use throughout atomic physics in applications such as absorption spectroscopy and laser cooling1,2 makes it imperative for incoming students to gain a firm practical understanding of these lasers. This publication builds upon the seminal work by Wieman3, updating components, and providing a video tutorial. The setup, frequency locking and performance characterization of an ECDL will be described. Discussion of component selection and proper mounting of both diodes and gratings, the factors affecting mode selection within the cavity, proper alignment for optimal external feedback, optics setup for coarse and fine frequency sensitive measurements, a brief overview of laser locking techniques, and laser linewidth measurements are included.
Physics, Issue 86, External Cavity Diode Laser, atomic spectroscopy, laser cooling, Bose-Einstein condensation, Zeeman modulation
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Activating Molecules, Ions, and Solid Particles with Acoustic Cavitation
Authors: Rachel Pflieger, Tony Chave, Matthieu Virot, Sergey I. Nikitenko.
Institutions: UMR 5257 CEA-CNRS-UM2-ENSCM.
The chemical and physical effects of ultrasound arise not from a direct interaction of molecules with sound waves, but rather from the acoustic cavitation: the nucleation, growth, and implosive collapse of microbubbles in liquids submitted to power ultrasound. The violent implosion of bubbles leads to the formation of chemically reactive species and to the emission of light, named sonoluminescence. In this manuscript, we describe the techniques allowing study of extreme intrabubble conditions and chemical reactivity of acoustic cavitation in solutions. The analysis of sonoluminescence spectra of water sparged with noble gases provides evidence for nonequilibrium plasma formation. The photons and the "hot" particles generated by cavitation bubbles enable to excite the non-volatile species in solutions increasing their chemical reactivity. For example the mechanism of ultrabright sonoluminescence of uranyl ions in acidic solutions varies with uranium concentration: sonophotoluminescence dominates in diluted solutions, and collisional excitation contributes at higher uranium concentration. Secondary sonochemical products may arise from chemically active species that are formed inside the bubble, but then diffuse into the liquid phase and react with solution precursors to form a variety of products. For instance, the sonochemical reduction of Pt(IV) in pure water provides an innovative synthetic route for monodispersed nanoparticles of metallic platinum without any templates or capping agents. Many studies reveal the advantages of ultrasound to activate the divided solids. In general, the mechanical effects of ultrasound strongly contribute in heterogeneous systems in addition to chemical effects. In particular, the sonolysis of PuO2 powder in pure water yields stable colloids of plutonium due to both effects.
Chemistry, Issue 86, Sonochemistry, sonoluminescence, ultrasound, cavitation, nanoparticles, actinides, colloids, nanocolloids
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Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
Authors: Alison X. Xie, Kelli Lauderdale, Thomas Murphy, Timothy L. Myers, Todd A. Fiacco.
Institutions: University of California Riverside, University of California Riverside, University of California Riverside.
Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a “calcium roadmap” is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.
Neuroscience, Issue 85, astrocyte, plasticity, mGluRs, neuronal Firing, electrophysiology, Gq GPCRs, Bolus-loading, calcium, microdomains, acute slices, Hippocampus, mouse
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Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology
Authors: F. Aura Kullmann, Stephanie L. Daugherty, William C. de Groat, Lori A. Birder.
Institutions: University of Pittsburgh School of Medicine, University of Pittsburgh School of Medicine.
We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions. The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release. The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.
Medicine, Issue 90, Krebs, species differences, in vitro, smooth muscle contractility, neural stimulation
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Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles
Authors: Eva Wagner, Sören Brandenburg, Tobias Kohl, Stephan E. Lehnart.
Institutions: Heart Research Center Goettingen, University Medical Center Goettingen, German Center for Cardiovascular Research (DZHK) partner site Goettingen, University of Maryland School of Medicine.
In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.
Bioengineering, Issue 92, cardiac myocyte, atria, ventricle, heart, primary cell isolation, fluorescence microscopy, membrane tubule, transverse-axial tubule system, image analysis, image processing, T-tubule, collagenase
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Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in Saccharomyces cerevisiae
Authors: Melissa N. Patterson, Patrick H. Maxwell.
Institutions: Rensselaer Polytechnic Institute.
Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.
Microbiology, Issue 92, Aging, mutations, genome instability, Saccharomyces cerevisiae, fluctuation test, magnetic sorting, mother cell, replicative aging
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Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
Authors: Daniel E. Bradford, Katherine P. Magruder, Rachel A. Korhumel, John J. Curtin.
Institutions: University of Wisconsin-Madison.
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.
Behavior, Issue 91, Startle; electromyography; shock; addiction; uncertainty; fear; anxiety; humans; psychophysiology; translational
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The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy
Authors: Nigel Tse, Marco Morsch, Nazanin Ghazanfari, Louise Cole, Archunan Visvanathan, Catherine Leamey, William D. Phillips.
Institutions: University of Sydney, Macquarie University, University of Sydney.
The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.
Neuroscience, Issue 94, neuromuscular, motor endplate, motor control, sarcopenia, myasthenia gravis, amyotrophic lateral sclerosis, morphometry, confocal, immunofluorescence
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Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone
Authors: Sandra Zehentmeier, Zoltan Cseresnyes, Juan Escribano Navarro, Raluca A. Niesner, Anja E. Hauser.
Institutions: German Rheumatism Research Center, a Leibniz Institute, German Rheumatism Research Center, a Leibniz Institute, Max-Delbrück Center for Molecular Medicine, Wimasis GmbH, Charité - University of Medicine.
Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.
Developmental Biology, Issue 98, Image analysis, neighborhood analysis, bone marrow, stromal cells, bone marrow niches, simulation, bone cryosectioning, bone histology
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Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
Authors: James J. Jun, André Longtin, Leonard Maler.
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
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Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI
Authors: Trecia A. Brown, Joseph S. Gati, Sarah M. Hughes, Pam L. Nixon, Ravi S. Menon, Stephen G. Lomber.
Institutions: University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario, University of Western Ontario.
Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.
Neuroscience, Issue 84, Central Nervous System, Ear, Animal Experimentation, Models, Animal, Functional Neuroimaging, Brain Mapping, Nervous System, Sense Organs, auditory cortex, BOLD signal change, hemodynamic response, hearing, acoustic stimuli
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Making Sense of Listening: The IMAP Test Battery
Authors: Johanna G. Barry, Melanie A. Ferguson, David R. Moore.
Institutions: MRC Institute of Hearing Research, National Biomedical Research Unit in Hearing.
The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated. When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli. We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.
Neuroscience, Issue 44, Listening skills, auditory processing, auditory psychophysics, clinical assessment, child-friendly testing
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Recapitulation of an Ion Channel IV Curve Using Frequency Components
Authors: John R. Rigby, Steven Poelzing.
Institutions: University of Utah.
INTRODUCTION: Presently, there are no established methods to measure multiple ion channel types simultaneously and decompose the measured current into portions attributable to each channel type. This study demonstrates how impedance spectroscopy may be used to identify specific frequencies that highly correlate with the steady state current amplitude measured during voltage clamp experiments. The method involves inserting a noise function containing specific frequencies into the voltage step protocol. In the work presented, a model cell is used to demonstrate that no high correlations are introduced by the voltage clamp circuitry, and also that the noise function itself does not introduce any high correlations when no ion channels are present. This validation is necessary before the technique can be applied to preparations containing ion channels. The purpose of the protocol presented is to demonstrate how to characterize the frequency response of a single ion channel type to a noise function. Once specific frequencies have been identified in an individual channel type, they can be used to reproduce the steady state current voltage (IV) curve. Frequencies that highly correlate with one channel type and minimally correlate with other channel types may then be used to estimate the current contribution of multiple channel types measured simultaneously. METHODS: Voltage clamp measurements were performed on a model cell using a standard voltage step protocol (-150 to +50 mV, 5mV steps). Noise functions containing equal magnitudes of 1-15 kHz frequencies (zero to peak amplitudes: 50 or 100mV) were inserted into each voltage step. The real component of the Fast Fourier transform (FFT) of the output signal was calculated with and without noise for each step potential. The magnitude of each frequency as a function of voltage step was correlated with the current amplitude at the corresponding voltages. RESULTS AND CONCLUSIONS: In the absence of noise (control), magnitudes of all frequencies except the DC component correlated poorly (|R|<0.5) with the IV curve, whereas the DC component had a correlation coefficient greater than 0.999 in all measurements. The quality of correlation between individual frequencies and the IV curve did not change when a noise function was added to the voltage step protocol. Likewise, increasing the amplitude of the noise function also did not increase the correlation. Control measurements demonstrate that the voltage clamp circuitry by itself does not cause any frequencies above 0 Hz to highly correlate with the steady-state IV curve. Likewise, measurements in the presence of the noise function demonstrate that the noise function does not cause any frequencies above 0 Hz to correlate with the steady-state IV curve when no ion channels are present. Based on this verification, the method can now be applied to preparations containing a single ion channel type with the intent of identifying frequencies whose amplitudes correlate specifically with that channel type.
Biophysics, Issue 48, Ion channel, Kir2.1, impedance spectroscopy, frequency response, voltage clamp, electrophysiology
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Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats
Authors: Aaron M. Johnson, Emerald J. Doll, Laura M. Grant, Lauren Ringel, Jaime N. Shier, Michelle R. Ciucci.
Institutions: University of Wisconsin, University of Wisconsin.
Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations. The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training. To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 μg of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging). Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1). An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.
Neuroscience, Issue 54, ultrasonic vocalization, rat, aging, Parkinson disease, exercise, 6-hydroxydopamine, voice disorders, voice therapy
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Habituation and Prepulse Inhibition of Acoustic Startle in Rodents
Authors: Bridget Valsamis, Susanne Schmid.
Institutions: University of Western Ontario.
The acoustic startle response is a protective response, elicited by a sudden and intense acoustic stimulus. Facial and skeletal muscles are activated within a few milliseconds, leading to a whole body flinch in rodents1. Although startle responses are reflexive responses that can be reliably elicited, they are not stereotypic. They can be modulated by emotions such as fear (fear potentiated startle) and joy (joy attenuated startle), by non-associative learning processes such as habituation and sensitization, and by other sensory stimuli through sensory gating processes (prepulse inhibition), turning startle responses into an excellent tool for assessing emotions, learning, and sensory gating, for review see 2, 3. The primary pathway mediating startle responses is very short and well described, qualifying startle also as an excellent model for studying the underlying mechanisms for behavioural plasticity on a cellular/molecular level3. We here describe a method for assessing short-term habituation, long-term habituation and prepulse inhibition of acoustic startle responses in rodents. Habituation describes the decrease of the startle response magnitude upon repeated presentation of the same stimulus. Habituation within a testing session is called short-term habituation (STH) and is reversible upon a period of several minutes without stimulation. Habituation between testing sessions is called long-term habituation (LTH)4. Habituation is stimulus specific5. Prepulse inhibition is the attenuation of a startle response by a preceding non-startling sensory stimulus6. The interval between prepulse and startle stimulus can vary from 6 to up to 2000 ms. The prepulse can be any modality, however, acoustic prepulses are the most commonly used. Habituation is a form of non-associative learning. It can also be viewed as a form of sensory filtering, since it reduces the organisms' response to a non-threatening stimulus. Prepulse inhibition (PPI) was originally developed in human neuropsychiatric research as an operational measure for sensory gating7. PPI deficits may represent the interface of "psychosis and cognition" as they seem to predict cognitive impairment8-10. Both habituation and PPI are disrupted in patients suffering from schizophrenia11, and PPI disruptions have shown to be, at least in some cases, amenable to treatment with mostly atypical antipsychotics12, 13. However, other mental and neurodegenerative diseases are also accompanied by disruption in habituation and/or PPI, such as autism spectrum disorders (slower habituation), obsessive compulsive disorder, Tourette's syndrome, Huntington's disease, Parkinson's disease, and Alzheimer's Disease (PPI)11, 14, 15 Dopamine induced PPI deficits are a commonly used animal model for the screening of antipsychotic drugs16, but PPI deficits can also be induced by many other psychomimetic drugs, environmental modifications and surgical procedures.
Neuroscience, Issue 55, Startle responses, rat, mouse, sensory gating, sensory filtering, short-term habituation, long-term habituation, prepulse inhibition
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Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach
Authors: Simeon J. Morgan, Antonio G. Paolini.
Institutions: La Trobe University.
Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7. Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed). Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.
Neuroscience, Issue 64, Physiology, auditory, hearing, brainstem, stimulation, rat, abi
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A Low Cost Setup for Behavioral Audiometry in Rodents
Authors: Konstantin Tziridis, Sönke Ahlf, Holger Schulze.
Institutions: University of Erlangen-Nuremberg.
In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in naïve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in naïve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).
Neuroscience, Issue 68, Physiology, Anatomy, Medicine, otolaryngology, behavior, auditory startle response, pre-pulse inhibition, audiogram, tinnitus, hearing loss
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Applications of EEG Neuroimaging Data: Event-related Potentials, Spectral Power, and Multiscale Entropy
Authors: Jennifer J. Heisz, Anthony R. McIntosh.
Institutions: Baycrest.
When considering human neuroimaging data, an appreciation of signal variability represents a fundamental innovation in the way we think about brain signal. Typically, researchers represent the brain's response as the mean across repeated experimental trials and disregard signal fluctuations over time as "noise". However, it is becoming clear that brain signal variability conveys meaningful functional information about neural network dynamics. This article describes the novel method of multiscale entropy (MSE) for quantifying brain signal variability. MSE may be particularly informative of neural network dynamics because it shows timescale dependence and sensitivity to linear and nonlinear dynamics in the data.
Neuroscience, Issue 76, Neurobiology, Anatomy, Physiology, Medicine, Biomedical Engineering, Electroencephalography, EEG, electroencephalogram, Multiscale entropy, sample entropy, MEG, neuroimaging, variability, noise, timescale, non-linear, brain signal, information theory, brain, imaging
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Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
Authors: Helmar Waiczies, Martin Guenther, Julia Skodowski, Stefano Lepore, Andreas Pohlmann, Thoralf Niendorf, Sonia Waiczies.
Institutions: A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, Max Delbrück Center for Molecular Medicine.
Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.
Molecular Biology, Issue 73, Immunology, Cellular Biology, Physiology, Anatomy, Biomedical Engineering, Hematology, nuclear magnetic resonance, NMR, Fluorine, dendritic cells, migration, lymph nodes, magnetic resonance imaging, MRI, magnetic resonance spectroscopy, MRS, spectroscopy, imaging, cell tracking, clinical techniques
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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Authors: Nikki M. Curthoys, Michael J. Mlodzianoski, Dahan Kim, Samuel T. Hess.
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
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Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish
Authors: Hans Maaswinkel, Liqun Zhu, Wei Weng.
Institutions: xyZfish.
Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.
Behavior, Issue 82, neuroscience, Zebrafish, Danio rerio, anxiety, Shoaling, Pharmacology, 3D-tracking, MK801
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How to Ignite an Atmospheric Pressure Microwave Plasma Torch without Any Additional Igniters
Authors: Martina Leins, Sandra Gaiser, Andreas Schulz, Matthias Walker, Uwe Schumacher, Thomas Hirth.
Institutions: University of Stuttgart.
This movie shows how an atmospheric pressure plasma torch can be ignited by microwave power with no additional igniters. After ignition of the plasma, a stable and continuous operation of the plasma is possible and the plasma torch can be used for many different applications. On one hand, the hot (3,600 K gas temperature) plasma can be used for chemical processes and on the other hand the cold afterglow (temperatures down to almost RT) can be applied for surface processes. For example chemical syntheses are interesting volume processes. Here the microwave plasma torch can be used for the decomposition of waste gases which are harmful and contribute to the global warming but are needed as etching gases in growing industry sectors like the semiconductor branch. Another application is the dissociation of CO2. Surplus electrical energy from renewable energy sources can be used to dissociate CO2 to CO and O2. The CO can be further processed to gaseous or liquid higher hydrocarbons thereby providing chemical storage of the energy, synthetic fuels or platform chemicals for the chemical industry. Applications of the afterglow of the plasma torch are the treatment of surfaces to increase the adhesion of lacquer, glue or paint, and the sterilization or decontamination of different kind of surfaces. The movie will explain how to ignite the plasma solely by microwave power without any additional igniters, e.g., electric sparks. The microwave plasma torch is based on a combination of two resonators — a coaxial one which provides the ignition of the plasma and a cylindrical one which guarantees a continuous and stable operation of the plasma after ignition. The plasma can be operated in a long microwave transparent tube for volume processes or shaped by orifices for surface treatment purposes.
Engineering, Issue 98, atmospheric pressure plasma, microwave plasma, plasma ignition, resonator structure, coaxial resonator, cylindrical resonator, plasma torch, stable plasma operation, continuous plasma operation, high speed camera
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