Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.
22 Related JoVE Articles!
A Tactile Automated Passive-Finger Stimulator (TAPS)
Institutions: Duquesne University, McMaster University.
Although tactile spatial acuity tests are used in both neuroscience research and clinical assessment, few automated devices exist for delivering controlled spatially structured stimuli to the skin. Consequently, investigators often apply tactile stimuli manually. Manual stimulus application is time consuming, requires great care and concentration on the part of the investigator, and leaves many stimulus parameters uncontrolled. We describe here a computer-controlled tactile stimulus system, the Tactile Automated Passive-finger Stimulator (TAPS), that applies spatially structured stimuli to the skin, controlling for onset velocity, contact force, and contact duration. TAPS is a versatile, programmable system, capable of efficiently conducting a variety of psychophysical procedures. We describe the components of TAPS, and show how TAPS is used to administer a two-interval forced-choice tactile grating orientation test.
Corresponding Author: Daniel Goldreich
Medicine, Neuroscience, Issue 28, tactile, somatosensory, touch, cutaneous, acuity, psychophysics, Bayesian, grating orientation, sensory neuroscience, spatial discrimination
Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp
Institutions: The George Washington University, Clarkson University.
The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient's ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in vitro
driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e.
a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.
Bioengineering, Issue 84, oil-flow visualization, vocal fold polyp, three-dimensional flow separation, aerodynamic pressure loadings
Methods to Explore the Influence of Top-down Visual Processes on Motor Behavior
Institutions: Rutgers University, Rutgers University, Rutgers University, Rutgers University, Rutgers University.
Kinesthetic awareness is important to successfully navigate the environment. When we interact with our daily surroundings, some aspects of movement are deliberately planned, while others spontaneously occur below conscious awareness. The deliberate component of this dichotomy has been studied extensively in several contexts, while the spontaneous component remains largely under-explored. Moreover, how perceptual processes modulate these movement classes is still unclear. In particular, a currently debated issue is whether the visuomotor system is governed by the spatial percept produced by a visual illusion or whether it is not affected by the illusion and is governed instead by the veridical percept. Bistable percepts such as 3D depth inversion illusions (DIIs) provide an excellent context to study such interactions and balance, particularly when used in combination with reach-to-grasp movements. In this study, a methodology is developed that uses a DII to clarify the role of top-down processes on motor action, particularly exploring how reaches toward a target on a DII are affected in both deliberate and spontaneous movement domains.
Behavior, Issue 86, vision for action, vision for perception, motor control, reach, grasp, visuomotor, ventral stream, dorsal stream, illusion, space perception, depth inversion
Construction and Characterization of a Novel Vocal Fold Bioreactor
Institutions: University of Delaware, University of Delaware.
engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro
platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.
Bioengineering, Issue 90, vocal fold; bioreactor; speaker; silicone membrane; fibrous scaffold; mesenchymal stem cells; vibration; extracellular matrix
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Institutions: Freie Universität Berlin.
Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102
repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4
, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.
Biophysics, Issue 88, bacteriorhodopsin, channelrhodopsin, attenuated total reflection, proton transfer, protein dynamics, infrared spectroscopy, time-resolved spectroscopy, step-scan, membrane proteins, singular value decomposition
Using the Threat Probability Task to Assess Anxiety and Fear During Uncertain and Certain Threat
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
Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials
Institutions: Illinois Wesleyan University.
Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, an understanding of how perceptions of social exclusion develop over the course of a social interaction is important for advancing treatments aimed at lessening the harmful costs of being excluded. To date, most scientific examinations of social exclusion have looked at exclusion after a social interaction has been completed. While this has been very helpful in developing an understanding of what happens to a person following exclusion, it has not helped to clarify the moment-to-moment dynamics of the process of social exclusion. Accordingly, the current protocol was developed to obtain an improved understanding of social exclusion by examining the patterns of event-related brain activation that are present during social interactions. This protocol allows greater precision and sensitivity in detailing the social processes that lead people to feel as though they have been excluded from a social interaction. Importantly, the current protocol can be adapted to include research projects that vary the nature of exclusionary social interactions by altering how frequently participants are included, how long the periods of exclusion will last in each interaction, and when exclusion will take place during the social interactions. Further, the current protocol can be used to examine variables and constructs beyond those related to social exclusion. This capability to address a variety of applications across psychology by obtaining both neural and behavioral data during ongoing social interactions suggests the present protocol could be at the core of a developing area of scientific inquiry related to social interactions.
Behavior, Issue 93, Event-related brain potentials (ERPs), Social Exclusion, Neuroscience, N2, P3, Cognitive Control
Disrupting Reconsolidation of Fear Memory in Humans by a Noradrenergic β-Blocker
Institutions: University of Amsterdam.
The basic design used in our human fear-conditioning studies on disrupting reconsolidation includes testing over different phases across three consecutive days. On day 1 - the fear acquisition
phase, healthy participants are exposed to a series of picture presentations. One picture stimulus (CS1+) is repeatedly paired with an aversive electric stimulus (US), resulting in the acquisition of a fear association, whereas another picture stimulus (CS2-) is never followed by an US. On day 2 - the memory reactivation
phase, the participants are re-exposed to the conditioned stimulus without the US (CS1-), which typically triggers a conditioned fear response. After the memory reactivation we administer an oral dose of 40 mg of propranolol HCl, a β-adrenergic receptor antagonist that indirectly targets the protein synthesis required for reconsolidation by inhibiting the noradrenaline-stimulated CREB phosphorylation. On day 3 - the test phase, the participants are again exposed to the unreinforced conditioned stimuli (CS1- and CS2-) in order to measure the fear-reducing effect of the manipulation. This retention test is followed by an extinction procedure and the presentation of situational triggers to test for the return of fear. Potentiation of the eye blink startle reflex is measured as an index for conditioned fear responding. Declarative knowledge of the fear association is measured through online US expectancy ratings during each CS presentation. In contrast to extinction learning, disrupting reconsolidation targets the original fear memory thereby preventing the return of fear. Although the clinical applications are still in their infancy, disrupting reconsolidation of fear memory seems to be a promising new technique with the prospect to persistently dampen the expression of fear memory in patients suffering from anxiety disorders and other psychiatric disorders.
Behavior, Issue 94, Fear memory, reconsolidation, noradrenergic β-blocker, human fear conditioning, startle potentiation, translational research.
In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices
Institutions: Fraunhofer Institute for Ceramic Technologies and Systems, Technische Universität Dresden, Globalfoundries Fab 8, Globalfoundries Fab 1.
The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ
experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ
electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.
Engineering, Issue 100, Time-dependent dielectric breakdown, reliability, copper interconnect, degradation kinetics, in situ TEM, ultra-low-k (ULK) material
Shock Wave Application to Cell Cultures
Institutions: Innsbruck Medical University, Goethe-University Hospital.
Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro
experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo
conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (“mechanotransduction").
Bioengineering, Issue 86, shock wave therapy (SWT), cell culture, mechanotransduction, human umbilical vein endothelial cells (HUVECs), In Vitro shock wave trials (IVSWT)
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
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
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
Differential Imaging of Biological Structures with Doubly-resonant Coherent Anti-stokes Raman Scattering (CARS)
Institutions: University of California, Davis, University of California, Davis.
Coherent Raman imaging techniques have seen a dramatic increase in activity over the past decade due to their promise to enable label-free optical imaging with high molecular specificity 1
. The sensitivity of these techniques, however, is many orders of magnitude weaker than fluorescence, requiring milli-molar molecular concentrations 1,2
. Here, we describe a technique that can enable the detection of weak or low concentrations of Raman-active molecules by amplifying their signal with that obtained from strong or abundant Raman scatterers. The interaction of short pulsed lasers in a biological sample generates a variety of coherent Raman scattering signals, each of which carry unique chemical information about the sample. Typically, only one of these signals, e.g. Coherent Anti-stokes Raman scattering (CARS), is used to generate an image while the others are discarded. However, when these other signals, including 3-color CARS and four-wave mixing (FWM), are collected and compared to the CARS signal, otherwise difficult to detect information can be extracted 3
. For example, doubly-resonant CARS (DR-CARS) is the result of the constructive interference between two resonant signals 4
. We demonstrate how tuning of the three lasers required to produce DR-CARS signals to the 2845 cm-1
CH stretch vibration in lipids and the 2120 cm-1
CD stretching vibration of a deuterated molecule (e.g. deuterated sugars, fatty acids, etc.) can be utilized to probe both Raman resonances simultaneously. Under these conditions, in addition to CARS signals from each resonance, a combined DR-CARS signal probing both is also generated. We demonstrate how detecting the difference between the DR-CARS signal and the amplifying signal from an abundant molecule's vibration can be used to enhance the sensitivity for the weaker signal. We further demonstrate that this approach even extends to applications where both signals are generated from different molecules, such that e.g. using the strong Raman signal of a solvent can enhance the weak Raman signal of a dilute solute.
Cellular Biology, Issue 44, Raman scattering, Four-wave mixing, Coherent anti-Stokes Raman scattering, Microscopy, Coherent Raman Scattering
A Novel Method for Assessing Proximal and Distal Forelimb Function in the Rat: the Irvine, Beatties and Bresnahan (IBB) Forelimb Scale
Institutions: University of California, San Francisco.
Several experimental models of cervical spinal cord injury (SCI) have been developed recently to assess the consequences of damage to this level of the spinal cord (Pearse et al.
, 2005, Gensel et al.
, 2006, Anderson et al.
, 2009), as the majority of human SCI occur here (Young, 2010; www.sci-info-pages.com). Behavioral deficits include loss of forelimb function due to damage to the white matter affecting both descending motor and ascending sensory systems, and to the gray matter containing the segmental circuitry for processing sensory input and motor output for the forelimb. Additionally, a key priority for human patients with cervical SCI is restoration of hand/arm function (Anderson, 2004). Thus, outcome measures that assess both proximal and distal forelimb function are needed. Although there are several behavioral assays that are sensitive to different aspects of forelimb recovery in experimental models of cervical SCI (Girgis et al.
, 2007, Gensel et al.
, 2006, Ballerman et al.
, 2001, Metz and Whishaw, 2000, Bertelli and Mira, 1993, Montoya et al.
, 1991, Whishaw and Pellis, 1990), few techniques provide detailed information on the recovery of fine motor control and digit movement.
The current measurement technique, the Irvine, Beatties and Bresnahan forelimb scale (IBB), can detect recovery of both proximal and distal forelimb function including digit movements during a naturally occurring behavior that does not require extensive training or deprivation to enhance motivation. The IBB was generated by observing recovery after a unilateral C6 SCI, and involves video recording of animals eating two differently shaped cereals (spherical and doughnut) of a consistent size. These videos were then used to assess features of forelimb use, such as joint position, object support, digit movement and grasping technique.
The IBB, like other forelimb behavioral tasks, shows a consistent pattern of recovery that is sensitive to injury severity. Furthermore, the IBB scale could be used to assess recovery following other types of injury that impact normal forelimb function.
Neuroscience, Issue 46, spinal cord injury, recovery of function, forelimb function, neurological test, cervical injuries
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
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System
Institutions: Ohio University.
Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1
, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2
(Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3
. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5
. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8
. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9
, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8
. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.
Medicine, Issue 59, neuroscience, muscle, electromyography, physiology, TMS, strength, motor control. sarcopenia, dynapenia, lumbar
Method to Measure Tone of Axial and Proximal Muscle
Institutions: Oregon Health and Science University, Queen Square, Oregon Health and Science University.
The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1
, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.
Medicine, Issue 58, Muscle Tone, Posture, Stiffness, Motor Control
Extinction Training During the Reconsolidation Window Prevents Recovery of Fear
Institutions: Mt. Sinai School of Medicine, New York University , New York University .
Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2
. Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3
. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4
. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9
. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16
, although this may vary depending on the strength and age of the memory15
. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11
. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14
. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e.
, extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11
. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16
, or declarative18
memories leads to interference with the original memory trace14
. We outline below a novel protocol used to block fear recovery in humans.
Neuroscience, Issue 66, Medicine, Psychology, Physiology, Fear conditioning, extinction, reconsolidation, emotional memory, spontaneous recovery, skin conductance response
Echo Particle Image Velocimetry
Institutions: University of New Hampshire.
The transport of mass, momentum, and energy in fluid flows is ultimately determined by spatiotemporal distributions of the fluid velocity field.1
Consequently, a prerequisite for understanding, predicting, and controlling fluid flows is the capability to measure the velocity field with adequate spatial and temporal resolution.2
For velocity measurements in optically opaque fluids or through optically opaque geometries, echo particle image velocimetry (EPIV) is an attractive diagnostic technique to generate "instantaneous" two-dimensional fields of velocity.3,4,5,6
In this paper, the operating protocol for an EPIV system built by integrating a commercial medical ultrasound machine7
with a PC running commercial particle image velocimetry (PIV) software8
is described, and validation measurements in Hagen-Poiseuille (i.e.
, laminar pipe) flow are reported.
For the EPIV measurements, a phased array probe connected to the medical ultrasound machine is used to generate a two-dimensional ultrasound image by pulsing the piezoelectric probe elements at different times. Each probe element transmits an ultrasound pulse into the fluid, and tracer particles in the fluid (either naturally occurring or seeded) reflect ultrasound echoes back to the probe where they are recorded. The amplitude of the reflected ultrasound waves and their time delay relative to transmission are used to create what is known as B-mode (brightness mode) two-dimensional ultrasound images. Specifically, the time delay is used to determine the position of the scatterer in the fluid and the amplitude is used to assign intensity to the scatterer. The time required to obtain a single B-mode image, t
, is determined by the time it take to pulse all the elements of the phased array probe. For acquiring multiple B-mode images, the frame rate of the system in frames per second (fps) = 1/δt
. (See 9 for a review of ultrasound imaging.)
For a typical EPIV experiment, the frame rate is between 20-60 fps, depending on flow conditions, and 100-1000 B-mode images of the spatial distribution of the tracer particles in the flow are acquired. Once acquired, the B-mode ultrasound images are transmitted via an ethernet connection to the PC running the PIV commercial software. Using the PIV software, tracer particle displacement fields, D(x,y)
[pixels], (where x and y denote horizontal and vertical spatial position in the ultrasound image, respectively) are acquired by applying cross correlation algorithms to successive ultrasound B-mode images.10
The velocity fields, u(x,y)
[m/s], are determined from the displacements fields, knowing the time step between image pairs, ΔT
[s], and the image magnification, M
. The time step between images ΔT
= 1/fps + D(x,y)/B
, where B
[pixels/s] is the time it takes for the ultrasound probe to sweep across the image width. In the present study, M = 77[μm/pixel], fps
= 49.5[1/s], and B
= 25,047[pixels/s]. Once acquired, the velocity fields can be analyzed to compute flow quantities of interest.
Mechanical Engineering, Issue 70, Physics, Engineering, Physical Sciences, Ultrasound, cross correlation, velocimetry, opaque fluids, particle, flow, fluid, EPIV
Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers
Institutions: University of Michigan Medical School, University of Michigan Medical School, University of Michigan Medical School, University of Michigan Medical School.
Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.
Bioengineering, Issue 100, Muscle physiology, skeletal muscle, single muscle fiber, permeabilized, cross-sectional area, isometric force, specific force
Appetitive Associative Olfactory Learning in Drosophila Larvae
Institutions: University of Konstanz, University of Fribourg.
In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila
larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative
learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4
. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6
. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila
larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10
larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14
. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9
. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9
Neuroscience, Issue 72, Developmental Biology, Neurobiology, Biochemistry, Molecular Biology, Physiology, Behavior, Drosophila, fruit fly, larvae, instar, olfaction, olfactory system, odor, 1-octanol, OCT, learning, reward, sugar, feeding, animal model