Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.
19 Related JoVE Articles!
Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
Production of Haploid Zebrafish Embryos by In Vitro Fertilization
Institutions: University of Notre Dame.
The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro
is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.
) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.
Developmental Biology, Issue 89, zebrafish, haploid, in vitro fertilization, forward genetic screen, saturation, recessive mutation, mutagenesis
Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue
Institutions: Duke University , Hokkaido University.
Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ
hybridization to mRNAs within cells. Here we present a radioactive in situ
hybridization method modified from Clayton et al.
that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5
. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7
. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9
, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.
Neuroscience, Issue 62, in situ hybridization, radioactive, riboprobes, vertebrate, embryo, silver emulsion, darkfield, genetics
Assessing Differences in Sperm Competitive Ability in Drosophila
Institutions: University of California, Irvine.
Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e.
selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1
. Sperm competition represents the competition between males after copulating with the same female 2
, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3
. For example, wild-caught D. melanogaster
females usually contain sperm from 2-3 males 4
. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7
. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8
. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9
, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster
Developmental Biology, Issue 78, Molecular Biology, Cellular Biology, Genetics, Biochemistry, Spermatozoa, Drosophila melanogaster, Biological Evolution, Phenotype, genetics (animal and plant), animal biology, double-mating experiment, sperm competitive ability, male fertility, Drosophila, fruit fly, animal model
Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila
Institutions: Rutgers University, University of California, Davis, Rutgers University.
Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (≡24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila
have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila
is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila
. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila
exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila
. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila
manifesting altered circadian or sleep properties.
Neuroscience, Issue 43, circadian rhythm, locomotor activity, Drosophila, period, sleep, Trikinetics
Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks
Institutions: Freie Universität Berlin.
Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.
Developmental Biology, Issue 87, songbird, molecular sexing, PCR, individual marking, down feather, DNA extraction, sample storage, zebra finch, buccal swabs, saliva, gender
A Simple Method of Mouse Lung Intubation
Institutions: Johns Hopkins Bloomberg School of Public Health, Oregon Health Sciences University.
A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.
Medicine, Issue 73, Biomedical Engineering, Anatomy, Physiology, Surgery, Respiratory System, Respiratory Tract Diseases, pulmonary function, chronic, longitudinal studies, airway resistance, trachea, lung, clinical techniques, intubation, cannula, animal model
A Novel Rescue Technique for Difficult Intubation and Difficult Ventilation
Institutions: Children’s Hospital of Michigan, St. Jude Children’s Research Hospital.
We describe a novel non surgical technique to maintain oxygenation and ventilation in a case of difficult intubation and difficult ventilation, which works especially well with poor mask fit.
Can not intubate, can not ventilate" (CICV) is a potentially life threatening situation. In this video we present a simulation of the technique we used in a case of CICV where oxygenation and ventilation were maintained by inserting an endotracheal tube (ETT) nasally down to the level of the naso-pharynx while sealing the mouth and nares for successful positive pressure ventilation.
A 13 year old patient was taken to the operating room for incision and drainage of a neck abcess and direct laryngobronchoscopy. After preoxygenation, anesthesia was induced intravenously. Mask ventilation was found to be extremely difficult because of the swelling of the soft tissue. The face mask could not fit properly on the face due to significant facial swelling as well. A direct laryngoscopy was attempted with no visualization of the larynx. Oxygen saturation was difficult to maintain, with saturations falling to 80%. In order to oxygenate and ventilate the patient, an endotracheal tube was then inserted nasally after nasal spray with nasal decongestant and lubricant. The tube was pushed gently and blindly into the hypopharynx. The mouth and nose of the patient were sealed by hand and positive pressure ventilation was possible with 100% O2
with good oxygen saturation during that period of time. Once the patient was stable and well sedated, a rigid bronchoscope was introduced by the otolaryngologist showing extensive subglottic and epiglottic edema, and a mass effect from the abscess, contributing to the airway compromise. The airway was secured with an ETT tube by the otolaryngologist.This video will show a simulation of the technique on a patient undergoing general anesthesia for dental restorations.
Medicine, Issue 47, difficult ventilation, difficult intubation, nasal, saturation
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds
Institutions: University of Antwerp.
The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e.
compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5
(for a review, see 6
). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8
. In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata
), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Medicine, Biophysics, Physiology, Anatomy, Functional MRI, fMRI, Magnetic Resonance Imaging, MRI, blood oxygenation level dependent fMRI, BOLD fMRI, Brain, Songbird, zebra finches, Taeniopygia guttata, Auditory Stimulation, stimuli, animal model, imaging
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
Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats
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
Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication
Institutions: Brigham Young University.
Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing material properties 1
. Normal voice production relies on healthy tissue and vocal folds, and occurs as a result of complex coupling between aerodynamic, structural dynamic, and acoustic physical phenomena. Voice disorders affect up to 7.5 million annually in the United States alone 2
and often result in significant financial, social, and other quality-of-life difficulties. Understanding the physics of voice production has the potential to significantly benefit voice care, including clinical prevention, diagnosis, and treatment of voice disorders.
Existing methods for studying voice production include in vivo
experimentation using human and animal subjects, in vitro
experimentation using excised larynges and synthetic models, and computational modeling. Owing to hazardous and difficult instrument access, in vivo
experiments are severely limited in scope. Excised larynx experiments have the benefit of anatomical and some physiological realism, but parametric studies involving geometric and material property variables are limited. Further, they are typically only able to be vibrated for relatively short periods of time (typically on the order of minutes).
Overcoming some of the limitations of excised larynx experiments, synthetic vocal fold models are emerging as a complementary tool for studying voice production. Synthetic models can be fabricated with systematic changes to geometry and material properties, allowing for the study of healthy and unhealthy human phonatory aerodynamics, structural dynamics, and acoustics. For example, they have been used to study left-right vocal fold asymmetry 3,4
, clinical instrument development 5
, laryngeal aerodynamics 6-9
, vocal fold contact pressure 10
, and subglottal acoustics 11
(a more comprehensive list can be found in Kniesburges et al. 12
Existing synthetic vocal fold models, however, have either been homogenous (one-layer models) or have been fabricated using two materials of differing stiffness (two-layer models). This approach does not allow for representation of the actual multi-layer structure of the human vocal folds 1
that plays a central role in governing vocal fold flow-induced vibratory response. Consequently, one- and two-layer synthetic vocal fold models have exhibited disadvantages 3,6,8
such as higher onset pressures than what are typical for human phonation (onset pressure is the minimum lung pressure required to initiate vibration), unnaturally large inferior-superior motion, and lack of a "mucosal wave" (a vertically-traveling wave that is characteristic of healthy human vocal fold vibration).
In this paper, fabrication of a model with multiple layers of differing material properties is described. The model layers simulate the multi-layer structure of the human vocal folds, including epithelium, superficial lamina propria (SLP), intermediate and deep lamina propria (i.e., ligament; a fiber is included for anterior-posterior stiffness), and muscle (i.e., body) layers 1
. Results are included that show that the model exhibits improved vibratory characteristics over prior one- and two-layer synthetic models, including onset pressure closer to human onset pressure, reduced inferior-superior motion, and evidence of a mucosal wave.
Bioengineering, Issue 58, Vocal folds, larynx, voice, speech, artificial biomechanical models
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
Endotracheal Intubation in Mice via Direct Laryngoscopy Using an Otoscope
Institutions: VA San Diego Healthcare System, University of California, San Diego, University of California, San Diego.
Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are necessary for whole animal experiments that require surgery under deep anesthesia or measurements of lung function. Tracheostomy has been the standard for intubating the airway in these mice to allow mechanical ventilation. Orotracheal intubation has been reported but has not been successfully used in many studies because of the substantial technical difficulty or a requirement for highly specialized and expensive equipment. Here we report a technique of direct laryngoscopy using an otoscope fitted with a 2.0 mm speculum and using a 20 G intravenous catheter as an endotracheal tube. We have used this technique extensively and reliably to intubate and conduct accurate assessments of lung function in mice. This technique has proven safe, with essentially no animal loss in experienced hands. Moreover, this technique can be used for repeated studies of mice in chronic models.
Medicine, Issue 86, lung physiology, endotracheal intubation, laryngoscopy, airway resistance, intubation technique
Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury
Institutions: University of Rouen, Karolinska Institutet, Rouen University Hospital, Amiens University Hospital.
Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.
Neuroscience, Issue 84, olfactory ensheathing cells, spinal cord injury, transplantation, larynx, recurrent laryngeal nerve, peripheral nerve injury, vocal cords
Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development
Institutions: College of William and Mary.
The zebra finch (Taeniopygiaguttata
) has become an increasingly important model organism in many areas of research including toxicology1,2
, and memory and learning4,5,6
. As the only songbird with a sequenced genome, the zebra finch has great potential for use in developmental studies; however, the early stages of zebra finch development have not been well studied. Lack of research in zebra finch development can be attributed to the difficulty of dissecting the small egg and embryo. The following dissection method minimizes embryonic tissue damage, which allows for investigation of morphology and gene expression at all stages of embryonic development. This permits both bright field and fluorescence quality imaging of embryos, use in molecular procedures such as in situ
hybridization (ISH), cell proliferation assays, and RNA extraction for quantitative assays such as quantitative real-time PCR (qtRT-PCR). This technique allows investigators to study early stages of development that were previously difficult to access.
Developmental Biology, Issue 88, zebra finch (Taeniopygiaguttata), dissection, embryo, development, in situ hybridization, 5-ethynyl-2’-deoxyuridine (EdU)
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Institutions: Institut Pasteur .
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3
. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7
Immunology, Issue 52, Polymerase fidelity, RNA virus, mutation frequency, mutagen, RNA polymerase, viral evolution
Determining 3D Flow Fields via Multi-camera Light Field Imaging
Institutions: Brigham Young University, Naval Undersea Warfare Center, Newport, RI.
In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and observed phenomena in fluid flows. Thus, a need exists for an accessible method capable of resolving three-dimensional (3D) data sets for a range of problems. We present a novel technique for performing quantitative 3D imaging of many types of flow fields. The 3D technique enables investigation of complicated velocity fields and bubbly flows. Measurements of these types present a variety of challenges to the instrument. For instance, optically dense bubbly multiphase flows cannot be readily imaged by traditional, non-invasive flow measurement techniques due to the bubbles occluding optical access to the interior regions of the volume of interest. By using Light Field Imaging we are able to reparameterize images captured by an array of cameras to reconstruct a 3D volumetric map for every time instance, despite partial occlusions in the volume. The technique makes use of an algorithm known as synthetic aperture (SA) refocusing, whereby a 3D focal stack is generated by combining images from several cameras post-capture 1
. Light Field Imaging allows for the capture of angular as well as spatial information about the light rays, and hence enables 3D scene reconstruction. Quantitative information can then be extracted from the 3D reconstructions using a variety of processing algorithms. In particular, we have developed measurement methods based on Light Field Imaging for performing 3D particle image velocimetry (PIV), extracting bubbles in a 3D field and tracking the boundary of a flickering flame. We present the fundamentals of the Light Field Imaging methodology in the context of our setup for performing 3DPIV of the airflow passing over a set of synthetic vocal folds, and show representative results from application of the technique to a bubble-entraining plunging jet.
Physics, Issue 73, Mechanical Engineering, Fluid Mechanics, Engineering, synthetic aperture imaging, light field, camera array, particle image velocimetry, three dimensional, vector fields, image processing, auto calibration, vocal chords, bubbles, flow, fluids