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Pubmed Article
Gene expression changes in the olfactory bulb of mice induced by exposure to diesel exhaust are dependent on animal rearing environment.
PLoS ONE
PUBLISHED: 01-01-2013
There is an emerging concern that particulate air pollution increases the risk of cranial nerve disease onset. Small nanoparticles, mainly derived from diesel exhaust particles reach the olfactory bulb by their nasal depositions. It has been reported that diesel exhaust inhalation causes inflammation of the olfactory bulb and other brain regions. However, these toxicological studies have not evaluated animal rearing environment. We hypothesized that rearing environment can change mice phenotypes and thus might alter toxicological study results. In this study, we exposed mice to diesel exhaust inhalation at 90 µg/m(3), 8 hours/day, for 28 consecutive days after rearing in a standard cage or environmental enrichment conditions. Microarray analysis found that expression levels of 112 genes were changed by diesel exhaust inhalation. Functional analysis using Gene Ontology revealed that the dysregulated genes were involved in inflammation and immune response. This result was supported by pathway analysis. Quantitative RT-PCR analysis confirmed 10 genes. Interestingly, background gene expression of the olfactory bulb of mice reared in a standard cage environment was changed by diesel exhaust inhalation, whereas there was no significant effect of diesel exhaust exposure on gene expression levels of mice reared with environmental enrichment. The results indicate for the first time that the effect of diesel exhaust exposure on gene expression of the olfactory bulb was influenced by rearing environment. Rearing environment, such as environmental enrichment, may be an important contributive factor to causation in evaluating still undefined toxic environmental substances such as diesel exhaust.
Authors: Patrick De Boever, Tijs Louwies, Eline Provost, Luc Int Panis, Tim S. Nawrot.
Published: 10-22-2014
ABSTRACT
The microcirculation consists of blood vessels with diameters less than 150 µm. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age. Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors. The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.
20 Related JoVE Articles!
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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
51458
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The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo
Authors: Thomas Hassenklöver, Ivan Manzini.
Institutions: University of Göttingen.
The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.
Neuroscience, Issue 92, Xenopus laevis, Anura, electroporation, single cell electroporation, sensory neurons, olfactory system, axon growth, glomerulus, olfactory bulb, olfactory map formation
52143
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A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation
Authors: Christine J. Fontaine, Bandhan Mukherjee, Gillian L. Morrison, Qi Yuan.
Institutions: Faculty of Medicine, Memorial University, University of Victoria.
Rat pups during a critical postnatal period (≤ 10 days) readily form a preference for an odor that is associated with stimuli mimicking maternal care. Such a preference memory can last from hours, to days, even life-long, depending on training parameters. Early odor preference learning provides us with a model in which the critical changes for a natural form of learning occur in the olfactory circuitry. An additional feature that makes it a powerful tool for the analysis of memory processes is that early odor preference learning can be lateralized via single naris occlusion within the critical period. This is due to the lack of mature anterior commissural connections of the olfactory hemispheres at this early age. This work outlines behavioral protocols for lateralized odor learning using nose plugs. Acute, reversible naris occlusion minimizes tissue and neuronal damages associated with long-term occlusion and more aggressive methods such as cauterization. The lateralized odor learning model permits within-animal comparison, therefore greatly reducing variance compared to between-animal designs. This method has been used successfully to probe the circuit changes in the olfactory system produced by training. Future directions include exploring molecular underpinnings of odor memory using this lateralized learning model; and correlating physiological change with memory strength and durations.
Neuroscience, Issue 90, lateralized odor learning, rats, memory, nose plug, olfactory bulb, piriform cortex, phosphorylated CREB
51808
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Construction of an Affordable and Easy-to-Build Zebrafish Facility
Authors: Candler Paige, Bailey Hill, Joseph Canterbury, Sarah Sweitzer, E. Alfonso Romero-Sandoval.
Institutions: Presbyterian College School of Pharmacy.
In vivo biomedical research is pivotal to translate in vitro findings into clinical advances. Small academic institutions with limited resources find it virtually impossible to build and maintain typical rodent facilities for research. Zebrafish research has been demonstrated to be a valuable alternative for in vivo research in pharmacology, physiology, development and genetic studies. This article demonstrates that a functional zebrafish facility can be built in an easy and affordable manner. We demonstrate that such a facility could be built in about one working day with minimal tools and expertise. The cost of the 27 1.8 L fish tank zebrafish facility constructed in this study was approximately $1,500. We estimate that the maintenance of an initial working 150 fish colony for 3 months is $1,000. This project involved students, who were introduced to aquaculturing of zebrafish for research proposes.
Basic Protocols, Issue 93, Zebrafish, in vivo, novice, practical, construction, affordable, aquarium, aquaculture, fish breeder racks.
51989
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Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines
Authors: Kenric J. Hoegler, Martin Distel, Reinhard W. Köster, John H. Horne.
Institutions: Pace University, University of California, San Diego, Braunschweig University of Technology.
In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.
Neuroscience, Issue 54, electroporation, zebrafish, olfactory bulb, Gal4 enhancer trap
2964
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Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo
Authors: Benjamin Sadrian, Huaiyang Chen, Qizhi Gong.
Institutions: University of California, Davis.
Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function during olfactory development.
Neuroscience, Issue 51, lentivirus, olfactory, sensory, neurons, genetics
2951
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Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves
Authors: Kimberly J. Schipke, S. D. Filip To, James N. Warnock.
Institutions: Mississippi State University.
The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.
Bioengineering, Issue 54, Mechanobiology, Bioreactor, Aortic Heart Valve, Organ Culture
3316
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An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus
Authors: Monica F. Poelchau, Xin Huang, Allison Goff, Julie Reynolds, Peter Armbruster.
Institutions: Georgetown University, The Ohio State University.
Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.
Genetics, Issue 93, Aedes albopictus Asian tiger mosquito, photoperiodic diapause, RNA-Seq de novo transcriptome assembly, mosquito husbandry
51961
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Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
Authors: Yves Molino, Françoise Jabès, Emmanuelle Lacassagne, Nicolas Gaudin, Michel Khrestchatisky.
Institutions: VECT-HORUS SAS, CNRS, NICN UMR 7259.
The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.
Medicine, Issue 88, rat brain endothelial cells (RBEC), mouse, spinal cord, tight junction (TJ), receptor-mediated transport (RMT), low density lipoprotein (LDL), LDLR, transferrin, TfR, P-glycoprotein (P-gp), transendothelial electrical resistance (TEER),
51278
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Whole-Body Nanoparticle Aerosol Inhalation Exposures
Authors: Jinghai Yi, Bean T. Chen, Diane Schwegler-Berry, Dave Frazer, Vince Castranova, Carroll McBride, Travis L. Knuckles, Phoebe A. Stapleton, Valerie C. Minarchick, Timothy R. Nurkiewicz.
Institutions: West Virginia University , West Virginia University , National Institute for Occupational Safety and Health.
Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter < 200 nm and a geometric standard deviation σg < 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5. A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis. In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.
Medicine, Issue 75, Physiology, Anatomy, Chemistry, Biomedical Engineering, Pharmacology, Titanium dioxide, engineered nanomaterials, nanoparticle, toxicology, inhalation exposure, aerosols, dry powder, animal model
50263
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Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Authors: Loretta Müller, Luisa E. Brighton, Johnny L. Carson, William A. Fischer II, Ilona Jaspers.
Institutions: The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill.
In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.
Cellular Biology, Issue 80, Epithelium, Cell culture models, ciliated, air pollution, co-culture models, nasal epithelium
50646
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A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Authors: Brian H. Smith, Christina M. Burden.
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g., food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides. We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
51057
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Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience
Authors: Hagit Turm, Diptendu Mukherjee, Doron Haritan, Maayan Tahor, Ami Citri.
Institutions: The Hebrew University of Jerusalem.
The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2. In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells. The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.
Behavior, Issue 90, Brain, behavior, RNA, transcription, nucleus accumbens, cocaine, high-throughput qPCR, experience-dependent plasticity, gene regulatory networks, microdissection
51642
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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice
Authors: Robert S. McNeill, Ralf S. Schmid, Ryan E. Bash, Mark Vitucci, Kristen K. White, Andrea M. Werneke, Brian H. Constance, Byron Huff, C. Ryan Miller.
Institutions: University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, University of North Carolina School of Medicine, Emory University School of Medicine, University of North Carolina School of Medicine.
Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.
Neuroscience, Issue 90, astrocytoma, cortical astrocytes, genetically engineered mice, glioblastoma, neural stem cells, orthotopic allograft
51763
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Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development
Authors: Russell A. Morton, Marvin R. Diaz, Lauren A. Topper, C. Fernando Valenzuela.
Institutions: University of New Mexico Health Sciences Center.
Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.
Medicine, Issue 89, fetal, ethanol, exposure, paradigm, vapor, development, alcoholism, teratogenic, animal, mouse, model
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Barnes Maze Testing Strategies with Small and Large Rodent Models
Authors: Cheryl S. Rosenfeld, Sherry A. Ferguson.
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g. bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g. distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e. random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g. shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
51194
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Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb
Authors: Wayne I. Doyle, Gary F. Hammen, Julian P. Meeks.
Institutions: UT Southwestern Medical Center, Washington University in St. Louis.
The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (<1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.
Neuroscience, Issue 90, vomeronasal organ, accessory olfactory bulb, ex vivo, mouse, olfaction
51813
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Protocol for Mosquito Rearing (A. gambiae)
Authors: Suchismita Das, Lindsey Garver, George Dimopoulos.
Institutions: Johns Hopkins University.
This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28°C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.
Cellular Biology, Issue 5, mosquito, malaria, infectious disease
221
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Choice and No-Choice Assays for Testing the Resistance of A. thaliana to Chewing Insects
Authors: Martin De Vos, Georg Jander.
Institutions: Cornell University.
Larvae of the small white cabbage butterfly are a pest in agricultural settings. This caterpillar species feeds from plants in the cabbage family, which include many crops such as cabbage, broccoli, Brussel sprouts etc. Rearing of the insects takes place on cabbage plants in the greenhouse. At least two cages are needed for the rearing of Pieris rapae. One for the larvae and the other to contain the adults, the butterflies. In order to investigate the role of plant hormones and toxic plant chemicals in resistance to this insect pest, we demonstrate two experiments. First, determination of the role of jasmonic acid (JA - a plant hormone often indicated in resistance to insects) in resistance to the chewing insect Pieris rapae. Caterpillar growth can be compared on wild-type and mutant plants impaired in production of JA. This experiment is considered "No Choice", because larvae are forced to subsist on a single plant which synthesizes or is deficient in JA. Second, we demonstrate an experiment that investigates the role of glucosinolates, which are used as oviposition (egg-laying) signals. Here, we use WT and mutant Arabidopsis impaired in glucosinolate production in a "Choice" experiment in which female butterflies are allowed to choose to lay their eggs on plants of either genotype. This video demonstrates the experimental setup for both assays as well as representative results.
Plant Biology, Issue 15, Annual Review, Plant Resistance, Herbivory, Arabidopsis thaliana, Pieris rapae, Caterpillars, Butterflies, Jasmonic Acid, Glucosinolates
683
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Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region
Authors: Kin Chiu, Wui Man Lau, Ho Tak Lau, Kwok-Fai So, Raymond Chuen-Chung Chang.
Institutions: The University of Hong Kong - HKU.
Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.
Issue 7, Neuroscience, brain, dissection
269
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