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.
25 Related JoVE Articles!
Measuring Diffusion Coefficients via Two-photon Fluorescence Recovery After Photobleaching
Institutions: University of Rochester, University of Rochester.
Multi-fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient (or analogous transport parameters) of macromolecules, and can be applied to both in vitro
and in vivo
biological systems. Multi-fluorescence recovery after photobleaching is performed by photobleaching a region of interest within a fluorescent sample using an intense laser flash, then attenuating the beam and monitoring the fluorescence as still-fluorescent molecules from outside the region of interest diffuse in to replace the photobleached molecules. We will begin our demonstration by aligning the laser beam through the Pockels Cell (laser modulator) and along the optical path through the laser scan box and objective lens to the sample. For simplicity, we will use a sample of aqueous fluorescent dye. We will then determine the proper experimental parameters for our sample including, monitor and bleaching powers, bleach duration, bin widths (for photon counting), and fluorescence recovery time. Next, we will describe the procedure for taking recovery curves, a process that can be largely automated via LabVIEW (National Instruments, Austin, TX) for enhanced throughput. Finally, the diffusion coefficient is determined by fitting the recovery data to the appropriate mathematical model using a least-squares fitting algorithm, readily programmable using software such as MATLAB (The Mathworks, Natick, MA).
Cellular Biology, Issue 36, Diffusion, fluorescence recovery after photobleaching, MP-FRAP, FPR, multi-photon
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry
Institutions: University of Exeter.
A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
Biophysics, Issue 91, differential scanning fluorimetry, dissociation constant, protein-ligand interactions, StepOne, cooperativity, WcbI.
Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR
Institutions: Spedali Civili di Brescia.
T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106
cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.
Immunology, Issue 94, B lymphocytes, primary immunodeficiency, real-time PCR, immune recovery, T-cell homeostasis, T lymphocytes, thymic output, bone marrow output
Operant Procedures for Assessing Behavioral Flexibility in Rats
Institutions: St. Mary's College of Maryland, University of British Columbia.
Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one’s environment (i.e.
, behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer’s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.
Behavior, Issue 96, executive function, behavioral flexibility, prefrontal cortex, strategy shifting, reversal learning, behavioral neuroscience, schizophrenia, operant
A Small Volume Bioassay to Assess Bacterial/Phytoplankton Co-culture Using WATER-Pulse-Amplitude-Modulated (WATER-PAM) Fluorometry
Institutions: University of Alberta.
Conventional methods for experimental manipulation of microalgae have employed large volumes of culture (20 ml to 5 L), so that the culture can be subsampled throughout the experiment1–7
. Subsampling of large volumes can be problematic for several reasons: 1) it causes variation in the total volume and the surface area:volume ratio of the culture during the experiment; 2) pseudo-replication (i.e.,
replicate samples from the same treatment flask8
) is often employed rather than true replicates (i.e.,
sampling from replicate treatments); 3) the duration of the experiment is limited by the total volume; and 4) axenic cultures or the usual bacterial microbiota are difficult to maintain during long-term experiments as contamination commonly occurs during subsampling.
The use of microtiter plates enables 1 ml culture volumes to be used for each replicate, with up to 48 separate treatments within a 12.65 x 8.5 x 2.2 cm plate, thereby decreasing the experimental volume and allowing for extensive replication without subsampling any treatment. Additionally, this technique can be modified to fit a variety of experimental formats including: bacterial-algal co-cultures, algal physiology tests, and toxin screening9–11
. Individual wells with an alga, bacterium and/or co-cultures can be sampled for numerous laboratory procedures including, but not limited to: WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, microscopy, bacterial colony forming unit (cfu) counts and flow cytometry. The combination of the microtiter plate format and WATER-PAM fluorometry allows for multiple rapid measurements of photochemical yield and other photochemical parameters with low variability between samples, high reproducibility and avoids the many pitfalls of subsampling a carboy or conical flask over the course of an experiment.
Environmental Sciences, Issue 97, Phytoplankton, microalgae, co-culture, bacteria, WATER-Pulse-Amplitude-Modulated (WATER-PAM) fluorometry, photosynthetic yield, chlorophyll, microtiter plate assay, high-throughput bioassay
Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-γ-Dependent Cell Autonomous Immunity
Institutions: New York Medical College.
the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.
Immunology, Issue 97, Toxoplasma, macrophages, innate immunity, intracellular pathogen, immune evasion, infectious disease, forward genetics, parasite
Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses
Institutions: University of Washington, University of Washington, Walter Reed Army Institute of Research, Henry M. Jackson Foundation.
fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.
Immunology, Issue 99, HIV-1, Recombinant, Mutagenesis, Viral replication fitness, Growth competition, Fitness calculation
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Institutions: Rice University.
It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.
Genetics, Issue 97, recombinase polymerase amplification, isothermal amplification, quantitative, diagnostic, HIV-1, viral load
Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment
Institutions: West Virginia University, West Virginia University, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, N. Bud Grossman Center for Memory Research and Care, University of Minnesota, GRECC, VA Medical Center, West Virginia University.
The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer’s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer’s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.
Behavior, Issue 100, Spatial learning, spatial reference memory, Morris water maze, Alzheimer’s disease, behavior, tau, hippocampal-dependent learning, rTg4510, Tg2576, strain background, transgenic mouse models
Barnes Maze Testing Strategies with Small and Large Rodent Models
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
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Basics of Multivariate Analysis in Neuroimaging Data
Institutions: Columbia University.
Multivariate analysis techniques for neuroimaging data have recently received increasing attention as they have many attractive features that cannot be easily realized by the more commonly used univariate, voxel-wise, techniques1,5,6,7,8,9
. Multivariate approaches evaluate correlation/covariance of activation across brain regions, rather than proceeding on a voxel-by-voxel basis. Thus, their results can be more easily interpreted as a signature of neural networks. Univariate approaches, on the other hand, cannot directly address interregional correlation in the brain. Multivariate approaches can also result in greater statistical power when compared with univariate techniques, which are forced to employ very stringent corrections for voxel-wise multiple comparisons. Further, multivariate techniques also lend themselves much better to prospective application of results from the analysis of one dataset to entirely new datasets. Multivariate techniques are thus well placed to provide information about mean differences and correlations with behavior, similarly to univariate approaches, with potentially greater statistical power and better reproducibility checks. In contrast to these advantages is the high barrier of entry to the use of multivariate approaches, preventing more widespread application in the community. To the neuroscientist becoming familiar with multivariate analysis techniques, an initial survey of the field might present a bewildering variety of approaches that, although algorithmically similar, are presented with different emphases, typically by people with mathematics backgrounds. We believe that multivariate analysis techniques have sufficient potential to warrant better dissemination. Researchers should be able to employ them in an informed and accessible manner. The current article is an attempt at a didactic introduction of multivariate techniques for the novice. A conceptual introduction is followed with a very simple application to a diagnostic data set from the Alzheimer s Disease Neuroimaging Initiative (ADNI), clearly demonstrating the superior performance of the multivariate approach.
JoVE Neuroscience, Issue 41, fMRI, PET, multivariate analysis, cognitive neuroscience, clinical neuroscience
Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department
Institutions: Universit Catholique de Louvain Cliniques Universitaires Saint-Luc.
Carbon dioxide (CO2
) along with oxygen (O2
) share the role of being the most important gases in the human body. The measuring of expired CO2
at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2
for the organism, the relevance of measuring expired CO2
, the differences between arterial and expired CO2
, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2
measurement is due to lack of correct knowledge concerning the physiopathology of CO2
by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.
Medicine, Issue 47, capnography, CO2, emergency medicine, end-tidal CO2
Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons
Institutions: National Institutes of Health, Bethesda.
FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green
fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.
Neuroscience, Issue 50, Spine, FRAP, hippocampal neurons, live cell imaging, protein mobility
Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy
Institutions: University of California San Diego - UCSD.
Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, ΔG°H2O
, for a representative integral membrane protein from E. coli
. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.
Bioengineering, Issue 50, tryptophan, peptides, Gibbs free energy, protein stability, vesicles
Measuring the Kinetics of mRNA Transcription in Single Living Cells
Institutions: Bar-Ilan University.
The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo
is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3
In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4
Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6
Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo
, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8
The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo
, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9
To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5
In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.
Cell Biology, Issue 54, mRNA transcription, nucleus, live-cell imaging, cellular dynamics, FRAP
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
Morris Water Maze Test for Learning and Memory Deficits in Alzheimer's Disease Model Mice
Institutions: University of British Columbia.
The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1
. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4
. In addition, the MWM testing environment reduces odor trail interference 5
. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer’s Disease 6, 7
. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer’s disease transgenic model mice.
Neuroscience, Issue 53, Morris Water Maze, spatial memory testing, hippocampal dependent learning, Alzheimer's Disease
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
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Investigating the Neural Mechanisms of Aware and Unaware Fear Memory with fMRI
Institutions: University of Alabama at Birmingham.
Pavlovian fear conditioning is often used in combination with functional magnetic resonance imaging (fMRI) in humans to investigate the neural substrates of associative learning 1-5
. In these studies, it is important to provide behavioral evidence of conditioning to verify that differences in brain activity are learning-related and correlated with human behavior.
Fear conditioning studies often monitor autonomic responses (e.g. skin conductance response; SCR) as an index of learning and memory 6-8
. In addition, other behavioral measures can provide valuable information about the learning process and/or other cognitive functions that influence conditioning. For example, the impact unconditioned stimulus (UCS) expectancies have on the expression of the conditioned response (CR) and unconditioned response (UCR) has been a topic of interest in several recent studies 9-14
. SCR and UCS expectancy measures have recently been used in conjunction with fMRI to investigate the neural substrates of aware and unaware fear learning and memory processes 15
. Although these cognitive processes can be evaluated to some degree following the conditioning session, post-conditioning assessments cannot measure expectations on a trial-to-trial basis and are susceptible to interference and forgetting, as well as other factors that may distort results 16,17
Monitoring autonomic and behavioral responses simultaneously with fMRI provides a mechanism by which the neural substrates that mediate complex relationships between cognitive processes and behavioral/autonomic responses can be assessed. However, monitoring autonomic and behavioral responses in the MRI environment poses a number of practical problems. Specifically, 1) standard behavioral and physiological monitoring equipment is constructed of ferrous material that cannot be safely used near the MRI scanner, 2) when this equipment is placed outside of the MRI scanning chamber, the cables projecting to the subject can carry RF noise that produces artifacts in brain images, 3) artifacts can be produced within the skin conductance signal by switching gradients during scanning, 4) the fMRI signal produced by the motor demands of behavioral responses may need to be distinguished from activity related to the cognitive processes of interest. Each of these issues can be resolved with modifications to the setup of physiological monitoring equipment and additional data analysis procedures. Here we present a methodology to simultaneously monitor autonomic and behavioral responses during fMRI, and demonstrate the use of these methods to investigate aware and unaware memory processes during fear conditioning.
Neuroscience, Issue 56, fMRI, conditioning, learning, memory, fear, contingency awareness, neuroscience, skin conductance
High-throughput Fluorometric Measurement of Potential Soil Extracellular Enzyme Activities
Institutions: Colorado State University, Oak Ridge National Laboratory, University of Colorado.
Microbes in soils and other environments produce extracellular enzymes to depolymerize and hydrolyze organic macromolecules so that they can be assimilated for energy and nutrients. Measuring soil microbial enzyme activity is crucial in understanding soil ecosystem functional dynamics. The general concept of the fluorescence enzyme assay is that synthetic C-, N-, or P-rich substrates bound with a fluorescent dye are added to soil samples. When intact, the labeled substrates do not fluoresce. Enzyme activity is measured as the increase in fluorescence as the fluorescent dyes are cleaved from their substrates, which allows them to fluoresce. Enzyme measurements can be expressed in units of molarity or activity. To perform this assay, soil slurries are prepared by combining soil with a pH buffer. The pH buffer (typically a 50 mM sodium acetate or 50 mM Tris buffer), is chosen for the buffer's particular acid dissociation constant (pKa) to best match the soil sample pH. The soil slurries are inoculated with a nonlimiting amount of fluorescently labeled (i.e.
C-, N-, or P-rich) substrate. Using soil slurries in the assay serves to minimize limitations on enzyme and substrate diffusion. Therefore, this assay controls for differences in substrate limitation, diffusion rates, and soil pH conditions; thus detecting potential enzyme activity rates as a function of the difference in enzyme concentrations (per sample).
Fluorescence enzyme assays are typically more sensitive than spectrophotometric (i.e.
colorimetric) assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light; so caution is required when handling fluorescent substrates. Likewise, this method only assesses potential enzyme activities under laboratory conditions when substrates are not limiting. Caution should be used when interpreting the data representing cross-site comparisons with differing temperatures or soil types, as in situ
soil type and temperature can influence enzyme kinetics.
Environmental Sciences, Issue 81, Ecological and Environmental Phenomena, Environment, Biochemistry, Environmental Microbiology, Soil Microbiology, Ecology, Eukaryota, Archaea, Bacteria, Soil extracellular enzyme activities (EEAs), fluorometric enzyme assays, substrate degradation, 4-methylumbelliferone (MUB), 7-amino-4-methylcoumarin (MUC), enzyme temperature kinetics, soil
Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins
Institutions: San Diego State University, San Diego State University, San Diego State University, San Diego State University, San Diego State University, Argonne National Laboratory, Broad Institute.
Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.
Immunology, Issue 100, phenomics, phage, viral metagenome, Multi-phenotype Assay Plates (MAPs), continuous culture, metabolomics