The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.
19 Related JoVE Articles!
Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans
Institutions: Deakin University.
Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.
Neuroscience, Issue 88, taste, overweight and obesity, dietary fat, fatty acid, diet, fatty food liking, detection threshold
Isolation and Culture of Human Fungiform Taste Papillae Cells
Institutions: Monell Chemical Senses Center, New York University College of Dentistry, AFB International.
Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices1,2,3,4
. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d5,6,7,8
. We recently reported on a successful method for the extended culture of rodent taste cells9
. We here report for the first time the establishment of an in vitro
culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro
Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al
Neuroscience, Issue 63, Fungiform, taste cells, cell culture, gustducin, calcium imaging, molecular biology, human fungiform papillae
Physiological Recordings and RNA Sequencing of the Gustatory Appendages of the Yellow-fever Mosquito Aedes aegypti
Institutions: United States Department of Agriculture.
Electrophysiological recording of action potentials from sensory neurons of mosquitoes provides investigators a glimpse into the chemical perception of these disease vectors. We have recently identified a bitter sensing neuron in the labellum of female Aedes aegypti
that responds to DEET and other repellents, as well as bitter quinine, through direct electrophysiological investigation. These gustatory receptor neuron responses prompted our sequencing of total mRNA from both male and female labella and tarsi samples to elucidate the putative chemoreception genes expressed in these contact chemoreception tissues. Samples of tarsi were divided into pro-, meso- and metathoracic subtypes for both sexes. We then validated our dataset by conducting qRT-PCR on the same tissue samples and used statistical methods to compare results between the two methods. Studies addressing molecular function may now target specific genes to determine those involved in repellent perception by mosquitoes. These receptor pathways may be used to screen novel repellents towards disruption of host-seeking behavior to curb the spread of harmful viruses.
Molecular Biology, Issue 94, Gustation, insect, Aedes aegypti, electrophysiology, mosquito, RNA-seq, qRT-PCR, taste, chemosensory
Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons
Institutions: Heinrich Heine University Düsseldorf.
Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis
subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.
Neuroscience, Issue 92, Neurosciences, two-photon microscopy, patch-clamp, UV-flash photolysis, mouse, hippocampus, caged compounds, glutamate, brain slice, dendrite, sodium signals
Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor
Institutions: Université de Bourgogne.
Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs' coding properties and their modulation at the membrane level.
Neuroscience, Issue 101, Neuroscience, electrophysiology, perforated patch-clamp, olfactory sensory neurons, gene-targeted mouse, transduction, pharmacology.
High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity
Institutions: Monell Chemical Senses Center.
Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11
. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e.
receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system.
Neuroscience, Issue 88, Firefly luciferase, Renilla Luciferase, Dual-Glo Luciferase Assay, olfaction, Olfactory receptor, Odorant, GPCR, High-throughput
Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay
Institutions: KU Leuven.
For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors (GPCRs). The onset of a reverse pharmacology assay is the cloning and subsequent transfection of a GPCR of interest in a cellular expression system. The heterologous expressed receptor is then challenged with a compound library of candidate ligands to identify the receptor-activating ligand(s). Receptor activation can be assessed by measuring changes in concentration of second messenger reporter molecules, like calcium or cAMP. The fluorescence-based calcium mobilization assay described here is a frequently used medium-throughput reverse pharmacology assay. The orphan GPCR is transiently expressed in human embryonic kidney 293T (HEK293T) cells and a promiscuous Gα16
construct is co-transfected. Following ligand binding, activation of the Gα16
subunit induces the release of calcium from the endoplasmic reticulum. Prior to ligand screening, the receptor-expressing cells are loaded with a fluorescent calcium indicator, Fluo-4 acetoxymethyl. The fluorescent signal of Fluo-4 is negligible in cells under resting conditions, but can be amplified more than a 100-fold upon the interaction with calcium ions that are released after receptor activation. The described technique does not require the time-consuming establishment of stably transfected cell lines in which the transfected genetic material is integrated into the host cell genome. Instead, a transient transfection, generating temporary expression of the target gene, is sufficient to perform the screening assay. The setup allows medium-throughput screening of hundreds of compounds. Co-transfection of the promiscuous Gα16
, which couples to most GPCRs, allows the intracellular signaling pathway to be redirected towards the release of calcium, regardless of the native signaling pathway in endogenous settings. The HEK293T cells are easy to handle and have proven their efficacy throughout the years in receptor deorphanization assays. However, optimization of the assay for specific receptors may remain necessary.
Cellular Biology, Issue 89, G protein-coupled receptor (GPCR), calcium mobilization assay, reverse pharmacology, deorphanization, cellular expression system, HEK293T, Fluo-4, FlexStation
Denver Papillae Protocol for Objective Analysis of Fungiform Papillae
Institutions: Denver Museum of Nature & Science, University of Colorado Denver.
The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller & Reedy’s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller & Reedy’s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller & Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.
Neuroscience, Issue 100, Fungiform Papillae, Taste, Gustatory, Quantification, Tongue, FP, Dichotomous Key, Nutrition, Method
Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates
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
Proboscis Extension Response (PER) Assay in Drosophila
Institutions: Yale University.
Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.
Neuroscience, Issue 3, Drosophila, behavior, taste, proboscis, extension
Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport
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),
Testing Drosophila Olfaction with a Y-maze Assay
Institutions: UMR-6265 CNRS, UMR-1324 INRA, Université de Bourgogne.
Detecting signals from the environment is essential for animals to ensure their survival. To this aim, they use environmental cues such as vision, mechanoreception, hearing, and chemoperception through taste, via direct contact or through olfaction, which represents the response to a volatile molecule acting at longer range. Volatile chemical molecules are very important signals for most animals in the detection of danger, a source of food, or to communicate between individuals. Drosophila melanogaster
is one of the most common biological models for scientists to explore the cellular and molecular basis of olfaction. In order to highlight olfactory abilities of this small insect, we describe a modified choice protocol based on the Y-maze test classically used with mice. Data obtained with Y-mazes give valuable information to better understand how animals deal with their perpetually changing environment. We introduce a step-by-step protocol to study the impact of odorants on fly exploratory response using this Y-maze assay.
Neuroscience, Issue 88, environmental effects (biological, animal and plant), genetics (animal and plant), life sciences, animal biology, behavioral sciences, Y-maze, olfaction, adult, choice, behavior, Drosophila melanogaster
Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology
Institutions: California Institute of Technology, California Institute of Technology, Massachusetts Institute of Technology, University of Minnesota.
Ideal cell-free expression systems can theoretically emulate an in vivo
cellular environment in a controlled in vitro
This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3
To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo
cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli
based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5
The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli
, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6
Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7
The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8
Accompanying mathematical models are available.9,10
The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."
Cellular Biology, Issue 79, Bioengineering, Synthetic Biology, Chemistry Techniques, Synthetic, Molecular Biology, control theory, TX-TL, cell-free expression, in vitro, transcription-translation, cell-free protein synthesis, synthetic biology, systems biology, Escherichia coli cell extract, biological circuits, biomolecular breadboard
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (https://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids
Institutions: Washington University, Washington University, Washington University.
Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13
C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13
C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2
, we grow cells with 13
C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4
, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third
, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2
. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1
. In this protocol, we will use Cyanothece
51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.
Molecular Biology, Issue 59, GC-MS, novel pathway, metabolism, labeling, phototrophic microorganism
Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises
Institutions: University of Kentucky, University of Toronto.
The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+
on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.
Neuroscience, Issue 47, Invertebrate, Crayfish, neurophysiology, muscle, anatomy, electrophysiology
Technique to Collect Fungiform (Taste) Papillae from Human Tongue
Institutions: College of Dentistry, New York University, School of Medicine, Washington University in St. Louis, Veterans Affairs Medical Center, University of Pennsylvania-School of Medicine, Monell Chemical Senses Center, Monell Chemical Senses Center.
The sense of taste is critical for human life. It informs the body about the quality of food that will be potentially ingested and stimulates metabolic processes that prepare the alimentary canal for digestion. Steady progress is being made towards understanding the early biochemical and molecular events underlying taste transduction (for a review, Breslin and Spector, 20081
). However, progress to date has largely resulted from animal models. Yet, since marked differences in receptor specificity and receptor density vary among species, human taste transduction will only be understood by using human taste tissue. Here we describe a biopsy technique to collect human fungiform papillae, visible as rounded pink anterior structures, about 0.5 mm in diameter that contain taste buds. These biopsied papillae are used for several purposes including the isolation of viable taste bud cells, in situ
hybridization, immunohistochemistry and, through techniques of molecular biology, the identification of taste-specific novel proteins.
JoVE Medicine, Issue 42, tongue, human, taste cells, fungiform papillae, biopsy
Loading Drosophila Nerve Terminals with Calcium Indicators
Institutions: University of Texas Health Science Center at San Antonio (UTHSCSA).
Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+
-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+
changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+
and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+
changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+
indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+
to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+
-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+
dynamics. Dextran-conjugation helps prevent Ca2+
indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.
Neuroscience, Issue 6, Drosophila, neuron, imaging
Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell
Institutions: Duke University Medical Center, University of North Carolina - Chapel Hill, Renovo Neural Incorporated.
Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.
Cellular Biology, Issue 101, Gut-Brain neurobiology, 3D electron microscopy, serial block-face scanning electron microscopy, chemosensory cell, neuropod, nutrient-sensing, gastrointestinal chemosensation