Soutenu par USDA-NIFA/AFRI 2010-65105-20582, NSF 0918177, et l&#39;incorporation de recherche Bedoukian.

<html> 1. Insectes <ol><li> Nombril orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae) colonie dans notre laboratoire proviennent de mites recueillies auprès des amandiers à Fresno, en Californie. Notre colonie d&#39;insectes est maintenue dans l&#39;environnement des chambres (Percival Inc, IA) à 28 ± 2 ° C, 75 ± 10% d&#39;humidité relative, et sous 16h08 h (lumière: obscurité) photo régime. </li><li> Mites émergents sont collectées quotidiennement, sexué, et transféré à des boîtes en plastique (669 ml boîte à lunch, 13 x 13 cm, hauteur, 4,5 cm, Rubbermaid) fourni avec jusqu&#39;à 10 couches de serviettes papier imbibé d&#39;eau (Thirsty ultra absorbants, 27,9 x 27,9 cm ; Safeway). Couvre Box sont perforés pour permettre la circulation de l&#39;air. Cet arrangement permet ~ 100% HR. </li><li> Pour des mesures électrophysiologiques nous utilisons 2-4 jours après l&#39;émergence des papillons anciens, qui sont maintenus sous une forte humidité. </li></ol> 2. Préparation électrophysiologiques <ol><li> Mites (mâles ou femelles) sont doucement poussé dans une pipette (200 uL, USA Scientific Inc) qui est coupé par le haut pour avoir un diamètre de l&#39;extrémité de grande taille (environ 2 mm). Un papillon de nuit est doucement poussé par l&#39;autre bout avec un talon de papiers tissu humidifié jusqu&#39;au antennes et une partie de la tête dépasser de l&#39;extrémité. </li><li> Tête exposée est immobilisé en couvrant la tête avec de l&#39;argile non-séchage (Claytoon, Van Aken international, CA) en laissant une petite partie d&#39;un œil et deux antennes exposés. Cette préparation est placée sur une plate-forme de l&#39;EAG Micromanipulateur MP-12 (Syntech, Allemagne) entre les deux porte-électrodes. </li><li> Nous utilisons électrode en verre fabriqués à partir de 1,0 mm tube capillaire en borosilicate avec filament (WPI Inc, FL) et a tiré dans un modèle P-97 micropipette extracteur (Sutter Instruments, CA) pour obtenir des microélectrodes de moins d&#39;un micromètre de pointe. Les électrodes sont de retour rempli sensillum lymphatiques sonnerie (Kaissling et Thorson, 1980). Une électrode empale la surface exposée de l&#39;oeil sous le microscope (Leica MZ75) et sert de référence, tandis que l&#39;électrode d&#39;enregistrement accueille les deux antennes. Afin de faciliter le contact, distale 1-2 segments antennaires sont coupés avant d&#39;être inséré dans l&#39;électrode d&#39;enregistrement. Pour les signaux à partir des enregistrements individuels sensilles, nous avons légèrement modifier cette configuration dans laquelle l&#39;électrode d&#39;enregistrement est empalée sous le microscope (Olympus BX51WI; 800x de grossissement). Les potentiels d&#39;action sont enregistrés en insérant l&#39;électrode de verre à la base d&#39;un sensillum. Les signaux sont amplifiés par une haute impédance de pré-amplificateur (Syntech, Allemagne) et introduit dans une UB-IDAC être analysées hors ligne avec AUTOSPIKE logiciel (Syntech, Allemagne). Signaux AC sont filtrés passe-bande entre 100 -10 000 Hz et les potentiels d&#39;action sont extraits par ordinateur en utilisant un algorithme AUTOSPIKE qui distingue leur pic à creux amplitudes du bruit. Les réponses des neurones individuels sont calculés comme l&#39;augmentation (ou diminution) de la fréquence des potentiels d&#39;action (impulsions par seconde) sur la fréquence spontanée. </li></ol> 3. Stimuli chimiques et le protocole de stimulation <ol><li> Produits chimiques de haute pureté sont utilisés. Un des principaux constituants du mélange de phéromones femelles de la orangeworm nombril, (Z, Z) -11,13-hexadecadienal a été obtenu à partir de Bedoukian Research Inc, CT. Les produits chimiques sont dilués, p / v, avec du verre-hexane distillé pour faire le point sur les solutions dilutions pg / pl et 10 décimal sont faites. Une aliquote (10 pi) d&#39;un produit chimique de relance dissous dans l&#39;hexane est chargé sur une bande de papier filtre Whatman (8x40 mm), le solvant est évaporé en agitant pendant 10 s sous une hotte et la bande est placée dans un polypropylène 5 ml seringue (seringues BD, NJ) à partir de laquelle les différents volumes sont éjectés. Hexane seul et une seringue vide servir de témoin. </li><li> La préparation se déroule dans un flux d&#39;air humidifié délivré par le contrôleur de stimulation Syntech (CS-55 modèle; Syntech, Allemagne) à 20 ml / sec à laquelle une impulsion de stimulation de 4 mL / s est ajouté pendant 500 ms. Les signaux sont enregistrés pendant 10 s, 2 s de départ avant le début de l&#39;impulsion de stimulation. Préparation antennaire est stimulé avec 500 ms d&#39;impulsion au cours de laquelle ca. 2 ml de l&#39;air purifié à partir d&#39;une seringue en polypropylène 5 ml contenant le stimulus est ajouté sur le flux d&#39;air principal. Un écart d&#39;au moins 1 min ou plus, suivant les réponses de haut, est autorisé entre les stimulations. </li></ol> 4. Les résultats représentatifs Antennes de papillons retenue (figure 1) stimulées par composante phéromone femelle, (Z, Z) -11,13-hexadecadienal générer des réponses à la dose dépendante robustes (figure 2). Cette composante majeure de phéromone suscite également dose-dépendante les réponses excitatrices à long trochoïdes sensilles (figure 3A) tel qu&#39;il apparaît dans les traces d&#39;enregistrements unitaires (figure 3B). <img src="/files/ftp_upload/2489/2489fig1.jpg" alt="Figure 1" /> Figure 1. Live nombril papillon orangeworm sobre et exposé de l&#39;antenne load/2489/2489fig2.jpg &quot;alt =&quot; Figure 2 &quot;/&gt; Figure 2. Réponses à la dose dépendante EAG a suscité en réponse à (Z, Z) -11,13-hexadecadienal, un constituant majeur des phéromones femelles. <img src="/files/ftp_upload/2489/2489fig3.jpg" alt="Figure 3" /> Figure 3. Trichoid Sensilles chez le mâle nombril papillon orangeworm répondre à (Z, Z) -11,13-hexadecadienal de façon dose dépendante. (A) Antenne du mâle nombril orangeworm papillon est multi-segmenté et chaque segment est orné d&#39;un grand nombre de cheveux comme des structures, des sensilles. Un microscope électronique à balayage, montrant les détails dans l&#39;encart (barres d&#39;échelle sont de 200 et 50 uM, respectivement). (B) extracellulaire unitaire des enregistrements à partir d&#39;un sensillum trichoid.

<ol>
	<li>Kaissling, K. -. E., Thorson, J., Sattelle, D. B., Hall, L. M., Hildebrand, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+olfactory+sensilla:+structural,+chemical+and+electrical+aspects+of+the+functional+organization.">Insect olfactory sensilla: structural, chemical and electrical aspects of the functional organization.</a> Receptors for Neurotransmitters. Hormones and Pheromones in Insects. , 261-282 (1980).</li></ol>

Cette nouvelle méthode développée pour retenir un nombril papillon orangeworm direct pour mesurer des signaux olfactifs s&#39;est avéré être robuste et très fiable. Nous avons régulièrement recours à cette méthode pour isoler et identifier de nouveaux appâts à partir de substrats naturels comme l&#39;hôte d&#39;amandes et les pistaches.

<table border="1"><tbody><tr><td> Nom du produit </td><td> Catalogue # / Modèle </td><td> Société </td><td> Commentaire </td></tr><tr><td> Microscope </td><td> BX51WI modèle </td><td> Olympus, USA </td><td></td></tr><tr><td> Microscope stéréo </td><td> MZ75 </td><td> Leica Microsystems Inc USA </td><td></td></tr><tr><td> 1,0 mm borosilicate tubes capillaires avec des filaments </td><td> 1B100F-3 </td><td> WPI Inc, FL </td><td></td></tr><tr><td> Micropipette extracteur </td><td> P-97 </td><td> Sutter Instruments, CA </td><td></td></tr><tr><td> Stimulation du contrôleur </td><td> CS-55 modèle </td><td> Syntech, Allemagne </td><td></td></tr><tr><td> Haute impédance de pré-amplificateurs (Sonde universelle single ended) </td><td></td><td> Syntech, Allemagne </td><td></td></tr><tr><td> Amplificateur / acquisition de données système (USB-IDAC) </td><td></td><td> Syntech, Allemagne </td><td></td></tr><tr><td> Micromanipulateur EAG MP-12 </td><td></td><td> Syntech, Allemagne </td><td></td></tr><tr><td> (Z, Z) -11,13-hexadecadienal </td><td></td><td> Bedoukian Research Inc, CT. </td><td></td></tr><tr><td> Papier filtre Whatman </td><td> 1001070 </td><td> Whatman USA </td><td></td></tr><tr><td> 5 ml seringue en polypropylène </td><td> 309633 </td><td> Seringues BD, NJ </td><td></td></tr><tr><td> embout de pipette (200 uL) </td><td> 1111-0806 </td><td> USA Scientific Inc </td><td></td></tr><tr><td> 669 ml boîte à lunch, 13 x 13 cm, hauteur, 4,5 cm, </td><td></td><td> Rubbermaid </td><td></td></tr><tr><td> Assoiffé ultra absorbants, 27,9 x 27,9 cm </td><td></td><td> Safeway </td><td></td></tr><tr><td> Non desséchant d&#39;argile </td><td> 18150 </td><td> Claytoon, Van Aken international, CA </td><td></td></tr><tr><td> Chambre de l&#39;environnement </td><td> I-30BLL modèle </td><td></td><td></td></tr></tbody></table>

electrophysiological measurements from a moth olfactory system

Insecte systèmes olfactifs offrent des occasions uniques pour l&#39;enregistrement odorant réponses induites dans les formes de electroantennograms (EAG) et les enregistrements sensillum unique (SSR), qui sont résumées les réponses de tous les neurones récepteurs olfactifs (Orns) situé sur l&#39;antenne et de ceux logés dans différents sensilles, respectivement. Ces approches ont été exploitées pour obtenir une meilleure compréhension de la communication chimique des insectes. Les stimuli identifiés peuvent ensuite être utilisés soit comme attractifs ou répulsifs dans des stratégies de gestion pour les insectes ravageurs.

Watch this Scientific Journal Video about Electrophysiological Measurements from a Moth Olfactory System at JoVE.com

Electrophysiological Measurements from a Moth Olfactory System

Insecte systèmes olfactifs offrent des occasions uniques pour l&#39;enregistrement odorant réponses induites dans les formes de electroantennograms (EAG) et les enregistrements sensillum unique (SSR), qui sont résumées les réponses de tous les neurones récepteurs olfactifs (Orns) situé sur l&#39;antenne et de ceux logés dans différents sensilles, respectivement.

Mesures électrophysiologiques à partir d&#39;un système de Moth olfactive

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

electrophysiological-measurements-from-a-moth-olfactory-system

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JoVE Journal

Neuroscience

13.6K vues.  University of California, Davis. Insecte systèmes olfactifs offrent des occasions uniques pour l'enregistrement odorant réponses induites dans les formes de electroantennograms (EAG) et les enregistrements sensillum unique (SSR), qui sont résumées les réponses de tous les neurones récepteurs olfactifs (Orns) situé sur l'antenne et de ceux logés dans différents sensilles, respectivement.

Vidéo: Electrophysiological Measurements from a Moth Olfactory System

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

Enregistrements électrophysiologiques de la voie des fibres Géant de D. melanogaster</em

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

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Neurosciences

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

Isoler les cellules souches nasal olfactif des rongeurs ou des êtres humains

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Mesures électrophysiologiques et d&#39;analyse de la nociception chez les nourrissons humains

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

Odorant induites réponses enregistrées à partir de neurones récepteurs olfactifs en utilisant la technique de la pipette d&#39;aspiration

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Appétitive apprentissage olfactif associatif en<em&gt; Drosophila</em&gt; Les larves

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Un indicateur moléculaire de l&#39;adaptation à long terme olfactive dans<em&gt; C. elegans</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

En Vivo mesures électrophysiologiques sur les nerfs sciatiques souris

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

Enregistrement électrophysiologique De<em&gt; Drosophila</em&gt; Labellar Goût Sensilles

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

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.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Le système olfactif comme un modèle pour étudier des modèles axonale croissance et la morphologie<em&gt; In Vivo</em

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 ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Mont entiers étiquetage des Cilia dans le système olfactif principal de souris

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...