Unterstützt durch USDA-NIFA/AFRI 2010-65105-20582, NSF 0918177 und Bedoukian Forschung Incorporation.

<html> 1. Insekten <ol><li> Navel orangeworm, Amyelois transitella (Walker) (Lepidoptera: Pyralidae) Kolonie in unserem Labor von Motten aus Mandelbäume in Fresno, CA gesammelt entstanden. Unsere Insekten Kolonie ist in Klimakammern (Percival Inc, IA) bei 28 ± 2 ° C, 75 ± 10% relative Luftfeuchtigkeit, und unter 16.08 h (Licht: dunkel) Foto-Regimes. </li><li> Aufstrebende Motten werden täglich gesammelt sexed, und an Kunststoffboxen (669 mL Lunchbox, 13 x 13 cm, Höhe, 4,5 cm, Rubbermaid) versehen mit bis zu 10 Schichten von Wasser eingeweicht Papierhandtücher (Thirsty Ultra-saugfähige, 27,9 x 27,9 cm ; Safeway). Box umfasst sind perforiert, um die Luftzirkulation zu ermöglichen. Diese Anordnung bietet ~ 100% RH. </li><li> Für elektrophysiologische Messungen verwenden wir 2-4 Tage alten Motten nach dem Auflaufen, die unter hoher Luftfeuchtigkeit gehalten werden. </li></ol> 2. Elektrophysiologische Vorbereitung <ol><li> Moths (männliche oder weibliche) werden sanft in eine Pipettenspitze (200 ul, USA Scientific Inc), die von oben geschnitten wird, um eine große (ca. 2 mm) Spitze Durchmesser haben geschoben. Eine Motte wird sanft durch das andere Ende mit einem befeuchteten Papiertuch Stub geschoben, bis die Antennen und einen Teil des Kopfes von der Spitze herausragen. </li><li> Exposed Kopf wird durch das Bedecken des Kopfes mit einem nicht trocknenden Ton (Claytoon, Van Aken International, CA) verlässt ein kleiner Teil eines Auges und zwei Antennen ausgesetzt immobilisiert. Dieses Präparat ist auf einer Plattform des EAG Micromanipulator MP-12 (Syntech, Deutschland) zwischen den beiden Elektrodenhalter platziert. </li><li> Wir verwenden Glaselektrode von 1,0 mm Borosilikatglas Kapillarrohr mit Filament (WPI Inc, FL) hergestellt und zog in einem Modell P-97 Mikropipette Puller (Sutter Instruments, CA) auf Mikroelektroden von weniger als einem Mikrometer Spitze zu erhalten. Die Elektroden sind zurück mit Sensillum Lymphe ringer (Kaissling und Thorson 1980) gefüllt. Eine Elektrode spießt den exponierten Bereich des Auges unter dem Mikroskop (Leica MZ75) und dient als Referenz, während die Aufzeichnung Elektrode nimmt die beiden Antennen. Zur Erleichterung der Kontaktaufnahme sind distal 1-2 Fühlerglied Segmente, bevor sie in die Aufnahme-Elektrode eingesetzt geschnitten. Für Aufnahmen Signale von einzelnen Sensillen wir etwas ändern diese Einstellung, wobei die Aufnahme Elektrode unter dem Mikroskop (Olympus BX51WI; 800x Vergrößerung) ist aufgespießt. Aktionspotenziale werden durch Einfügen der Glaselektrode an der Basis eines Sensillum aufgezeichnet. Die Signale werden durch eine hohe Impedanz-Vorverstärker (Syntech, Deutschland) und in einen UB-IDAC analysiert werden off-line mit AUTOSPIKE Software (Syntech, Deutschland) amplifiziert. AC-Signale werden Bandpass zwischen 100 -10000 Hz und Aktionspotentiale gefiltert werden per Computer mit einem AUTOSPIKE Algorithmus, der die peak-to-Trough-Amplituden unterscheidet sich von Lärm extrahiert. Responses von einzelnen Neuronen werden als die Zunahme (oder Abnahme) der Aktionspotential-Frequenz (Spikes pro Sekunde) über die spontane Frequenz berechnet. </li></ol> 3. Chemische Reize und Stimulation Protocol <ol><li> Chemikalien von höchster Reinheit eingesetzt werden. Ein wesentlicher Bestandteil der weiblichen Pheromon Mischung aus dem Nabel orangeworm, (Z, Z) -11,13-hexadecadienal wurde von Bedoukian Research Inc, CT erhalten. Chemicals verdünnt sind, w / v, mit Glas-destilliertem Hexan Stammlösungen von 10 ug / ul und dekadischen Verdünnungen machen gemacht. Ein Aliquot (10 l) eines Reizes Chemikalie in Hexan gelöst auf ein Whatman-Filterpapier Streifen (8x40 mm) eingelegt ist, wird das Lösungsmittel durch leichtes Schütteln für 10 s unter einer Abzugshaube verdampft und der Streifen ist in einem 5 mL Polypropylen platziert Spritze (BD Spritzen, NJ), aus dem verschiedene Mengen ausgestoßen werden. Hexan allein und eine leere Spritze dienen als Kontrolle. </li><li> Die Zubereitung ist in einem befeuchteten Luftstrom durch die Syntech Stimulus-Controller (CS-55-Modell; Syntech, Deutschland) geliefert statt bei 20 ml / sec, zu dem ein Reiz Puls 4 ml / s für 500 ms hinzugefügt wird. Die Signale werden für 10 s aufgezeichnet, beginnend 2 s vor dem Beginn des Stimulus Puls. Fühlerglied Vorbereitung wird mit 500 ms Puls, während der ca. stimuliert. 2 mL der gereinigten Luft aus einem 5 mL Polypropylen-Spritze mit der Reiz auf die Hauptstraße Luftstrom aufgenommen. Ein Abstand von mindestens 1 min oder mehr, nach hohen Antworten wird zwischen Stimulationen erlaubt. </li></ol> 4. Repräsentative Ergebnisse Antennen verhalten Motten (Abbildung 1) mit weiblichen Pheromon-Komponente stimuliert, (Z, Z) -11,13-hexadecadienal erzeugen robust dosisabhängige Reaktionen (Abbildung 2). Diese große Pheromon-Komponente auch entlockt dosisabhängig exzitatorische Antworten von langen trichodea Sensillen (Abb. 3A) als in Spuren Einheit Aufnahmen (Abbildung 3B) angezeigt. <img src="/files/ftp_upload/2489/2489fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Live Nabel orangeworm Motte zurückhaltend und Antenne exponierte load/2489/2489fig2.jpg &quot;alt =&quot; Bild 2 &quot;/&gt; Abbildung 2. Dosisabhängige EAG hervorgerufenen Reaktionen als Antwort auf (Z, Z) -11,13-hexadecadienal, eine große weibliche Pheromon Bestandteil. <img src="/files/ftp_upload/2489/2489fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Sensillen trichoid bei männlichen Nabel orangeworm Motte zu reagieren (Z, Z) -11,13-hexadecadienal in einer dosisabhängigen Weise. (A)-Antenne der männlichen Nabel orangeworm Motte ist Multi-segmentiert und jedes Segment ist mit einer großen Anzahl von Haaren wie Strukturen, Sensillen geschmückt. Eine rasterelektronenmikroskopische Aufnahme, die Details im Einschub (Scale Bars sind 200 und 50 pM bzw.). (B) Extrazelluläre Single-Unit-Aufnahmen von einer trichoid Sensillum.

<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>

Dieses neuartige Verfahren für die Fesselung von einer Live-Nabel orangeworm Motte olfaktorischen Signale zu messen entwickelt hat sich als robust und sehr zuverlässig. Wir setzen routinemäßig diese Methode zur Isolierung und Identifizierung neuartiger Lockstoffe aus natürlichen Wirt Substraten wie Mandeln und Pistazien.

<table border="1"><tbody><tr><td> Material Name </td><td> Katalog # / Model </td><td> Firma </td><td> Kommentar </td></tr><tr><td> Mikroskop </td><td> BX51WI Modell </td><td> Olympus, USA </td><td></td></tr><tr><td> Stereo-Mikroskop </td><td> MZ75 </td><td> Leica Microsystems Inc. USA </td><td></td></tr><tr><td> 1,0 mm Borosilikatglas Kapillarrohr mit Filament </td><td> 1B100F-3 </td><td> WPI Inc, FL </td><td></td></tr><tr><td> Micropipette puller </td><td> P-97 </td><td> Sutter Instruments, CA </td><td></td></tr><tr><td> Stimulus-Controller </td><td> CS-55 Modell </td><td> Syntech, Deutschland </td><td></td></tr><tr><td> Hohe Impedanz-Vorverstärker (Universal Single ended-Sonde) </td><td></td><td> Syntech, Deutschland </td><td></td></tr><tr><td> Verstärker / Datenerfassungssystem (USB-IDAC) </td><td></td><td> Syntech, Deutschland </td><td></td></tr><tr><td> EAG Micromanipulator MP-12 </td><td></td><td> Syntech, Deutschland </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> Whatman-Filterpapier </td><td> 1001070 </td><td> Whatman USA </td><td></td></tr><tr><td> 5 mL Polypropylen-Spritze </td><td> 309633 </td><td> BD Spritzen, NJ </td><td></td></tr><tr><td> Pipettenspitze (200 ul) </td><td> 1111-0806 </td><td> USA Scientific Inc </td><td></td></tr><tr><td> 669 mL Lunchbox, 13 x 13 cm, Höhe, 4,5 cm, </td><td></td><td> Rubbermaid </td><td></td></tr><tr><td> Thirsty Ultra-saugfähige, 27,9 x 27,9 cm </td><td></td><td> Safeway </td><td></td></tr><tr><td> Non-Trocknung Ton </td><td> 18150 </td><td> Claytoon, Van Aken International, CA </td><td></td></tr><tr><td> Klimakammer </td><td> I-30BLL Modell </td><td></td><td></td></tr></tbody></table>

electrophysiological measurements from a moth olfactory system

Insect olfaktorischen Systeme bieten einzigartige Möglichkeiten für die Aufnahme Riechstoff-induzierten Reaktionen in den Formen der Elektroantennogramme (EAG) und Single Sensillum Aufnahmen (SSR), die Antworten zusammengefasst sind von allen Geruchsrezeptor Neuronen (ORNs) an der Antenne befindet und von denen in einzelnen untergebracht Sensillen, jeweils. Diese Ansätze wurden für ein besseres Verständnis von Insekten chemische Kommunikation genutzt. Die identifizierten Stimuli kann dann entweder als Lockmittel oder Repellents in Managementstrategien für Schadinsekten eingesetzt werden.

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

Electrophysiological Measurements from a Moth Olfactory System

Insect olfaktorischen Systeme bieten einzigartige Möglichkeiten für die Aufnahme Riechstoff-induzierten Reaktionen in den Formen der Elektroantennogramme (EAG) und Single Sensillum Aufnahmen (SSR), die Antworten zusammengefasst sind von allen Geruchsrezeptor Neuronen (ORNs) an der Antenne befindet und von denen in einzelnen untergebracht Sensillen, jeweils.

Elektrophysiologische Messungen von einer Moth Riechsystem

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

Nanyang Technological University

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JoVE Journal

Neuroscience

13.7K Aufrufe.  University of California, Davis. Insect olfaktorischen Systeme bieten einzigartige Möglichkeiten für die Aufnahme Riechstoff-induzierten Reaktionen in den Formen der Elektroantennogramme (EAG) und Single Sensillum Aufnahmen (SSR), die Antworten zusammengefasst sind von allen Geruchsrezeptor Neuronen (ORNs) an der Antenne befindet und von denen in einzelnen untergebracht Sensillen, jeweils.

Serie: 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.

Elektrophysiologischen Ableitungen aus dem Riesen-Fiber Pathway der 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" ...

Forschung

Neurowissenschaften

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.

Isolating Nasal Olfaktorische Stammzellen von Nagetieren oder Menschen

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.

Elektrophysiologische Messungen und Analyse von Nozizeption in Human Infants

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-induzierten Reaktionen aus olfaktorischen Rezeptor-Neuronen mit der Saugpipette Technik aufgenommen

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.

Appetitive Assoziative olfaktorischen Lernens in<em&gt; Drosophila</em&gt; Larven

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

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.

In-vivo-Elektrophysiologische Messungen an Maus Ischiasnerven

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.

Elektrophysiologische Aufnehmen von<em&gt; Drosophila</em&gt; Labellar Geschmack Sensillen

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

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Isolation von unterschiedlichen Zellpopulationen aus sich entwickelnden Kleinhirn durch Mikrodissektion

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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.

Das olfaktorische System als Modell zur Axonal Wachstumsmuster und Morphologie Studieren<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.

Ganze Berge Kennzeichnung von Cilia im Hauptriechsystem der Mäuse

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