Wir möchten die Mitglieder des Heiligen Lab danken. Diese Arbeit wurde durch das Nationale Institut für Taubheit und Communication Disorders (R01 DC005964 zu THE) und NSF IGERT 0548890 (HAA) unterstützt. Tierversuche wurden in Übereinstimmung mit den Vereinigten Staaten Animal Welfare Acts und National Institutes of Health Richtlinien durchgeführt und waren von der Washington University Animal Care und Verwenden Committee genehmigt.

Teil 1. Elektrische Recording Instrumentation <ol><li> Wir verwenden eine planare Multielektroden array (ALA Scientific Instruments) mit 10 pm flache Titan-Nitrid-Elektroden mit Siliziumnitrid isoliert. 60 Elektroden sind in zwei Bereichen, bestehend aus einem 5x6-Gitter mit Mitte-zu-Zentrum-Abstand von 30 um angeordnet. (Andere Konfigurationen sind ebenfalls möglich; Elektroden mit einer Aufnahme Fläche 10 um im Durchmesser sind besser als größere Elektroden in Bezug auf die Isolierung der Aktivität einzelner Neuronen.) </li><li> Elektrische Signale werden verstärkt (1000x Verstärkung) mit einem MEA 1060 Verstärker (ALA Scientific Instruments). Die Signale werden bei 10 kHz mit einer Datenerfassungskarte (National Instruments) und kundenspezifische Software zur Datenerfassung (kann man auch kaufen kommerzielle Software) erworben. </li><li> Das Gewebe wird auf den MEA durch ein Nylonnetz (53 um), die eine benutzerdefinierte Einsatz, der genau passt in die Kultur Rohr Ring des MEA angebracht gehalten wird. </li></ol> Part 2. Flüssige Lieferung Instrumentation <ol><li> Das Gewebe erfordert eine kontinuierliche Superfusion, die wir garantieren, indem Ringer-Lösung (115 mM Natriumchlorid, 5 mM Kaliumchlorid, 2 mM Calciumchlorid, 2 mM Magnesiumchlorid, 25 mM Natriumhydrogencarbonat, 10 mM HEPES und 10 mM D-(+ )-Glucose) aus einer HPLC-Pumpe. Stimuli werden durch das Anwenden Injektionen in eine HPLC-Ventil und das Umschalten zwischen Flow-Through und die Schleife vorgestellt. Sowohl die HPLC-Pumpe und die Injektion des Roboters Spritzenpumpe mit Ringer-Lösung geliefert. </li><li> Ringer-Lösung wird in einem beheizten Wasserbad (ISOTEMP 105) bei 42 ° C während des gesamten Versuchs gehalten und wird kontinuierlich mit 95% O 2 / 5% CO 2 geleitet. Eine separate Flasche Ringerlösung ohne Glukose ist für die Sicherung der Spritzenpumpe vorbereitet. Ringer-Lösung ist, um das Gewebe mit A 307 HPLC-Pumpe (Gilson) mit einer Rate von 3 ml / min geliefert. In einem typischen Experiment 5-6 Stunden, 1 L der einzelnen Ringer-Lösung ausreichend. </li><li> Während des Experiments wird das Gewebe kontinuierlich mit 37 ° C Ringer-Lösung superfundiert. Eine Heizung Sonde befindet sich direkt oberhalb des Gewebes positioniert. </li><li> Stimuli werden geliefert mit dem Gilson-215-Liquid Handler Roboterarm. Stimuli werden in Gummi-capped Glasfläschchen gespeichert und unter der Kontrolle von Gilson-Software, liefert der Roboterarm die Reize auf die Durchblutung line während konstantem Durchfluss und Druck gehalten werden. </li><li> Der Zustand der HPLC-Ventil (loop engagierte / nicht aktiviert) wird durch eine elektrische Spannung, die durch den Erwerb Computer als einen zusätzlichen Kanal zur Synchronisation mit der neuronalen Aktivität zu gewährleisten aufgezeichnet vertreten. </li></ol> Teil 3. Vorbereiten des MEA und Stimulus Lieferumfang Gerät <ol><li> Vor Durchführung der Dissektion, füllen Sie die MEA gut mit BSA in dH2O für mindestens 5 Minuten bei Raumtemperatur. </li><li> Spülen Sie die MEA mit warm-sprudelte Ringer-Lösung und setzen Sie die MEA auf Eis. </li><li> Prime der Liquid Handler und HPLC-Pumpe nach den Anweisungen des Herstellers. </li></ol> Teil 4. VNO Dissection <ol><li> Vor der Durchführung der Dissektion, Fill 2 60x15 mm Petrischalen mit sprudelte Ringer-Lösung und auf Eis stellen. Auch Fill 2 Sylgard beschichteten Petrischalen von der gleichen Größe mit Ringer-Lösung und auf Eis stellen. Gefühlt einer zusätzlichen 50 mL von warm-sprudelte Ringer für den Einsatz während der Präparation. </li><li> Die Maus wird über CO 2 Ersticken durch Genickbruch folgte geopfert. </li><li> Machen bilateralen Einschnitte von der Seite des Mundes, die parallel zur Kiefer-Linie, auf der Rückseite des Kopfes. Join the 2 Schnitte entlang der Rückseite des Halses und entfernen Sie den dorsalen Teil des Kopfes, so dass der Unterkiefer an der Maus. </li><li> Mit einer feinen Schere, schneiden Sie zwischen dem Gewebe und beiderseits der oberen Kieferknochen, um das Gewebe aus dem Knochen zu trennen. </li><li> Mit Nr. 3 Zangen, ziehen Sie die Gaumengewebe in die Nasenhöhle aussetzen. </li><li> Mit einer kleinen Schere oder ein Skalpell, zwischen und auf beiden Seiten der oberen 2 Schneidezähne, die vomeronasalen Kapsel aus dem Knochen frei geschnitten. </li><li> Mit Nr. 3 Zangen, nehmen Sie den vomeronasalen Kapsel durch den flachen Knochen und in der Petrischale auf Eis. Die Schritte oben durch Eintauchen sollte in weniger als 3 Minuten durchgeführt werden. </li><li> Die Schale unter einem Stereomikroskop mit Auflicht beleuchtet. Entfernen Sie die knöchernen Kapsel von einem VNO in einer Zeit mit Nr. 3 Zangen. Verwenden Sie eine Hand an den knöchernen Kapsel durch Ergreifen von der kaudalen Ende und die andere Hand zu schälen den Knochen weg von der VNO, kümmert sich nicht um die VNO Schaden zu stabilisieren. </li><li> Wenn der Knochen entfernt ist, übertragen Sie die VNO eine Sylgard beschichtete Petrischale. Verwenden Sie die restlichen Teller für die anderen VNO. Schalten Sie die Beleuchtung Durchlicht. </li><li> Mit Mikro-Schere oder Zange Nr. 5, trennen Sie die VNO aus dem Blutgefäß durch Schneiden entlang der Kante des VNO. An dieser Stelle werden Siehaben die neuronalen Blatt vollständig aus dem Blutgefäß getrennt. </li><li> Legen Sie die VNO mit dem internen (dendritischen) nach oben. Mit Nr. 3 Zangen, Anker der VNO der Sylgard an einer Ecke des Gewebes. Schälen Sie die neuralen Epithel von der Basalmembran mit einem kleinen Fragment eines Teflon-beschichtete Rasierklinge mit einer Klammer gehalten. Halten Sie die Klinge in einem flachen Winkel (&lt;30 ° aus der Schale Oberfläche) am besten ist. </li><li> Mit einer Glaspipette, Übertragung des Stückes von neuralen Epithel aus der Schale der MEA. Position der VNO auf der Elektrode Feld mit den dendritischen Seite nach oben. Entfernen Sie überschüssiges Ringer-Lösung mit einer Pipette. Legen Sie die Mesh-Gewebe Inhaber in den Brunnen des Arrays der VNO in Position zu halten, und füllen Sie mit gekühlten Ringer-Lösung. </li></ol> Teil 5. Aufnahme <ol><li> Legen Sie die MEA in einer MEA 1060 Verstärker und die Position der Heizung Sonde direkt über das Gewebe. </li><li> Um zu bestätigen, richtige Heizung Aufsetzen der Sonde, mit Ringer-Lösung mit 50 mM Kalium-(substituierte für äquimolare Natrium) für 2 Sekunden zu stimulieren. Passen Sie die Positionierung, um die Latenz der neuronalen Antwort auf die Kalium-Lösung zu minimieren. </li><li> Superfuse das Gewebe mit beheiztem Ringer-Lösung für 45 Minuten bis 1 Stunde vor der Aufnahme, damit das Gewebe sich zu akklimatisieren. </li><li> Elektrische Signale werden auf die Festplatte mithilfe von benutzerdefinierten Recording-Software gespeichert. </li></ol> Teil 6. Data Analysis <ol><li> Offline-Daten-Analyse kann mit dem rohen Elektrode Aufnahmen werden. Zum Beispiel messen wir die Feuerrate Veränderungen der aufgezeichneten Zellen in Reaktion auf bestimmte Liganden. Die daraus resultierenden Datenmengen sind groß und kann auf viele verschiedene Fragen zu beantworten sein; Vertrautheit mit einem Programmier-oder Analyse Sprache (C, Matlab, R, Python, o.ä.) erforderlich ist. </li></ol> Teil 7. Repräsentative Ergebnisse Dieses Präparat ist in der Regel stabil genug, um von 6 Stunden aufnehmen, was eine große Menge von Daten (6 Stunden Aufnahmezeit von 60 Elektroden). <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="Abbildung 1" border="0" /> Abbildung 1. Eine Momentaufnahme der gleichzeitige Tätigkeit in einer Hälfte des Arrays angezeigt. Das Zeitfenster reicht 400 ms. Bitte <a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 1 zu sehen. <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="Abbildung 2" border="0" /> Abbildung 2. In einem typischen Experiment, arbeiten wir mit speziellen Verbindungen von Interesse zu wecken für 10 Sekunden. Diese Spur ist ein Beispiel für eine Reaktion auf die 100-fach verdünnt weiblichen Maus Urin. Bitte <a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">klicken Sie hier,</a> um eine größere Version der Abbildung 2 zu sehen.

<ol>
	<li>Dulac, C., Torelo, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+pheromone+signals+in+mammals:+from+genes+to+behaviour.">Molecular detection of pheromone signals in mammals: from genes to behaviour.</a> Nat Rev Neurosci. 4, 551-562 (2003).</li><li>Holy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+Vomeronasal+neurons+to+natural+stimuli.">Responses of Vomeronasal neurons to natural stimuli.</a> Science. 289, 1569-1572 (2000).</li><li>Nodari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+steroids+as+natural+ligands+of+mouse+pheromone-sensing+neurons.">Sulfated steroids as natural ligands of mouse pheromone-sensing neurons.</a> J Neurosci. 28, 6407-6418 (2008).</li></ol>

Multielektroden Array-Aufnahmen ermöglichen es uns die Möglichkeit, die gleichzeitige Tätigkeit von einer großen Population von Neuronen zu überwachen. Dies ist ein nützliches Werkzeug für die Erforschung der Eigenschaften eines sensorischen Systems, insbesondere des VNO. Im Gegensatz zu den wichtigsten olfaktorischen System, erkennt das VNO Reize in der flüssigen Phase. Darüber hinaus ist das VNO ein heterogenes Gewebe. Es gibt ungefähr 300 verschiedene Arten von Rezeptoren in der Maus 1, vermutlic...

multielectrode array recordings of the vomeronasal epithelium

Understanding neuronalen Schaltkreisen erfordert Methoden, um aus vielen Neuronen gleichzeitig aufzunehmen. Für<em&gt; In-vitro-</em&gt; Studien, eine derzeit verfügbaren Technologie ist planar Multielektroden Array (MEA) Aufnahme. Hier dokumentieren wir die Verwendung von MEAs zur Maus Vomeronasalorgan (VNO), die eine wesentliche Rolle bei der Erkennung von Pheromonen und soziale Signale über eine vielfältige Bevölkerung von sensorischen Neuronen exprimiert Hunderte von Arten von Rezeptoren zu untersuchen. Die Kombination MEA-Aufnahme mit einem Roboter-Liquid Handler auf chemische Reize liefern, kann die sensorische Reaktionen einer großen und vielfältigen Bevölkerung von Neuronen aufgenommen werden. Die Vorbereitung ermöglicht es uns, die intakte Neuroepithel des VNO von der Maus zu entfernen und zu stimulieren mit einer Batterie von Chemikalien oder potentiellen Liganden bei der Überwachung der elektrischen Aktivität der Neuronen für mehrere Stunden. Daher dient diese Technik als eine nützliche Methode zur Beurteilung der Liganden-Aktivität sowie die Erforschung der Eigenschaften von Rezeptorneuronen. Wir präsentieren Ihnen die Techniken erforderlich, um die vomeronasalen Epithel, MEA Aufnahme und chemische Stimulation vorzubereiten.

Watch this Scientific Journal Video about Multielectrode Array Recordings of the Vomeronasal Epithelium at JoVE.com

Multielectrode Array Recordings of the Vomeronasal Epithelium

Multielektroden Array (MEA)-Aufnahmen stellen eine Methode zur Untersuchung der elektrischen Aktivität des großen Populationen von Neuronen. Hier präsentieren wir die Details eines MEA Vorbereitung auf die von der Maus vomeronasalen Epithel aufzeichnen, während gleichzeitig die Förderung der Gewebe.

Multielektroden Array Recordings der Vomeronasal Epithel

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

multielectrode-array-recordings-of-the-vomeronasal-epithelium

Research

JoVE Journal

Neuroscience

12.1K Aufrufe.  Washington University School of Medicine. Multielektroden Array (MEA)-Aufnahmen stellen eine Methode zur Untersuchung der elektrischen Aktivität des großen Populationen von Neuronen. Hier präsentieren wir die Details eines MEA Vorbereitung auf die von der Maus vomeronasalen Epithel aufzeichnen, während gleichzeitig die Förderung der Gewebe.

Serie: Multielectrode Array Recordings of the Vomeronasal Epithelium

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

Single-Einheit In-vivo- Aufnahmen aus dem Chiasma der Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Forschung

Neurowissenschaften

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" ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Präparation einer Maus Eye für einen ganzen Berg von retinalen Pigmentepithel

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

Multi-Elektroden-Array Recordings neuronaler Lawinen in organotypischen Kulturen

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

Imaging Pheromone Sensing in a Mouse Vomeronasal Akute Gewebeschnitt Vorbereitung

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

Imaging neuronalen Antworten in Scheiben Zubereitungen von Vomeronasalorgan Ausdruck eines genetisch kodierte Kalzium-Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first neural circuit in the AOS pathway, called the accessory olfactory bulb (AOB), plays an important role in establishing sex-typical behaviors such as territorial aggression and mating. This small (&#60;1 mm3) circuit possesses the capacity to distinguish unique behavioral states, such as sex, strain, and stress from chemosensory cues in the secretions and excretions of conspecifics. While the compact organization of this system presents unique opportunities for recording from large portions of the circuit simultaneously, investigation of sensory processing in the AOB remains challenging, largely due to its experimentally disadvantageous location in the brain. Here, we demonstrate a multi-stage dissection that removes the intact AOB inside a single hemisphere of the anterior mouse skull, leaving connections to both the peripheral vomeronasal sensory neurons (VSNs) and local neuronal circuitry intact. The procedure exposes the AOB surface to direct visual inspection, facilitating electrophysiological and optical recordings from AOB circuit elements in the absence of anesthetics. Upon inserting a thin cannula into the vomeronasal organ (VNO), which houses the VSNs, one can directly expose the periphery to social odors and pheromones while recording downstream activity in the AOB. This procedure enables controlled inquiries into AOS information processing, which can shed light on mechanisms linking pheromone exposure to changes in behavior.

Ex Vivo Preparations of the Intact Vomeronasal Organ and Accessory Olfactory Bulb

The mouse accessory olfactory bulb (AOB) has been difficult to study in the context of sensory coding. Here, we demonstrate a dissection that produces an ex vivo preparation in which AOB neurons remain functionally connected to their peripheral inputs, facilitating research into information processing of mouse pheromones and kairomones.

Ex vivo Zubereitungen der Intact Vomeronasalorgan und akzessorischen olfaktorischen Bulbus

The mouse accessory olfactory system (AOS) is a specialized sensory pathway for detecting nonvolatile social odors, pheromones, and kairomones. The first ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In eingehenden Physiologische Analyse definierter Zellpopulationen bei der akuten Gewebeschnitten der Maus Vomeronasalorgans

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

Elektrophysiologische Methode für Ganzzell-Spannungsklemmen Aufnahmen von<em&gt; Drosophila</em&gt; Fotorezeptoren

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Fokale Macropatch Aufnahmen der synaptischen Ströme aus der Drosophila larvalen neuromuskulären Synapse

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...