Nous tenons à remercier les membres du Laboratoire Saint. Ce travail a été soutenu par l&#39;Institut national des troubles Surdité et Communication (R01 DC005964 à LA) et NSF IGERT 0548890 (AHA). Expériences sur les animaux ont été effectuées en conformité avec les lois des États-Unis la protection des animaux et des National Institutes of Health et de directives ont été approuvées par les soins des animaux de Washington University et le Comité d&#39;utilisation.

Partie 1. Instrumentation enregistrement électrique <ol><li> Nous utilisons un large multiélectrodes planaires (ALA Scientific Instruments) avec 10 um électrodes plates de nitrure de titane isolé avec du nitrure de silicium. 60 électrodes sont disposées dans deux domaines constitué d&#39;un quadrillage 5x6 avec le centre à centre espacement de 30 um. (D&#39;autres configurations sont également possibles; électrodes avec une surface d&#39;enregistrement 10 m de diamètre sont mieux que les grandes électrodes en termes d&#39;isoler l&#39;activité des neurones individuels.) </li><li> Les signaux électriques sont amplifiées (amplification 1000x) en utilisant un amplificateur de MEA 1060 (ALA Scientific Instruments). Les signaux sont acquis à 10 kHz en utilisant une carte d&#39;acquisition de données (National Instruments) et de logiciels personnalisés d&#39;acquisition de données (on peut également acheter des logiciels commerciaux). </li><li> Le tissu est maintenu sur l&#39;AEM par un filet de nylon (53 um), qui est attaché à une insertion personnalisé qui s&#39;adapte parfaitement à l&#39;intérieur de l&#39;anneau tubulaire de la culture de la MEA. </li></ol> Partie 2. Instrumentation distribution de liquide <ol><li> Le tissu nécessite surfusion continue, que nous garantissons en fournissant une solution de Ringer (115 de chlorure de sodium mM, 5 mM de chlorure de potassium, 2 chlorure de calcium mm, 2 de chlorure de magnésium mm, 25 bicarbonate de sodium mM, 10 mM d&#39;HEPES et 10 mM de D-(+ )-glucose) à partir d&#39;une pompe HPLC. Les stimuli sont présentés en effectuant des injections dans une vanne de CLHP et de commutation entre les actions accréditives et de la boucle. Tant la pompe HPLC et seringue d&#39;injection du robot sont fournis avec la solution de Ringer. </li><li> La solution de Ringer est maintenu dans un bain d&#39;eau chauffée (ISOTEMP 105) à 42 ° C toute l&#39;expérience et est continuellement à barboter avec 95% d&#39;O 2 / 5% CO 2. Un ballon séparé de Ringer sans glucose est préparé pour la sauvegarde de la pompe à seringue. Solution de Ringer est délivrée au tissu en utilisant une pompe HPLC 307 (Gilson) à un taux de 3 ml / min. Dans un typique 5-6 expérimentation heures, 1 L de solution de Ringer chaque est suffisante. </li><li> Tout au long de l&#39;expérience, le tissu est continuellement perfusées avec 37 ° C une solution de Ringer. Une sonde radiateur est positionné directement au-dessus du tissu. </li><li> Les stimuli sont livrés avec le bras de Gilson 215 Handler liquide robotisé. Les stimuli sont stockés dans des flacons en verre de caoutchouc plafonnés, et sous le contrôle des logiciels de Gilson, le bras robotique fournit les stimuli à la ligne de perfusion alors que le taux de débit et pression constantes sont maintenues. </li><li> L&#39;état de la vanne HPLC (boucle engagés / non engagé) est représenté par une tension électrique, qui est enregistré par l&#39;ordinateur d&#39;acquisition comme un canal supplémentaire pour assurer la synchronisation avec l&#39;activité neuronale. </li></ol> Partie 3. Préparation de la MEA et dispositif de livraison de stimulation <ol><li> Avant de procéder à la dissection, remplissez le MEA bien avec BSA dans dH2O pendant au moins 5 minutes à température ambiante. </li><li> Rincez à l&#39;aide de la MEA warm-buller solution de Ringer et le lieu de la MEA sur la glace. </li><li> Premier gestionnaire de liquide et pompe HPLC selon les instructions du fabricant. </li></ol> Partie 4. Dissection VNO <ol><li> Avant d&#39;effectuer la dissection, remplissez 2 60x15 mm boîtes de Pétri avec une solution de Ringer et barbotage placer sur la glace. Remplissez également deux Sylgard revêtement des boîtes de Petri de la même taille avec une solution de Ringer et le placer sur la glace. Réfrigérer une 50mL supplémentaire de warm-buller Ringer pour une utilisation lors de la dissection. </li><li> La souris est sacrifiée par asphyxie CO 2 suivie par dislocation cervicale. </li><li> Faire des incisions bilatérales du côté de la bouche, parallèle à la ligne de la mâchoire, à l&#39;arrière de la tête. Rejoignez les deux coupes le long du dos de la nuque et retirez la partie dorsale de la tête, laissant la mâchoire inférieure attachée à la souris. </li><li> Avec des ciseaux fins, coupés entre le tissu et de chaque côté de l&#39;os maxillaire supérieur pour séparer les tissus de l&#39;os. </li><li> En utilisant une pince # 3, décoller le tissu palais à exposer la cavité nasale. </li><li> L&#39;utilisation de petits ciseaux ou un scalpel, couper entre et de chaque côté de la tige 2 dents de devant pour libérer la capsule voméronasal de l&#39;os. </li><li> En utilisant une pince # 3, ramasser la capsule voméronasal par l&#39;os plat et le placer dans la boîte de Pétri sur la glace. Les étapes à travers l&#39;immersion devrait être effectué en moins de 3 minutes. </li><li> Placer le plat sous un stéréomicroscope, éclairé par la lumière réfléchie. Retirez la capsule osseuse d&#39;un VNO à un moment à l&#39;aide n ° 3 pinces. Utilisez une main pour stabiliser la capsule osseuse par serrage de l&#39;extrémité caudale et l&#39;autre main pour éplucher l&#39;os loin du VNO, en prenant soin de ne pas endommager le VNO. </li><li> Lorsque l&#39;os est enlevé, le transfert du VNO à une boîte de Pétri Sylgard couché. Utilisez le plat reste pour le VNO autres. Mettre l&#39;éclairage à lumière transmise. </li><li> L&#39;utilisation de micro-ciseaux ou # 5 forceps, séparer le VNO à partir du vaisseau sanguin en coupant le long du bord de tIl VNO. À ce stade, vous aurez la feuille de neurones complètement séparé du vaisseau sanguin. </li><li> Placez le VNO à la place de côté (dendritiques) interne. En utilisant une pince # 3, ancrer le VNO à l&#39;Sylgard à un coin du tissu. Peler l&#39;épithélium neuronal de la lame basale en utilisant un petit fragment d&#39;une lame de rasoir en téflon tenue avec une pince. En tenant la lame à un angle faible (&lt;30 ° de la surface de plat) est le meilleur. </li><li> En utilisant une pipette en verre, le transfert de la pièce de l&#39;épithélium neural de l&#39;antenne à l&#39;AEM. Position du VNO sur le dessus de l&#39;électrode sur le terrain avec le côté jusqu&#39;à dendritiques. Retirer l&#39;excès de solution de Ringer avec une pipette. Placez le porte-mailles du tissu dans le puits de la matrice de tenir le VNO en place, et le remplir d&#39;une solution de Ringer réfrigérée est. </li></ol> Partie 5. Enregistrement <ol><li> Placez la MEA dans un MEA 1060 amplificateur et la position de la sonde chauffe directement au-dessus du tissu. </li><li> Pour confirmer le placement correct de sonde de chauffage, de stimuler avec une solution de Ringer contenant 50 mM de potassium (substitué pour le sodium équimolaire) pendant 2 secondes. Ajustez le positionnement de minimiser la latence de la réponse neuronale à la solution de potassium. </li><li> Superfuse le tissu avec une solution de Ringer chauffée pendant 45 minutes à 1 heure avant l&#39;enregistrement pour permettre au tissu de s&#39;acclimater. </li><li> Les signaux électriques sont sauvegardées sur le disque en utilisant un logiciel d&#39;enregistrement personnalisés. </li></ol> Partie 6. Analyse des données <ol><li> Hors analyse des données peut être fait en utilisant les enregistrements d&#39;électrode premières. Par exemple, nous mesurons les changements cadence de tir de cellules enregistrées en réponse à des ligands particulier. Les ensembles de données qui en résultent sont grands et peuvent être utilisés pour traiter de nombreuses questions différentes; familiarité avec un langage de programmation ou d&#39;analyses (C, Matlab, R, Python, ou similaire) est nécessaire. </li></ol> Partie 7. Les résultats représentatifs Cette préparation est généralement assez stable pour enregistrer à partir de 6 heures, ce qui donne une grande quantité de données (6 heures d&#39;enregistrement de 60 électrodes). <a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg"><img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="Figure 1" border="0" /></a> Figure 1. Un instantané de l&#39;activité simultanée à travers une moitié du tableau est montré. La fenêtre de temps s&#39;étend de 400 ms. <a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg"><img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="Figure 2" border="0" /></a> Figure 2. Dans une expérience typique, nous stimulons par des composés d&#39;intérêt particulier pour les 10 secondes. Cette trace est un exemple d&#39;une réponse à 100 fois l&#39;urine de souris dilué femelle.

<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>

Enregistrements array multiélectrodes nous permettent la possibilité de surveiller l&#39;activité simultanée d&#39;une population importante de neurones. C&#39;est un outil utile pour sonder les propriétés d&#39;un système sensoriel, en particulier le VNO. Contrairement au système olfactif principal, le VNO détecte les stimuli en phase liquide. En outre, le VNO est un tissu hétérogène. Il ya environ 300 types différents de récepteurs chez la souris une, sans doute avec des profils de réponse di...

multielectrode array recordings of the vomeronasal epithelium

Comprendre les circuits neuronaux nécessite des méthodes pour enregistrer à partir de nombreux neurones simultanément. Pour<em&gt; In vitro</em&gt; Les études, une seule technologie actuellement disponible est planaire array multiélectrodes (MEA) d&#39;enregistrement. Ici, nous documenter l&#39;utilisation des AME pour étudier la souris voméronasal organe (VNO), qui joue un rôle essentiel dans la détection des phéromones et des indices sociaux via une population diversifiée de neurones sensoriels exprimant des centaines de types de récepteurs. Combinant l&#39;enregistrement MEA avec un gestionnaire robotique de liquide pour administrer des stimuli chimiques, les réponses sensorielles d&#39;une population importante et diversifiée de neurones peuvent être enregistrées. La préparation nous permet d&#39;enlever le neuroépithélium intacte du VNO de la souris et stimuler avec une batterie de produits chimiques ou des ligands potentiels tout en surveillant l&#39;activité électrique des neurones pendant plusieurs heures. Par conséquent, cette technique sert comme une méthode utile pour évaluer l&#39;activité du ligand ainsi que d&#39;explorer les propriétés des neurones récepteurs. Nous présentons les techniques nécessaires pour préparer l&#39;épithélium voméronasal, l&#39;enregistrement des AEM, et une stimulation chimique.

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

Multielectrode Array Recordings of the Vomeronasal Epithelium

Multiélectrodes array (MEA) enregistrements fournissent une méthode pour étudier l&#39;activité électrique des grandes populations de neurones. Ici, nous présentons les détails d&#39;une préparation de la MEA pour enregistrer à partir de l&#39;épithélium de souris voméronasal tout en stimulant le tissu.

Enregistrements tableau multiélectrodes de l&#39;épithélium Vomeronasal

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

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12.1K vues.  Washington University School of Medicine. Multiélectrodes array (MEA) enregistrements fournissent une méthode pour étudier l'activité électrique des grandes populations de neurones. Ici, nous présentons les détails d'une préparation de la MEA pour enregistrer à partir de l'épithélium de souris voméronasal tout en stimulant le tissu.

Vidéo: 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.

Une seule unité In vivo Enregistrements à partir du chiasma optique du 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 ...

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Neurosciences

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" ...

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.

Dissection d&#39;un oeil de la souris pour un mont entiers de l&#39;épithélium pigmentaire rétinien

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.

Enregistrements tableau multi-électrodes des Avalanches neuronale dans des cultures organotypiques

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.

Imagerie phéromones détection dans une souris Vomeronasal tissus Préparation Trancher aiguë

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.

Réponses d&#39;imagerie neuronale dans les préparations d&#39;organe voméronasal Slice Exprimant un capteur de calcium codées génétiquement

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 préparatifs de l&#39;ampoule Vomeronasal organes et accessoire olfactive Intact

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.

En profondeur Physiological Analyse des populations de cellules définies dans le tissu Tranches aiguë de l&#39;Vomeronasal Organ Souris

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.

Méthode électrophysiologique pour les enregistrements de la pince à tension intégrale à partir de<em&gt; Drosophila</em&gt; Photorécepteurs

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.

Focal Macropatch enregistrements de courants synaptiques de la jonction neuromusculaire larves de drosophile

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