Vorremmo ringraziare i membri del Laboratorio Santo. Questo lavoro è stato sostenuto dall&#39;Istituto Nazionale per la Sordità ei disturbi della comunicazione (R01 DC005964 a LA) e NSF IGERT 0.548.890 (HAA). Gli esperimenti sugli animali sono stati eseguiti in conformità con gli Atti del benessere degli animali Stati Uniti e gli Istituti Nazionali di Sanità e le linee guida sono state approvate dalla cura degli animali Washington University e Comitato Usa.

Parte 1. Registrazione Strumentazione elettrica <ol><li> Usiamo una schiera planare multielectrode (ALA Strumenti scientifici) con 10 elettrodi in nitruro di titanio micron piatto isolato con nitruro di silicio. 60 elettrodi sono disposti in due campi formato da una griglia 5x6 con centro a centro distanza di 30 micron. (Altre configurazioni sono possibili; elettrodi con una superficie di registrazione 10 micron di diametro, sono migliori rispetto alle grandi elettrodi in termini di isolare l&#39;attività dei singoli neuroni.) </li><li> I segnali elettrici sono amplificati (amplificazione 1000x) utilizzando un MEA 1060 amplificatore (ALA Strumenti scientifici). I segnali vengono acquisiti a 10 kHz utilizzando una scheda di acquisizione dati (National Instruments) e personalizzati software di acquisizione dati (si può anche acquistare il software commerciale). </li><li> Il tessuto viene tenuto premuto sulla MEA da una maglia di nylon (53 micron), che è collegato a un inserto personalizzata che si adatta perfettamente all&#39;interno dell&#39;anello cultura tubo del MEA. </li></ol> Parte 2. Consegna Strumentazione liquido <ol><li> Il tessuto richiede superfusione continuo, che possiamo garantire, fornendo la soluzione di Ringer (115 cloruro di sodio mm, 5 mm cloruro di potassio, 2 cloruro di calcio mM, 2 cloruro di magnesio mm, 25 bicarbonato di sodio mm, 10 HEPES mM e 10 mM D-(+ )-glucosio) da una pompa HPLC. Gli stimoli sono presentati eseguendo iniezioni in una valvola di HPLC e il passaggio tra flow-through e il ciclo. Sia la pompa HPLC e pompa a siringa del robot di iniezione sono forniti con la soluzione di Ringer. </li><li> Ringer è tenuto in un bagno di acqua riscaldata (Isotemp 105) a 42 ° C durante tutto l&#39;esperimento ed è continuamente bollito con il 95% O 2 / 5% di CO 2. Un fiasco separata di Ringer senza glucosio è preparato per sostenere la pompa a siringa. Ringer viene consegnato al tessuto utilizzando un HPLC 307 pompa (Gilson) ad una velocità di 3 ml / min. In un tipico esperimento di 5-6 ore, 1 litro di soluzione ogni Ringer è sufficiente. </li><li> Tutto l&#39;esperimento, il tessuto è in continuo superfused con 37 ° C la soluzione di Ringer. Una sonda riscaldamento è posizionata direttamente sopra il tessuto. </li><li> Gli stimoli vengono consegnati con il liquido Gilson 215 braccio robotico Handler. Gli stimoli vengono memorizzati in gomma-capped fiale di vetro, e sotto il controllo del software Gilson, il braccio robotico offre gli stimoli alla linea di perfusione, mentre la portata e pressione costanti sono mantenute. </li><li> Lo stato della valvola HPLC (anello impegnato / non impegnati) è rappresentato da una tensione elettrica, che viene registrata dal computer di acquisizione come un canale supplementare per garantire la sincronizzazione con l&#39;attività neuronale. </li></ol> Parte 3. Preparazione della MEA e dispositivo di erogazione Stimolo <ol><li> Prima di eseguire la dissezione, riempire il MEA bene con BSA in dH2O per almeno 5 minuti a temperatura ambiente. </li><li> Sciacquare la MEA utilizzando warm-bolle Ringer soluzione e posizionare il MEA sul ghiaccio. </li><li> Primo il gestore di liquido e pompe HPLC secondo le istruzioni del produttore. </li></ol> Parte 4. VNO Dissection <ol><li> Prima di eseguire la dissezione, riempire 2 piatti 60x15 millimetri Petri con bollito soluzione di Ringer e posto sul ghiaccio. Anche riempire 2 Sylgard piatti rivestiti Petri della stessa dimensione con la soluzione di Ringer e posto sul ghiaccio. Freddo uno 50mL aggiuntivo di warm-bollito Ringer per l&#39;uso durante la dissezione. </li><li> Il mouse è sacrificato tramite CO 2 asfissia seguita da dislocazione cervicale. </li><li> Fai incisioni bilaterali dal lato della bocca, che corre parallela alla linea della mascella, alla parte posteriore della testa. Partecipa al 2 tagli lungo la parte posteriore del collo e togliere la parte dorsale della testa, lasciando la mascella inferiore attaccato al mouse. </li><li> Utilizzando forbici, tagliare tra il tessuto e ai lati della mascella superiore per separare il tessuto dall&#39;osso. </li><li> Utilizzando # 3 pinze, staccare il tessuto palato per esporre la cavità nasale. </li><li> Utilizzando piccole forbici o un bisturi, tagliare tra e su entrambi i lati della tomaia 2 denti anteriori per liberare la capsula vomeronasale dall&#39;osso. </li><li> Utilizzando # 3 pinze, prendere la capsula vomeronasale dalle ossa piatte e posto nella scatola di Petri sul ghiaccio. I passi attraverso l&#39;immersione deve essere eseguita in meno di 3 minuti. </li><li> Porre la capsula sotto uno stereomicroscopio, illuminata con luce riflessa. Rimuovere la capsula ossea da un VNO alla volta utilizzando # 3 pinze. Usare una mano per stabilizzare la capsula ossea da presa a partire dalla fine caudale e l&#39;altra mano per sbucciare l&#39;osso dalla VNO, facendo attenzione a non danneggiare la VNO. </li><li> Quando l&#39;osso viene rimosso, trasferire il VNO Sylgard ad un piatto rivestito di Petri. Utilizzare il piatto rimanenti per il VNO altri. Passa l&#39;illuminazione a luce trasmessa. </li><li> Utilizzando micro-forbici o pinze # 5, separare il VNO dal vaso sanguigno, tagliando lungo il bordo del VNO. A questo punto sihanno il foglio neurali completamente separato dal vaso sanguigno. </li><li> Posizionare il VNO con l&#39;interno (dendritiche) fino laterali. Utilizzando # 3 pinze, ancora il VNO al Sylgard in un angolo del tessuto. Sbucciate l&#39;epitelio neurale dalla lamina basale con un piccolo frammento di rivestita in teflon lama di rasoio tenuto con una fascetta. Tenendo la lama con un angolo basso (&lt;30 ° rispetto alla superficie del piatto) è il migliore. </li><li> Utilizzando una pipetta di vetro, trasferire il pezzo di epitelio neurale dal piatto alla MEA. Posizionare il VNO sulla parte superiore del campo di elettrodi con il lato alto dendritiche. Rimuovere la soluzione di Ringer in eccesso con una pipetta. Posizionare il supporto del tessuto mesh nel pozzo della matrice per tenere il VNO a posto, e riempire con soluzione di Ringer refrigerata è. </li></ol> Parte 5. Registrazione <ol><li> Posizionare il MEA in un amplificatore 1060 MEA e posizionare la sonda riscaldamento direttamente sopra il tessuto. </li><li> Per confermare il posizionamento corretto della sonda riscaldamento, stimolare con la soluzione di Ringer contenente 50 mM di potassio (sostituito sodio equimolari) per 2 secondi. Regolare il posizionamento di ridurre al minimo la latenza della risposta neurale alla soluzione di potassio. </li><li> Superfuse il tessuto con una soluzione di Ringer riscaldata per 45 minuti a 1 ora prima della registrazione per consentire il tessuto si adatti. </li><li> I segnali elettrici vengono salvati su hard disk utilizzando software personalizzato di registrazione. </li></ol> Parte 6. Analisi dei dati <ol><li> Offline analisi dei dati può essere fatto utilizzando le registrazioni elettrodo prime. Per esempio, si misura la variazione dei tassi di cottura delle cellule registrate in risposta a ligandi particolare. I set di dati risultanti sono grandi e possono essere utilizzate per affrontare molte questioni diverse, la familiarità con un linguaggio di programmazione o di analisi (C, Matlab, R, Python, o simili) è richiesto. </li></ol> Parte 7. Rappresentante Risultati Questa preparazione è in genere abbastanza stabile per registrare da per 6 ore, producendo una grande quantità di dati (6 ore di registrazione da 60 elettrodi). <img src="/files/ftp_upload/1845/1845fig1tmb.jpg" alt="Figura 1" border="0" /> Figura 1. Un&#39;istantanea di attività simultanee su una metà della matrice è mostrato. La finestra temporale si estende su 400 ms. Si prega di <a href="https://www.jove.com/files/ftp_upload/1845/1845fig1.jpg">cliccare qui</a> per vedere una versione più grande della figura 1. <img src="/files/ftp_upload/1845/1845fig2tmb.jpg" alt="Figura 2" border="0" /> Figura 2. In un tipico esperimento, stimoliamo con particolari composti di interesse per 10 secondi. Questa traccia è un esempio di una risposta a 100 volte l&#39;urina diluita topo femmina. Si prega di <a href="https://www.jove.com/files/ftp_upload/1845/1845fig2.jpg">cliccare qui</a> per vedere una versione più grande della figura 2.

<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>

Registrazioni serie Multielectrode ci permette la possibilità di monitorare l&#39;attività simultanea di una grande popolazione di neuroni. Questo è un utile strumento per sondare le proprietà di un sistema sensoriale, in particolare il VNO. A differenza del sistema olfattivo principale, il VNO rileva stimoli in fase liquida. Inoltre, il VNO è un tessuto eterogeneo. Ci sono circa 300 tipi diversi di recettori nel topo 1, presumibilmente con profili di risposta diversa. Registrazioni MEA combinato con il ...

multielectrode array recordings of the vomeronasal epithelium

Comprensione circuiti neurali richiede metodi per registrare da molti neuroni contemporaneamente. Per<em&gt; In vitro</em&gt; Studi, una tecnologia attualmente disponibile è planare serie multielectrode (MEA) di registrazione. Qui documentare l&#39;uso di MEA per studiare il mouse organo vomeronasale (VNO), che svolge un ruolo essenziale nella rilevazione dei feromoni e segnali sociali attraverso una popolazione diversificata di neuroni sensoriali che esprimono centinaia di tipi di recettori. Combinando la registrazione MEA con un gestore robotica liquido per fornire stimoli chimici, le reazioni sensoriali di una popolazione vasta e diversificata di neuroni possono essere registrati. La preparazione ci permette di rimuovere il neuroepitelio intatta del VNO dal mouse e stimolare con una batteria di sostanze chimiche o di ligandi potenziali monitorando l&#39;attività elettrica dei neuroni per diverse ore. Pertanto, questa tecnica serve come un metodo utile per valutare l&#39;attività legante così come esplorare le proprietà dei neuroni. Vi presentiamo le tecniche necessarie per preparare l&#39;epitelio vomeronasale, registrazione MEA, e la stimolazione chimica.

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

Multielectrode Array Recordings of the Vomeronasal Epithelium

Multielectrode array (MEA), le registrazioni forniscono un metodo per studiare l&#39;attività elettrica di ampie popolazioni di neuroni. Qui, vi presentiamo i dettagli di una preparazione MEA per registrare dal epitelio del mouse vomeronasale mentre simultaneamente stimola il tessuto.

Array registrazioni Multielectrode dell&#39;epitelio 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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JoVE Journal

Neuroscience

12.1K Views.  Washington University School of Medicine. Multielectrode array (MEA), le registrazioni forniscono un metodo per studiare l'attività elettrica di ampie popolazioni di neuroni. Qui, vi presentiamo i dettagli di una preparazione MEA per registrare dal epitelio del mouse vomeronasale mentre simultaneamente stimola il tessuto.

Video: Multielectrode Array Recordings of the Vomeronasal Epithelium

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.

Le registrazioni elettrofisiologiche dal Percorso di Fiber Gigante 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" ...

Ricerca

Neuroscienze

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.

Dissezione di un occhio mouse per un monte intero di dell&#39;epitelio pigmentato retinico

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-array di elettrodi Recordings Valanghe neuronali in Culture organotipica

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 preparazione dei tessuti Vomeronasal acuta Slice mouse

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.

Le risposte di imaging neuronale in preparazione Fetta di organo vomeronasale Esprimere un sensore di calcio geneticamente codificato

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 preparati della lampadina vomeronasale Organo e accessori olfattivo intatto

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.

Approfondita fisiologica Analisi di popolazioni di cellule definite nel acuta del tessuto Fette del mouse Vomeronasal Organo

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

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

Subcellulari patch-clamp dal dominio Somatodendritic di nigral dopamina neuroni

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

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.

Metodo elettrofisiologico per le registrazioni a morsetto di tensione a cellule intere da<em&gt; Drosophila</em&gt; Fotorecettori

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.

Macropatch focale registrazioni delle correnti sinaptiche da giunzione neuromuscolare larvale della drosofila

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