Ringraziamo il fondo di Vita, Università di Oklahoma Undergraduate Research Program Opportunità, e il Dipartimento di Zoologia per finanziare questo lavoro. Ringraziamo anche il Dott. Hoefnagels Marielle aiuto editoriale.

1. Pre-registrazione, di preparazione degli animali dal vivo <ol><li> Rimuovere lo scorpione dalla sua casa vaso e metterlo in un pre-raffreddata contenitore di vetro; prossimo, posizionare il vaso contenente lo scorpione in un ambiente freddo (-5 ° C) per un minimo di due minuti. Questo tempo può variare a seconda dell&#39;età, le dimensioni, specie, ecc scorpione dello specifico oggetto di studio. Ma in generale, la fase di congelamento ambiente è completo una volta che l&#39;animale è immobile. </li><li> Dopo due minuti, togliere il immobilizzato scorpione del contenitore in vetro freddo, e posizionarlo lato ventrale su un vetrino da microscopio. Fissare il pungiglione, la coda, le gambe e pedipalpi con argilla modellabile (a base di olio, Van Aken Plastalina). </li><li> Fai una piattaforma su cui mettere le scorpione pectine. Regolare la lunghezza della piattaforma e la larghezza in base alle dimensioni del pecten. Tipicamente, le nostre piattaforme sono costruite in vetro microscopio copertura e sono circa 10 (L) x 18 (W) mm. </li><li> Aderire a doppia faccia nastro adesivo alla piattaforma pecten, taglio del nastro (se necessario) per adattarsi. </li><li> Per effettuare una camera pectinal, mettere tre dei quattro lati piattaforma in pre-cera fusa (usiamo chip di cera d&#39;api 100%) ad una profondità di 3-4 mm. Noi sciogliere la cera mettendo il chip all&#39;interno di un piatto di vetro di Petri, che si trova su un piatto caldo. Una volta che la cera sui bordi della camera pectinal viene raffreddato a temperatura ambiente, questo dovrebbe creare muri di cera di circa 1 mm di altezza. </li><li> Sull&#39;animale, individuare il punto di inserimento pectinal, e posto il non-cerata bordo della piattaforma (lato nastro-up) appena posteriormente ad essa. In seguito, stabilizzare la piattaforma (in modo che non si sposta più avanti) premendo delicatamente la sua destra e di sinistra bordi periferici contro l&#39;argilla utilizzata per contenere le gambe dell&#39;animale. </li><li> Usa punta sottile pinzetta per posizionare accuratamente il pecten o pectine all&#39;interno della camera. In particolare, utilizzando pinze, tenere premuto il pecten lungo la spina dorsale pectinal, e delicatamente portare i pecten fuori da sotto il vetro di copertura e abbassarlo sul pavimento della piattaforma adesivo. Stabilire un pecten-to-tape legame con attenzione pressione sulla colonna vertebrale pectinal. </li><li> Ora applicare cera fusa (usiamo un pre-riscaldato spatola metallica) per il non-cerata bordo della piattaforma. Lo scopo è duplice, per fissare il pecten e da includere completamente la camera per la futura introduzione di olio minerale. Prestare attenzione quando si applica la cera sopra pecten dell&#39;animale, in quanto è possibile danneggiare il pettine con la cera che è troppo caldo. </li><li> La costruzione della camera pecten è completa. Quindi, stabilire una connessione con elettrodo indifferente emolinfa dell&#39;animale inserendo un filo d&#39;argento tra due segmenti della coda. </li></ol> 2. Registrazione simultanea extracellulare e stimolazione chimica <ol><li> Prepariamo le 24 h animale prima di effettuare extracellulare, in punta di registrazioni, che, tra le altre ragioni (regolazione animale cioè in posizione fissa), permette ora di fare su misura pipette stimolante di uno specifico (sensillum-dipendente) in punta di diametro. Usiamo un estrattore micropipetta per raggiungere tali specifiche. Idealmente, il diametro delle pipette è ≈ 2 micron più grande del diametro dei pori sensillar. Noi regolare le nostre specifiche micropipetta di conseguenza per ogni animale. </li><li> 1 prima registrazione h, si aggiungono 5 ml di olio minerale per la camera pectinal, sommergendo il pecten sott&#39;olio. Poi, abbiamo la posizione dell&#39;animale sotto un potente microscopio, che da tempo gli obiettivi di lavoro a distanza e epi-illuminazione. </li><li> Per stimolare chimicamente piolo sensilli, introduciamo tastants acquosa (es. acido citrico e sale) per mezzo di olio. In primo luogo, si usa un riempitivo micropipetta per iniettare il stimolante nella pipetta stimolante. Nota: tutte le soluzioni stimolante dovrebbe contenere un elettrolita per la conduttanza elettrica. </li><li> Successivamente, dopo aver posizionato la pipetta stimolante su un micromanipolatore (nella registrazione rig), inserire l&#39;elettrodo di registrazione nel smussato, estremità aperta della pipetta stimolante. E &#39;importante per assicurare il contatto tra l&#39;elettrodo metallico e la soluzione stimolante elettrolitico. </li><li> Una volta che la preparazione è a massa (tramite l&#39;elettrodo indifferente), abbasseranno la pipetta attraverso il mezzo del petrolio e su un poro sensillar. La durata della stimolazione chimica per sensillum pectinal può variare da fino a trenta minuti per non più di un secondo. In generale, possiamo campione spine multiple (&gt; 30) per ogni pipetta. Nel caso in cui la pipetta stimolante si intasa di detriti minuti, semplicemente scambiare la pipetta intasato per un nuovo, pipetta inutilizzato. </li></ol> 3. Preparazione di un animale per più giorni di sperimentazione <ol><li> Se l&#39;animale deve essere usato per un periodo di tempo prolungato, si consiglia di riutilizzare la camera pectinal e sostituendo la goccia di olio minerale per ogni sessione di registrazione. Dopo la sessione di registrazione di un dato giorno, rimuovere l&#39;animale dalla registrazione di set-up ed effettuare un lavaggio con olio minerale. </li><li> Specifically, aggiungere un&#39;altra goccia (5 mL) di olio minerale per la camera, e rimuovere tutto l&#39;olio. Questo aiuta a minimizzare la presenza di residui stimolante, che può avere trapelato della pipetta durante la sessione di registrazione. </li><li> Prima della prossima sessione, aggiungere una goccia di olio, come descritto in precedenza. </li></ol> 4. Rappresentante dei risultati: A seconda dell&#39;apparecchio di controllo utilizzato (ad esempio l&#39;amplificatore particolare, la digitalizzazione di hardware, ecc), un risultato rappresentativo è una registrazione extracellulare con un S: rapporto N di almeno 3:1. Costruzione di successo della camera di olio minerale è necessario per isolare stimolazione chimica per sensilli singoli, in quanto fornisce un mezzo per la distribuzione di tastants chimici polari (a base d&#39;acqua) 8. Se c&#39;è l&#39;olio troppo poco o niente, la soluzione stimolante si diffonderà dal sensillum registrato al suo sensilli vicini, che rischia di causare un prolungato attacco incontrollata di stimolazione chimica 8.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

Abbiamo già pubblicato questi metodi in due riviste, Knowlton e Gaffin (2009) e Knowlton e Gaffin (in stampa).

Il protocollo sopra descritto come preparare un deserto prateria scorpione (Paruroctonus utahensis) per lo studio elettrofisiologico. In particolare, si mostra come costruire una camera di controllo extracellulare punta-registrazioni di pectinal neuroni chemosensoriali sott&#39;olio. Perché l&#39;olio e l&#39;acqua non si mescolano, è possibile isolare stimolazione chimica (con acqua a base di stimolanti) per sensilli singoli. Va sottolineato che P. utahensis, è un animale relativamente piccolo (≈ 2,5-5 cm) e l&#39;applicazione di questo protocollo per animali più grandi dimensioni possono richiedere un aggiustamento molti, come le dimensioni della piattaforma dell&#39;animale, da camera, e la quantità di olio applicato. Si consiglia la realizzazione di studi pilota per testare la minima quantità di olio necessario per la consegna controllata a base d&#39;acqua stimolanti chimici. Inoltre, abbiamo riscontrato alcun effetto di olio sulla linea di base dell&#39;attività neurale all&#39;interno pectinal sensilli 8. Questo dovrebbe essere confermata per sistemi modello anche altri, prima di valutare chemoresponsiveness. Questo metodo può essere utilizzato in combinazione con un altro metodo per testare gli effetti di interazione tra chemoresponse sensilli nel campo sensoriale. Per esempio, è possibile punta di record (in olio) da un sensillum come base-record (tramite un elettrodo di tungsteno) da un sensillum vicina 9. Tale configurazione di registrazione possono essere utilizzati per valutare anche di costituzione chimica stimolante sensillum influenza l&#39;attività neurale della base-registrato sensillum. Ad oggi, nessuno ha testato questa per pectinal sensilli scorpione, e resta una questione aperta per gli altri sistemi chemosensoriali pure. Per riassumere, pensiamo che la punta di registrazione metodo del petrolio sotto i progressi nel campo di applicazione indagini elettrofisiologiche nelle basi neurali di gustazione artropodi. In questo manoscritto, mettiamo a disposizione un protocollo per la preparazione degli animali per questo metodo, e speriamo che fornisce una solida base per ulteriori studi di sistemi sensoriali nervoso periferico.

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electrophysiology of scorpion peg sensilla

Descriviamo una modifica di una esistente punta-registrazione 1,2 tecnica elettrofisiologico per indagare breve, come il peg-sensoriale sensilli 3,4. Sulla revisione a medio ventrale superficie di tutti gli scorpioni sono due appendici chiamate pectine, che hanno campi densi di meccano-e chemosensoriali peg sensilli 5,6. Un metodo per la valutazione chemoresponsiveness di questi sensilli utilizza un elettrodo di tungsteno per extracellulare registrare l&#39;attività neurale all&#39;interno di un sensillum come odorizzante volatile è introdotto nel campo sensoriale 5,7. I limiti di questo metodo sono lento i dati raccolti e stimolante introduzione incontrollata di, e la rimozione da, il campo piolo. Per superare questi limiti, abbiamo sviluppato una nuova punta-registrazione tecnica che utilizza l&#39;olio minerale non polare come un mezzo attraverso il quale fornire a base d&#39;acqua tastants a singoli peg sensilli 8,9. Noi abbiamo applicato con successo questo metodo per ottenere chemoresponses sensillar di acido citrico, etanolo, e sale. Qui descriviamo il protocollo sperimentale per uno studio 9. Pensiamo che questo nuovo metodo può essere utile per lo studio delle proprietà di risposta di altri sistemi artropodi chemosensoriali, compresi quelli degli insetti 10, 11 e crostacei 12.

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

Questo articolo descrive un metodo per isolare elettrofisiologico stimolazione chimica per i singoli sensilli via extracellulare, in punta di registrazioni in olio minerale.

Elettrofisiologia di Scorpion Peg sensilli

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.9K Views.  University of Oklahoma. Questo articolo descrive un metodo per isolare elettrofisiologico stimolazione chimica per i singoli sensilli via extracellulare, in punta di registrazioni in olio minerale.

Video: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

FMRI simultanea ed Elettrofisiologia nel cervello roditori

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Ricerca

Neuroscienze

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Elettrofisiologia II: extracellulare fabbricazione di elettrodi di aspirazione

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

Coltura ed Elettrofisiologia di Celle NRCC patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Nanoinjection associato e test Elettrofisiologia allo schermo per la bioattività dei composti con il Drosophila melanogaster Sistema Fiber Giant

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Un isolato semi-intatte Preparazione del mouse vestibolare epitelio sensoriale di Elettrofisiologia e ad alta risoluzione microscopia a due fotoni

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

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

Elettrofisiologici Registrazione da<em&gt; Drosophila</em&gt; Labellar Taste Sensilla

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

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Real-time Elettrofisiologia: utilizzando protocolli a circuito chiuso per sonda neuronali Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Elettrofisiologia su isolati tronco cerebrale, midollo spinale Ammassi di Newborn Roditore Consente Neural Network respiratoria registrazione Uscita

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Ganglio spirale Neuron Espianto cultura e Elettrofisiologia su Multi Electrode Array

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

L&#39;incubazione prolungata di acuta neuronale tessuto per Elettrofisiologia e Calcio imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...