L&#39;autore desidera ringraziare Peter Castro e Matteo Dinday per i loro primi sforzi per stabilire zebrafish in laboratorio. Questo lavoro è stato finanziato dal National Institutes of Health EUREKA sovvenzione (# R01NS079214-01).

1. Uovo di produzione e raccolta <ol><li> Zebrafish allevamento segue le procedure standard descritte in precedenza 8. In breve, zebrafish adulti vengono impostate in acquari di riproduzione con divisori in atto. Quando le luci in sala si accendono la mattina seguente, divisori vengono rimossi da acquari di riproduzione e pesci sono autorizzati circa 20 a 60 minuti di tempo di accoppiamento indisturbato. </li><li> Uova da vasche di allevamento sono raccolti in un colino e sciacquato con acqua uovo. Uova vengono poi trasferiti ad una piastra di Petri con acqua uovo. Uova non fecondate e detriti vengono rimossi con una pipetta di trasferimento. </li><li> Luogo piatto Petri contenente le uova raccolte in un incubatore (28-32 ° C). Dopo le uova si schiudono, circa due giorni dopo la fecondazione (dpf), rimuovere corion e altri detriti con una pipetta di trasferimento. </li><li> Il giorno desiderato post-fertilizzazione (3-8 dpf) rimuovere piastra Petri contenente larve liberamente nuoto e posto sul banco del laboratorio a temperatura ambiente. </li></ol><ol><li> Utilizzando un estrattore micropipetta, tirare un mm 1,2 diametro esterno in vetro borosilicato capillare in due aghi e conservare in un piatto 150 millimetri Petri o scatola vuota pipetta da posa su rampe Silly Putty. Aghi può essere tirato in anticipo. Diversa OD capillari di vetro possono essere utilizzati a seconda del tipo di amplificatore headstage disponibili. </li><li> Ritardare l&#39;microelettrodo con soluzione di registrazione extracellulare (2 M NaCl) utilizzando una siringa da 1 ml con una siringa Nalgene filtro (4 mm) e Microfil filamento (Warner Precision Instruments) allegata. Agitare o picchiettare il bolo verso la punta dell&#39;ago fino a quando non ci sono bolle o non ancora. </li></ol> 3. Immobilizzazione in Agar <ol><li> Preparare una soluzione fresca di 1,2% agar a basso punto di fusione in acqua uovo. Soluzione da agar in un set da bagno di acqua a ~ 37 ° C. </li><li> Preparare un vetrino con la camera di registrazione e mettere in freezer (5-10 minuti). La camera di registrazione dovrebbe MATCh quello utilizzato sulla piattaforma elettrofisiologia. Usiamo un basso profilo aperto bagno camera di diamante immagini da Warner Instruments (RC-26GLP). </li><li> Usando una pipetta Pasteur o il trasferimento, mettere un pesce zebra larvale in una goccia d&#39;acqua uovo in un piatto pulito piastra di Petri. Per questo gocciolina, aggiungere una goccia di soluzione contenente un anestetico (0,02% tricaine) e agente paralizzante (1 mg / ml α-bungarotossina). </li><li> Monitorare zebrafish per la perdita di movimento (da 5 a 10 minuti). </li><li> Rimuovere il coprioggetto / camera di registrazione dal congelatore. Posizionare camera sul palco di uno stereomicroscopio. Usando una pipetta di trasferimento, mescolare alcuni millilitri di 1,2% di agarosio a basso punto di fusione con la gocciolina e la soluzione di trasferimento (con pesce) al coprioggetto / registrazione da camera. </li><li> Utilizzando un puntale o con ago sottile smussato, larve posizione sotto lo stereomicroscopio in modo che la parte dorsale del pesce è esposto alla superficie del gel di agarosio. Lasciar indurire agarosio (5-10 minuti), quindi utilizzando un trasferimento spatola piatta del b agarbloccare ad una camera di registrazione impostato su una piattaforma elettrofisiologia. </li></ol> 3. Campo di registrazione extracellulare <ol><li> Aggiungere 2-5 ml di supporti di registrazione zebrafish alla camera di registrazione. Se droga cambiamenti sono necessari, camera profumato con soluzione di registrazione ad una velocità di circa 1 ml / min. No ossigenazione della soluzione di registrazione è necessaria. </li><li> Inserire un microelettrodo (circa 1 micron di diametro punta, 2-7 MW) nel headstage amplificatore montato su un tridimensionale micromanipolatore. Controllare che il micromanipolatore è in una posizione appropriata per consentire una gamma di movimento e regolazione. Portare la punta microelettrodo nel piano di vista del microscopio, alto fuori fase, e con regolazione grossolana inferiore ad un punto appena sopra e leggermente davanti alla testa della zebrafish. </li><li> Con l&#39;amplificatore in modalità &quot;current-clamp&quot; zero dell&#39;elettrodo. </li><li> Uso della regolazione grossolana abbassare la punta dell&#39;elettrodo in modo che tocchi solo la surface del zebrafish, leggermente davanti proencefalo. </li><li> Usare passi di regolazione fine avanzare la punta dell&#39;elettrodo fino perfora la pelle del zebrafish. Lasciar depositare e poi avanzare l&#39;elettrodo alcuni micron nel proencefalo. </li><li> Registrare l&#39;attività elettrica in current-clamp mode utilizzando il software Axoscope. I dati vengono acquisiti ad una frequenza di campionamento di 10 kHz. </li></ol>

<ol>
	<li>Clark, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing+zebrafish+for+behavioral+genetics.">Stressing zebrafish for behavioral genetics.</a> Reviews in Neuroscience. 22 (1), 49 (2011).</li><li>Rinkwitz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+integrative+system+for+neurogenomics+and+neurosciences.">Zebrafish: an integrative system for neurogenomics and neurosciences.</a> Progress in Neurobiology. 93 (2), 231 (2011).</li><li>Penberthy, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+human+disease.">The zebrafish as a model for human disease.</a> Frontiers in Bioscience. 7, d1439 (2002).</li><li>Letamendia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+automated+high-throughput+system+for+zebrafish+in+vivo+screenings.">Development and validation of an automated high-throughput system for zebrafish in vivo screenings.</a> PLoS One. 7, e36690 (2012).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nature Genetics. 26 (2), 216 (2000).</li><li>Haffter, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+development+of+the+zebrafish+inner+ear+and+lateral+line.">Mutations affecting development of the zebrafish inner ear and lateral line.</a> Development. 123, 1 (1996).</li><li>Suster, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Molecular Biology. 561, 41 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, 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=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pentylenetetrazole+induced+changes+in+zebrafish+behavior,+neural+activity+and+c-fos+expression.">Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.</a> Neuroscience. 131 (3), 759 (2005).</li><li>Baraban, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+mutagenesis+screen+to+identify+seizure-resistant+zebrafish.">A large-scale mutagenesis screen to identify seizure-resistant zebrafish.</a> Epilepsia. 48 (6), 1151 (2007).</li><li>Williams, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+radiotelemetry+to+evaluate+electrographic+seizures+in+rats+with+kainate-induced+epilepsy.">The use of radiotelemetry to evaluate electrographic seizures in rats with kainate-induced epilepsy.</a> Journal of Neuroscience Methods. 155 (1), 39 (2006).</li><li>Marsh, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+EEG+spikes+identify+the+region+of+electrographic+seizure+onset+in+some,+but+not+all,+pediatric+epilepsy+patients.">Interictal EEG spikes identify the region of electrographic seizure onset in some, but not all, pediatric epilepsy patients.</a> Epilepsia. 51 (4), 592 (2010).</li><li>Zhu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+application+of+modularly+assembled+zinc-finger+nucleases+in+zebrafish.">Evaluation and application of modularly assembled zinc-finger nucleases in zebrafish.</a> Development. 138 (20), 4555 (2011).</li></ol>

Nessun conflitto di interessi dichiarati.

Il metodo di registrazione extracellulare qui presentata consente un&#39;analisi molto sensibile e rapida di attività cerebrale. Queste registrazioni sono analoghi a elettroencefalografico (EEG) monitoraggio comunemente utilizzato per valutare la presenza di scarica elettrica anomala (cioè, sequestro) in modelli di roditori di epilessia 11 e 12 pazienti. Registrazioni extracellulari possono essere combinati con manipolazioni farmacologiche, come illustrato di seguito. Questi tipi di regi...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Agarosio basso punto di fusione </td><td> Fisher Scientific- </td><td> BP1360-100 </td><td> Sciogliere in mezzi embrioni a 1,2% </td></tr><tr><td> Supporti di registrazione </td><td> Fisher Scientific- </td><td> BP3581, P330-3, BP410-1, BP214-500, D16-1, C77-500 </td><td> 1 mM NaCl, 2,9 mM KCl, 10 mM di HEPES, 1,2 mM MgCl2, 10 mM destrosio, 2,1 mM CaCl 2 pH a circa 7,3 con NaOH 1 N </td></tr><tr><td> Tricaine </td><td> Argent Labs </td><td> MS-222 </td><td> 0,02% </td></tr><tr><td> α-bungarotossina </td><td> Bioscience Tocris </td><td> 2133 </td><td> 1 mg / ml </td></tr><tr><td> Vetro tubo capillare </td><td> Warner Instruments </td><td> G120TF-3 </td><td> Tirare per una resistenza di 2 -7 MW </td></tr><tr><td> Patch clamp amplificatore </td><td> Warner Instruments </td><td> PC-505B </td><td> Usiamo un amplificatore Warner in modalità current-clamp; guadagno fissato a 2 mV / pA e del filtro di Bessel fissato a 2K. Modelli comparabili può essere utilizzato in base alle istruzioni del produttore. </td></tr><tr><td> Filtro / amplificatore </td><td> Cygnus Tecnologia </td><td> FLA-01 </td><td> Usiamo un Cygnus pre-amplificatore; guadagno fissato a 10-20; Frequenza di taglio impostato a 1-2K, Filtro Notch IN. Modelli comparabili può essere utilizzato in base alle istruzioni del produttore. </td></tr><tr><td> Axon scheda A / D e il software Axoscope </td><td> Molecular Devices </td><td> Axon Digidata 1320A; Axoscope 8,2 </td><td> I dati vengono raccolti in Axoscope con gap-free modalità di acquisizione, campionamento a 10 kHz. Modelli comparabili e programmi possono essere utilizzati secondo le istruzioni del produttore. </td></tr><tr><td> Egg acqua </td><td&gt; Instant Ocean </td><td></td><td> 3 g immediata Oceano mare sale, 2 ml di blu di metilene 0,1% in 10 ml di acqua deionizzata </td></tr></tbody></table>

forebrain electrophysiological recording in larval zebrafish

L&#39;epilessia colpisce circa 3 milioni di persone negli Stati Uniti e fino a 50 milioni di persone in tutto il mondo. Definito come il verificarsi di convulsioni non provocate spontanee, epilessia può essere acquisita a seguito di un insulto al cervello o una mutazione genetica. Gli sforzi per sequestri modello su animali hanno principalmente utilizzato acquisito insulti (farmaci convulsivanti, stimolazione o lesione cerebrale) e manipolazioni genetiche (atterramento antisenso, ricombinazione omologa o transgenesi) nei roditori. Zebrafish sono un sistema modello vertebrato 1-3 che potrebbe fornire una valida alternativa al roditore ricerca basata epilessia. Zebrafish sono ampiamente utilizzati nello studio dei vertebrati genetica o di sviluppo, presentano un elevato grado di somiglianza genetica ai mammiferi ed esprimere omologhi per ~ 85% di umani noti singolo gene mutazioni epilessia. A causa delle loro piccole dimensioni (4-6 mm di lunghezza), larve di zebrafish può essere mantenuta in volumi di fluido a partire da 100 pl durante lo sviluppo precoce e ARRAYED in multi-pozzetti. I reagenti possono essere aggiunti direttamente alla soluzione in cui gli embrioni sviluppano, semplificando la somministrazione del farmaco e consentendo un rapido screening in vivo di composti di prova 4. Oligonucleotidi sintetici (Morpholinos), mutagenesi, zinc finger nucleasi e approcci transgenici possono essere utilizzati per generare rapidamente knockdown gene o mutazione in zebrafish 5-7. Queste proprietà permettersi studi zebrafish senza precedenti analisi statistica vantaggio potere roditori nello studio di disturbi neurologici come l&#39;epilessia. Poiché il &quot;gold standard&quot; per la ricerca l&#39;epilessia è quello di monitorare e analizzare le scariche elettriche anomale che hanno origine in una struttura cerebrale centrale (ad esempio, convulsioni), un metodo per registrare in modo efficiente l&#39;attività cerebrale in larvale zebrafish è descritto qui. Questo metodo è un adattamento dei tradizionali tecniche di registrazione extracellulari e permette stabile monitoraggio a lungo termine di attività cerebrale in larve di zebrafish intatto. Sregistrazioni ampie sono mostrate per crisi acute indotte da bagno applicazione di farmaci convulsivanti e sequestri spontanei registrati in un pesce geneticamente modificato.

Esempi di elettrografico sequestro-come scarico registrata nel proencefalo di agar-embedded larve zebrafish sono mostrati nella Figura 1. Grande ampiezza multi-picco di scarico scoppio in questi campioni è stata evocata da bagno applicazione di un farmaco convulsivante, 40 pilocarpina mM (in A, 6 dpf) o 1 picrotossina mM (in B, 8 dpf). In queste registrazioni, immobilizzato e agar-embedded zebrafish sono continuamente monitorati per un massimo di 90 min. Pesce rimangono vitali in queste condizioni di r...

Watch this Scientific Journal Video about Forebrain Electrophysiological Recording in Larval Zebrafish at JoVE.com

Forebrain Electrophysiological Recording in Larval Zebrafish

Un metodo semplice per registrare potenziali di campo extracellulari nel proencefalo larvale zebrafish è descritto. Il metodo fornisce un robusto<em&gt; In vivo</em&gt; Lettura di sequestro un&#39;attività simile. Questa tecnica può essere utilizzata con le larve di zebrafish geneticamente modificati portando epilessia geni correlati o convulsioni evocate dalla somministrazione di farmaci convulsivanti.

Proencefalo registrazione elettrofisiologica in larvale Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

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17.0K Views.  University of California, San Francisco. Un metodo semplice per registrare potenziali di campo extracellulari nel proencefalo larvale zebrafish è descritto. Il metodo fornisce un robusto In vivo Lettura di sequestro un'attività simile. Questa tecnica può essere utilizzata con le larve di zebrafish geneticamente modificati portando epilessia geni correlati o convulsioni evocate dalla somministrazione di farmaci convulsivanti.

Video: Forebrain Electrophysiological Recording in Larval Zebrafish

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

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Neuroscienze

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Registrazione elettrofisiologica nel cervello di intatto Zebrafish adulti

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

A Method for Systematic Electrochemical and Electrophysiological Evaluation of Neural Recording Electrodes

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant characteristics.&#160;This precludes a rational selection of a particular implant as optimal for a particular application and the development of new materials based on the most critical performance parameters.&#160;This article develops a protocol for in vitro and in vivo testing of neural recording electrodes.&#160;Recommended parameters for electrochemical and electrophysiological testing are documented with the key steps and potential issues discussed.&#160;This method eliminates or reduces the impact of many systematic errors present in simpler in vivo testing paradigms, especially variations in electrode/neuron distance and between animal models.&#160;The result is a strong correlation between the critical in vitro and in vivo responses, such as impedance and signal-to-noise ratio.&#160;This protocol can easily be adapted to test other electrode materials and designs.&#160;The in vitro techniques can be expanded to any other nondestructive method to determine further important performance indicators.&#160;The principles used for the surgical approach in the auditory pathway can also be modified to other neural regions or tissue.

Different electrode coatings affect neural recording performance through changes to electrochemical, chemical and mechanical properties. Comparison of electrodes in vitro is relatively simple, however comparison of in vivo response is typically complicated by variations in electrode/neuron distance and between animals. This article provides a robust method to compare neural recording electrodes.

Un metodo per la sistematica elettrochimici ed elettrofisiologiche di valutazione dei neurali registrazione elettrodi

New materials and designs for neural implants are typically tested separately, with a demonstration of performance but without reference to other implant ...

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 ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord transezione nel larvale Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Registrazione simultanea elettrofisiologici e micro-iniezioni di agenti inibitori nel cervello del roditore

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, oviposition, and mating. Myriad chemicals of varying volatilities have been identified as natural odorants that activate insect Olfactory Receptor Neurons (ORNs). However, studying the olfactory responses to low-volatility odorants has been hampered by an inability to effectively present such stimuli using conventional odor-delivery methods. Here, we describe a procedure that permits the effective presentation of low-volatility odorants for in vivo Single-Sensillum Recording (SSR). By minimizing the distance between the odor source and the target tissue, this method allows for the application of biologically salient but hitherto inaccessible odorants, including palmitoleic acid, a stimulatory pheromone with a demonstrated effect on ORNs involved in courtship and mating behavior1. Our procedure thus affords a new avenue to assay a host of low-volatility odorants for the study of insect olfaction and pheromone communication.

Electrophysiological Recording from Drosophila Trichoid Sensilla in Response to Odorants of Low Volatility

The overall goal of this protocol is to demonstrate how to present odorants of low volatility for single-sensillum recording from Drosophila olfactory receptor neurons that respond to long-chain cuticular pheromones.

Registrazione elettrofisiologica da<em&gt; Drosophila</em&gt; Senso Trichoide in risposta a Odoranti di bassa volatilità

Insects rely on their sense of smell to guide a wide range of behaviors that are critical for their survival, such as food-seeking, predator avoidance, ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolamento e l'estrazione di RNA dei neuroni, macrofagi e Microglia da cervelli di Zebrafish larvale

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...