I finanziamenti forniti dalla National Science Foundation (IOS-1050701 a BAC), il National Institutes of Health (NS54174 di SM e F30DC0111907 di AML-W.), Il Uehara Memorial Foundation e la Japan Society per la promozione della scienza (G2205 di TK ). Ringraziamo Julian Meeks per il suo sostegno e la guida in tema di registrazioni extracellulari assonale. Ringraziamo Carl Hopkins per la fornitura di una camera di registrazione prototipo.

<ol>
	<li>Meeks, J. P., Jiang, X., Mennerick, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+potential+fidelity+during+normal+and+epileptiform+activity+in+paired+soma-axon+recordings+from+rat+hippocampus.">Action potential fidelity during normal and epileptiform activity in paired soma-axon recordings from rat hippocampus.</a> J. 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Nessun conflitto di interessi dichiarati.

Una volta padroneggiata, questa tecnica permetterà uno a bersaglio i neuroni identificati, tra cui i singoli assoni, per registrazioni in vivo in molti sistemi modello. Inoltre, questa tecnica permette di registrare attendibilmente uscita spike da neuroni con caratteristiche anatomiche uniche che rendono tradizionali metodi di registrazione in vivo impegnativo. Abbiamo utilizzato questa tecnica per registrare da Ela &quot;piccole cellule&quot; in mormyrid pesci debolmente elettrico. I precedenti tentativi di studiare le proprietà di sintonia di &quot;piccole celle&quot; non hanno avuto successo a causa di condizioni di ripresa difficili 11, 14. Caratteristiche anatomiche simili creano ostacoli all&#39;ottenimento di registrazioni singola unità da molti uditivo vertebrati diversi e neuroni electrosensory 8-10. Per superare queste sfide nel nostro sistema, abbiamo approfittato del fatto che &quot;piccole celle&quot; sono le uniche cellule in ELA che proiettano a Elp. Così, trasporto retrogrado di colorante collocato in Elp limita etichettatura in ELA &quot;piccolocellulari &quot;somas e assoni. etichettatura fluorescente degli assoni consentito il posizionamento degli elettrodi preciso vicino al assoni etichettati, effettuare registrazioni singola unità di cellule identificate possibili nonostante somas inaccessibili. Abbiamo tentato registrazioni somatiche, ma non hanno avuto successo, probabilmente a causa delle circostanti sinapsi engulfing 11 , 17. Tuttavia il marchio somatica era chiaramente visibile suggerendo che questa tecnica potrebbe essere utilizzato per mirare registrazioni somatiche in altri tipi cellulari e altri circuiti. etichettatura fluorescente di neuroni attraverso trasporto retrogrado in vivo è stato usato per guidare registrazioni mirate in vitro 19-21 . Una tecnica simile è stata utilizzata per la mirata in registrazioni dal vivo motoneuroni zebrafish midollo spinale 22. nostro lavoro rappresenta una nuova espansione di questo approccio, in cui sia l&#39;etichettatura e la registrazione sono fatte in vivo all&#39;interno del cervello. nostro metodo dimostra che in vivo etichettatura delle aree del sistema nervoso centrale con un retrogrado tcorridore può essere ampliato per lo studio di altri circuiti intatti con i neuroni di proiezione allo stesso modo selettivo. Per esempio, in elaborazione uditiva mammiferi, il collicolo inferiore (IC) serve come un centro importante relè per ingressi da più strutture rhombencephalic 23. Iniezione di colorante nella IC avrebbe etichettare selettivamente le cellule di proiezione da ciascuno di questi nuclei. Il collicolo superiore (SC) ha una funzione simile per la visione 24. Preparazioni midollo spinale sono particolarmente adatti per questa tecnica, come il midollo spinale è facilmente accessibile, iniezione colorante può verificarsi lontano dal sito di registrazione, e può essere combinato con registrazione intracellulare e riempimento dei neuroni selezionati per acquisire informazioni più dettagliate anatomico 25. Infine, tratto-tracing è una tecnica ben consolidata utilizzata in tutto il sistema nervoso centrale per mappare circuiteria complessa 26. Il nostro metodo può essere utilizzato per aggiungere informazioni funzionali a questi studi come è stato fatto wIndicatori di calcio-sensibili esima in gatto corteccia visiva 27. L&#39;intervento, che è ben stabilita, affidabile, e regolarmente utilizzati per ciechi in vivo registrazioni 16, deve essere completato con minimo sanguinamento e nessun danneggiamento della superficie del cervello per consentire l&#39;animale e il tessuto di sopravvivere. Con la pratica, l&#39;applicazione chirurgia e colorante può essere completato in 30-45 minuti. Abbiamo etichettato con successo &quot;piccole cellule&quot; assoni nel 67% dei preparativi. La maggior parte dei preparativi sono solo 1 o 2 assoni etichettati visibili, ma alcuni hanno ben 8. Delle 119 unità etichettati tentato, abbiamo ottenuto le registrazioni singola unità da 26 unità distribuite su 12 preparati (Tabella 2). Pertanto, i dati sono stati raccolti da 41% di preparazioni assoni etichettati per una percentuale di successo del 28%. L&#39;aspetto critico di applicazione colorante viene etichettando profondità. Inserimento superficiale del filo di tungsteno comporterà il colorante essendo aw lavatoay. Tuttavia, se la penetrazione è troppo profonda, assoni etichettati non saranno visibili per il targeting. Inoltre, alcuni danni meccanici alla cella deve verificarsi per la tintura di essere adeguatamente ripreso 25, 28. Tuttavia, troppi danni uccide le cellule. Abbiamo tentato di etichettatura con altri coloranti e altri metodi (Tabella 2), tra cui l&#39;etichettatura anterograda attraverso iniezione colorante in ELA in coppia con la registrazione da assoni etichettati a Elp (Tabella 3). Ipotizziamo che l&#39;etichettatura anterograda non è riuscita perché l&#39;etichettatura è stata limitata alle cellule con danni somatici, rendendoli insensibili. Inoltre, i terminali presinaptici possono essere stati disturbati. Al contrario, marcatura retrograda minimizza sia di queste preoccupazioni. La quantità e la posizione di applicazione colorante possono essere modificati secondo la particolare circuito studiato. Etichettatura massima si verifica con la maggiore concentrazione di colorante, che abbiamo realizzato con fili di tungsteno rivestiti. Tuttavia, per l&#39;etichettatura di assoni con deproiezioni ep, colorante può venire fuori un ago di tungsteno in quanto è avanzato. In questi casi, la pressione di iniezione sarebbe più appropriato. Assorbimento della tintura e il trasporto sono rapidi, con assoni etichettato la nostra preparazione e sia ben visibile fin da 2 ore dopo l&#39;iniezione e ulteriori assoni etichettati appaiono più tardi sei ore dopo l&#39;iniezione. Pertanto, l&#39;etichettatura e la registrazione può essere realizzato in un solo giorno, eliminando le difficoltà tecniche connesse con la chirurgia sopravvivenza. Timing varierà per ogni applicazione a seconda della distanza necessaria per il trasporto colorante. Un altro aspetto critico è il posizionamento degli elettrodi. È importante immettere il tessuto vicino al sito dell&#39;assone marcato per evitare l&#39;ostruzione della punta. Per il tessuto denso come in ELA, un lungo, gambo sottile sulla elettrodo di registrazione minimizza eccesso movimento del tessuto circostante. Se una registrazione di successo non si ottiene al primo tentativo, ripetere con elettrodi freschi fino a una registrazione si ottiene o il papue è interrotto al punto in cui l&#39;assone non è più visibile. Tuttavia, è anche importante per velocemente l&#39;elettrodo vicino alla assone per minimizzare la quantità di esposizione fluorescente, che può causare fototossicità, candeggio e possono influenzare le proprietà fisiologiche della cellula 29-31. Una volta che un segmento di assone viene aspirato con successo nella elettrodo di registrazione, le registrazioni possono essere ottenuti per diverse ore. Se le unità sono sempre persi in meno di 1 ora, considerare la possibilità di punte degli elettrodi più piccoli per evitare l&#39;assone di scivolare fuori. D&#39;altra parte, troppo piccola di una punta può provocare l&#39;intasamento, bassa segnale-rumore, o danni al assone. Una diminuzione costante nel picco di ampiezza e il &#39;ritorno&#39; di una unità con l&#39;aggiunta di aspirazione è un&#39;indicazione che la punta è troppo grande. Troppo aspirazione può causare danni irreparabili al assone. Una soluzione è quella di consentire una piccola perdita nella linea aria quindi la pressione lentamente ritorna a zero. Rapidamente equalizzazione Thpressione e si tradurrà in un parente-esteriore &#39;push&#39; transitoria che può espellere l&#39;assone. Sebbene questa tecnica rappresenta un grande vantaggio per l&#39;ottenimento di registrazioni mirate di neuroni di proiezione identificati, non sarà utile per distinguere interneuroni locali, come colorante sarebbe assorbito da tutti i tipi di cellule nel sito di iniezione. Teoricamente, l&#39;uso di più fluorofori con siti di iniezione può consentire questo metodo per essere espanso. Per esempio, il confronto di singolo-versus doppia etichettatura potrebbe essere utilizzata per distinguere interneuroni da neuroni di proiezione successivo alla doppia iniezioni in due punti del circuito 32. Allo stesso modo, traccianti retrogradi può essere combinato con altre tecniche di imaging avanzate, come ad esempio due fotoni di imaging, come è stato fatto recentemente in diamante mandarino Alta centro Vocal (HVC) 32. Inoltre, le regioni di registrazione sono limitate a quelle vicino alla superficie quando si usano microscopi epifluorescenza, come siamo stati solo in grado di risolvere 1 &amp;mu; strutture m entro i primi 30 micron di tessuto. Tuttavia, questa profondità potrebbe essere esteso mediante l&#39;uso di altre tecniche di microscopia, come la microscopia a due fotoni 33 o oggettiva-accoppiato planare illuminazione microscopia 34. Complessivamente, questa tecnica rappresenta un avanzamento importante nello studio dei circuiti neurali in vivo perché può essere utilizzato per registrare da singoli neuroni in molti circuiti diversi in una varietà di sistemi modello - compresi quelli che sono relativamente inaccessibile.

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

L&#39;obiettivo generale di questo metodo è quello di registrare le risposte-unità singole da una popolazione di neuroni identificati. Nelle registrazioni elettrofisiologiche in vivo di singoli neuroni sono fondamentali per la comprensione di come i circuiti neurali funzionano in condizioni naturali. Tradizionalmente, queste registrazioni sono state effettuate &#39;cieco&#39;, intendendo l&#39;identità della cellula registrata è sconosciuto all&#39;inizio della registrazione. Identità cellulare può essere determinata successivamente via intracellulare 1, juxtacellular 2 o loose-patch 3 ionoforesi di colorante, ma queste registrazioni non può essere pre-mirati a specifici neuroni in regioni con tipi di cellule funzionalmente eterogenee. Proteine ​​fluorescenti possono essere espressi in modo specifico tipo cellulare permettendo visivamente a guida singola cellula elettrofisiologia 4-6. Tuttavia, ci sono molti sistemi modello per cui questi strumenti genetici non sono disponibili. Anche in sistemi modello geneticamente accessibili, il PRO desideratomoter può essere sconosciuto o neuroni geneticamente omogenee può avere diversi modelli di proiezione. Allo stesso modo, vettori virali sono stati utilizzati per etichettare specifici sottogruppi di neuroni di proiezione 7, ma l&#39;uso di questo metodo è limitato dalla tossicità e la mancanza di specificità trans-sinaptica. Pertanto, sono necessari ulteriori tecniche che offrono specifiche di pre-visualizzazione per registrare da singoli neuroni identificati in vivo. Pre-visualizzazione del neurone bersaglio è particolarmente utile per le difficili condizioni di registrazione, per cui le registrazioni monocellulari classici sono spesso proibitivo 8-11. La nuova tecnica descritta in questo articolo utilizza il trasporto retrogrado di un colorante fluorescente applicato utilizzando aghi di tungsteno per etichettare rapidamente e selettivamente uno specifico sottoinsieme di cellule all&#39;interno di una particolare regione del cervello in base alle loro uniche proiezioni assonali, fornendo in tal modo un segnale visivo per ottenere registrazioni elettrofisiologiche mirate dai neuroni identificati in un intatto circuito withina vertebrato CNS. Il romanzo progresso più significativo del nostro metodo è l&#39;uso di marcatori fluorescenti per indirizzare specifici tipi cellulari in un sistema modello accessibile non geneticamente. Pesci debolmente elettrici sono un sistema ottimo modello per lo studio di circuiti neurali in sveglio, comportandosi animali 12. Abbiamo utilizzato questa tecnica per studiare l&#39;elaborazione sensoriale da &quot;piccole celle&quot; nel nucleo exterolateral anteriore (ELA) di pesci debolmente elettrico mormyrid. &quot;Piccole celle&quot; sono ipotizzate per essere in tempo comparatore neuroni importanti per rilevare differenze submillisecond nei tempi di arrivo dei picchi presinaptici 13. Tuttavia, le caratteristiche anatomiche come mielina denso, sinapsi engulfing, e corpi cellulari piccoli hanno reso estremamente difficile per registrare da queste cellule con metodi tradizionali 11, 14. Qui mostriamo che il nostro nuovo metodo etichette selettivamente queste cellule nel 28% dei preparativi, consentendo affidabili, registrazioni e caratte robustirizzazione delle risposte a stimoli electrosensory.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Trasporto retrogrado di colorante fluorescente etichetta una sotto-popolazione di neuroni basati su proiezione anatomica. Assoni con etichetta possono essere mirati visivamente<em&gt; In vivo</em&gt;, Permettendo registrazione extracellulare da assoni identificati. Questa tecnica facilita la registrazione quando i neuroni non possono essere etichettati con la manipolazione genetica o sono difficili da isolare con &#39;cieco&#39;<em&gt; In vivo</em&gt; Approcci.

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

retrograde-fluorescent-labeling-allows-for-targeted-extracellular

Research

JoVE Journal

Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

19.8K Views.  Washington University in St. Louis. Trasporto retrogrado di colorante fluorescente etichetta una sotto-popolazione di neuroni basati su proiezione anatomica. Assoni con etichetta possono essere mirati visivamente In vivo, Permettendo registrazione extracellulare da assoni identificati. Questa tecnica facilita la registrazione quando i neuroni non possono essere etichettati con la manipolazione genetica o sono difficili da isolare con 'cieco' In vivo Approcci.

Video: Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

Doppio registrazioni somatica da Gonadotropina-Releasing Hormone (GnRH) Neuroni Identificato da una proteina fluorescente verde (GFP) in fette ipotalamici

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

Ricerca

Neuroscienze

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

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

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

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

Singola unità In vivo Registrazioni dal chiasma ottico di Rat

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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

Impianto chirurgico di elettrodi neurali cronici per la registrazione di attività singola unità e segnali Electrocorticographic

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

La registrazione simultanea dei segnali di calcio da parte dei neuroni identificati e comportamento alimentare di Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Caricamento in corso retrogrado di Nervi, tratti e radici spinali con le tinture fluorescenti

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

La registrazione dell&#39;attività elettrica dai neuroni identificati nel cervello intatto di pesci transgenici

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

Intracellulare di registrazione, sensoriale Campo Mapping, e la coltura neuroni identificata nel Leech, Hirudo medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

Etichettatura retrograda di cellule gangliari della retina in Zebrafish adulti con coloranti fluorescenti

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

Una guida per<em&gt; In vivo</em&gt; Singola unità di registrazione da Optogenetically Identificato corticale inibitorio interneuroni

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

Perforato Patch-clamp registrazione di topo neuroni sensoriali olfattivi In neuroepithelium intatto: Analisi funzionale di neuroni Esprimendo un Identificato odorizzante Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...