This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

Etica Dichiarazione: procedure che coinvolgono soggetti animali sono stati approvati dalla cura e l&#39;uso Comitato Istituzionale Animal (IACUC) presso la Wayne State University. 1. Preparazione della soluzione sperimentale <ol><li> Preparare la soluzione di dissezione 1 giorno a 1 settimana prima l&#39;esperimento vero e proprio. Utilizzare una soluzione tampone HEPES per la dissezione della retina a causa della sua forte capacità di buffering a temperature inferiori a 16. Mescolare tutti i prodotti chimici, come segue (in mm): 115 NaCl, KCl 2.5, 2.5 CaCl 2, 1.0 MgCl 2, 10 HEPES, 28 glucosio. Regolare il pH a 7,4 con NaOH. Conservare la soluzione in frigorifero fino a 1 settimana. </li><li> La soluzione di registrazione è medio Ames &#39;, che è un fluido spinale cerebrale artificiale (aCSF) progettato per preparazioni retina 17. Il giorno dell&#39;esperimento, pesare polvere Ames &#39;(4,4 g per 500 ml di soluzione) in un tubo. Inoltre, pesare NaHCO 3 (0,95 g per 500 ml di soluzione) e mescolare con la Apolvere mes. </li><li> Preparare la soluzione pipetta intracellulare per le registrazioni di patch clamp dal giorno dell&#39;esperimento. Mescolare tutti i prodotti chimici, come segue (in mm): 111 K-gluconato, 1.0 CaCl 2, 10 HEPES, 1.1 EGTA, 10 NaCl, 1.0 MgCl 2, 5 ATP-Mg, e 1,0 GTP-Na. Aggiustare il pH a 7,2 con KOH. Filtrare la soluzione pipetta con un filtro a siringa convenzionale. Fai ~ 500 microlitri aliquote e memorizzarli in un congelatore o un congelatore. </li></ol> 2. Preparazione per la Giornata di Experiment <ol><li> La notte prima dell&#39;esperimento, posizionare un topo in una gabbia (C57BL / 6J o background simile, 4 - 8 settimane di vita, di sesso maschile), in uno spazio buio per O / N adattamento al buio. </li><li> Preparazione per la dissezione: <ol><li> Riempire dissezione soluzione (100-200 ml) in un bicchiere di vetro, posizionarlo sul ghiaccio, e la bolla con l&#39;ossigeno per almeno 10 min. </li><li> Avviare ossigenazione della scatola buia per la conservazione di preparazione della retina. </li><li> Preparare vetrini in plastica con guide di grasso per il riutilizzopreparazioni fetta fonodale e mettono ciascuno di essi in un piatto di plastica da 35 mm. </li><li> Collegare un nuovo lama di rasoio per un chopper (affettatrice tessuto). </li><li> Tagliare una membrana filtrante in due metà. Questo è per affettare retinica. </li><li> Allineare gli strumenti di dissezione e di trasferimento pipette vicino al microscopio da dissezione. L&#39;area di lavoro deve essere ben organizzato in modo che ogni strumento può essere facilmente accessibile al buio. </li></ol></li><li> Preparazione per le registrazioni di patch clamp: <ol><li> Sciogliere la polvere Ames &#39;e NaHCO 3 in acqua distillata (Ames 4,4 g e NaHCO 3 0.95g / 500 ml DDH 2 O). Medio Bubble Ames &#39;ossigeno misto (95% O 2 e 5% CO 2) per almeno 30 minuti, mentre il riscaldamento a una temperatura di registrazione (~ 35 ° C). Regolare il pH a 7,4 con NaHCO 3 mentre ancora ribolle. </li><li> Scongelare la soluzione pipetta interno durante la dissezione. Dopo lo scongelamento, aggiungere 0,01% solforodamina B per la colorazione intracellulare. </li></ol></li></ol> 3. retina Dissection <ol><li> In una stanza buia, eutanasia il mouse utilizzando anidride carbonica e pneumotorace bilaterale. Dopo 2 - 3 min usando l&#39;anidride carbonica, il mouse perderà coscienza. </li><li> Quando il mouse non risponde ad una presa di coda, enucleare rapidamente gli occhi e metterli in una soluzione di dissezione a freddo in un piatto di plastica. Eseguire il pneumotorace bilaterale per assicurare la morte dell&#39;animale. Memorizzare rapidamente l&#39;occhio nel piatto nella scatola buia. Utilizzare un visualizzatore a infrarossi per condurre la procedura nel buio. NOTA: In alternativa, isoflurano, decapitazione, o dislocazione cervicale può essere utilizzato per l&#39;eutanasia. Il metodo di eutanasia dovrebbe seguire le linee guida del National Institute of Health (NIH) e / o la cura e l&#39;uso degli animali Comitato Istituzionale. </li><li> In una stanza buia, regolare il microscopio da dissezione e illuminazione ad infrarossi. Al microscopio, trasferire un occhio dal suo piatto in un piatto di plastica 10 centimetri won la soluzione dissezione raffreddato. Continuously bolla ossigeno attraverso il tubo posto sul fondo del piatto di plastica. Regolare il volume del flusso in modo che non sia troppo basso, ma assicurarsi che non è sufficiente a disturbare la dissezione. </li><li> Cornea e obiettivo la rimozione: <ol><li> Fare un&#39;incisione sulla parte superiore della cornea con un coltello micro-chirurgica. Tenendo il nervo ottico all&#39;altra estremità del bulbo oculare impedisce l&#39;occhio da movimento. Estendere il taglio utilizzando un paio di forbici chirurgiche. Tagliare la cornea con un po &#39;sclera intorno ad esso. </li><li> Afferra l&#39;obiettivo con una pinza sottile e tirare lentamente la lente fuori. Se il vitreo è troppo appiccicoso per rimuovere la lente, tagliare il vitreo con un paio di forbici. </li><li> Una volta che l&#39;occhio-tazza è fatta, versare lentamente il freddo, la soluzione dissezione ossigenata in oculare con una piccola pipetta di trasferimento. NOTA: Il bulbo oculare si sgonfia rapidamente dopo un incisione nella cornea. È importante mantenere la forma dell&#39;occhio come deformare l&#39;occhio durante questo proprocedura danneggia la retina e provocare il distacco di retina. </li></ol></li><li> Identificare i lati dorsale e ventrale della retina: NOTA: A causa della distribuzione non uniforme del verde e la luce ultravioletta (UV) Coni 18, che identifica una specifica area della retina è importante per le registrazioni di risposta di luce. <ol><li> Identificare la linea che attraversa l&#39;oculare retinica passando vicino alla testa del nervo ottico. Questa linea passa per lo più attraverso il lato ventrale del nervo ottico; lato compreso il nervo ottico è il lato dorsale, mentre l&#39;altro è ventrale 19. Vedere questo punto di riferimento chiaramente sotto spettatori infrarossi. </li><li> A seconda dello scopo dell&#39;esperimento, fare un taglio sul lato inutili. Questo sarà utile dopo aver isolato la retina dalla oculare. </li></ol></li><li> Rimozione del vitreo: <ol><li> Facoltativamente, incubare l&#39;occhio-tazza con ialuronidasi (0,5 mg / ml) per 15 min. </li><li> Rimuovere il vitreo con una pinza extra-fini. Il vitreo è principalmenteattaccato al fondo e il bordo esterno dell&#39;occhio tazza. Rimuovere delicatamente senza toccare o frugando la retina. Come il vitreo potrebbe essere strettamente collegato al oculare, rimuovere delicatamente per evitare il distacco della retina dall&#39;occhio-coppa. Continuare a tirare fino a quando non la tensione si fa sentire. NOTA: Questo passaggio è particolarmente importante per sezionare la retina del mouse; Tuttavia, non è un problema per le retine di altre specie come il ratto o salamander. </li></ol></li><li> Isolare la retina dall&#39;occhio-coppa. Afferra la sclera e staccare delicatamente la retina con la parte posteriore del forcipe. NOTA: Whole-mount preparati retina può essere fatto dalla retina isolata. Fare tre o quattro tagli ai bordi e appiattire la retina, o tagliare la retina in diversi pezzi. </li><li> Tagliare la retina e scartare il superfluo metà della retina (punto 3.5). Nello stesso piatto di plastica, tagliare la retina rimanente in due pezzi. Per ogni pezzo della retina, tagliare i bordi per effettuare unalastra della retina, e tagliare i bordi di piegatura. </li><li> Trasferire una lastra retinica su una lastra di vetro usando un grande pipetta di trasferimento (~ 2 ml). Aspirare la soluzione in eccesso con una piccola pipetta, utilizzare un pezzo di carta da filtro per appiattire la lastra, e quindi inserire rapidamente un pezzo della membrana filtrante sulla parte superiore del tessuto. <ol><li> Usando una piccola pipetta di trasferimento, una goccia di soluzione di dissezione sulla membrana del filtro. Attendere ~ 15 sec finché la soluzione estende su membrana. Durante questo tempo, il tessuto retinico si attacchi alla membrana filtrante. </li><li> Versare soluzione dissezione più freddo sotto e intorno al filtro. Il tessuto retinico con la membrana del filtro galleggerà. Afferra la membrana filtro con una pinza e posizionarlo nella camera di taglio. Versare dissezione soluzione sopra il tessuto, e memorizzare la preparazione in scatola buia. NOTA: La procedura descritta è critica. È importante eseguire questi passaggi rapidamente per garantire che i bastoni tessuto retinico ai mem filtribrane e non si secca. </li></ol></li><li> Utilizzando un chopper, tagliare il tessuto retinico a fette. Circa 200 micron di spessore è fattibile per gli studi di patch clamp. </li><li> Posizionare un coprioggetto di plastica con guide di grasso nella camera di taglio. Trasferire una fetta retina sopra le rotaie grasso su un vetrino coprioggetto di plastica. Ruotare la fetta 90 ° in modo che la sezione trasversale è visibile. Premere la membrana del filtro verso il basso sul vetrino, quindi, coprire i lati della carta da filtro con grasso. <ol><li> Dopo la fetta è immobilizzato su un coprioggetto di plastica, afferrare il coprioggetto e trasferirlo in un piatto di plastica da 35 mm. Versare soluzione dissezione a freddo sulla preparazione della retina e immergere la preparazione. Conservare ogni piatto nella scatola scura, che dovrebbe essere ossigenato continuamente. NOTA: L&#39;intera procedura deve essere fatto con cura in modo che la membrana filtro non è deformata. Altrimenti, la fetta retina separa facilmente dalla membrana del filtro. </li></ol></li></ol><ol><li> Preparazione di registrazione: <ol><li> Prime i tubi di perfusione con media Ames &#39;. Lasciare tutte le bolle di passare attraverso quando si riempie il tubo. </li><li> Fai pipette di registrazione patch clamp con un estrattore. Per riempire una pipetta con la soluzione di pipetta, tuffo nella parte posteriore di una pipetta per 1 minuto fino a quando la punta della pipetta è riempita. Successivamente, riempire ~ 1/3 della pipetta utilizzando un filler micro-pipetta. Conservare ogni pipetta in una scatola pipetta umido. </li><li> Accendere l&#39;apparecchio per le registrazioni di patch clamp; incluso il computer, amplificatore, telecamera CCD, e microscopio. </li></ol></li><li> Spegnere la luce della stanza. Mettere una preparazione fetta della retina sulla camera palco microscopio con un visualizzatore a infrarossi. Dopo che è immobilizzato, iniziare perfusione continua. Impostare la temperatura perfusione a 33 a 37 ° C. </li><li> Visualizza la superficie fetta con la fotocamera CCD. Focus sul luogo di destinazione in cui il ce bersagliotipi ll risiedono. Selezionare una cellula sana, cercando di patch clamp registrazione (ad esempio, dovrebbe avere superficie liscia di aspetto e la forma delle cellule buona). </li><li> Inserire una pipetta di registrazione in un supporto pipetta. Avanzare la pipetta alla preparazione fetta. Quando è vicino alla preparazione fetta (~ 2 mm sopra), trovare la punta della pipetta con il microscopio. Una volta che il puntale è visibile al microscopio, spostare la punta verso la cella di destinazione. </li><li> Impostare l&#39;amplificatore. Regolare la pipetta a 0 mV / 0 pA (azzeramento) e avviare un impulso elettrico continua di ~ 5 mV a ~ 10 Hz. Controllare la resistenza pipetta; resistenza ideale è compresa tra 5 e 10 MW per la maggior parte dei neuroni della retina. </li><li> Inizia soffia fuori dalla punta. Utilizzare un boccaglio, o utilizzare una siringa, per applicare pressione positiva quando la pipetta immerge nella soluzione del bagno. Continuo soffiare la soluzione interna finché la punta è sulla superficie della cellula bersaglio. <ol><li> Quando la pressione positiva fa una piccola fossetta thsuperficie cellulare e, avanzare la punta leggermente e smettere di soffiare fuori. Verificare la resistenza pipetta, che dovrebbe aumentare. Se è in continuo aumento, lasciarlo e monitorare la resistenza fino a raggiungere&gt; 1 GΩ (gigaseal). Se la resistenza non aumenta spontaneamente, applicare delicatamente pressione negativa fino a diventare un gigaseal. </li></ol></li><li> Dopo il gigaseal è raggiunto, modificare il potenziale tenendo a -70 mV. Quindi, applicare ad intermittenza pressione negativa alla rottura della membrana all&#39;interno della punta della pipetta. Quando viene effettuata la configurazione whole-cell, la resistenza pipetta può essere compreso tra 500 MW e 1 GΩ, e la corrente capacitiva è visto. A volte correnti postsinaptiche spontanee possono essere osservati. </li><li> Registrare il rapporto IV da -80 a +40 mV. Diversi tipi di canali voltaggio-dipendenti vengono attivati ​​a seconda del tipo di cellula. </li><li> Record luce evocata correnti sinaptiche o tensioni. </li></ol>

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The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the ...

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

La retina è la porta verso il sistema visivo. Per comprendere i meccanismi di elaborazione dei segnali visivi, indaghiamo funzioni di rete neurale della retina. I neuroni della retina nella rete comprendono numerosi sottotipi. Più di 10 sottotipi di cellule bipolari, cellule gangliari, e le cellule amacrine sono stati identificati da studi morfologici. Più sottotipi di neuroni della retina sono pensati per codificare le caratteristiche distinte di segnalazione visiva, come il movimento e il colore, e la forma più percorsi neurali. Tuttavia, i ruoli funzionali di ciascun neurone nel trattamento del segnale visivo non sono pienamente compresi. Il metodo di patch clamp è utile per affrontare questa questione fondamentale. Qui, un protocollo per registrare risposte sinaptiche luce evocati nel topo neuroni della retina con delle patch clamp registrazioni in condizioni di oscurità adattato è fornito. Gli occhi del mouse sono dark-adattato O / N e preparazioni fetta della retina vengono sezionati in una stanza buia con illuminazione ad infrarossi e gli spettatori. Luce infrarossa non lo faattivare fotorecettori mouse e conserva quindi la loro capacità di risposta luce. Patch clamp viene utilizzato per registrare le risposte luce evocata nei neuroni della retina. Un colorante fluorescente viene iniettato durante le registrazioni per caratterizzare sottotipi morfologiche neuronali. Questa procedura permette di determinare le funzioni fisiologiche di ogni neurone nella retina mouse.

Una preparazione fetta rappresentativo è mostrato in Figura 1. La preparazione fetta è in posizione diritta, mostrando fotorecettori alle cellule gangliari in una superficie piana, e nessun distacco dalla carta da filtro. Se la porzione è inclinato, solo una parte della preparazione è a fuoco, il che rende difficile identificare una cella appropriata di patch serraggio. Per le registrazioni, è importante scegliere un buon soma, che di solito ha una superficie lucida, una forma rotonda ancora, e non...

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Research

JoVE Journal

Neuroscience

8.4K Views.  Wayne State University School of Medicine. We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Video: Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

Analizzando le risposte di topo neuroni olfattivi sensoriali che utilizzano l&#39;aria in fase di registrazione Electroolfactogram

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

Ricerca

Neuroscienze

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp Misure di capacità e Ca 2 + Imaging terminazioni nervose della retina singola in Fette

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

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

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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 ...

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 ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Registrazioni Whole-cell patch-clamp nel cervello fette

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Patch clamp di celle Starburst amacrine in un appartamento di montaggio Preparazione di Deafferentated mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

Riepilogo

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Capitoli in questo video

Analizzando le risposte di topo neuroni olfattivi sensoriali che utilizzano l'aria in fase di registrazione Electroolfactogram

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Trasfezione delle cellule gangliari della retina di topo In vivo Elettroporazione

Patch-clamp Misure di capacità e Ca^{2 +} Imaging terminazioni nervose della retina singola in Fette

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

Retrograda Etichettatura fluorescente Consente mirata extracellulare singola unità di registrazione da parte dei neuroni identificati

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

Registrazioni Whole-cell patch-clamp nel cervello fette

Patch clamp di celle Starburst amacrine in un appartamento di montaggio Preparazione di Deafferentated mouse Retina

Sezione tecnica del Patch Clamp per l'analisi di plasticità indotta su apprendimento