This work is supported by a National Science Foundation award (1145581) to LWG.

NOTA: L&#39;intera procedura è stata condotta in conformità con le linee guida del National Institutes of Health, come approvato dalla cura e uso degli animali Comitato della University of Illinois a Chicago. 1. Soluzioni e Media Cultura Preparati <ol><li> Conservare tutte le soluzioni a -20 ° C per un massimo di sei mesi. Tenere il terreno di coltura a 4 ° C per un massimo di 3 mesi. </li><li> Preparare 100 ml di coltura mescolando 1 ml di soluzione di pen-strep e 1 ml di ITSX (Insulin-transferrina-Selenio-supplementazione) a 100 ml con DMEM. </li><li> Preparare la soluzione enzimatica mescolando 250 ml di DMEM, 2,5 ml di 100 mM CaCl 2, e 2,5 ml di 50 mM EDTA. </li><li> Preparare la soluzione inattivazione mescolando 225 ml di DMEM, 25 ml di FBS, 625 mg di albumina, e 625 mg di inibitore della tripsina. </li><li> Preparare la soluzione di un Locke di 154 mM NaCl, 5.6 mM KCl, 5,0 HEPES mM, 3,6 mM NaHCO3, 5,6 mM di glucosio. Regolare il pH a 7,3 con NaOH. </li><li>Preparare una soluzione di poli-D-lisina (PDL) di 50 mg / ml. Utilizzare una concentrazione finale di 1 mg / ml a lamelle cappotto. </li><li> Preparare una soluzione extracellulare di 140 mM NaCl, KCl 5 mM, 2 mM CaCl 2, 1 mM MgCl 2, 10 mM HEPES-NaOH, e glucosio 10 mM. Regolare il pH a 7,3 con NaOH con una osmolarità di ~ 310 nmol / kg. </li><li> Preparare una soluzione di pipetta di 50 mM NaCl, TEACL 100 mM, KCl 5 mM, 2 mM CaCl 2, 1 mM MgCl 2, e HEPES 10 mM. Regolare il pH a 7,3 con NaOH con una osmolarità di ~ 290 nmol / kg. </li></ol> Isolamento 2. ghiandola surrenale <ol><li> Autoclave un paio di grandi e piccole forbici dissezione e due paia di pinze. Indossare i guanti durante l&#39;intero processo e posizionare il vassoio dissezione su un secchio di ghiaccio. </li><li> Utilizzare i topi prese a Day 0-3. Euthanize animali con una pressione di CO 2 serbatoio seguita da decapitazione. </li><li> Utilizzare il set più piccolo di forbici per tagliare lungo la schiena del cucciolo che segue la colonna vertebrale da cervi<html> cal per caudale regione. Assicuratevi di tagliare superficialmente sotto la pelle e non penetrare nella cavità del corpo. </li><li> Avanti, pelle a buccia lontano da spina dorsale in modo che la muscolatura sia esposto ad avere una visione chiara della colonna vertebrale. Da qui, fare un taglio transectional al midollo spinale cervicale e poi fare due tagli paralleli lungo i lati della colonna vertebrale. </li><li> Con un paio di pinze che tengono il corpo dell&#39;animale, utilizzare l&#39;altra coppia di pinze per afferrare colonna vertebrale e buccia indietro della colonna vertebrale fino reni sono esposti. </li><li> In questo momento, visualizzare le ghiandole surrenali sulla parte superiore dei reni. Poi posizionare accuratamente i due ghiandole di ogni animale in un tubo conico riempito con soluzione di Locke. NOTA: A volte le ghiandole si attacchi alle pinze. Se questo è il caso, utilizzare delicatamente un altro paio di pinze per aiutare nella rimozione delle ghiandole delle pinze e verso la soluzione di Locke. Le ghiandole possono essere mantenuti in questa condizione per un massimo di 2 ore. </li></ol>e &quot;&gt; 3. Tissue digestione <ol><li> Nel frattempo, bolla soluzione enzimatica con 5% di CO 2 + 95% O 2 per 15 minuti prima dell&#39;uso. Assicurarsi che la soluzione equilibrata si trasforma da un colore rosa brillante nel colore rosa / arancio. </li><li> Aggiungere la papaina alla soluzione enzimatica appena bolle ad una concentrazione finale di 20-25 unità / ml. Incubare ciascuna coppia di ghiandole di ciascun animale per 40-60 minuti a 37 ° C nella soluzione enzimatica con papaina. </li><li> Aggiungere 75% di soluzione di inattivazione a ciascuna provetta e incubare a 37 ° C per altri 10 min. Questo incubazione inattiva l&#39;attività enzimatica della papaina. </li><li> Dopo i 10 minuti di incubazione con la soluzione di inattivazione, trasferire delicatamente le ghiandole in una provetta nuova etichetta e poi lavare le ghiandole 3x con la cultura dei media. </li></ol> 4. cellulare Dissociazione e placcatura <ol><li> Dopo il lavaggio, posizionare i due ghiandole in 200 ml di media cultura e poi titolare delicatamente le ghiandole egiù attraverso la punta della pipetta con una pipetta 200 microlitri fino a quando non vi è più un grosso pezzo di tessuto nella soluzione. NOTA: Quando titolazione, mantenere ghiandole nella parte inferiore del tubo con una forza verso il basso dalla punta della pipetta 200 microlitri e impegnare il tessuto delicatamente e lentamente. Essere certi di utilizzare lenti, continui colpi, mai pipettaggio veloce o improvviso. </li><li> Aggiungere un ulteriore coltura 250 microlitri per portare il volume totale dei terreni di coltura fino a 450 ml. </li><li> Piatto 60 ml di questa soluzione di cellule contenenti su ciascuno dei sette coprioggetto PDL-pre-rivestite 12 mm, che sono collocati in un piatto di coltura 60 x 15 mm. </li><li> Una volta placcato, mettere il piatto in incubatrice che è impostato a 37 ° C e mantenuta con un costante flusso / fornitura di 5% di CO 2 per 30 minuti al fine di garantire un ambiente per la vitalità cellulare. Dopo l&#39;incubazione, aggiungere delicatamente 5 ml di terreni di coltura pre-riscaldato nel piatto cultura e restituire il piatto di nuovo nel incubatore per 24 ore prima di cellula-addettoregistrazioni d. NOTA: in genere, le cellule possono essere utilizzate per le registrazioni elettrofisiologiche per un massimo di 4 giorni. </li></ol> 5. cellulare Identificazione e Gigaohm Seal Formazione <ol><li> Posizionare il coprioggetto 12 millimetri nella soluzione extracellulare. Dal momento che l&#39;intero ghiandole surrenali sono utilizzati per la preparazione delle cellule, mescolare le cellule cromaffini (in genere molto luminose con un marrone / colorazione beige e una forma quasi perfetta circolare (Figura 1) in coltura con cellule corticali (più piccole e dimmer di cellule cromaffini). </li><li> Tirare le pipette di patch in quattro fasi utilizzando una programmabile P-97 estrattore. Immergere la punta della pipetta in una cera fusa, che contribuirà a ridurre la capacità del vetro e poi sparano lucidare la punta di &quot;blow-away&quot;, questa cera dall&#39;interno della punta della pipetta. NOTA: Quando riempito con una soluzione di patch pipetta, pipette di patch in genere hanno una resistenza di circa 2 MW. </li><li> Avvicinarsi alla pipetta di patch vicino alla cella all&#39;interno diversi microns. Per ottenere una configurazione cell-schiera, cercare qualsiasi lieve deformazione cellula durante l&#39;avvicinamento della pipetta di patch più vicino alla cella. Esercitare una leggera aspirazione fino ad ottenere un sigillo GΩ. </li></ol> 6. cellulari-Attached Recordings di capacità <ol><li> Una volta che un sigillo GΩ si rivela, effettuare registrazioni cellulari-attached attraverso l&#39;uso di un amplificatore EPC-7 plus patch-clamp e un amplificatore lock-in analogico a due fasi (vedi tabella allegata delle attrezzature). Applicare una sinusoide con ampiezza di 50 mV (rms) e la frequenza di 20 kHz dall&#39;amplificatore lock-in. </li><li> Impostare il filtro di uscita dell&#39;amplificatore lock-in a 1 costante di tempo msec e 24 db. Impostare la fase dell&#39;amplificatore lock-in modo tale che variazioni di capacità transitori prodotte da impulsi di aspirazione dolci appaiono solo nella traccia senza proiezione nel tracciato cerotto conduttanza (Ri) cerotto capacità (Im) (Figura 2). NOTA: Si prega di vedere i dettagli per la calibrazione del sistema in reFerence 19. </li><li> Identificare le tipiche variazioni di capacità di passi &quot;verso il basso&quot; (Figura 3). NOTA: Una registrazione solido sarà considerata come una &quot;scala&quot; tipo somiglianza con molti passi verso il basso, che indica interiorizzare singole vescicole. Il processo di patching serve da stimolo meccanico in quanto attuale azione può essere in genere registrato dalla maggior parte delle cellule. </li><li> Nome file come singole cartelle compresa la data: gg / mm / anno e ogni registrazione cell-attached come propria numerazione individuale (xx) all&#39;interno di quel file. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Misure di capacità cellulari iscritti richiedono diversi passaggi critici al fine di ottenere con successo le registrazioni di alta qualità: 1) cellule vitali e sane preparate dalle ghiandole surrenali; 2) il rivestimento PDL dei coprioggetto; 3) formazione sigillo gigaohm; 4) livello di rumore del sistema; e 5) correzione di fase. Per la chirurgia animali, le modifiche si può potenzialmente fare è quello di regolare l&#39;approccio chirurgico al meglio destrezza vestito e per evitare da...

Trasmissione sinaptica è mediata da esocitosi delle vescicole sinaptiche, contenenti neurotrasmettitori, e queste vescicole deve subire riciclaggio endocitica locale all&#39;interno del nervo terminale per mantenere la comunicazione neuronale a lungo termine. Dato il ruolo essenziale della trasmissione sinaptica nel cervello, la comprensione del macchinario molecolare che costituisce il ciclo delle vescicole sinaptiche è un fondamento essenziale verso una migliore comprensione nella comunicazione cellulare nel suo complesso. Tra sistemi modello delle cellule, la cellula cromaffini surrenale ha fornito alcune delle l&#39;intuizione più definitiva nel meccanismo molecolare alla base riciclo delle vescicole sinaptiche. Esocitosi, la fase finale di rilascio di neurotrasmettitore, è stato immensamente studiata ed esaminata attraverso l&#39;uso della cella cromaffini surrenale 1,2. In realtà, la maggior parte dei giocatori molecolari che orchestrano la formazione, il targeting, attracco, e la fusione dei granuli secretori sono stati identificati grazie alla applicazione di divtecniche di Erse nelle cellule cromaffini 1. Inoltre, fornendo l&#39;opportunità di consentire la risoluzione di una singola vescicola dei macchinari proteina coinvolta nella esocitosi, la cella cromaffini rimane un modello potente per affrontare le questioni della fusione delle vescicole 3. Misure di capacità cellulari iscritti sono stati utilizzati nella risoluzione di singolo-fusione delle vescicole durante esocitosi 3. Esocitosi di vescicole piccole come ~ 60 nm di diametro è stato dimostrato di essere rilevato da membrana cellulare misurazioni ammissione con la tecnica del patch clamp in configurazione cell allegato 4-7. Accettazione è definita come una misura di quanto facilmente un circuito o un dispositivo consentirà una corrente di flusso; è l&#39;inverso di impedenza. Pertanto, le misure di ammissione forniscono una comprensione della capacità di membrana. Questo si ottiene l&#39;incorporazione della membrana vescicolare nella membrana plasmatica; questa incorporazione rivela cambiamenti di superficiezona 8. Ogni vescicola fusione provoca un aumento graduale di capacità di membrana 9,10. Inoltre, questa misura ammettenza fornisce la conduttanza della membrana e la fusione dei pori conduttanza durante un evento esocitotico 3. Poiché questa tecnica ha fornito uno strumento unico di identificare cinetica singoli-vescicole durante esocitosi, il nostro laboratorio ha recentemente applicato questo concetto per rilevare endocitosi di singole vescicole 11,12. Il nostro interesse specifico è clatrina-mediata endocitosi (CME), che è stato considerato come una componente fondamentale di pulizia in molte cellule 13 e come via principale per endocitosi delle vescicole sinaptiche nei terminali neuronali 14,15. CME è conosciuto per essere biologicamente importante, tuttavia, la sua cinetica non restino ben capito a causa di limitazioni tecniche di monitoraggio degli eventi endocitiche singolari. Date le somiglianze nei meccanismi di esocitosi tra le cellule cromaffini e neuroni 1, è plausible che i meccanismi di fissione nelle cellule cromaffini può probabilmente applicarsi a endocitosi delle vescicole sinaptiche nei neuroni. Le misure di capacità di cella-attached sono stati utilizzati per monitorare singoli eventi endocitiche e analizzare la cinetica di fissione, che la maggior parte dei metodi non sono in grado di risolvere. Nelle nostre registrazioni cellulari-attached, un&#39;onda sinusoidale a 20 kHz si trova sopra il potenziale di detenzione, e la corrente di uscita è suddiviso in membrana conduttanza in un canale e capacità di membrana in un altro canale da una a due fasi amplificatore lock-in 16 18. Dai cambiamenti nella conduttanza di membrana e capacità, si può calcolare la cinetica di fissione poro, che probabilmente corrispondente al collo membrana tubolare che collega la vescicola internalizzazione alla membrana plasmatica prima vescicola pinch-off. Collettivamente, questa tecnica ci dà l&#39;opportunità di esaminare i meccanismi regolatori della vescicola fissione durante CME.

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
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			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
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		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
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			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
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			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
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		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
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			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

La vitalità delle cellule e la qualità di tenuta gigaohm sono critici nel determinare la qualità delle registrazioni capacità della cella collegata. Pertanto, è fondamentale per procurare un coltura cellulare efficace ed efficiente prima di registrazioni elettrofisiologiche, e le cellule vitali tipiche sono illustrate nella figura 1. Practice e il tempo sarà utile nel raggiungimento di un sigillo gigaohm con alta qualità. Se si può vedere chiaramente deformazione cella quando la pipetta di patch...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Metodi per cellulari-attached Misure di capacità in mouse surrenale cromaffini cellulare

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

Research

JoVE Journal

Neuroscience

8.7K Views.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Video: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultrasound-Guided Microiniezione nel proencefalo mouse In Utero A E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Ricerca

Neuroscienze

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-topo Imaging neuroanatomici Risonanza Magnetica

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

In elettroporazione in vivo di Retina topo in via di sviluppo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

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 ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Metodi per l&#39;auto-somministrazione per via endovenosa in un modello murino

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

Metodi per la modulazione e analisi di NF-kB-dipendente adulti neurogenesi

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

In Misure Vivo elettrofisiologici sui nervi mouse sciatico

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Immagini dal vivo della mitosi in via di sviluppo embrionali di topo Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.

Uno schermo comportamentale per le convulsioni indotte dal calore in modelli murini di epilessia

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Due metodi di peeling per l'isolamento dei compartimenti cellulari fotorecettori nella retina del topo per l'analisi delle proteine

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...