<p class="jove_content"&gt; Ringraziamo il Dott. Yuval Gottlieb-Dror per l&#39;illustrazione elettroporazione. Questo lavoro è stato sostenuto da sovvenzioni a DSD presso l&#39;Istituto Nazionale per la Psicobiologia in Israele e dal programma di cooperazione di ricerca Niedersachsen-Israele e da sovvenzioni per AK da The Israel Science Foundation, il Ministero Israele di salute, e il centro di eccellenza del Patrimonio-Legacy Biomedical Science collaborazione.</p&gt;

1. Romboencefalo elettroporazione 1.1 gestione Egg <ol><li> Mettere le uova orizzontalmente in un incubatore umidificato (37-38,5 ° C). Gli embrioni sono elettroporati dopo 65-70 ore di incubazione, quando raggiungono 16-17 (HH) stadio (25-30 somiti). </li><li> Togliere le uova dal termostato, rimangono in posizione orizzontale. </li></ol> 1.2 Preparativi <ol><li> Tirare vetro capillari (0,5 mm di diametro). </li><li> Collegare piegati a forma di L oro Genetrodes elettrodi (3 mm di diametro) con un supporto adattatore per il generatore di impulsi. Parametri di elettroporazione sono 25 volt, 5 numeri di impulsi, 45 ms lunghezza dell&#39;impulso, 300 msec intervalli di impulso. </li><li> Preparare una pipetta a bocca, 10 ml della siringa, strisce parafilm, una spazzola morbida, forbici dissezione nitide e 25 ml di soluzione PBS sterile. Tale importo è di solito sufficiente per gestire un vassoio di uovo (18 uova). </li><li> Preparare una miscela adeguata di plasmidi di DNA (5 mg / mL ciascuno) eaggiungere circa 0.025% Veloce colorante verde per visualizzare l&#39;iniezione del DNA. Solitamente un volume di 4 ml è sufficiente per l&#39;iniezione di un vassoio. </li></ol> 1.3 windowing, iniezione ed elettroporazione <ol><li> Pulire i gusci d&#39;uovo con il 70% di etanolo. </li><li> Maneggiare un uovo alla volta a ridurre al minimo il tempo di esposizione embrionale all&#39;aria. </li><li> Fare un buco al polo uovo con le estremità forbici e togliere 3-4 ml di albumina con la siringa. Non rimuovere una maggiore quantità di albumina in quanto riduce la vitalità. </li><li> Aprire una piccola finestra ovale (2,5 x 2 dimensioni cm) al centro superiore del guscio delle uova usando le forbici dissezione. </li><li> Gocciolare qualche goccia di PBS sopra l&#39;embrione per prevenire la secchezza durante tutto il processo. </li><li> Caricare una piccola quantità della miscela DNA nel capillare di vetro usando una pipetta a bocca. </li><li> Posizionare l&#39;embrione con la sua estremità posteriore verso di voi. Nessuna iniezione di inchiostro è richiesta in questa fase, l&#39;embrione è chiaramente visibile. Evitare aumenti di iniezione d&#39;inchiostrosopravvivenza embrionale. Penetrare il romboencefalo caudale tenendo il capillare a ~ 45 °. Non rimuovere la membrana da tutto l&#39;embrione. Iniettare la soluzione di DNA con cura nel lume romboencefalo ed espirare senza formare bolle d&#39;aria. Soluzione di DNA diffonde anteriormente. </li><li> Subito dopo l&#39;iniezione del DNA, posizionare gli elettrodi paralleli al preciso AP e la posizione romboencefalo DV si mira a bersaglio. Spingere gli elettrodi leggermente ventrale senza toccare il cuore, in quanto può ridurre la vitalità. Pulse il elettroporatore. Elettrodi devono essere coperti con bolle per indicare conducibilità elettrica (Figura 1A). </li><li> Rimuovere con attenzione gli elettrodi e gocciolare qualche goccia di PBS per raffreddare l&#39;embrione e per rimuovere le bolle. Sigillare l&#39;uovo apertura correttamente con una striscia di parafilm per evitare l&#39;essiccazione durante l&#39;incubazione. Lavare elettrodi in PBS con la spazzola morbida. </li><li> Mettere uova in incubatrice per il tempo desiderato. La sopravvivenza degli embrioni è di circa il 90% 2-3 giorni dopoelettroporazione, e riduce fino al 50% 12 giorni dopo l&#39;elettroporazione. </li></ol> 2. Analisi di embrioni 2.1 preparazione Flat-mount e immunofluorescenza <ol><li> Flat-mount preparativi del cervello posteriore possono essere eseguite fino a E6.5 di visualizzare facilmente gli assoni al microscopio o di uno stereo-microscopio. </li><li> Sezionare con cura l&#39;embrione separandola dalla membrane che circondano e vasi sanguigni. Posizionare l&#39;embrione in un piccolo silicone - rivestito piastra di Petri contenente PBS. Attaccare l&#39;embrione al piatto con perni di tungsteno rivolti dorsalmente. Eseguire una fenditura dorsale nel romboencefalo tettoia parapioggia con tagliente capillari di vetro. </li><li> Usando le pinzette taglienti e micro-forbici tenere la parte superiore della testa e tirare con molta attenzione il tessuto hindbrain dal rostrale caudale. Dopo separazione della romboencefalo eliminare i rimanenti tessuti ventrali raggiungere una preparazione pulita. </li><li> Incubare la romboencefalo con 4Paraformaldeide soluzione% (PFA) per 1-2 ore a temperatura ambiente (RT). Lavare il romboencefalo con PBT (PBS/0.1% Triton X-100) per rimuovere PFA. </li><li> Per immunofluorescenza in hindbrains piatto montati aggiungere 500 ml di soluzione di bloccaggio (5% di siero di capra in PBT e incubare per 2 ore a RT Incubare con 1 m anticorpo ON a 4 ° C. Lavare con PBT (15 min x 3). Incubare con il 2 ° anticorpo per 2 ore a temperatura ambiente. Lavare con PBT (15 min x 3). </li><li> Posizionare il romboencefalo fronte dorsalmente su un vetrino. Aggiungi mezzi di montaggio fluorescenti sul cervello posteriore. Utilizzare vetro capillari o aghi di tungsteno per appiattire il romboencefalo sulla diapositiva. </li><li> Aggiungere piccole quantità di grasso ad ogni angolo per impedire spremitura del tessuto dal vetrino. Collocare il vetrino sulla parte superiore del cervello posteriore e di evitare bolle d&#39;aria. Per aderenza aggiungere smalto per coprire i bordi di vetro. </li></ol> 2.2 Cryo-sezioni ed immunofluorescenza <ol><li> Cryo-sezioni del tronco encefalico can essere eseguita in qualsiasi momento per visualizzare traiettorie assonali. Eppure, dopo E6.5 questo dovrebbe essere il metodo preferito per la difficoltà di visualizzare assoni nell&#39;embrione piatto montata, che diventa troppo spesso. Inoltre, la dimostrazione di sinapsi richiede sezionamento del tessuto e una vista di alto ingrandimento al microscopio. </li><li> Sezionare l&#39;embrione e rimuovere tutti i tessuti circostanti. Risciacquare con PBS. Tagliare l&#39;embrione alla parte caudale del midollo utilizzando micro-forbici e pinzette taglienti. Fai una vasta incisione tra il cervelletto e il mesencefalo e rimuovere l&#39;intero tronco encefalico. Il tessuto deve contenere il midollo, ponte e cervelletto. </li><li> Fissare il tronco encefalico in PFA 4% per il pernottamento (ON) a 4 ° C, lavare con PBS (10 min x 3), e incubare con il 30% di saccarosio per ON a 4 ° C fino al naufragio. </li><li> Incorpora il romboencefalo in un crio-stampo con ottobre composto (Optimal Cutting Temperature), e metterlo a -20 ° C fino a congelamento, come descritto altrove 11. </li><li&gt; Posizionare il blocco di ottobre nel criostato in corrispondenza dell&#39;asse della sezione desiderata. Larghezza della sezione è di 12 micron. </li><li> Asciugare i vetrini. Aggiungere 100 microlitri soluzione bloccante in ogni diapositiva per 2 ore a temperatura ambiente. Aggiungere 100 ml di anticorpi (diluito in soluzione di blocco fino alla concentrazione desiderata). Il periodo di incubazione per 1 ° e 2 ° anticorpi è ON a 4 ° C o 2 ore a temperatura ambiente, rispettivamente. Per ogni periodo di incubazione, aggiungere delicatamente striscia parafilm sulla parte superiore della diapositiva per prevenire la secchezza. Lavare con PBS (10 min x 3) tra gli anticorpi. Se necessario, aggiungere 1:1.000 Dapi (4&#39;-6-Diamidino-2-fenilindolo), diluito in PBS per 20 minuti a RT. Risciacquare con PBS. </li><li> Montare il vetrino con mezzi di montaggio fluorescenti. Lasciate asciugare per 1 ora prima dell&#39;analisi. </li></ol>

<ol>
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<p class="jove_content"&gt; Nessun conflitto di interessi dichiarati.</p

In elettroporazione ovo è uno strumento fattibile, affidabile ed efficace per esaminare le specifiche delle cellule e la guida assonale durante pulcino sviluppo del sistema nervoso 20. In questo protocollo si descrive una modalità di elettroporazione in romboencefalo pulcino a E2.75 utilizzando elementi enhancer che consentano l&#39;etichettatura condizionale di interneuroni specifici. Questa strategia è combinato con il sistema di recepimento PiggyBac-mediata di inserire geni estranei ne...

Romboencefalo rappresenta un mozzo relè chiave del sistema nervoso comunicando tra il sistema nervoso centrale e periferico tramite ascendente e discendente reti neuronali. Esso regola le funzioni di base tra cui la respirazione, la coscienza, l&#39;udito, e la coordinazione motoria 1-3. Durante lo sviluppo embrionale precoce, la romboencefalo vertebrati viene transitoriamente suddiviso lungo il suo asse antero-posteriore (AP) in rhombomeres ripetitivi, in cui si formano diversi tipi di cellule neuronali e generano molteplici centri nuclei del tronco cerebrale 4. Il cervello posteriore è diviso lungo il suo asse dorso-ventrale (DV) in una piastra basale e alari, in cui progenitori neuronali discrete diventano specificati e differenziano in distinti luoghi DV 3,5,6. Come il primo AP e specifiche DV modelli neuronali sono di istituzione dei circuiterie tronco cerebrale funzionale è in gran parte sconosciuto. Di acquisire conoscenze su questo fondamentalequestione, gli strumenti sono necessari al fine di etichettare specifici sottoinsiemi di neuroni nel cervello posteriore e presto per tracciarne la traiettoria degli assoni e la connettività nelle fasi più avanzate. Abbiamo precedentemente utilizzati specifici elementi enhancer, e un sistema di espressione condizionale Cre / loxP-based per il monitoraggio traiettoria assonale delle dorsali interneuroni spinali nei primi embrione di pollo 7-9. Nel manoscritto attuale abbiamo preso di mira il cervello posteriore e aggiornato il paradigma sperimentale per l&#39;etichettatura di fine interneuroni romboencefalo embrionali, gli assoni e ai loro obiettivi sinaptici, utilizzando una strategia di elettroporazione modificato e il PiggyBac - trasposizione del DNA mediata. La nostra nuova strategia permette la codifica dei sottotipi neuronali distinti in un lato del cervello posteriore e il tracciamento delle loro proiezioni assonali e siti sinaptici nelle varie fasi embrionali, da 2 a 12 giorni dopo l&#39;elettroporazione. In base a questo metodo, abbiamo etichettato la dorsale più a sottogruppo di interneuroni romboencefalo (dA1/Atoh1 + </sup&gt; celle) e ha rivelato due controlaterale ascendenti modelli di proiezione assonale, ogni deriva da una posizione diversa AP e allunga in un funicolo distinto. sono stati trovati DA1 assoni al progetto e formano sinapsi nei nuclei uditivi, mesencefalo e in strati multipli del cervelletto 10. La combinazione di pulcino elettroporazione, genetica rintracciamento di neuroni e analisi di siti per proiezioni molto stadi avanzati di sviluppo prevede una piattaforma unica per studiare la formazione di reti neuronali nel cervello e per chiarire i meccanismi molecolari che presiedono circuito.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
		 </tr>
		 <tr>
			<td>L-shaped gold Genetrodes 3 mm electrodes</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0162 </td>
		 </tr>
		 <tr>
			<td>pulse generator, ECM 830</td>
			<td>BTX, Harvard Apparatus</td>
			<td>45-0002</td>
		 </tr>
		 <tr>
			<td>OCT (Optimal Cutting Temperature) Compound</td>
			<td>Tissue-Tek
				Sakura</td>
			<td>4583 O.C.T. Compound </td>
		 </tr>
		 <tr>
			<td>Nail Polish</td>
			<td>From Any Commercial Supplier </td>
			<td></td>
		 </tr>
		</tbody>
 </table>

electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo

Elettroporazione del pulcino embrionale tubo neurale ha molti vantaggi come essere veloce ed efficiente per l&#39;espressione di geni estranei in cellule neuronali. In questo manoscritto forniamo un metodo che dimostra univocamente come l&#39;elettroporazione di DNA nel cervello posteriore aviaria in E2.75, al fine di etichettare specificamente un sottoinsieme di progenitori neuronali, e come seguire le loro proiezioni assonali e traguardi sinaptiche in Gran fase avanzata di sviluppo, fino a E14.5. Abbiamo utilizzato nuovi strumenti genetici tra cui specifici elementi enhancer, Cre / Lox - plasmidi base e il sistema di recepimento DNA PiggyBac-mediata di guidare l&#39;espressione della GFP in un sottotipo di cellule romboencefalo (la dorsale più sottogruppo di interneuroni, DA1). Traiettorie e target di DA1 assoni assonale sono seguiti a inizio e fine fasi embrionali in varie regioni del tronco encefalico. Questa strategia contribuisce tecniche avanzate per il targeting di cellule di interesse nel romboencefalo embrionale e per il TRACing formazione circuito a più stadi di sviluppo.

Questo protocollo è stato recentemente utilizzato per scoprire i modelli assonale e siti di proiezione di DA1 sottogruppo di interneuroni nella romboencefalo pulcino 10. Per etichettare specificamente questi assoni, un elemento enhancer (Atoh1), che è stata precedentemente caratterizzata da specifici per i neuroni spinali DI1 8,12,13, è stato confermato da esprimere in romboencefalo DA1 cellule 10. L&#39;elemento è stato clonato a monte di Cre ricombinasi e co-elettroporato a E2.75 i...

Watch this Scientific Journal Video about Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo at JoVE.com

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Come le reti neuronali sono stabiliti nel cervello embrionale è una questione fondamentale nella neurobiologia dello sviluppo. Qui abbiamo combinato una tecnica di elettroporazione con nuovi strumenti genetici, come ad esempio Cre / Lox-plasmidi e PiggyBac-mediata sistema trasposizione del DNA nel romboencefalo aviaria di etichettare interneuroni dorsali e monitorare le loro proiezioni assonali e traguardi sinaptiche a vari stadi di sviluppo.

Elettroporazione del romboencefalo tracciare traiettorie assonale e Obiettivi Synaptic in embrione di pollo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Research

JoVE Journal

Neuroscience

10.8K Views.  The Hebrew University of Jerusalem. Come le reti neuronali sono stabiliti nel cervello embrionale è una questione fondamentale nella neurobiologia dello sviluppo. Qui abbiamo combinato una tecnica di elettroporazione con nuovi strumenti genetici, come ad esempio Cre / Lox-plasmidi e PiggyBac-mediata sistema trasposizione del DNA nel romboencefalo aviaria di etichettare interneuroni dorsali e monitorare le loro proiezioni assonali e traguardi sinaptiche a vari stadi di sviluppo.

Video: Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Etichettatura e cellule Imaging nel romboencefalo Zebrafish

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Ricerca

Neuroscienze

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

Produzione di estratto di Chick dell&#39;embrione per la coltivazione delle cellule della cresta neurale Murine staminali

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Veloce micro-ionoforesi di glutammato e GABA: un utile strumento per indagare l&#39;integrazione Synaptic

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

Uso di pHluorin per valutare la dinamica dei recettori di guida axon nella coltura cellulare e nell'embrione del pulcino

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

Il sistema olfattivo come modello per studiare gli schemi di crescita assonale e Morfologia<em&gt; In Vivo</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultanea a due fotoni<em&gt; In Vivo</em&gt; Imaging di Synaptic ingressi e degli obiettivi postsinaptici nella corteccia mouse retrosplenial

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

L'uso di traccia Eyeblink condizionamento classico per valutare la disfunzione Hippocampal in un modello del ratto dei disturbi dello spettro fetale alcol

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

Uso di zinco sinaptica istochimica per rivelare diverse regioni e lamine nel cervello in via di sviluppo ed adulto

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

Combinando optogenetica con microRNA artificiali per caratterizzare gli effetti del Gene atterramento su funzione presinaptica all'interno di circuiti neuronali intatto

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...