Abbiamo gentilmente Ringraziamo il Dott. John Hayes per gli script, Drs. Eric Bradley e Christopher Del Negro per l&#39;assistenza con la microscopia confocale, Drew Hughes, Laura Odorizzi, Alex Garafalo, Rebecca Lowden, e Liz MacMurray per il loro lavoro nello sviluppo del progetto e la fornitura di dati preliminari, il dottor Greg Smith per suggerimenti utili per l&#39;analisi statistica. Questo lavoro è stato finanziato da una sovvenzione del NIH (NINDS IR15N5067566-01) per MSS e un Howard Hughes Medical Institute Science Education Sovvenzione al College of William and Mary.

Nessun conflitto di interessi dichiarati.

Con i suoi tipi di cellule ben caratterizzati che si conservano in tutti i vertebrati, la retina fornisce un modello utile per lo studio dei processi molecolari-cellulari che regolano tipo di specifica e la differenziazione cellulare. Coltura cellulare primaria offre un metodo potente per indagare un&#39;ampia gamma di processi tra espressione genica, dinamica della proteina, e di calcio e attività elettrica a livello di singola cellula risoluzione. Qui vi presentiamo una tecnica semplice per colture cellulari primarie...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td></td></tr><tr><td></td><td></td><td></td><td> Per le dissezioni e coltivando </td></tr><tr><td> BD Falcon Facile Grip tessuti piatti Cultura, 35 mm </td><td> Pescatore </td><td> 08-772A </td><td></td></tr><tr><td> Monouso in polistirolo piastre di Petri, 60 x 15 mm </td><td> Pescatore </td><td> 0875713A </td><td></td></tr><tr><td> 35 millimetri di superficie Nunclon piastre Petri (con Airvent) </td><td> Pescatore </td><td> 12-565-91 </td><td></td></tr><tr><td> Dumont fine Pinza (Dumostar # 55) </td><td> Pescatore </td><td> NC9341917 </td><td></td></tr><tr><td> Cellattice Micro-governato coprioggetto in plastica, 25 mm </td><td> Pescatore </td><td> 50-313-17 </td><td></td></tr><tr><td> Ethyl-m-aminobenzoate metansolfonato Salt (MS-222) </td><td> MP Biomedicals, LLC </td><td> 103106 </td><td></td></tr><tr><td> Gentamicina solfato </td><td> Enzo Life Sciences </td><td> 380-003-G025 </td><td></td></tr><tr><td> Gibco tripsina (1:250) in polvere </td><td> Life Technologies </td><td> 27250-018 </td><td></td></tr><tr><td> Collagenasi B da Clostridium histolyticum </td><td> Roche </td><td> 11088831001 </td><td></td></tr><tr><td> Penicillina-Streptomicina </td><td> Gibco </td><td> 15140-122 </td><td></td></tr><tr><td> Nilo Azzurro Solfato (opzionale) </td><td> Pfaltz &amp; Bauer </td><td> N05550 </td><td></td></tr><tr><td> 500 ml vuoto filtro / System Storage bottiglia, 0,22 micron pori 33,2 centimetri 2 membrana CN </td><td> Corning </td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ Td&gt; <td></td><td> Per Imaging di calcio </td></tr><tr><td> Fluo-4 AM 1 Soluzione mM in DMSO, Cell permanente </td><td> Life Technologies </td><td> F-14217 </td><td></td></tr><tr><td> Soluzione Pluronic F-127 10% in acqua </td><td> Life Technologies </td><td> P-6866 </td><td></td></tr><tr><td> LSM 510 Sistema microscopio confocale </td><td> Zeiss </td><td> Modello fuori produzione </td><td></td></tr><tr><td> Reagente Bloccante </td><td> Roche </td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> Per ibridazione fluorescente in situ (FISH) </td></tr><tr><td> Anti-Digoxigenin-POD, Frammenti Fab </td><td> Roche </td><td> 11207733910 </td><td></td></tr><tr><td> Anti-DNP-HRP Anticorpo </td><td> Perkin-Elmer </td><td> NEL747A </td><td></td></tr><tr><td> Cy3NHS estere </td><td> GE Healthcare </td><td> PA13101 </td><td></td></tr><tr><td> NHS-Fluoresceina </td><td> Thermo Scientific </td><td> 46409 </td><td></td></tr><tr><td> Formammide, deionizzata </td><td> Amresco </td><td> 0606-950ml </td><td></td></tr><tr><td> Torula RNA, Tipo IX </td><td> Sigma-Aldrich </td><td> R3629 </td><td></td></tr><tr><td> Sale di sodio eparina, dalla mucosa intestinale suina </td><td> Sigma-Aldrich </td><td> H3393-250KU </td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich </td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> Tabella 2. Reagenti e attrezzature specifiche. </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> Soluzione </td><td> Riferimento </td><td> Contenuto </td></tr><tr><td> Cella Cultura Media </td><td> Chang e Spitzer, 2009 54 </td><td> 116 mM di NaCl 0,67 mM KCl 2 mM CaCl 2 1,31 mM MgSO4 4,6 mM Tris 1% (v: v) di penicillina / streptomicina Regolare il pH a 7,8 con HCl. Filtrare sterilizzare passando attraverso un filtro 0,22 micron CN. Conservare a 4 ° C. </td></tr><tr><td> Calcio-Magnesio gratuita Media (CMF) </td><td> Gu et al, 1994 42,. Gu e Spitzer, 1995 55 </td><td> 116 mM di NaCl 0,67 mM KCl 4,6 mM Tris 0,4 mM EDTA 1% (v: v) di penicillina / streptomicina Regolare il pH a 7,8 con HCl. Filtrare sterilizzare passando attraverso un filtro 0,22 micron CN. Conservare a 4 ° C. </td></tr><tr><td> Acid Buffer maleico (MAB) </td><td> SIVE et al., 2000 53 </td><td> 100 mM di acido maleico 150 mM NaCl (pH 7,5). </td></tr><tr><td> Marc Ringers Modified (MMR), 10X </td><td> SIVE et al., 2000 53 </td><td> 100 mM NaCl mM KCl 1 mM MgSO4 2 mM CaCl 2 5 mM HEPES pH regolato a 7,4 con NaOH 0,1 X. MMR contiene inoltre 50 mg / ml gentamicina solfato e si corregge il pH a 7,4 con NaOH. </td></tr><tr><td> Soluzione MEMFA, 10X </td><td> SIVE et al., 2000 53 </td><td> MOPS 0,1 M (pH 7,4) 2 mM EGTA 1 mM MgSO4 3,7% di formaldeide Una soluzione 10X, senza formaldeide, può essere conservato a 4 ° C. La formaldeide è aggiunto il fresco (1/10 del volume di una madre standard 37%). </td></tr><tr><td> In tampone di ibridazione in situ </td><td> SivE et al., 2000 53 </td><td> 50% formammide 5X SSC 1 mg / ml di RNA Torula 100 mg / ml di eparina Soluzione 1X Denhart di 0,1% Tween 20 0,1% CHAPS 10 mM EDTA. </td></tr></tbody></table> Solutions. * Gentamicina, un antibiotico, è utilizzato nelle nostre soluzioni MMR, mentre la penicillina e la streptomicina sono utilizzate nelle nostre terreni di coltura. </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

Il processo attraverso il quale la regione anteriore della piastra neurale dà luogo alla retina dei vertebrati continua ad essere uno degli obiettivi principali della ricerca, sia clinica e di base. Oltre alla ovvia rilevanza medica per capire e trattare malattie retiniche, lo sviluppo della retina dei vertebrati continua a servire come sistema modello importante ed elegante per la determinazione neuronale tipo comprensione e differenziazione 1-16. La retina neurale si compone di sei tipi di cellule discrete (ganglio, fotorecettori amacrine, orizzontale,, cellule bipolari e cellule gliali Müller) disposte a strati stereotipati, un modello che è in gran parte conservata tra tutti i vertebrati 12,14-18. Mentre studiava la retina di un embrione in via di sviluppo è chiaramente intatta necessario per capire come questo organo complesso si sviluppa da una sporgenza del prosencefalo in una struttura a strati, ci sono molte domande che beneficiano from impiegando approcci utilizzando colture primarie di cellule presunte cellule retiniche 7,19-23. Ad esempio, analizzando le cellule da tessuti rimossi e dissociati in diverse fasi permette di distinguere lo stato di specificazione di singole cellule a diversi stadi di sviluppo, cioè, il destino delle cellule in assenza di interazioni con i tessuti vicini 8,19-22 ,24-33. Coltura cellulare primaria permette anche al ricercatore di trattare la cultura con reagenti specifici e analizzare i risultati a livello di singola cellula 5,8,21,24,27-30,33-39. Xenopus laevis, un sistema classico modello per lo studio dei prime fasi dello sviluppo neurale 19,27,29,31-32,40-42, serve come un sistema particolarmente adatto per coltura di cellule della retina primario 10,38,43-45. Presunto tessuto retinico è accessibile sin dalle prime fasi di sviluppo, subito dopo l&#39;induzione neurale 25,38,43. Inoltre, dato thad ogni cellula nell&#39;embrione contiene una fornitura di tuorlo, cellule retiniche possono essere coltivate in un mezzo molto semplici definiti costituiti da una soluzione salina tamponata, eliminando così gli effetti confondenti di incubazione o altri prodotti a base di sieri 10,24,44-45 . Tuttavia, l&#39;isolamento del tessuto retinico dai tessuti circostanti e la successiva elaborazione è impegnativo. Qui, presentiamo un metodo per la dissezione e la dissociazione di cellule retiniche in Xenopus laevis che verranno utilizzati per preparare colture cellulari primarie che, a sua volta, essere analizzati per attività di calcio e l&#39;espressione genica a risoluzione di singole cellule. Mentre l&#39;argomento presentato in questo documento è l&#39;analisi di transienti di calcio spontanee, la tecnica è ampiamente applicabile ad una vasta gamma di domande di ricerca e approcci (Figura 1).

Esempi di successo sezionati vescicole ottiche (fase 25) e retine (fase 35) sono mostrati nelle figure 2E e 2J. Mentre questo protocollo può essere utilizzato in diverse fasi di sviluppo, è fondamentale per ottenere solo tessuto retinico per assicurare la precisione per ulteriori esperimenti. Rimuovere con cura l&#39;epidermide in tutte le fasi e garantire che i pinza non forare la retina. In fase di 35 anni o più, la lente può essere vista come uno strato trasparente sopra la retina e può essere r...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis fornisce un sistema modello ideale per studiare il destino specificazione delle cellule e la funzione fisiologica delle singole cellule retiniche in colture cellulari primarie. Presentiamo qui una tecnica per la dissezione e la generazione di tessuti retinici colture cellulari primarie che vengono esposte ad attività calcio e analizzati mediante ibridazione in situ.

Dissezione, Cultura, e analisi di<em&gt; Xenopus laevis</em&gt; Tessuto embrionale della retina

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

dissection-culture-analysis-xenopus-laevis-embryonic-retinal

Research

JoVE Journal

Neuroscience

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

13.8K Views.  College of William and Mary. Xenopus laevis fornisce un sistema modello ideale per studiare il destino specificazione delle cellule e la funzione fisiologica delle singole cellule retiniche in colture cellulari primarie. Presentiamo qui una tecnica per la dissezione e la generazione di tessuti retinici colture cellulari primarie che vengono esposte ad attività calcio e analizzati mediante ibridazione in situ.

Video: Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissezione e la cultura di neuroni embrionali commissurali da midollo spinale

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Ricerca

Neuroscienze

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection e cultura primaria di neuroni embrionali di topo ippocampale e corticale

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

Dissezione di un occhio mouse per un monte intero di dell&#39;epitelio pigmentato retinico

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

Lectina a base di isolamento e la coltura del mouse motoneuroni embrionali

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

Isolamento e la coltura di cellule embrionali neuronali di ratto: un protocollo veloce

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Culture Explant neurali di<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

Isolamento e coltura di cellule endoteliali dal embrionali Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

Un test comportamentale semplice for Testing Funzione visiva in<em&gt; Xenopus laevis</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

Registrazione neuronale attività Temperatura indotta attraverso il monitoraggio dei cambiamenti di calcio nel bulbo olfattivo di<em&gt; Laevis Xenopus</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparazioni e protocolli per l'intera cella Patch Clamp registrazione dei neuroni Tectal Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...