We graciously thank Dr. John Hayes for scripts; Drs. Eric Bradley and Christopher Del Negro for assistance with confocal microscopy; Drew Hughes, Laura Odorizzi, Alex Garafalo, Rebecca Lowden, and Liz MacMurray for their work in developing the project and providing preliminary data; Dr. Greg Smith for helpful suggestions on statistical analysis. This work was supported by an NIH grant (NINDS IR15N5067566-01) to MSS and a Howard Hughes Medical Institute Science Education Grant to the College of William and Mary.

No conflicts of interest declared.

With its well-characterized cell types that are conserved across all vertebrates, the retina provides a useful model for studying the molecular-cellular processes governing cell type specification and differentiation. Primary cell culture affords a powerful method for investigating a wide range of processes including gene expression, protein dynamics, and calcium and electrical activity at the level of single cell resolution. Here we present a straightforward technique for primary cell culture from dissected presumptive ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Dissections and Culturing</td>
 </tr>
 <tr>
 <td>BD Falcon Easy Grip Tissue Culture Dishes, 35 mm</td>
 <td>Fisher</td>
 <td>08-772A</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable Polystyrene Petri Dishes, 60 x 15 mm</td>
 <td>Fisher</td>
 <td>0875713A</td>
 <td></td>
 </tr>
 <tr>
 <td>35 mm Nunclon Surface Petri Dishes (with Airvent)</td>
 <td>Fisher</td>
 <td>12-565-91</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont Fine Forceps (Dumostar #55)</td>
 <td>Fisher</td>
 <td>NC9341917 </td>
 <td></td>
 </tr>
 <tr>
 <td>Cellattice Micro-ruled plastic coverslip, 25 mm</td>
 <td>Fisher</td>
 <td>50-313-17 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl-m-Aminobenzoate Methanesulfonate Salt (MS-222)</td>
 <td>MP Biomedicals, LLC</td>
 <td>103106 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gentamicin Sulfate</td>
 <td>Enzo Life Sciences</td>
 <td>380-003-G025 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gibco Trypsin (1:250) Powder</td>
 <td>Life Technologies</td>
 <td>27250-018 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B from Clostridium histolyticum</td>
 <td>Roche</td>
 <td>11088831001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td></td>
 </tr>
 <tr>
 <td>Nile Blue Sulfate (optional)</td>
 <td>Pfaltz &amp; Bauer</td>
 <td>N05550 </td>
 <td></td>
 </tr>
 <tr>
 <td>500 ml Vacuum Filter/Storage Bottle System, 0.22 &mu;m Pore 33.2 cm2 CN Membrane</td>
 <td>Corning</td>
 <td>430758</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Calcium Imaging</td>
 </tr>
 <tr>
 <td>Fluo-4 AM 1 mM Solution in DMSO, Cell Permanent</td>
 <td>Life Technologies</td>
 <td>F-14217</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F-127 10% Solution in Water </td>
 <td>Life Technologies</td>
 <td>P-6866</td>
 <td></td>
 </tr>
 <tr>
 <td>LSM 510 Confocal Microscope System </td>
 <td>Zeiss</td>
 <td>Model Discontinued</td>
 <td></td>
 </tr>
 <tr>
 <td>Blocking Reagent</td>
 <td>Roche</td>
 <td>11096176001</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>For Fluorescent in situ hybridization (FISH)</td>
 </tr>
 <tr>
 <td>Anti-Digoxigenin-POD, Fab Fragments</td>
 <td>Roche</td>
 <td>11207733910</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-DNP-HRP Antibody </td>
 <td>Perkin-Elmer</td>
 <td>NEL747A</td>
 <td></td>
 </tr>
 <tr>
 <td>Cy3 NHS ester</td>
 <td>GE Healthcare</td>
 <td>PA13101</td>
 <td></td>
 </tr>
 <tr>
 <td>NHS-Fluorescein </td>
 <td>Thermo Scientific</td>
 <td>46409</td>
 <td></td>
 </tr>
 <tr>
 <td>Formamide, Deionized</td>
 <td>Amresco</td>
 <td>0606-950ML</td>
 <td></td>
 </tr>
 <tr>
 <td>Torula RNA, Type IX</td>
 <td>Sigma-Aldrich</td>
 <td>R3629</td>
 <td></td>
 </tr>
 <tr>
 <td>Heparin Sodium Salt, from Porcine Intestinal Mucosa</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>Table 2. Specific reagents and equipment.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td><table border="1">
 <tbody>
 <tr>
 <td>Solution</td>
 <td>Reference</td>
 <td>Contents</td>
 </tr>
 <tr>
 <td>Cell Culture Medium</td>
 <td>Chang and Spitzer , 200954</td>
 <td>116 mM NaCl 
 0.67 mM KCl 
 2 mM CaCl2 
 1.31 mM MgSO4 
 4.6 mM Tris 
 1 % (v:v) Penicillin/Streptomycin 
 Adjust pH to 7.8 with HCl. 
 Filter sterilize by passing through a 0.22 &mu;m CN filter. 
 Store at 4 &deg;C.</td>
 </tr>
 <tr>
 <td>Calcium-Magnesium Free Medium (CMF)</td>
 <td>Gu et al., 199442; Gu and Spitzer, 199555</td>
 <td>116 mM NaCl 
 0.67 mM KCl 
 4.6 mM Tris 
 0.4 mM EDTA 
 1 % (v:v) Penicillin/Streptomycin 
 Adjust pH to 7.8 with HCl. 
 Filter sterilize by passing through a 0.22 &mu;m CN filter. 
 Store at 4 &deg;C. </td>
 </tr>
 <tr>
 <td>Maleic Acid Buffer (MAB)</td>
 <td>Sive et al., 200053 </td>
 <td>100 mM maleic acid 
 150 mM NaCl (pH 7.5).</td>
 </tr>
 <tr>
 <td>Marc's Modified Ringers (MMR), 10X</td>
 <td>Sive et al., 200053 </td>
 <td>100 mM NaCl 
 mM KCl 
 1 mM MgSO4 
 2 mM CaCl2 
 5 mM HEPES 
 pH adjusted to 7.4 with NaOH 
 0.1X MMR also contains 50 mg/ml gentamicin sulfate and pH is adjusted to 7.4 with NaOH.</td>
 </tr>
 <tr>
 <td>MEMFA Solution, 10X</td>
 <td>Sive et al., 200053</td>
 <td>0.1 M MOPS (Ph 7.4) 
 2 mM EGTA 
 1 mM MgSO4 
 3.7% formaldehyde 
 A 10X solution, without formaldehyde, can be stored at 4 &deg;C. Formaldehyde is added fresh (1/10 volume of a standard 37% stock). </td>
 </tr>
 <tr>
 <td>In situ Hybridization Buffer</td>
 <td>Sive et al., 200053</td>
 <td>50% Formamide 
 5X SSC 
 1 mg/ml Torula RNA 
 100 mg/ml Heparin 
 1X Denhart's Solution 
 0.1% Tween 20 
 0.1% CHAPS 
 10 mM EDTA.</td>
 </tr>
 </tbody>
</table>
Solutions. *Gentamicin, an antibiotic, is used in our MMR solutions while penicillin and streptomycin are used in our culture media.</td>
 </tr>
 </tbody>
</table>

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

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 basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Examples of successfully dissected optic vesicles (stage 25) and retinae (stage 35) are shown in Figures 2E and 2J. While this protocol can be used at various stages of development, it is critical to obtain only retinal tissue to ensure accuracy for further experiments. Carefully remove the epidermis at all stages and ensure that your forceps do not puncture the retinal tissue. In stage 35 or older, the lens can be seen as a clear layer on top of the retina and can be removed by cautious scraping ...

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 provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

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

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

13.8K Views.  College of William and Mary. Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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