Methods for Investigating TRP Channel Gating.

A complete characterization of temperature -and voltage-activated TRP channel gating requires a precise determination of the absolute probability of opening in a wide range of voltages, temperatures, and agonist concentrations. We have achieved this in the case of the TRPM8 channel expressed in Xenopus laevis oocytes. Measurements covered an extensive range of probabilities and unprecedented applied voltages up to 500 mV. In this chapter, we describe animal care protocols of patch-clamp pipette preparation, temperature control methods, and analysis of ionic currents to obtain reliable absolute open channel probabilities.

Hargreaves does not evaluate nociception following a surgical laparotomy in Xenopus leavis frogs.

The present study was performed to determine the effectiveness of the Hargreaves test for the evaluation of nociception in frogs, more precisely to determine if cutaneous thresholds to a radiant heat stimulus would increase with analgesics following an abdominal laparotomy performed under general anaesthesia. Non breeding female Xenopus leavis frogs (3 groups (non-anaesthetized, anaesthetized with tricaine methanesulfonate (MS222), with or without an abdominal laparotomy) were used to evaluate the effectiveness of the Hargreaves test. Cutaneous thresholds were evaluated at baseline and following anaesthetic recovery (over 8 h) at six different body locations. Increased reaction times were observed in the gular area only at 1 h post-recovery following a MS222 bath immersion in frogs with (p < 0.02) and without the abdominal laparotomy (p < 0.002). In conclusion, the Hargreaves test does not provide an adequate test to evaluate nociception induced by an abdominal laparotomy and consequently cannot be used to evaluate analgesics in X. leavis frogs.

Dissection of Xenopus laevis neural crest for in vitro explant culture or in vivo transplantation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration.

Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation, through the coordinated activity of numerous growth factor signalling pathways, including the Wnt, Fgf, Bmp, Notch and TGF-ß pathways. Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, using pharmacological or genetic approaches, reduces the level of cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/ß-catenin signalling and the activation of one of its main downstream targets, fgf20 (ref. 7), which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.

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 differentiation(1-16). The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and Müller 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).

Evaluation of the gross and histologic reactions to five commonly used suture materials in the skin of the African clawed frog (Xenopus laevis).

Surgical harvest of Xenopus laevis oocytes for developmental research is a common procedure that requires closure of a 0.5- to 2.0-cm incision with suture material. Although such harvests are a frequent practice, little published information exists to provide guidance regarding the most appropriate suture material for wound closure in laboratory amphibians. To determine which suture material elicits the least response in amphibian skin, we used Xenopus laevis as a model to investigate the gross and histologic tissue reactions to 5 commonly used suture materials-3-0 silk, monofilament nylon, polydioxanone, polyglactin 910, and chromic gut. The skin reacted in 3 ways to suture material, showing edema, epidermal changes, and inflammation. Although the gross reactions to monofilament nylon, polydioxanone, and polyglactin 910 were clinically indistinguishable and were associated with lowest gross reaction scores, monofilament nylon elicited the least histologic reaction and therefore seems to be the most appropriate choice for use in amphibian skin.

Cholinergic currents in Xenopus oocytes transplanted with human brain membranes

Corrientes colinérgicas de cerebro humano fueron registradas en oocitos de Xenopus laevis trasplantados con membranas de cerebro humano procedentes de dos zonas diferentes, la corteza frontal y el hipocampo. Las corrientes registradas fueron activadas por el receptor nicotínico o por el receptor nicotínico o muscarínico de la acetilcolina. Se probaron los efectos de diferentes agonistas nicotínicos como acetilcolina, nicotina y yoduro de 1,1-dimetil-4-fenil-piperazinio (DMPP), y antagonistas del receptor nicotínico como a-bungarotoxina y d-tubocurarina en los oocitos transplantados. Detectamos cuatro clases de cinéticas de corrientes nicotínicas. Las diferencias en la amplitud y en la carga eléctrica total de las corrientes provocadas por varios agonistas en el rango de potencial mantenido no fueron significativas, excepto en el caso del DMPP a un potencial mantenido de -90 mV. Nuestros resultados indican que las formas alfa4beta2, alfa3beta4 y alfa7 son los principales receptores nicotínicos en el cerebro humano

Cholinergic human brain currents were recorded in Xenopus laevis oocytes transplanted with human cerebral membranes from two different zones, the frontal cortex and the hippocampus. The recorded currents were supported by the nicotinic or the muscarinic acetylcholine receptor. We tested the effects of a number of several nicotinic agonists acetylcholine, nicotine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and the nicotinic receptor antagonists a-bungarotoxin and d-tubocurarine on the transplanted oocytes. We detected four kinds of nicotinic current kinetics. The differences in the amplitude and in the total electric charge of the currents elicited by various agonists at a range of holding potentials were not significant, except in the case of DMPP at a holding potential of -90 mV. Our results indicate that alpha4beta2, alpha3beta4 and alpha7 are the main nicotinic receptors in human brain

Muscle formation in regenerating Xenopus froglet limb.

A spike, a resultant regenerate made after amputation of a Xenopus froglet limb, has no muscle tissue. This muscle-less phenotype was analyzed by molecular approaches, and the results of analysis revealed that the spike expresses no myosin heavy chain or Pax7, suggesting that neither mature muscle tissue nor satellite cells exist in the spike. The regenerating blastema in the froglet limb lacked some myogenesis-related marker genes, myoD and myf5, but allowed implanted muscle precursor cells to survive and differentiate into myofiber. Implantation of hepatocyte growth factor (HGF) -releasing cell aggregates rescued this muscle-less phenotype and induced muscle regeneration in Xenopus froglet limb regenerates. These results suggest that failure of regeneration of muscle is due to a disturbance of the early steps of myogenesis under a molecular cascade mediated by HGF/c-met. Improvement of muscle regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic tissue regeneration in amphibians and other vertebrates.

Postoperative analgesics in South African Clawed frogs (Xenopus laevis) after surgical harvest of oocytes.

Heightened awareness for the welfare of earlier-evolved laboratory species has prompted increasing inquiries by institutional animal care committees, investigators, and laboratory animal veterinarians regarding the need for post-surgical analgesics in laboratory Xenopus. Basic research into the mechanisms and regulation of pain in Rana pipiens has demonstrated the clinical potential of opioid, alpha2-adrenergic, and non-opioid analgesic agents in amphibians. However, clinical studies using objectively established indices of amphibian pain, or pharmacological studies in either Rana pipiens or laboratory Xenopus have not been conducted. As discussed above, comparison of limited lethality data suggests that the safety index for these agents is quite narrow in Rana pipiens. Analgesic use in laboratory Xenopus has the added risk of drowning due to over sedation. Drug doses extrapolated from such studies and intended to provide pain relief in Xenopus should therefore be considered very carefully. An additional concern for laboratory Xenopus is that the effects of these agents on amphibian oogenesis, oocyte quality, and embryogenesis are unknown. As the numbers of laboratory Xenopus used in basic and biomedical research continues to increase, clinical studies that address all of these issues cannot come too soon.

Demonstration of cells possessing tolerance-inducing activity in Xenopus laevis rendered tolerant perimetamorphically.

J-strain (JJ) Xenopus laevis is easily made tolerant to semixenogeneic (X laevis x X borealis: JB) adult skin grafted onto immunologically competent larvae at stages 53-54. If the larvae are thymectomized shortly before skin grafting (late Txd), tolerance will never be induced. This suggests that suppression of the immune response to JB skin is mediated by a population of thymus-derived cells. In an attempt to confirm the presence of these cells, the spleen and tolerated JB skin were tested for their tolerance-inducing activity, by grafting either spleen or skin and a new JB skin piece simultaneously to late Txd JJ larva (secondary host). The results clearly indicated that both spleen and skin possessed the ability to induce tolerance. The injection of 1 x 10(5) splenocytes resulted in induction of tolerance in more than 80% of secondary hosts, and the rate of tolerance induction increased in relation to the injected cell number. Furthermore, tolerance was induced in about 90% of the animals by injecting as few as 100 cells isolated from the tolerated skin. Immunohistochemical observation of grafted skin showed that many host-derived T cells were distributed around the epidermal basal lamina. These results indicate that the cells with suppressive activity are a population of T cells that have differentiated in metamorphosing thymuses and have been released peripherally.

[Mechanically dependent heterotopias of the axial rudiments in clawed toad embryos]. / Mekhanozavisimye geterotopii osevykh zachatkov u zarodyshei shportsevoi liagushki.

Relaxation of tangential tensions was achieved in Xenopus laevis embryos at the stage of early gastrula by introducing radial cuts and insertion of ventral tissue wedges from the embryos at the similar stage. Velocity and direction of movement of the cellular material was compared in the normal and operated embryos using vital marking methods. We observed the formation of complexes of axial organs in atypical positions: after dorsomedial cuts, along the medial line and in the lateral blastopore lips; after anteroposterior (animal-vegetal) cuts, also on the ventral side; after lateral cuts (incisions), on the contralateral side of embryos. Ultrastructure of cells from the ventral region, which participate in the formation of neural plate, resembles that of the neural plate of the normal embryo as early as 4 h after surgery. We interpret these heterotopias of axial rudiments as the result of overtensions in relaxed tissues. The presence of overtensions was confirmed by tissue separation after local incisions. In all experimental series, axial rudiments were elongated in the direction of maximal overtension. Movements of the cellular material both in the normal and in the operated embryos were directed towards maximal tension.