Distribution and corticosteroid regulation of glucocorticoid receptor in the brain of Xenopus laevis

Glucocorticoids (GCs) play essential roles in physiology, development, and behavior that are mediated largely by the glucocorticoid receptor (GR). Although the GR has been intensively studied in mammals, very little is known about the GR in nonmammalian tetrapods. We analyzed the distribution and GC regulation of GR in the brain of the frog Xenopus laevis by immunohistochemistry. GR-immunoreactive (GR-ir) cells were widely distributed, with the highest densities in the medial pallium (mp; homolog of the mammalian hippocampus), accumbens, anterior preoptic area (POA; homolog of the mammalian paraventricular nucleus), Purkinje cell layer of the cerebellum, and rostral anterior pituitary gland (location of corticotropes). Lower but distinct GR-ir was observed in the internal granule cell layer of the olfactory bulbs, dorsal and lateral pallium, striatum, various subfields of the amygdala, bed nucleus of the stria terminalis (BNST), optic tectum, various tegmental nuclei, locus coeruleus, raphe nuclei, reticular nuclei, and the nuclei of the trigeminal motor nerves. Treatment with corticosterone (CORT) for 4 days significantly decreased GR-ir in the POA, mp, medial amygdala (MeA), BNST, and rostral pars distalis. Treatment with the corticosteroid synthesis inhibitor metyrapone (MTP) also significantly reduced GR-ir in the POA, mp, MeA and BNST, but not in the rostral pars distalis. Replacement with a low dose of CORT in MTP-treated animals reversed these effects in brain. Thus, chronic increase or decrease in circulating corticosteroids reduces GR-ir in regions of the frog brain. Our results show that the central distribution of GR-ir and regulation by corticosteroids are highly conserved among vertebrates.     

A developmental analysis of periodic albinism in the amphibian Xenopus laevis

The periodic albino of Xenopus laevis displays a transitory presence of black melanin pigment in the embryo but looses this during tadpole development. This mutation, involving a recessive allele, affects melanogenesis in dermal melanophore pigment cells. It has been suggested that the mutation is intrinsic to the melanophore cell itself or, alternatively, reflects malfunction in the neuroendocrine system that regulates melanophore cell function. This latter system, involving pituitary melanotrope cells which produces α-melanophore stimulating hormone (α-MSH), is responsible for stimulating the production and dispersion of melanin pigment in dermal melanophores. The purpose of the present study was to determine to which degree the albinism is intrinsic to the melanophore or involves neuroendocrine malfunction. Experiments involved transplantation of presumptive melanophores from wild-type to albino embryos, and vice versa, immunocytochemical analysis of the albino neuroendocrine system and the creation of wild-type/albino parabiotic animals to determine if the neuroendocrine system of the albino can support melanotrope cell function. We show that the albino has a functional neuroendocrine system and conclude that the defect in the albino primarily affects the melanophore cell, possibly rendering it incapable of responding to α-MSH. It is also apparent from our results that in later stages of development the cellular environment of the melanotrope cell does become important to its development, but the nature of the critical cellular factors involved remains to be determined.     

Restorative Regeneration of Digital Tips in the African Clawed Frog (Xenopus laevis Daudin)

Localized, specialized structures are carried on the tips of digits of many amniote and certain non‐amniote tetrapods. The pes of some members of Pipidae represents a rare example among tetrapods of differential expression of digital tip form (clawed vs. non‐clawed digits). As a step towards understanding how such localized forms are generated and maintained, we conducted a series of amputation experiments and observed, through the process of regeneration, the potential for reconstitution, at the tissue and organ level, of the different digit tip morphologies. Results of this study reveal that immediately following metamorphosis specialized digit tip structures are regenerated with a high degree of structural and spatial accuracy by a process that essentially replicates normal development in recently metamorphosed Xenopus laevis froglets. Furthermore, this regenerative capacity is maintained in juveniles 4 months beyond metamorphosis, and also in adults of 1 year or more in age, indicating that metamorphosis‐specific conditions do not exclusively facilitate regeneration of digit tips. In addition, regenerative capacity is maintained through repeated bouts of amputation and regeneration, indicating deep‐seated digit identity and retention of the distinct digit tip developmental programs within the digits. Together, these data suggest that the developmental programing responsible for the formation of the discrete digital tip morphologies is located regionally within each digit, and that it is retained through time. Our results suggest that Xenopus can serve as a model organism for exploring the molecular underpinnings of digit tip formation because regeneration leads to morphologically identical structures to those of the original digit tips. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Preparation of dissociated Zebrafish spinal neuron cultures

The method described here explains a simple protocol for how to prepare dissociated Zebrafish spinal neuron cultures. The neurons grow fast in a simple culture medium and at room temperature. Considering the advantages afforded by the optical transparency of the Zebrafish embryo combined with the powerful molecular perturbation techniques available, this technique has potential to further advance molecular analysis of axon growth and guidance.     

Novel properties of the depolarization-induced endogenous sodium conductance in the Xenopus laevis oocyte

 It has been shown by means of the two-microelectrode voltage-clamp technique that in membranes of Xenopus laevis oocytes a Na⁺-selective permeability can be activated by long-lasting or repetitive depolarization (R.T. Kado and C. Baud, Journal of Physiology, Paris, 77:1113–1117, 1981). In this study the permeability in inside-out giant membrane patches with diameters of 20–30 μm was analysed. Once induced, the Na⁺ permeability has a voltage-dependent open probability that increases with positive potentials and half-maximally activates at about 0 mV. Sudden changes of membrane potential elicit transient currents with strongly voltage-dependent time constants of from less than 1 ms at –150 mV to several hundreds of milliseconds at positive potentials. In contrast to the on-cell configuration, the permeability ceases completely within a few minutes in the cell-free inside-out configuration. This rundown can be prevented by including MgATP, but not Mg²⁺ or ATP alone, in the intracellular solution. Intracellular Mg²⁺ ions, in addition to being a co-factor for ATP in the activation process, decrease the permeability in a dose-dependent manner. Steady-state fluctuation analysis gave no evidence that an increased noise level is caused by open–close kinetics of an ion channel, suggesting that the single-channel conductance is below 1 pS if a channel-like structure is the origin of the endogenous Na⁺ permeability. Received: 14 July 1998 / Received after revision: 4 November 1998 / Accepted: 18 January 1999     

Particle tracking model of electrophoretic morphogen movement reveals stochastic dynamics of embryonic gradient

Some developmental events rely on an electrophoretic force to produce morphogenetic gradients. To quantitatively explore the dynamics of this process, we constructed a stochastic model of an early phase of left–right patterning: serotonin movement through the gap junction‐coupled blastomeres of the Xenopus embryo. Particle‐tracking simulations showed that a left–right gradient is formed rapidly, quickly reaching a final stable level. The voltage difference was critical for producing a morphogen gradient of the right steepness; gap junctional connectivity and morphogen mass determined the timing of the gradient. Endogenous electrophoresis drives ～50% of the particles across more than one cell width, and ～20% can travel across half the embryo. The stochastic behavior of the resulting gradients exhibited unexpected complexity among blastomeres' morphogen content, and showed how spatiotemporal variability within individual cells resulted in robust and consistent gradients across the embryonic left–right axis. Analysis of the distribution profile of gradient gain values made quantitative predictions about the conditions that result in the observed background level of laterality defects in unperturbed frog embryos. This work provides a general model that can be used to quantitatively analyze the unexpectedly complex dynamics of morphogens in a wide variety of systems. Developmental Dynamics 238:1923–1935, 2009. © 2009 Wiley‐Liss, Inc.     

青蒿素对蛙卵表达的克隆内向整流钾电流的抑制作用

采用分子和这及膜片钳技术，观测了青蒿素对非注蛙卵表达的克隆内向整流钾通道的影响。当灌注不同浓度青蒿素时，青蒿素呈剂量依赖关系降低Ｋｉｒ２．１钾通道在非注蛙卵细胞膜的功能表达。青蒿素阻断Ｋｉｒ２．１钾通道亦呈电压依赖性。     

Ca2+ modulates an unspecific cation conductance in olfactory cilia of Xenopus laevis

Summary Olfactory neurones of Xenopus laevis were studied by the patch clamp technique under voltage-clamp conditions. Isolated receptor cells were obtained by dissociating the olfactory mucosa in a Ca²⁺-free solution. Usually some of the resulting isolated olfactory cells lost all of their cilia during the dissociation procedure. Comparing the currents of cells with cilia to those of cells without cilia, a marked difference was found. When all known voltage-gated currents except the Ca²⁺-current were blocked, cells without cilia showed the voltage-gated Ca²⁺-current alone whereas cells with cilia clearly had an additional conductance g_c. It could be activated in two ways, either by Ca²⁺ entry through Ca²⁺-channels or by Ca²⁺ entry through the Na/Ca-exchanger working in the reversed mode at positive membrane potentials. This ciliar conductance g_c had its reversal potential at 0 mV. Replacing extracellular Cl^- by isethionate on the one hand, and Na⁺ by Cs⁺ or N-methyl-D-glucamine on the other showed that g_c was permeable for cations but not for Cl^-. In conclusion, there appears to be a Ca²⁺-dependent unselective cation conductance on the cilia of olfactory neurones. The probable role of g_c as the last step in an IP₃/Ca mediated transduction path-way is suggested.     

Pathways of NH3/NH 4 + permeation across Xenopus laevis oocyte cell membrane

While acid loading with extracellular NH₄Cl solutions usually first alkalinizes the cells through NH₃ influx, and acidifies only when NH₄Cl is removed, Xenopus oocytes became immediately acidic upon NH₄Cl addition and the cells did not acidify further upon its removal. Since NH₄Cl solutions also collapsed the membrane potential (V _m) and resistance (R _m), we conclude that primarily NH ₄ ⁺ entered the cells where it liberated H⁺, with NH₃ being trapped in intracellular lipid stores. To identify the NH ₄ ⁺ permeation pathway we have used K⁺ channel blockers (Ba²⁺, Cs⁺, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La³⁺, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of V _m and R _m. This suggests, that NH ₄ ⁺ passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH ₄ ⁺ fluxes appeared to be asymmetric. NH ₄ ⁺ influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.     

Pharmacological and Physiological Characterization of Murine Homomeric β3 GABAA Receptors

γ-Aminobutyric acid (GABA_a) receptor β3 subunits were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp. Injected oocytes exhibited an increased resting membrane conductance and more depolarized membrane potentials compared to uninjected control cells. Oocytes expressing β3 subunits were insensitive to GABA and muscimol, but pentobarbitone increased the membrane conductance in a concentration-dependent manner. The membrane current response to pentobarbitone reversed at the Cl^?equilibrium potential and at relatively high concentrations (>500 μM), a rebound CI^? current was induced following the removal of pentobarbitone. In transfected human embryonic kidney (HEK) cells, the rebound current amplitude was reduced by desensitizing the β3 receptor with increased durations of ligand application. Both picrotoxin (0.5 nM to 10 μM) and Zn²⁺ (10 nM to 100 μM) reduced the resting membrane conductance for β3 cDNA-injected oocytes. These oocytes were insensitive to flurazepam (5 μM) and alphaxalone (10 μM), but responded with increased membrane conductance to propofol (10 μM) and pregnanolone (50 nM to 5 μM). The antagonists, bicuculline (10 μM) and strychnine (50 nM to 100 μM), also induced conductance increases in a concentration dependent manner; however, glycine (1 mM) was inactive. It was concluded that β3 subunits form spontaneously opening ion channels that can be up-regulated by some allosteric modulators, principally by pentobarbitone and propofol and, surprisingly, by bicuculline and strychnine, whilst picrotoxin and Zn²⁺ acted as antagonists. Computer modelling of some kinetic schemes was used to describe the rebound current observed in transfected HEK cells. This indicated that pentobarbitone, after modulation of the conductance, is potentially capable of further binding to the β3 receptor complex ‘driving’ the receptor into one or more desensitized states. This phenomenon may be of some importance for native neuronal GABA_A receptors, where pentobarbitone can also evoke rebound current activation.