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PubMed1999

Retinal ganglion cell dysfunction induced by hypoxia and glutamate: potential neuroprotective effects of beta-blockers.

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2-50 microM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 microM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 microM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

Action Potentials↗
PubMed2002

Relative contributions of bipolar cell and amacrine cell inputs to light responses of ON, OFF and ON-OFF retinal ganglion cells.

Light-evoked postsynaptic currents (lePSCs) were recorded from ON, OFF and ON-OFF ganglion cells in dark-adapted salamander retinal slices under voltage clamp conditions, and the cell morphology was examined using Lucifer yellow fluorescence with confocal microscopy. The current-voltage relations of the lePSCs in all three types of ganglion cells are approximately linear within the cells' physiological range. The average chloride/cation conductance ratio (Deltag(Cl)(NR)/Deltag(C)(NR)) of the lePSCs is near 3, suggesting that ganglion cell light responses are associated with a greater postsynaptic conductance change at the amacrine-ganglion cell inhibitory synapses than at the bipolar-ganglion cell excitatory synapses. By comparing the charge transfer of lePSCs in normal Ringer's and in picrotoxin+strychnine+Imidazole-4-acidic acid, we found that the GABAergic and glycinergic amacrine-bipolar cell feedback synapses decreased the light-induced glutamatergic vesicle release from bipolar cells to all ganglion cells, and the degree of release reduction varied widely from ganglion cell to ganglion cell, with a range of 3-28 fold.

Ambystomatidae↗
PubMed1998

Amino acid neurotransmitters in the retina: a functional overview.

Physiological and pharmacological mechanisms of glutamatergic, GABAergic and glycinergic synapses in the tiger salamander retina were studied. We used immunocytochemical and autoradiographic methods to study localizations of these neurotransmitters and their uptake transporters; and electrophysiological methods (intracellular, extracellular and whole cell patch electrode recordings) to study the light responses, miniature postsynaptic currents and neurotransmitter-induced postsynaptic currents in various retinal neurons. Our results are consistent with the following scheme: Glutamate is used by the photoreceptor and bipolar cell output synapses and the release of glutamate is largely mediated by calcium-dependent vesicular processes. The postsynaptic glutamate receptors in DBCs are L-AP4 receptors, in HBCs, HCs and ganglion cells are the kainate/AMPA and NMDA receptors. Subpopulations of HCs make GABAergic synapses on cones and gate chloride condunctance through GABAA receptors. GABAergic HCs do not make feedforward synapses on bipolar cell dendrites and the neurotransmitter identity of the HCs making feedforward synapses is unknown. Subpopulations of amacrine cells make GABAergic synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and GABA gates chloride conductances in theses cells. Glycinergic amacrine cells make synapses on bipolar cell synaptic terminals, other amacrine cells and ganglion cells and glycine opens postsynaptic chloride channels. Glycinergic interplexiform cells make synapses on bipolar cells in the outer retina and glycine released from these cells open chloride channels in bipolar cell dendrites.

Ambystoma↗
PubMed1999

Endothelial nitric oxide synthase (eNOS) is localized to Müller cells in all vertebrate retinas.

The distribution of endothelial nitric oxide synthase immunoreactivity (eNOS-IR) was investigated in the retinas of all phylogenetic vertebrate classes by using a monoclonal eNOS antibody. Confocal light microscopy showed immunoreactive labeling in Müller cells of fish, frog, salamander, turtle, chicken, rat, ground squirrel, and monkey retina. In vascularized retinas (rat, monkey), astrocytes and some blood vessels were also stained. Furthermore, eNOS-IR was localized to axon terminals of turtle and fish horizontal cells. These observations are the first to show the presence of eNOS-IR in Muller glia and horizontal cell structures of the vertebrate retina.

Animals↗
PubMed1980

Microperoxisomes and catalase peroxidatic activity in the pancreas of two amphibian species (Salamandra salamandra L. and Rana esculenta L.).

The fine structure of microperoxisomes (anucleoid peroxisomes) and catalase peroxidatic activity in salamander and frog pancreas have been investigated following glutaraldehyde fixation and incubation in a modified NOVIKOFF-GOLDFISCHER alkaline DAB-medium. Reactive microperoxisomes ranging in diameter from 0.16 to 0.76 micrometer were identified in both exocrine (acinar and ductular centro-acinar cells) and insular (B-, A-, D-cells) pancreatic tissue, as well as in the nerve fibers and endings. Their incidence was considerably higher in the exocrine parenchyma, where sometimes in frog they accumulated in clusters. Individual microperoxisomes showed close spatial relationship and occasionally membrane continuities with smooth surfaced endoplasmic reticulum and zymogen granules. They were also localized in the proximity or in direct contact with the nuclear envelope, mitochondria, GOLGI condensing vacuoles, lipid inclusions and plasma membrane. The morphological findings support the microperoxisomes origin from the agranular endoplasmic reticulum and their possible discharge into the extracellular spaces through an exocytosis-like mechanism. The cytochemical observations are discussed in relation with the concepts on catalase biosynthesis and peroxisomes functional significance.

Animals↗
PubMed1985

Cytochemical study of K+-dependent p-nitrophenyl-phosphatase activity in the urinary bladder of Salamandra salamandra.

The ultrastructural Na+-K+-ATPase localization in the salamander urinary bladder has been studied by the histochemical techniques of Ernst (1972) and Mayahara et al. (1980). Reaction products have been specially found on the basal and lateral membranes of granular and mitochondria-rich cells. The variable presence of artifacts, often accompanying Pb-capture phosphatase cytochemistry, is discussed. Additional data on the active ion transport have been obtained by using electrophysiological techniques.

4-Nitrophenylphosphatase↗
PubMed1986

Light and electron microscopic study on complex carbohydrates in the testis of Salamandra salamandra L. (Amphibia, Urodela).

The distribution of complex carbohydrates was studied in the testis of the European fire salamander, Salamandra salamandra, by light- and electron-microscopic methods. The basal laminae and fibrous structures in the connective tissue between the lobules are PAS-positive. After alcianblue staining (at pH = 2.8), acid mucopolysaccharides could be demonstrated in steroid hormone-producing cells in the interstitial tissue between lobules containing spermatids, spermatozoa, and lobules after spermiation, as well as in most of the Sertoli cells in lobules after spermiation. In all spermatogenic stages from secondary spermatocytes to mature sperms, dictyosome-like structures and flat vesicles showed a distinct contrast enhancement, as did parts of the acrosome after treatment with the phosphotungstic acid-chromic acid method for electron microscopy.

Acrosome↗
PubMed1998

Patch-clamp, ion-sensing, and glutamate-sensing techniques to study glutamate transport in isolated retinal glial cells.

We have described how a combination of electrical, ion-sensing, and glutamate-sensing techniques has advanced our understanding of glutamate uptake into isolated salamander retinal glial cells. The next steps in understanding glutamate transport will inevitably depend strongly on molecular biological methods, as described elsewhere in this book, but will also require more detailed study of transporters in their normal environment, perhaps by using patch-clamping or imaging techniques to study cells in situ.

ATP-Binding Cassette Transporters↗
PubMed2004

How the mesencephalic locomotor region recruits hindbrain neurons.

This chapter summarizes experiments which were designed to reveal how repetitive electrical stimulation of the mesencephalic locomotor region (MLR) recruits nearby hindbrain neurons into activity, such that locomotion can ensue in the tiger salamander, A. tigrinum. The MLR stimulus strength was subthreshold or near-threshold for locomotor movements to ensue. Such relatively weak stimulation of the MLR produced locomotor movements after a relatively long delay, which featured neuronal interactions in the hindbrain. MLR-evoked spike responses of single hindbrain neurons were recorded before locomotor movements began. This allowed consideration of the build-up of the hindbrain neuronal activity, which was subsequently impressed upon the spinal cord such as to evoke locomotor movements. Each train of MLR stimulus pulses evoked monosynaptic responses in but a small proportion of the hindbrain's neurons. Rather, oligosynaptic responses were routinely evoked, even in the "input" neurons that were activated monosynaptically. Consecutive stimulus volleys recruited a given neuron after a variable number of synaptic translations. It is argued that the hindbrain's input neurons excited a much larger number of other hindbrain neurons. By this means, an MLR-evoked, short-lived propagating wave of excitation (i.e., approximately 2-4 successive synaptic activations) can be spread throughout the hindbrain.

Ambystoma↗
PubMed2002

An alternative internal splicing site defines new Ikaros isoforms in Pleurodeles waltl.

The Ikaros gene encodes a family of transcription factors which plays a crucial role in hematopoiesis. To improve our knowledge about the immune system of Pleurodeles waltl, we sequenced the cDNA coding for the Ik-1 isoform of that salamander and analyzed its tissue expression by semi-quantitative RT-PCR. Ikaros transcripts are abundant in the thymus and the spleen, thereby confirming that these organs are, respectively, the primary and secondary lymphoid tissues of Pleurodeles. Analysis of the isoforms produced by this animal revealed two isoforms characteristic of amphibians in which an alternative internal splicing site deletes the 3' half of exon 3 which interestingly comprises the first Zn finger of Ikaros. Ikaros transcripts were found at the earliest stages of development of Pleurodeles indicating that Ikaros has a function at the very early lymphopoietic stages. Moreover, the presence of Ikaros transcripts in spermatozoa suggests that this protein could have another and yet unknown function.

Alternative Splicing↗
PubMedDate not supplied

Noradrenergic and peptidergic innervation of the amphibian spleen: comparative studies.

Spleens from representatives of the three amphibian orders were examined using sucrose-potassium phosphate-glyoxylic acid (SPG) histofluorescence to detect catecholamines and immunocytochemistry to detect several neural antigens. Nerve fibers are scattered throughout the spleens of adult salamanders (Taricha torosa, Notophthalmus viridescens, and Ambystoma mexicanum). A less abundant but similarly diffuse pattern of innervation characterizes the spleen of the caecilian, Typhlonectes sp. The spleen of the adult frog, Xenopus laevis, is separated into clearly defined compartments of red pulp and white pulp, much as is seen in the mammalian spleen. As in mammals, sympathetic innervation of the Xenopus spleen is noradrenergic (NA) and confined to the white pulp. The white pulp of Xenopus spleen also contains fibers which stain for neuropeptide Y and substance P. The spleen of the anuran, Rana pipiens, is also highly compartmentalized, with tyrosine hydroxylase positive fibers in proximity to blood vessels. These findings provide an anatomical substrate for neural-immune interactions in the Amphibia.

Amphibians↗
PubMedDate not supplied

Axolotl (Ambystoma mexicanum) lymphocytes produce and are growth-inhibited by transforming growth factor-beta.

Recombinant (r)TGF-beta 5, an isoform of TGF-beta thus far identified only in the frog, Xenopus' laevis, inhibited phytohemagglutinin (PHA)-induced mitogenesis of salamander (axolotl) splenocytes and thymocytes, and T cell growth factor-induced proliferation of splenic lymphoblasts. This inhibition could be reversed by incubating (r)TGF-beta 5 with an anti-TGF-beta 5 antibody, but not with an antibody directed against TGF-beta 2, another Xenopus-produced TGF-beta isoform. Acid-treated supernatants from cultures of PHA-stimulated axolotl splenocytes (PHA-SNs) inhibited proliferation of mink lung fibroblasts (MLF), cells whose growth is sensitive to all known isoforms of TGF-beta. This inhibition was reversed by incubating the supernatants with the anti-pan-specific TGF-beta antibody which neutralizes TGF-beta 1, TGF-beta 2, TGF-beta 3 and TGF-beta 5. This inhibition was unaffected by antibodies that specifically neutralize either TGF-beta 1, TGF-beta 2, TGF-beta 3 or TGF-beta 5. Acid-treated axolotl PHA-SNs also inhibited proliferation of PHA-stimulated freshly harvested axolotl spleen cells. Once again, this inhibition could be reversed by treating supernatants with an anti-pan-specific TGF-beta antibody but not with anti-TGF-beta 2 or anti-TGF-beta 5 antibodies. All these data are consistent with the hypothesis that axolotl lymphocytes secrete a previously unknown TGF-beta isoform.

Ambystoma↗
PubMedDate not supplied

Characterization of beta-adrenergic receptors in fish and amphibian lymphoid organs.

Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity beta-adrenergic receptors specific for beta-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian beta1 and beta2-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another beta-AR subtype, or a serotonergic receptor.

Amphibians↗
PubMed2000

cDNA sequence and tissue-specific expression of a basic liver-type fatty acid binding protein in adult zebrafish (Danio rerio).

We have determined the nucleotide sequence for a cDNA clone derived from zebrafish (Danio rerio) that codes for a fatty acid binding protein (FABP). Amino acid sequence similarity of the zebrafish FABP was highest to FABPs isolated from the livers of catfish, chicken, salamander and iguana. The open-reading frame of the zebrafish FABP cDNA codes for a protein of 14.0 kDa with a calculated isoelectric point of 8.8. The zebrafish liver-type FABP (L-FABP) cDNA hybridized to single restriction fragments of total zebrafish genomic DNA digested with the restriction endonucleases HaeIII or EcoRI suggesting that a single copy of the L-FABP gene is present in the zebrafish genome. Northern blot analysis demonstrated that the zebrafish L-FABP cDNA hybridized to a transcript of 700 nucleotides in total zebrafish RNA. In situ hybridization and emulsion autoradiography revealed that the L-FABP was expressed exclusively in the liver of the adult zebrafish. Based on amino acid sequence similarity, the isoelectric point and its tissue-specific pattern of expression, we conclude that this zebrafish FABP belongs to the basic liver-type FABPs only found, thus far, in non-mammalian vertebrates.

Amino Acid Sequence↗
PubMed2000

The mechanics of air-breathing in anuran larvae: implications to the development of amphibians in microgravity.

Because of their rapid development, amphibians have been important model organisms in studies of how microgravity affects vertebrate growth and differentiation. Both urodele (salamanders) and anuran (frogs and toads) embryos have been raised in orbital flight, the latter several times. The most commonly reported and striking effects of microgravity on tadpoles are not in the vestibular system, as one might suppose, but in their lungs and tails. Pathological changes in these organs disrupt behavior and retard larval growth. What causes malformed (typically lordotic) tadpoles in microgravity is not known, nor have axial pathologies been reported in every flight experiment. Lung pathology, however, has been consistently observed and is understood to result from the failure of the animals to inflate their lungs in a timely and adequate fashion. We suggest that malformities in the axial skeleton of tadpoles raised in microgravity are secondary to problems in respiratory function. We have used high speed videography to investigate how tadpoles breathe air in the 1G environment. The video images reveal alternative species-specific mechanisms, that allow tadpoles to separate air from water in less that 150 ms. We observed nothing in the biomechanics of air-breathing in 1G that would preclude these same mechanisms from working in microgravity. Thus our kinematic results suggest that the failure of tadpoles to inflate their lungs properly in microgravity is due to the tadpoles' inability to locate the air-water interface and not a problem with the inhalation mechanism per se.

Animals↗
PubMed2000

Emesis and space motion sickness in amphibians.

Amphibians possess the ability to vomit in response to a variety of stimuli that provoke emesis in mammals. Pharmacological studies have establish that the ejection of gastric contents and the basic mechanism for vomiting have been phylogenetically conserved among these tetrapods. As part of on-going comparative studies on emesis in vertebrates, we previously documented that some postmetamorphic anurans and salamander larvae experience motion-induced emesis when exposed to the provocative stimulus of parabolic aircraft flight. However, more recent experiments suggest that there are strict conditions for inducing emesis in amphibians exposed to parabolic flight and that amphibians are not as sensitive to this stimulus as mammals. Further studies on emesis in lower vertebrates may help us understand the processes that cause emesis in abnormal gravitational regimes.

Amphibians↗
PubMed2000

Allozymic variation of Hynobius kimurae Dunn (Amphibia, Caudata).

An electrophoretic survey was conducted to examine genetic divergence among 21 populations of a lotic-breeding salamander Hynobius kimurae from Honshu, Japan. Genetically H. kimurae proved to be specifically distinct from H. naevius. Hynobius kimurae is divided genetically into two groups of local populations, and the five populations from the eastern area are genetically distinct from the remaining populations of the central and western areas. Less prominent genetic differentiation was revealed between the western and central populations. Separation of the eastern and central-western groups are discussed in relation to the formation of the Japanese mainland.

Animals↗