Ultrastructure and immunocytochemistry of the neuroepithelial bodies in the lung of the tiger salamander, Ambystoma tigrinum (Urodela, Amphibia).

Light and electron microscopy of the lungs of Ambystoma tigrinum (Urodela) revealed a relatively complex pattern of the neuroendocrine (NE) cells. In the apical parts of smaller septa single NE cells not associated with nerve fibres were covered and surrounded by pneumocytes. The larger septa possessed small areas of ciliated epithelium, in which the NE cells were grouped in a form of neuroepithelial bodies (NEB) consisting of 3-5 cells and covered by goblet cells. NE cells possessed a large nucleus with patches of condensed chromatin, clear cytoplasm, and membrane-bound vesicles of variable morphology and size, containing an electron dense interior surrounded by a lucent space. The size of these dense core vesicles (DCV) ranged from 70-140 nm, while rarely the larger ones exhibited a diameter of 300-600 nm. In some NEB a second type of NE cells was observed for the first time in an amphibian species: these cells communicated with the air space and exhibited on their surface microvilli and a single modified cilium with a 8 + 1 microtubule arrangement. Their cytoplasm contained two types of DCV: dense core granules with a diameter of 140-260 nm and vesicles 320-700 nm in diameter with a moderately electron dense interior. The NEB were associated with intracorpuscular, sensory nerve terminals morphologically afferent and efferent. By immunocytochemistry, the NE cells revealed the presence of serotonin, met-enkephalin, and leu-enkephalin. A paracrine and chemoreceptor role is proposed for NEB of Ambystoma tigrinum.     

Immunolocalisation of nitric oxide synthase isoforms in the epidermis of the tiger salamander, Ambystoma tigrinum

A. tigrinum: Immunoreactivity for isoforms of nitric oxidase synthase is found in the flash cells and outer-deep epidermal cell layers of the tiger salamander A. tigrinum. Despite the absence of physiological data we assume NO may be lumped together as cytocrine regulators in the amphibian epidermis.     

The projection from the olfactory epithelium to the olfactory bulb in the salamander, Ambystoma tigrinum

Summary Odor quality may be represented as a topographic code of responses of receptor cells throughout the olfactory epithelium, with this code conveyed to the central nervous system by a topographic projection from the olfactory epithelium to the olfactory bulb. There is good evidence for topographic differences in odor-induced receptor cell activity in the tiger salamander but there is no evidence for a topographic epithelium-to-bulb projection in this species. In the present study ³H-leucine autoradiography was used to trace the projections of olfactory receptor neurons in the tiger salamander. Thirteen animals received small injections of tritiated leucine into different regions of the dorsal or the ventral olfactory epithelium, or into the ventrolateral, vomeronasal organ. The results show that the anterior-to-posterior axes in the dorsal and ventral epithelia are represented along the ventral-to-dorsal axis in the rostral end of the olfactory bulb. The vomeronasal organ projects to the caudal end of the bulb. We conclude that the central projection of the olfactory epithelium in the tiger salamander is topographically organised only along the antero-posterior axis and not the medio-lateral axis. Thus epithelial receptor cell activity along the antero-posterior axis would be represented in the glomerular layer of the bulb by activity along its ventro-dorsal axis.     

Regional differences in cell density and cell genesis in the olfactory epithelium of the salamander,Ambystoma tigrinum

Summary The olfactory epithelium undergoes continuous regeneration. The present quantitative study uses tritiated thymidine autoradiography to investigate regional differences in the rate of olfactory epithelial cell genesis in the tiger salamander. There was a significant gradient in the incorporation of thymidine from the posterior to the anterior in the nasal cavity: the posterior epithelium underwent cell genesis much faster than the anterior. Additionally, the posterior epithelium was thinner and contained fewer cells than the anterior, although the proportions of receptor, supporting and basal cells remained about the same throughout the epithelium. After 5 or 20 days most of the labelled cells were found in the basal cell layer, although there were a few labelled supporting cells. This confirms observations in other species that there are two populations of dividing cells in the olfactory epithelium: the basal cells which give rise to receptor cells, and the supporting cells. The gradients in epithelial thickness, receptor cells, and the rate of cell genesis parallel a gradient in responsiveness to odorants observed in electrophysiological studies (Mackay-Sim et al. 1982; Mackay-Sim and Shaman 1984). The significance of these anatomical and physiological gradients is presently unclear.     

A light and scanning electron microscopic study of the basilar papilla in the salamander,Ambystoma tigrinum

The basilar papilla and basilar recess of Ambystoma tigrinum have been investigated by light and scanning electron microscopy. The recess is an evagination of the lagena, and is invested externally by dense periotic connective tissue, except over a thin area of one wall abutting against a periotic diverticulum communicating with the periotic sac. The surface and histological features of the non-sensory lining epithelium are described. The basilar papilla occupies both slopes of an elevation adjacent to the thin wall of the recess, and consists of 40 to 80 sensory cells interposed between sustentacular cells. The sensory cells are innervated by 30 to 40 nerve fibers exhibiting two ranges of diameter, and they are capped by typical ciliary bundles that are taller at the center of the receptor than at its periphery. Bundles in the proximal and distal halves of the papilla are polarized, respectively, toward the saccule and toward the thin wall in contact with the periotic diverticulum; this divergent pattern of polarization has not been reported previously in the basilar papilla of other vertebrates. A tectorial body overlies only the bundles in the distal half of the receptor, and is attached to both the neuroepithelium and the opposite wall of the recess. Functional considerations are discussed, and comparisons are made with conditions reported in frogs and toads. The findings suggest that the basilar papilla and recess in caudate and anuran amphibians arose from common precursors and probably function in a similar manner.     

Reproducible proliferative responses of salamander (Ambystoma mexicanum) lymphocytes cultured with mitogens in serum-free medium.

There are several reports that proliferative responses (tritiated thymidine incorportation (^3HTdR)) of salamander splenocytes cultured with phytohemagglutinin-P (PHA) or concanavalin A (Con A) in 1% fetal bovine serum (FBS)-supplemented medium are either statistically insignificant or never approach the magnitude typically observed in similarly treated cultures of frog lymphocytes. The present study confirms these findings, but also reports highly significant and reproducible PHA-induced proliferation of axolotl splenocytes and thymocytes when the medium is supplemented with 0.25% bovine serum albumin (BSA) rather than 1% FBS. In one study, splenocytes from six of six axolotls cultured in BSA-supplemented medium displayed a dose-dependent response to PHA with stimulation indices (SLs) ranging from 4.2 to 14.1. In contrast, SLs of PHA-treated cells from the same animals, cultured in parallel in FBS-supplemented medium, ranged from 0.8 to 3.0. In a kinetic study (cells harvested from days 3–7), maximal proliferation in BSA-supplemented medium was noted after 5 days; cells cultured in parallel in FBS-containing medium were not responsive to the mitogen at any time point. Although axolotl splenocytes do not exhibit PHA-stimulated growth in FBS-supplemented medium, they are reproducibly stimulated in this serum-containing medium by phorbol 12-myristate, 13-acetate (PMA). This suggests that FBS may interfere with (or does not support) some early step(s) in lectin-induced signalling, rather than with proliferation itself.     

Discrimination and generalization among reagent grade odorants by tiger salamanders (Ambystoma tigrinum)

Using an intense light as a negative reinforcer, two groups of tiger salamanders (Ambystoma tigrinum) were trained to respond to odorant-air mixtures of butyl acetate (8.9×10^?5M) or butyl alcohol (6.7×10^?5M). Then, they were given discrimination tests on which butyl acetate and butyl alcohol were presented successively, and generalization tests on which animals trained with butyl acetate were tested for generalization with propyl and amyl acetate, and the animals trained with butyl alcohol were tested with propyl and amyl alcohol. The salamanders then received either olfactory bulbectomies or sham surgeries. Post-surgical trials were given using the training odorant stimulus. Results indicated that salamanders discriminated between butyl acetate and butyl alcohol and that they generalized among acetates or alcohols chemically similar to their respective training odorant but not to filtered air. The immediate loss of conditioned response following bulbectomy but not sham surgery suggests that indeed olfactory cues were conditioned stimuli during the preceding phases of the experiment. Overall, these results are consistent with electrophysiological studies of olfaction in these animals.     

Nitric oxide in the amphibian (Ambystoma tigrinum) lateral line

Nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d) histochemistry was investigated in the axolotl (Ambystoma tigrinum) lateral line. Hair cells of neuromast organs of the head skin and neurons of the postotic ganglia showed a significant NADPH-d reaction. Multiunit recording of neuromast afferent activity was also performed. Nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) produced an initial slight excitation followed by a significant inhibition of the resting discharge of neuromast afferent neurons. In contrast N(G)-nitro-L-arginine (L-NOARG) produced non-significant actions on the afferent neurons discharge. These findings suggest that afferent neurons and hair cells of the lateral line produce nitric oxide that plays an active role in the mechanisms sustaining basal spike discharge in afferent neurons.     

Heterogeneity in the distal nephron of the salamander (Ambystoma tigrinum): A correlated structure function study of isolated tubule segments

Studies on isolated perfused tubules of the tiger salamander (Ambystoma tigrinum) have shown that the distal nephron is heterogeneous with respect to function (Stoner, 1977). In this study, the initial portion of the distal tubule (diluting segment) exhibited a voltage, positive in the lumen, and a net absorption of chloride. Since the chloride was transported against an electrochemical gradient, its transport was active. More distad, the junctional segment exhibited a lumen-negative voltage and sodium, rather than chloride, was transported actively. More recently Delaney and Stoner (1981) have demonstrated in vitro that the collecting duct of this species also has a lumen-negative voltage which is probably associated with active sodium reabsorption. The primary objective of the present paper was to correlate the morphology of the diluting and junctional segments of the Ambystoma distal tubules with the physiologic data from the same isolated perfused tubules. The results indicate that the morphological heterogeneity previously demonstrated in distal tubules of Necturus exists with respect to both structure and function in Ambystoma. The cell types found in the amphibian distal nephron appear to be homologous to those seen in the mammalian nephron.     

Neuroepithelial bodies in the lung of Melanophryniscus stelzneri stelzneri (Anura, Bufonidae)

Electron microscopy of the lungs of Melanophryniscus stelzneri stelzneri (Anura) revealed the presence of a complex pattern of corpuscular cells (CCs). The respiratory surface over the septa presents small areas where the CCs are grouped forming neuroepithelial bodies (NEBs). These corpuscular structures can also be localized in the inner layer of the lung wall. Although in both cases NEBs protrude slightly into the airway lumen, they are separated from the airway lumen and the basal connective tissue by thin apical and basal cytoplasmic processes of neighbouring pneumocytes. Ultrastructurally, the CCs possess a large nucleus, clear cytoplasm and vesicles of variable morphology and size, containing an electron dense material surrounded by a lucent space in some cases. The size of these dense-core vesicles (DCVs) ranged from 40 to 100 nm. The NEBs are associated with afferent and efferent terminal nerves. These types of nerve endings are located between the CCs and in the basal part of the NEBs. The location of the NEBs in strategic positions on the septa and in the wall of the lung, the presence of the DCVs in the basolateral region of the CCs, the occurrence of synaptic contacts between nerve endings and the CCs and the occurrence of capillaries close to the NEBs, suggest a receptosecretory function for NEBs in the lung of M.s. stelzneri.     

Osteological characteristics of the Setouchi salamander Hynobius setouchi (Urodela,Hynobiidae)

This study described the detailed osteological features of the Setouchi salamander Hynobius setouchi. H. setouchi, which is endemic to central Japan, was recently delineated from the wide range of H. nebulosus species based on molecular and external morphological characteristics. However, the osteological features of the species belonging to the genus Hynobius have not been examined, which has hindered the detailed understanding of morphological diversity in the genus. To address this problem, this study elucidated the osteological features of the salamander using micro-computed tomography scanning and whole-mount double staining methods. The articular, a part of the hypobranchial I, a part of coracoid, pubis, carpus, and tarsus were cartilaginous in small-sized individuals and were partially or completely ossified in large-sized individuals. The degree of ossification varied in the operculum, basibranchial II, and ascending process of the palatoquadrate. However, ossification was not associated with body size. The lacrimal, ossified manus, and ossified pes exhibited varying degrees of articulation or fusion irrespective of body size. Moreover, the cranium and tarsus of H. setouchi exhibited several unique characteristics. These osteological characteristics will aid in revising the taxonomy and phylogeny of the Hynobius species.     

Intracellular injection of vital dyes into single cells in the salamander olfactory epithelium

Intracellular vital dye injection was used to examine the morphology of single sustentacular and receptor cells and the developmental fate of individual basal cells in the olfactory epithelium of the tiger salamander. In acute experiments, Lucifer yellow injections were used to identify single basal, receptor or sustentacular cells on the basis of their overall morphology. Dye-coupling between a number of the different epithelial cells was observed. Progeny of basal cells were examined by following labeled cells for up to 2 weeks using intracellular injection of rhodamine-labeled dextran. These experiments indicate that some olfactory epithelial cells are dye-coupled and that dye-filled basal cells can undergo division and migration.     

Temporary and selective anosmia in tiger salamanders (Ambystoma tigrinum) caused by chemical treatment of the olfactory epithelium

Tiger salamanders (Ambystoma tigrinum) were trained to respond to two dissimilar odorants (i.e., cyclohexanone, dimethyl disulfide) but not to a third odorant (n-butanol). When the training criterion was met, the animals were anesthetized and given nasal lavages of saline or odorants in saline. Lavage with cyclohexanone decreased responding to cyclohexanone but not dimethyl disulfide, and vice-versa, in later behavioral tests. Likewise, ethyl acetoacetate lavage produced selective response decrements to cyclohexanone. Lavage with n-butanol had no effect on responding to presentations of either cyclohexanone or dimethyl disulfide. The effects of lavage increased with increasing odorant concentration. Thus, lavage with 0.05 M cyclohexanone or ethyl acetoacetate produced more persistent response decrements than lavage with 0.01 M concentrations of either odorant. Such results are consistent with several testable interpretations, including the possibility the response decrements could reflect the nature of odorant-receptor interactions.     

Ultrastructure and innervation of neuroepithelial bodies in the lungs of newborn cats

Neuroepithelial bodies (NEB) occur throughout the airway mucosa and alveolar parenchyma of kitten lungs. In the bronchi, they are often situated on top of a cartilage plate. They form compact corpuscles containing 10–20 corpuscular cells and appear covered with a layer of flattened Claracells. Kitten NEB are occasionally observed to display mitosis of the corpuscular epithelial cells. A prominent blood capillary lies at their basal pole. The corpuscular cells contain numerous dense core vesicles (DCV), whose number and diameter remain unchanged with age. Kitten NEB are innervated by nerve fibres that “loop” through the corpuscle and form morphologically afferent as well as efferent nerve endings. The nerve endings display afferent synaptic junctions with the corpuscular cells and sometimes run in clusters, so that they contact each other. Many nerve endings undergo spontaneous degeneration. We conclude that kitten NEB are well adapted to function as chemoreceptors and as endocrine or paracrine organs. Their chemoreceptor activity could be modulated by axon reflexes since their afferent nerve endings are often continuous with the efferent ones, as well as by interneural modulation since nerve endings often form clusters. In addition, kitten NEB innervation appears to involute rapidly soon after birth. This may indicate that their chemoreceptor function is only of primary importance during gestation and at birth. However, the secretory function of kitten NEB, as evidenced by the unchanged numbers and dimensions of their DCV, seems to remain steady throughout life.© 1993 Wiley-Liss, Inc.     

Organization of the serotoninergic system in the brain of two amphibian species,Ambystoma mexicanum (Urodela) and Typhlonectes compressicauda (Gymnophiona)

An immunocytochemical investigation was made of the distribution of serotonin (5-HT) in the brain of larval and adult Ambystoma mexicanum and adult Typhlonectes compressicauda. Immunoreactive perikarya can be identified in the caudal diencephalon (paraventricular organ and infundibular nucleus), in the ventral mesencephalon (interpeduncular nucleus) and in the raphe of the rhombencephalon. Immunopositive fibers and terminal arborizations are widely distributed, extending from the whole telencephalon to the spinal lemniscus area. However, the retinorecipient structures of the thalamus and mesencephalon are either very weakly innervated (Ambystoma) or completely immunonegative (Typhlonectes). The habenular system also exhibits very few 5-HT-positive structures. The major serotoninergic neuron clusters, in both Urodela and Gymnophiona, tend to gather, from the paraventricular organ to the raphe, on both sides of the sagittal plane, showing no tendency to “lateralization”. A new interpretation of the limited development of the serotoninergic system in amphibians is given.     

Voltage sensitive,high-conductance chloride channels in the luminal membrane of cultured pulmonary alveolar (type II) cells

The properties of ion channels in patches from the luminal membrane of cultured monolayers of type II alveolar epithelial cells from adult rat lung have been studied. In excised patches, prepared as described to yield the outside-out configuration (with cesium ions in the bathing solutions to suppress currents arising from potassium channels), we observed anion-selective channels with a conductance of 350–400 pS, and burst lengths lasting seconds. When patches were bathed in solutions with equal chloride concentrations, channels opened and closed spontaneously at membrane voltages close to zero, but tended to close when the potential was shifted by ±10 mV, particularly in the negative direction. Other anions could pass through these channels with a permeability sequence of I(1.5)>Br(1.02)=>Cl(1.00)>NO₃(0.9)>gluconate(0.6) but there was a very low permeability for sodium (P _Na/P _Cl=0.015±0.006). Calcium ions reduced channel conductance. The channels probably have a role to play in salt absorption by this epithelium, but insufficient information is available about how sodium crosses it for the role of the anion channels in transport to be defined with confidence.