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PubMed1982

A fine-structural study of interstitial cell changes in the testes of Necturus maculosus during a portion of the annual cycle, and possible evidence for local feedback control by seminiferous epithelium.

The developmental stages of the interstitial cells of Leydig in Necturus maculosus were studied in testes of animals obtained in November and December. As a consequence of the caudal to cranial progression of the wave of spermatogenesis, samples obtained from cranial, central, and caudal portions of these testes exhibit regional differences in the morphology of their Leydig cells. In these samples, fibroblast-like stromal cells with large, dense, elongated nuclei and thin sheets of cytoplasm surround the cranial seminiferous lobules that usually contain spermatozoa. Such stromal cells possess a small juxtanuclear Golgi apparatus, profiles of smooth and rough endoplasmic reticulum, and mitochondria with tubular and shelf-like cristae. In the central area of the testis, interstitial cells with oval nuclear profiles and a finer chromatin pattern surround degenerating seminiferous lobules. The abundant cytoplasm of these cells contains predominantly smooth endoplasmic reticulum interspersed with a few lipid droplets. These cells contain mitochondria that are packed with tubular cristae. Inthe caudal part of the testis, the Leydig cells have a round nucleus with finely dispersed chromatin. Numerous lipid droplets accumulate in the ample cytoplasm, which is filled with smooth endoplasmic reticulum. The juxtanuclear Golgi apparatus becomes enlarged; associated with it are vesicles with an electron-dense content. The cristae of the mitochondria are more numerous and have tubules of 50-nm diameter mixed with arrays of small tubules 25-30 nm in diameter. The appearance of morphologically mature interstitial cells of Leydig only adjacent to degenerated seminiferous lobules inthe caudal portion of the testis suggests the possibility of local feedback control of Leydig-cell development.

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PubMed2004

Morphology of brachial segments in mudpuppy (Necturus maculosus) spinal cord studied with confocal and electron microscopy.

The isolated brachial spinal cord of Necturus maculosus is useful for studies of neural networks underlying forelimb locomotion, but information about its cellular morphology is scarce. We addressed this issue by using confocal and electron microscopy. Remarkably, the central region of gray matter was aneural and consisted exclusively of a tenuous meshwork of glial fibers and large extracellular spaces. Somata of motoneurons (MNs) and interneurons (INs), labeled by retrograde transport of fluorescent tracers from ventral roots and axons in the ventrolateral funiculus, respectively, were confined within a gray neuropil layer abutting the white matter borders, whereas their dendrites projected widely throughout the white matter. About one-third of labeled INs were found contralaterally, with axons crossing ventral to a thick layer of ependyma surrounding the central canal. Lateral MN dendrites proliferated under the pial surface to form a dense, thin (1-2 microm) plexus immediately beneath a thin layer of glial fibrillary acidic protein-positive glia limitans. The latter contained arrays of unusual tubular structures (diameter 200-400 nm, length 3 microm) that resembled mitochondria but lacked double membranes or cristae. Dorsal roots (DRs) produced dense presynaptic arbors within a wedge-shaped afferent termination zone medial to the dorsal root entry, within which dendrites of MNs and INs mingled with dense collections of synaptic boutons. Our data suggest that a major fraction of synaptic interactions takes place within the white matter. This study provides a detailed foundation for designing electrophysiological experiments to study the neural circuits involved in locomotor pattern generation.

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PubMed1988

Ultrastructure of taste cells and synapses in the mudpuppy Necturus maculosus.

Taste buds in the mudpuppy Necturus maculosus were examined with electron microscopy. Three cell types (dark, light, and basal) were identified and reconstructed from serial thick sections. Dark and light cells extend from the basal lamina to the surface of the tongue. The apical process of the dark cells was usually quite lamellar when viewed in cross section, in contrast to light cells, whose apical process appeared more cylindrical. Basal cells are situated at the base of the bud and do not extend processes to the surface of the tongue. The cytoplasm of basal cells contains numerous clear and dense-cored vesicles. Small, spinelike processes (2-3 microns in length) project outward from the basal cells into the cytoplasm of the surrounding tast receptor cells. Morphologically, basal cells in mudpuppy taste buds resemble Merkel cells. Unmyelinated afferent nerve fibers enter the taste bud at the base and course through the lower portion of the bud. Synapses were found between taste receptor cells and nerve fibers, between basal cells and nerve fibers, and between basal cells and taste receptor cells. Over 65% of the synapses observed in the mudpuppy taste bud involved the basal cell. These findings suggest that basal cells play some role in chemosensory signal processing or integration of the taste response.

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PubMed1992

Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus.

To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.

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PubMed1993

Merkel-like basal cells in Necturus taste buds contain serotonin.

Several types of cells have been identified in vertebrate taste buds, including dark cells, light cells, intermediate cells, type III cells, and basal cells. The physiological roles of these cell types are not well understood, especially those of basal cells. In this paper we show that there are two types of basal cells in taste buds from Necturus maculosus. One type of basal cell is an undifferentiated cell, presumably a stem cell. By combining light microscopic immunocytochemistry with electron microscopy, we show that the other type of basal cell is positive for serotonin-like immunoreactivity and that these cells have ultrastructural features similar to those found in cutaneous Merkel cells. Based on these findings, and the fact that the Merkel-like taste cells have been shown to make synaptic contacts with adjacent taste cells and with innervating nerve fibers, we conclude that these Merkel-like basal taste cells are serotonergic interneurons.

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PubMed1992

Autocrine and paracrine role of steroids during spermatogenesis: studies in Squalus acanthias and Necturus maculosus.

Due to a cystic mode of spermatogenesis and zonal testes, the spiny dogfish shark (Squalus acanthias) and salamander (Necturus maculosus) are advantageous for studying the relationship between steroidogenesis and germ cell development. Direct analysis of steroidogenic enzymes and steroid receptors in staged tissue samples, together with light and electron microscopic observation, suggests that androgens and estrogens produced by Sertoli cells or Leydig cells immediately before or after spermiation serve as intratesticular regulators of events specific to premeiotic stages of spermatogenesis. These correlative data are consistent with limited observations in mammalian testis and provide a conceptual framework for direct testing of functional interactions between spermatogenesis and steroidogenesis in future studies.

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PubMed1985

Neuropeptides in the gastrointestinal canal of Necturus maculosus. Distribution and effects on motility.

The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut. Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum. Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa. The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like-bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity. It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.

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PubMed1996

Serotonergic modulation of the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

The aims of the present study were to: (1) study the role of serotonin (5-HT) in modulating the central pattern generator (CPG) underlying locomotion in the mudpuppy (Necturus maculatus); (2) investigate whether there is an intrinsic spinal serotonergic system. These aims were achieved by the use of pharmacological and immunocytochemical methods. To study modulation of the locomotor pattern and rhythm, we applied 5-HT, its uptake blocker zimelidine, and a variety of 5-HT receptor agonists and antagonists to an in vitro brainstem-spinal cord preparation isolated from the mudpuppy. The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial plexus. The spinal CPG for locomotion was activated chemically by adding NMDA to the superfusing solution. During locomotion, bipolar electromyographic (EMG) recordings were made unilaterally from flexor and extensor ulnae muscles. 5-HT on its own did not induce locomotion, but it did have a profound modulatory effect on NMDA-induced locomotion. 5-HT produced a dose-dependent increase in the overall cycle duration and enhanced the EMG burst duration. Use of zimelidine indicated that there is an endogenous release of 5-HT which modulated the locomotor rhythm. The endogenous release was antagonized by 5-HT1/5-HT2 receptor antagonist methiothepin. Immunocytochemical analysis, in which the entire spinal cord of the mudpuppy was used, revealed that there were more than one type of spinal serotonergic neuron. They were differentiated according to the cell diameter, shape, and arborization pattern of their processes. These neurons were located within the central gray matter ventrolateral to the central canal. Our results suggest that 5-HT plays an important role in modulating the locomotor CPG in the mudpuppy, by acting through a well-developed spinal serotonergic system. This is in contrast to what has been reported in higher vertebrates, where serotonergic innervation is derived from supraspinal structures.

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PubMed1993

A mechanism for isotonic fluid flow through the tight junctions of Necturus gallbladder epithelium.

During isotonic fluid flow, Necturus gallbladder epithelium mediates net fluxes of paracellular probes by a convective process. We show here that the paracellular system is modeled by permeation through three populations of channels: (i) convective parallel-sided ones of width 7.7 nm (ii) small diffusive ones of radius approximately 0.6 nm, and (ii) large diffusive ones of radius exceeding 50 nm. The reflexion coefficient of the convective channels is very low and the calculated osmotic flow rate is close to zero when compared with the observed fluid absorptive rate of 2 x 10(-6) cm/sec. Analysis reveals that the convective channels behave as though closed to back-diffusion of probes; if this is due to solvent drag then very high fluid velocities are required, acting through minute areas. There are no transjunctional gradients that could drive the flow, and so the fluid must be propelled through the channel by components of the junction. We propose a mechanism based upon an active junctional peristalsis which allows discrimination on the basis of molecular size, in which the channels are always occluded at some point and so back-diffusion cannot occur. There is no local gradient of salt distal to the junctions and therefore the osmotic permeability of the membranes is irrelevant. High fluid velocities are not required, and the flow can occur over a substantial fraction of the junction. The mechanism must involve motile and contractile elements associated with the junction for which there is already considerable evidence.

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PubMed1993

Necturus gallbladder epithelial cell volume regulation and inhibitors of arachidonic acid metabolism.

Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.

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PubMed1981

Capacitive and inductive low frequency impedances of Necturus gallbladder epithelium.

The electrical impedance of Necturus gallbladder epithelium was analysed in the frequency range 0.24 Hz to 6,323 Hz. Under control conditions (NaCl-Ringer's on both sides), the impedance function yields a semicircle with depressed center. When serosal Na+ was replaced by K+, an inductive low frequency (LF) component appeared in the impedance locus. With KCl-Ringer on the mucosal side a second circular arc was observed at frequencies below 1 Hz. The resistive parts of the capacitive and inductive LF components increased after application of TAP+ to the mucosal side. Both LF features were abolished after application of 5 mM TEA+ to the mucosal medium as well as after acidification of the mucosal side. The LF components were depressed by addition of 5 mM Ba2+ to the mucosal solution. As TEA+ blocks apical K+ channels (Van Driessche and Gögelein 1978), it is concluded that the capacitive as well as the inductive LF components are related to transcellular K+ flow. With KCl-Ringer on the mucosal side, mucosa negative potentials increased the equivalent resistance and decreased the equivalent capacitance of the LF impedance. With serosal KCl-Ringer, negative potentials evoked a capacitive component which overlapped with the inductive component observed at open circuit conditions. Positive potentials, however, abolished the capacitive as well as the inductive LF component, elicited by mucosal or serosal KCl-Ringer, respectively. These results demonstrate that serosa to mucosa directed K+ flow causes an inductive LF feature and that mucosa negative potentials elicit a capacitive LF component.

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PubMed1985

Electrophysiological studies on lateral intercellular spaces of Necturus gallbladder epithelium.

The lateral intercellular spaces of Necturus gallbladder epithelium were punctured with double-barrelled ion selective microelectrodes in order to determine the ion concentrations of lateral space fluid and the contribution of the lateral spaces to transepithelial resistance. Neither under control conditions, nor after diluting the bathing fluids to increase the rate of volume absorption, nor during passage of direct current of 200 microA/cm2, were any reliable concentration differences observed between lateral space fluid and external bathing fluids. These observations suggest that water can follow salt transport without requiring osmotic concentration gradients of greater than 1 or 2 mosmol/l and indicate that recently observed high values of water permeability must still be considered as underestimates. After developing a test to recognize and exclude leaky punctures, the contribution of the lateral spaces to transepithelial resistance could be determined. It amounted to around 29%. This value agrees well with results from recent impedance measurements which were performed under control conditions in the same preparation.

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PubMed1982

Square wave pulse analysis of cellular and paracellular conductance pathways in Necturus gallbladder epithelium.

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 omega cm2 respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 micro F/cm2 respectively. The fact that the resistance values are 4-15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients.

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PubMed1983

Reinvestigation of the transepithelial P.D. in the proximal tubule of Necturus kidney.

Published values of transepithelial potential differences (VTE) in the proximal tubule of Necturus vary from approximately 0 to approximately -15 mV. In view of this disparity, we reinvestigated VTE. Our measured VTE was on the average -1.0 mV in early convolutions and +0.6 mV in terminal proximal segments. In the course of this study, we considered five distinct causes of artifacts. 1) Tip and pre-tip potentials: their occurrence was minimized by using Ringer's filled microelectrodes. 2) Interstitial tip localizations: the position of the tip was ascertained by the shift in potential, resulting in response to peritubular perfusion with gluconate solutions. 3) Leaky impalements: VTE responses to gluconate, input resistance determinations and the presence of positive VTE's rule out the leak hypothesis. 4) Zeroline shifts between pre- and postimpalement stages, and 5) Spontaneous VTE drifts in the positive direction, due to gradual passage of the tip from cell to lumen, or in the negative direction, resulting from tip contact with the lower cellular layer. All five causes of artifacts may be involved in the controversy regarding past VTE estimates.

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PubMed1982

Relations between intracellular ion activities and extracellular osmolarity in Necturus gallbladder epithelium.

The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in the Necturus gallbladder. The intracellular activities of K+ and Cl- in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K+, Cl- and some unidentified anions. Surprisingly, the apparent K+ concentration remained high: at an external osmolarity of 64 mOsm the intracellular K+ concentration averaged 95 mM. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na+, K+ and Cl- were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na+ and Cl- were equal. The data were consistent with Na+ and Cl- ions initially remaining inside the cell and a cell membrane Lp of 10(-3) cm sec-1 osm-1, which is close to the values determined by Spring and co-workers (K.R. Spring, A. Hope & B.E. Persson, 1981. In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190-200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K+ was only 0.1-0.2 times the change observed in Na+ and Cl- activities, and suggests that K+ ions leave the cell during the osmotically induced H2O efflux and enter with an induced H2O influx. The coupling is between 98 and 102 mmoles liter-1. Various explanations for the anomalous behavior of intracellular K+ ions are considered. A discussion of the apparent coupling between K+ and H2O, observed in nonsteady states, and its effects on the distribution of K+ and H2O across the cell membrane in the steady states, is presented.

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PubMed1984

Volume regulation by Necturus gallbladder: basolateral KCl exit.

Swelling of the epithelial cells of Necturus gallbladder caused by an 18% reduction in the osmolality of the mucosal bath is followed by rapid volume readjustment. This volume regulatory decrease requires Cl and is sensitive to the K and Cl gradients across the basolateral cell membrane. Volume regulatory decrease is not inhibited by amiloride, SITS, ouabain or bicarbonate removal. The process is blocked by bumetanide in the serosal bath. Measurement of the intracellular activities of K and Cl and the rate of volume regulation under five different experimental conditions showed that KCl exited from the cell across the basolateral membrane with a stoichiometry of 3 K to 2 Cl. This KCl exit process appears to be transiently activated following the reduction in osmolality of the mucosal perfusate.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1987

Regulation of K+ channels in the basolateral membrane of Necturus oxyntic cells.

Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands from Necturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67 +/- 21 pS in 24% of the patches and channels of smaller conductance, 33 +/- 6 pS in 56% of the patches. Channels in both classes were highly selective for K+ over Na+ and Cl-, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca2+ in the range between 5 and 500 nM, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca2+. The presence of K+ channels in the basolateral membrane which are regulated by these known "second messengers" can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.

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PubMed1987

Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane.

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

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