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Relationship between Müller cell responses, a local transretinal potential, and potassium flux.

1. In the Necturus retina, light-evoked field potentials, Müller (glial) cell responses, and extracellular potassium ion concentration ([K+]0) were recorded and found to exhibit the three-way correlation characteristic of these variables elsewhere in the nervous system. 2. Müller cell responses to a flashed spot or annulus consist primarily of slow depolarizations at both light onset and offset. The responses are maximum to 0.5-mm-diameter spots and decrease with larger diameters. Responses to stimulus intensity and flicker were also used to characterize Müller cell behavior. 3. In response to long-duration stimuli, the initial Müller cell depolarization is followed by a very slow hyperpolarization, which is likely the origin of slow PIII. 4. A new extracellular potential is described, the M-wave, the basic properties of which suggest that it is generated by Müller cells. Moreover, the M-wave and Müller cells show remarkably similar behavior to a wide variety of stimulus parameters. 5. In the proximal retina, [K+]0 increases at both light onset and offset with a time course similar to that of Müller cell depolarizing responses. This K+ increase also behaves similarly to the Müller cell depolarization in response to changes in stimulus parameters. 6. It is concluded that light stimulation leads to an increase in [K+]0 in the proximal retina and that this increase depolarizes Müller cells whose associated currents, in turn, generate the M-wave.

Animals↗

Effect of changes in hydrostatic pressure in peritubular capillaries on the permeability of the proximal tubule.

The effect of increased hydrostatic pressure in the peritubular vessels on net sodium reabsorption from the proximal tubule was examined in the Necturus. An increase in the pressure gradient of 2.0 cm H(2)O across the wall of the proximal tubule, produced by ligation of the postcaval vein was associated with a marked reduction in net reabsorption and an increased back flux of water and electrolytes. This change was accompanied by a slight, but significant drop in the transepithelial electrical potential but not by an alteration in the steady-state chemical gradient. These studies highlight the importance of changes in the permeability characteristics of the proximal tubule on net sodium transport.

Animals↗

Mechanics of lung ventilation in a large aquatic salamander, siren lacertina

Lung ventilation in Siren lacertina was studied using X-ray video, measurements of body cavity pressure and electromyography of hypaxial muscles. S. lacertina utilizes a two-stroke buccal pump in which mixing of expired and inspired gas is minimized by partial expansion of the buccal cavity during exhalation and then full expansion after exhalation is complete. Mixing is further reduced by the use of one or two accessory inspirations after the first, mixed-gas cycle. Exhalation occurs in two phases: a passive phase in which hydrostatic pressure and possibly lung elasticity force air out of the lungs, and an active phase in which contraction of the transverse abdominis (TA) muscle increases body cavity pressure and forces most of the remaining air out. In electromyograms of the lateral hypaxial musculature, the TA became active 200-400 ms before the rise in body cavity pressure, and activity ceased at peak pressure. The TA was not active during inspiration, and no consistent activity during breathing was noted in the external oblique, internal oblique and rectus abdominis muscles. The finding that the TA is the primary expiratory muscle in S. lacertina agrees with findings in a previous study of another salamander, Necturus maculosus. Together, these results indicate that the use of the TA for exhalation is a primitive character for salamanders and support the hypothesis that the breathing mechanism of salamanders represents an intermediate step in evolution between a buccal pump, in which only head muscles are used for ventilation, and an aspiration pump, in which axial muscles are used for both exhalation and inhalation.

Journal Article↗

Effect of volume expansion on the paracellular flux of lanthanum in the proximal tubule.

Although studies of volume expansion (VE) in the Necturus suggest a major role for paracellular flux in reabsorption by the proximal tubule, results from morphologic or electrophysiologic studies of the effect of VE on the rat proximal tubule suggest only a minor role for paracellular transport. In the present study, during in vivo microperfusion, lanthanum was used as an extracellular marker to determine bidirectional paracellular flux in the rat proximal tubule before and during 10% VE. Lanthanum itself did not affect proximal tubule reabsorption (delta 0.7 +/- 3.3 nl/min, LaCl3 versus saline infusion, n = 7). When lanthanum was added to the luminal perfusate, paracellular lanthanum efflux from the lumen to the interstitium was 28.9 +/- 6.6 pg/min per mm, n = 7. Subsequent VE significantly decreased the paracellular lumen-to-interstitium efflux to 12.8 +/- 8.3 pg/min per mm concomitant with a 49% decrease in proximal fluid reabsorption (delta -2.6 +/- 0.9 nl/min per mm, P < 0.05). When lanthanum was infused interstitially, by means of a chronically implanted matrix, there was significant paracellular lanthanum influx from the interstitium into the lumen (143.9 +/- 18.6 pg/min per mm, n = 4). Subsequent VE significantly increased this interstitium-to-lumen influx to 212.1 +/- 29.2 pg/min per mm as proximal reabsorption was significantly decreased by 58% (delta -2.8 +/- 0.8 nl/min per mm, P < 0.05). Thus, VE affects bidirectional paracellular flux in a manner that would decrease proximal reabsorption; paracellular efflux from the lumen to the interstitium was decreased, whereas paracellular influx from the interstitium to the lumen was increased.

Absorption↗

Human psychophysical analysis of receptive field-like properties. I. A new transient-like visual response using a moving windmill (Werblin-type) target.

A human transient-like visual response has been revealed psychophysically with a rotating windmill target, similar to that used by Werblin in his recent studies of the retina of Necturus. The windmill pattern evoked complex perceptual phenomena (important in and of themselves) which were minimized by using a high luminance for the "criterion" flashing test field (relative to its increment threshold), and by moving the four-vaned windmill pattern at a rate of two complete revolutions per second. The transient response, herein defined as the difference in threshold between the stationary and moving windmill conditions, was greatest when the windmill vanes fell completely within the sensitization (inhibition-like) portion of the Westheimer function determined at the same retinal locus. The test has been developed so that it may be readily adapted to available clinical equipment for future diagnostic purposes .

Differential Threshold↗

Effects of antiinflammatory agents and prostaglandins on acid and bicarbonate secretions in the amphibian-isolated gastric mucosa.

Effects of antiinflammatory agents and prostaglandins on H+ and HCO-3 secretions and electrical properties were investigated in the amphibian-isolated gastric mucosa. Gastric HCO-3 transport was studied in Rana temporaria fundus, in which H+ secretion had been inhibited with the histamine H2-receptor antagonists metiamide or cimetidine (10(-3) M), and in Necturus antrum, which secreted HCO-3 spontaneously. Hydrocortisone (100-500 microgram/ml) had no effect on H+ or HCO-3 secretion in the fundus. Indomethacin (10(-4) M) was a considerably more potent inhibitor of HCO-3 secretion than of H+ secretion in the fundus and also inhibited HCO-3 transport in the antrum. Fenclofenac (3 x 10(-3) M) almost abolished fundic HCO-3 transport and also depressed H+ secretion. There was a marked fall in transmucosal potential difference and a decrease in electrical resistance in fenclofenac-treated mucosae whereas indomethacin had less effect on electrical properties at the concentrations used here. The prostaglandins, E2, 16,16-dimethyl E2 and I2 all inhibited H+ secretion but only 16,16-dimethyl E2 stimulated HCO-3 secretion. The inhibitory action of indomethacin on HCO-3 secretion was prevented by co-administration of 16,16-dimethyl PGE2 (10(-6) M). It is proposed that the inhibitory action of nonsteroidal antiinflammatory drugs and the stimulatory action of some prostaglandins on HCO-3 secretion contributes to their ulcerogenic and anti-ulcer actions on the gastric mucosa.

Animals↗

Microscopic investigation of structure and function in living epithelial tissues.

In this paper we shall illustrate the utility of direct microscopic methods for studying living epithelia. Beginning with an exposition on the available strategies for visualization of unstained biological materials, the rationale that leads to the choice of differential interference-contrast optics for examination of epithelia is illustrated. Findings from toad urinary bladder, Necturus gallbladder, and rabbit cortical collecting tubule are reviewed. Emphasis on renal structures is provided with a report on work in progress on proximal tubule volume regulation and on structural examination of the isolated perfused macula densa. Conclusions are drawn with respect to the advantages and shortcomings of discussed optical methods and with respect to the choice of model epithelia.

Animals↗

Thyroid hormone receptors in perennibranchiate amphibians.

Thyroid hormone has long been known to induce metamorphosis in amphibians. The understanding of the molecular steps controlling the completion of metamorphosis has nevertheless been hampered by the complexity of this event. The comparison of organisms in which metamorphosis does or does not occur, may provide clues into the molecular cascade that control it. Up to now the available data suggest that perennibranchiate amphibians retain their larval characters mainly because their tissues do not respond to thyroid hormones. In such a context the recent identification of a thyroid hormone receptor alpha in the perennibranchiate Proteus anguinus is provocative (Ho Huynh et al., Int. J. Dev. Biol. 40:537-543, 1996). In the present paper, we provide evidences that this recently described sequence is in fact a sequence from Xenopus laevis. Indeed, we identified the authentic thyroid hormone receptors of both alpha and beta types in two perennibranchiate species Necturus maculosus and Proteus anguinus. The various controls required to ascertain the authenticity of a developmental gene cloned by PCR or RT-PCR analysis are presented. The results reported in the present paper are relevant with phylogenetical analysis. This induces our team to conclude that the Proteus TR alpha sequence reported by Ho Huynh et al. (1996) reflects a contamination of the RT-PCR by Xenopus laevis material.

Amphibians↗

Localization of gap junctions and tracer coupling in retinal Müller cells.

Physiological studies have demonstrated the existence of direct intercellular communication, presumably mediated by gap junctions, both between neurons and between glial cells in the vertebrate retina. We localized gap junctions in the retinas of rat, goldfish, and mudpuppy by using antisera directed against proteins that make up the connexon channels in two tissues from which connexins have been isolated: liver (connexin 32; CX32) and heart (connexin 43; CX43). Although the antiserum against CX32 stained liver gap junctions, it did not reveal any staining in rat or goldfish retina. The antiserum against CX43 stained gap junctions associated with the intercalated disk in rat heart and also stained gap junctions between pigment epithelium cells in rat, goldfish, and mudpuppy retina. Anti-CX43 also stained gap junctions between Müller cells in goldfish and mudpuppy retina but not in rat retina. Intracellular injections of the tracer Neurobiotin into Müller cells in the mudpuppy retina revealed that these glial cells are extensively tracer coupled. Staining with the tracer formed a syncytium of thin processes surrounding every neuron from the outer limiting membrane to the inner limiting membrane. Confocal microscopy demonstrated that the Müller cells were in close apposition with one another at every level of the retina. However, CX43 immunoreactivity was heaviest at the outer limiting membrane, where the apical processes of Müller cells are located. Some anti-CX43 staining was observed at the level of the outer nuclear layer and the inner plexiform layer but not in the ganglion cell layer or at the Müller cell end feet forming the inner limiting membrane.

Animals↗

Structure and functional connections of presynaptic terminals in the vertebrate retina revealed by activity-dependent dyes and confocal microscopy.

The fluorescent dyes sulforhodamine 101 (SR 101) and FM1-43 were used as activity-dependent dyes (ADDs) to label presynaptic terminals in the retinas of a broad range of animals, including amphibians, mammals, fish, and turtles. The pattern of dye uptake was studied in live retinal preparations by using brightfield, fluorescence, and confocal microscopy. When bath-applied to the retina-eyecup, these dyes were avidly sequestered by the presynaptic terminals of virtually all rods, cones, and bipolar and amacrine cells; ganglion cell dendrites and horizontal cells lacked significant dye accumulation. Other structures stained with these dyes included pigment epithelial cells, cone outer segments, and Müller cell end-feet. Studies of dye uptake in dark- and light-adapted preparations showed significant differences in the dye accumulation pattern in the inner plexiform layer (IPL), suggesting a dynamic, light-modulated control of endocytotic activity. Presynaptic terminals in the IPL could be segregated on the basis of volume: bipolar varicosities in the IPL were typically larger than those of amacrine cells. The combination of retrograde labeling of ganglion cells and presynaptic terminal labeling with ADDs served as the experimental preparation for three-dimensional reconstruction of both structures, based on dual detector, confocal microscopy. Our results demonstrate a new approach for studying synaptic interactions in retinal function. These findings provide new insights into the likely number and position of functional connections from amacrine and bipolar cell terminals onto ganglion cell dendrites.

Adaptation, Ocular↗

Mudpuppy retinal ganglion cell morphology revealed by an HRP impregnation technique which provides Golgi-like staining.

A new technique of retrograde labeling of ganglion cells with horseradish peroxidase (HRP) has been developed, based on orbital injections of HRP combined with a detergent (lysolecithin). When injections are followed by an appropriate survival time, dense staining of a small number of widely scattered cells results in Golgi-like filling of each neuron. This technique, as well as a variation which causes mass staining of ganglion cell somas, has been used to analyze the morphology of mudpuppy retinal ganglion cells. Morphological analysis has relied on computer reconstruction techniques for display, analysis of dendritic sublamination pattern, and morphometric analysis of the dendrites and soma. Based on morphological criteria, the mudpuppy retina contains a rich variety of ganglion cell types which vary according to soma placement, dendritic field size, polar vs. non-polar dendritic fields, dendritic branching pattern, and dendritic sublamination. The mudpuppy retina contains both conventional and displaced ganglion cells: the latter constitute about 15% of the total ganglion cell population. Both conventional and displaced ganglion cells show morphological diversity of dendritic sublamination branching pattern; cells from each group have a dendritic branching pattern confined to either distal or proximal divisions of the inner plexiform layer, whereas other cells have dendrites which branch in both sublaminae. Using morphological criteria, two subtypes of ganglion cells were identified, which have a distinctive branching pattern and dendritic tree size. The size and distribution of ganglion cell somas were analyzed from retinas in which mass staining of ganglion cells was present. The total number of ganglion cells was estimated at approximately 14,500 cells per retina. There was a tendency for soma size and density to decrease near the optic disk. The somas of displaced ganglion cells are smaller than their conventional counterparts, at the same retinal eccentricity. The somas of all HRP filled cells swell when compared to those of unstained fixed and freshly dissected retinas. The degree of swelling is proportional to the length of exposure to HRP. Cell swelling was evident for both retrograde labeling and intracellularly injected HRP. This artifact of HRP staining could influence the interpretation of studies in which quantitative differences in soma sizes are based on the use of HRP labeling.

Animals↗

Adrenomedullin in nonmammalian vertebrate pancreas: an immunocytochemical study.

Adrenomedullin (AM) immunoreactive cells have been identified, by immunocytochemical methods, in the endocrine pancreas of seven nonmammalian vertebrate species, belonging to the cartilaginous and bony fish, amphibian, reptilian, and bird classes. The frequency and distribution of the pancreatic AM cells vary among the different animals. In most species, these cells are found scattered mainly among the exocrine component, with a few present in the islet-like structures. The distribution of AM cells in both fish species and Xenopus shows an inverse pattern, since almost every AM cell is located in the islets. In addition, the colocalization of AM with other classical pancreatic peptide immunoreactivities has been analyzed. In numerous cells, AM immunoreactivity did not colocalize with the other hormones, suggesting that AM-producing cells might constitute a new endocrine cell type in the pancreas of many species. Nevertheless, in other cells a species-specific pattern of colocalizations with insulin, somatostatin, glucagon, and pancreatic polypeptide was found, indicating that complex interactions among all these hormones may occur. In conclusion, AM represents a new regulatory peptide of the endocrine nonmammalian vertebrate pancreas, which is possibly involved in the modulation of insulin secretion and other pancreatic functions.

Adrenomedullin↗

Proadrenomedullin N-terminal 20 peptide (PAMP) immunoreactivity in vertebrate juxtaglomerular granular cells identified by both light and electron microscopy.

The gene for adrenomedullin (AM), a multifunctional peptide hormone, is expressed in mammalian renal tissue and has been shown to stimulate renin release. The exact cell source of this peptide and its gene-related partner, proadrenomedullin N-terminal 20 peptide (PAMP), in kidney is still uncertain. In the present study we have identified PAMP-immunoreactive cells in the kidney of different mammalian species, including man, by light microscopy. In addition, these cells have been further studied in mouse kidney by both light and electron microscopic techniques. At the light microscopic level, PAMP immunolabeling is preferentially located in the subendothelial cells of the enlarged glomerular afferent arterioles, that is, in the juxtaglomerular cells. However, these cells do not show immunolabeling for AM. At the electron microscopic level, the immunostaining appears inside the renin-containing secretory granules of the juxtaglomerular cells. These results confirm the direct link between renin and the AM peptide family and provide a morphological basis for studying the potential modulatory function of AM and PAMP in the control of renin activity. In contrast, neither AM nor PAMP immunoreactivities were detected in the kidney of nonmammalian vertebrates, other than in blood vessels of particular species, providing a new phylogenetic difference in the juxtaglomerular apparatus between mammalian and nonmammalian vertebrates.

Adrenomedullin↗

Automated electron microscope tomography of frozen-hydrated chromatin: the irregular three-dimensional zigzag architecture persists in compact, isolated fibers.

The potential of electron microscope tomography as a tool for obtaining three-dimensional (3D) information about large macromolecular assemblies is greatly extended by automation of data collection. With the implementation of automated control of tilting, focusing, and digital image recording described here, tilt series of frozen-hydrated specimens can be collected with the requisite low dose. Long chromatin fibers were prepared in 90 mM monovalent ions to maintain a fully compact conformation, and after vitrification were completely contained within the ice layer. Tilt series of this material were recorded at 5 degrees tilt increments between +60 degrees and -60 degrees, with a cumulative dose of approximately 35 e-/A2 for the series. This extremely low dose data was successfully aligned, then reconstructed by weighted backprojection. The underlying architecture of the fibers is an irregular 3D zigzag of interconnected nucleosomes, with the linker DNA between successive nucleosomes in a largely extended conformation. The visualization of this structural motif within long, frozen-hydrated chromatin fibers at relatively high salt extends our previous studies on small fragments at low ionic strength and is in agreement with the observation of this architecture in chromatin fibers in situ in sectioned nuclei.

Animals↗

Monoaminergic neurons of the mudpuppy retina.

The mudpuppy retina was investigated with the histofluorescence method of Falck and Hillarp in normal animals and in animals injected intraocularly with alpha-methylnoradrenaline, 5,6-dihydroxytryptamine, or a combination of the two drugs. Catecholaminergic amacrine cells were found to form a thin layer of terminals at the border between the inner nuclear and the inner plexiform layers. Catecholaminergic interplexiform cells were not found. Indoleamine-accumulating amacrine cells were also observed. They are fifteen to twenty times more numerous than the catecholaminergic cells, and their terminals occur diffusely throughout the inner plexiform layer. In a number of eyes the majority of the indoleamine-accumulating terminals were eliminated with intraocular injections of the neurotoxin, 5,7-dihydroxytryptamine, but the reproducibility of this effect was not consistent. Intravitreal injections of 5,6 dihydroxytryptamine were used to label both types of neurons for electron microscopy. They were found to make conventional type synapses on amacrine cells and, less frequently, on bipolar cells.

5,6-Dihydroxytryptamine↗

Heterochromatic DNA in Triturus (Amphibia, Urodela). I. A satellite DNA component of the pericentric C-bands.

We have studied the structure, genome organization, chromosomal location, conservation across species and transcription on lampbrush chromosomes, of an AT-rich satellite DNA component of the newt, Triturus vulgaris meridionalis. The satellite (Sat G), originally isolated by gradient centrifugation, represents about 2% of the vulgaris genome and comprises a highly repetitive sequence family (HindIII family), whose monomers have been cloned. The repeat units are about 330 bp long, as measured on gels, and a cloned unit (pTvm1) is 310 bp long, as shown by sequencing. Abundant clusters of the HindIII family sequences are located within the pericentric heterochromatin (i.e. the C-bands placed at both sides of, and at a certain distance from, the centromeres) in most chromosomes. Both the sequence family and its overall pattern of chromosomal distribution are conserved within the genus Triturus, despite a few species-specific differences. The great majority of the HindIII family sequences are unexpressed on lampbrush chromosomes; they reside within pericentric, condensed segments of the chromosome axis ("loopless bars"). Only a few sequences are transcribed on some loops, suggesting that transcription promotion does not depend on the satellite sequences themselves.

Ambystoma↗

Galanin activates an inwardly rectifying potassium conductance in mudpuppy atrial myocytes.

Galanin- and bethanechol-activated K+ currents have been studied in mudpuppy atrial myocytes. The galanin and bethanechol K+ currents were time-dependent and inwardly rectifying. In GTP gamma S, the galanin and bethanechol currents were reduced progressively as G-protein gated K+ channels became activated. GDP beta S inhibited agonist-induced outward currents. We conclude that galanin and bethanechol activate the same or a very similar inwardly rectifying K+ conductance and that activation of a G protein is required.

Animals↗

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. I. Experimental procedures.

A method has been developed to determine rapidly and simultaneously the resistance of the tight junctions, the resistance of the lateral intercellular space, and the resistances and capacitances of the apical and basolateral cell membrane in leaky epithelia by alternating current spectroscopy. The present paper describes the experimental procedures. Multi-frequency sine-wave currents are simultaneously applied across the tissue and the transepithelial and intracellular voltage responses are recorded with shielded microelectrodes, digitized, and stored in a computer. Using Fast Fourier Transform techniques the frequency-dependent transepithelial impedance and an apparent basal cell membrane impedance are then calculated from the voltage and current waveform. By fitting appropriate model circuits to the data the above listed individual resistances can be deduced. The model calculations and the fit results are described in the subsequent paper [14]. In the present design the analysis covers the frequency range between 2.5 Hz and 12.5 kHz. The minimal measuring time is in the order of 1-2s.

Animals↗