Biomedical subjects

S D Roper

Publications and source records attributed to S D Roper.

50 records · Page 3Linked to original sources

PubMed1991

Mediation of responses to calcium in taste cells by modulation of a potassium conductance.

Calcium salts are strong taste stimuli in vertebrate animals. However, the chemosensory transduction mechanisms for calcium are not known. In taste buds of Necturus maculosus (mud puppy), calcium evokes depolarizing receptor potentials by acting extracellularly on the apical ends of taste cells to block a resting potassium conductance. Therefore, divalent cations elicit receptor potentials in taste cells by modulating a potassium conductance rather than by permeating the cell membrane, the mechanism utilized by monovalent cations such as sodium and potassium ions.

Animals↗

PubMed1991

Ca(2+)-dependent chloride conductance in Necturus taste cells.

This report describes the occurrence and localization of a Ca(2+)-dependent chloride conductance in taste cells of Necturus maculosus. Lingual epithelium from Necturus was removed with blunt dissection and mounted in a modified Ussing chamber which allowed individual taste cells to be impaled with intracellular micropipettes. Solutions in the mucosal and serosal chambers could be changed independently and the properties of apical and basolateral membranes tested separately. Action potentials in taste cells, elicited by brief depolarizing current pulses passed through the intracellular recording microelectrode, provided an accurate description of whether voltage-dependent conductances had been blocked or unmasked by the experimental conditions. We found that Ca2+ influx during the action potential triggers a prolonged depolarization due to Ca(2+)-dependent conductance changes, particularly in the presence of TEA to block repolarizing K+ currents. This afterdepolarization could last up to 7 sec and is due, in part, to a Ca(2+)-dependent Cl- conductance. Other Ca(2+)-dependent channels such as Ca(2+)-dependent K+ channels or nonselective cation channels may also contribute to the afterpotential. Calcium-dependent conductance channels were situated on apical and basolateral membranes of the taste cells. We speculate that Ca(2+)-dependent Cl- channels may play a role in discriminating chloride salts from salts of other anions and may help shape receptor cell responses elicited by taste stimuli.

Action Potentials↗

PubMed1990

Chemotransduction in Necturus taste buds, a model for taste processing.

The taste bud in Necturus serves as a good model for taste mechanisms in vertebrates. The large size of taste cells and relative accessibility of the tissue for detailed electrophysiological and ultrastructural studies makes this species well-suited for studying taste transduction. Important features of taste transduction that have been learned from investigations in Necturus are that voltage-gated potassium channels are preferentially distributed on the apical membrane of taste cells; voltage-gated potassium channels allow K ions to enter the cell when taste buds are stimulated with K salts; some chemical stimuli act by closing K channels, thereby eliciting depolarizing receptor potentials in taste cells. Many of these findings have been confirmed and extended in other animals, including mammals. Furthermore, recent evidence from experiments in Necturus suggests that there is a considerable degree of synaptic coupling among taste cells. This synaptic coupling could form the basis for signal processing and integration in the peripheral sensory organs of taste, the taste buds.

Animals↗

PubMed1989

Distribution of ion channels on taste cells and its relationship to chemosensory transduction.

The presence and regional localization of voltage-gated ion channels on taste cells in Necturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na+, K+, or Ca2+ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (i) voltage-gated K+ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na+ and Ca2+ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membranes; (iii) there is a K+ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K+ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K+ conductance may underlie receptor potentials ellicted by rapid stimuli.

Animals↗

PubMed1989

The cell biology of vertebrate taste receptors.

New technologies in neurophysiology and ultrastructural research are bringing about rapid advances in our understanding of taste, particularly at the cellular level. The model of chemosensory processing in the taste bud presented here can now be explored in great detail. The synaptic organization of the taste bud indicates a potential for intriguing peripheral integrative mechanisms, including cross-talk between taste cells, summation of chemoreceptor responses by interneurons (basal cells) in the taste bud, and centrifugal control of taste buds via efferent input from the CNS. Figure 2 summarizes these findings. The existence of voltage-gated ionic channels on taste cells and their unequal distribution in apical and basolateral membrane suggests mechanisms for chemosensory transduction: A primary event in the transduction process for many taste stimuli is likely to be the closure of apical potassium channels, thus leading to a depolarizing receptor potential. The closure of these apical potassium channels is probably mediated via cyclic nucleotides or intracellular Ca2+.

Animals↗

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.

Animals↗

PubMed1988

Ultrastructure of mouse vallate taste buds: III. Patterns of synaptic connectivity.

We have used serial high voltage electron micrographs and computer-generated, three-dimensional reconstructions to study morphological relationships and patterns of synaptic connectivity in taste buds from the circumvallate papillae of the mouse. The intragemmal arborizations of 40 sensory nerve fibers were examined from 7 taste buds that were sectioned serially. We identified the synaptic connections from taste cells onto the reconstructed nerve fibers and classified the presynaptic taste cells based on previously established ultrastructural criteria. From these data we were able to extract the following information for the reconstructed nerve fibers: (1) the morphology of intragemmal nerve fibers and their arborizations within the taste bud, (2) the total number of synaptic connections from taste bud cells onto the nerve fibers, and (3) the taste cell types associated with each of the synapses. Fifty-six synapses were studied. Synapses were often found to be located at either the branch points or terminations of nerve fiber processes. The maximum number of taste cells observed to synapse onto a single nerve fiber was 5. Several nerve fibers had no apparent synapses. Dark cells (type I), intermediate cells, and light cells (type II) all formed synaptic connections with sensory nerve fibers. In no cases did dark cells and light cells synapse onto the same sensory nerve fiber. Our observation that any given nerve fiber receives its synaptic input from morphologically similar taste cells provides evidence for specificity in taste bud synaptic connections. We speculate that the observed pattern of synaptic connections is related to taste bud function. Since all of the synapses onto a given nerve fiber are from morphologically similar taste cells, we postulate that there is a correlation between taste cell morphology and sensory responsiveness. Intracellular electrophysiological studies on taste cells, in which responses to focally applied chemical stimuli are followed by characterization of the ultrastructural features of the same taste cells, will prove or disprove this hypothesis.

Afferent Pathways↗

PubMed1988

Membrane properties of isolated mudpuppy taste cells.

The voltage-dependent currents of isolated Necturus lingual cells were studied using the whole-cell configuration of the patch-clamp technique. Nongustatory surface epithelial cells had only passive membrane properties. Small, spherical cells resembling basal cells responded to depolarizing voltage steps with predominantly outward K+ currents. Taste receptor cells generated both outward and inward currents in response to depolarizing voltage steps. Outward K+ currents activated at approximately 0 mV and increased almost linearly with increasing depolarization. The K+ current did not inactivate and was partially Ca++ dependent. One inward current activated at -40 mV, reached a peak at -20 mV, and rapidly inactivated. This transient inward current was blocked by tetrodotoxin (TTX), which indicates that it is an Na+ current. The other inward current activated at 0 mV, peaked at 30 mV, and slowly inactivated. This more sustained inward current had the kinetic and pharmacological properties of a slow Ca++ current. In addition, most taste cells had inwardly rectifying K+ currents. Sour taste stimuli (weak acids) decreased outward K+ currents and slightly reduced inward currents; bitter taste stimuli (quinine) reduced inward currents to a greater extent than outward currents. It is concluded that sour and bitter taste stimuli produce depolarizing receptor potentials, at least in part, by reducing the voltage-dependent K+ conductance.

Action Potentials↗

PubMed1987

Ultrastructure of apical specializations of taste cells in the mudpuppy, Necturus maculosus.

The first interaction of taste stimuli with lingual chemoreceptors occurs on the apical membrane of taste cells, since only that portion is exposed to the oral cavity. To gain better insight into this interaction, we examined the pore region of taste buds in Necturus maculosus with scanning electron microscopy (SEM), transmission electron microscopy, and high-voltage electron microscopy. SEM of the pore reveals a patchwork distribution of three morphologically distinct types of apical specializations: long and branched (LB) microvilli, short and unbranched (SU) microvilli, and bundles of stereocilia. As demonstrated in thin and thick sections, LB microvilli are specializations of dark cells, SU microvilli are the apical specializations of light cells, and stereocilia arise from a cell that has the cytoplasmic markers characteristic of light cells. When left in place, the pore mucus completely covers the SU microvilli and partially covers the LB microvilli. However, stereocilia project above the surface and thus are highly exposed to taste stimuli in the oral cavity. These three morphologically distinct types of apical specializations may reveal functional differences among taste cells. The initial interaction between chemical stimulus and taste cell, and possibly chemoreceptor specificity itself, may be influenced by the morphology of the apical ending.

Animals↗

PubMed1987

Passive and active membrane properties of mudpuppy taste receptor cells.

1. Intracellular recordings were obtained from taste receptor cells and surface epithelial cells of isolated mudpuppy lingual epithelium. 2. Surface epithelial cells had a mean resting potential of -40.2 +/- 8.9 mV, a mean input resistance of 40.3 +/- 11.3 M omega, and a linear current-voltage (I-V) relationship. Taste receptor cells had a mean resting potential of -61.7 +/- 15 mV, a mean input resistance of 380.3 +/- 177.2 M omega, and the I-V relationship showed pronounced outward rectification; the outward rectification persisted in high-K+ saline, but was abolished by tetraethylammonium bromide (TEA). 3. Surface epithelial cells responded to depolarizing current injection with only passive membrane potential changes. Taste receptor cells responded to brief pulses of depolarizing current injection with regenerative action potentials characterized by an abrupt rising phase, an inflexion on the falling phase, and a prolonged after-potential. 4. The abrupt rising phase of the action potential was blocked by tetrodotoxin (TTX), suggesting that voltage-gated Na+ currents are responsible for the rising phase. 5. Long-duration action potentials were elicited from cells treated with TEA to block outward K+ currents and with TTX to block Na+ currents, and from cells bathed in isotonic CaCl2. These results suggest that the active membrane response contains a significant Ca2+ component. 6. The after-potential was blocked or greatly reduced by the addition of Ca2+ channel blockers to the bathing medium. In contrast, addition of TEA to the bathing medium greatly enhanced the after-potential. These data suggest that a significant portion of the after-potential is Ca2+ mediated. 7. The mean reversal potential for the after-potential (-76.8 +/- 6.0 mV) was significantly different from the mean reversal potential for the undershoot of the action potential (-86 +/- 5.6 mV). Superfusion with TEA reduced the reversal potential of the after-potential to -42.3 +/- 8.2 mV and abolished the undershoot. These results suggest that the after-potential results from at least two conductances, one which is blocked by TEA and the other which is Ca2+ dependent and involves ions other than, or in addition to K+. 8. Our data suggest that taste receptor cells, unlike surface epithelial cells, possess voltage-gated Na+, Ca2+, and K+ channels, as well as Ca2+-mediated channels. The role of the Ca2+ channels may be in part to regulate release of transmitter onto nerve terminals. The role of the other conductances in taste transduction is unknown.

Action Potentials↗

PubMed1987

Dye-coupling in taste buds in the mudpuppy, Necturus maculosus.

Electrical coupling in taste buds and in non-taste lingual epithelium in the mudpuppy was examined by injecting cells with a fluorescent dye, Lucifer yellow. Lucifer yellow coupling has been shown to indicate the presence of electrical junctions between cells. Lucifer yellow-filled taste cells usually have an elongate shape. Cells were an average of 111 microns long and were 13 microns in diameter at the widest region (nucleus). In taste buds, from a sample of 105 impalements we detected Lucifer yellow coupling in 21 cases: dye-coupled pairs of cells were observed in 17 cases, and trios of cells in 4 cases. Larger subsets of coupled cells (greater than 3) were not observed. Dye-coupled cells were usually equally intensely stained. In non-taste epithelium, we examined dye-coupling in the superficial and basal layers. Extensive Lucifer yellow coupling was found in the basal layer (15/15 cases). The number of cells coupled to the dye-injected cell varied from 3 to 5. In the superficial epithelium, dye-coupling was rare (1/45 cases). No dye-coupling was observed between epithelial cells and taste cells at the taste pore region. We conclude that strong electrical coupling in groups of 2-3 cells occurs in the mudpuppy taste buds. Coupling may occur selectively between identical types of taste cells (dark, light, etc.), but this remains to be determined. Electrical coupling also exists among basal epithelial cells but not in the superficial epithelial layers.

Animals↗

PubMed1986

Ultrastructure of mouse vallate taste buds: II. Cell types and cell lineage.

The lifespan of cells in the mouse taste bud was examined with high-voltage electron microscopic (HVEM) autoradiography (ARG) after giving a single injection of 3H-thymidine. Animals were killed at 1 hour, 6 hours, 12 hours, 24 hours, and then daily up through 10 days postinjection. Lingual tissues were prepared for HVEM ARG so that we could identify and characterize labeled cells. Four categories of taste cells were identified: basal, dark, intermediate, and light cells. Basal cells were polygonal cells located near the basolateral sides of the taste buds and were characterized primarily by the presence of filaments attached to the nuclear envelope. Dark and light cells had the typical features described by previous authors. Intermediate cells had features in between those of dark and light cells. Over 90% of the cells labeled in the first 2 days following injection of 3H-thymidine were basal cells. Labeled dark cells appeared 6 hours after injection, reached their peak incidence at the fourth day postinjection, and then gradually decreased. Labeled intermediate cells were identified after the appearance of dark cells (12 hours) and reached a peak incidence at the fifth day after injection of 3H-thymidine. Lastly, labeled light cells were first observed on the fourth day postinjection and continued to increase until the tenth day, when they constituted 45% of the labeled cells. These data support the hypothesis that there is one cell line in the mouse vallate taste bud that undergoes morphological changes in its lifespan.

Animals↗

PubMed1985

Ultrastructure of mouse vallate taste buds. I. Taste cells and their associated synapses.

The ultrastructural features of murine vallate taste bud cells and their associated synapses have been examined in thin and thick sections with conventional transmission electron microscopy and high-voltage electron microscopy. Computer-assisted reconstructions from serial sections were utilized to aid in visualization of taste bud cell-nerve fiber synapses. We have classified taste bud cells on the basis of previously established criteria-namely, size of the nucleus, shape and density of chromatin, density of cytoplasm, and presence or absence of dense-cored or clear vesicles, other cytoplasmic organelles, and synaptic foci. Both dark cells and light cells are present, as well as cells with intermediate morphological characteristics. Synapses were observed from taste bud cells onto nerve fiber processes. In virtually all instances, synapses are associated with the nuclear region of the taste cell. These synapses are characterized by the presence of 40-70 nm clear vesicles embedded in a thickened presynaptic membrane separated from the postsynaptic membrane by a 16-30 nm cleft. Synapses are not unique to any particular cell type. Dark, intermediate, and light cells all synapse onto nerve fibers. Two general types of synapses exist: spot (or macular) and fingerlike. In the latter, the postsynaptic region of the neuronal process protrudes into an invagination of the taste cell membrane. Differences in synaptic morphology are not correlated with taste cell type. In some cases a single taste cell was observed to possess both macular and fingerlike synapses adjacent to one another, forming a synaptic complex onto a single neuronal process. On the basis of the presence of synaptic contacts, we conclude that both "dark" and "light" cells are gustatory receptors.

Animals↗

PubMed1983

Accuracy of regeneration of vagal parasympathetic axons.

The degree of accuracy with which regenerating preganglionic parasympathetic fibers can restore their original connections was examined in the frog, Rana pipiens. The normal motor pool of the vagus nerve was determined by labeling the vagal cardiac branch fibers with horseradish peroxidase (HRP). Cardiac neurons form a relatively compact subgroup within the rostral half of the vagal motor column and represent less than 5% of the total vagal motor pool. Five to 14 weeks after crushing the vagus nerve, HRP labeling revealed that a more extensive population of vagal motor neurons has reinnervated the cardiac branch. The regenerated cardiac motor neuron pool is about twice as numerous as normal but is still centered in the rostral half of the vagal motor column. These experiments show that regeneration of the cardiac branch of the vagus nerve is neither a random process nor one which is completely accurate. Some degree of accuracy is maintained during regeneration following a crush lesion. Neurons nearer the original cardiac motor pool are more likely to reinnervate the cardiac branch than are vagal motor neurons located at some distance from the original cardiac pool.

Animals↗