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R C Gesteland

Publications and source records attributed to R C Gesteland.

At least 19 recordsLinked to original sources
PubMed1994

Guanine nucleotides modulate steady-state inactivation of voltage-gated sodium channels in frog olfactory receptor neurons.

The voltage for half-inactivation (V1/2) of Na+ currents in frog olfactory receptor neurons (ORNs) under whole-cell voltage clamp showed a shift to more negative potentials with time. Inclusion of guanosine triphosphate (GTP) or its nonhydrolyzable analogue, guanosine-5'-O-3-thiotriphosphate (GTP-gamma-S), which activates G proteins, in the recording pipette, not only gave a more positive V1/2, but also reduced and delayed the negative shift observed in the absence of nucleotides. Guanosine-5'-O-2-thiodiphosphate (GDP-beta-S), a nonhydrolyzable analogue that prevents the binding of GTP to G proteins, did not affect the V1/2 significantly by itself but blocked the positive shift induced by GTP. Since the steady-state activation was not affected, our results indicate that a G protein or a G-protein-dependent process may be important in regulating the steady-state inactivation of Na+ channels in ORNs of the frog.

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PubMed1994

An electrophysiological survey of frog olfactory cilia.

Individual olfactory receptor neurons vary widely in their responses to odorants. Olfactory stimulus reception occurs in the cilia of the receptor neurons. Thus, the variability among individual neurons could in part be due to differences among the olfactory cilia. We have quantified the known conductance properties of each of 117 frog olfactory cilia. From a strictly qualitative viewpoint, the cilia were very homogeneous. All but a few of them had a basal conductance in the absence of odorants and second messengers, conductances stimulated by cytoplasmic cyclic AMP and by Ca2+ and a conductance measured in the presence of ATP and stimulated by GTP gamma S. However, the magnitudes of the conductances varied widely among the cilia. Amplitudes of the cyclic-AMP- and Ca(2+)-activated ciliary currents correlated strongly with one another across the 117 cilia and 24 frogs studied, suggesting that expression of the underlying channels may be co-regulated. None of the conductance properties correlated strongly with ciliary length, a marker of cell maturity. Given cytoplasmic MgATP as substrate, ciliary adenylate cyclase apparently produced cyclic AMP, which in turn gated membrane channels and increased the ciliary conductance. In some cilia, MgATP alone caused a very large increase in conductance. In others, there was little effect unless GTP gamma S, which increases cyclase activity, was also added. No effect of cytoplasmic inositol trisphosphate on ciliary conductance was detectable.

Adenosine Triphosphate↗
PubMed1991

Somatic sodium channels of frog olfactory receptor neurones are inactivated at rest.

Membrane excitability of acutely isolated olfactory receptor neurones (ORNs) of the grass frog (R. pipiens) was studied with the use of the whole-cell "tight-seal" patch recording technique. ORNs of the frog had a mean resting membrane potential of -52 mV, a mean input resistance of 1-2 G omega, and a mean capacitance of 4.5 pF. In the majority of cells examined (over 70%), short duration (several milliseconds) action potentials were elicited at the end of a hyperpolarising pulse (off-spike) or following hyperpolarization of the membrane potential by injection of current. Under voltage-clamp conditions, a fast inward current followed by an outward current could be evoked upon depolarisation of the membrane. The fast inward current decayed with a time constant of 1-2 ms, with an e-fold decrease per 52 mV increase in voltage, and was blocked by the selective voltage-dependent sodium channel blocker tetrodotoxin (0.5-1 microM). Steady-state inactivation studies revealed that the mean voltage for half-inactivation (V1/2) was -82 mV (range -72 to -98 mV), which indicates that the voltage-dependent Na+ channels in the cell body or soma of frog ORNs are not available for conducting currents at the resting membrane potential. This finding raises the possibility that voltage-dependent Na+ channels may not play a significant role in sensory transduction at the soma. Our results indicate that ORNs of the frog are very efficient in transducing signals towards the brain since currents generated at the cilia will be directed towards depolarising the axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Transmembrane currents in frog olfactory cilia.

We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.

Action Potentials↗
PubMed1991

Calcium-activated chloride conductance in frog olfactory cilia.

We have measured the effects of cytoplasmic Ca2+ on the conductance of single cilia excised from frog olfactory receptor neurons. When free cytoplasmic Ca2+ is buffered at 0.1 microM, ciliary conductance is low. As Ca2+ is increased, ciliary conductance increases. Maximal conductance averages sevenfold higher than that measured in the absence of Ca2+. We estimate that the K1/2 for Ca2+ activation is 5 microM; the dose-response curve indicates some positive cooperativity of Ca2+ binding. Activation by Ca2+ is rapid and fully reversible. Most of the Ca(2+)-activated current is carried by Cl- and persists in the absence of Na+ and K+. The Cl- channel inhibitor 3',5-dichlorodiphenylamine-2-carboxylate (300 microM) reduces the Ca(2+)-activated current by 90%. Odorants induce a Ca2+ influx in some olfactory receptor neurons, but the consequences of this influx for neuronal function are not well understood. Our findings allow us to predict that a Ca2+ influx would increase the permeability of the olfactory cilia to Cl-. How this would affect the neuronal potential is uncertain, since the equilibrium potential for Cl- in olfactory receptor neurons is unknown.

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PubMed1990

Ultrastructural organization of receptor cell axons in frog olfactory nerve.

Repeated odorant or electrical stimulation of the frog olfactory nerve leads to long-lasting reduction of excitability of the receptor neurons. In the turtle olfactory nerve electrical stimulation causes an increase in extracellular potassium concentration due to the efflux of potassium from active axons. Elevated potassium concentration depolarizes an axon membrane and inactivates it. If axons travel parallel to each other in the nerve for extended distances, changes in ionic concentration due to activity in one axon will reduce excitability of its neighbors. This phenomenon may have effects like those of lateral inhibition among neighbors first described in Limulus and may act as a filter with a long time constant, for the nervous message. The amphibian olfactory nerve consists of densely packed, small-diameter, unmyelinated fibers. In this study we have examined the ultrastructure of the frog (Rana pipiens) olfactory nerve in longitudinal and cross-sections to determine whether axons follow a sinuous course and change neighbors often along the length of the olfactory nerve, or whether they follow parallel trajectories and thus tend to stay close to the same neighbors. We have found that axons have a very straight course within the nerve and that axons tend to remain adjacent to the same individual axons over long distances. We think that the anatomical substrate of the olfactory nerve favors strong inhibitory axon-axon interaction.

Animals↗
PubMed1990

Organization of projections from olfactory epithelium to olfactory bulb in the frog, Rana pipiens.

One hypothesis for the coding of olfactory quality is that regions of the olfactory epithelium are differentially sensitive to particular odor qualities and that this regional sensitivity is conveyed to the olfactory bulb in a topographic manner by the olfactory nerve. A corollary to this hypothesis is that there is a sufficiently orderly connection between the epithelium and the olfactory bulb to convey this topographical coding. Thus we examined topography in the projection from epithelium to bulb in the frog, which has been the subject of numerous electrophysiological studies but has not yet been examined using modern neuroanatomical techniques. The tracer WGA-HRP was applied to the ventral or to the dorsal olfactory epithelium, or both. Anterograde transport of label to the olfactory bulb was seen after as few as 2 days; label was still present in the bulb as long as 21 days postinjection. In cases where WGA-HRP was applied to the entire epithelium, there was dense anterograde labelling of the ipsilateral olfactory bulb. In addition, a small medial portion of the contralateral bulb was labelled. Injections limited to either the ventral or dorsal epithelium produced patterns of anterograde labelling in the glomerular layer of the olfactory bulb, which varied with the size and location of the injection. With very large injections in either the dorsal or ventral epithelium, label appeared to be evenly distributed in the glomerular layer. With smaller injections in the ventral epithelium, there was heavier labelling in the lateral than in the medial portions of the glomerular layer, although light labelling was found in all regions of the glomerular layer. In contrast, injection sites restricted to the dorsal epithelium produced more anterograde labelling in the medial than lateral portions of the glomerular layer. These patterns extended throughout the dorsal-ventral extent of the bulb. Within the limits of the anterograde tracing technique used, we were unable to detect any systematic relationship between the pattern of labelling in the glomerular layer and the medial-lateral or rostral-caudal location of the injection site in either the ventral or dorsal epithelium. We conclude that in the frog, as in other amphibia, there is only a limited degree of topographic order between the epithelium and the olfactory bulb.

Animals↗
PubMed1987

Comparative study of immature and mature olfactory receptor cells in adult frogs.

We describe a method to produce frog olfactory epithelium with a population of developmentally synchronized receptor cells suitable for electrophysiological studies. The epithelium is ablated with ZnSO4 and allowed to regenerate for 10 days. Generation of new cells is then blocked by continuous treatment with hydroxyurea. Since new receptor cells are generated beginning on the 6th day after ablation, it is reasonable to assume that receptor cells in the preparation originated between the 6th and 10th days following ablation. The age range of these cells would be no more than 5 days. The cells develop normally and in relative synchrony. Olfactory epithelia with developmentally synchronized receptor cells were used for a physiological study of response properties as a function of developmental age. Developmental stages of receptor cells were confirmed by fine-structure analysis. There were no differences in the polarities or shapes of electro-olfactograms (EOGs) recorded from olfactory epithelia composed of immature or mature receptor cells. However, amplitudes of the main negative components in EOGs recorded from epithelia composed of mature receptor cells were generally higher than those from epithelia composed of immature cells. The majority of immature olfactory receptor cells had low spontaneous activity or none at all. Mature olfactory receptor cells had a variety of frequencies of spontaneous activity ranging from less than one to more than 70 spikes/min. Extracellular single unit activity recordings showed that, contrary to what has been observed in rat embryos, olfactory receptor cells in regenerating epithelial of adult frogs do not go through a stage in which they respond to all odorants.

Animals↗
PubMed1986

Suppression of mitotic activity and synchronization of cell development in olfactory epithelium.

Prolonged continuous application of hydroxyurea to frog olfactory epithelium suppresses mitotic activity in this tissue without side effects. After ZnSO4-induced coagulation necrosis of the olfactory epithelium, application of hydroxyurea prevents the usual epithelial regeneration. A method for obtaining frog olfactory epithelium with a developmentally synchronized neuronal population is introduced. The epithelium is first ablated by perfusion of the nasal cavity with ZnSO4. Then the epithelium is allowed to regenerate for 6 days, after which continuous introduction into the nasal cavity of hydroxyurea suppresses mitotic activity and thus the generation of new cells in the tissue. Cells formed during the 6 days prior to hydroxyurea treatment continue to develop. We found that the olfactory receptor cells are generated beginning on the 6th day after ablation, and so the epithelia contain a highly synchronized population of neurons all generated within about 24 h.

Animals↗
PubMed1985

Cell and explant culture of olfactory chemoreceptor cells.

An in vitro system for the study of maturation of rat and chick embryonic olfactory receptor cells is presented. A variety of dissociating agents, culture media and substrata were tried in attempts to obtain a preparation of mature living olfactory receptor cells readily visible in the microscope. Maturation was judged by the development of axons greater than 1 mm long, by the presence of cilia at the end of the dendrites and, in the rat, by the presence of immunohistochemically demonstrable olfactory marker protein, a protein present only in olfactory receptor cells. By these criteria, dissociated cells did not mature in vitro, though occasional bipolar cells with relatively short axons were seen. In explant culture, small fragments of rat tissue were positive for all 3 criteria after 6 days. In 9-day cultures, the axons had grown up to 3 mm long in both rat and chick cultures. Olfactory bulb fragments co-cultured close to the olfactory epithelium had no influence on the direction of outgrowth of axons from the olfactory receptor cells. Preliminary experiments with intracellular electrodes on the fragment cultures suggest that there are two cell types in the epithelium; one with a potential of -25 to -30 mV and, the other, -12 to -15 mV.

Animals↗
PubMed1984

Transduction physiology of olfactory receptor cilia.

Electro-olfactograms (EOGs) evoked by 8 odorants from frog olfactory epithelia during ciliary regrowth and during epithelial regeneration were analyzed. During ciliary regrowth following detergent-induced ciliary removal, EOG amplitudes initially increase proportionately with ciliary length. EOGs reach maximal amplitudes after 2 days of growth, when cilia are 40 micron long. Therefore olfactory transduction sites are located primarily on cilia rather than on the dendrite terminal and most of the receptor current enters through the proximal portion of the cilium. Zinc sulfate lavage of the nasal cavity causes selective necrosis of the receptor epithelium. During epithelial regeneration, EOGs increase linearly with time from 13 days after zinc lavage, the time of first cilium emergence, through 30 days. The rate of increase is different for different odorants. At 30 days and within a period of a few days, EOG amplitudes increase abruptly, then asymptote. Thus the development of receptors for different substances occurs at different rates and occurs in two steps. The transition between the two developmental states is coincident with arrival of receptor axon terminals at the central nervous system and with the immobilization of the ciliary contractile apparatus. Since there is continual generation of new receptor neurons throughout life, EOGs recorded in a normal nose reflect a complex combination of the differing receptor processes of cells of differing developmental stages.

Animals↗
PubMed1983

Dissociation of frog olfactory epithelium.

We report a method for producing cell suspensions from frog olfactory epithelium. The tissue is incubated for 45 min at room temperature in a solution which causes dissociation of the epithelium. The solution is an isotonic saline buffered to maintain a pH of 10.3 and a free Ca2+ ion concentration of 10(-6) M. The method uses no degradative enzymes, except for a brief DNase treatment. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, glandular cells, and respiratory epithelial cells. The cells are viable as judged by vital staining, ciliary motility, and synthesis of RNA. Some types of cells lose their normal columnar shapes and become rounded in the suspension.

Animals↗
PubMed1982

Changes in morphology and physiology of olfactory receptor cilia during development.

Ciliated olfactory receptor neurons in vertebrates turn over throughout life. We show that these neurons bear different types of cilia at different developmental stages; cilia on newly differentiating cells are short and motile; cilia on mature cells are longer and immotile; Mg2+ and adenosine 5'-triphosphate are requisite for ciliary motion; stimulation with odorants can induce synchronous motion and that this process is mediated by Ca2+. We propose that receptor neurons have two distinguishable developmental states. In the first, before the growing axon establishes synaptic connection to the brain, the cells bear motile cilia and are generally irritable. In the second, the cilia are long and immotile and the cells can distinguish between odorants.

Animals↗
PubMed1982

Postnatal proliferation and maturation of olfactory bulb neurons in the rat.

Mitral cells are formed prenatally whereas most granule cells originate postnatally. Material was taken from 2-day-old, 14-day-old, 28-day-old, and adult rat olfactory bulbs and processed for rapid Golgi or Cresyl Violet staining. We show that the number of granule cell bodies/mitral cell body increases from 7.0 to 46.3 during the first two weeks of life; most mitral cells appear morphologically functional during the first postnatal week; few granule cells appear to be functional until the second postnatal week; and the number of short axon interneurons increases dramatically during the second postnatal week. We conclude the newborn rats have an intact afferent pathway from olfactory receptors to primary cortex that lacks the extensive interneuronal circuitry characteristic of adults.

Animals↗
PubMed1982

Response properties of mitral cells in the olfactory bulb of the neonatal rat.

Activity was recorded from mitral cells in newborn to six-day-old rat pups during odorous stimulation. Twenty-eight neurons were studied in pups with unopened nasal cavities which sampled stimuli during intermittent periods of inhalation. Forty-six neurons were studied in pups with opened nasal cavities which were stimulated by delivering odorants directly to the olfactory epithelia. We show that mitral cells are selectively excited by different odorants on the day pups are born; prior to the maturation of bulb interneurons, the responses of neonatal mitral cells are time-locked to the inhalation cycle; neonatal mitral cells preserve the temporal patterns of activity exhibited by receptor neurons during stimulation with different concentrations of odorants; and the response patterns of mitral cells differ qualitatively between newborn and adult rats. We conclude that receptor-to-mitral cell synapses are functional in newborn rat pups and that the activity of this afferent pathway is modulated by the pups' respiratory behavior. We argue that without interneurons, mitral cells repeat the temporal code exhibited by receptor neurons and do not produce the types of response patterns characteristic of neurons in the adult rat olfactory bulb.

Action Potentials↗
PubMed1982

Development of olfactory receptor neuron selectivity in the rat fetus.

Olfactory receptor neurons begin to differentiate from stem cells on day E10 of embryonic life in the rat. By day E16, the receptor epithelium is well populated. On this day single neuron action potentials could be recorded with some ease and the electro-olfactogram was well developed. The receptor neurons were functional in that they responded to the vapors of odorous substances. However, they were not selective. Each cell responded to nearly all of the substances in the stimulus set. The first synaptic connections between receptors and mitral cells are established on day E18. The olfactory marker protein is reported to appear first in the receptors on the same day. By day E21, single unit responses changed dramatically. The cells became selective, responding to about half of the substances in our set. The electro-olfactogram reached its limiting amplitude well before this time.

Action Potentials↗
PubMed1981

Dissociation of frog olfactory epithelium with N-ethylmaleimide.

Treatment of frog olfactory epithelium with 8 mM N-ethylmaleimide for 2 min results in extensive dissociation of the epithelium. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, respiratory epithelial cells, and cells of Bowman's glands. The cells in suspension exhibit the same morphologies seen in histological sections of intact epithelium.

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