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D Schild

Publications and source records attributed to D Schild.

At least 19 recordsLinked to original sources
PubMed1996

Second messenger signaling in olfactory transduction.

Olfactory receptor neurons respond to odorants with G-protein mediated increases in the concentration of cyclic adenosine 3',5'-monophosphate (cAMP) and/or inositol 1,4,5-trisphospahte (InsP3). These two second messengers directly regulate opening of cAMP- and InsP3-regulated conductances localized to the apical transduction compartments of the cell (cilia and olfactory knob). In the presence of physiological concentrations of extracellular Ca2+, these second messenger regulated conductances mediate influx of Ca2+ into the olfactory neuron resulting in large, localized increases in intracellular Ca2+ ([Ca2+]i). A significant advance in our understanding of the molecular mechanisms of olfaction is the recent realization that this increase in [Ca2+]i plays an important role as a "third messenger" in olfactory transduction. Second messenger dependent increases in [Ca2+]i cause opening of ciliary Ca(2+)-activated Cl-, cation and/ or K+ channels that can carry a large percentage of the generator current, thus amplifying the signal substantially. As a result of this sequence of events, the generator potential in olfactory neurons can be depolarizing, leading to excitation of the neuron, or hyperpolarizing, leading to suppression of basal action potential firing rate. This dual effect of odorants on olfactory neurons may play an important role in quality coding and in the ability to detect low concentrations of odorants, particularly in complex mixtures.

Animals↗
PubMed1996

A transient, RCK4-like K+ current in cultured Xenopus olfactory bulb neurons.

A transient K+ current in cultured olfactory bulb neurons of Xenopus tadpoles was studied using the whole-cell patch-clamp technique. The current, which was resistant to 80 mM tetraethylammoniumchloride (TEA) and 10 nM charybdotoxin but blocked by 5 mM 4-aminopyridine (4-AP), activated between -60 and -40 mV and showed time- and voltage-dependent inactivation. Its peak amplitude was nearly independent of the extracellular K+ concentration ([K+]o) in the range of 0.05 to 10 mM, indicating that its conductance increased upon increasing [K+]o. The transient K+ current showed a slow recovery from inactivation with the time for half-maximum recovery from a conditioning pulse to 80 mV for 1 s varying from 100 ms to 500 ms. Complete recovery required as much as 5-10 s at -80 mV, but could be speeded up at hyperpolarized potentials. The current resembles the RCK4 (Kv1.4) current of rat neurons except that its recovery from inactivation was independent of [K+]o. High-frequency stimulation (20-67 Hz) of the neurons with short (5 ms) voltage pulses resulted in a frequency-dependent, progressive inactivation of the transient K+ current. This suggests that, during phasic responses of olfactory bulb neurons, inactivation of the transient K+ current occurs and may lead to lengthening of action potentials and facilitation of synaptic transmission.

Action Potentials↗
PubMed1996

Glutamate-mediated release of Ca2+ in mitral cells of the olfactory bulb.

1. Effects of glutamate on intracellular Ca2+ concentration ([Ca2+]i) were investigated in cultured olfactory bulb neurons of Xenopus laevis tadpoles. We imaged [Ca2+]i in these cells with the use of a confocal laser scanning microscope and the calcium indicator dyes Fluo3 and FuraRed. 2. In the standard bath solution, application of glutamate through a pipette resulted in an increase of [Ca2+]i in both mitral/ tufted (M/T) cells and interneurons. The increase occurred in all compartments of the cells, although in a nonhomogenous way. 3. In an ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-buffered bath solution (Ca(2+)-free Ringer solution), glutamate reproducibly induced an increase of [Ca2+]i in M/T cells but not in interneurons. This increase in [Ca2+]i had the following properties: 1) it was delayed with respect to the combined ionotropic and metabotropic response to glutamate, 2) in some cases it stopped before the end of the glutamate application, and 3) it was not affected by D,L-2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione added to the bath. It was interpreted as a release of Ca2+ from intracellular calcium stores. 4. Of 47 M/T cells that showed a glutamate-mediated release of [Ca2+]i, 46 also showed Ca2+ influx through ionotropic glutamate receptors, so that intracellular release of [Ca2+]i appeared to be associated with the presence of glutamate-gated ion channels. However, of 110 M/T cells showing an ionotropic response to glutamate, only 46 showed a glutamate-mediated release of [Ca2+]i. 5. The glutamate-mediated release of [Ca2+]i in the dendrites was higher than that in the soma. No indications for calcium waves were found. 6. Quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) mimicked the effect of glutamate on the release of [Ca2+]i, with potencies quisqualate > glutamate > > ACPD. N-acetyl-aspartyl-glutamate did not induce release of [Ca2+]i. The release was partly blocked by (+)-alpha-methyl-4-carboxyphenylglycine. 7. In conclusion, some but not all M/T cells of the olfactory bulb of X. laevis show a glutamate-mediated and predominantly dendritic increase of [Ca2+]i that is associated with glutamategated channels. The pharmacological properties of the corresponding metabotropic glutamate receptor (mGluR) resemble those of the mGluR1/5 receptors in rat.

Animals↗
PubMed1996

Glutamate and N-acetylaspartylglutamate block HVA calcium currents in frog olfactory bulb interneurons via an mGluR2/3-like receptor.

1. We investigated the glutamate-mediated modulation of voltage-activated calcium currents in cultured interneurons of the olfactory bulb of Xenopus laevis, with the use of standard patch-clamp whole-cell recordings. 2. Glutamate and N-acetylaspartylglutamate (NAAG) activated (2S,1'R,2'R,3'R)-2-(2,3-dicarboxy-cyclopropyl)glycin-sens iti ve metabotropic glutamate receptors at olfactory bulb interneurons and thereby inhibited calcium influx through presynaptic voltage-gated calcium channels. 3. The glutamate- or NAAG-mediated blockage of calcium channels showed indistinguishable dose-response curves, with K1/2 = 388 and 350 nM for glutamate and NAAG, respectively. At saturating concentrations of glutamate or NAAG, approximately 40% of the maximum calcium currents were blocked. 4. It is concluded that glutamate, which excites interneurons at olfactory bulb reciprocal synapses, also causes a reduction of gamma-aminobutyric acid-mediated feedback inhibition at these synapses by activating a metabotropic-glutamate-receptor-2/3-like receptor.

Animals↗
PubMed1995

Different spatial patterns of [Ca2+] increase caused by N- and L-type Ca2+ channel activation in frog olfactory bulb neurones.

1. The intracellular calcium concentration ([Ca2+]i) in cultured olfactory bulb neurones of Xenopus laevis tadpoles was imaged using the calcium indicator dyes fluo-3 and Fura Red as well as a laser scanning microscope. 2. Upon extracellular application of brief pulses of a solution with high potassium concentration (high [K+]o), an increase in [Ca2+]i occurred in all neurones observed. During the first 2 days in culture this increase was highest. At later stages (more than 2 days in culture) the increase in [Ca2+]i was non-homogeneous and highest in the dendritic processes. 3. Nifedipine (10 microM) reduced the high [K+]o-induced increase in [Ca2+]i. The reduction was greatest in somata and proximal dendrites. 4. With nifedipine in the bath, the high [K+]o-induced increase of [Ca2+]i was further reduced by the application of omega-conotoxin GVIA (1 microM). The omega-conotoxin-sensitive Ca2+ influx occurred predominantly on dendritic processes. 5. Noradrenaline (NA), as well as the alpha 2-adrenergic receptor agonist clonidine, reduced the high [K+]o-induced increase of [Ca2+]i. This reduction occurred mainly on dendritic processes. 6. Our results suggest a highly non-homogeneous spatial distribution of voltage-gated Ca2+ channels in cultured olfactory bulb neurones. L-type channels were found mainly on somata and their density seemed to decrease on the dendrites with increasing distance from the soma. In contrast, nifedipine-insensitive N-type channels were mainly observed on dendrites and were blocked by omega-conotoxin. NA, as well as clonidine, markedly blocked Ca2+ influx through dendritic N-type Ca2+ channels.

Adrenergic alpha-Agonists↗
PubMed1995

Imaging of L-type Ca2+ channels in olfactory bulb neurones using fluorescent dihydropyridine and a styryl dye.

We have imaged the fluorescence of dihydropyridine-Bodipy (fDHP) in cultured olfactory bulb neurones in order to investigate the subcellular distribution of L-type calcium channels in these neurones. The neurones were stained with both fDHP and the voltage-sensitive styryl dye RH414. The fluorescence emission maxima of these dyes were in the green and red ranges of the spectrum, respectively, and were recorded by the 2 photomultiplier channels of a laser scanning microscope. The fDHP images were ratioed with the RH414 images taken simultaneously. The resulting ratio images revealed the spatial distribution of the surface density of L-type calcium channels. This density was highest on somata, in particular at the base of dendrites, and decreased with the distance from this maximum. Two classes of dendritic L-type channel distributions were observed: one with a homogeneously low density and another one with a characteristic gradient of the L-type channel density along proximal dendrites. This subcellular localization of L-type calcium channels is discussed in the light of specific functional roles.

Animals↗
PubMed1995

Suppression of a new allele of the yeast RAD52 gene by overexpression of RAD51, mutations in srs2 and ccr4, or mating-type heterozygosity.

The RAD52 gene of Saccharomyces cerevisiae is involved both in the recombinational repair of DNA damage and in mitotic and meiotic recombination. A new allele of rad52 has been isolated that has unusual properties. Unlike other alleles of rad52, this allele (rad52-20) is partially suppressed by an srs2 deletion; srs2 mutations normally act to suppress only rad6 and rad18 mutations. In addition, although haploid rad52-20 strains are very X-ray sensitive, diploids homozygous for this allele are only slightly X-ray sensitive and undergo normal meiosis and meiotic recombination. Because rad52-20 diploids homozygous for mating type are very X-ray sensitive, mating-type heterozygosity is acting to suppress rad52-20. Mating-type heterozygosity suppresses this allele even in haploids, because sir mutations, which result in expression of the normally silent mating-type cassettes, were identified among the extragenic revertants of rad52-20. A new allele of srs2 and alleles of the transcriptional regulatory genes ccr4 and caf1 were among the other extragenic revertants of rad52-20. Because other researchers have shown that the RAD51 and RAD52 proteins interact, RAD51 on a high copy number plasmid was tested and found to suppress the rad52-20 allele, but RAD54, 55 and 57 did not suppress. The RAD51 plasmid did not suppress rad52-1. The rad52-20 allele may encode a protein that has low affinity binding to the RAD51 protein. To test whether the selected revertants suppressed rad52-20 by elevating the expression of RAD51, an integrated RAD51-lacZ fusion was genetically crossed into each revertant. Because none of the revertants increased the level of RAD51-lacZ, the revertants must exert their effect by one or more mechanisms that are not mediated by RAD51.

Alleles↗
PubMed1995

InsP3 causes an increase in apical [Ca2+]i by activating two distinct current components in vertebrate olfactory receptor cells.

1. Effects of inositol-1,4,5-trisphosphate (InsP3) applied through a patch pipette to Xenopus laevis olfactory receptor cells (ORCs) were studied using the patch-clamp technique in conjunction with calcium imaging with fura-2. 2. InsP3 activated, first, a novel voltage-independent Ca2+ current (ICa) and, second, a nonselective cation current (Icat). 3. The activation of these currents occurred at different intracellular calcium concentrations, and the activation of either current led to a marked increase of [Ca2+]i in the dendritic knob. 4. The results suggest that InsP3 might act as a second messenger in vertebrate olfactory receptor cells by activating, through different mechanisms, a plasma membrane Ca2+ conductance (ICa) and a nonselective cation conductance (Icat).

Animals↗
PubMed1994

Sodium/calcium exchanger in olfactory receptor neurones of Xenopus laevis.

Ca2+ ions enter neurones through various types of calcium and cation channels. The mechanisms by which Ca2+ ions are spatially buffered and expelled from neurones have been studied considerable less. Using calcium imaging in conjunction with the patch clamp technique, we investigated the Na/Ca exchanger in olfactory neurones and found evidence for its localization on the dendrite. It is suggested that this tends to decouple increases in [Ca2+]i occurring in the transduction compartments of the cell from processes in the soma.

Animals↗
PubMed1994

Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red.

The intracellular calcium concentration [Ca2+]i in olfactory receptor neurones of Xenopus laevis was imaged with high spatial and temporal resolution. A new method using a mixture of the calcium indicator dyes Fluo-3 and Fura-Red was employed. The fluorescence patterns in two wavelength bands were measured on the emission side of a confocal laser scanning microscope, and the ratio R of the fluorescence intensities was taken as an estimate of [Ca2+]i. When the neurones were depolarized by elevating the extracellular potassium concentration [K+]o they showed one of three types of responses: a fast increase in [Ca2+]i, a slow increase in [Ca2+]i, or no change in [Ca2+]i. The fast increase in [Ca2+]i took place in the soma compartment. For at least 4 s after the onset of depolarization the calcium distribution in the dendrite remained essentially unchanged. To study the fast increase with high time resolution, line scan images were taken. The neurones were depolarized for brief periods applying a solution containing high [K+] onto the soma from an application pipette. The fast increase in [Ca2+]i began with a delay of about 200 ms and went from the resting concentration to about 110 nM above resting concentration. Following the depolarization, recovery from elevated [Ca2+]i to resting levels had a time constant of about 15 s. The slow response seemed to depend on the removal of [Na+] from the bath rather than on the elevated [K+] in the bath. The response was also observed with Cd2+, Ni2+, and Co2+ (1.5 mM each) in the bath.(ABSTRACT TRUNCATED AT 250 WORDS)

Aniline Compounds↗
PubMed1994

Amiloride-insensitive cation conductance in Xenopus laevis olfactory neurons: a combined patch clamp and calcium imaging analysis.

We used digital calcium imaging with Fura-2 in conjunction with the tight-seal whole-cell patch clamp technique to describe a novel cation conductance in olfactory neurons of the clawed toad Xenopus laevis. Substitution of extracellular Ca2+ and Na+ was used as a tool to change [Ca2+]i. When [Ca2+]i was increased to about 450 nM, a conductance gcat activated that was permeable for cations. Upon gcat activation, an increase in [Ca2+]i occurred in the dendritic knob. Once activated, gcat showed no further dependence upon [Ca2+]i. Icat is shown to be different from the current activated by a mixture of the odorants citralva and amyl acetate. We conclude that there are two different cation conductances in the peripheral compartments of olfactory neurons in X. laevis.

Amiloride↗
PubMed1993

Ciliary cation conductances in olfactory receptor cells of the clawed toad Xenopus laevis.

One transduction pathway in olfactory receptor neurons is a cascade of receptors, a G-protein, adenylate cyclase, cAMP, and a cyclic nucleotide-activated cation conductance. Here, we show that this conductance is also present in olfactory cells of Xenopus laevis. With optical recordings from the cell's dendritic knob, we show that this conductance, when activated by odors, leads to an increase of intracellular calcium. It is further shown that there is a second cation conductance on the cilia of these cells which is modulated by calcium and can be activated by the application of odorants.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Standing calcium gradients in olfactory receptor neurons can be abolished by amiloride or ruthenium red.

Digital imaging and the patch clamp technique were used to investigate the intracellular calcium concentration in olfactory receptor neurons using the Ca2+ indicator dyes fura-2 and fura-2/AM. The spatial distribution of Cai2+ as well as its modification by the drugs Amiloride and Ruthenium Red were studied. Resting calcium concentrations in cells loaded with fura-2/AM were between 10 and 200 nM. In cells that were loaded with the pentapotassium salt of fura-2 through the patch pipette, calcium concentrations were in the same range if ATP was added to the pipette solution. Otherwise, Ca2+ reached concentrations of approximately 500 nM. Most of the observed cells showed a standing gradient of calcium, the calcium concentrations in the distal dendritic end of the cell being higher than in the soma. In some cells, the gradient was markedly reduced or abolished by adding either Amiloride or Ruthenium Red to the bath solution. In a few cells, neither drug had any effect upon the gradient. It is suggested that the inhomogenous spatial distribution of intracellular calcium in olfactory cells of Xenopus laevis is brought about by an influx of calcium ions through two different calcium permeable conductances in the peripheral compartments of the cells. The fact that only either Ruthenium Red or Amiloride abolished the standing calcium gradient further suggested that the two conductances blocked were presumably not coexpressed in the same cells.

Amiloride↗
PubMed1993

Effects of nitric oxide upon olfactory receptor neurones in Xenopus laevis.

We have studied olfactory receptor neurones under voltage-clamp conditions using the patch clamp technique. Application of sodium nitroprusside (SNP) activated a cation conductance if the GTP concentration in the pipette was in the millimolar range. With 10 microM GTP, SNP had no effect on the holding current but it abolished the normally occurring wash-out of responses to odorants. This effect could also be obtained by adding 150 nM cGMP to the pipette solution. We conclude that there is a NO/cGMP system in Xenopus' olfactory neurones and that cGMP plays an important role in the transduction process in that it guarantees ongoing responsiveness to odorants.

Animals↗
PubMed1992

Identification of RAD16, a yeast excision repair gene homologous to the recombinational repair gene RAD54 and to the SNF2 gene involved in transcriptional activation.

The RAD54 gene of Saccharomyces cerevisiae is involved in the recombinational repair of DNA damage. The predicted amino acid sequence of the RAD54 protein shows significant homologies with the yeast SNF2 protein, which is required for the transcriptional activation of a number of diversely regulated genes. These proteins are 31% identical in a 492-amino acid region that includes presumed nucleotide and Mg2+ binding sites. We noted previously that the SNF2 protein also shares homology with a partial open reading frame (ORF) that was reported with the sequence of an adjacent gene. This ORF also shares homology with the RAD54 protein. To test whether this ORF is involved in transcriptional activation or DNA repair, yeast strains deleted for part of it have been isolated. These strains do not show a Snf-like phenotype, but they are UV sensitive. This gene has been identified as RAD16, a gene involved in the excision repair of DNA damage. Analysis of the rad16 deletion mutations indicates that RAD16 encodes a non-essential function and is not absolutely required for excision repair. Outside the region of homology to RAD54 and SNF2, the predicted RAD16 protein contains a novel cysteine-rich motif that may bind zinc and that has been found recently in eleven other proteins, including the yeast RAD18 protein. The homologies between RAD16, RAD54 and SNF2 are also shared by several additional, recently isolated yeast and Drosophila genes.

Adenosine Triphosphatases↗