Biomedical subjects

Detlev Schild

Publications and source records attributed to Detlev Schild.

17 recordsLinked to original sources

PubMed2006

Important contribution of alpha-neurexins to Ca2+-triggered exocytosis of secretory granules.

Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three alpha-neurexin genes show a severe reduction of synaptic release. Although analyses of alpha-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+ channels, it remained unclear whether alpha-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult alpha-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that alpha-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for alpha-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.

Animals↗

PubMed2006

Finite-particle tracking reveals submicroscopic-size changes of mitochondria during transport in mitral cell dendrites.

The mechanisms of molecular motor regulation during bidirectional organelle transport are still uncertain. There is, for instance, the unsettled question of whether opposing motor proteins can be engaged in a tug-of-war. Clearly, any non-synchronous activation of the molecular motors of one cargo can principally lead to changes in the cargo's shape and size; the cargo's size and shape parameters would certainly be observables of such changes. We therefore set out to measure position, shape and size parameters of fluorescent mitochondria (during their transport) in dendrites of cultured neurons using a finite-particle tracking algorithm. Our data clearly show transport-related submicroscopic-size changes of mitochondria. The observed displacements of the mitochondrial front and rear ends are consistent with a model in which microtubule plus- and minus-end-directed motor proteins or motors of the same type but moving along anti-parallel microtubules are often out-of-phase and occasionally engaged in a tug-of-war. Mostly the leading and trailing ends of mitochondria undergo similar characteristic movements but with a substantial time delay between the displacements of both ends, a feature reminiscent of an inchworm-like motility mechanism. More generally, we demonstrate that observing the position, shape and size of actively transported finite objects such as mitochondria can yield information on organelle transport that is generally not accessible by tracking the organelles' centroid alone.

Animals↗

PubMed2006

ATP activates both receptor and sustentacular supporting cells in the olfactory epithelium of Xenopus laevis tadpoles.

Nucleotides and amino acids are acknowledged categories of water-borne olfactory stimuli. In previous studies it has been shown that larvae of Xenopus laevis are able to sense amino acids. Here we report on the effect of ATP in the olfactory epithelium (OE) of Xenopus laevis tadpoles. First, ATP activates a subpopulation of cells in the OE. The ATP-sensitive subset of cells is almost perfectly disjoint from the subset of amino acid-activated cells. Both responses are not mediated by the well-described cAMP transduction pathway as the two subpopulations of cells do not overlap with a third, forskolin-activated subpopulation. We further show that, in contrast to amino acids, which act exclusively as olfactory stimuli, ATP appears to feature a second role. Surprisingly it activated a large number of sustentacular supporting cells (SCs) and, to a much lower extent, olfactory receptor neurons. The cells of the amino acid- and ATP-responding subsets featured differences in shape, size and position in the OE. The latencies to activation upon stimulus application differed markedly in these subsets. To obtain these results two technical points were important. We used a novel dextran-tetramethylrhodamine-backfilled slice preparation of the OE and we found out that an antibody to calnexin, a known molecular chaperone, also labels SCs. Our findings thus show a strong effect of ATP in the OE and we discuss some of the possible physiological functions of nucleotides in the OE.

Adenosine Triphosphate↗

PubMed2005

In situ background estimation in quantitative fluorescence imaging.

Fluorescence imaging of bulk-stained tissue is a popular technique for monitoring the activities in a large population of cells. However, a precise quantification of such experiments is often compromised by an ambiguity of background estimation. Although, in single-cell-staining experiments, background can be measured from a neighboring nonstained region, such a region often does not exist in bulk-stained tissue. Here we describe a novel method that overcomes this problem. In contrast to previous methods, we determined the background of a given region of interest (ROI) using the information contained in the temporal dynamics of its individual pixels. Since no information outside the ROI is needed, the method can be used regardless of the staining profile in the surrounding tissue. Moreover, we extend the method to deal with background inhomogeneities within a single ROI, a problem not yet solved by any of the currently available tools. We performed computer simulations to demonstrate the accuracy of our method and give example applications in ratiometric calcium imaging of bulk-stained olfactory bulb slices. Converting the fluorescence signals into [Ca2+] gives resting values consistent with earlier single-cell staining results, and odorant-induced [Ca2+] transients can be quantitatively compared in different cells. Using these examples we show that inaccurate background subtraction introduces large errors (easily in the range of 100%) in the assessment of both resting [Ca2+] and [Ca2+] dynamics. The proposed method allows us to avoid such errors.

Algorithms↗

PubMed2005

Stem cell-coated titanium implants for the partial joint resurfacing of the knee.

The goal of the present study was to evaluate the partial surface replacement of the knee with stem cell-coated titanium implants and to provide a basis for a successful treatment of large osteochondral defects. Mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates of adult sheep. Round titanium implants with a diameter of 2 x 7.3 mm were seeded with autologous MSC and inserted into an osteochondral defect in the medial femoral condyle. As controls, defects received either an uncoated implant or were left untreated. Nine animals with 18 defects were sacrificed after 6 months. Histological evaluation was performed by intravital polychrome fluorescent labelling, intravital perfusion with Indian ink, microradiographs and differential staining with toluidine blue. The quality of regenerated cartilage was assessed by in situ hybridization of collagen type II and immunohistochemistry of collagen types I and II. In 50% of the cases, defects treated with MSC-coated implants showed a complete regeneration of the subchondral bone layer. In these cases collagen type II and only traces of collagen type I were detected. A high level of collagen type II mRNA expression compared to articular cartilage indicates regenerating hyaline-like cartilage. A total of 50% of MSC-coated and uncoated implants failed to osseointegrate and formation of fibrocartilage was observed. Untreated defects as well as defects treated with uncoated implants demonstrated incomplete healing of subchondral bone and formation of fibrous cartilage. A modified histological score according to Wakitani significantly demonstrated better results for cell-coated implants (8.8+/-6.4) than for uncoated implants (5.5+/-3.9) and for untreated defects (2.8+/-2.5). Our results demonstrate that, in a significant number of cases, a partial joint resurfacing of the knee with stem cell-coated titanium implants occur. A slow bone and cartilage regeneration and an incomplete healing in half of the MSC-coated implants are limitations of the presented method. To improve our approach and optimize the experimental parameters, further investigations are needed prior to clinical application.

Animals↗

PubMed2005

Individual olfactory sensory neurons project into more than one glomerulus in Xenopus laevis tadpole olfactory bulb.

An essential step in the coding of odorants is the way olfactory sensory neurons (OSNs) convey their information to the olfactory bulb. This projection determines how the specificities of OSNs are mapped onto the spatial activity patterns of the olfactory bulb (OB). Despite the fact that virtually nothing is known about how individual OSN axons project to glomeruli, it is generally believed that OSNs always project to one glomerulus each. Our recent findings in tadpoles of Xenopus laevis challenge this view. By injection of a tracer into individual OSNs, we show for the first time that axons typically project into more than one glomerulus and that they do so in a characteristic way. Upon entering the olfactory bulb, an axon bifurcates to give two primary branches. Each of these branches bifurcates again to give two subbranches, thus resulting in four subbranches per OSN. The two subbranches of each primary branch project into two different glomeruli. Variations of this characteristic innervation pattern include the innervation of three and, occasionally, one glomerulus. In any case, and independent of the number of glomeruli innervated by an OSN, each glomerulus receives at least two axonal branches of the same OSN.

Animals↗

PubMed2004

Sizing-up finite fluorescent particles with nanometer-scale precision by convolution and correlation image analysis.

Determining the positions, shapes and sizes of finite living particles such as bacteria, mitochondria or vesicles is of interest in many biological processes. In fluorescence microscopy, algorithms that can simultaneously localize such particles as a function of time and determine the parameters of their shapes and sizes at the nanometer scale are not yet available. Here we develop two such algorithms based on convolution and correlation image analysis that take into account the position, orientation, shape and size of the object being tracked, and we compare the precision of the two algorithms using computer simulations. We show that the precision of both algorithms strongly depends on the object's size. In cases where the diameter of the object is larger than about four to five times the beam waist radius, the convolution algorithm gives a better precision than the correlation algorithm (it leads to more precise parameters), while for smaller object diameters, the correlation algorithm gives superior precision. We apply the convolution algorithm to sequences of confocal laser scanning micrographs of immobile Escherichia coli bacteria, and show that the centroid, the front end, the rear end, the left border and the right border of a bacterium can be determined with a signal-to-noise-dependent precision down to approximately 5 nm.

Algorithms↗

PubMed2004

Classes and narrowing selectivity of olfactory receptor neurons of Xenopus laevis tadpoles.

In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.

Amino Acids, Essential↗

PubMed2004

Cascades of response vectors of olfactory receptor neurons in Xenopus laevis tadpoles.

Olfactory receptor neurons (ORNs) of Xenopus laevis tadpoles respond to water-born stimuli such as amino acids. Their sensitivity spectra with respect to amino acids have recently been shown to become more selective over ontogenetic stages [Manzini & Schild (2004) J. Gen. Physiol., 123, 99-107]. In this paper, we undertake a theoretical analysis of this data set and determine the correlational relationships among odorant responses represented as binary response vectors. We first show that, on the one hand, the number of 204 ORN classes (out of 283 recorded ORNs) cannot be explained by a random expression pattern of olfactory receptors (ORs). On the other hand, this number does not appear to be reconcilable with the idea that individual ORNs express one type of OR each. The covariance matrix of stimulus responses shows that the responses to some stimuli are correlated to those of others. Furthermore, the response vectors show positive as well as negative correlations among each other. While the positive correlations can partly be explained by the differing response frequencies to the odorants used, the negative ones cannot. Finally, we analyse the similarity among responses using the Hamming distance as a distance measure, the result being that most response vectors differ from others by small Hamming distances. Such vectors are shown to form pattern cascades, possibly reflecting a decreasing number of ORs being expressed over ontogenetic stages.

Amino Acid Sequence↗

PubMed2003

Organization of glomeruli in the main olfactory bulb of Xenopus laevis tadpoles.

Structural and functional investigations were carried out to study olfactory glomeruli in the main olfactory bulb (OB) in tadpoles of the clawed frog, Xenopus laevis. Calcium imaging of odor response patterns of OB neurons revealed that the synapses within the glomeruli are functional. Tracing axons of individual olfactory receptor neurons (ORNs), dendrites of mitral/tufted (M/T) cells and processes of periglomerular interneurons indicate that the glomerular architecture is solely determined by terminal branches of ORN axons and tufts of M/T primary dendrites. The small population of periglomerular neurons forms wide-field arborizations that always extend over many glomeruli, enter the glomeruli, but lack any glomerular tufts. Antibodies to synaptophysin indicate a high density of synapses within glomeruli, which was further confirmed at the ultrastructural level and quantified to approximately 0.5 synaptic sites per microm(2). Combining immunocytochemistry and ultrastructural investigations, we show that glomeruli in Xenopus laevis tadpoles lack any cellular borders. Glomeruli are surrounded neither by periglomerular somata nor by glial processes. Taken together, our results demonstrate that olfactory glomeruli in Xenopus laevis tadpoles (1) are fully functional, (2) are spheroidal neuropil aggregations of terminal tufts of ORNs and tufts of primary dendrites of M/T cells, and (3) are not enwrapped by a border formed by juxtaglomerular cells.

Animals↗

PubMed2003

cAMP-independent olfactory transduction of amino acids in Xenopus laevis tadpoles.

Whether odorants are transduced by only one or more than one second messenger has been a long-standing question in olfactory research. In a previous study we started to address this question mainly by using calcium imaging in the olfactory bulb. Here, we present direct evidence for our earlier conclusions using the calcium imaging technique in the mucosa slice. The above question can now unambiguously be answered. We show that some olfactory receptor neurons (ORNs) respond to stimulation with amino acids with an increase of the intracellular calcium concentration [Ca2+]i. In order to see whether or not these responses were mediated by the cAMP transduction pathway we applied forskolin or the membrane-permeant cAMP analogue pCPT-cAMP to the olfactory epithelium. The ensemble of ORNs that was activated by amino acids markedly differed from the ensemble of neurons activated by forskolin or pCPT-cAMP. Less than 6 % of the responding ORNs showed a response to both amino acids and the pharmacological agents activating the cAMP transduction pathway. We conclude that ORNs of Xenopus laevis tadpoles have both cAMP-dependent and cAMP-independent olfactory transduction pathways and that most amino acids are transduced in a cAMP-independent way.

Amino Acids↗

PubMed2003

Multidrug resistance transporters in the olfactory receptor neurons of Xenopus laevis tadpoles.

Olfactory receptor neurons (ORNs) are the only class of neurons that is directly exposed to the environment. Therefore, they need to deal with xenobiotic and potentially cytotoxic substances. Here we show for the first time that ORNs possess transporter systems that expel xenobiotics across the plasma membrane. Using calcein and calcium-indicator dyes as xenobiotics, we demonstrate that ORNs appear to express the multidrug resistance P-glycoprotein (MDR1) and multidrug resistance-associated proteins (MRP). This endows ORNs with the ability to transport a large number of substrates including calcium-indicator dyes and calcein across their plasma membranes. Conversely, blocking P-glycoprotein and MRP increases the net uptake of these dyes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Neuronal representation of odourants in the olfactory bulb of Xenopus laevis tadpoles.

When an odourant enters the nose, olfactory receptor neurons (ORNs) convey information about it to the olfactory bulb (OB), where this information is processed and where the first central representations of the odourant are generated. In this paper we show how odourants are represented by ensembles of OB neurons, in particular mitral cells (MCs) which are the output neurons of the OB. We were able to demonstrate for the first time that the intracellular calcium concentrations ([Ca2+]i) in the somata of these neurons undergo specific changes and that different stimuli are represented by different neuronal [Ca2+]i patterns. The similarity of patterns was assessed by cross-correlation analysis. We further show that noradrenaline (NA), which is reported to be involved in olfactory memory formation and to modulate synaptic transmission at dendrodendritic synapses in the OB, profoundly changes the representation of odourants at the level of MCs.

Animals↗

PubMed2002

Odorant responses of Xenopus laevis tadpole olfactory neurons: a comparison between preparations.

We used a slice preparation of the olfactory epithelium of Xenopus laevis tadpoles to record odorant responses of olfactory receptor neurons (ORNs) and compared these to odorant responses recorded in isolated ORNs. The maximum recording time in the slice was considerably longer than in isolated ORNs, which is essential when many odorants are to be tested. No odorant-induced responses could be obtained from isolated ORNs recorded in the on-cell mode, while recordings in the slice (on-cell and whole-cell) as well as previously reported perforated-patch recordings in isolated ORNs of the same species () were successful, though qualitatively different. In the slice preparation, amino acids as well as an extract from Spirulina algae always induced excitatory responses, while, in a previous study on isolated ORNs, responses were either excitatory or inhibitory. The results of this study show that ORNs obtained using different preparation techniques can give markedly different responses upon the application of odorants. Our experiments indicate that the slice preparation combined with the on-cell configuration of the patch-clamp technique is the method of choice for testing many odorants on individual ORNs.

Action Potentials↗

PubMed2002

cAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons.

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch-clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well-known cAMP-mediated one. The differential processing of cAMP-mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium-imaging of olfactory bulb neurons using a novel nose-olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid-sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin-sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid- and cAMP-mediated responses are processed differentially at the level of the olfactory bulb.

Amino Acids↗

PubMed2002

Anisotropic diffusion in mitral cell dendrites revealed by fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) can be used to measure kinetic properties of single molecules in drops of solution or in cells. Here we report on FCS measurements of tetramethylrhodamine (TMR)-dextran (10 kDa) in dendrites of cultured mitral cells of Xenopus laevis tadpoles. To interpret such measurements correctly, the plasma membrane as a boundary of diffusion has to be taken into account. We show that the fluorescence data recorded from dendrites are best described by a model of anisotropic diffusion. As compared to diffusion in water, diffusion of the 10-kDa TMR-dextran along the dendrite is slowed down by a factor 1.1-2.1, whereas diffusion in lateral direction is 10-100 times slower. The dense intradendritic network of microtubules oriented parallel to the dendrite is discussed as a possible basis for the observed anisotropy. In somata, diffusion was found to be isotropic in three dimensions and 1.2-2.6 times slower than in water.

Animals↗

PubMed2002

Aggregation of f-actin in olfactory glomeruli: a common feature of glomeruli across phyla.

Using fluorophore-conjugated phalloidin, we show that filamentous (F)-actin is strongly aggregated in olfactory glomeruli within primary olfactory centers of vertebrates and insects. Our comparative study demonstrates that aggregation of F-actin is a common feature of glomeruli across phyla, and is independent of glomerular architecture and/or the presence or absence of cellular borders around glomeruli formed by neurons or glial cells. The distribution of F-actin in axonal and dendritic compartments within glomeruli, however, appears different between vertebrates and insects. The potential role of the actin-based cytoskeleton in synaptic and structural plasticity within glomeruli is discussed.

Actins↗