12 research outputs found
Basic coding activities of populations of Xenopus laevis olfactory receptor neurons recorded with a fast confocal line illumination microscope
Das Geruchssystem ist in der Lage, mittels sogenannter kombinatorischer Kodierung einen hochdimensionalen Geruchsraum durch eine begrenzte Anzahl von olfaktorischen Rezeptorneuronen (ORN) abzutasten. Hierbei weisen verschiedene ORN-Klassen eine breite und gleichzeitig spezifische GeruchssensitivitÀt auf, durch welche ein geruchsspezifisches Antwortmuster auf Populationen von Mitral-/Tufted Zellen (M/T) des bulbus olfactoris (OB) abgebildet wird. Neueren Untersuchungen zufolge sind diese Antwortmuster nicht notwendigerweise statisch, sondern enthalten Information in ihrer zeitlichen Entwicklung. Im OB von Larven des Krallenfrosches Xenopus laevis wurde herausgefunden, dass sowohl GeruchsidentitÀt als auch -Konzentration besser vorhergesagt wird durch M/T Antwortlatenzmuster als durch durchschnittliche Feuerraten.
Diese Arbeit befasst sich mit der Messung von ORN-AktivitĂ€t auf verschiedenen raumzeitlichen Skalen. Auf der Ebene von ORN Populationen wurde mit Hilfe von konfokaler Mikroskopie und [Ca2+] -sensitiven Fluoreszenzfarbstoffen untersucht, in wie weit Latenzmuster auftreten. Es wurde gezeigt, dass Latenzmuster im Unterschied zu M/T Zellen eine geringere Vorhersagekraft fĂŒr die Geruchsstoffkonzentration besitzen als Feuerratenmuster. AuĂerdem wiesen Ensemble-Feuerraten einen gröĂeren dynamischen Bereich bezĂŒglich der Geruchsstoffkonzentration auf als Latenzen.
Durch eine Kombination von schneller (1,25 kHz) [Ca2+] -Bildgebung und whole-cell Patch-Clamp Technik in einzelnen ORNs wurde die zeitliche Entwicklung der dreidimensionalen intrazellulÀren Ca2+ -Konzentration wÀhrend eines Depolarisationspulses gemessen. Mit Hilfe von pixelweiser Angleichung eines numerischen Modells wurden Ballungen spannungsabhÀngiger Ca2+ KanÀle (VGCC) auf der OberflÀche von ORN-Somata lokalisiert. Da der durchschnittliche gemessene VGCC-Kalziumioneneinstrom einen geringen Beitrag im Vergleich zum Ca2+ Generatorstrom darstellt (<80 pA bzw. geschÀtzt 900 pA), erklÀrte sich, warum einzelne Aktionspotentiale nicht mittels [Ca2+] Bildgebung gemessen werden konnten.
BezĂŒglich VGCC-HĂ€ufung und möglicher Kolokalisation mit KaliumkanĂ€len hoher LeitfĂ€higkeit (BK) wurde der Effekt von BK Blocker Iberiotoxin auf ORN-Reizantworten untersucht. In einer Untergruppe aller ORNs wurde eine Verringerung der Antwortamplituden nach Anwendung von Iberiotoxin festgestellt.
Aus den gezeigten Ergebnissen wurde geschlossen, dass eine wichtige Funktion von Glomeruli im OB die Konversion von Geruchsinformation zwischen Feuerratenkodierung und Latenzkodierung sein mĂŒsse
miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly
Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis
miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly
Airway mucociliary regeneration and function are key players for airway defense and
are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in
COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes
and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial
mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or
infective insults. miR0449â/â mice (both alleles are deleted) showed impaired ciliated epithelial
regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense
inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously
in aged miR449â/â mice. We identified Aurora kinase A and its effector target HDAC6 as key
mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is
downregulated upon miR449 overexpression in vitro and upregulated in miR449â/â mouse lungs.
Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied
by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and
coverage in miR449â/â cells, consistent with its tubulin-deacetylating function. Altogether, our study
establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis
Preparation of olfactory cilia.
<p>(AâD) SEM micrographs, (A) Top view onto the nostrils of a Xenopus laevis tadpole, (B) one nostril, at the bottom of which a lawn of sensory clila can be seen (C, D), (E) detached cilia (arrow heads) on a Poly-L-Lysine-coated coverslip, imaged with a 100Ă objective using DIC.</p
Distribution of diffusion coefficients.
<p>Blue: histogram of best-fit results for diffusion coefficients of fluorescein from 35 cilia, with mean and standard deviation as dark gray line and light gray area, respectively. The diffusion coefficient of fluorescein in aqueous solution at 25°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039628#pone.0039628-Culbertson1" target="_blank">[14]</a> is shown in red, while values corrected for a range of (23±1)°C are shown in green.</p
FRAP scanning protocol and sample data.
<p>(A) Schematic of the three-phase scanning protocol showing the position of the illumination line in pixel coordinates. In the first phase, several full frames are acquired to determine initial fluorescence. Half-frames are acquired in the second phase at high frame rate (488 f/s) for photobleaching in the lower half of the cilium. The third phase records the fluorescence redistribution due to diffusion at a low frame rate (28 f/s). Image acquisition (blue) is delayed in respect to the mirror position signal (green) for mirror response linearity. Time axis is not to scale, number of images reduced for simplicity. (BâH) Sample frames from all FRAP phases show evolution of fluorescence distribution, scale bar 5 , frame times relative to first frame. (B) Initial fluorescence. (C,D) First and last half-frame of the bleaching phase, upper half not imaged and displayed as black. (E) First full frame of the recovery phase shows inhomogeneous fluorescence distribution. (F) Mostly homogeneous distribution after 9 frames in the recovery phase. (G) Last frame of the recovery phase. (H) 2D pixel mask used for maximum projection of 2D intensities onto 1D position on cilium. (I) Projected intensity plots (dots) for selected frames (blue: data from frame B, green: bleaching phase (tâ=â51 ms), red: E, cyan: F), and corresponding best-fits (solid lines, for full data see Fig. 3B).</p
Fit of 1D diffusion model to experimental data.
<p>(A) Experimental data, shown as 1D fluorescence distribution over frame numbers for a full experiment, with normalized fluorescence () color-coded according to color map. FRAP phases (see Fig. 2A) indicated above. (B) Corresponding data from the best-fit result of the 1D diffusion model. (C) Residuals between A and B, using a smaller colormap range.</p
Detection of silent cells, synchronization and modulatory activity in developing cellular networks
Developing networks in the immature nervous system and in cellular cultures are characterized by waves of synchronous activity in restricted clusters of cells. Synchronized activity in immature networks is proposed to regulate many different developmental processes, from neuron growth and cell migration, to the refinement of synapses, topographic maps, and the mature composition of ion channels. These emergent activity patterns are not present in all cells simultaneously within the network and more immature "silent" cells, potentially correlated with the presence of silent synapses, are prominent in different networks during early developmental periods. Many current network analyses for detection of synchronous cellular activity utilize activity-based pixel correlations to identify cellular-based regions of interest (ROIs) and coincident cell activity. However, using activity-based correlations, these methods first underestimate or ignore the inactive silent cells within the developing network and second, are difficult to apply within cell-dense regions commonly found in developing brain networks. In addition, previous methods may ignore ROIs within a network that shows transient activity patterns comprising both inactive and active periods. We developed analysis software to semi-automatically detect cells within developing neuronal networks that were imaged using calcium-sensitive reporter dyes. Using an iterative threshold, modulation of activity was tracked within individual cells across the network. The distribution pattern of both inactive and active, including synchronous cells, could be determined based on distance measures to neighboring cells and according to different anatomical layers
Activity Correlation Imaging: Visualizing Function and Structure of Neuronal Populations
For the analysis of neuronal networks it is an important yet unresolved task to relate the neurons' activities to their morphology. Here we introduce activity correlation imaging to simultaneously visualize the activity and morphology of populations of neurons. To this end we first stain the network's neurons using a membrane-permeable [Ca2+] indicator (e.g., Fluo-4/AM) and record their activities. We then exploit the recorded temporal activity patterns as a means of intrinsic contrast to visualize individual neurons' dendritic morphology. The result is a high-contrast, multicolor visualization of the neuronal network. Taking the Xenopus olfactory bulb as an example we show the activities of the mitral/tufted cells of the olfactory bulb as well as their projections into the olfactory glomeruli. This method, yielding both functional and structural information of neuronal populations, will open up unprecedented possibilities for the investigation of neuronal networks