No description available.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus project axons from the supraoptic nucleus to the posterior pituitary gland, where they secrete either oxytocin or vasopressin into the circulation. Oxytocin is important for delivery at birth and is essential for milk ejection during suckling. Vasopressin primarily promotes water reabsorption in the kidney to maintain body fluid balance. The profile of oxytocin and vasopressin secretion is principally determined by the pattern of action potentials initiated at the cell bodies in the hypothalamus. MNCs principally secrete hormones from terminals in the pituitary, but the secretion also occurs from their dendrites in the supraoptic nucleus, where they diffuse and affect the neighbouring cells. Mechanisms controlling the oxytocin and vasopressin secretion from MNCs have been extensively studied over the last decades and it is assumed that the relationship between Ca2+ signalling, secretion from dendrites, and the firing patterns is essential in understanding the magnocellular neurosecretory system. In this project, we combine mathematical analysis and experimental measurements of Ca2+ activity of MNCs of transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein (AVP-eGFP) or oxytocin-monomeric red fluorescent...

No description available.

No description available.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.

No description available.

No description available.

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves and show spontaneous calcium activity in vivo and in vitro. They express purinergic receptors and calcium transients can be evoked by ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10-16 to 10-4 mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18% ± 1.65 (mean ± SEM) of the sMF (N=48 experiments). ATP significantly increased calcium activity even at 10-16 mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1μM; A-317491, 1μM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence. Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca2+ response in cultured sMF at very low concentrations, most likely involving ionotropic P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.:1. Introduction............................................................................ 1 1.1. Anatomy and histology of the human urinary bladder..................... 1 1.1.1. Anatomy of the human urinary bladder..................................... 1 1.1.2. Structure of the human urinary bladder wall............................... 2 1.2. Normal bladder function and bladder dysfunction.......................... 3 1.2.1 Normal bladder function......................................................... 3 1.2.2 Sensory aspect.................................................................... 4 1.2.3 Overactivity or hypersensitivity of bladder.................................. 5 1.3 The role of functional cell types and interaction in urinary bladder... 6 1.3.1 The role of urothelium.......................................................... 7 1.3.2Theroleofsuburotheliamyofibroblast...................................... 7 1.3.3Theroleofdetrusorsmoothmusclecells.................................. 9 1.3.4 Possible interactions in urinary bladder cell types........................ 10 1.4 ATP function and Purinergic signalling in bladder........................... 11 1.5 Spontaneous activity of bladder................................................... 13 2. Objective.................................................................................. 15 3. Material and methods............................................................... 16 3.1. Ethics Statement........................................................................ 16 3.2. Cell preparation.......................................................................... 16 3.3. Solutions and chemicals............................................................. 19 3.4. Intracellular calcium measurements............................................. 20 2.4.1. Preparing cells for Calcium Imaging.......................................... 20 2.4.2. Preparing workspace of calcium imaging................................... 20 2.4.3. Calcium imaging recording...................................................... 22 3.5 Data analysis with automated Fluorescence analysis..................... 22 3.6 Confocal Immunofluorescence.................................................... 25 3.7 Statistics................................................................................. 26 4. Results.................................................................................. 27 4.1 Spontaneous calcium activity of sMF........................................... 27 4.2 ATP effects on calcium response in sMF...................................... 27 4.3 Analysis of purinergic receptors involved.................................... 30 3.3.1 Agonist stimulation.............................................................. 30 3.3.2 Signal inhibition by specific antagonists................................... 31 4.4 Confocal immunofluorescence of purinergic receptors.................. 32 5. Discussion............................................................................. 34 5.1 Myofibroblast identification....................................................... 34 5.2 Spontaneous activity in the bladder............................................ 36 5.3 ATP modulated calcium activity in sMF....................................... 37 5.4 purinergic signalling in sMF........................................................ 39 6. Summary................................................................................ 42 7. References.............................................................................. 45 Declaration............................................................................. 50 Acknowledgements................................................................. 51

From a physical perspective biological cells consist of active soft matter that exist in a thermodynamic state far from equilibrium. Not only in muscles but also during cell proliferation, wound healing, embryonic development, and many other physiological tasks, generation of forces on the scale of whole cells is required. To date, cellular contractions have been ascribed to adhesion dependent processes such as myosin driven stress fiber formation and the development of focal adhesion complexes. In this thesis it is shown for the first time that contractions can occur independently of focal adhesions in single suspended cells. To measure mechanical properties of suspended cells the Optical Stretcher – a dualbeam laser trap – was used with phase contrast video microscopy which allowed to extract the deformation of the cell for every single frame. For fluorescence imaging confocal laser scanning microscopy was employed. The ratio of the fluorescence of a temperature sensitive and a temperature insensitive rhodamine dye was utilized to determine the temperatures inside the optical trap during and after Optical Stretching. The rise in temperature at a measuring power of 0.7W turned out to be enough to open a temperature sensitive ion channel transfected into an epithelial cell line. In this way a massive Ca2+ influx was triggered during the Optical Stretcher experiment. A new setup combining Optical Stretching and confocal laser scanning microscopy allowed fluorescence imaging of these Ca2+ signals while the cells were deformed by optically induced surface forces, showing that the Ca2+ influx could be manipulated with adequate drugs. This model system was then employed to investigate the influence of Ca2+ on the observed contractions, revealing that they are partially triggered by Ca2+. A phenomenological mathematical model based on the fundamental constitutive equation for linear viscoelastic materials extended by a term accounting for active contractions allowed to quantify the activity of the measured cells. The skewness and the median of the strain distributions were shown to depend on the activity of the cells. The introduced model reveals that even in measurements, that seemingly are describable by passive viscoelasticity, active contractililty might be superimposed. Ignoring this effect will lead to erroneous material properties and misinterpretation of the data. Taken together, the findings presented in this thesis demonstrate that active processes are an essential part of cellular mechanics and cells can contract even independently of adhesions. The results provide a method that allows to quantify active contractions of suspended cells. As the proposed model is not based on specific assumptions on force generating processes, it paves the way for a thorough investigation of different influences, such as cytoskeletal structures and intra-cellular signaling processes, to cellular contractions. The results present an important contribution for better mechanical classification of cells in future research with possible implications for medical diagnosis and therapy.

Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits.