Impaired mass transfer characteristics of blood borne vasoactive species such as ATP in regions such as an arterial bifurcation have been hypothesized as a prospective mechanism in the aetiology of atherosclerotic lesions. Arterial endothelial (EC) and smooth muscle cells (SMC) respond differentially to altered local hemodynamics and produce coordinated macro-scale responses via intercellular communication. Using a computationally designed arterial segment comprising large populations of mathematically modelled coupled ECs & SMCs, we investigate their response to spatial gradients of blood borne agonist concentrations and the effect of micro-scale driven perturbation on the macro-scale. Altering homocellular (between same cell type) and heterocellular (between different cell types) intercellular coupling we simulated four cases of normal and pathological arterial segments experiencing an identical gradient in the concentration of the agonist. Results show that the heterocellular calcium (Ca2+) coupling between ECs and SMCs is important in eliciting a rapid response when the vessel segment is stimulated by the agonist gradient. In the absence of heterocellular coupling, homocellular Ca2+ coupling between smooth muscle cells is necessary for propagation of Ca2+ waves from downstream to upstream cells axially. Desynchronized intracellular Ca2+ oscillations in coupled smooth muscle cells are mandatory for this propagation. Upon decoupling the heterocellular membrane potential, the arterial segment looses the inhibitory effect of endothelial cells on the Ca2+ dynamics of underlying smooth muscle cells. The full system comprising hundreds of thousands of coupled nonlinear ordinary differential equations simulated on the massively parallel Blue Gene architecture. The use of massively parallel computational architectures shows the capability of this approach to address macro-scale phenomena driven by elementary micro-scale components of the system.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism.

The plasma membrane electrical activities of neurons that secrete gonadotropin releasing hormone (GnRH), referred to as GnRH neurons hereafter, have been studied extensively. A couple of mathematical models have been developed previously to explain different aspects of these activities including spontaneous spiking and responses to stimuli such as current injections, GnRH, thapsigargin (Tg) and apamin. The goal of this paper is to develop one single, minimal model that accounts for the experimental results reproduced by previously existing models and results that were not accounted for by these models. The latter includes two types of membrane potential bursting mechanisms and the associated calcium oscillations in the cytosol. One of them has not been reported in experimental literatures on GnRH neurons and is thus regarded as a model prediction. Other improvements achieved in this model include the incorporation of a more detailed description of calcium dynamics in a three dimensional cell body with the ion channels evenly distributed on the cell surface. Although the model is mainly based on data collected in cultured GnRH cell lines, we show that it is capable of explaining some properties of GnRH neurons observed in several of other preparations including mature GnRH neurons in hypothalamic slices. One potential explanation is suggested. A phenomenological reduction of this model into a simplified form is presented. The simplified model will facilitate the study of the roles of plasma membrane electrical activities on the pulsatile release of GnRH by these neurons when it is coupled with a model of pulsatile GnRH release based on the autoregulation mechanism. / Science, Faculty of / Mathematics, Department of / Graduate

The contractile state of microcirculatory vessels is a major determinant of the blood pressure of the whole systemic circulation. Continuous bi-directional communication exists between the endothelial cells (ECs) and smooth muscle cells (SMCs) that regulates calcium (Ca2+) dynamics in these cells. This study presents theoretical approaches to understand some of the important and currently unresolved microcirculatory phenomena. Agonist induced events at local sites have been shown to spread long distances in the microcirculation. We have developed a multicellular computational model by integrating detailed single EC and SMC models with gap junction and nitric oxide (NO) coupling to understand the mechanisms behind this effect. Simulations suggest that spreading vasodilation mainly occurs through Ca2+ independent passive conduction of hyperpolarization in RMAs. Model predicts a superior role for intercellular diffusion of inositol (1,4,5)-trisphosphate (IP3) than Ca2+ in modulating the spreading response. Endothelial derived signals are initiated even during vasoconstriction of stimulated SMCs by the movement of Ca2+ and/or IP3 into the EC which provide hyperpolarizing feedback to SMCs to counter the ongoing constriction. Myoendothelial projections (MPs) present in the ECs have been recently proposed to play a role in myoendothelial feedback. We have developed two models using compartmental and 2D finite element methods to examine the role of these MPs by adding a sub compartment in the EC to simulate MP with localization of intermediate conductance calcium activated potassium channels (IKCa) and IP3 receptors (IP3R). Both models predicted IP3 mediated high Ca2+ gradients in the MP after SMC stimulation with limited global spread. This Ca2+ transient generated a hyperpolarizing feedback of ~ 2-3mV. Endothelium derived hyperpolarizing factor (EDHF) is the dominant form of endothelial control of SMC constriction in the microcirculation. A number of factors have been proposed for the role of EDHF but no single pathway is agreed upon. We have examined the potential of myoendothelial gap junctions (MEGJs) and potassium (K+) accumulation as EDHF using two models (compartmental and 2D finite element). An extra compartment is added in SMC to simulate micro domains (MD) which have NaKα2 isoform sodium potassium pumps. Simulations predict that MEGJ coupling is much stronger in producing EDHF than alone K+ accumulation. On the contrary, K+ accumulation can alter other important parameters (EC Vm, IKCa current) and inhibit its own release as well as EDHF conduction via MEGJs. The models developed in this study are essential building blocks for future models and provide important insights to the current understanding of myoendothelial feedback and EDHF.

No description available.

The tightly regulated openings of the cardiac ryanodine receptor (RyR2) help to ensure that intracellular Ca2+- release from the sarcoplasmic reticulum (SR) can only occur when heart contractions are required. Usually this process is self-regulatory, where Ca2+ both activates and inhibits release of further Ca2+ from the SR. In the progression of heart failure some of this control is lost and in rest periods Ca2+ can leak from the SR into the cytosol. Recent evidence has suggested that Zn2+- dyshomeostasis may contribute to SR Ca2+- leak but the underlying mechanism is unclear. Using single channel electrophysiological studies in combination with live cell imaging of HEK 293 and fibroblasts, this study reveals that Zn2+, along with Ca2+ and the inhibitor Mg2+, plays a physiological role in the grading of Ca2+- release via RyR2. Importantly the data reveal that pathophysiological concentrations of Zn2+ (> 100pM) within the cytosol remove the requirement of Ca2+ to activate RyR2, resulting in irregular channel activity even in the presence of Mg2+. This increase in channel open probability due to Zn2+ is known to be associated with increased Ca2+- release events such as Ca2+ sparks suggesting that Zn2+ is a regulator of the SR Ca2+-leak current. A potential source of releasable Zn2+, which could modulate RyR2 activity in cardiomyocytes, are the acidic organelles (endosomes and lysosomes). This study provides key evidence that the two pore channels (TPCs), which are expressed on the surface of these organelles, are candidate channels for ligand-gated release of Zn2+. Importantly this research demonstrates that dysregulated Zn2+ homeostasis, resulting in elevated Zn2+ within the lysosome, has severe consequences upon cellular Ca2+- release from fibroblasts, which is primarily the result of Zn2+ acting as a pore blocker of TPC2. Together these data reveal a key role of Zn2+ as a second messenger which can regulate intracellular Ca2+- release in both health and disease.

Les activités électriques ectopiques à l’origine des épisodes de fibrillation atriale pourraient être dues à des échanges calciques anormaux dans les cardiomyocytes (CM) de veine pulmonaire (VP). Le cycle du calcium des CM de VP a donc été caractérisé et comparé à celui des CM d’oreillette gauche (OG) et de ventricule gauche (VG). Des outils ont été développés pour mesurer la régularité d’organisation des réseaux de tubules transverses et la contractilité des CM de VP. Contrairement aux CM d’OG et de VG, l’organisation hétérogène du réseau de tubules dans la population des CM de VP conduit à une grande variabilité de forme des transitoires calciques et d’amplitude de contraction. Au sein des VP, ces différents types de CM sont regroupés en îlots. La fréquence des libérations calciques spontanées est également plus grande dans les CM de VP que dans ceux d’OG et de VG. La population des CM de VP présente une dynamique calcique aux caractéristiques uniques pouvant être source d’arythmies. / Ectopic foci leading to atrial fibrillation episodes might be due to abnormal calcium handling by the pulmonary vein (PV) cardiomyocytes (CM). Therefore, the calcium cycle of PV CM was characterized and compared to those of left atria (LA) and left ventricle (LV) CM. Some tools have been developed to measure the organization of transverse tubular networks and contractility of PV CM. Unlike LA and LV CM, the heterogeneous organization of the tubular networks in the PV CM population leads to wide ranges of calcium transient shapes and contraction amplitudes. Within the whole PV, these different types of CM are gathered in islets. The frequency of spontaneous calcium release is also higher in PV CM than in LA and LV CM. The special features of the calcium handling properties of the PV CM population could be a source of arrhythmias.

Computational Neuroscience deals with spatio-temporal scales which vary considerably.For example interactions at synaptic contact regions occur on the scale of nanometers and nanoseconds to milliseconds (micro-scale) whereas networks of neurons can measure up to millimeters and signals are processed on the scale of seconds (macro-scale). Whole-cell calcium dynamics models (meso-scale) mediate between the multiple spatio-temporal scales. Of crucial importance is the calcium propagation mediated by the highly complex endoplasmic reticulum network. Most models do not account for the intricate intracellular architecture of neurons and consequently cannot resolve the interplay between structure and calcium-mediated function. To incorporate the detailed cellular architecture in intracellular Calcium models, a novel mesh generation methodology has been developed to allow for the efficient generation of computational meshes of neurons with a three-dimensionally resolved endoplasmic reticulum. Mesh generation routines are compiled into a versatile and fully automated reconstruct-and-simulation toolbox for multi-scale physics to be utilized on high-performance or regular computing infrastructures. First-principle numerical simulations on the neuronal reconstructions reveal that intracellular Calcium dynamics are effected by morphological features of the neurons, for instance a change of endoplasmic reticulum diameter leads to a significant spatio-temporal variability of the calcium signal at the soma. / Math & Science Education

The fundamental attribute of neurons is their cellular electrical excitability, which is based on the expression of a plethora of ligand- and voltage-gated membrane channels that give rise to prominent membrane currents and membrane potential variations that represent the biophysical substrate underlying the transfer and integration of information at the cellular level. Dendrites have both an electrical and a biochemical character, which are closely linked. In contrast, glial cells are non-electrically excitable but nevertheless display a form of excitability that is based on variations of the Ca2+ concentration in the cytosol rather than electrical changes in the membrane. Cytoplasmic Ca2+ serves as an intracellular signal that is responsible for controlling a multitude of cellular processes. The key to this pleiotropic role is the complex spatiotemporal organization of the [Ca2+]i rise evoked by extracellular agonists, which allows selected effectors to be recruited and specific actions to be initiated. Ca2+ handling in the cell is maintained by operation of multiple mechanisms of Ca2+ influx, internal release, diffusion, buffering and extrusion. Ca2+ tends to be a rather parochial operator with a small radius of action from its point of entry at the cytoplasm resulting in the concept of microdomains. Dendritic Ca2+ signaling have been shown to be highly compartmentalized and astrocytic processes have been reported to be constituted by hundreds of microdomains that represent the elementary units of the astrocyte Ca2+ signal, from where it can eventually propagate to other regions of the cell. The astrocyte Ca2+ elevation may thus act as intra and intercellular signal that can propagate within and between astrocytes, signaling to different regions of the cell and to different cells. The spatio-temporal features of neuron-to-astrocyte communication, results from diverse neurotransmitters and signaling pathways that converge and cooperate to shape the Ca2+ signal in astrocytes. Alterations in Ca2+ homeostasis have been shown to be associated with major pathological conditions of the brain such as epilepsy, ischemia and neurodegenerative diseases. Although there are evidences of Ca2+ rise in hippocampal neurons in in vitro models of epilepsy (Pal et al., 1999; Limbrick et al., 2001), there is no information on the Ca2+ regulatory mechanisms operating in discrete compartments of the epileptic neuron following Ca2+ influx through voltage gated calcium channels (VGCCs). In the first part of the work, the spatial and temporal profiles of depolarization induced changes in the intracellular Ca2+ concentration in the dendrites of cultured autaptic hippocampal pyramidal neurons rendered epileptic experimentally have been addressed. Our in vitro epilepsy model consisted of hippocampal neurons in autaptic culture that were grown in the presence of kynurenate and high Mg2+, and subsequently washing the preparation free of the blockers. To understand the differences in Ca2+ handling mechanisms in different compartments of a control neuron and the kynurenate treated neuron, a combination of whole-cell patch-clamp recording and fast Ca2+ imaging methods using the Ca2+ indicator Oregon Green 488 BAPTA-1 was applied. All our analysis was focused on localized regions in the dendrite that showed pronounced Ca2+ transients upon activation of high voltage activated (HVA) Ca2+ channels. The spatial extent of Ca2+ signals suggested the presence of distinct dendritic compartments that respond to the depolarizing stimulus. Further, the local Ca2+ transients were observed even in the presence of NMDA and AMPA receptor antagonists, suggesting that the opening of VGCCs primarily triggered the local Ca2+ changes. The prominent changes in intracellular Ca2+ observed in these dendritic regions appear to be sites where Ca2+ evoked dendritic exocytosis (CEDE) takes place. Since cellular Ca2+ buffers determine the amplitude and diffusional spread of neuronal Ca2+ signals, quantitative estimates of the time-dependent spread of intracellular Ca2+ in the dendritic compartments in the control and treated neurons were done using image processing techniques. Physiological changes in Ca2+ channel functioning were also induced by kynurenate treatment and one such noticeable difference was the observation of Ca2+ dependent inactivation in the treated neurons. We provide evidences of localized Ca2+ changes in the dendrites of hippocampal neurons that are rendered epileptic by kynurenate treatment, suggesting that these sites are more vulnerable (Padmashri et al., 2006). This might contribute to the epileptiform activity by local changes in cellular and membrane properties in complex ways that remains to be clearly understood. Status Epilepticus (SE), stroke and traumatic brain injury are all associated with large increases in extracellular glutamate concentrations. The concentration of glutamate in the extracellular fluid is around 3-4 µM and astrocytes are primarily responsible for the uptake of glutamate at the synapses. The extracellular levels of glutamate has been shown to increase dramatically (16 fold) in human SE suggesting an important role of glutamate in the mechanism of seizure activity and seizure related brain damage (Carlson et al., 1992). Several other studies have also shown a persistent increase in extracellular glutamate concentration to potentially neurotoxic concentrations in the epileptogenic hippocampus (During and Spencer, 1993; Sherwin, 1999; Cavus et al., 2005). We addressed the problem related to the effects of prolonged glutamate pretreatment on Ca2+ signaling in an individual astrocyte and its adjoining astrocyte (astrocyte pair), rather than on a syncytium of astrocytes in culture. Individual astrocytes may have functional domains that respond to an agonist through distinct receptor signaling systems. These are difficult to observe in studies that are done on glial syncytium because of spatial limits of image capture. This was examined with simultaneous somatic patch-pipette recording of a single astrocyte to evoke voltage-gated calcium currents, and Ca2+ imaging using the Ca2+ indicator Oregon Green 488 BAPTA-1 to identify the Ca2+ microdomains. Transient Ca2+ changes locked to the depolarization were observed in certain compartments in the astrocyte processes of the depolarized astrocyte and the responses were more pronounced in the adjoining astrocyte of the astrocyte pair. The Ca2+ transient amplitudes were enhanced on pretreatment of cells with glutamate (500 µM for 20 minutes). Estimation of local Ca2+ diffusion coefficients in the astrocytic processes indicated higher values in the adjoining astrocyte of the glutamate pretreated group. In order to understand the underlying mechanisms, we performed the experiments in the presence of different blockers for the metabotropic glutamate receptor, inositol 1,4,5 triphosphate (IP3) receptors and gap junctions. Ca2+ transients recorded on pretreatment of cells with glutamate showed attenuated responses in the presence of the metabotropic glutamate receptor (mGluR) antagonist α-Methyl(4-Carboxy-Phenyl) Glycine (MCPG). Intracellular heparin (an antagonist of IP3 receptor) introduced in the depolarized astrocyte did not affect the Ca2+ transients in the heparin loaded astrocyte, but attenuated the [Ca2+]i responses in the adjoining astrocyte suggesting that IP3 may be the transfer signal. The uncoupling agent 1-Octanol attenuated the [Ca2+]i responses in the adjoining cell of the astrocyte pair in both the control and glutamate pretreated astrocytes indicating the role of gap junctional communication. The findings of [Ca2+]i responses within discrete regions of astrocytic processes suggest that astrocytes may be comprised of microdomains whose properties are altered by glutamate pretreatment. The data also indicates that glutamate induced alterations in Ca2+ signaling in the astrocyte pair may be mediated through phospholipase C (PLC), IP3, internal Ca2+ stores, VGCCs and gap junction channels (Padmashri and Sikdar, 2006). Neuronal (EAAC-1) and glial (GLT-1 and GLAST) glutamate transporters facilitate glutamate reuptake after synaptic release. Transgenic mice with GLT-1 knockout display spontaneous epileptic activity (Tanaka et al., 1997) and loss of glial glutamate transporters using chronic antisense nucleotide administration was reported to result in elevated extracellular glutamate levels and neurodegeneration characteristic of excitotoxity (Rothstein et al., 1996). Dysfunction of glutamate transporters and the resulting increase of glutamate have been speculated to play an important role in infantile epilepsies (Demarque et al., 2004). We examined the effects of pretreatment with glutamate in the presence of the glutamate transport inhibitor threo-β-hydroxy-aspartate (TBHA) and in Na+-free extracellular medium to understand whether this resulted in any alteration in the astrocytic intracellular Ca2+ dynamics following activation of voltage gated calcium channels. The Ca2+ responses were found to be attenuated in both the cases indicating that the elevated levels of extracellular glutamate due to blockade of glutamate transporters may influence the responses mediated by the astrocytic glutamate receptors. Our studies indicate that the heightened extracellular glutamate concentration is not gliotoxic in our experimental system, although it may have a profound effect on altering the activity of surrounding neurons which was not addressed in the present work. Several studies have indicated that neurons control the level of gap junction mediated communication between astrocytes (Giaume and McCarthy, 1996; Rouach et al, 2000). All our earlier studies were done on process bearing astrocytes that were co-cultured with neurons. We have addressed the question as to whether the spatio-temporal changes in [Ca2+]i in astrocyte pairs differ if the astrocytes are cultured in the absence of neurons. The results indicate that there is indeed a significant reduction in the responses that are evoked in response to the depolarization pulse in the adjoining cell of the astrocyte pair. These experiments demonstrate that neurons in the cocultures may selectively enhance the Ca2+ responses possibly by increasing the coupling between the two cells.