The contribution of astrocytes to the pathophysiology of AD (Alzheimer's disease) and the molecular and signalling mechanisms that potentially underlie them are still very poorly understood. However, there is mounting evidence that calcium dysregulation in astrocytes may be playing a key role. Intercellular calcium waves in astrocyte networks in vitro can be mechanically induced after Aβ (amyloid β-peptide) treatment, and spontaneously forming intercellular calcium waves have recently been shown in vivo in an APP (amyloid precursor protein)/PS1 (presenilin 1) Alzheimer's transgenic mouse model. However, spontaneous intercellular calcium transients and waves have not been observed in vitro in isolated astrocyte cultures in response to direct Aβ stimulation in the absence of potentially confounding signalling from other cell types. Here, we show that Aβ alone at relatively low concentrations is directly able to induce intracellular calcium transients and spontaneous intercellular calcium waves in isolated astrocytes in purified cultures, raising the possibility of a potential direct effect of Aβ exposure on astrocytes in vivo in the Alzheimer's brain. Waves did not occur immediately after Aβ treatment, but were delayed by many minutes before spontaneously forming, suggesting that intracellular signalling mechanisms required sufficient time to activate before intercellular effects at the network level become evident. Furthermore, the dynamics of intercellular calcium waves were heterogeneous, with distinct radial or longitudinal propagation orientations. Lastly, we also show that changes in the expression levels of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and S100B are affected by Aβ-induced calcium changes differently, with GFAP being more dependent on calcium levels than S100B.

Network signaling through astrocyte syncytiums putatively contribute to the regulation of a number of both physiological and pathophysiological processes in the mammalian central nervous system. As such, an understanding of the underlying mechanisms is critical to determining any roles played by signaling through astrocyte networks. Astrocyte signaling is primarily mediated by the propagation of intercellular calcium waves (ICW) in the sense that paracrine signaling results in measurable intracellular calcium transients. Although the molecular mechanisms are relatively well known, there is confl icting data regarding the mechanism by which the signal propagates through the network. Experimentally there is evidence for both a point source signaling model in which adenosine triphosphate (ATP) is released by an initially activated astrocyte only, and a regenerative signaling model in which downstream astrocytes release ATP. We modeled both conditions as a simple lumped parameter phenomenological diffusion model and show that the only possible mechanism that can accurately reproduce experimentally measured results is a dual signaling mechanism that incorporates elements of both proposed signaling models. Specifi cally, we were able to accurately simulate experimentally measured in vitro ICW dynamics by assuming a point source signaling model with a downstream regenerative component. These results suggest that seemingly confl icting data in the literature are actually complimentary, and represents a highly effi cient and robustly engineered signaling mechanism.Keywords: astrocytes, glial cells, calcium waves, diffusion, mathematical modeling, cell signaling INTRODUCTIONAstrocytes in the central nervous system possess a full compliment of membrane receptors and ion channels that allow them to respond to intercellular signals from both other astrocytes and from neurons on a millisecond time scale (Barres et al., 1990;MacVicar and Tse, 1988;Newman and Zahs, 1997;Salm and McCarthy, 1990;Serrano et al., 2006;Usowic et al., 1989;Winshop et al., 2007). First shown in culture (Charles et al., 1991;Cornell-Bell et al., 1990), the principal mechanism of long distance signaling in astrocyte networks are intercellular calcium waves (ICW) mediated by the extracellular diffusion of adenosine 5′-triphosphate (ATP) and related purines interacting with the P2Y family of membrane receptors (Coco et al., 2003;Guthrie et al., 1999). This causes a rise in intracellular calcium levels, which is part of a cascade that can result in the vesicular release of gliotransmitters, in particular ATP, that can signal other downstream astrocytes (i.e., thereby propagating the calcium wave) and neurons (Kang et al., 1998;Nedergaard, 1994;Parpura et al., 1994;Verkhratsky et al., 1998). More recently, ICW have been shown to occur in situ in brain slices (Dani et al., 1992;Newman and Zahs, 1997;Schipke et al., 2002) and in vivo in the rat neocortex using two photon microscopy (Hirase et al., 2004). Although the physiological and pathophysiological roles...

We introduce a framework for simulating signal propagation in geometric networks (networks that can be mapped to geometric graphs in some space) and developing algorithms that estimate (i.e., map) the state and functional topology of complex dynamic geometric networks. Within the framework, we define the key features typically present in such networks and of particular relevance to biological cellular neural networks: dynamics, signaling, observation, and control. The framework is particularly well suited for estimating functional connectivity in cellular neural networks from experimentally observable data and has been implemented using graphics processing unit high-performance computing. Computationally, the framework can simulate cellular network signaling close to or faster than real time. We further propose a standard test set of networks to measure performance and compare different mapping algorithms.

Abstract-Retinal Mu¨ller glial cells, in addition to providing homeostatic support to retinal neurons, have been shown to engage in modulation of neuronal activity and regulate vasomotor responses in the retina, among other functions. Calcium-mediated signaling in Mu¨ller cells has been implicated to play a significant role in the intracellular and intercellular interactions necessary to carry out these functions. Although the basic molecular mechanisms of calcium signaling in Mu¨ller cells have been described, the dynamics of calcium responses in Mu¨ller cells have not been fully explored. Here, we provide a quantitative characterization of calcium signaling in an in vitro model of Mu¨ller cell signaling using the rMC-1 cell line, a well-established line developed from rat Mu¨ller cells. rMC-1 cells displayed robust intracellular calcium transients and the capacity to support calcium transient-mediated intercellular calcium waves with signaling dynamics similar to that reported for Mu¨ller cells in in situ retinal preparations. Furthermore, pharmacological perturbations of intracellular calcium transients with thapsigargin and intercellular calcium waves with purinergic receptor antagonists and gap junction blockers (PPADS and FFA, respectively) suggest that the molecular mechanisms that underlie calcium signaling in rMC-1 cells has been conserved with those of Mu¨ller cells. This model provides a robust in vitro system for investigating specific mechanistic hypotheses of intra-and intercellular calcium signaling in Mu¨ller cells.