Recent evidence demonstrates that low-frequency oscillations of intracellular calcium on timescales of seconds to minutes drive distinct aspects of neuronal development, but the mechanisms by which these calcium transients are coupled to signaling cascades are not well understood. Here we test the hypothesis that spontaneous electrical activity activates protein kinase A (PKA). We use live-cell indicators to observe spontaneous and evoked changes in cAMP levels and PKA activity in developing retinal neurons. Expression of cAMP and PKA indicators in neonatal rat retinal explants reveals spontaneous oscillations in PKA activity that are temporally correlated with spontaneous depolarizations associated with retinal waves. In response to short applications of forskolin, dopamine, or high-potassium concentration, we image an increase in cAMP levels and PKA activity, indicating that this second-messenger pathway can be activated quickly by neural activity. Depolarization-evoked increases in PKA activity were blocked by the removal of extracellular calcium, indicating that they are mediated by a calcium-dependent mechanism. These findings demonstrate for the first time that spontaneous activity in developing circuits is correlated with activation of the cAMP/PKA pathway and that PKA activity is turned on and off on the timescale of tens of seconds. These results show a link between neural activity and an intracellular biochemical cascade associated with plasticity, axon guidance, and neural differentiation.

Neurons undergo long term, activity dependent changes that are mediated by activation of second messenger cascades. In particular, calcium-dependent activation of the cyclic-AMP/Protein kinase A signaling cascade has been implicated in several developmental processes including cell survival, axonal outgrowth, and axonal refinement. The biochemical link between calcium influx and the activation of the cAMP/PKA pathway is primarily mediated through adenylate cyclases. Here, dual imaging of intracellular calcium concentration and PKA activity was used to assay the role of different classes of calcium-dependent adenylate cyclases (ACs) in the activation of the cAMP/PKA pathway in retinal ganglion cells (RGCs). Surprisingly, depolarization-induced calcium-dependent PKA transients persist in barrelless mice lacking AC1, the predominant calcium-dependent adenylate cyclase in RGCs, as well as in double knockout mice lacking both AC1 and AC8. Furthermore, in a subset of RGCs, depolarization-induced PKA transients persist during the inhibition of all transmembrane adenylate cyclases. These results are consistent with the existence of a soluble adenylate cyclase that plays a role in calcium-dependent activation of the cAMP/PKA cascade in neurons.

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A new reconstructed Discontinuous Galerkin (rDG) method, based on orthogonal basis/test functions, is developed for fluid flows on unstructured meshes. Orthogonality of basis functions is essential for enabling robust and efficient fully-implicit Newton-Krylov based time integration. The method is designed for generic partial differential equations, including transient, hyperbolic, parabolic or elliptic operators, which are attributed to many multiphysics problems. We demonstrate the method's capabilities for solving compressible fluid-solid systems (in the low Mach number limit), with phase change (melting/solidification), as motivated by applications in Additive Manufacturing (AM). We focus on the method's accuracy (in both space and time), as well as robustness and solvability of the system of linear equations involved in the linearization steps of Newton-based methods. The performance of the developed method is investigated for highly-stiff problems with melting/solidification, emphasizing the advantages from tight coupling of mass, momentum and energy conservation equations, as well as orthogonality of basis functions, which leads to better conditioning of the underlying (approximate) Jacobian matrices, and rapid convergence of the Krylov-based linear solver.

A characteristic feature of developing neural circuits is that they are spontaneously active. There are several examples, including the retina, spinal cord, and hippocampus, where spontaneous activity is highly correlated among neighboring cells, with large depolarizing events occurring with a periodicity on the order of minutes. One likely mechanism by which neurons can "decode" these slow oscillations is through activation of second messenger cascades that either influence transcriptional activity or drive posttranslational modifications. Here, we describe recent experiments where imaging has been used to characterize slow oscillations in the cAMP/PKA second messenger cascade in retinal neurons. We review the latest techniques in imaging this specific second messenger cascade, its intimate relationship with changes in intracellular calcium concentration, and several hypotheses regarding its role in neurodevelopment.