Precise 3D modulation of electro-optical parameters during neurotransmitter uncaging experiments with neurons in vitro

Ruthenium\u2013bipyridinetriphenylphosphine\u2013GABA (RuBi\u2013GABA) is a caged compound that allows studying the neuronal transmission in a specific region of a neuron. The inhibitory neurotransmitter \u3b3-aminobutyric acid (GABA) is bound to a caged group that blocks the interaction of the neurotransmitter with its receptor site. Following linear\u2014one-photon (1P)\u2014and non-linear\u2014multi-photon\u2014absorption of light, the covalent bond of the caged molecule is broken, and GABA is released. Such a controlled release in time and space allows investigating the interaction with its receptor in four dimensions (X,Y,Z,t). Taking advantage of this strategy, we succeeded in addressing the modulation of GABAA in rat cerebellar neurons by coupling the photoactivation process, by confocal or two-photon excitation microscopy, with the electrophysiological technique of the patch-clamp in the whole-cell configuration. Key parameters have been comprehensively investigated and correlated in a temporally and spatially confined way, namely: photoactivation laser power, time of exposure, and distance of the uncaging point from the cell of interest along the X, Y, Z spatial coordinates. The goal of studying specific biological events as a function of controlled physical parameters has been achieved

Super-resolution 3D maps to study the antisecretory factor's effects on different GABAa receptor subunits in rat cerebellar granule cells in vitro

The Antisecretory Factor (AF) is an endogenous protein that inhibits intestinal hypersecretion and various inflammation disorders in vivo. AF has been detected in many mammalian tissues and plasma, but its mechanisms of action are essentially unknown. Previously, we studied the pharmacological action of the AF on GABAA receptors in cerebellar granule cells, comparing the electrophysiological response evoked by two-photon mediated release of caged GABA compounds before and after the administration of AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed an increase in the GABAA receptor responses, particularly in those containing the α6 subunit. To figure out the interactions of AF with GABAA receptors in the same cellular model (cerebellar granule cells), we combined immunofluorescence subunits’ staining with confocal and super-resolution microscopy. In particular, we took advantage of an innovative imaging technique that combines stimulated emission depletion (STED) with fluorescence lifetime microscopy (FLIM) to collect super-resolution 3D maps of different subunits distributed on the neuron cell membrane. We explored different approaches to analyze super-resolution fluorescence images obtained by labeling α1 and α6 subunits before and after 1-hour incubation with AF-16. Comparing pre- and post-treatment maps, we found differences in how different subunit populations respond to AF treatment. We critically analyzed these new experimental findings with our previous electrophysiological data to widen the knowledge of the mechanisms of interaction between GABAA receptor subunits and AF protein

Involvement of GABA(A) receptors containing alpha(6) subtypes in antisecretory factor activity on rat cerebellar granule cells studied by two-photon uncaging

The antisecretory factor (AF) is an endogenous protein that counteracts intestinal hypersecretion and various inflammation conditions in vivo. It has been detected in many mammalian tissues and plasma, but its mechanisms of action are largely unknown. To study the pharmacological action of the AF on different GABA(A) receptor populations in cerebellar granule cells, we took advantage of the two-photon uncaging method as this technique allows to stimulate the cell locally in well-identified plasma membrane parts. We compared the electrophysiological response evoked by releasing a caged GABA compound on the soma, the axon initial segment and neurites before and after administering AF-16, a 16 amino acids long peptide obtained from the amino-terminal end of the AF protein. After the treatment with AF-16, we observed peak current increases of varying magnitude depending on the neuronal region. Thus, studying the effects of furosemide and AF-16 on the electrophysiological behaviour of cerebellar granules, we suggest that GABA(A) receptors, containing the alpha(6) subunit, may be specifically involved in the increase of the peak current by AF, and different receptor subtype distribution may be responsible for differences in this increase on the cell

Current challenges and future trends in analytical fragility and vulnerability modeling

none13siThe lack of empirical data regarding earthquake damage or losses has propelled the development of dozens of analytical methodologies for the derivation of fragility and vulnerability functions. Each method will naturally have its strengths and weaknesses, which will consequently affect the associated risk estimates. With the purpose of sharing knowledge on vulnerability modeling, identifying shortcomings in the existing methods, and recommending improvements to the current practice, a group of vulnerability experts met in Pavia (Italy) in April 2017. Critical topics related to the selection of ground motion records, modeling of complex real structures through simplified approaches, propagation of aleatory and epistemic uncertainties, and validation of vulnerability results were discussed, and suggestions were proposed to improve the reliability and accuracy in vulnerability modeling.mixedSilva V.; Akkar S.; Baker J.; Bazzurro P.; Castro J.M.; Crowley H.; Dolsek M.; Galasso C.; Lagomarsino S.; Monteiro R.; Perrone D.; Pitilakis K.; Vamvatsikos D.Silva, V.; Akkar, S.; Baker, J.; Bazzurro, P.; Castro, J. M.; Crowley, H.; Dolsek, M.; Galasso, C.; Lagomarsino, S.; Monteiro, R.; Perrone, D.; Pitilakis, K.; Vamvatsikos, D

Framework for seismic hazard analysis of spatially distributed systems

The analysis of seismic risk to multiple systems of spatially distributed infrastructures presents new challenges in the characterisation of the seismic hazard input. For this purpose a general procedure entitled “Shakefield” is established, which allows for the generation of samples of ground motion fields for both single scenario events, and for stochastically generated sets of events needed for probabilistic seismic risk analysis. For a spatially distributed infrastructure of vulnerable elements, the spatial correlation of the ground motion fields for different measures of the ground motion intensity is incorporated into the simulation procedure. This is extended further to consider spatial cross-correlation between different measures of ground motion intensity. In addition to the characterisation of the seismic hazard from transient ground motion, the simulation procedure is extended to consider secondary geotechnical effects from earthquake shaking. Thus the Shakefield procedure can also characterise the effects site amplification and transient strain, and also provide estimates of permanent ground displacement due to liquefaction, slope displacement and coseismic fault rupture