Browsing by Author "Vaz, Sandra H."
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- Caffeine has a dual influence on NMDA receptor-mediated glutamatergic transmission at the hippocampusPublication . Silva Martins, Robertta; D.M., Rombo; Ribeiro, Joana; Meneses, Carlos; Peralva Borges Martins, Vladimir Pedro; Ribeiro, Joaquim A.; Vaz, Sandra H.; Cussa Kubrusly, Regina Celia; Sebastião, Ana MCaffeine, a stimulant largely consumed around the world, is a non-selective adenosine receptor antagonist, and therefore caffeine actions at synapses usually, but not always, mirror those of adenosine. Importantly, different adenosine receptors with opposing regulatory actions co-exist at synapses. Through both inhibitory and excitatory high-affinity receptors (A(1)R and A(2)R, respectively), adenosine affects NMDA receptor (NMDAR) function at the hippocampus, but surprisingly, there is a lack of knowledge on the effects of caffeine upon this ionotropic glutamatergic receptor deeply involved in both positive (plasticity) and negative (excitotoxicity) synaptic actions. We thus aimed to elucidate the effects of caffeine upon NMDAR-mediated excitatory post-synaptic currents (NMDAR-EPSCs), and its implications upon neuronal Ca(2+)homeostasis. We found that caffeine (30-200 mu M) facilitates NMDAR-EPSCs on pyramidal CA1 neurons from Balbc/ByJ male mice, an action mimicked, as well as occluded, by 1,3-dipropyl-cyclopentylxantine (DPCPX, 50 nM), thus likely mediated by blockade of inhibitory A(1)Rs. This action of caffeine cannot be attributed to a pre-synaptic facilitation of transmission because caffeine even increased paired-pulse facilitation of NMDA-EPSCs, indicative of an inhibition of neurotransmitter release. Adenosine A(2A)Rs are involved in this likely pre-synaptic action since the effect of caffeine was mimicked by the A(2A)R antagonist, SCH58261 (50 nM). Furthermore, caffeine increased the frequency of Ca(2+)transients in neuronal cell culture, an action mimicked by the A(1)R antagonist, DPCPX, and prevented by NMDAR blockade with AP5 (50 mu M). Altogether, these results show for the first time an influence of caffeine on NMDA receptor activity at the hippocampus, with impact in neuronal Ca(2+)homeostasis.
- SIGAA: signaling automated analysis: a new tool for Ca2+ signaling quantification using ratiometric Ca2+ dyesPublication . Lopes, Rafael Faria; Gonçalves-Ribeiro, Joana; Sebastião, Ana M.; Meneses, Carlos; Vaz, Sandra H.Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte–astrocyte and astrocyte–neuron interactions occur through cytoplasmic Ca2+ level changes that are assessed to determine cell function and response (i.e., drug testing). The evaluation of alterations in intracellular Ca2+ levels primarily relies on fluorescence imaging techniques, performed through video recording of cells incubated with Ca2+-sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) encompassing a single cell, it is possible to draw conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA—signaling automated analysis, for astrocyte ROI-based fluorescent imaging. This tool is specifically tailored for two wavelengths excited dyes by using two inputs of Ca2+ signaling recorded frames/videos and outputting a set of features relevant to the experiment’s conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. Subsequently, SIGAA extracts intracellular Ca2+ evolution functions for all identified ROIs detects function transients, and estimates a set of features for each signal. These features closely resemble those obtained through traditional methods and software used thus far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.