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During the last years we have developed acquisition and analysis software for electrophysiology and imaging. Thanks to the use of this software we have been able to contribute with original data. Below, there is a list of available software from our lab. Similarly, we have developed equioment for data acquisiton.
Software:
- Gapless acquisition system of electrophysiological signals (programmed in Visual Basic and adapted to INDEC Systems interfases)
- Sofware lockin amplifier (programmed in Visual Basic and adapted to INDEC Systems interfases, designed originally by Prof. Julio Fernández)
- Software for acquisition of voltammetric signals (Visual Basic, and INDEC interfases)
- Acquisition system in Igor Pro, based on NAtional Instruments interfases (PCI 6052) or NIDAQmx.
- Software lockin amplifier based on Igor Pro and National Instruments boards
- Image acquistion software programmed in Igor Pro for cameras ORCA and PC-9100-01 (Hamamatsu DCAM) or QE (PCO Imaging). Based on drivers SIDX (Bruxton Corp)
Hardware:
- Picoamperometer for electrochemical detection of released subtances
- Intacellular amplifier (based on a design by Prof. López Barneo)
- Carbon fiber electrode puller
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Using imaging techniques to study synaptic transmission |
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For the study of exo- and endocytosis in neurons from the Central Nervous System (CNS) we use synaptic proteins tagged with Green Fluorescence Protein (GFP). Because of the difficulties to apply capacitance methods to monitor exo- and endocytosis at the level of single vesicles we use SynaptopHluorin (SpH) (Miesenbock et al, Nature 394: 192-5, 1998). This protein is obtained by tagging GFP to the carboxi terminal of synaptobrevin. This makes the GFP to be sequestred inside synaptic vesicles
This scheme shows the principle behind SpH and its application in the study of synaptic activity. The acidic interior of the vesicle (pH = 5.5) leads to a quenched SpH molecule. Upon fusion, protons scape to the extracellular medium, balancing the intravesicular pH to the external pH (7.4). Sph are unquenched and light up. On fusion pore reclosure or endocytosis teaking place somewhere in the terminal, SpH are reacidified by the proton pump, giving rise again to a SpH quenched molecule. The rapid lighting up of SpH contrats to the slower reacidification process. With the method, aspects of endocytosis that remain silent when using cell membrana capacitance measurements, are reveal nicely by this method. |
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Patch amperometry measures the patch membrane capacitance. In this mode the sensitivity of capacitance measurements increases more than 10 fold,  compare to whole cell recordings, because the capacitance in cell attached is dominated by the small capacitance of the membrane patch. Release of the exocytotic fusion events occurs into the patch pipette. Therefore, To monitor release of catecholamines from these granules the electrochemical detector needs to be placed inside the patch pipette (figure)
Inverted amplifier settings in patch amperometry. The bath is driven with the Vcommand of the "head stage" of the patch clamp amplifier and the ground electrode is located in the pipette filling solution. This makes a better reference for the carbon fiber.
Permanent and reversible fusion events measured in chromaffin cells in patch amperometry. Reversible fusion events were obtained by increasing the extracellular (only in the patch) calcium concentration to tens of mM. (see Nature Cell Biology 1, 40-44, 1999)
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