Computational protocol: Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

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Protocol publication

[…] Purine biosensors (Sarissa Biomedical, Coventry, United Kingdom) were used to detect extracellular levels of either adenosine (ADO) or inosine (INO). Each biosensor included a platinum wire 50 μm in diameter and 0.5 mm in length, coated with a permselective layer and an enzymatic matrix surrounding the platinum electrode. For the ADO biosensors, the enzymatic layer contained nucleoside phosphorylase, xanthine oxidase, and adenosine deaminase, whereas the INO biosensors lacked adenosine deaminase and were sensitive to adenosine. Amperometric measurements started when hydrogen peroxide oxidized the platinum wire to create an electrical signal proportional to the purine concentration. Measurements with adenosine biosensors gave a total purinergic concentration of adenosine and its metabolites, referred to as ADO’. In contrast, INO sensors were used to measure the response to inosine and its metabolites. We thus obtained a specific ADO signal by subtracting the signal of INO sensors from ADO sensors; approximately 55% of the signal from the ADO/INO biosensor was specific to adenosine in our conditions. Before use, all biosensors were rehydrated in a buffer containing (in mM): 100 NaCl, 50 Tris, 20 glycerol, 1 MgCl2, and 1 CaCl2. All biosensor sets were calibrated at the beginning and end of each experiment with 10 μM adenosine or inosine. The ADO and INO recordings were performed after a 30 min stabilization period. The sensors were inserted sequentially during a 3-minute period in the regions of interest in aCSF containing the following concentrations of glucose: first 1 mM, followed by 5 mM, and again 1 mM. Each recording at a different concentration was separated by 20 min. Biosensors were placed in contact with the surface of the slice by micromanipulators, which was visually confirmed as a slight deformation on its surface, indicating that the electrode was properly embedded. During the experiment, microelectrode biosensors were polarized at +500 mV versus Ag/AgCl. Purine concentrations were assessed by subtracting the electrical signal of the sensor when inserted into the region of interest from the mean of the electrical signals measured 30 s before and after the insertion. Biosensor signals were acquired with an amperometric detector (AMU 110; Radiometer Analytical, Villeurbanne, France) using an acquisition board (Digidata 1322A; Molecular Devices) attached to a computer running the pCLAMP 9.2 software package (Molecular Devices). Analyses were performed using the Clampfit (Molecular Devices), SigmaPlot (Systat) and Python software programs. […]

Pipeline specifications

Software tools pCLAMP, SigmaPlot
Applications Miscellaneous, Patch-clamp
Chemicals Adenosine, Glucose, Purines