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.