Figure 4. DBI is released by satellite glia cells.

A, Detection of DBI by ELISA in the extracellular media from total dissociated DRG culture (containing all the cell types dissociated from the ganglion – ‘mixed DRG culture’) and from purified SGC culture (see Methods; ‘purified SGCs’). Measurements were made after 30 min incubation in the control medium and in the medium with high extracellular [K⁺] (150 mM; ‘High-K⁺’). B–G, detection of an endozepine release by the cultured purified SGCs using the ‘reporter’ HEK293 cells transfected with α1, β2, and γ2 subunits of GABA_A receptors and a halide-sensitive EYFP mutant (H148Q/I152L; EYFP-QL). B, micrographs depicting co-culture of the reporter HEK293 cells (green) with the purified primary SGC culture. C, Schematic of the experimental timeline. D, example of the experiment: reporter HEK293 cells alone (black line) or in co-culture with the SGCs (red line) are imaged in the presence of 5 mM extracellular iodide. After introduction of the I⁻-containing solution, the perfusion is stopped for 5 min to allow for releasable molecules to accumulate. When GABA_A receptors are activated, I⁻ enters the cells and produces EYFP-QL fluorescence quenching. GABA (5 μM) is added at the end of experiment to authenticate the fluorescence quenching. E–G, Mean data for the EYFP-QL quenching of the reporter HEK293 cells only (black symbols) or in co-culture with the SGCs (blue symbols) in control conditions (E) or in the presence of the GABA_A receptor blocker, bicuculline (50 μM; F) or the benzodiazepine antagonist, flumazenil (8 μM; G). **, ***, indicate significant difference from the reporter HEK293 cells only at p<0.01, or p<0.001, respectively; unpaired t-test).