Fig. 6: PKA activity mediates downscaling of GABAergic synapses.
a Primary hippocampal neurons from Nbea +/+ mice stimulated with GABA upon DMSO or PKA-inhibitor KT5720 treatment, labeled for endogenous VGAT (green), endogenous gephyrin (red), and surface GABAARγ2 (blue) using specific antibodies. Arrows depict examples of colocalized signals. b Synaptic area (µm2) of triple-colocalized puncta in (a) compared between control and GABA (DMSO ***p = 0.0009; KT5720 p = 0.7661). 3 independent experiments; DMSO (control: n = 31 cells; GABA: n = 31 cells) or PKA-inhibitor KT5720 (control: n = 31 cells; GABA: n = 28 cells). c mIPSCs recorded in hippocampal neurons at DIV 12–14 treated with DMSO or PKA-inhibitor KT5720 after pre-stimulation with 100 µM GABA for 4 h. d Average mIPSCs amplitude from (c) in pA comparing neurons treated with DMSO or PKA-inhibitor KT5720 (***p = 0.0003). 3 independent experiments DMSO n = 20 cells, KT5720 n = 23 cells. e mIPSC frequency from (c) in Hz comparing Nbea +/+ and Nbea +/- neurons (p = 0.6097). 3 independent experiments DMSO n = 20 cells, KT5720 n = 23 cells. f Internalization assay in cultured hippocampal neurons conducted during treatment with DMSO (control) or PKA-inhibitor KT5720 (1 µM) and stained for cell surface GABAA Rγ2 (green) and internalized GABAARγ2 (red). g Intensity of internalized GABAARγ2 receptors, quantified relative to the number of surface receptors treated either with DMSO or PKA-inhibitor KT5720 (****p < 0.0001) 3 independent experiments, n = 28 neurons. Scale bars (a, f): 2 µm. Data are represented as mean ± SD. Statistical significance determined using One-way ANOVA (b) or unpaired Student’s t-test (d, e, g).
