Fig 2: Characterization of interneurons used for SPT of a7-nAChRs at 21 div.(A) Fluorescent a-BT (red) selectively labels a7-nAChRs in interneurons positive for vesicular inhibitory amino acid transporter (VIAAT;?). Neighboring neurons show no somatic VIAAT staining (•) but are surrounded by GABAergic synapses. (B) Fluorescent a-BT clusters are opposed to the presynaptic marker Synapsin-1, but larger clusters are presumably extrasynaptic (). (C–D) Predominant perisynaptic localization of fluorescent a-BT clusters, as shown by their apposition () to mCherry-Homer1c and EGFP-gephyrin clusters in living interneurons transfected with one of these markers. (E–F) Segregation of mCherry-Homer1c and EGFP-gephyrin (co-transfected by magnetofection at 11 div) between excitatory and inhibitory postsynaptic sites, as shown by immunofluorescence staining for VIAAT (E–E') and vGluT1 (F–F') at 21 div. Note the selective opposing of the labeled terminals to the corresponding postsynaptic marker. Image was enlarged to depict the pixel array detected by the CCD camera. Scale bars: A, 40 µm; B–F, 5 µm.

Fig 3: OGD induces rapid GABAAR dispersal from inhibitory postsynaptic sites(A) dSTORM images of VGAT-positive inhibitory synapses under control conditions or following a 5-min OGD insult. Surface GABAARs (?2; teal) and gephyrin (magenta) are shown.(B) Localization maps of images in (A) showing individual localization points of GABAARs (teal) and gephyrin (magenta). The shaded magenta region delineates the gephyrin scaffold boundary. GABAAR localizations that overlap with the gephyrin scaffold are represented in dark blue.(C–E) Quantification of the (C) gephyrin scaffold boundary area, (D) mean gephyrin localization density, and (E) GABAAR localization enrichment within the synaptic region in control and OGD conditions; n = 58–78 synapses per condition.Values represent mean ± SEM. **p < 0.01; Mann-Whitney tests (C–E).

Fig 4: VGAT puncta density decreases in dentate gyrus (DG), area CA3, and area CA1 two weeks following noise exposure. Schematic granule cell in DG (a), pyramidal neuron in CA3 (b), and pyramidal neuron in CA1 (c), depicting organization of inputs corresponding to the layers on the right, which are images of VGAT labeling in CA1 at 400x magnification. Scale bar, 50 µm. Mean (±SEM) VGAT puncta density (per 104 µm2; normalized to respective control) in the dentate gyrus (DG) (a), area CA3 (b), and area CA1 (c) two weeks following noise exposure is shown on respective right panels. #When compared to the controls (dashed line), p = 0.05. mld: distal region of molecular layer; mlm: middle region of molecular layer; mlp: proximal region of molecular layer; g: granule cell layer; h: hilus; s.o: stratum oriens; s.p: stratum pyramidale; s.l: stratum lucidum; s.r: stratum radiatum; s.rp: proximal region of stratum radiatum; s.rd: distal region of stratum radiatum; s.lm: stratum lacunosum-moleculare.

Fig 5: GABAAR-?2 serine 327 regulates OGD-dependent GABAAR declustering(A) Hippocampal neuronal lysates analyzed following a 30-min OGD insult with and without CaN inhibitors; CsA (5 µM) and FK506 (5 µM). Phospho-GABAAR-?2 was normalized to total GABAAR-?2. n = 5–6.(B)Hippocampal crude membrane lysates from mice following sham or CA/CPR procedure. Phospho-GABAAR-?2 was normalized to total GABAAR-?2. n = 5 animals per condition.(C) Hippocampal neuronal lysates analyzed at 3-min intervals up to a maximum of a 15-min OGD insult. Phospho-GABAAR-?2 was normalized to total GABAAR-?2. n = 5.(D) Confocal images of dendritic segments from hippocampal neurons expressing super-ecliptic pHluorin (SEP)-tagged GABAAR-?2 and labeled for gephyrin and VGAT. Neurons expressing WT or Mut ?2-S327 were treated with control or OGD conditions for 30 min. Arrows, VGAT-positive synapses.(E) Quantification of synapse cluster area and density from (D); n = 30–36 neurons.Values represent mean ± SEM. **p < 0.01 and ****p < 0.0001; one-way ANOVA; Bonferroni post hoc test (A, C, and E) or t test (B). See also Figure S5.