Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity

Abstract

GABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABA_ARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABA_AR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Keywords: GABA(A) receptor; OGD; SIM; calcineurin; calpain; dSTORM; gephyrin; inhibitory synapse; ischemia; super-resolution microscopy.

Figure 1.. Ischemic insult induces rapid removal of synaptic GABA_ARs and gephyrin from inhibitory synaptic sites

(A) Confocal images of hippocampal neurons labeled for synaptic and surface GABA_AR-γ2, gephyrin, and VGAT. Neurons were treated with control or OGD conditions for 30 min. Arrows, VGAT-positive synapses. (B) Quantification of cluster area and density from (A) for dendritic and somatic compartments, n = 35–36 neurons per condition. (C) Representative images of CA1 hippocampal slices from sham or CA/CPR mice labeled for GABA_AR-γ2, gephyrin, and VGAT. (D) Quantification of cluster area and density from (C) for S. radiatum and pyramidale regions; n = 5 mice per condition. Values represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Student’s t test (B and D). See also Figure S1.

Figure 2.. GABA_ARs and presynaptic active zone proteins are lost prior to gephyrin during excitotoxic-induced inhibitory synapse disassembly

(A) 3D-SIM maximum projections of hippocampal neuronal dendrites labeled for surface GABA_ARs, gephyrin, and VGAT. Arrows, VGAT-positive synapses. Neurons were fixed at 5-min intervals up to 25 min of OGD. (B) Quantification of GABA_AR, gephyrin, and VGAT compartment volumes at 5-min intervals throughout the 25-min OGD insult. n = 158–166 synapses. Colored stars compare statistical significance to the control condition. (C) Quantification of percentage of synapses (VGAT +ve) with GABA_ARs or gephyrin present at each time point (bars). VGAT density is plotted as a percentage normalized to the control (line). n = 158–166 synapses. (D) 3D-SIM maximum projections of hippocampal neuronal dendrites labeled for VGAT and the active zone proteins RIM1 (left) or Bassoon (right). Arrows, VGAT-positive synapses. (E and F) Quantification of VGAT compartment volumes with either (E) RIM1 or (F) Bassoon compartment volumes at 5-min intervals throughout the 25-min OGD insult. n = 115–122 synapses. Colored stars compare statistical significance to the control condition. Values represent mean or percentage of neuron population ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001; one-way ANOVA; Bonferroni post hoc test (B, E, and F). See also Figure S2.

Figure 3.. Rapid nanoscale alterations to GABA_AR sub-synaptic domains (SSDs) immediately following OGD induction

(A) 3D-SIM maximum projections of individual dendritic inhibitory synapses labeled for synaptic and surface GABA_ARs, gephyrin, and VGAT. Neurons were fixed at 5-min intervals up to a maximum 25 min of OGD. Arrows, individual GABA_AR SSDs. Scale bar represents 500 nm. (B) Schematic of SSD parameters measured by 3D-SIM segmentation. (C and D) Quantification of the (C) mean SSD volume and (D) mean number of SSDs per compartment. n = 158–166 synapses; stars compare statistical significance to the control condition. (E) 3D-SIM maximum projections of individual somatic inhibitory synapses labeled for synaptic and surface GABA_ARs, gephyrin, and VGAT. Neurons were fixed at 5-min intervals up to a maximum 25 min of OGD. Arrows, individual GABA_AR SSDs. Scale bar represents 500 nm. (F–H) Quantification of the (F) compartment volume, (G) mean SSD volume, and (H) mean number of SSDs per compartment. n = 115–122 synapses; stars compare statistical significance to the control condition. Values represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; one-way ANOVA; Bonferroni post hoc test (C, D, and F–H). See also Figure S3.

Figure 4.. OGD induces rapid GABA_AR dispersal from inhibitory postsynaptic sites

(A) dSTORM images of VGAT-positive inhibitory synapses under control conditions or following a 5-min OGD insult. Surface GABA_ARs (γ2; teal) and gephyrin (magenta) are shown. (B) Localization maps of images in (A) showing individual localization points of GABA_ARs (teal) and gephyrin (magenta). The shaded magenta region delineates the gephyrin scaffold boundary. GABA_AR 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) GABA_AR 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).

Figure 5.. Calcineurin activity is required for OGD-induced GABA_AR nanoscale reorganization and removal from synapses

(A) Confocal images of dendritic segments from hippocampal neurons labeled for synaptic GABA_ARs, gephyrin, and VGAT. Neurons were treated with control or OGD conditions for 30 min in the presence of DMSO (DM), cyclosporin A (CsA) (5 μM), or FK506 (5 μM). Arrows, VGAT-positive synapses. (B) Quantification of synapse cluster area and density from images in (A); n = 27–33 neurons. (C) 3D-SIM maximum projections of individual synapses labeled for synaptic GABA_ARs, gephyrin, and VGAT. Neurons were treated with control or OGD conditions for 5 min in the presence of DM or CsA. Arrows, individual GABA_AR SSDs. Scale bars represent 500 nm. (D) Quantification of compartment volume, mean SSD volume, and mean SSD number per compartment for gephyrin and GABA_ARs. n = 159–163 synapses. (E) dSTORM images of VGAT-positive inhibitory synapses labeled for surface GABA_ARs (orange) and gephyrin (green). Neurons were treated with control or OGD conditions for 5 min in the presence of DM or CsA. Localization maps of dSTORM images show localization points for GABA_ARs and gephyrin. The shaded green region delineates the gephyrin scaffold boundary. GABA_AR localizations that overlap with gephyrin are represented in dark orange. (F) Quantification of the GABA_AR localization enrichment in the synaptic region following control, OGD 5 min, and OGD 5 min + CsA treatments; n = 65–84 synapses per condition. Values represent mean ± SEM. **p < 0.01 and ****p < 0.0001; one-way ANOVA; Bonferroni post hoc test (B, D, and F). See also Figure S4.

Figure 6.. GABA_AR-γ2 serine 327 regulates OGD-dependent GABA_AR 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-GABA_AR-γ2 was normalized to total GABA_AR-γ2. n = 5–6. (B)Hippocampal crude membrane lysates from mice following sham or CA/CPR procedure. Phospho-GABA_AR-γ2 was normalized to total GABA_AR-γ2. 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-GABA_AR-γ2 was normalized to total GABA_AR-γ2. n = 5. (D) Confocal images of dendritic segments from hippocampal neurons expressing super-ecliptic pHluorin (SEP)-tagged GABA_AR-γ2 and labeled for gephyrin and VGAT. Neurons expressing WT or Mut γ2-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.

Figure 7.. Calcineurin and calpain inhibition are both required to preserve inhibitory synaptic structure following OGD

(A) Hippocampal neuronal lysates analyzed following a 30-min OGD insult in the absence and presence of a calpain inhibitor (MDL-28170, Calp-i 100 μM). Cleaved gephyrin levels (50 kDa) were normalized to full-length gephyrin levels (~100 kDa); n = 5. (B) Hippocampal neuronal lysates analyzed at 3-min intervals up to 15 min of OGD. Cleaved gephyrin levels were normalized to full-length gephyrin levels; n = 5. (C) 3D-SIM maximum projections of inhibitory synapses labeled for surface GABA_ARs, gephyrin, and VGAT. Neurons were treated with control or OGD conditions for 20 min in the presence of DMSO (DM) or Calp-i. Arrows, individual GABA_AR SSDs. Scale bars represent 500 nm. (D) Quantification of the mean SSD volume and number per compartment for gephyrin and GABA_ARs. n = 130–133 synapses. (E) Confocal images of dendrites from hippocampal neurons labeled for surface GABA_ARs, gephyrin, and VGAT. Neurons were treated with control or OGD conditions for 30 min in the presence of CsA, Calp-i, or both. Arrows, VGAT-positive synapses. (F) Quantification of cluster area and density from images in (E); n = 35–36 neurons per condition. Values represent mean ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001; one-way ANOVA; Bonferroni post hoc test (A, B, D, and F). See also Figure S6.