For this, we assessed changes in synaptic markers (VGLUT1, GAD65/67, GABAAR 2, and GABAAR 5) in both ipsi- and contralateral hemispheres at 24 hours after MCAO compared to BDNFflox/flox/CX3CR1CreERT2+/? sham animals. synapse preservation at 24 hours after MCAO. Collectively, these observations unravel microglia-derived BDNF as the transmission transducer linking microglia and neurons, to activate ERK1/2 and GSK3 pathways to influence glutamatergic and GABAergic synapse integrity through gephyrin phosphorylation. RESULTS OGD causes glutamatergic and GABAergic synapse down-regulation To understand the mechanisms of BDNF action in ischemia, we started with an in vitro model of ischemia, OGD, in organotypic hippocampal slice cultures obtained from transgenic mice that express myristoylated enhanced green fluorescent protein (GFP) in a subset of CA1 pyramidal neurons and analyzed synaptic changes in the CA1 area after OGD (4 min) and recovery at 90 min and 24 hours. First, we confirmed that we experienced induced hypoxia with OGD by measuring hypoxia-inducible factor 1 (HIF1) expression ( 0.05 and ** 0.01, two-tailed indie Students test). Total spine density (spines per micrometer of dendrite): Control, 1.76 0.08 (= CCK2R Ligand-Linker Conjugates 1 8); OGD, 1.25 0.11 (= 8). (C) Example images of maximum intensity projections of area CA1 immunostained for gephyrin and VGAT in control cultures versus 90 min following OGD. Scale bars, 2 m. (D) Quantification of quantity of gephyrin clusters per confocal stack (consisting of CCK2R Ligand-Linker Conjugates 1 five 512 pixelCbyC512 pixel planes each; *** 0.001, two-tailed indie Students test; gephyrin cluster values were normalized to control). Mean number [arbitrary models (A.U.)]: Control, 1.00 0.03 (= 10 slices); OGD, 0.40 0.05 (= 10 slices). (E) Quantification of the total volume of gephyrin cluster (*** 0.001, two-tailed indie Students test). Mean volume (A.U.): Control, 1.00 0.05; OGD, 0.53 0.03. Data are shown as means CCK2R Ligand-Linker Conjugates 1 SD (= 30 cells). (F) Cumulative probability histogram and means SEM amplitude (** 0.01, Kolmogorov-Smirnov test). (F) Cumulative probability histogram and means SEM for IEIs of mEPSCs (* 0.05, Kolmogorov-Smirnov test). (F) Sample mEPSC traces from control cells and OGD cells. (G) Cumulative probability histogram and means SEM amplitude of mIPSCs ( 0.05, Kolmogorov-Smirnov test). (G) Cumulative probability histogram and means SEM for IEIs of mIPSCs (* 0.05, Kolmogorov-Smirnov test). (G) Sample mEPSC traces from control cells and OGD cells. Data are shown as means SD. Next, we evaluated OGD-induced changes at GABAergic synapses in area CA1 at 90 min and 24 hours following OGD. We immunolabeled inhibitory presynaptic vesicular GABA transporter (VGAT) and postsynaptic inhibitory scaffolding protein gephyrin. We found a significant overall down-regulation in gephyrin cluster density 90 min following OGD compared to control (Fig. 1, C to E) in the stratum radiatum. However, after 24 hours, gephyrin cluster density remained significantly reduced, while cluster volume had recovered to baseline (fig. S1, B and B) much Rabbit Polyclonal to GSK3beta like untreated cells. To determine whether these morphological changes were accompanied by a functional deficit, we recorded AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs) from CA1 pyramidal neurons within 24 hours after OGD (Fig. 1, F and F). We found that the input resistance and resting membrane potential of CA1 hippocampal pyramidal cells in OGD and sister untreated cells were comparable, suggesting that OGD does not affect receptor open probability or intracellular chloride concentration. However, we found a significant decrease in the mEPSC amplitude in OGD-treated slices compared to control (Fig. 1F). Similarly, the interevent interval (IEI) of CCK2R Ligand-Linker Conjugates 1 mEPSC of OGD cells was increased compared to control (Fig. 1F). Together, dendritic spine loss is usually mirrored by functional loss of excitatory synapses 24 hours after OGD. Subsequently, to determine whether inhibitory circuitry was also affected, we recorded GABAA-mediated miniature inhibitory postsynaptic current (mIPSC) within 24 hours of OGD induction. mIPSC analysis showed no observable changes in the amplitude of.