Fig. 4

Secreted Clu promotes excitatory synaptic function in vitro. a Western blotting of Clu in conditioned medium of primary astrocyte infected with AAV-GFAP-GFP (GFP) or AAV-GFAP-Clu (Clu) at 9 or 12 days post infection (d.p.i.). b mEPSC sample traces from primary neurons co-cultured with primary astrocytes infected with AAV-GFAP-GFP or AAV-GFAP-Clu at 12–13 DIV (scale bar, 40 pA/500 ms). c Cumulative distribution of inter-event interval from all mEPSCs events of 17 neurons co-cultured with GFP astrocytes and 18 neurons co-cultured with Clu astrocytes (left) and average mEPSC frequency (right). d Cumulative distribution of amplitude (left) and average amplitude (right) in neurons co-cultured with GFP (n = 17) or Clu (n = 18) astrocytes. e Western blot analysis of Clu in lysate of HEK293 cells transfected with Clu or vector control (Con) plasmid and in conditioned medium of Clu-transfect HEK293 cells. f mEPSC sample traces from primary cultured neurons treated with control conditioned medium (Con CM) or Clu CM at 14–16 DIV (scale bar, 20 pA/2 s). g Cumulative distribution of inter-event interval from all mEPSCs events of 23 neurons treated with Con CM and 21 neurons treated with Clu CM (left) and average mEPSC frequency (right). h Cumulative distribution of amplitude (left) and average amplitude (right) in neurons treated with Con CM (n = 23) or Clu CM (n = 21). i Representative Confocal images of cultured neurons triple-stained with anti-vGlut1, -PSD95 and -MAP2 antibodies and counterstained with DAPI. Lower panel is a zoomed-in view of a dendritic segment. Colocalized pre- (vGluT1) and post-synaptic (PSD95) puncta reveal sites of excitatory synapses. Scale bar, 10 μm (top), 1 μm (bottom). j quantification of synaptic density. k Gene Ontology analysis of upregulated genes in Clu vs. control CM treated neuronal cultures. All data are presented as mean ± SEM. *p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (Student’s t test)