Fig 2: Increased NMDAR, but not AMPAR, currents at hippocampal synapses of juvenile and adult Lrfn3−/− mice.a Increased NMDAR currents at SC-CA1 synapses in the hippocampus of juvenile Lrfn3−/− mice (P21–24), as indicated by the ratio of NMDAR- to AMPAR-EPSCs. (n = 8 slices from 6 mice [WT] and 9, 5 [KO]; *p < 0.05, Student’s t-test). b Increased currents of GuN2B-containing NMDARs at SC-CA1 synapses of Lrfn3−/− mice (P21–23), as shown by the sensitivity of the currents to ifenprodil (GluN2B-specific antagonist). (n = 11 neurons from 6 mice [WT] and 11, 5 [KO]; *p < 0.05 (last five points), Student’s t-test). c Increased ratio of NMDAR- to AMPAR-mediated synaptic transmission at SC-CA1 synapses in adult Lrfn3−/− mice (P68–79), as indicated by the initial slopes of NMDAR-fEPSPs plotted against those for AMPAR-fEPSPs. (n = 9 slices from 5 mice [WT] and 13, 5 (KO); *p < 0.05, **p < 0.01, ***p < 0.001, two-way RM-ANOVA with Bonferroni test). d Dissection of AMPAR-fEPSPs by plotting initial fEPSP slopes against fiber volley amplitudes (left) or stimulation intensities (right), or plotting fiber volley amplitudes against stimulation intensities (middle). (n = 9, 5 [WT] and 13, 5 [KO]; ns, not significant, two-way RM-ANOVA). e Dissection of NMDAR-fEPSPs by plotting initial fEPSP slopes against fiber volley amplitudes (left) or stimulation intensities (right), or plotting fiber volley amplitudes against stimulation intensities (middle). (n = 9, 5 [WT] and 13, 5 [KO], two-way RM-ANOVA [genotype p for left/middle/right panels are 0.03/0.71/0.02]; *p < 0.05 and **p < 0.01 indicate results of Bonferroni tests). Error bars represent the SEM.

Fig 3: Distinct fluoxetine-induced changes in GluA1 and GluN2B phosphorylation in the WT and Lrfn3−/− hippocampus.a Levels of GluA1 and GluN2B phosphorylation at different amino acid residues in the hippocampus (crude synaptosomes) of WT and Lrfn3−/− mice (2–3 months) chronically treated with vehicle or fluoxetine (5 mg/kg/d; 7 days; i.p.). It is noteworthy that fluoxetine treatment increased GluA1 Ser-831/845 and GluN2B S1303 phosphorylation without affecting total protein levels. Immunoblot signals from WT and KO mice normalized to α-tubulin signals were used to obtain KO/WT ratio values. (n = 3 mice [WT-V/vehicle], 3 [WT-F/fluoxetine], 3 [KO-V], and 3 [KO-F], *p < 0.05, **p < 0.01, Student’s t-test). b Levels of GluA1 S831/S845 and GluN2B S1303 phosphorylation in chronic vehicle/fluoxetine-treated WT and Lrfn3−/− mice. All four samples were run on the same gel for two-way ANOVA comparisons. (n = 3 mice [WT-V/vehicle], 3 [WT-F/fluoxetine], 3 [KO-V], and 3 [KO-F], *p < 0.05, **p < 0.01; ns, not significant, two-way RM-ANOVA with Tukey’s test (except for WT-F and KO-F in p-GluN2B S1303 where Student’s t-test was used to obtain #P < 0.05)). Error bars represent the SEM.

Fig 4: Negative regulation of GLT-1 by astrocytes HDAC2 leads to dysfunction of glutamate reuptake in the synaptic cleft. In normal condition, the acetylation of histones in astrocytes facilitates the transcriptional regulation of GLT-1. Glutamate in the synaptic cleft is rapidly absorbed into astrocytes to maintain excitability of synapses. The increase of HDAC2 in astrocytes after SAH results in the deacetylation of histones and inhibits the transcription expression of GLT-1. The decrease of GLT-1 expression will lead to the impairment of glutamate reuptake in astrocytes and the long-term accumulation of glutamate in the synaptic space, resulting the dephosphorylation of ionized glutamate receptors GluN2B and GluA1 on the postsynaptic membrane. These eventually result in the long-term inhibition of synaptic excitability and DCI. DCI, delayed cognitive impairment.

Fig 5: Effects of selective HDAC2 inhibitor and GLT-1 inhibitor on DCI in SAH mice. (A) The SAH mice were intraperitoneally administered with HDAC2 inhibitor (2 mg/kg) and GLT-1 inhibitor (WAY-213613, 1 mg/kg) two weeks after surgery once every other day. The expression of GLT-1, HDAC2, p-GluN2B and p-GluA1 in hippocampus was detected after SAH at 8 weeks. N=5 animals in each group. (B) Forced swimming test and (C) sugar water preference test were used to evaluate the depressive behavior of SAH mice treated with HDAC2 inhibitor (Santacruzamate A) and GLT-1 inhibitor (WAY-213613). **, P<0.01, vs. the sham group; #, P<0.05 and ##, P<0.01, vs. the indicated groups. (D) Morris water maze test was used to detect the escape latency for reflecting spatial learning memory, *, P<0.01, and **, P<0.01, vs. the indicated groups. (E) Reference memory was detected by recording the time in target quadrant of Morris water maze test. **, P<0.01, vs. the sham group; #, P<0.05, vs. the indicated groups. N=8 mice per group.