Fig 2: The changes of interstitial glutamate concentration, astrocytic glutamate reuptake function, and the expression of GluN2B, GluA1 and glutamate transporters in hippocampus after SAH at 8 weeks. (A) The concentration of interstitial glutamate in the hippocampus of the mice was detected by HPLC-MS/MS. **, P<0.01, the SAH group vs. the sham group. N=8 mice per group. (B) Glutamate uptake assay on the hippocampal astrocytes sorted by immunomagnetic beads. **, P<0.01, the SAH group vs. the sham group. N=6 mice per group. (C,D) Western blot was performed to detect the expression of p-GluN2B/GluN2B, p-GluA1/GluA1, GLT-1, and GLAST protein in hippocampal tissue in the sham group and the SAH group at 1 week before and 8 weeks after SAH. **, P<0.01, vs. the indicated groups. N=5 animals in each group.

Fig 3: 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.

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.