Fig 2: (A) Representative images of immunohistochemistry of Iba1 in the DG (left) and CA1 (right) regions of WT (upper panel) and ROR?t Tg (lower panel) mice. (B) Semi-quantitative results of Iba1 immunofluorescence in DG (left) and CA1 (right) of WT and ROR?t Tg mice (n = 3 per group, *P < 0.05). (C) Density of Iba1+ microglia in the DG (left) and CA1 (right) of WT and ROR?t Tg mice (n = 3 per group, *P < 0.05). Microglial activity and density were significantly lower in the DG of ROR?t Tg compared with WT mice. (D) Representative images of immunohistochemistry for GFAP in DG (left) and CA1 (right) regions of WT (upper panel) and ROR?t Tg (lower panel) mice. (E) Semi-quantitative results of GFAP immunofluorescence in the DG (left) and CA1 (right) of WT and ROR?t Tg mice (n = 3 per group). (F) Density of GFAP+ astrocytes in the DG (left) and CA1 (right) of WT and ROR?t Tg mice (n = 3 per group). There were no significant differences in astrocyte activity or density between ROR?t Tg and WT mice. (G) Representative images of immunohistochemistry of DCX (green) and DAPI (blue) in DG regions of WT (upper panel) and ROR?t Tg (lower panel) mice. (H) Density of DCX+ immature neurons in the DG of WT and ROR?t Tg mice (n = 4 per group). (I) Western blot of synaptic molecules (NR2A, NR2B, PSD-93, and PSD-95) in the hippocampi of WT (left) and ROR?t Tg (right) mice. For normalization of proteins in each sample, a-tubulin was used as a control (the lowest band). Each lane corresponds to a sample derived from one mouse (n = 3 per group). (J) Comparison of relative expression levels of NR2A, NR2B, PSD-93, and PSD-95 between WT and ROR?t Tg mice (n = 3 per group). Scale bars = 50 µm

Fig 3: Simultaneous over-expression of NR2B and knockdown of PSD95 induce dendritic branching in mature hippocampal neurons. A, Cultured hippocampal neurons were transfected with a magnetofection-based method at 15 DIV with GFP alone (control; left image), GFP plus shRNA-PSD95 (shRNA-PSD95; middle image), or GFP plus NR2B and shRNA-PSD95 (NR2B+shRNA-PSD95; right image). At 20 DIV, cultures were fixed and images taken. Scale bar is 25 µm. Inset: Magnified views of boxed areas showing examples of dendritic branches. Note that neurons that express NR2B+shRNA-PSD95 display a more complex dendritic architecture compared to control or shRNA-PSD95-expressing cells. B–C, Quantification of the average number of secondary (B) and tertiary (C ) processes: neurons expressing NR2B+shRNA-PSD95 have more branches relative to control neurons and to cells transfected with shRNA-PSD95. D–E, Total outgrowth (D) and Sholl analysis (E) of hippocampal neurons expressing the different constructs as indicated. For each condition, at least 20 neurons, obtained from 3 independent experiments, were analyzed. Figures show Mean ± SEM. *** p<0.001 (ANOVA).

Fig 4: mTBI increases exocyst complex assembly and interaction between Exo70 and GluN2B in the PSD fraction. Two-month-old male mice were subjected to mTBI and hippocampal PSD/nonPSD fractions were obtained. 50 and 300 µg of PSD (a) and nonPSD (c) protein samples, respectively, were immunoprecipitated using a specific Exo70 polyclonal antibody. Beads were recovered by brief centrifugation. Samples were resolved in 6% SDS-PAGE and transferred to PVDF membranes. Signal intensity was normalized with Exo70 band intensity. Both hippocampi of each mouse were used. Membranes were stripped and tested again with the indicated antibodies. b, d GluN2A, GluN2B, GluR1, GluR2, Sec6, and Sec10 densitometric analysis from PSD and nonPSD immunoprecipitations. The analysis shows the assembly level of the exocyst complex and Exo70-GluN2B increased interaction after mTBI. Values represent means ± SEM, n = 3 mice per experimental group. Statistical differences were determined by an unpaired t-test comparing Sham and mTBI. *p < 0.05

Fig 5: Expression of NMDAR subunits NR2A and NR2B, and MAGUKs PSD95 and SAP102, at different stages of development. A, Total protein extracts were obtained from hippocampal neurons at 2, 5, 7, 12, 15, 18, and 20 DIV and immunoblotted with antibodies against NR2B, NR2A, PSD95, SAP102, or N-cadherin as loading control. B, Results of qRT-PCR run with primers designed to determine mRNA levels for NR2B, NR2A, PSD95, or SAP102 at the same developmental stages; results were normalized against levels of GAPDH mRNA. Note that NR2B expression decreases with development, while NR2A and PSD95 increases. Expression of SAP102 remains relatively stable at these developmental stages. C, Immunodetection of endogenous NR2B, NR2A, PSD95, or SAP102 (green) and co-localization with presynaptic marker Bassoon or Piccolo (red) at 5 and 20 DIV. Insets show magnified views of boxed areas showing examples of synaptic clusters located in close apposition to presynaptic clusters.