Fig 2: Overexpression of human NR2B induced changes in song motif structure. (a) Average number of syllables per song motif. (b) Mean number of introductory notes per song motif. (c) Song motif duration. (d) Intersyllable gap durations. (e) Average of all syllable durations (bird Red08 of the control group appeared to be an outlier, which was confirmed by a Dixon’s test in R in both presurgery (p = 0.02) and postsurgery (p < 0.0001) conditions, and thus not included in the statistical analyses of the entire group. Each line is one animal presurgery and postsurgery, mean ± SE, with p values determined using paired Wilcoxon signed-rank tests.

Fig 3: Effects of Aß1-6A2V(D) on synaptic integrity.Biochemical study on APPSwe/PS1dE9 mice intranasally treated with Aß1-6A2V(D) every 48 h for 5 months. The levels of the synaptic proteins NR2A, NR2B, GluA1, GluA2 and PSD-95 were analyzed by western blot. Two representative animals from each group are included in the figure. The densitometric analysis showed a significant increase in the levels of GluA1 (*p = 0.015) and GluA2 (**p = 0.007) and a trend toward an increase in the levels of NR2A, NR2B and PSD-95 in mice treated with Aß1-6A2V(D) (red columns, n = 10) compared to animals treated with saline solution (blue columns, control group, n = 10).

Fig 4: CCI promotes s1R-mediated associations of a2d1-2 proteins with NR1 subunits. (A) CD1 WT sham-operated (n = 4), CD1 WT CCI (n = 4), CD1 s1R-/- sham (n = 4) and CD1 s1R-/- CCI (n = 4) were sacrificed 7 days after surgery and PAG synaptosomal fractions were prepared. NR1 subunits were immunoprecipitated (IP) from the solubilized membrane preparations, and the presence of co-precipitated (A) NR1, (B) NR2A, (C) NR2B, (D) a2(d1), (E) (a2)d1, and (F) (a2)d2 proteins was assessed in Western blots (WB). The bars represent the mean ± SD of three measurements. Data are computed relative to the WT mice and in the absence of CCI (assigned the arbitrary value of 1). The arrows refer to the comparison and * indicates significant difference of the CCI group relative to the CD1 WT or CD1 s1R-/- group: p < 0.05. Details as in Figure 4. (G) CD1 WT mice: effect of S1RA on the association of a2d1-2 proteins with NR1 subunits promoted by CCI (n = 6). S1RA (3 nM) was injected icv 7 days after CCI surgery in three CD1 WT mice. The animals were sacrificed 30 min later to obtain the synaptosomal fraction from the PAG. The NR1 subunits were immunoprecipitated from the solubilized membrane preparations, and the co-precipitated a2(d1), a2(d2), (a2)d1, and (a2)d2 were analyzed in Western blots: * indicating significant difference relative to the CCI group, p < 0.05. Sham operated CD1 WT mice (n = 3) served as control to the effect of CCI (for further details see Section 2).

Fig 5: Proposed molecular mechanism underlying PNDs. Surgical trauma induces transient neuroinflammation by triggering the systemic inflammatory response. Then neuroinflammation causes long-lasting downregulation of synaptic NR2A and NR2B subunits in the dorsal hippocampus that is associated with synaptic NR2A and NR2B dysfunction, which is manifested by impaired structural plasticity of dendritic spine and decreased activated Rac1 (Rac1–GTP), phospho-cofilin, and Arp3 protein expressions. Ultimately, the persistent hypofunction of NR2A and NR2B leads to the development of long-lasting PNDs