Fig 2: Effect of SH, CN and ESI protocol on NMDA receptor subunits in the crude membrane fraction of the mPFC of PND35 male (top) and female (middle) rats. Protein levels of GluN1 (A), GluN2A (B), GluN2B (C), GluN2A/GluN2B ratio (D) in male rats, and GluN1 (E), GluN2A (F), GluN2B (G), GluN2A/GluN2B ratio (H) in female rats are expressed as percentages of SH-CTRL and represent the mean±S.E.M. of six rats per group (GluN2A and GluN2A/GluN2B ratio: 1 outlier in SH-ESI males). Representative immunoblots (bottom) are shown for each protein in the respective panels (I, J). *p<0.05, **p<0.01, ***p<0.001 vs. SH-CTRL, $p<0.05, $$p<0.01, $$$p<0.001 vs CN+CTRL (Two-way ANOVA followed by Tukey’s multiple comparisons test). SH, Standard Housing; CN, Communal Nesting; CTRL, controls no ESI; ESI, Early Social Isolation.

Fig 3: CP2 treatment improves synaptic function and LTP in 3xTg-AD mice. A) Experimental setup for stimulation-evoked local field potential (LFP) measurement in the hippocampal acute slice. The stimulation (Stim.) electrode was placed at the Schaffer collaterals and the recording (Rec.) electrode was placed at the striatum radiatum in the CA1 region. To induce LTP, three tetanic stimulations (100 Hz, 60μs-pulse width for 1 s) were applied with 3-s intervals. In the slice, LTP was induced and maintained over 30–50 min. Slope of EPSPs was measured and results normalized to the average value established during the 30 min baseline period. Recording continued for at least 60 min following a tetanic stimulation and the first 30 min was used to calculate the LTP. B) CP2 treatment improves LTP formation in CP2-treated 3xTg-AD female mice. Traces represent mean±SEM per time. N= 2-3 slices from 3-5 mice per group. C) LTP intensity in CP2-treated female 3xTg-AD mice compared to vehicle-treated counterparts at 30 min after the stimulation. N= 2-3 slices from 3-5 mice per group. D) Representative western blot images of protein markers involved in synaptic function of dendritic spines in the brain tissue of CP2- and vehicle-treated female 3xTg-AD mice. N= 4–7 mice per group. E) Quantification of western blot data from (D) showing a percent of changes in protein expression after CP2 treatment in NTG and 3xTg-AD mice, relative to the levels in vehicle-treated NTG counterparts. Significant increase was observed in levels of synaptophysin (Syn), PSD95, and GluA1 and GluN2B phosphorylation. No changes were detected in total levels of GluA1 and GluN2B. F) Representative western blot images of protein markers involved in synaptic function of dendritic spines in the brain tissue of CP2- and vehicle-treated male 3xTg-AD mice. N= 4–7 mice per group. G) Quantification of western blot data from (F) showing a percent of changes in protein expression after CP2 treatment in NTG and 3xTg-AD mice, relative to the levels in vehicle-treated NTG counterparts. Significant increase was observed in levels of Syn and PSD95, with no improvement in GluN2B phosphorylation. Differences between individual groups were analyzed by one-way ANOVA, with Fisher‘s LSD post-hoc test. Data are presented as mean±SEM. *p < 0.05, **p < 0.01.

Fig 4: Blockade of GluN2B subunits restores the nicotine-induced increase in cofilin activity. (A) Repeated nicotine exposure increased the expression of both surface- and intracellular GluN2B subunits. N = 4 rats per group. (B) Bilateral intra-CPu infusion of the GluN2B subunit antagonist, Ro 25-6981 (Ro), was confirmed by reconstructing microinjection placements. (C) Blockade of the GluN2B subunits with Ro elevated the repeated nicotine-induced decrease in pCofilin-IR. N = 3 rats per group. *p < 0.05

Fig 5: Increased excitability of PV+ interneurons in Htr3a-/- mice by NMDAR upregulation. (A) Significant increase in GluN2B immunoreactivity (green) in the PV+ (red) neurons in the CA1 region of Htr3a-/- mice (bottom row), compared with controls (top row). Scale bar: 50 µm. (B) Quantified data showing a significant increase of GluN2B in PV+ neurons in the CA1 region of Htr3a-/- mice. (Student's t test, p = 0.0005, n = 93 cells from 3 WT mice, n = 100 cells from 3 KO mice). (C) Representative traces of evoked AMPAR- (held at -70 mV) and NMDAR-current (held at +40 mV) in PV+ neurons in the CA1 region of Htr3a-/- and WT mice. Scale bar: 20 pA, 100 ms. (D) Normal AMPAR current but increased NMDAR current in PV+ neurons in the CA1 region of Htr3a-/- mice. (For AMPAR current, two-way ANOVA, F1, 140 = 1.831, p = 0.0001; for NMDAR current, two-way ANOVA, F1, 140 = 31.27, p < 0.0001. n = 13 cells from 3 WT mice, n = 9 cells from 3 KO mice). (E) Voltage traces evoked by 300 pA current injection before and after D-APV (50 µM) treatment of Htr3a-/- and WT mice. Scale bars represent 100 ms, 20 Mv. (F) Firing number plotted against depolarizing currents. (n = 9 cells from 3 WT mice, n = 12 cells from 3 KO mice; two-way ANOVA, WT vs. KO, F1,171= 25.95, p < 0.0001; two-way ANOVA by matching factors, WT vs. WT APV, F1,8= 9.451, p = 0.0152; two-way ANOVA by matching factors, KO vs. KO APV, F1,11= 10.60, p = 0.0077; two-way ANOVA, WT vs. KO APV, F1,19= 0.3283, p = 0.5734). Data are presented as boxplots (median and 5th-95th percentile whiskers), or as mean ± SEM., * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001.