Fig 1: Expression of subunits forming AMPAR (GluA1, GluA2) and NMDAR (GluN2A, GluN2B) in neurons of the cerebral cortex derived from Satb-deficient, Satb-null, and control mice. (A,B)—Immunostaining of GluA1 (green color), GluA2 (red color) subunits of AMPA receptors (A) and GluN2A (green color), GluN2B (red color) of NMDA receptors (B) of cortical neurons from control (Satb+/+ * NexCre/+), Satb-deficient (Satbfl/+ * NexCre/+) and Satb-null (Satb2fl/fl * NexCre/+) mice. (C–F)—The effects of Satb- and Satb2-deletions on the level of GluA1 (C), GluA2 (D), GluN2A (E), and GluN2B (F) subunits. Intensity levels of surface-expressed receptor subunits were determined by confocal imaging. We analyzed individual neurons which had fluorescence of Alexa Fluor 633 (GluA1, green color), Alexa Fluor 488 (GluA2, red color) and Alexa Fluor-555 (GluN2B, red color), and Alexa Fluor-594 (GluN2A, green color). The quantitative data reflecting the level of subunits expression are presented as fluorescence intensity values in summary bar charts (mean ± SEM). The values were averaged by 300 ± 50 neurons for each column. Statistical significance was assessed using paired t-test. The results obtained after immunostaining well agree with the data of fluorescent Ca2+ measurements presented in Figure 4 and Figure 5. After Ca2+ measurements, the cells were fixed and stained by the antibodies. We used the scans from three independent view fields for each experimental group. n/s—data not significant (p > 0.05); * p < 0.05; ** p < 0.01; and *** p < 0.001, comparing Satb-deficient group and Satb-null group with control mice. Significance indicated by a horizontal bar—comparison of Satb-deficient with Satb-null mice.
Fig 2: Expression and co-localization of NR2A and transient receptor potential melastatin-related 2 (TRPM2) in the rat hippocampus. (A) Protein amounts of TRPM2 in the hippocampal dorsal CA1 region of rats. (B) Protein amounts of NR2A in the hippocampal dorsal CA1 region of rats. (C) Effects of Pur on NR2A and TRPM2 co-localization in the hippocampal dorsal CA1 region, examined by immunofluorescence double staining. Representative micrographs are shown at ×200. Results were mean ± SEM (n = 6). **P < 0.01 vs. Sham; ##P < 0.01 vs. BCCAO; $$P < 0.01 vs. BCCAO + Pur (150 mg/kg/day).
Fig 3: Deletion in Satb1 and Satb2 affects the expression of genes, encoding subunits of excitatory and inhibitory neurotransmitters produced by the cortical cell cultures. (A,B)—alteration in the expression of genes, encoding subunits of NMDAR (Grin1, Grin2a, Grin2b), AMPAR (Gria1, Gria2), KAR (Grik1, Grik2) and GABA(A)R (Gabra1) in the cortical neurons in Satb-deficient and Satb-null mice with deletions of transcription factors Satb1 (A) and Satb2 (B). Data obtained on four (n = 3) different cell cultures are presented. All values are given as mean ± SEM. Gene expression level was normalized to reference gene Gapdh and was presented relating control (neurons from Satb+/+ * NexCre/+-mice), which was considered as 1 (dashed line). Total RNA was obtained from 10 DIV cultures. Statistical analyses were performed by paired t-test. Comparison of gene expression was performed between Satb-deficient and Satb-null mice. The data were significant: n/s—data not significant (p > 0.05); * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001.
Fig 4: Short-term NMDAR overactivation increases interactions between STIM2 (but not STIM1) and GluN2A or GluN2B in cortical neurons in vitro. A, B Representative Western blots from Co-IP experiments and B quantification of interactions between STIM proteins and NMDAR subunits before and after the NMDA and glycine treatment of neurons. Immunoprecipitation was performed with anti-GluN2B, anti-GluN2A, and anti-IgG antibodies. The input represents the cell lysate. The fractions were immunoblotted with the indicated antibodies. The Co-IP bands were normalized to the level of the loading control (i.e., bands obtained after Western blot with the antibody that was used for immunoprecipitation). C Representative deconvoluted confocal microscopy images of STIM2 with GluN2B before and after the NMDA and glycine treatment of neurons. The co-immunostaining of STIM2 (red), GluN2B (green), MAP2 (magenta), and nuclei (blue) is shown. The first “merge” column shows STIM2 labeling overlapped with GluN2B labeling, and the last column shows its higher magnification. All images were taken from a single optical section in the middle of the cell. Scale bar = 20 µm and 1 µm. Quantification of co-localization points was performed using Manders’ co-localization coefficient (M1). M1 was calculated from the entire field of view (Total), single cell (Cell), and dendrites (Dendrite). The data are expressed as the mean ± SEM of three-to-four independent experiments. *p < 0.05, ***p < 0.001 (unpaired t test)
Fig 5: STIM2 knockdown decreases the co-localization of GluN1, GluN2A, and GluN2B with EEA1, a marker of early endosomes, in dendrites after short-term NMDAR overactivation. Representative deconvoluted confocal microscopy images of NMDAR subunits and EEA1 in dendrites from neurons that were transduced with scrRNA or shStim2 C and quantification of co-localization points using Manders’ co-localization coefficient (M1). A–C The co-immunostaining of GluN1, GluN2A, and GluN2B (red) with EEA1 (green) is shown. The “merge” columns show the labeling of NMDAR subunits overlapped with EEA1 labeling. All images were taken from a single optical section. Scale bar = 10 µm and 1 µm. The data are expressed as the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (1-way Anova)
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