Fig 2: NMDAR regulates store operated calcium entry (SOCE) in murine platelets. (A) Determination of intracellular Ca2+ concentration [(Ca2+)IC] of platelets in buffer containing 1 mM Ca2+ for SOCE measurement (n = 22–23). (B) Determination of [Ca2+]IC of platelets in buffer containing 5 mM EGTA for store-release measurement (n = 16–20). (C) Determination of SOCE and store-release in NMDAR deficient platelets after stimulation with thrombin compared to respective WT control platelets (n = 5–6). (D) Representative measurements of [Ca2+]IC in the spectral fluorimeter after thrombin stimulation. (E) Determination of SOCE and store-release in Grin1fl/fl-Pf4-Cre+platelets after stimulation with collagen-related peptide (CRP) compared to respective WT control platelets (n = 5). (F) Representative measurements of [Ca2+]IC in the spectral fluorimeter after CRP stimulation. (G) Determination of the [Ca2+]IC using a sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA-2) inhibitor thapsigargin following Ca2+ administration (n = 4–5). (H) Representative measurements of intracellular Ca2+ flux in the spectral fluorimeter after SERCA-2 inhibition via thapsigargin and Ca2+ exposure. Depicted are mean values + s.e.m.; *P < 0.05, ***P < 0.001 using two-way ANOVA followed by Sidak's multiple comparison test.

Fig 3: Datumetine effects on NMDAR signalling.(a): representative immunoblots of GluN1, CamKIIα, pCamKIIα-T286, CREB, BDNF and actin expression in the cell lysates of the hippocampus of experimental animals;(b): quantification of relative GluN1 expression;(c): quantification of relative CamKIIα expression;(d): quantification of relative pCamKIIα-T286 expression;(e): quantification of relative CREB expression; and(f): quantification of relative BDNF expression.(n = 4 per group, *p < 0.05, **p < 0.01, ***p < 0.001 one-way ANOVA with Tukey post-hoc test).

Fig 4: Effects of PCDH7 overexpression in hippocampal slices. (A) Representative images of hippocampal CA1 pyramidal neurons overexpressing either GFP or PCDH7 + GFP in cultured hippocampal slices after biolistic transfection for 1 ~ 4 days. Images were taken from basal dendrites. Scale bar 10 μm. (B) Sample traces and quantification of evoked NMDAR mediated EPSCs from paired transfected (with either GFP or PCDH7) and neighboring untransfected hippocampal CA1 pyramidal neurons in organotypic brain slice cultures. Each black circle represents current amplitude of one pair of neurons. Blue circle represents mean amplitude ± SEM. Recordings were performed 3–4 days after transfection. 3 independent experiments, n = 11–14 pairs of neurons. **p < 0.01, Paired t test. (C) and (D) Representative images of hippocampal CA1 pyramidal neurons expressing c-terminal GFP tagged PCDH7 (PCDH7-GFP) with either DsRed (C) or c-terminal tdTomato tagged SHANK3 (SHANK3-tdTomato) (D). Both whole cell (top) and zoomed in basal dendrites (bottom) are shown. Scale bar 10 μm.

Fig 5: PCDH7 mRNA and protein expression in mouse brain. (A) Dual label in situ hybridization of Pcdh7 (red) and Grin1 (blue), Dapb (bacterial gene as a negative control gene; blue), or Actb (universal positive control gene; blue) in P14 mouse brain. Scale bar 20 μm. Insets show zoomed in images. (B) Dual label in situ hybridization of Pcdh7 (red) and Pvalb, Sst, Vip, or Npy (blue) in hippocampal CA3 or cortex layer V regions of P14 mouse brain. Scale bar 20 μm. Insets show zoomed in images. (C) Timecourse of protein expression in wildtype mouse cortex. Western blots for PCDH7 and other neuronal proteins (N-Cadherin, PSD95, GluN1) were run with 50 μg of RIPA lysate collected from forebrain of mouse at indicated developmental timepoints. (D) Synaptic fractionation and western blot for PCDH7 and other neuronal proteins was performed on adult mouse brain lysate, SY synaptosome fraction, PSD postsynaptic density enriched fraction. Western blots were run with 10 μg of lysate from indicated fraction.