Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Post-embedding immunogold EM of V1 L5 sections from P21 mice showed GluN1, GluN2A, and GluN2B subunits in presynaptic axon terminals (at) and postsynaptic spines (s) (arrowheads). Synapses were identified ultrastructurally and quantified if immunogold-labeled (see Methods). (B) Quantification of immunogold particle locations from morphologically identified excitatory synapses, pooled across three mice, revealed that GluN1 was distributed similarly across pre- and postsynaptic compartments (postsynaptic: 126/235 immunoparticles = 54%, χ p = 0.27, n = 102 excitatory synapses). GluN2A was enriched postsynaptically (138/174 = 79%, χ p < 0.001, n = 90), whereas GluN2B showed a more modest postsynaptic bias (105/177 = 59%, χ p < 0.05, n = 90). Error bars: normalized square root of counts. (C) GluN2B was ∼2× more likely than GluN2A to be found presynaptically (72 vs. 36 puncta; p < 0.001; Monte Carlo randomization of particle identities across pooled locations; see Methods). Puncta outside ± 60 nm are not shown. Error bars were computed by error propagating from (B).

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Labeling

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Schematic of Emx1, NR1, and Ai9 alleles in WT (Emx1 cre/+; NR +/+ ;Ai9 tdTomato/+ ) and global NMDAR deletion mice (Emx1 cre/+; NR1 flox/flox ;Ai9 tdTomato/+ ). (B) Representative confocal image of a coronal slice from a juvenile (P10) NMDAR deletion mouse showing widespread tdTomato fluorescence, indicating broad Emx1-Cre–mediated recombination across cortical layers. (C) Representative agarose gel confirming the presence of Emx1-Cre, NR1 floxed or WT alleles, and Ai9 tdTomato reporter in control and NMDAR deletion animals.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Fluorescence, Agarose Gel Electrophoresis, Control

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Representative confocal image of a 50-μm-thick coronal brain section from an Emx1 cre/+ ;NR1 +/+ ;Ai9 tdTomato/+ mouse (NMDAR WT), labelled for NeuN (grayscale, left), GluN1 (blue), and tdTomato (red). Arrowheads indicate example cells. (B) Same as in (A), but from an Emx1 cre/+ ;NR1 flox/flox ;Ai9 tdTomato/+ mouse (NMDAR Deletion). (C and D) NMDARs were efficiently deleted in tagged cells, as evidenced by colocalization in L5 being reduced from 89 ± 3% in NMDAR WT mice to 6 ± 1% in NMDAR Deletion animals (Mann-Whitney p < 0.001), where colocalization was calculated as the fraction of tdTomato-positive cells that were GluN1-positive. Although we focus on L5 here, NMDAR deletion was similarly efficacious in other cortical layers (see Dryad data set). Quantification of cell densities (cells per 10,000 μm cortex) expressing GluN1 only (blue), tdTomato only (red), or both GluN1 and tdTomato (purple) across cortical layers in NMDAR WT ( N = 7 animals, 1–4 cortical sections per animal) and NMDAR Deletion mice ( N = 4 animals, 3 cortical sections per animal). Percentage next to SEM bars denotes proportion of cells within the respective layer expressing either GluN1 only, tdTomato only, or both GluN1 and tdTomato.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: MANN-WHITNEY, Expressing

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Two-photon images of patched neurons in acute slices from a WT littermate (top, Emx1 cre/+; NR +/+ ;Ai9 tdTomato/+ ) and from a global NMDAR deletion mouse (bottom, Emx1 cre/+; NR1 flox/flox ;Ai9 tdTomato/+ ). Left: tdTomato fluorescence tagged recombined cells. Right: Sample 2-photon images of patched cells, with MNI-NMDA puff pipette and laser uncaging spot indicated (*). (B) Example uncaging-evoked EPSC traces evoked by laser uncaging of MNI-NMDA at the sites indicated in (A) (average of 10 sweeps). (C) Quantification of uncaging-evoked EPSCs confirmed efficient elimination of functional NMDARs in Cre-expressing pyramidal cells in the global deletion model. In NMDAR deletion animals, tdTomato-positive PCs (Del tdTomL, n = 31) showed no detectable responses to NMDA uncaging, in contrast to neighboring tdTomato-negative presumed interneurons (Del tdTomL, n = 16). In WT littermates, tdTomato-positive PCs (WT tdTomL, n = 19) responded robustly, with amplitudes indistinguishable from tdTomato-negative interneurons (WT tdTomL, n = 9). Student’s t test.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Fluorescence, Transferring, Functional Assay, Expressing

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) In this sample whole-cell recording (top) from a triple-transgenic Emx1 Cre/+ ;NR1 flox/flox ;Ai9 tdTomato/+ mice (bottom), NMDARs were globally deleted, which we denote ‘Del’ below. Recordings obtained from Emx1 Cre/+ ;NR1 +/+ ;Ai9 tdTomato/+ littermates (images not shown) are denoted ‘WT’. (B) In this representative PC recording from a WT L5 mouse, AP5 reversibly reduced mEPSC frequency (top, KS test p < 0.001) but not amplitude (bottom, p = 0.09), consistent with reduced presynaptic release. Sample traces scale bars: 200 ms, 10 pA. Insets show mEPSC traces averaged over periods indicated in blue and light blue. Scale bars: 5 ms, 5 pA. (C) For the sample PC in (A) obtained from a Del mouse, AP5 had no effect on mEPSC frequency (top, KS test p = 0.82) or amplitude (bottom, p = 0.9), suggesting that the effect of AP5 in WT requires presynaptic NMDARs. Scale bars as in (B). (D) AP5 robustly reduced mEPSC frequency in PCs from WT mice relative to no-drug controls (ANOVA p < 0.01), but had no detectable effect in PCs from Del mice (p = 0.52), consistent with presynaptic NMDAR control of spontaneous release. Baseline mEPSC frequencies were indistinguishable (WT: 2.7 ± 0.2 Hz, n = 62; Del: 2.8 ± 0.1 Hz, n = 86; Mann Whitney p = 0.40), and mEPSC amplitudes were unaffected.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Transgenic Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: The GluN2B-selective NMDAR antagonist Ro 25-6981 significantly reduced mEPSC frequency without affecting mEPSC amplitude in WT animals (Welch’s ANOVA, p < 0.05; Bonferroni-corrected post hoc comparisons), indicating that GluN2B-containing NMDARs regulate spontaneous release. This effect mirrors prior findings demonstrating presynaptic NMDAR control of spontaneous transmission at V1 L5 PC → PC synapses. , , This effect of Ro 25-6981 is consistent with prior literature showing that pre- but not postsynaptic NMDARs at V1 L5 PC → PC synapses are sensitive to GluN2B-specific blockers at this age. , , However, in slices from Emx1 cre/+ ; NR1 flox/flox ;Ai9 tdTomato/+ mice (see Methods) with GluN1 globally deleted (“Del”), Ro 25-6981 had no effect and was indistinguishable from controls. Together, these results support the conclusion that spontaneous excitatory release onto V1 L5 pyramidal neurons is regulated by presynaptic, GluN2B-containing NMDARs.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Control, Transmission Assay

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Schematic of neonatal viral injection (P0–P2) delivering Cre recombinase and an mCherry reporter (AAV-eSYN-mCherry-T2A-iCre-WPRE) or EGFP reporter (AAV-eSYN-EGFP-T2A-iCre-WPRE) to excitatory neurons in primary visual cortex (V1) of NR1 flox/flox mice. Created in BioRender. Rannio, S. (2026) https://BioRender.com/27fmwdc (B) Representative confocal image showing sparse mCherry expression in V1 at P21, including labeling of a few L5 PCs with easily identified by their prominent apical dendrites.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Injection, Expressing, Labeling

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) Left: mCherry fluorescence identifying PCs expressing AAV9-eSYN-mCherry-T2A-iCre-WPRE (see Methods). Cell outlines are based on Alexa 488 fills. Right: Two-photon image of V1 L5 PCs filled with Alexa Fluor 488, indicating the position of the laser uncaging site (*). MNI-NMDA was locally puffed near the uncaging location (“MNI”). (B) Ten-ms-long 405-nm laser pulses uncaged NMDA, resulting in NMDAR-mediated currents in PC1 but no detectable response in PC2 (averages of 10 sweeps shown), demonstrating that injection of AAV9-eSYN-mCherry-T2A-iCre-WPRE into NR1 fl/fl neonates (see Methods) deleted NMDARs in mCherry-tagged PCs. (C) Sparse viral delivery of Cre recombinase into NR1 flox/flox neonates reliably abolished uncaging-evoked NMDAR currents (uEPSCs) in V1 L5 PCs ( n WT = 26, n Del = 15, Student’s t test). Each data point represents the mean of 10–30 sweeps. (D) From P10 onward, mCherry-positive PCs (red) showed no response to NMDA uncaging, whereas WT PCs (blue) responded robustly. Connected symbols indicate simultaneously recorded cell pairs. Each recording from a deleted PC was paired with a WT PC as a positive control for successful uncaging.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: Fluorescence, Expressing, Injection, Positive Control

Journal: bioRxiv

Article Title: Distinct NMDA Receptor Pools Determine Diverse Forms of Cortical Plasticity

doi: 10.64898/2026.02.14.705915

Figure Lengend Snippet: (A) In this sample quadruple recording (top), PC1 but not PC2 had intact NMDARs following sparse viral deletion in NR1 flox/flox mice (see Methods). Fluorescent labelling allowed identification of NMDAR-deleted neurons (bottom). (B) In WT PC1, AP5 reversibly reduced mEPSC frequency (top, KS test p < 0.001) without affecting amplitude (bottom, p = 0.15), indicating a presynaptic reduction in release. Sample traces scale bars: 200 ms, 10 pA. Insets show averaged mEPSC traces. Scale bars: 5 ms, 5 pA. (C) In the paired NMDAR-deleted PC2, AP5 similarly reduced frequency (top, KS test p < 0.001) but not amplitude (bottom, p = 0.15). With postsynaptic NMDARs deleted, the remaining AP5 effect excludes a postsynaptic role. Scale bars as in (B) (D) AP5 consistently reduced frequency in WT and NMDAR-deleted PCs relative to controls (ANOVA p < 0.001), demonstrating that NMDARs controlling spontaneous release were not postsynaptic. Baseline mEPSC frequencies were indistinguishable (WT: 2.9 ± 0.2 Hz, n = 63; Del: 2.9 ± 0.3 Hz, n = 38, Mann Whitney p = 0.62), and amplitudes were unaffected.

Article Snippet: C57BL/6J (Strain 000664), homozygous Emx1cre/cre (005628), homozygous NR1 flox/flox (005246), and Ai9 reporter animals (007909) were obtained from The Jackson Laboratory.

Techniques: MANN-WHITNEY

Journal: Biomedicines

Article Title: Effects of Continuous Theta Burst Stimulation on Behavior and NMDA Receptor Subunits in the Trimethyltin-Induced Alzheimer’s-like Disease Model

doi: 10.3390/biomedicines14020391

Figure Lengend Snippet: Representative immunoblots and quantitative data of Western blot analysis of target proteins expression in hippocampal P2 fraction: ( A ) vGlut1, ( B ) NR1, ( C ) NR2A, ( D ) NR2B. Data were statistically analyzed by one-way ANOVA, followed by Tukey’s post hoc test, and expressed as a percentage of the mean values of the CONT group ± SEM (dots in the graphs represent values of individual animals). The control group is shown in the representative blot images above the graphs but was not included as a separate bar in the graphs to maintain clarity in the main comparisons. Symbols indicate significant differences compared to control: ** p < 0.01, *** p < 0.001, **** p < 0.0001, or Sham cTB. S group: ### p < 0.001, #### p < 0.0001, number of animals per group: 4–6. The can be downloaded at .

Article Snippet: NR1 , Cell Signaling Technology, Danvers, MA, USA, #5704 RRID: AB_1904067 , rabbit monoclonal; 1:1000.

Techniques: Western Blot, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Protective effects of Gryllus bimaculatus (Gb) extract on abnormal expression levels of glutamatergic and GABAergic synaptic proteins in the valproic acid (VPA)-induced autism spectrum disorder (ASD) mouse brain tissues. Immunoblot analyses for GRM5, vGluT1, NMDA R1, GABA R1α, and VGAT proteins were performed on prefrontal cortex (PFC) tissue lysates collected at embryonic day 15 (E15) (A) , postnatal day 3 (P3) (B) , and P40 (C) from mice subjected to various treatment combinations. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Expressing, Western Blot, Saline, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Regulatory effects of Gryllus bimaculatus (Gb) extract on excitatory and inhibitory neuronal activity in primary cortical neurons from valproic acid (VPA)-treated embryonic mice. (A) Schematic representation of primary cortical neuron cultures derived from embryonic mouse brains. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (B,D) Immunoblot analyses of NMDA R1, vGluT1, GRM5, GABA R1α, VGAT, NLGN3, NRXN1, and Tuj-1 in cultured primary cortical neuron lysates. Equal amounts of protein were loaded per lane, with β-tubulin used as a loading control. The bars represent fold-changes in the densitometric values of individual protein bands relative to the corresponding β-tubulin band densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant). (C) Confocal microscopy images of cortical neurons from various experimental groups. Cells were cultured for 7 days, fixed, and subsequently immunostained for vGluT1 (red), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm.

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Activity Assay, Derivative Assay, Saline, Western Blot, Cell Culture, Control, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Astrocytic gatekeeping of neural circuitry and synaptic balance in an autism mouse model: mechanistic insights beyond Gryllus bimaculatus extract-derived therapy

doi: 10.3389/fcell.2025.1677851

Figure Lengend Snippet: Crucial role of astrocytes in excitatory and inhibitory (E/I) neurotransporter activities in Gryllus bimaculatus (Gb) extract-treated mixed cultures from valproic acid (VPA)-treated mouse brain. (A) Schematic representation of three different types of mixed culture systems derived from embryonic and postnatal mouse brains: Type 1, astrocytes from each treatment group combined with neurons from untreated mice; Type 2, astrocytes from untreated mice combined with neurons from each treatment group; Type 3, astrocytes and neurons both derived from the same treatment group. Astrocytes from postnatal day 3 mouse brains were seeded for 7 days, followed by the addition of cortical neurons from embryonic day 15 mouse brains onto astrocytes monolayers for an additional 7 days. (B–D) Confocal microscopy images of the different types of mixed cultures. Cells were fixed and immunostained for Tuj-1 (green) and GFAP (purple), with nuclei counterstained using DAPI (blue). Scale bar: 50 μm. Experimental groups included CTL (saline, n = 8); VPA (600 mg/kg VPA, n = 8); VPA + Gb 5 (600 mg/kg VPA + 5 g/kg Gb extract, n = 8); VPA + Gb 10 (600 mg/kg VPA + 10 g/kg Gb extract, n = 8); Gb 5 (5 g/kg Gb extract, n = 8); Gb 10 (10 g/kg Gb extract, n = 8). (E) Western blots analysis of type III mixed culture. Cell lysates were immunoblotted for Tuj-1, GFAP, synaptophysin, NMDA receptor 1 (NMDA R1), GABA receptor 1α (GABA R1α), EAAT1, and EAAT2. Equal amounts of protein were loaded per each lane, with β-actin serving as the loading control. Bars represent fold-changes in the densitometric values of the bands relative to the corresponding β-actin densities. Control values were normalized to 1 (mean ± SEM, n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with VPA alone; ns , not significant).

Article Snippet: Antibodies against synaptic markers, including NLGN1 (#NBP2-42192), NLGN2 (#NBP2-41299), NLGN3 (#NBP2-42200), SHANK3 (#NBP1-47610), the NMDA receptor NR1 subunit (#NB300-114) from Novus Biologicals (Centennial, CO, United States) and NRXN1 (#PA5-79764) was from Thermo Fisher Scientific (Waltham, MA, United States).

Techniques: Derivative Assay, Confocal Microscopy, Saline, Western Blot, Control