Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design (top) and time course of memory formation (bottom). Performance of EXP rats was robust and long-lasting with forgetting occurring only after 60 days (group × delay interaction: F 2,50 = 3.52; P < 0.05); * P < 0.05. ( B ) Immunoblots (top) and quantitative analysis (bottom) of NMDAR subunit expression in PSD-enriched membrane fractions from the OFC of FP and EXP rats. A progressive increase in the GluN2A/GluN2B ratio occurred as memory consolidated in the OFC of EXP rats (day 15, long-term memory; day 30, remote memory). At day 60, memory forgetting was associated with an increased contribution of GluN2B-containing NMDARs (group × delay interaction: F 3,40 = 7.20; P < 0.001); * P < 0.05. ( C ) Experimental design investigating memory persistence. EXP rats interacted socially with a demonstrator fed with cumin-flavored chow. Odor control (OC) rats were only able to smell cumin from a jar. ( D ) Enhanced preference for cumin-flavored food remained stable over 30 days in EXP rats but faded over the same time period in OC rats (group × delay interaction: F 2,45 = 4.44; P < 0.05). Performance of OC rats at day 30 was similar to that of FP controls, which interacted with a demonstrator fed with plain food. ( E ) Quantitative analysis of NMDAR subunit expression in PSD-enriched membrane fractions from the OFC of FP and OC rats tested at various intervals (days 1, 15, and 30) following social interaction. No redistribution of synaptic GluN2A and GluN2B subunits occurred over time in OC rats (group × delay interaction: F 2,43 = 0.013; P > 0.98, NS). * P < 0.05, **** P < 0.0001. (A), (B), (D), and (E), n = 4 to 11 rats per group.

Article Snippet: Prevention of NMDAR surface redistributions was achieved using an antibody-based x-link strategy in which cultured hippocampal neurons were exposed for 30 min to high concentrations (0.08 mg/ml) of rabbit polyclonal immunoglobulins directed against extracellular epitopes of GluN2B subunits (RRID:AB_2040028; catalog number: AGC-003, Alomone Labs; epitopes corresponding to residues 323-337 of the GluN2B subunit, Jerusalem, Israel) to aggregate the receptors and limit their movements within the membrane plane, as previously described and figs. S17 to S21) ( ).

Techniques: Western Blot, Expressing, Membrane, Control

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design. Rats were injected chronically into the OFC with aCSF (vehicle) or NMDAR antagonists (AP5, TCN-201, or Ifenprodil) during the early (from day 1 to day 11) or late (from day 16 to day 26) post-acquisition periods and tested at day 30 following social interaction. ( B ) Early, but not late, post-encoding blockade of GluN2B subunits by intra-OFC infusions of ifenprodil impaired remote memory retrieval at day 30. The opposite pattern was observed when antagonizing GluN2A-NMDARs with TCN-201. Both early and late OFC infusions of the nonselective NMDAR antagonist AP5 impaired remote memory retrieval (early treatment effect: F 3,32 = 6.73, P < 0.05; late treatment effect: F 3,37 = 4.09, P < 0.05). * P < 0.05 versus aCSF (vehicle control), n = 8 to 13 rats per group. ( C ) Experimental design investigating relearning. Animals infused with aCSF (vehicle) or AP5 into the OFC during the early (from day 1 to day 11) or late (from day 16 to day 26) post-acquisition periods were submitted to a second interaction with a different flavor (cocoa) 1 week later (day 37). This novel memory for cocoa was assessed 30 days later to enable the establishment of remote memory in the OFC (day 67, choice between cocoa and cinnamon). ( D ) Groups previously injected with aCSF or AP5 during the early or the late post-acquisition periods exhibited a similar acquired preference for cocoa (group × delay: F 1,15 = 0.51, P > 0.48, NS, n = 8 to 13 rats per group). The dotted line in (B) and (D) represents innate preference of FP control rats.

Article Snippet: Prevention of NMDAR surface redistributions was achieved using an antibody-based x-link strategy in which cultured hippocampal neurons were exposed for 30 min to high concentrations (0.08 mg/ml) of rabbit polyclonal immunoglobulins directed against extracellular epitopes of GluN2B subunits (RRID:AB_2040028; catalog number: AGC-003, Alomone Labs; epitopes corresponding to residues 323-337 of the GluN2B subunit, Jerusalem, Israel) to aggregate the receptors and limit their movements within the membrane plane, as previously described and figs. S17 to S21) ( ).

Techniques: Injection, Control

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A and B ) Early, but not late, hippocampal inactivation with 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) prevented the consolidation-induced rearrangement of cortical GluN2A and GluN2B subunit–containing NMDARs in the OFC ( F 1,16 = 9.86; P < 0.001); * P < 0.05, n = 5 rats per group. ( C to E ), Memory strength (one versus four social interactions) modulated the post-acquisition kinetics of hippocampal and cortical involvement upon memory retrieval at day 7. CNQX-induced disruption of hippocampal activity after one interaction was no longer observed after four interactions (D, treatment × interaction number: F 2,37 = 4.519; P < 0.05); * P < 0.05, n = 6 to 9 rats per group). OFC dependency manifested after four, but not one, interactions (E, F 2,39 = 12.60; P < 0.0001); ** P < 0.01, n = 7 to 9 rats per group). ( F ) Immunoblots (top) and corresponding quantitative analysis (bottom). An increase in the GluN2A-/GluN2B-NMDAR ratio was observed in the OFC at day 7 after four, but not one, interactions ( F 2,13 = 16.51; P = 0.0003); ** P < 0.01, *** P < 0.001, n = 5 to 7 rats per group. ( G and H ) Accordingly, blocking GluN2A-containing NMDARs with TCN-201 during the early post-encoding phase impaired remote memory retrieval at day 30 while being ineffective in the single interaction condition ( F 1,26 = 17.22; P = 0.0003); * P < 0.05, ** P < 0.01, n = 8 rats per group; data from are shown for comparison).

Article Snippet: Prevention of NMDAR surface redistributions was achieved using an antibody-based x-link strategy in which cultured hippocampal neurons were exposed for 30 min to high concentrations (0.08 mg/ml) of rabbit polyclonal immunoglobulins directed against extracellular epitopes of GluN2B subunits (RRID:AB_2040028; catalog number: AGC-003, Alomone Labs; epitopes corresponding to residues 323-337 of the GluN2B subunit, Jerusalem, Israel) to aggregate the receptors and limit their movements within the membrane plane, as previously described and figs. S17 to S21) ( ).

Techniques: Disruption, Activity Assay, Western Blot, Blocking Assay, Comparison

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A ) Experimental design investigating memory specificity. ( B ) Intra-OFC infusions of the GluN2B-NMDAR antagonist ifenprodil at the time of the second interaction (cumin) impaired remote memory for this second flavor while sparing remote memory for the first one (cocoa) (treatment × flavor pair: F 1,36 = 11.90; P = 0.0014). The nonselective NMDAR antagonist AP5 exhibited a similar profile ( F 1,32 = 17.31; P = 0.0002). The GluN2A-NMDAR antagonist TCN-201 was ineffective regardless of the flavor pair (main treatment effect: F 1,34 = 0.28; P = 0.59, NS). ( C ) Similar effects of AP5 infusions into the OFC were obtained when the two cumin/thyme and cocoa/cinnamon flavor pairs were counterbalanced (flavor × treatment: F 1,24 = 16.36, P < 0.001). ( D ) Experimental design investigating encoding processes. ( E ) Intra-OFC infusions of AP5, TCN-201, and ifenprodil before social interaction did not impair performance of rats tested 4 days (D4) later (treatment effect: F 3,26 = 1.207, P > 0.33, NS). ( F ) Experimental design investigating CaMKII contribution. ( G ) Intra-OFC infusions of the CaMKII inhibitor CN21 impaired formation of a newly encoded memory without destabilizing a previously encoded information undergoing consolidation ( F 2,49 = 23.92; P < 0.0001). ( H ) (Top) Efficacy of intra-OFC infusions of GluN2A- and GluN2B-NMDAR antagonists to impair remote memory retrieval at day 30 as a function of the specific phases of systems consolidation. Graph is based on findings from and (B). (Bottom) Schematic of the GluN2A- and GluN2B-NMDAR redistributions at cortical synapses as enduring memories mature in the cortex. Size variation of NMDAR pictograms represents changes in the functional contribution of GluN2A- (in brown) and GluN2B-NMDARs (in pink). (B), (C), (E), and (G), n = 6 to 12 rats per group; * P < 0.05, **** P < 0.0001.

Article Snippet: Prevention of NMDAR surface redistributions was achieved using an antibody-based x-link strategy in which cultured hippocampal neurons were exposed for 30 min to high concentrations (0.08 mg/ml) of rabbit polyclonal immunoglobulins directed against extracellular epitopes of GluN2B subunits (RRID:AB_2040028; catalog number: AGC-003, Alomone Labs; epitopes corresponding to residues 323-337 of the GluN2B subunit, Jerusalem, Israel) to aggregate the receptors and limit their movements within the membrane plane, as previously described and figs. S17 to S21) ( ).

Techniques: Functional Assay

Journal: Science Advances

Article Title: Synaptic rearrangement of NMDA receptors controls memory engram formation and malleability in the cortex

doi: 10.1126/sciadv.ado1148

Figure Lengend Snippet: ( A to D ) Intra-OFC infusion of an anti-GluN2B, but not of an anti-GluN2A, antibody during the early post-encoding period (A) impaired remote memory retrieval assessed 30 days later (B, group × treatment interaction: F 1,38 = 16.10; P = 0.0003) and abolished the increase in GluN2A-/GluN2B-NMDAR ratio observed in the OFC of control rats (D, F 2,19 = 10.45; P = 0.0009); * P < 0.05, ** P < 0.01, n = 8 to 9 rats per group. ( E to H ) Intra-OFC delivery of the anti-GluN2B antibody during the late post-encoding period (E) prevented forgetting (F, F 2,32 = 7.67; P = 0.0019) and the decrease of the GluN2A-/GluN2B-NMDAR ratio in the OFC (H, F 2,18 = 5.88; P = 0.0109) normally seen in control rats at day 60. * P < 0.05, # P < 0.05, n = 10 to 14 rats per group. Immunoblots in (C) and (G) served for quantification of respective GluN2A-/GluN2B-NMDAR ratios.

Article Snippet: Prevention of NMDAR surface redistributions was achieved using an antibody-based x-link strategy in which cultured hippocampal neurons were exposed for 30 min to high concentrations (0.08 mg/ml) of rabbit polyclonal immunoglobulins directed against extracellular epitopes of GluN2B subunits (RRID:AB_2040028; catalog number: AGC-003, Alomone Labs; epitopes corresponding to residues 323-337 of the GluN2B subunit, Jerusalem, Israel) to aggregate the receptors and limit their movements within the membrane plane, as previously described and figs. S17 to S21) ( ).

Techniques: Control, Western Blot

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . ( left ) Representative traces of the evoked fEPSP from control (black), control+AP5 [50µM] (green), nIH (red) and nIH +AP5 [50µM] (blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in control, control+AP5, nIH and nIH+AP5. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in control slices vs control+AP5 [50 µM]. B . ( top ) Representative image for GluN1. (bottom) The graph shows GluN1 did not change protein expression after nIH exposure compared with control. (two tailed t-test, t=0.63; df=4.1; P=0.55). C . (top) Western blot picture for GluN2A. (bottom) Comparison for both conditions show GluN2A decreased the protein content levels after nIH. (two tailed t-test, t=4.017; df=6.43; P=0.006). D . ( top ) Representative immunoblot image for GluN2B. (bottom) Comparison of both conditions show increased GluN2B levels after nIH (two tailed t-test, t=3.43; df=5.78; P=0.014). E . GluN2B/GluN2A comparison ratio. F . ( left ) Representative traces of the evoked fEPSP control +TCN [5 µM] (orange), control + ifenprodil [5 µM] (cyan) and control + both drugs (olive) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS and ( right ) Comparison of fEPSP slope represented as percent change from baseline at 60 min after TBS (one way ANOVA, F (3,20) =43.52; P<0.001). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . G . ( left ) Representative traces of the evoked fEPSP from nIH+TCN [5 µM] (dark yellow) and nIH +ifenprodil [5 µM] (light blue) in baseline conditions prior to TBS (1) and following TBS (2). ( middle ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS (two tailed t-test, t=12.46; df=4.59; P=0.001). Red dashed line represents the mean slope of the fEPSP 60 min following TBS in nIH slices from . Scale bars for A, F and G: 10 msec x 0.2 mV. The box-plot parameters indicate mean ± S.E. The analysis was performed for A to E using unpaired two-tailed t-test with Welch’s correction and for F and G the analysis was performed using one-way ANOVA followed by Bonferroni post hoc. **P<0.01, ***P<0.001 and ****P<0.0001).

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Expressing, Two Tailed Test, Western Blot, Comparison

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . Malondialdehyde (MDA) content was measured in hippocampal homogenates from control, nIH Saline and 10-Mn. (one way ANOVA, F (2,12) =11.53; P=0.0016). B . (top) Representative blot of nuclear HIF1a performed from control, nIH Saline and IH 10-Mn mice. (bottom) Quantification of HIF1a expression (one way ANOVA, F (2,12) =5.76; P=0.017). C . (top) Immunoblot of NOX2. (bottom) Significant differences was found in hippocampal homogenate from nIH Saline compared to control and nIH Mn (one way ANOVA, F (2,15) =9.26; P=0.0024). D . (top) Representative image of NOX4. ( bottom ) Comparison of NOX4 expression between control, nIH Saline and nIH Mn . (one way ANOVA, F (2,9) =11.46; P=0.0034). E . ( left ) Representative blot of GluN2A from control, nIH Saline and nIH Mn mice. ( right ). Quantification of GluN2A expression. (one way ANOVA, F (2,15) =6.63; P=0.0086). F . ( left ) Immunoblot of GluN2B. ( right ) Significant differences were found in hippocampal homogenate from nIH Saline vs control and nIH Mn . (one way ANOVA, F (2,12) =6.88; P=0.01). G . (top) Representative traces of evoked fEPSP from nIH Mn (purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn . ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in nIH vs nIH Mn slices. (two tailed t-test, t=3.41; df=10.61; P=0.0061). Dashed lines represent the mean slope of the fEPSP 60 min following TBS in control (black dashed line) and nIH (red dashed line) slices from . H . (top) Representative traces of the evoked fEPSP from nIH Mn in presence the ifenprodil [5 µM] (light purple) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to the slope before TBS in nIH Mn in presence the ifenprodil. ( right ) fEPSP slope represented as percent change from baseline at 60 min after TBS in IH+ifenprodil vs nIH Mn in presence of ifenprodil. (two tailed t-test, t=3.99; df=6.99; P=0.019). Blue dashed line represents the mean slope of the fEPSP 60 min following TBS in ifenprodil treated nIH slices from . For G and H, Scale bars 10 msec x 0.2 mV. The analysis was performed for A to F using one-way ANOVA followed by Bonferroni post hoc and for G and F, the analysis was performed using unpaired two-tailed t-test with Welch’s correction. *P<0.05, **P<0.01, and ***P<0.001.

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Saline, Expressing, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: A Consequence of Immature Breathing induces Persistent Changes in Hippocampal Synaptic Plasticity and Behavior: A Role of Pro-Oxidant State and NMDA Receptor Imbalance

doi: 10.1101/2023.03.21.533692

Figure Lengend Snippet: A . (top) Representative traces of the evoked fEPSP from adult control (black) and adult mice were exposed to neonatal IH (red) in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS in adult control vs Adult nIH (two tailed t-test, t=5.70; df=9.04; P=0.003). B . (top) Representative trace of the evoked from control+ TCN-213 [5 µM] and control + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to baseline prior to TBS. ( right bottom ) fEPSP comparison at 60 min following TBS in control +TCN and control+ ifenprodil (two tailed t-test, t=7.43; df=4.65; P=0.0009). Black dashed line represents the mean slope of the fEPSP 60 min following TBS in control slices from . C . (top) Representative trace of the evoked response from Adult nIH + TCN-213 [5 µM] and Adult nIH + ifenprodil [5 µM] in baseline conditions prior to TBS (1) and following TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time relative to baseline prior to TBS. ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min following TBS (two tailed t-test, t=4.72; df=4.06; P=0.0064). Red dashed line represents the mean slope of the fEPSP after 60 min following TBS in control slices from . D . (top) Representative traces of the evoked fEPSP from adult mice was exposure to neonatal IH+10 days of MnTMPyP (Adult nIH-Mn ) and adult mice receive MnTMPyP after exposure to neonatal IH (Adult REC-Mn ) in baseline conditions before TBS (1) and after TBS (2). ( left bottom ) Mean fEPSP slope plotted as a function of time and relative to slope before TBS in Adult nIH-Mn vs Adult REC-Mn ( right bottom ) fEPSP slope represented as percent change from baseline at 60 min after TBS Adult nIH-Mn vs Adult REC-Mn (two tailed t-test, t=3.79; df=8.16; P=0.0051). E. Representative image of GluN1. ( bottom ) Quantification shows GluN1 protein expression is not changed in Adult nIH , Adult nIH-Mn or 10+Mn exposures compared to control (one way ANOVA, F (3,16)=1.14 ; P=0.93; N=5). F . (top) Representative blot of GluN2A performed from adult mice unexposed, Adult nIH , Adult nIH-Mn or Adult REC-Mn . ( bottom ). Quantification of GluN2A expression from adult control, Adult nIH , Adult nIH-Mn or 10+Mn. (one way ANOVA, F (3,12)=8.31 ; P=0.0029, N=4). G . (top) Immunoblot of GluN2B. ( bottom ) Significant differences were found in hippocampal homogenates from adult control, Adult nIH , Adult nIH-Mn or Adult REC-Mn (one way ANOVA, F (3,12)=4.91 ; P=0.018, N=4). The box plot parameters indicate mean ± S.E. The analysis was performed for A-D using unpaired two-tailed t-test with Welch’s correction. The analysis was performed for E-G using one-way ANOVA followed by Bonferroni post hoc. *P=0.05, **P=0.01, ***P=0.001 and N.S= no significant. Scale bars for A,B, F and G= 10 msec x 0.2 mV

Article Snippet: Membranes were incubated under constant shaking with primary antibodies: anti rabbit GluN1 (1:2000; Abcam Cat# ab109182, RRID:AB_10862307) anti-rabbit GluN2A (1:2000; Cell Signaling Technology Cat# 4205, RRID:AB_2112295), anti-rabbit GluN2B (1:2000; Cell Signaling Technology Cat# 14544, RRID:AB_2798506), anti-rabbit NOX2 (1:2000; Abcam Cat# ab129068, RRID:AB_11144496) anti-rabbit NOX4 (1:500; Novus Cat# NB110-58851B, RRID:AB_1217375 and anti-mouse GAPDH (1: 10.000; Abcam Cat# ab8245, RRID:AB_2107448).

Techniques: Control, Two Tailed Test, Comparison, Expressing, Western Blot

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: The changes of interstitial glutamate concentration, astrocytic glutamate reuptake function, and the expression of GluN2B, GluA1 and glutamate transporters in hippocampus after SAH at 8 weeks. (A) The concentration of interstitial glutamate in the hippocampus of the mice was detected by HPLC-MS/MS. **, P<0.01, the SAH group vs. the sham group. N=8 mice per group. (B) Glutamate uptake assay on the hippocampal astrocytes sorted by immunomagnetic beads. **, P<0.01, the SAH group vs. the sham group. N=6 mice per group. (C,D) Western blot was performed to detect the expression of p-GluN2B/GluN2B, p-GluA1/GluA1, GLT-1, and GLAST protein in hippocampal tissue in the sham group and the SAH group at 1 week before and 8 weeks after SAH. **, P<0.01, vs. the indicated groups. N=5 animals in each group.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Concentration Assay, Expressing, Tandem Mass Spectroscopy, Western Blot

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: Effects of selective HDAC2 inhibitor and GLT-1 inhibitor on DCI in SAH mice. (A) The SAH mice were intraperitoneally administered with HDAC2 inhibitor (2 mg/kg) and GLT-1 inhibitor (WAY-213613, 1 mg/kg) two weeks after surgery once every other day. The expression of GLT-1, HDAC2, p-GluN2B and p-GluA1 in hippocampus was detected after SAH at 8 weeks. N=5 animals in each group. (B) Forced swimming test and (C) sugar water preference test were used to evaluate the depressive behavior of SAH mice treated with HDAC2 inhibitor (Santacruzamate A) and GLT-1 inhibitor (WAY-213613). **, P<0.01, vs. the sham group; #, P<0.05 and ##, P<0.01, vs. the indicated groups. (D) Morris water maze test was used to detect the escape latency for reflecting spatial learning memory, *, P<0.01, and **, P<0.01, vs. the indicated groups. (E) Reference memory was detected by recording the time in target quadrant of Morris water maze test. **, P<0.01, vs. the sham group; #, P<0.05, vs. the indicated groups. N=8 mice per group.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: Negative regulation of GLT-1 by astrocytes HDAC2 leads to dysfunction of glutamate reuptake in the synaptic cleft. In normal condition, the acetylation of histones in astrocytes facilitates the transcriptional regulation of GLT-1. Glutamate in the synaptic cleft is rapidly absorbed into astrocytes to maintain excitability of synapses. The increase of HDAC2 in astrocytes after SAH results in the deacetylation of histones and inhibits the transcription expression of GLT-1. The decrease of GLT-1 expression will lead to the impairment of glutamate reuptake in astrocytes and the long-term accumulation of glutamate in the synaptic space, resulting the dephosphorylation of ionized glutamate receptors GluN2B and GluA1 on the postsynaptic membrane. These eventually result in the long-term inhibition of synaptic excitability and DCI. DCI, delayed cognitive impairment.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Expressing, De-Phosphorylation Assay, Inhibition

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: Electron micrograph depicting a layer III dlPFC spine receiving a synapse (white arrows) from an axonal bouton. Post-embedding immunoEM preparation reveals immunogold particles (black arrowheads) labeling NMDAR-GluN2B in the post-synaptic density. Adapted from .

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A , Confocal microscopy fluorescent images depicting layer III dlPFC single-labeled for NMDAR-GluN2B using the Alomone rabbit anti-GluN2B (cat #AGC-003) antibody. B , A control section incubated in a separate aliquot of primary antibody solution used in A , but with the addition of the Alomone blocking peptide (cat #BLP-GC003). The negligible labeling observed after imaging with the same parameters as A suggests that the paratope region of the antibody has very low non-specific interactions in our tissue. C , Electron micrograph from a control section of tissue with the omission of the primary antibody (1°) but all other procedures intact. The image was captured at the edge of the tissue “coming into plane”, where antibody penetration is at its greatest, often producing some noise or background level labeling. D , Electron micrograph from the same batch of tissue, from a section treated with both the primary antibody and secondary antibody (1°), and all other procedures held constant. The image is taken near the edge of the tissue “coming into plane” (right), where antibody penetration is typically greatest, often producing some noise or background level labeling.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Confocal Microscopy, Labeling, Control, Incubation, Blocking Assay, Imaging

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: Tiled images obtained via confocal microscopy of immunolabeled PV (red), CB (yellow), CR (magenta), and NMAR-GluN2B (cyan) in A25 ( A ) and dlPFC ( B ) across all laminar compartments. Images were systematically sampled from layer III. For each image, we isolated the NMDAR-GluN2B channel. Then we segmented i) NMDAR-GluN2B+ pyramidal-like neurons, as judged by morphology; and ii) manually selected immunonegative regions of tissue with no labeled NMDAR-GluN2B processes. We measured the mean intensity (MI) in all of these traces, and took the mean for each category (GluN2B+ pyramidal neurons or immunonegative background). We then used the other channels to segment the somata of PV, CB, and CR neurons. Then the MI was measured for each PV, CB, and CR neuron. We then used the mean NMDAR-GluN2B expression of the pyramidal neurons and of the immunonegative “neuropil” regions to create a normalized index of expression for each inhibitory neuron, where 0 was the average across sampled neuropil regions, and 1 was the average across sampled pyramidal-like neurons. This index forms the y-axis for C-F . Individual circles for each plot represent a cell, and these data were pooled across images after normalization. We divided the index into four equal bins from [0,1], delineated by dotted lines (Negative, at or below the average MI across sampled immunonegative regions; Weak; Moderate; or Strong, which was defined as at or above the average MI across sampled pyramidal neurons). Compiled data for Monkey 1 in A25 ( C , One-way ANOVA, F(5,562)=6.314, p<0.0001, with post-hoc Tukey test) and dlPFC ( D One-way ANOVA, F(4,701)=35.97, p<0.0001, with post-hoc Tukey test), and for Monkey 2 in A25 ( E, One-way ANOVA, F(4,1693)=75.90, p<0.0001, with post-hoc Tukey test) and dlPFC ( F, One-way ANOVA, F(3,542)=30.23, p<0.001, with post-hoc Tukey test) . G, Mean percent across cases of CBP+ inhibitory neurons by type that fell into Negative, Weak, Moderate, or Strong bins. CB, calbindin; CBP, calcium-binding protein; CR, calretinin; PV, parvalbumin; WM, white matter. *, p < 0.05; **, p< 0.01, *** p< 0.001; ****, p<0.0001

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Confocal Microscopy, Immunolabeling, Isolation, Labeling, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A , Electron micrograph depicting spines in A25 layer III. A1 , An A25 spine (sp, pseudocolored yellow) with NMDAR-GluN2B immunogold particles (green arrowheads) in the postsynaptic density of a synapse (black arrows) formed by an axonal bouton (ax, pseudocolored blue). The spine contains a spine apparatus, an extension of the smooth endoplasmic reticulum (SER, pseudocolored pink) in the spine neck. A2-A3, A25 spines with NMDAR-GluN2B adhered to extrasynaptic membranes (orange arrowheads), near the SER. In A3, the extrasynaptic NMDAR-GluN2B is apposed to a structure consistent with glial morphology (gl, pseudocolored green), and the bouton contains a presynaptic cytoplasmic NMDAR-GluN2B (blue arrowhead) among the vesicles. B, Electron micrographs depicting spines in dlPFC layer III. B1-B2, dlPFC spines containing synaptic NMDAR-GluN2B (green arrowheads), and cytosolic NMDAR-GluN2B (grey arrowheads), which are likely being trafficked. B3, A dlPFC spine with an extrasynaptic NMDAR-GluN2B in the spine neck, apposed to a structure consistent with glial morphology, and near a spine apparatus in the spine neck. C, Nested pie charts depicting location of NMDAR-GluN2B immunogold particles in spines of A25 (left), and dlPFC (right) in Monkey 1 (M1, inside) and Monkey 2 (M2, outside). Percent of NMDAR-GluN2B immunogold particles found in the cytoplasm (grey), post-synaptic density (green), perisynaptic membrane (yellow-green), and extrasynaptic membrane (orange) of GluN2B+ spines. D, Plot depicting the location of membrane-bound GluN2B immunogold particles, in relation to the synapse, averaged across M1 and M2. Error bars depict standard deviation. One-way ANOVA, F(5,6=67.71, p<0.001, with post-hoc Tukey test). *, p < 0.05; **, p< 0.01, *** p< 0.001; ****, p<0.0001; scale bars, 200nm. ax,axon; gl, glial process; mit, mitochondria; mvb, multivesicular body; SER, smooth endoplasmic reticulum spine apparatus; sp, spine.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Membrane, Standard Deviation

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A , Electron micrographs of A25 spines (pseudocolored yellow), receiving synapses (denoted by black arrows) formed by axon terminals (pseudocolored blue). NMDAR-GluN2B are prominent expressed in the synapse (green arrowheads), or at cytosolic locations (grey arrowheads). B , A25 spines with prominent perisynaptic NMDAR-GluN2B (yellow-green arrowheads). NMDAR-GluN2B were classified as perisynaptic when within ∼100nm of membrane distance from the synapse. C, A25 spines with NMDAR-GluN2B expressed in the extrasynaptic membrane (orange arrowheads). D , A25 spines with extrasynaptic NMDAR-GluN2B apposed to glial-like processes (pseudocolored green), which sometimes also express NMDAR-GluN2B ( e.g. , D2 ). Presynaptic cytosolic NMDAR-GluN2B (blue arrowheads) are occasionally evident ( e.g. , D4 ). E-H, same as above but for dlPFC spines. Scale bars, 200nm; ax, axon; mit, mitochondria; sp, spine

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Membrane

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A, Plot of the major Feret’s diameter of spines in A25 and dlPFC that were GluN2B+ and GluN2B- in plane. Error bars depict standard deviation. One-way ANOVA with post-hoc Tukey test, F(7,1343)=26.69, p<0.001. B , Swarm plot depicting the shortest distance from membrane bearing the NMDAR-GluN2B to the nearest spine apparatus SER membrane for all membrane-bound GluN2B immunogold particles found in spines. Swarm plot also depicts mean and standard deviation. *, p < 0.05; **, p< 0.01, *** p< 0.001; ****, p<0.0001;

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Standard Deviation, Membrane

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: An A25 spine (sp, pseudocolored yellow) with prominent NMAR-GluN2B synaptic labeling (green arrowhead), and presynaptic NMDAR-GluN2B in the bouton (ax, pseudocolored blue) in the cytosol (darker blue arrowheads) amidst the vesicles, or on the extrasynaptic bouton membrane (lighter blue arrowhead). Black arrows denote the boundaries of the perforated synapse.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Labeling, Membrane

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A, Electron micrographs from layer III A25. A1 , A putative excitatory dendrite, labeled with MAP2+ non-nickel immunoperoxidase diaminobenzidine (smudge-like precipitate, double-headed arrows), expressing an extrasynaptic NMDAR-GluN2B (orange arrowhead); A2-A3 , NMDAR-GluN2B at extrasynaptic (orange arrowheads), cytoplasmic (grey arrowheads), or near-synaptic locations (grey-green striped arrowhead) in putative excitatory dendrites. B , Electron micrographs from layer III dlPFC. B1, A putative excitatory dendrite, with a spine in plane, expressing cytoplasmic and extrasynaptic NMDAR-GluN2B. B2, A putative excitatory dendrite, labeled with MAP2, expressing extrasynaptic and cytoplasmic NMDAR-GluN2B. C, Nested pie charts depicting the percent of NMDAR-GluN2B immunogold particles found at cytoplasmic, extrasynaptic, perisynaptic, and synaptic locations in MAP2+ dendritic shafts in A25 (left) and dlPFC (right) of Monkey 1 (outside) and Monkey 2 (inside). Synapses on the shaft of MAP2+ dendrites were rare, and synaptic NMDAR-GluN2B on MAP2+ shaft synapses were extremely rare (<0.2% of all immunogold particles in all areas analyzed). D, Mean percent of extrasynaptic NMDAR-GluN2B immunogold particles across all MAP2+ dendrites in A25 and dlPFC. One-way ANOVA with post-hoc Tukey test, F(3,4) = 1691, p<0.0001. *, p<0.05; pink pseudocolor, SER spine apparatus; black arrows, synapse; scale bars, 200nm; ax, axon; dend, dendrite; MAP2, microtubule-associated protein-2; mit, mitochondria; sp, spine;

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Labeling, Expressing

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A , A MAP2+ putative excitatory dendrite (pseudocolored yellow) with NMDAR-GluN2B labeling at intracellular (grey arrowheads) and extrasynaptic (orange arrowheads) locations. B,C , Insets from A .

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A, A panorama of stitched electron micrographs depicting a pyramidal-like soma and dendrites (psuedocolored yellow), including nucleus (pseudocolored plum) and its axon initial segment (pseudocolored blue). NMDAR-GluN2B are prevalently expressed in extrasynaptic membranes (orange arrowheads) at the soma and proximal processes. Black boxes depict inset locations for subsequent panels. B-G, Insets from A depicting extrasynaptic NMDAR-GluN2B at higher magnification. H, Inset from A selected to emphasize the antibody labeling specificity of the labeled neuron compared to the surrounding neuropil, as well as to emphasize nuclear labeling. Few NMDAR-GluN2B immunoparticles are evident in the surrounding neuropil, while the pyramidal-like soma densely expresses cytosolic NMDAR-GluN2B immunoparticles (grey arrowheads). NMDAR-GluN2B immunoparticles are also present in the nucleus (pseudocolored plum, white arrowhead). I, Inset from A depicting several more extrasynaptic NMDAR-GluN2B on a basal dendrite, as well as a symmetric synapse (double arrowheads) formed on the soma (axon pseudocolored red). Scale bars in B-I , 200nm.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Antibody Labeling, Labeling

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: Electron micrographs depicting high likelihood inhibitory dendrites in A25 ( A ) and dlPFC ( B ). Dendrites were deemed high likelihood inhibitory dendrites by the lack of spines in plane, and the presence of two or more asymmetric synapses formed on the dendritic shaft, as inhibitory dendrites in the cortex are sparsely spiny or aspiny . A1-A3, Examples of NMDAR-GluN2B immunogold labeling in high likelihood inhibitory dendrites found in layer III A25. B1-B2, Examples of NMDAR-GluN2B immunogold labeling in high likelihood inhibitory dendrites found in layer III dlPFC. C, Nested pie charts depicting location of NMDAR-GluN2B immunogold particles in high likelihood dendrites of A25 (left) and dlPFC (right) for Monkey 1 (inside) and Monkey 2 (outside). No statistically significant differences were detected between dlPFC and A25. Scale bars, 200nm. ax, axon; dend, dendrite; gl, glial process; mit, mitochondria.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Labeling

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A-B , Maximum projection Images of layer III A25 ( A ) and dlPFC ( B ) obtained via confocal microscopy depicting multiple immunofluorescence labeling for PV (red), CB (yellow), CR (magenta), and NMDAR-GluN2B (cyan). Color-coded arrows depict inhibitory neurons, and double-headed arrows indicate CB+ pyramidal neurons. C-D, Stacked bar charts depicting mean proportion of PV (red), CB (yellow), and CR (magenta) that fell into negative, weak, moderate or strong NMDAR-GluN2B expression bins as determined by the mean intensity (MI) in the NMDAR-GluN2B channel. GluN2B-negative is defined by MI at or below the average amount in immunonegative sampled neuropil regions. GluN2B-strong is defined by MI at or above the average found in morphologically identified pyramidal-like neurons expressing NMDAR-GluN2B. See for more detailed information about the inhibitory neuron analysis. CB, calbindin; CR, calretinin; PV, parvalbumin.

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Confocal Microscopy, Immunofluorescence, Labeling, Expressing

Journal: bioRxiv

Article Title: Contrasting patterns of extrasynaptic NMDAR-GluN2B expression in macaque subgenual cingulate and dorsolateral prefrontal cortices

doi: 10.1101/2025.02.05.636752

Figure Lengend Snippet: A, Schematic depicting a spine (yellow), receiving a synapse from a glutamatergic bouton (blue), with an astrocytic leaflet (green) near the synapse. A synaptic NMDAR-GluN2B is present in the synapse (green), and an extrasynaptic NMDAR-GluN2B (orange) is present near the astrocytic process. B, A more magnified view showing that the bouton releases glutamate (grey circles) toward the postsynaptic density (thick black band), where the green synaptic NMDAR-GluN2B is engaged and calcium ions (pink), as well as sodium (Na+) ions, flow into the spine. AMPA receptors in the post-synaptic density also allow influx of sodium ions (Na+), though these are emphasized less as they are not the focus of the current work. Incoming calcium ions can trigger feedforward calcium release( ; ), via 1) direct calcium-mediated calcium release from the smooth endoplasmic reticulum (SER) via activation of primarily Ryanodine receptors (RyR), and 2) by cAMP magnification of calcium release, whereby calcium activates AC to produce cAMP which activates PKA signaling. PKA in turn phosphorylates the SER calcium channels RyR and IP3R to further increase calcium release. Glutamate escaping out of the synaptic cleft is sequestered into the astrocyte via the EAAT, where it can be converted to glutamine. In the dlPFC, calcium influx can also lead to a reduction in delay-related firing via SK3 channels (not shown). C, If the EAATs are perturbed or downregulated, then glutamate can more readily engage extrasynaptic NMDA-GluN2B. Evidence suggests that there may be glial pathology in A25 (i.e. the SGC) during states of depression [ reviewed in ( ; ; ; )]. Given the prevalence of extrasynaptic NMDA-GluN2B we have found in the present study, we hypothesize that these may contribute to A25 hyperactivity observed in depression, perhaps by engaging feedforward calcium mechanisms and increasing depolarization (yellow arrow). D , Rapid acting antidepressants that antagonize NMDA receptors may work in part by blocking extrasynaptic NMDAR-GluN2B in the SGC . E, Calcium is normally tightly regulated by cytosolic buffers (like calbindin and mitochondria), and by phosphodiesterases (which catabolize cAMP). Loss of this regulation with aging and/or inflammation( ; ; ) can dysregulate feedforward calcium signaling. Very high levels of cytosolic calcium can activate calpain-2, which cleaves and disinhibits GSK3β and cdk5, kinases that hyperphosphorylate tau, producing toxic species like pT217Tau . Further post-translational modifications lead to tau fibrillation and the formation of neurofibrillary tangles. AC, adenylyl cyclase; cAMP, cyclic adenosine monophosphate; cdk5, cyclin-dependent kinase 5; EAAT, excitatory amino acid transporter; GSK3β, glycogen synthase kinase 3 beta; IP3R, inositol triphosphate receptor; PKA, protein kinase A; SER, smooth endoplasmic reticulum spine apparatus; RyR, ryanodine receptor

Article Snippet: One section was run in tandem with the above, but incubated in antibody dilution buffer containing the Alomone NMDAR-GluN2B primary antibody, but with the addition of the blocking peptide for the NMDAR-GluN2B antigen (Alomone, cat# BLP-GC003), at 10x the concentration of the primary antibody ( ).

Techniques: Activation Assay, Blocking Assay

Journal: BMC Complementary and Alternative Medicine

Article Title: Tong Luo Jiu Nao ameliorates Aβ 1–40 -induced cognitive impairment on adaptive behavior learning by modulating ERK/CaMKII/CREB signaling in the hippocampus

doi: 10.1186/s12906-015-0584-9

Figure Lengend Snippet: Effects of drugs administration on the expression levels of memory-related molecules in the hippocampus. Subpart (A) is the expression levels of mAchR M1 receptor; subpart (B) is the expression levels of phosphor-NMDAR1 and NMDAR1 proteins; subpart (C) is the expression levels of phosphor-NMDAR2B and NMDAR2B proteins; subpart (D) is the expression levels of phosphor-ERK1/2 and ERK1/2 proteins; subpart (E) is the expression levels of phosphor-CaMKII and CaMKII proteins; subpart (F) is the expression levels of phosphor-CREB and CREB proteins;. All data are expressed as means ± SEM, n=3. Significant differences *p<0.05, **p<0.01, ***p<0.001; compared with the sham; #p<0.05, ##p<0.01 compared with the Aβ 1–40 .

Article Snippet: Antibodies for Phospho-NMDAR1 (Ser890), NMDAR1 (D65B7), Phospho-NMDAR2B (Tyr1070), NMDAR2B, Phospho-CaMKII (Thr286), CaMKII (pan), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2) (137 F5), Phospho-CREB (Ser133) and CREB (48H2) were obtained from Cell Signaling Technology (Cell Signaling, USA).

Techniques: Expressing