Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) Examples of VE-2A, VE-2B, VE-2BΔ7, and VE three hours after exit from the ER immunostained for SAP102 (red), and synaptophysin (Sp; pseudocolored blue), and merged. VE-2B, and VE-2BΔ7 demonstrate significant clustering compared to VE (quantified in E), and targeting to synaptophysin (quantified in D). (B) Higher magnifications of clusters from VE-2B and VE-2BΔ7 seen in A (indicated by boxes) demonstrate roughly equivalent colocalization to SAP102 (quantified in C) and synaptophysin (quantified in D; scale bars 1 µm). (C) VE-2B and VE-2BΔ7 co-localized with postsynaptic SAP102 at 2X background (52–255 inclusive gray scale). By one way Anova (P<0.01) there was a significant group difference. Using Tukey’s post hoc pairwise comparisons, both VE-2B and VE-2BΔ7 pixel overlap with SAP102 was significantly greater than VE (p<0.05), however VE-2B and VE-2BΔ7 were not significantly different than eachother. This relationship between VE-2B and VE-2BΔ7 overlap with SAP102 was only obtained when analyzing pixel overlap at 2X background. (D) The percent of overlap of VE-2B, and VE-2BΔ7 with synaptophysin was significantly greater than VE (one-way Anova with post hoc pairwise comparisons to VE * p<0.05). Surprisingly, VE-2A was not significantly different than VE among the 4 groups in a post hoc comparison. VE-2B targeted to synaptophysin significantly better than VE-2A (** p<0.05) but no differently than VE-2BΔ7. These relative differences were the same regardless of the green or blue threshold. The same results were obtained at this time point after ER release using synapsin as the presynaptic marker (data not shown). (E) Clustering was measured using Zeiss LSM510 image analysis software. Average intensity was calculated from each intensity graph of 20–30 dendrites for a total of 839.5 µm (VE-2B), 750.4 µm (VE-2BΔ7), and 776.0 µm (VE). A cluster was defined as being more than twice the average intensity of each dendrite for equal to or greater than 0.4 µm. The average number of clusters per µm ± SEM is plotted in E. There was a significant effect of group by one-way Anova. Post hoc comparisons indicated Both VE-2B and VE-2BΔ7 showed significantly more clustering than VE (p<0.05), and were not significantly different from each other. (F) Examples of immunogold labeling with i14 α-VSVG antibody (10 nm; arrowheads) and α-NR2A/B antibody (5 nm; arrows) indicate localization of VE-2B at synapses 3 hours after release from the ER (pre, presynaptic terminal; post, postsynaptic process). Scale bar is 100 nm. Quantification of 10 nm gold indicated that 10 of 47 synapses were labeled within 0–100 nm, and 18 of 47 (38.3%) synapses showed immunogold labeling within 0–500 nm of the postsynaptic density.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Marker, Software, Labeling

Journal: PLoS ONE

Article Title: Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse

doi: 10.1371/journal.pone.0039585

Figure Lengend Snippet: (A) VE-2B and VE-2BΔ7 were allowed to exit the ER for 45 minutes and then immunostained for surface expression with I1 antibody and presynaptic terminals with anti-synapsin. Yellow arrows in VE-2BΔ7 indicate surface puncta not in the vicinity of synapsin (right panels). At 45 minutes after ER exit, only about 30% of VE-2B and VE-2BΔ7 puncta in dendrites showed any immunostaining with i1 antibody. (B) The VE and VE-2BΔ7 surface puncta more than 1 µm away from synapsin were significantly greater in relative number than VE-2B (left panel). The surface VE-2BΔ7 within 0.3 µm is similar to VE-2B but not significantly different from VE, while VE-2B within 0.3 µm is significantly different than VE (one-way Anova considering VE, VE-2B, and VE-2BΔ7 in the >1.0 micron bins and then in the <0.3 micron bins, then pairwise post hoc comparisons; p<0.05). Centroids and distances were calculated with images thresholded at 2X mean background. Percent pixel overlap of green puncta with synapsin in the same data set showed no difference in the total VE-2B and VE-2BΔ7 at any threshold and trended toward increased synaptic localization at 45 minutes after permissive temperature, but did not reach significance when compared to VE, as was apparent at 3 hours (one-way Anova, p = 0.11; right panel, indicated as ‘total’). Green and red images from the same data set also were merged and color-thresholded for yellow to define the surface population. Percent overlap of yellow puncta with synapsin (blue) was then assessed for VE, VE-2B, and VE-2BΔ7 (right panel, indicated as ‘surface’). VE-2BΔ7 surface pixel overlap with synapsin trended toward a decrease compared to VE-2B at 2X background but not significantly until thresholded at 3X background (one-way Anova, post hoc comparison p<0.05). (C) Model of trafficking of NR2B. NR2B forms hetero-oligomers with NR1 subunits at the level of the ER , but the NR2 distal C-terminus is necessary and sufficient to confer significant synaptic localization , . NR2A/B clusters with SAP102 early in the secretory pathway, and significantly so at the level of the cis-medial- Golgi. PSD-95 is added as part of the NR2B/NR1-SAP102 complex as soon as the TGN. NR2B/NR1-SAP102 complexes may be cotransported to the vicinity of the synapse, and also cotransported at least in-part along dendrites via Kif-17, mLin-2/Cask, mLin7, mLin10, and SAP97 in a poly-protein complex [see ] and added to postsynaptic structures. The NR2B/SAP102/PSD-95 association does not appear to be essential for immediate synaptic targeting, but is required for maintenance of position on the synaptic surface.

Article Snippet: Neurons were incubated with primary antibodies for 1 hour using the following dilutions: anti-GM130, 1∶200; anti-TGN38, 1∶200; anti-PSD-95, 1∶500 (T60); anti-PSD-95, 1∶150 (TL); anti-SAP102, 1∶500 (JH62514); anti-SAP102, 1∶200 (Alomone); anti-Synapsin, 1∶500; anti-Synaptophysin, 1∶500; anti-VSVG, 1∶3 (i1 hybridoma).

Techniques: Expressing, Immunostaining

Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images from naïve or 5HT6-GGECO1-expressing cultured mouse neurons (from cortex and CA1) showing the distribution of GABA-B1 receptors (magenta) in primary cilia positive for AC3 (green). Intensity profiles from lines drawn along the cilia are shown to the right and further show the confinement of GABA-B1 receptors to the cilia base of cultured neurons. B. Quantification of GABA-B1 receptor enrichment at the cilia base in naïve cultured neurons or cultured cortical and hippocampal neurons expressing 5HT6-GGECO1 (naïve 2,76±0,37; Cx 8,18±0,91; Hipp 4,94±0,44 arbitrary units). GABA-B1 positive segment length was measured for each group and found significantly different (naïve, Cx and Hipp, in µm: 2,78±0,37; 8,18±0,91; 5,03±0,39; mean±SEM; Kruskal-Wallis ANOVA followed by multiple comparisons, naïve n=12, Cx n=22, Hipp n=38, for A & B). C. Simultaneous recording of cytoplasmic (grey) and ciliary (pink) calcium activities from a neuron before and after stimulation with 100nM baclofen. Activation of metabotropic GABA receptors was without effect on the frequency of somatic events while ciliary signaling was increased by the agonist. Positive deviations from baseline were computed over time to produce ciliary activity count traces (black, lower panel). D. Averaged activity counts corresponding to neurons in control (blue) baclofen (100nM, black) and TTX (1 µM) + baclofen (100nM, grey) showing an increase of activity after stimulation with baclofen. E. Quantification of activity changes was performed in a pair wise fashion, comparing the averaged activity during initial vs final period (control) and baseline vs baclofen (either in the absence of presence of TTX). Time lacked an effect on activity (control P=0,4332) while baclofen stimulation led to a significant increase in both - /+ TTX conditions (respectively P=0,0002 and P=0,0039; two tailed Wilcoxon matched-pairs signed rank test) F. To test whether abolition of action potentials had an impact on spontaneous ciliary activity, recordings in control and TTX (initial 20 minutes both groups) were compared. Statistical analysis showed no difference (P=0,1468 Mann-Whitney two tailed t-test). G. Immunofluorescence images showing the distribution of primary cilia (AC3; green) and synapses (Synapsin II; magenta) in cultured cortical neurons. Boxed areas are magnified to the right. Scatter plot to the left shows Synapsin II fluorescence intensity at primary cilia and random locations within the same sample (P=0,3696 Wilcoxon matched pairs signed rank test).

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence, Expressing, Cell Culture, Activation Assay, Activity Assay, Control, Two Tailed Test, MANN-WHITNEY, Fluorescence

Journal:

Article Title: Long Term Synaptic Depression That Is Associated with GluR1 Dephosphorylation but Not ?-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Internalization *

doi: 10.1074/jbc.M803431200

Figure Lengend Snippet: Neither GluR1 nor GluR2/3 total surface expression is changed 45 min following cLTD treatment, as determined by cross-linking assay. A, representative blots of X-linked (25.0 μg of total protein loaded) and un-X-linked (5.0 μg of total protein loaded) samples for control and cLTD conditions probed with antibodies specific for GluR1 (left group) and GluR2/3 (right group). B, representative blots of un-X-linked and X-linked (5.0μg of total protein loaded for both) samples probed with antibodies specific for PP2Aα (top bands) and synapsin (bottom bands). C and D, graphs of cumulative data comparing X-linked control and X-linked cLTD samples probed with antibodies specific for GluR1 (C) and GluR2/3 (D)(n = 8). E and F, data from C and D normalized to the transferrin receptor. G and H, graphs of cumulative data comparing the percent surface expression (see “Experimental Procedures” for explanation of calculation) between control and cLTD conditions for GluR1 (G) and GluR2/3 (H)(n = 8). I and J, graphs of cumulative data comparing un-X-linked and X-linked samples probed with antibodies specific for PP2Aα (I) and synapsin (J)(n = 8). No significant differences were observed for any of the comparisons as determined by Student's paired t test. Values represent the mean ± S.E.

Article Snippet: Primary antibodies were as follows: GluR1 and GluR2/3 (Chemicon) used at 1:3000 in BSA, phospho-Ser-845 and phospho-Ser-831 (PhosphoSolutions) used at 1:1000 in BSA, PP2Aα (BD Pharmingen) used at 1:5000 in milk, synapsin, NR2B, and NR1 (PhosphoSolutions) used at 1:2000 in BSA, the α subunit of calcium/calmodulin-dependent protein kinase II (αCaMKII) (BD Pharmingen) used at 1:3000 in BSA, phospho-Thr-286 (PhosphoSolutions) used at 1:3000 in milk, PSD-95 (Affinity Bioreagents) used at 1:5000 in BSA, and transferrin receptor (Zymed Laboratories Inc.) used at 1:1000 in milk.

Techniques: Expressing