Fig 2: Additional features of SynapseJ allow for identification of synaptic puncta with increased discrimination. (A) Specifying a minimum number of pixel overlap of 6 or more, removes some overlapping puncta (arrow), while other puncta remain (arrowhead), in this image of anti-bassoon (magenta) and anti-gephyrin (cyan) from BLA. Scale bar is 10 µm for the panels in panel (A). (B) In this image of anti-bassoon from SNc, applying the subtract background with a radius of 10 efficiently removed background staining while not modifying the brightly labeled puncta. Scale bar is 10 µm, and applies to panels in panels (B–D). (C) Original immunostaining for NR1 (cyan), tyrosine hydroxylase (blue) and YFP in SNc from the same area as the bassoon in panel (B). (D) The merge images for the original staining (left), identified overlapping anti-bassoon/anti-gephyrin puncta from the entire panel (center), and identified overlapping YFP+ PPN axonal anti-bassoon puncta with TH+ SNc neuronal anti-NR1 puncta (right) demonstrate the utility of SynapseJ in identifying neuronal sub-class limited expression of synaptic markers.

Fig 3: Adeno-associated virus (AAV)-mediated ablation of GluN1 in the hippocampus. (A) Schematic illustration of AAV-CMV-eGFP and AAV-CMV-Cre-eGFP construct. (B) AAV expression is verified by eGFP (reporter) fluorescence in the hippocampus. Scale bar = 100 µm.

Fig 4: Activity-dependent expression of SK2 and GluN1. (A,B) Western blot and bar graph demonstrating hippocampal NMDAR-GluN1 expression. (C,D) Representative immunoblot and bar graph illustrating hippocampal SK2 expression. (E) Fluorescence image (DAPI) showing the track of the cannula guide and cannula for intrahippocampal injections. Scale bar = 0.5 mm (E; B,D; *p < 0.05, **p < 0.01).

Fig 5: SK channel potentiation impacts hippocampal a-actinin expression. (A–B) Fluorescence images illustrating a-actinin expression in the hippocampus (so, stratum oriens; pyr, pyramidal layer; sr, stratum radiatum; mol, molecular layer; gr, granule cell layer, and hil, hilus). Scale bar = 0.4 mm (A), 100 µm (B). (C) Bar graph representation of a-actinin fluorescence intensity in the hippocampus (One-Way ANOVA). Schematic illustration of SK2 modulation of CaMKII spine anchorage: (D) synaptic pCaMKII is structurally linked with to the GluN2B sub-unit of NMDAR and is anchored to the dendritic spine cytoskeleton (F-actin) by a-actinin. In NMDAR-mediated synaptic potentiation, synaptic accumulation of pCaMKIIa/ß is facilitated by a-actinin. (E) SK channel potentiation induced a loss CaMKII and a-actinin expression (C; **p < 0.01).