Fig. 4

The novel clusters of NeuN⁺ puncta are formed from synaptophysin-positive presynaptic, swollen terminals and MAP2 positive postsynaptic structures. A In the CA1 region of the hippocampus of a 12-month old DPP6-KO, immunofluorescence for NeuN (green, 1:500, Millipore) and synaptophysin (red, 1:500, Sigma) show complete colocalization in clusters of the novel large puncta. On the right is a high magnification image showing the colocalized NeuN and synaptophysin in the individual, large puncta of the cluster. Scale bar = 50 μm. sp, stratum pyramidale; sr, stratum radiatum. B Immunogold labeling for NeuN and synaptophysin in the deep region of the CA1 (probably stratum lacunosum-moleculare) of the DPP6-KO mouse. 20 nm gold for NeuN is concentrated within the large swellings (arrows in low magnification image; N in high magnification images, Bi and Bii), where it colocalizes very well with 10 nm gold (arrows in high magnification images; all 10 nm gold particles in the image are indicated with the arrows) for synaptophysin. Both gold sizes are relatively uncommon outside of the swellings. The positions of swellings in Bi and Bii are indicated on the low magnification, left micrograph, which has at least 10 swellings (arrows). Scale bars in Bi and Bii are 500 nm. C, D In the CA1 of the 12-month old DPP6-KO hippocampus, immunofluorescence for NeuN (green, 1:500, Millipore) and MAP2 (red, 1:500, Millipore) are partly colocalized in the large novel puncta clusters, with MAP2 on the periphery of most NeuN⁺ puncta and throughout some of them, as evident in the higher magnification (D); MAP2 also is evident in the apical dendrites of the CA1 pyramidal neurons that overlap with the clusters. Nuclei were counterstained with DAPI in blue. Scale bar = 5 μm. sp, stratum pyramidale; sr, stratum radiatum. E Immunogold labeling in the CA1 stratum radiatum of the 12-month DPP6-KO mouse. 20 nm gold for NeuN is concentrated within the large swellings (N), while 10 nm gold for MAP2 is mostly absent from the swellings except for a few small clusters. In contrast, the MAP2 labeling is highest in the large apical dendrites (ad) and smaller postsynaptic structures (p) including spines (sp); but presynaptic terminals (t) show little labeling. The right micrographs show higher magnifications of the swelling in the upper left micrograph. The lower left inset is magnified from the lower left micrograph. Scale bars on the left are 2 micrometers, the bar in the inset is 500 nm, and the right scale bars are 400 and 200 nm for the top and bottom micrographs, respectively