Image Quantification and Presentation.

All analysis was done on nonmanipulated, raw images in ImageJ. For quantification of puncta number and size, the cell-fill channel (GFP) was thresholded and then used to create a mask tracing the border of the cell. Only secondary and tertiary dendrites were included, and their total length was measured per image. Then the channel for each type of punctum was thresholded and counted within the cell-fill mask using the “analyze particles” function. The number and size of each punctum were recorded. The number of puncta was divided by the length of the corresponding dendrite, and both punctum density and punctum shape were quantified per image. Spines were counted manually. Only secondary and tertiary dendrites were included, and their total length was measured per image. The total spine number was divided by the length of the corresponding dendrite per image. The length between the tip of each spine and the junction between the spine and the dendrite and the width of the head of each spine were measured manually on about 50 randomly picked spines per image. The shape of each spine was calculated by dividing the width by the length of each spine. This ratio was compared between all WT and KO spines. For expression analysis comparing full-length and truncated OGT, the laser intensity varied slightly between images. The differences were minor and were not consistently lower or higher for either construct and did not affect the interpretation of the experiment. For quantifying PSD-95 and OGT overlap, both channels were first thresholded. Thereafter, a mask was created for every PSD-95 puncta by using the “analyze particles” function. Any positive OGT signal within the mask was counted as positive overlap. Whereas all analysis was done on raw, unmanipulated images, for image presentation purposes, most images shown in the figures were improved post hoc. Within the same experiment, all changes were done in the same way between WT and KO images. The manipulations were applied in Photoshop and MS Paint and included: levels and brightness/contrast and filtering (“despeckle” and “Gaussian blur”).