Fig. 3

Synaptic changes in limbic regions during disease. a Subregions within the three limbic regions (schematic diagram), including the hippocampal CA1, ventral medial hypothalamus (VMH), and basolateral amygdala (BLA), were assessed by immunofluorescence (IF) labelling for synapses. The right panels display the subregions labeled with phalloidin (green), where the dotted boxes mark the specific regions assessed by higher magnification confocal microscopy. b Immunofluorescence assessment of total synapses by MAP2 and synaptophysin in the CA1, VMH, and BLA in uninfected controls (UN) relative to infected (INF) samples from the pre-clinical and clinical onset phases. c, d The quantification of MAP2 (c) and synaptophysin (d) in multiple fields of view of immunofluorescence images represented in b. Each dot represents a field of view. e Western blotting analysis of synaptophysin in the three brain regions at both time points with the lower panels showing the Coomassie stain for total protein as the loading control. f The quantification of synaptophysin normalized to the total protein. g Western blotting analysis of PSD95 in the three brain regions at both disease time points with the bottom panels showing Coomassie stain for loading control. h The quantification of PSD95 after normalizing to total protein. c, d, f, h Unpaired Student’s t-test was used to analyze the disease-related change in each marker relative to its respective UN control. Data (biological replicates) are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001