(A) Representative images illustrating native VGluT3 expression within CA1/3 stratum pyramidale (SP) at postnatal day 5, 15, and 30 (p5,15,30) as indicated. Group data for terminal density counts in CA1 and CA3 throughout development are plotted at right (n = 6–19 sections from three to six animals per age were examined; individual observations (n) along with means are provided). (B) Representative high resolution Airyscan images illustrating colocalization of VGluT3 signal in CA1 perisomatic axon terminals with CB1R signal (upper row, (B1) as well as GAD1 and VGAT signals (second row, (B2) but not Syt2 labelled terminals (third row, (B3); SO, stratum oriens; SR, stratum radiatum). Widefield as well as digitally magnified images from boxed regions are provided showing merged and split channels for each set of markers, with DAPI labeling, as indicated. Filled arrowheads highlight terminals showing colocalization while open arrowheads highlight terminals lacking colocalization. Colocalization of VGluT3 with each marker is quantified in the box plots (B4) with data normalized to total CB1R+ terminal counts (left plot) or total VGluT3+ terminal counts (middle and right plots; 1200–6580 terminals from n = 8–14 sections in each staining combination, from three mice were evaluated). (C) Representative Airyscan images and group data illustrating conserved VGluT3 expression at all putative release sites along continuous individual axon segments. Three individual axon segments are highlighted in the CA1 SP widefield image and a segment from one of these axons (boxed region, (C1) is digitally magnified. Group data plotted at right summarize the percentage of putative release sites along individual axons from CB1R+/VGluT3+ cells that are VGluT3+ (pie chart and box plot, normalized to number of boutons in each axon segment) as well as the correlation between total number of boutons scored per length of each continuous axon examined (IBI, interbouton interval; 510 boutons were evaluated from n = 28 axon segments across three mice). (D) An additional example of conserved VGluT3 expression across all putative release sites of a continuous axon segment within SO with merged and split channels as indicated. For statistical analysis *p<0.05, **p<0.01, ***p<0.001. Scalebar in A = 10 µm; in B, C, D = 5 µm; in C1 = 1 µm.