The classical complement cascade participates in the pruning of terminal fields in control mice and may be disrupted in E3–E12 diet mice. (A–C) Terminal field volumes of the IX, CT, and GSP in C1q and C3 knockout mice (colored symbols) at P15, P30, and adulthood (P60). They are shown with developmental terminal field data from controls and E3–E12 diet mice (gray lines and symbols) from Fig. 1, D–F. The number of C3 and C1q knockout animals are P15 (5, 4, respectively), P30 (5, 6, respectively), and P60 (5, 5, respectively). (D) qPCR data for C1qa during development in the geniculate ganglion in controls and in E3–E12 diet mice. Both groups show an age-dependent peak expression of C1qa, but the expression is delayed in E3–E12 diet mice compared with controls. Geniculate ganglia for two animals/group were pooled and analyzed as a single sample. The number of animals/groups are P9 controls (n = 4), P15 controls and E3–E12 diet (7, 6, respectively), P25 controls and E3–E12 diet (3, 4, respectively), and P60 controls and E3–E12 diet (3, 3, respectively). NA, not available. (E and F) Photomicrographs through the rostral NST of a control P25 mouse showing widespread C1q⁺ staining (E, green) and P2X2⁺ staining (F, red), indicating gustatory terminal field location and staining for both antibodies. The number of P15, P25, and adult control and E3–12 diet mice analyzed for C1q⁺ immunohistochemistry was n = 5/group. (G) The core region (densest P2X2⁺ staining) is outlined by thin white lines in E and F, illustrating that the core gustatory projection is nearly devoid of C1q. Thicker white bars denote the rostral border of the NST. Scale bar in F = 200 µm. (H–J) Electron micrographs from the rostral NST in a P25-d control mouse immunostained for C1q and P2X2. (H) The magenta line outlines a microglia cell (N, nucleus) that contains immunopositive gold staining for C1q. The box outlined in the lower right portion of the micrograph shows a P2X2⁺ stained terminal with C1q⁺ labeling on and near the terminal. (I) An enlarged view of the box outlined in the lower right panel of H. (J) A P2X2⁺ terminal (T) labeled with numerous C1q⁺ gold profiles. Short black and white arrows point to C1q⁺ gold profiles in H–J. The large black arrow in H points to the P2X2⁺ terminal shown in I. The thin black arrow in H points to a P2X2⁺ terminal, but without C1q⁺ staining. Scale bars in H–J = 350 nm. (K) IL-34⁺ staining in the rostral NST in P22 control and E3–E12 diet mice (n = 5, 9, respectively) and in P40 control and E3–E12 diet mice (n = 6, 8, respectively). (L) IL-33⁺ staining in the rostral NST in P22 control and E3–E12 diet mice (n = 6, 6, respectively) and in P40 control and E3–E12 diet mice (n = 8, 6, respectively). For all dietary and age group comparisons, statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. Data in A–D, K, and L are shown as mean ± SEM. * denotes P < 0.05. Significant statistical comparisons for B are P15: E3–E12 diet mice with C1qKO, P = 0.003; P30: controls with C1qKO, P = 0.0005; and P60: controls with C1qKO, P = 0.0006; for C, they are P15: controls with C3KO, P = 0.0006 and E3–E12 diet mice with C1qKO, P = 0.01; P30: controls with C1qKO, P = 0.001 (also see Table S4). (D) P15 control versus P15 E3–E12 diet, P = 0.02. (E–G) C1q within microglia and located in the core region: P15 control versus adult control, P = 0.02; C1q within microglia but located within the surround region: P15 control versus adult control, P = 0.01; P15 E3–E12 diet versus adult E3–E12 diet, P = 0.008; C1q not within microglia but located in the core region: P15 control versus P15 E3–E12 diet, P = 0.01; C1q not within microglia and located in the surround region: P15 control versus P15 E3–E12 diet, P = 0.01. (K) P22 control versus P22 E3–E12 diet, P = 0.0001; P22 control versus P40 control, P = 0.0009. (L) P22 control versus P40 control, P = 0.01; P40 control versus P40 E3–E12 diet, P = 0.0008. One observation/animal was obtained and analyzed for data shown in A–C, K, and L.