Age- and diet-related differences in microglia densities in the rostral NST begin at E9.5 and extend into adulthood and have functional and molecular differences that may contribute to terminal field development and maintenance. (A) Horizontal sections through a P15 control brainstem showing the NST to be rich in microglia. White lines show the border of the NST and arrows point to the solitary tract. Scale bar = 500 µm. R, rostral; L, lateral. (B) Microglia in a P15 and an adult (P60) control and E3–E12 mouse NST (white lines show the rostral NST border). Scale bar = 200 µm. R, rostral; L, lateral. (C and D) Microglia densities of individual animals and their means in the (C) dorsal and (D) ventral zones of the rostral NST. The number of animals analyzed for each group in the dorsal and ventral zones, respectively, are as follows: P15 controls (n = 11, 7); adult controls (n = 11, 9); P15 E3–E12 diet mice (n = 11, 7); and adult E3–E12 diet mice (n = 10, 6). (E) Percent of total microglia in the rostral NST in P22 control (n = 5) and E3–E12 (n = 5) mice scored in each of five categories to assess microglia morphology and lysosome activity. Microglia in controls are larger with fewer dendrites than E3–E12 diet mice. (F) Microglia densities of individual animals and their means in the caudal zone of the NST in P15 controls (n = 12); adult controls (n = 10); P15 E3–E12 diet mice (n = 9); and adult E3–E12 diet mice (n = 8). (G, a and b) Photomicrographs of images through the whole anterior portion of an E9.5 control (a) and E9.5 E3–E12 diet embryo (b). Arrows point to microglia in these CX3CR1 reporter mice. Inset panels (a i and b i) show higher magnifications of the respective area in the brain denoted by the magenta rectangles. The number of embryos examined were four controls and six E3–E12 diet mice. Scale bars = 100 µm. OV, optic vesicle; SC, spinal cord; V, ventral; D, dorsal; R, rostral; C, caudal. (H) Photomicrographs of images of an E12.5 control embryo (a and a i) and an E9.5 E3–E12 diet embryo (b and b i). Inset panels show higher magnifications of the respective area in the brain denoted by the magenta rectangle. The oval outlined in white denotes the location of the medulla. 8 controls and 13 E3–E12 diet animals were examined at E12.5. Scale bars = 500 µm. SC, spinal cord; V, ventral; D, dorsal; R, rostral; C, caudal. (I) Microglia densities of individual E12.5 embryos and their means in the medulla of the NST in controls (n = 8) and E3–E12 diet mice (n = 13). (J) Photomicrographs of microglia in the dorsal zone of the rostral NST in an E18.5 control (left panel) and E3–E12 diet (right panel) mouse. Scale bar = 20 µm. (K) Microglia densities of individual E18.5 embryos and their means in the dorsal and ventral zones of the rostral NST, the caudal NST, and the dLGN. The number of animals analyzed for each group in the four brain areas are: dorsal NST: E18.5 controls (n = 7), E3–E12 diet (n = 6); ventral NST: E18.5 controls (n = 7), E3–E12 diet (n = 5); caudal NST: E18.5 controls (n = 6), E3–E12 diet (n = 6); dLGN: E18.5 controls (n = 5), E3–E12 diet (n = 8). (L) Heat map showing upregulation (increased) or downregulation (decreased) of genes from microglia in P15 E3–E12 diet mice compared with P15 controls. The functional categories are shown to the right of the heat maps. Data were obtained from three independent replicates where the NST was isolated from six E3–E12 diet mice and six control mice/replicate, all at P15. (M) Photomicrograph of a microglia cell (green) within the P2X2+ terminal field. Arrows point to engulfed material (yellow) by the microglia. (N) Higher magnification of only the microglia in M showing the engulfed P2X2+ label (yellow) and CD68+ (blue) label denoting lysosomes. (O) 3D rendering of the microglia with the engulfed P2X2+ label (red) and lysosomes (transparent purple). (O i) Higher magnification of 3D-rendered microglia within the magenta rectangle shown in O. Scale bars for M–O = 3 µm. (P) Percent of large cell microglia occupied by engulfed P2X2+ label in P15 controls and E3–E12 diet mice (n = 6, 6, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). Data from the three regions of interest in the NST were summed and analyzed for each animal. For all dietary and age group comparisons, statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. Data in C–F, I, K, and P are shown as mean ± SEM. * denotes P < 0.05. (C) P15 control versus adult control, P = 0.0001; P15 E3–E12 diet versus adult E3 = E12 diet, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0001; adult control versus adult E3–E12 diet, P = 0.0001. (D) P15 control versus adult control, P = 0.0001; P15 E3–E12 diet versus adult E3 = E12 diet, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0003; adult control versus adult E3–E12 diet, P = 0.0006. (E) Category 2, P = 0.01; category 3, P = 0.009; category 4, P = 0.02. (F) P15 control versus adult control, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0003. (I) P = 0.004. (K) Dorsal NST, P = 0.0001; ventral NST, P = 0.02; caudal NST, P = 0.004. (P) P15 controls versus P15 E3–E12, P = 0.01; adult controls versus adult E3–E12, P = 0.003. Apart from the data shown in L and P, only one observation/animal was made. Embryonic atlases (Allen Mouse Brain Atlas, 2004; Chen et al., 2017) enabled the identification of the medulla in E12.5 embryos.
Figure 3.

Age- and diet-related differences in microglia densities in the rostral NST begin at E9.5 and extend into adulthood and have functional and molecular differences that may contribute to terminal field development and maintenance. (A) Horizontal sections through a P15 control brainstem showing the NST to be rich in microglia. White lines show the border of the NST and arrows point to the solitary tract. Scale bar = 500 µm. R, rostral; L, lateral. (B) Microglia in a P15 and an adult (P60) control and E3–E12 mouse NST (white lines show the rostral NST border). Scale bar = 200 µm. R, rostral; L, lateral. (C and D) Microglia densities of individual animals and their means in the (C) dorsal and (D) ventral zones of the rostral NST. The number of animals analyzed for each group in the dorsal and ventral zones, respectively, are as follows: P15 controls (n = 11, 7); adult controls (n = 11, 9); P15 E3–E12 diet mice (n = 11, 7); and adult E3–E12 diet mice (n = 10, 6). (E) Percent of total microglia in the rostral NST in P22 control (n = 5) and E3–E12 (n = 5) mice scored in each of five categories to assess microglia morphology and lysosome activity. Microglia in controls are larger with fewer dendrites than E3–E12 diet mice. (F) Microglia densities of individual animals and their means in the caudal zone of the NST in P15 controls (n = 12); adult controls (n = 10); P15 E3–E12 diet mice (n = 9); and adult E3–E12 diet mice (n = 8). (G, a and b) Photomicrographs of images through the whole anterior portion of an E9.5 control (a) and E9.5 E3–E12 diet embryo (b). Arrows point to microglia in these CX3CR1 reporter mice. Inset panels (a i and b i) show higher magnifications of the respective area in the brain denoted by the magenta rectangles. The number of embryos examined were four controls and six E3–E12 diet mice. Scale bars = 100 µm. OV, optic vesicle; SC, spinal cord; V, ventral; D, dorsal; R, rostral; C, caudal. (H) Photomicrographs of images of an E12.5 control embryo (a and a i) and an E9.5 E3–E12 diet embryo (b and b i). Inset panels show higher magnifications of the respective area in the brain denoted by the magenta rectangle. The oval outlined in white denotes the location of the medulla. 8 controls and 13 E3–E12 diet animals were examined at E12.5. Scale bars = 500 µm. SC, spinal cord; V, ventral; D, dorsal; R, rostral; C, caudal. (I) Microglia densities of individual E12.5 embryos and their means in the medulla of the NST in controls (n = 8) and E3–E12 diet mice (n = 13). (J) Photomicrographs of microglia in the dorsal zone of the rostral NST in an E18.5 control (left panel) and E3–E12 diet (right panel) mouse. Scale bar = 20 µm. (K) Microglia densities of individual E18.5 embryos and their means in the dorsal and ventral zones of the rostral NST, the caudal NST, and the dLGN. The number of animals analyzed for each group in the four brain areas are: dorsal NST: E18.5 controls (n = 7), E3–E12 diet (n = 6); ventral NST: E18.5 controls (n = 7), E3–E12 diet (n = 5); caudal NST: E18.5 controls (n = 6), E3–E12 diet (n = 6); dLGN: E18.5 controls (n = 5), E3–E12 diet (n = 8). (L) Heat map showing upregulation (increased) or downregulation (decreased) of genes from microglia in P15 E3–E12 diet mice compared with P15 controls. The functional categories are shown to the right of the heat maps. Data were obtained from three independent replicates where the NST was isolated from six E3–E12 diet mice and six control mice/replicate, all at P15. (M) Photomicrograph of a microglia cell (green) within the P2X2+ terminal field. Arrows point to engulfed material (yellow) by the microglia. (N) Higher magnification of only the microglia in M showing the engulfed P2X2+ label (yellow) and CD68+ (blue) label denoting lysosomes. (O) 3D rendering of the microglia with the engulfed P2X2+ label (red) and lysosomes (transparent purple). (O i) Higher magnification of 3D-rendered microglia within the magenta rectangle shown in O. Scale bars for M–O = 3 µm. (P) Percent of large cell microglia occupied by engulfed P2X2+ label in P15 controls and E3–E12 diet mice (n = 6, 6, respectively) and adult controls and E3–E12 diet mice (n = 7, 6, respectively). Data from the three regions of interest in the NST were summed and analyzed for each animal. For all dietary and age group comparisons, statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. Data in C–F, I, K, and P are shown as mean ± SEM. * denotes P < 0.05. (C) P15 control versus adult control, P = 0.0001; P15 E3–E12 diet versus adult E3 = E12 diet, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0001; adult control versus adult E3–E12 diet, P = 0.0001. (D) P15 control versus adult control, P = 0.0001; P15 E3–E12 diet versus adult E3 = E12 diet, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0003; adult control versus adult E3–E12 diet, P = 0.0006. (E) Category 2, P = 0.01; category 3, P = 0.009; category 4, P = 0.02. (F) P15 control versus adult control, P = 0.0001; P15 control versus P15 E3–E12 diet, P = 0.0003. (I) P = 0.004. (K) Dorsal NST, P = 0.0001; ventral NST, P = 0.02; caudal NST, P = 0.004. (P) P15 controls versus P15 E3–E12, P = 0.01; adult controls versus adult E3–E12, P = 0.003. Apart from the data shown in L and P, only one observation/animal was made. Embryonic atlases (Allen Mouse Brain Atlas, 2004; Chen et al., 2017) enabled the identification of the medulla in E12.5 embryos.

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