Terminal field volumes selectively decrease with age in control mice and increase in E3–E12 diet mice. Expanded terminal field sizes in the E3–E12 diet are associated with increased numbers of putative synapses. (A) Schematic of the peripheral gustatory system in a mouse showing the three nerves that carry taste information from taste buds on the tongue to the rostral portion of the NST. Cell somas of the three nerves are in the geniculate (CT and GSP) and petrosal (glossopharyngeal, IX) ganglia. The CT, GSP, and IX innervate taste buds located on the anterior tongue, palate, and posterior tongue, respectively. (B) Timeline of early embryonic dietary exposure period (sodium dietary restriction period denoted in yellow) and hallmarks of peripheral gustatory development. (C and H) Horizontal sections of labeled terminal fields of the IX (green), CT (red), GSP (blue), and the merged image (MERGE) in representative sections from the dorsal zone in P15 and adult control mice (C) and in adult control and adult E3–E12 diet mice (H). Refer to the color guide in the lower portion of C and H for overlap colors shown in the merged images. R, rostral; L, lateral. Scale bars in C and H = 200 µm. The photomicrographs used in C are duplicated for the dorsal zone in Fig. S1 A and the photomicrographs used in H are duplicated for the dorsal zone in Fig. S1 B. (D–G) Terminal field volumes of controls and E3–E12 diet mice for the IX (D), CT (E), and GSP (F) nerves and the overlapping fields of all three nerves (G). The numbers of mice/age for control and E3–E12 groups are, respectively: P15 = 5, 6; P20 = 6, 6; P25 = 5, 5; P30 = 5, 5; P45 = 4, 5; adult = 5, 5. (I) Terminal field volumes of controls and E3–E12 diet mice obtained through measurements of P2X2+ labeling in P40, P1, P5, and P10 mice. The numbers of mice/age for control and E3–E12 groups are, respectively: P40 = 3, 4; P1 = 5, 5; P5 = 5, 6; P10 = 5, 5. (J) Coronal sections of the right dLGN in adult control (n = 3) and E3–E12 diet mice (n = 3) showing the terminal field label for retinogeniculate projections from the contralateral (green) and ipsilateral (red) eye. Scale bar = 500 µm. D, dorsal; L, lateral. (K) Photomicrograph of an optical, horizontal section through the rostral NST. CT terminal field is shown in red, and PSD95+ labeling is shown in green. Examples of putative CT synapses are outlined by white circles. Scale bar (white) in center of left panel = 20 µm. R, rostral; L, lateral. (L) Volume of PSD+ and CT terminal field labeling within the region of interest in the NST of adult controls (PSD95, n = 7; CT, n = 6) and E3–E12 mice (PSD95, n = 7; CT, n = 6). (M) Volume of labels where PSD+ labeling was closely associated with CT terminals within the region of interest in the NST of controls (PSD95, n = 7; CT, n = 6) and E3–E12 mice (PSD95, n = 7; CT, n = 6). Data in D–G and I are shown as mean ± SEM. All statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. * denotes P < 0.05. Terminal field volumes (D–G)—IX: P30 control versus E3–E12, P = 0.02; CT: P values at P15, P30, P50, and P60 are 0.002, 0.0000043, 0.0000046, and 0.000001, respectively; GSP: P values at P15, P30, P50, and P60 are 0.000008, 0.0000018, 0.00004, and 0.0000017, respectively. (I) P = 0.003 for comparison at P40. (L) P = 0.02 for both comparisons. (M) P = 0.04. One observation/animal was made for all measures of terminal field volumes and immunohistochemistry.
Figure 1.

Terminal field volumes selectively decrease with age in control mice and increase in E3–E12 diet mice. Expanded terminal field sizes in the E3–E12 diet are associated with increased numbers of putative synapses. (A) Schematic of the peripheral gustatory system in a mouse showing the three nerves that carry taste information from taste buds on the tongue to the rostral portion of the NST. Cell somas of the three nerves are in the geniculate (CT and GSP) and petrosal (glossopharyngeal, IX) ganglia. The CT, GSP, and IX innervate taste buds located on the anterior tongue, palate, and posterior tongue, respectively. (B) Timeline of early embryonic dietary exposure period (sodium dietary restriction period denoted in yellow) and hallmarks of peripheral gustatory development. (C and H) Horizontal sections of labeled terminal fields of the IX (green), CT (red), GSP (blue), and the merged image (MERGE) in representative sections from the dorsal zone in P15 and adult control mice (C) and in adult control and adult E3–E12 diet mice (H). Refer to the color guide in the lower portion of C and H for overlap colors shown in the merged images. R, rostral; L, lateral. Scale bars in C and H = 200 µm. The photomicrographs used in C are duplicated for the dorsal zone in Fig. S1 A and the photomicrographs used in H are duplicated for the dorsal zone in Fig. S1 B. (D–G) Terminal field volumes of controls and E3–E12 diet mice for the IX (D), CT (E), and GSP (F) nerves and the overlapping fields of all three nerves (G). The numbers of mice/age for control and E3–E12 groups are, respectively: P15 = 5, 6; P20 = 6, 6; P25 = 5, 5; P30 = 5, 5; P45 = 4, 5; adult = 5, 5. (I) Terminal field volumes of controls and E3–E12 diet mice obtained through measurements of P2X2+ labeling in P40, P1, P5, and P10 mice. The numbers of mice/age for control and E3–E12 groups are, respectively: P40 = 3, 4; P1 = 5, 5; P5 = 5, 6; P10 = 5, 5. (J) Coronal sections of the right dLGN in adult control (n = 3) and E3–E12 diet mice (n = 3) showing the terminal field label for retinogeniculate projections from the contralateral (green) and ipsilateral (red) eye. Scale bar = 500 µm. D, dorsal; L, lateral. (K) Photomicrograph of an optical, horizontal section through the rostral NST. CT terminal field is shown in red, and PSD95+ labeling is shown in green. Examples of putative CT synapses are outlined by white circles. Scale bar (white) in center of left panel = 20 µm. R, rostral; L, lateral. (L) Volume of PSD+ and CT terminal field labeling within the region of interest in the NST of adult controls (PSD95, n = 7; CT, n = 6) and E3–E12 mice (PSD95, n = 7; CT, n = 6). (M) Volume of labels where PSD+ labeling was closely associated with CT terminals within the region of interest in the NST of controls (PSD95, n = 7; CT, n = 6) and E3–E12 mice (PSD95, n = 7; CT, n = 6). Data in D–G and I are shown as mean ± SEM. All statistical comparisons were multiple, unpaired t tests using the Holm–Sidak method for multiple comparisons. * denotes P < 0.05. Terminal field volumes (D–G)—IX: P30 control versus E3–E12, P = 0.02; CT: P values at P15, P30, P50, and P60 are 0.002, 0.0000043, 0.0000046, and 0.000001, respectively; GSP: P values at P15, P30, P50, and P60 are 0.000008, 0.0000018, 0.00004, and 0.0000017, respectively. (I) P = 0.003 for comparison at P40. (L) P = 0.02 for both comparisons. (M) P = 0.04. One observation/animal was made for all measures of terminal field volumes and immunohistochemistry.

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