Research ArticleResearch Article: New Research, Integrative Systems

Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression

Seol-Hee Kim, Stephen H. Hadley, Mikayla Maddison, Mayur Patil, Byeong Cha, Marian Kollarik and Thomas E. Taylor-Clark
eNeuro 14 February 2020, 7 (2) ENEURO.0494-19.2020; https://doi.org/10.1523/ENEURO.0494-19.2020

Article Figures & Data

Figures

  • Figure 1.
    Figure 1.

    tdTomato expression and α-TRPV1 immunoreactivity in vagal ganglia. A, Pirt-tdT. B, TRPV1-tdT. C, 5-HT3-tdT. D, Tac1-tdT. Native tdTomato expression (red) is shown on the left, with overlap with α-TRPV1 immunoreactivity on the right (green). Scale bar, 100 μm; insets show enlarged views of both jugular and nodose neurons. Data are representative of n = 3 animals for each strain.

  • Figure 2.
    Figure 2.

    Comparison of tdTomato expression and α-TRPV1 immunoreactivity in vagal ganglia of Pirt-tdT, 5-HT3-tdT, TRPV1-tdT, and Tac1-tdT. A, The relative contribution of jugular (orange bars) or nodose (blue bars) neurons to the vagal neuronal populations expressing tdTomato (tdT+), α-TRPV1 immunoreactivity (αV1+), or both tdTomato and α-TRPV1 immunoreactivity (tdT+αV1+). B, The percentage of tdTomato-expressing neurons in the nodose (blue) or jugular (orange) ganglia with α-TRPV1 immunoreactivity. C, The percentage of α-TRPV1-immunoreactive neurons in the nodose (blue) or jugular (orange) ganglia with tdTomato expression. Data are derived from n = 3 animals in each strain.

  • Figure 3.
    Figure 3.

    Neuronal diameters of tdTomato+ vagal neurons from Pirt-tdT, 5-HT3-tdT, TRPV1-tdT, and Tac1-tdT. A, Histogram of neuronal diameter of tdTomato+ vagal neurons from Pirt-tdT (n = 1040 neurons), 5-HT3-tdT (n = 850 neurons), TRPV1-tdT (n = 501 neurons), and Tac1-tdT (n = 330 neurons). B, Mean ± SD neuronal diameter of tdTomato+ vagal neurons from Pirt-tdT, 5-HT3-tdT, TRPV1-tdT, and Tac1-tdT. C, Mean ± SD neuronal diameter of tdTomato+ nodose neurons (upward triangles) compared with jugular neurons (downward triangles) from Pirt-tdT, 5-HT3-tdT, TRPV1-tdT, and Tac1-tdT. D, Histogram of neuronal diameter of tdTomato-expressing neurons with α-TRPV1 immunoreactivity (Pirt-tdT+αTRPV1+, black line) and tdTomato-expressing neurons without α-TRPV1 immunoreactivity (Pirt-tdT+αTRPV1−, dotted line) from Pirt-tdT. E, Mean ± SD neuronal diameter of tdTomato+ vagal neurons with (filled squares) and without (open squares) α-TRPV1 immunoreactivity from Pirt-tdT, 5-HT3-tdT, TRPV1-tdT, and Tac1-tdT. F, Mean ± SD neuronal diameter of tdTomato+ nodose (upward triangles) and jugular (downward triangles) neurons with (filled squares) and without (open squares) α-TRPV1 immunoreactivity from TRPV1-tdT. *Denotes significant difference (p < 0.05, ANOVA with Sidak’s multiple comparisons).

  • Figure 4.
    Figure 4.

    Native tdTomato expression, α-TRKA immunoreactivity and α-TRKB immunoreactivity in vagal ganglia. Top, TRPV1-tdT. Bottom, Tac1-tdT. Scale denotes 100 μm. Data are representative of n = 3 animals for each strain.

  • Figure 5.
    Figure 5.

    Native tdTomato expression, α-TRKA immunoreactivity, and α-TRPV1 immunoreactivity in vagal ganglia. Top, TRPV1-tdT. Bottom, Tac1-tdT. Scale bar, 100 μm. Data are representative of n = 3 animals for each strain.

  • Figure 6.
    Figure 6.

    Comparison of tdTomato expression, α-TRPV1 immunoreactivity, α-TRKA immunoreactivity, and α-TRKB immunoreactivity in vagal ganglia of TRPV1-tdT and Tac1-tdT. A, The relative contribution of jugular (orange bars) or nodose (blue bars) neurons to the vagal neuronal populations expressing α-TRKA immunoreactivity (αTRKA+), α-TRKB immunoreactivity (αTRKB+), α-TRPV1 immunoreactivity (αV1+), or tdTomato in either the Tac1-tdT or the TRPV1-tdT. B, The percentage of specific neuronal groups in the nodose (blue) or jugular (orange) ganglia with α-TRKA immunoreactivity. C, The percentage of specific neuronal groups in the nodose (blue) or jugular (orange) ganglia with α-TRKB immunoreactivity. D, The percentage of neurons expressing either α-TRKA immunoreactivity (αTRKA+) or α-TRKB immunoreactivity (αTRKB+) in the nodose (blue) or jugular (orange) ganglia with tdTomato expression in the Tac1-tdT ganglia. E, The percentage of neurons expressing either α-TRKA immunoreactivity (αTRKA+) or α-TRKB immunoreactivity (αTRKB+) in the nodose (blue) or jugular (orange) ganglia with tdTomato expression in the TRPV1-tdT ganglia. Data are derived from n = 3 animals in each strain.

  • Figure 7.
    Figure 7.

    Native tdTomato expression in cleared whole-mount medulla. A, Pirt-tdT. B, 5-HT3-tdT. C, TRPV1-tdT. D, Tac1-tdT. All medulla are displayed in the same dorsal aspect orientation. The following structures are identified: nTS, Pa5, Sp5, and TS. Scale bar, 1 mm.

  • Figure 8.
    Figure 8.

    tdTomato expression in serial coronal sections of the medulla in Pirt-tdT. A–T, Data presented from rostral to caudal, with labeling for the position relative to obex. The intensity of native tdTomato expression is shown in rainbow pseudocolor. The following structures are identified: area postrema (AP), cuneate nucleus (Cu), external cuneate nucleus (ECu), gracile nucleus (Gr), 12N, nTS, Pa5, Sp5, and TS. Scale bar, 400 μm. Data are representative of n = 8 animals.

  • Figure 9.
    Figure 9.

    tdTomato expression in serial coronal sections of the medulla in 5-HT3-tdT. A–T, Data presented from rostral to caudal, with labeling for the position relative to obex. The intensity of native tdTomato expression is shown in rainbow pseudocolor. The following structures are identified: nTS, Pa5, Sp5, and TS. Scale bar, 400 μm. Data are representative of n = 6 animals.

  • Figure 10.
    Figure 10.

    tdTomato expression in serial coronal sections of the medulla in TRPV1-tdT. A–T, Data presented from rostral to caudal, with labeling for the position relative to obex. The intensity of native tdTomato expression is shown in rainbow pseudocolor. The following structures are identified: area postrema (AP), DMX, 12N, nTS, Pa5, Sp5, and TS. Scale bar, 400 μm. Data are representative of n = 6 animals.

  • Figure 11.
    Figure 11.

    tdTomato expression in serial coronal sections of the medulla in Tac1-tdT. A–T, Data presented from rostral to caudal, with labeling for the position relative to obex. The intensity of native tdTomato expression is shown in rainbow pseudocolor. The following structures are identified: external cuneate nucleus (ECu), 12N, nTS, Pa5, Sp5, and TS. Scale bar, 400 μm. Data are representative of n = 5 animals.

  • Figure 12.
    Figure 12.

    Comparison of native tdTomato expression in coronal sections of the medulla. A–C, tdTomato expression in TRPV1-tdT (purple) is compared with tdTomato expression in Pirt-tdT (A; green), 5-HT3-tdT (B; green), and Tac1-tdT (C; green) and overlap of reporter expression is denoted in black. Data presented from rostral (left) to caudal (right), with labeling for the position relative to obex. Scale bar, 500 μm.

  • Figure 13.
    Figure 13.

    tdTomato expression in nTS subnuclei. A, Pirt-tdT at −330 μm (relative to obex). B, 5-HT3-tdT at −300 μm. C, TRPV1-tdT at −360 μm. D, Tac1-tdT at −330 μm. Left, Native tdTomato expression (red) merged with neurotrace labeling of intrinsic neurons (green). Right, tdTomato expression alone. The following structures are identified: area postrema (AP), central canal (CC), cuneate nucleus (Cu), DMX, 12N, parasolitary nucleus (Psol), SolC (C), SolCe (Ce), SolDL (DL), SolG (G), SolIM (IM), SolM (M), SolV (V), SolVL (VL), and TS. Scale bar, 300 μm.

  • Figure 14.
    Figure 14.

    tdTomato expression in dorsal medulla subnuclei along complete rostral–caudal axis. Data are mean native tdTomato intensity in SolC, SolG, SolM, SolDL, SolV, SolVL, area postrema (AP), DMX, and 12N from Pirt-tdT (purple), 5-HT3-tdT (cyan), TRPV1-tdT (red), and Tac1-tdT (green; n = 5–8 mice each). The gray shading represents the physical dimensions of each subnuclei in the rostral–caudal axis (positive values denote rostral of obex, negative values denote caudal of obex; Paxinos and Franklin, 2012).

  • Figure 15.
    Figure 15.

    Overlap of native tdTomato expression and α-TRPV1 immunoreactivity in the medulla of TRPV1-tdT and Tac1-tdT. A–C, TRPV1-tdT at −300 μm (relative to obex). D–F, Tac1-tdT at −240 μm. A, C, D, F, tdTomato expression (red) is compared with α-TRPV1 immunoreactivity (green) in the nTS (A–D) and the Pa5 (C–F), with merged image on the right. Scale bar, 250 μm. B, Quantification of tdTomato (red) and α-TRPV1 immunoreactivity (green) intensities along the line drawn in the merged image in A (TRPV1-tdT nTS), including area postrema (AP), dorsal motor nucleus of the vagus (10N), SolC (C), SolIM (IM), and SolM (M). E, Quantification of tdTomato (red) and α-TRPV1 immunoreactivity (green) intensities along the line drawn in the merged image in D (Tac1-tdT nTS), including AP, 10N, 12N, C, and M. Boxed area denotes overlap of tdTomato and α-TRPV1 immunoreactivity intensities along the border between SolC and SolM. Data are representative of n = 3 animals for each strain.

  • Figure 16.
    Figure 16.

    Intraganglionic injection of AAV vectors evokes robust reporter expression in vagal ganglia. A, Injection of AAV9-flex-GFP into TRPV1-tdT. tdTomato (red), and GFP (green) expression is compared with α-TRPV1 immunoreactivity (blue). B, Injection of AAV9-flex-GFP into Tac1-tdT. tdTomato (red) and GFP (green) expression is compared with α-TRPV1 immunoreactivity (blue). C, Coinjection of AAV9-flex-tdT and AAV9-GFP into TRPV1-Cre. tdTomato (red) and GFP (green) expression is compared. Scale bar, 100 μm.

  • Figure 17.
    Figure 17.

    Brainstem terminations of vagal TRPV1-expressing afferents labeled by unilateral intraganglionic injection of AAV9-flex-GFP into TRPV1-tdT. AAV-mediated GFP expression (green, enhanced by α-GFP immunoreactivity) is compared with ROSA26-mediated tdTomato expression (red, native) in coronal sections of the medulla. A, B, 3D reconstruction of medulla along entire rostral–caudal axis. A, Rostral aspect. B, Dorsal aspect. C–H, Coronal sections of medulla from rostral to caudal, with labeling for the position relative to obex. C, Pa5 at +300 μm (relative to obex). D, E, nTS at −200 μm (D) and −520 μm (E). The following structures are identified: area postrema (AP), DMX, SolC (C), SolCe (Ce), SolDL (DL), SolG (G), SolIM (IM), SolM (M), SolV (V), SolVL (VL), and TS. F, Pa5 at −520 μm. G, H, nTS at −820 μm. H, High-magnification image of SolC area identified by white box in G. Scale bars: A, B, 1 mm; C–G, 200 μm; H, 40 μm.

  • Figure 18.
    Figure 18.

    Brainstem terminations of vagal Tac1-expressing afferents labeled by unilateral intraganglionic injection of AAV9-flex-GFP into Tac1-tdT. AAV-mediated GFP expression (green, enhanced by α-GFP immunoreactivity) is compared with ROSA26-mediated tdTomato expression (red, native) in coronal sections of the medulla. A, B, 3D reconstruction of medulla along entire rostral–caudal axis. A, Rostral aspect. B, Dorsal aspect. C–H, Coronal sections of medulla from rostral to caudal, with labeling for the position relative to obex. C, nTS at +320 μm (relative to obex). D, High-magnification image of area identified by white box in C. E, F, nTS at obex (E) and −320 μm (F). G, Pa5 at −320 μm. H, nTS at −640 μm. The following structures are identified: area postrema (AP), central canal (CC), DMX, Pa5, SolC (C), SolCe (Ce), SolDL (DL), SolG (G), SolIM (IM), SolM (M), SolV (V), SolVL (VL), and TS. Scale bars: A, B, 1 mm; C, E, G, 200 μm; D, 50 μm.

  • Figure 19.
    Figure 19.

    Brainstem terminations of vagal TRPV1+ and TRPV1− afferents labeled by unilateral intraganglionic injection of AAV9-flex-tdT and AAV9-GFP into TRPV1-Cre. Cre-sensitive AAV-mediated tdTomato expression (red; enhanced by α-DsRed immunoreactivity) is compared with constitutively active AAV-mediated GFP expression (green, enhanced by α-GFP immunoreactivity) and neurotrace (blue) in coronal sections of the medulla (labeling for the position relative to obex). A–D, nTS from rostral to caudal. E, High-magnification image of SolC, SolM, and AP from C. F, High-magnification image of SolV, SolVL, and TS from C. G, Pa5 at −120 μm. The following structures are identified: area postrema (AP), DMX, SolC (C), SolCe (Ce), SolDL (DL), SolG (G), SolIM (IM), SolM (M), SolV (V), SolVL (VL), and TS. Scale bars: A, B, C, G, 200 μm; D, 100 μm; E, F, 40 μm.

  • Figure 20.
    Figure 20.

    nTS terminations of lung-specific TRPV1+ and Tac1+ afferents labeled with rAAV-flex-tdT. tdTomato expression enhanced by enhanced by α-DsRed immunoreactivity. A, B, Instillation of Cre-sensitive AAV into lungs of TRPV1-Cre. A, AAV-mediated tdTomato expression (red) identifying lung-specific TRPV1+ afferents in vagal ganglia (top) and merged with α-TRPV1 immunoreactivity (green; bottom). B, AAV-mediated tdTomato expression (red) in coronal sections of the medulla (labeling for the position relative to obex), counterstained with native autofluorescence (gray). C–E, instillation of Cre-sensitive AAV into lungs of Tac1-Cre. C, Composite image of serial coronal sections of the nTS (from −520 to −640 μm relative to obex), with tdTomato expression in lung-specific Tac1+ afferents labeled in pseudorainbow encoded by rostral–caudal position. D, E, High-magnification images of lung-specific Tac1+ afferents (red) in SolC at −520 μm, counterstained by neurotrace (green). D, Branching of afferent denoted by arrows. E, A single afferent makes putative synapses with two distinct intrinsic neurons, denoted by arrowheads. Contact SolC, SolM, and AP from C. F, High-magnification image of SolV, SolVL, and TS from C. The following structures are identified: area postrema (AP), dorsal motor nucleus of the vagus (10N), gracile fasciculus (g), 12N, SolC (C), SolCe (Ce), SolDL (DL), SolG (G), SolM (M), SolV (V), SolVL (VL), and TS. Scale bars: A, 50 μm; B, C, 100 μm; D, E, 10 μm.

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March/April 2020
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Mapping of Sensory Nerve Subsets within the Vagal Ganglia and the Brainstem Using Reporter Mice for Pirt, TRPV1, 5-HT3, and Tac1 Expression
Seol-Hee Kim, Stephen H. Hadley, Mikayla Maddison, Mayur Patil, Byeong Cha, Marian Kollarik, Thomas E. Taylor-Clark
eNeuro 14 February 2020, 7 (2) ENEURO.0494-19.2020; DOI: 10.1523/ENEURO.0494-19.2020
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