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Additional Supporting Information may be found in the online version of this article.

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cne23309-sup-0001-suppfig1a.tif13076K

Supporting Fig. 1 (magenta version of original Fig. 4). GHSR mRNA localization in the ventral medulla in relation to cardiovascular control centers, marked by TH and CART expression. Combined immunohistochemistry for Tyrosine Hydroxylase (TH) and Cocaine and Amphetamine Regulated Transcript (CART) and in situ hybridization (ISH) for GHSR on coronal sections of rat medulla. Sections at Bregma -10.80 mm (5A-C), -12.50 mm (5D-G), -12.80 mm (5H, I), -13.35 mm (J), -13.80 mm (K), -14.10 mm (L).

To allow direct comparison of the brightfield ISH signal with the immunofluorescence signals the brightfield image is inverted. GHSR mRNA is not expressed in the A5 area, which is identified by neurons that strongly express TH and CART (4A, B). The nearest GHSR mRNA expression is in the Superior Salivary nucleus (SuS, 5A, B). More caudally, GHSR mRNA expressing cells (yellow arrowheads in 4E, F, H and I), can occasionally be found near or within the area occupied by the rostroventrolateral medulla (RVLM), which is apparent by TH and CART expressing neurons (4D, E, F, H, I). However, GHSR mRNA is never co-localised with TH or CART in the RVLM. GHSR-positive neurons within the RVLM appear to be part of clusters of small cells that express to weak-to-moderate levels of GHSR mRNA and are frequently observed ventral to the AmbC (apparent by strong CART labelling; 4D, E, G, H). More caudally, the AmbC is no longer present, but similar clusters of cells are found dorsally from and intermingling with TH and CART expressing neurons in the caudoventrolateral medulla (CVLM; 4J, K, L, M). The majority of these cells presumably belong to the semi-compact (AmbC), loose (AmbL) and external (AmbE) formations of the nucleus ambiguus or the nucleus retroambiguus (RAmb). They generally do not express TH or CART (yellow arrowheads), with an exception (white arrowhead) shown in Fig 4L, M-O. Scale bars: 50 µm (M), 100 µm (J, K, L), 200 µm (B, C, E, F, G, I), 500 µm (A, D, H).

cne23309-sup-0002-suppfig1b.tif12953K

Supporting Fig. 1 (magenta version of original Fig. 4). GHSR mRNA localization in the ventral medulla in relation to cardiovascular control centers, marked by TH and CART expression. Combined immunohistochemistry for Tyrosine Hydroxylase (TH) and Cocaine and Amphetamine Regulated Transcript (CART) and in situ hybridization (ISH) for GHSR on coronal sections of rat medulla. Sections at Bregma -10.80 mm (5A-C), -12.50 mm (5D-G), -12.80 mm (5H, I), -13.35 mm (J), -13.80 mm (K), -14.10 mm (L).

To allow direct comparison of the brightfield ISH signal with the immunofluorescence signals the brightfield image is inverted. GHSR mRNA is not expressed in the A5 area, which is identified by neurons that strongly express TH and CART (4A, B). The nearest GHSR mRNA expression is in the Superior Salivary nucleus (SuS, 5A, B). More caudally, GHSR mRNA expressing cells (yellow arrowheads in 4E, F, H and I), can occasionally be found near or within the area occupied by the rostroventrolateral medulla (RVLM), which is apparent by TH and CART expressing neurons (4D, E, F, H, I). However, GHSR mRNA is never co-localised with TH or CART in the RVLM. GHSR-positive neurons within the RVLM appear to be part of clusters of small cells that express to weak-to-moderate levels of GHSR mRNA and are frequently observed ventral to the AmbC (apparent by strong CART labelling; 4D, E, G, H). More caudally, the AmbC is no longer present, but similar clusters of cells are found dorsally from and intermingling with TH and CART expressing neurons in the caudoventrolateral medulla (CVLM; 4J, K, L, M). The majority of these cells presumably belong to the semi-compact (AmbC), loose (AmbL) and external (AmbE) formations of the nucleus ambiguus or the nucleus retroambiguus (RAmb). They generally do not express TH or CART (yellow arrowheads), with an exception (white arrowhead) shown in Fig 4L, M-O. Scale bars: 50 µm (M), 100 µm (J, K, L), 200 µm (B, C, E, F, G, I), 500 µm (A, D, H).

cne23309-sup-0003-suppfig2.TIF12820K

Supporting Fig. 2 (magenta version of original Fig. 5). GHSR mRNA localization in the ventral medulla in relation to NK1R-expressing respiratory nuclei and cholinergic sensory interneurons. To allow direct comparison of the brightfield ISH signal with the immunofluorescence signals the brightfield image is inverted. Combined in situ hybridization (ISH) for GHSR and immunohistochemistry for neurokinin-1 receptor (NK1R; 5A-F, H, I, J) or acetyl choline transferase (ChAT; 5G, K) on coronal sections of rat medulla. Sections at Bregma -11.80 mm (A), -12.10 mm (B), -12.30 mm (C), -12.70 mm (D), -13.60 mm (E), -14.10 mm (F), -12.70 mm (G). Clusters of cells expressing weak-to-moderate levels of GHSR mRNA are found ventral to the AmbC and throughout areas associated with respiratory control, such as the Bötzinger complex (Bötz; 6A, B) , the preBötzinger complex (prB; 5C, H), the rostral and caudal ventrolateral respiratory group (RVRG; 5D, E) and (CVRG; 5F, J). NK1R expression varies along the rostral-caudal axis, with low levels in the rostral Bötz and RVRG, increasing levels toward the caudal Bötz (5A, B), and very high levels in (propriobulbar) neurons in the prB (5C, H) and in (bulbospinal) neurons in the RVRG (5E). NK1R-expressing neurons are often intermingled with TH-expressing neurons of the RVLM and CVLM (5B-F, I, J). No examples of co-expression of GHSR mRNA in NK1R expressing neurons were identified in the ventrolateral medulla. ChAT-staining marks small sensory interneurons in the medial RVLM (5G, K), in addition to large motor neurons in the AmbC (5G). GHSR mRNA expressing small neurons (yellow arrows) are intermingling with ChAT (magenta arrows) and TH expressing neurons in the RVLM, but GHSR mRNA is not co-expressed with either of these markers (5K). Scale bars: 50 µm (H, I, J), 100 µm (B, D, F, K), 200 µm (A, C, E, G) .

cne23309-sup-0004-suppfig3a.tif14884K

Supporting Fig. 3 (magenta version of original Fig 6). GHSR mRNA localization in the rostral NTS (rNTS) and the dorsal vagal complex (DVC) in relation to TH, CART and NK1R expression. Sections at Bregma -11.50 mm (6A), -13.20 mm (6B, F), -13.60 mm (6C, G, J), -13.85 mm (6D, H, K, L), -14.20 mm (6E, H). To allow direct comparison of the brightfield ISH signal with the immunofluorescence signals the brightfield image is inverted. NB: The tissues surrounding the DVC are more opaque (see Fig. 3), resulting in a higher relative background in the inverted image in these areas (e.g. Fig. 6C). CART immunoreactivity is very prominent in the solitary tract (TS; 6A) and in fibre projections throughout the DVC, clearly defining the boundaries of the NTS and DMNX (10). TH marks C2 adrenergic neurons (6B, F), A2 noradrenergic neurons (6C, D, E, G, H, I) in the cNTS and noradrenergic neurons in the area postrema (AP; C, D, J, K-O). In the rNTS, GHSR mRNA expressing neurons are found lateral to the TS, in the rostrolateral NTS (6A). In the DVC, neurons expressing GHSR mRNA are found in the DMNX (10; 6B, C, D, G, H), cNTS (6B-I) and the AP (6C, D, J, K, L). GHSR mRNA is not found in TH expressing neurons throughout the DVC, or in NK1R-expressing neurons in the AP (6L). A very large proportion of NK1R-positive neurons in the AP also express TH (6L-O). Scale bars: 50 µm (A, F, L-O'), 100 µm (B, G, H, I, J, K), 200 µm (C, D, E)

cne23309-sup-0005-suppfig3b.TIF10296K

Supporting Fig. 3 (magenta version of original Fig 6). GHSR mRNA localization in the rostral NTS (rNTS) and the dorsal vagal complex (DVC) in relation to TH, CART and NK1R expression. Sections at Bregma -11.50 mm (6A), -13.20 mm (6B, F), -13.60 mm (6C, G, J), -13.85 mm (6D, H, K, L), -14.20 mm (6E, H). To allow direct comparison of the brightfield ISH signal with the immunofluorescence signals the brightfield image is inverted. NB: The tissues surrounding the DVC are more opaque (see Fig. 3), resulting in a higher relative background in the inverted image in these areas (e.g. Fig. 6C). CART immunoreactivity is very prominent in the solitary tract (TS; 6A) and in fibre projections throughout the DVC, clearly defining the boundaries of the NTS and DMNX (10). TH marks C2 adrenergic neurons (6B, F), A2 noradrenergic neurons (6C, D, E, G, H, I) in the cNTS and noradrenergic neurons in the area postrema (AP; C, D, J, K-O). In the rNTS, GHSR mRNA expressing neurons are found lateral to the TS, in the rostrolateral NTS (6A). In the DVC, neurons expressing GHSR mRNA are found in the DMNX (10; 6B, C, D, G, H), cNTS (6B-I) and the AP (6C, D, J, K, L). GHSR mRNA is not found in TH expressing neurons throughout the DVC, or in NK1R-expressing neurons in the AP (6L). A very large proportion of NK1R-positive neurons in the AP also express TH (6L-O). Scale bars: 50 µm (A, F, L-O'), 100 µm (B, G, H, I, J, K), 200 µm (C, D, E)

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