Aldosterone-sensitive HSD2 neurons in mice

Abstract

Sodium deficiency elevates aldosterone, which in addition to epithelial tissues acts on the brain to promote dysphoric symptoms and salt intake. Aldosterone boosts the activity of neurons that express 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a hallmark of aldosterone-sensitive cells. To better characterize these neurons, we combine immunolabeling and in situ hybridization with fate mapping and Cre-conditional axon tracing in mice. Many cells throughout the brain have a developmental history of Hsd11b2 expression, but in the adult brain one small brainstem region with a leaky blood-brain barrier contains HSD2 neurons. These neurons express Hsd11b2, Nr3c2 (mineralocorticoid receptor), Agtr1a (angiotensin receptor), Slc17a6 (vesicular glutamate transporter 2), Phox2b, and Nxph4; many also express Cartpt or Lmx1b. No HSD2 neurons express cholinergic, monoaminergic, or several other neuropeptidergic markers. Their axons project to the parabrachial complex (PB), where they intermingle with AgRP-immunoreactive axons to form dense terminal fields overlapping FoxP2 neurons in the central lateral subnucleus (PBcL) and pre-locus coeruleus (pLC). Their axons also extend to the forebrain, intermingling with AgRP- and CGRP-immunoreactive axons to form dense terminals surrounding GABAergic neurons in the ventrolateral bed nucleus of the stria terminalis (BSTvL). Sparse axons target the periaqueductal gray, ventral tegmental area, lateral hypothalamic area, paraventricular hypothalamic nucleus, and central nucleus of the amygdala. Dual retrograde tracing revealed that largely separate HSD2 neurons project to pLC/PB or BSTvL. This projection pattern raises the possibility that a subset of HSD2 neurons promotes the dysphoric, anorexic, and anhedonic symptoms of hyperaldosteronism via AgRP-inhibited relay neurons in PB.

Keywords: 11-Beta-hydroxysteroid dehydrogenase type 2; Aldosterone; Angiotensin II; Dietary sodium; Dietary sodium deficiency; Dietary sodium deprivation; Mineralocorticoid receptor; Nucleus of the solitary tract; Salt appetite; Sodium appetite.

Figure 1.

(A-D) Diamidobenzidine (DAB) immunohistochemical staining for 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) at successively caudal levels of the dorsal medulla, with Nissl counterstain. (E-H) Fluorescence in situ hybridization for Hsd11b2 (red-orange) at similar levels in a separate mouse. Ubiquitin C mRNA (Ubc, green) and nuclear labeling (DAPI, blue) are shown for cytoarchitectural reference. Scale bars are 200 μm (A-D) and 100 μm (E-H). Other abbreviations: 4V, fourth ventricle; CC, central canal of the spinal cord; NTS, nucleus of the solitary tract; AP, area postrema; X, dorsal motor nucleus of the vagus nerve; XII, hypoglossal motor nucleus.

Figure 2.

(A) HSD2-immunoreactive neurons (red) contain nuclear immunoreactivity for the transcription factor Phox2b (green; double-labeling is yellow). HSD2 neurons are separate from Phox2b neuron populations labeled for tyrosine hydroxylase (TH, blue) and choline acetyltransferase (ChAT, magenta). (B-D) compare each pair of markers. (E-G) Nuclear immunoreactivity for Lmx1b (green) is present in many HSD2 neurons and other neurons across the dorsal NTS. Scale bars are 100 μm and apply to adjoining panels of similar size.

Figure 3.

(A-D) After systemic injection, Fluorogold (Fg, green) permeates the area postrema (AP) and the adjacent NTS, up to and including the distribution of HSD2-immunoreactive neurons (red). Retrograde Fg transport in peripheral axons also labels ChAT-immunoreactive (magenta) neurons in the dorsal vagal and hypoglossal motor nuclei. Scale bars are 100 μm. (E-H) Immunoglobulin G (IgG) immunofluorescence highlights linear structures in the area postrema and a large subpostremal region of the NTS up to and around many HSD2-immunolabeled (red) neurons particularly at the obex (A) and tail of the area postrema (G). Scale bar is 100 μm.

Figure 4.

A variety of gene expression in the NTS distinguishes HSD2 and surrounding neurons, evidence by fluorescence in situ hybridization (FISH), immunofluorescence, and GFP reporter expression. (A-C) HSD2 immunoreactivity (red) and Hsd11b2 mRNA (green) co-localize in the same NTS population. (D-F) Complementing their Phox2b-immunoreactivity in Figure 2, all HSD2-immunoreactive neurons and many dorsal vagal complex neurons, contain Phox2b mRNA (green). (G-F) Along with many other neurons in the NTS and elsewhere, all HSD2 neurons contain mRNA for the type 2 vesicular glutamate transporter (Vglut2/Slc17a6, green). (J-K) Mineralocorticoid receptor mRNA (Nr3c2, green) is expressed prominently in NTS neurons that contain mRNA for Hsd11b2 (red). (M-O) BAC transgenic mice expressing GFP following the angiotensin II receptor gene (Agtr1a-GFP) have a prominent cluster of green neurons in the NTS, many of which are immunoreactive for HSD2 (red). (P-R) We confirmed in C57BL6/J mice that mRNA for this receptor (Agtr1a, green) is expressed in a similar pattern in the medial NTS, and co-localizes with mRNA for Hsd11b2 (red). (S-X) HSD2 neurons (Hsd11b2 mRNA, red) co-localize with many cells from larger NTS populations expressing the neuropeptide precursors Cartpt (green, T-U) and Nxph4 (green, W-X). HSD2 neurons do not express a variety of other neuropeptide genes or Cre-reporters, including the L10GFP Cre-reporter for Pdyn (Y; note that the putative Pdyn NTS neurons are immunoreactive for Phox2b, blue) or mRNA for galanin (Gal, green Z), or cholecystokinin (Cck, green, A2). Complementing their lack of catecholaminergic and cholinergic synthetic enzyme markers in HSD2 neurons in Figure 2, Hsd11b2 does not co-localize with mRNA for the vesicular monoamine transporter 2 (VMAT2 / Slc18a2, green in B2) or vesicular acetylcholine transporter (VAChT / Slc18a3, green in C2). Scale bars are 20 μm (A), 25 μm (F, I, R and X) and 50 μm (L, O, R, U, Y, Z, A2, B2, C2) and apply to adjoining panels of similar size.

Figure 5.

Hsd11b2 Cre-driver nice crossed to separate strains of Cre-reporter nice: (A-C) Hsd11b2-Cre;R26-lsl-L10GFP, (D-G) Hsd11b2-Cre;R26-lsl-Ai9tdTomato, and (H-J) Hsd11b2^CreR26-lsl-Ai75tdTomato. (A, D, H) The cerebellum exhibits similar Cre-reporter expression in all three strains, with differences related to the expected intrcellular distribution of each fluorescent protein: L10GFP is tethered to ribosomes in cytoplasm; Ai9 tdTomato diffuses freely into dendrites and axons; and Ai75 tdTomato is targeted to the cell nucleus. In the NTS, the L10GFP reporter was sparse, with (B) one mouse expressing no GFP in any HSD2 neurons and (C) others containing a most one or few GFP-expressing HSD2 neurons per tissue section. The Ai9 reporter was expressed in virtually all HSD2 neurons (E) and many other cell types, including clusters of smalls cells and processes surrounding blood vessels (F) and small glial cells that appear similar to oligodendrocyte precursor cells (G). The Ai75 reporter filled the cell nucleus with intensely bright fluorescence in many cell types throughout the brain, including most HSD2 neurons (I), which in some cases were fewer in number and abnormal in appearance (see text). Scale bars are 1 mm (A,D,H) and 100 μm (all other panels).

Figure 6.

Experimental strategy for Cre-conditional labeling axonal projections from HSD2 neurons in the NTS to other brain regions (A). (B-F) a representative injection site from the cases we hand-traced to show the brain-wide pattern of axonal labeling in Figure 7 (with detail images in Supplemental Figure A4). (B) Nickel-DAB immunohistochemistry (black, NiDAB) reveals mCherry expression by Cre-expressing neurons within the injected region. (C) Nissl-counterstained and re-imaged image of the same tissue section clarifies background cytoarchitecture. (D-F) Cre-conditional expression of Syp-mCherry (red) co-localizes with HSD2-immunofluorescence (green) in a section adjacent to the NiDAB-stained NTS section shown in (B-C). Scale bars are 100 μm and apply to adjoining panels of similar size.

Figure 7.

We traced axonal projections of HSD2 neurons in a representative Hsd11b2-Cre mouse injected with Cre-conditional Syp-mCherry (injection site in Figure 6). These successive, caudal-to-rostral tracings show the full-brain distribution of Syp-mCh axons in this case. Each drawing was re-scaled for illustrative purposes, and a minimal set of labels are provided to highlight relevant landmarks, pathways, and target regions. Abbreviations: ac, anterior commissure; BSTvL, ventrolateral subregion of the bed nucleus of the stria terminalis; fx, fornix; gVII, genu of the seventh cranial nerve fascicles; mCeA, medial subdivision of the central nucleus of the amygdala; ml, medial lemniscus; NTS, nucleus of the solitary tract; PAGvL, ventrolateral subdivision/column of the periaqueductal gray matter; PBcL, central lateral parabrachial subnucleus; pLC, pre-locus coeruleus; PSTN, parasubthalamic nucleus; pyx, pyramidal decussation; Rt, medullary (intermediate) reticular formation; scp, superior cerebellar peduncle; sLHA, suprafornical lateral hypothalamic area; VIIn, seventh cranial nerve root; xscp, decussation of the superior cerebellar peduncle.

Figure 7.

We traced axonal projections of HSD2 neurons in a representative Hsd11b2-Cre mouse injected with Cre-conditional Syp-mCherry (injection site in Figure 6). These successive, caudal-to-rostral tracings show the full-brain distribution of Syp-mCh axons in this case. Each drawing was re-scaled for illustrative purposes, and a minimal set of labels are provided to highlight relevant landmarks, pathways, and target regions. Abbreviations: ac, anterior commissure; BSTvL, ventrolateral subregion of the bed nucleus of the stria terminalis; fx, fornix; gVII, genu of the seventh cranial nerve fascicles; mCeA, medial subdivision of the central nucleus of the amygdala; ml, medial lemniscus; NTS, nucleus of the solitary tract; PAGvL, ventrolateral subdivision/column of the periaqueductal gray matter; PBcL, central lateral parabrachial subnucleus; pLC, pre-locus coeruleus; PSTN, parasubthalamic nucleus; pyx, pyramidal decussation; Rt, medullary (intermediate) reticular formation; scp, superior cerebellar peduncle; sLHA, suprafornical lateral hypothalamic area; VIIn, seventh cranial nerve root; xscp, decussation of the superior cerebellar peduncle.

Figure 7.

We traced axonal projections of HSD2 neurons in a representative Hsd11b2-Cre mouse injected with Cre-conditional Syp-mCherry (injection site in Figure 6). These successive, caudal-to-rostral tracings show the full-brain distribution of Syp-mCh axons in this case. Each drawing was re-scaled for illustrative purposes, and a minimal set of labels are provided to highlight relevant landmarks, pathways, and target regions. Abbreviations: ac, anterior commissure; BSTvL, ventrolateral subregion of the bed nucleus of the stria terminalis; fx, fornix; gVII, genu of the seventh cranial nerve fascicles; mCeA, medial subdivision of the central nucleus of the amygdala; ml, medial lemniscus; NTS, nucleus of the solitary tract; PAGvL, ventrolateral subdivision/column of the periaqueductal gray matter; PBcL, central lateral parabrachial subnucleus; pLC, pre-locus coeruleus; PSTN, parasubthalamic nucleus; pyx, pyramidal decussation; Rt, medullary (intermediate) reticular formation; scp, superior cerebellar peduncle; sLHA, suprafornical lateral hypothalamic area; VIIn, seventh cranial nerve root; xscp, decussation of the superior cerebellar peduncle.

Figure 8:

HSD2 neurons project axons that terminate densely (red, mCherry) in highly specific subregion of the parabrachial complex (PB), in close association with other axons containing the neuropeptide AgRP (agouti-related peptide, magenta). We labeled a transcription factor marker (green, FoxP2-immunoreactivity) and catecholamine neuron marker for locus coeruleus (LC; immunoreactivity in blue for tyrosine hydroxylase, TH) for reference in this crowded, complex region of the brainstem. (A-C) HSD2 axons in the lateral PB terminate densely amid FoxP2 neurons within a narrow subregion of the PB central lateral subnucleus (PBcL), with minimal labeling in its dorsal lateral (dL) and external lateral (eL). (D-F) Caudally, HSD2 axons target the pre-locus coeruleus (pLC) FoxP2 neuron cluster flanking and streaking through the LC, while largely avoiding Barrington’s nucleus (Bar, D) and the most caudal-medial extent of FoxP2 neurons in this region (F). In both pLC (H-K) and PBcL (L-N), the axon terminal field of HSD2 axons is closely associated with a dense collection of axons and boutons immunoreactive for AgRP. All scale bars are 100 μm.

Figure 9:

(A-C) HSD2 axons (red, mCherry) projecting to the ventrolateral bed nucleus of the stria terminalis (BSTvL), beneath the anterior commissure form a dense terminal field ventral to the anterior commissure (ac) in rostrocaudal levels at and just rostral to its midline crossing. (D-G) show these axons at higher magnification to highlight their closely intertwined association with separate axons immunoreactive for calcitonin gene-related peptide (CGRP, green) or agouti gene-related peptide (AgRP, magenta). (H-K) show the HSD2 axon-terminal field in BSTvL and mRNA for the vesicular GABA transporter (Vgat/Slc32a1, magenta), and for the ubiquitously expressed Ubc (green) and DAPI nuclear counterstains, showing that HSD2 projections to BSTvL exclusively surround a population of putatively GABAergic neurons. Scale bars are 200 μm (A), 50 μm (D, G), and 20 μm (K) panels and apply to adjoining panels of similar size.

Figure 10.

Dual retrograde tracer injections reveal that most ipsilateral HSD2 neurons project to either PB or BST, not both. Large injections of cholera toxin B subunit (CTb, green) into the PB (A) and of Fluorogold (Fg, blue) into the BST (B) retrogradely labeled many HSD2 neurons in the NTS. (C-H) This example relies on a Cre-reporter to identify HSD2 neurons (Hsd11b2-Cre;Ai9-lsl-tdTomato), but immunolabeling HSD2 after dual retrograde tracer injections produced similar results in C57BL6/J mice (Table 4). Scale bars are 200 μm (A), 50 μm (D, G), and 20 μm (K) and apply to adjoining panels of similar size.

Figure 11.

Sagittal diagram summarizing the axonal projections of HSD2 neurons and genes and proteins that identify or distinguish them from surrounding NTS neurons.