The nucleus of the solitary tract (NTS) contains a unique subpopulation of aldosterone-sensitive neurons. These neurons express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) and are activated by sodium deprivation. They are located in the caudal NTS, a region which is densely innervated by the vagus nerve, suggesting that they could receive direct viscerosensory input from the periphery. To test this possibility, we injected the highly sensitive axonal tracer biotinylated dextran amine (BDA) into the left nodose ganglion in rats. Using confocal microscopy, we observed a sparse input from the vagus to most HSD2 neurons. Roughly 80% of the ipsilateral HSD2 neurons exhibited at least one close contact with a BDA-labeled vagal bouton, although most of these cells received only a few total contacts. Most of these contacts were axo-dendritic (approximately 80%), while approximately 20% were axo-somatic. In contrast, the synaptic vesicular transporters VGLUT2 or GAD7 labeled much larger populations of boutons contacting HSD2-labeled dendrites and somata, suggesting that direct input from the vagus may only account for a minority of the information integrated by these neurons. In summary, the aldosterone-sensitive HSD2 neurons in the NTS appear to receive a small amount of direct viscerosensory input from the vagus nerve. The peripheral sites of origin and functional significance of this projection remain unknown. Combined with previously-identified central sources of input to these cells, the present finding indicates that the HSD2 neurons integrate humoral information with input from a variety of neural afferents.

The purpose of this study was to identify brain sites that may be sensitive to the adrenal steroid aldosterone. After a survey of the entire brain for mineralocorticoid receptor (MR) immunoreactivity, we discovered unique clusters of dense nuclear and perinuclear MR in a restricted distribution within the nucleus of the solitary tract (NTS). These same cells were found to contain the glucocorticoid-inactivating enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), a signature of aldosterone-sensitive tissues. Immunoreactivity for various other NTS marker molecules failed to colocalize with HSD2 in these putative aldosterone target neurons, so they may represent a unique neuronal phenotype. Finally, the entire rat CNS was examined for evidence of HSD2 protein expression. Outside the NTS, HSD2-immunoreactive neurons were found in only two other sites: the ventrolateral division of the ventromedial hypothalamic nucleus and a few scattered neurons in the medial vestibular nucleus, just rostral to the NTS. HSD2 immunoreactivity was also found in the ependymal cells that form the subcommissural organ. In summary, few brain sites contain neurons that may be aldosterone sensitive, and only one of these sites, the NTS, contains neurons that express HSD2 and contain dense nuclear MR. The HSD2 neurons in the NTS may represent an important target for aldosterone action in the brain.

The HSD2 (11-beta-hydroxysteroid dehydrogenase type 2-expressing) neurons in the nucleus of the solitary tract (NTS) of the rat are aldosterone-sensitive and have been implicated in sodium appetite. The central nucleus of the amygdala (CeA) has been shown to modulate salt intake in response to aldosterone, so we investigated the connections between these two sites. A prior retrograde tracing study revealed only a minor projection from the HSD2 neurons directly to the CeA, but these experiments suggested that a more substantial projection may be relayed through the parabrachial nucleus. Small injections of cholera toxin beta subunit (CTb) into the external lateral parabrachial subnucleus (PBel) produced both retrograde cell body labeling in the HSD2 neurons and anterograde axonal labeling in the lateral subdivision of the CeA. Also, injections of either CTb or Phaseolus vulgaris leucoagglutinin into the medial subdivision of the CeA labeled a descending projection from the amygdala to the medial NTS. Axons from the medial CeA formed numerous varicosities and terminals enveloping the HSD2 neurons. Complementary CTb injections, centered in the HSD2 subregion of the NTS, retrogradely labeled neurons in the medial CeA. These bidirectional projections could form a functional circuit between the HSD2 neurons and the CeA. The HSD2 neurons may represent one of the functional inputs to the lateral CeA, and their activity may be modulated by a return projection from the medial CeA. This circuit could provide a neuroanatomical basis for the modulation of salt intake by the CeA.

The nucleus of the solitary tract (NTS) contains a unique subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). These neurons are mineralocorticoid-sensitive and are activated in association with salt appetite during sodium deficiency. In the absence of sodium deficiency, the HSD2 neurons and sodium appetite are both stimulated by chronic mineralocorticoid administration. After 7 days of treatment with deoxycorticosterone (2 mg/day), an increased number of HSD2 neurons became immunoreactive for the neuronal activity marker c-Fos. When given access to concentrated saline (3% NaCl), deoxycorticosterone-treated rats drank eight times more than vehicle-treated rats. Saline ingestion increased neuronal activation within the medial subdivision of the NTS, but the number of c-Fos-immunoreactive HSD2 neurons was reduced. This finding suggests that the HSD2 neurons are inhibited by signals directly related to saline ingestion, and not simply by the alleviation of sodium deficiency, which does not occur during mineralocorticoid administration.

Restricting dietary sodium promotes sodium appetite in rats. Prolonged sodium restriction increases plasma potassium (pK), and elevated pK is largely responsible for a concurrent increase in aldosterone, which helps promote sodium appetite. In addition to increasing aldosterone, we hypothesized that elevated potassium directly influences the brain to promote sodium appetite. To test this, we restricted dietary potassium in sodium-deprived rats. Potassium restriction reduced pK and blunted the increase in aldosterone caused by sodium deprivation, but did not prevent sodium appetite or the activation of aldosterone-sensitive HSD2 neurons. Conversely, supplementing potassium in sodium-deprived rats increased pK and aldosterone, but did not increase sodium appetite or the activation of HSD2 neurons relative to potassium restriction. Supplementing potassium without sodium deprivation did not significantly increase aldosterone and HSD2 neuronal activation and only modestly increased saline intake. Overall, restricting dietary sodium activated the HSD2 neurons and promoted sodium appetite across a wide range of pK and aldosterone, and saline consumption inactivated the HSD2 neurons despite persistent hyperaldosteronism. In conclusion, elevated potassium is important for increasing aldosterone, but it is neither necessary nor sufficient for activating HSD2 neurons and increasing sodium appetite.

Epithelial sodium channels (ENaCs) are strongly expressed in the circumventricular organs (CVOs), and these structures may play an important role in sensing plasma sodium levels. Here, the potent ENaC blocker amiloride was injected intraperitoneally in rats and 2h later, the c-Fos activation pattern in the CVOs was studied. Amiloride elicited dose-related activation in the area postrema (AP) but only ~10% of the rats showed c-Fos activity in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). Tyrosine hydroxylase-immunoreactive (catecholamine) AP neurons were activated, but tryptophan hydroxylase-immunoreactive (serotonin) neurons were unaffected. The AP projects to FoxP2-expressing neurons in the dorsolateral pons which include the pre-locus coeruleus nucleus and external lateral part of the parabrachial nucleus; both cell groups were c-Fos activated following systemic injections of amiloride. In contrast, another AP projection target--the aldosterone-sensitive neurons of the nucleus tractus solitarius which express the enzyme 11-β-hydroxysteriod dehydrogenase type 2 (HSD2) were not activated. As shown here, plasma concentrations of amiloride used in these experiments were near or below the IC50 level for ENaCs. Amiloride did not induce changes in blood pressure, heart rate, or regional vascular resistance, so sensory feedback from the cardiovascular system was probably not a causal factor for the c-Fos activity seen in the CVOs. In summary, amiloride may have a dual effect on sodium homeostasis causing a loss of sodium via the kidney and inhibiting sodium appetite by activating the central satiety pathway arising from the AP.

The area postrema (AP) is a circumventricular organ located in the dorsal midline of the medulla. It functions as a chemosensor for blood-borne peptides and solutes, and converts this information into neural signals that are transmitted to the nucleus tractus solitarius (NTS) and parabrachial nucleus (PB). One of its NTS targets in the rat is the aldosterone-sensitive neurons which contain the enzyme 11 β-hydroxysteroid dehydrogenase type 2 (HSD2). The HSD2 neurons are part of a central network involved in sodium appetite regulation, and they innervate numerous brain sites including the pre-locus coeruleus (pre-LC) and PB external lateral-inner (PBel-inner) cell groups of the dorsolateral pons. Both pontine cell groups express the transcription factor FoxP2 and become c-Fos activated following sodium depletion. Because the AP is a component in this network, we wanted to determine whether it also projects to the same sites as the HSD2 neurons. By using a combination of anterograde axonal and retrograde cell body tract-tracing techniques in individual rats, we show that the AP projects to FoxP2 immunoreactive neurons in the pre-LC and PBel-inner. Thus, the AP sends a direct projection to both the first-order medullary (HSD2 neurons of the NTS) and the second-order dorsolateral pontine neurons (pre-LC and PB-el inner neurons). All three sites transmit information related to systemic sodium depletion to forebrain sites and are part of the central neural circuitry that regulates the complex behavior of sodium appetite.

The paraventricular hypothalamic nucleus (PVH) contains many neurons that innervate the brainstem, but information regarding their target sites remains incomplete. Here we labeled neurons in the rat PVH with an anterograde axonal tracer, Phaseolus vulgaris leucoagglutinin (PHAL), and studied their descending projections in reference to specific neuronal subpopulations throughout the brainstem. While many of their target sites were identified previously, numerous new observations were made. Major findings include: 1) In the midbrain, the PVH projects lightly to the ventral tegmental area, Edinger-Westphal nucleus, ventrolateral periaqueductal gray matter, reticular formation, pedunculopontine tegmental nucleus, and dorsal raphe nucleus. 2) In the dorsal pons, the PVH projects heavily to the pre-locus coeruleus, yet very little to the catecholamine neurons in the locus coeruleus, and selectively targets the viscerosensory subregions of the parabrachial nucleus. 3) In the ventral medulla, the superior salivatory nucleus, retrotrapezoid nucleus, compact and external formations of the nucleus ambiguous, A1 and caudal C1 catecholamine neurons, and caudal pressor area receive dense axonal projections, generally exceeding the PVH projection to the rostral C1 region. 4) The medial nucleus of the solitary tract (including A2 noradrenergic and aldosterone-sensitive neurons) receives the most extensive projections of the PVH, substantially more than the dorsal vagal nucleus or area postrema. Our findings suggest that the PVH may modulate a range of homeostatic functions, including cerebral and ocular blood flow, corneal and nasal hydration, ingestive behavior, sodium intake, and glucose metabolism, as well as cardiovascular, gastrointestinal, and respiratory activities.

Using a double immunofluorescence procedure, we report the discovery of a novel group of fibrous astrocytes that co-express epithelial sodium channel (ENaC) γ-subunit protein along with glial acidic fibrillary protein (GFAP). These cells are concentrated along the borders of the sensory circumventricular organs (CVOs), embedded in the white matter (e.g., optic nerve/chiasm, anterior commissure, corpus callosum, pyramidal tract) and are components of the pia mater. In the CVOs, a compact collection of ENaC γ-immunoreactive glial fibers form the lamina terminalis immediately rostral to the organum vasculosum of the lamina terminalis (OVLT). Astrocyte processes can be traced into the median preoptic nucleus - a region implicated in regulation of sodium homeostasis. In the subfornical organ (SFO), ENaC γ-GFAP astrocytes lie in its lateral border, but not in the ventromedial core. In the area postrema (AP), a dense ENaC γ-GFAP glial fibers form the interface between the AP and nucleus tractus solitarius; this area is termed the subpostremal region. Antibodies against the ENaC α- or β-subunit proteins do not immunostain these regions. In contrast, the antibodies against the ENaC γ-subunit protein react weakly with neuronal cell bodies in the CVOs. Besides affecting glial-neural functions in the CVOs, the astrocytes found in the white matter may affect saltatory nerve conduction, serving as a sodium buffer. The ENaC γ-expressing astrocytes of the ventral medulla send processes into the raphe pallidus which intermingle with the serotoninergic (5-HT) neurons found in this region as well as with the other nearby 5-HT neurons distributed along ventral medullary surface.

The nucleus accumbens (NAc) is part of a forebrain system implicated in reward, motivation, and learning. NAc neurons become activated during various ingestive activities, including salt intake. A subset of neurons within the nucleus tractus solitarius (NTS) shows c-Fos activation during prolonged sodium deprivation in rats. These neurons express mineralocorticoid receptors and the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them selectively sensitive to aldosterone-an adrenal hormone that modulates sodium appetite. Here we tested whether these neurons project multisynaptically to the core or shell subregions of the NAc. Pseudorabies virus (PRV)-a retrograde transneuronal tracer-was injected into the NAc in rats and after 3-4 days PRV-infected HSD2 neurons were identified. PRV injections into the NAc core yielded greater numbers of PRV-labeled HSD2 neurons than did comparable injections into the NAc shell. Transneuronal labeling was also found in brainstem sites that receive direct projections from HSD2 neurons, namely, lateral parabrachial and prelocus coeruleus nuclei. In other experiments a retrograde neural tracer (cholera toxin beta-subunit) was injected into the NAc. Extensive retrograde labeling was found in the midline thalamus and frontal cortical regions, but no cells were labeled in the NTS or parabrachial region. These findings indicate that the HSD2 neurons project via a multisynaptic pathway to the NAc, which may be relayed sequentially through two sites: the dorsolateral pons and the paraventricular thalamic nucleus. HSD2 neurons may be part of an ascending pathway involved in the salt-seeking behavior of sodium-depleted rats.

The nucleus of the solitary tract (NTS) contains a subpopulation of neurons that express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2), which makes them uniquely sensitive to aldosterone. These neurons may drive sodium appetite, which is enhanced by aldosterone. Anterograde and retrograde neural tracing techniques were used to reveal the efferent projections of the HSD2 neurons in the rat. First, the anterograde tracer Phaseolus vulgaris leucoagglutinin was used to label axonal projections from the medial NTS. Then, NTS-innervated brain regions were injected with a retrograde tracer, cholera toxin beta subunit, to determine which sites are innervated by the HSD2 neurons. The HSD2 neurons project mainly to the ventrolateral bed nucleus of the stria terminalis (BSTvl), the pre-locus coeruleus (pre-LC), and the inner division of the external lateral parabrachial nucleus (PBel). They also send minor axonal projections to the midbrain ventral tegmental area, lateral and paraventricular hypothalamic nuclei, central nucleus of the amygdala, and periaqueductal gray matter. The HSD2 neurons do not innervate the ventrolateral medulla, a key brainstem autonomic site. Additionally, our tracing experiments confirmed that the BSTvl receives direct axonal projections from the neighboring A2 noradrenergic neurons in the NTS, and from the same pontine sites that receive major inputs from the HSD2 neurons (PBel and pre-LC). The efferent projections of the HSD2 neurons may provide new insights into the brain circuitry responsible for sodium appetite.

The transcription factor Forkhead box protein 2 (FoxP2) is expressed in two cell groups of the brainstem that have been implicated in sodium appetite regulation: the pre-locus coeruleus (pre-LC) and parabrachial nucleus--external lateral-inner subdivision (PBel-inner). Because the connections of these two groups are unknown, neuroanatomical tracing methods were used to define their central projections. The pre-LC outputs were first analyzed using an anterograde axonal tracer--Phaseolus vulgaris leucoagglutinin (PHAL) to construct a brain map. Next, we examined whether the FoxP2 immunoreactive (FoxP2+) neurons of the pre-LC contribute to these projections using a retrograde neuronal tracer--cholera toxin β-subunit (CTb). CTb was injected into selected brain regions identified in the anterograde tracing study. One week later the rats were killed, and brainstem sections were processed by a double immunohistochemical procedure to determine whether the FoxP2+ neurons in the pre-LC and/or PBel-inner contained CTb. FoxP2+ pre-LC neurons project to: (1) ventral pallidum; (2) substantia innominata and bed nucleus of the stria terminalis; (3) paraventricular, central medial, parafascicular, and subparafascicular parvicellular thalamic nuclei; (4) paraventricular (PVH), lateral, perifornical, dorsomedial (DMH), and parasubthalamic hypothalamic nuclei; and (5) ventral tegmental area (VTA), periaqueductal gray matter (PAG), dorsal and central linear raphe nuclei. FoxP2+ PBel-inner neurons project to the PVH and DMH, with weaker connections to the LHA, VTA, and PAG. Both the pre-LC and PBel-inner project to central sites implicated in sodium appetite, and related issues, including foraging behavior, hedonic responses to salt intake, sodium balance, and cardiovascular regulation, are discussed.

The HSD2 (11-beta-hydroxysteroid dehydrogenase-type 2 enzyme) containing neurons of the nucleus tractus solitarius (NTS) become activated during low-sodium and high-aldosterone states such as hypovolemia. This response may be due to hormonal and/or neural signals. Hormonal signals may activate neurons in the area postrema that innervate the HSD2 neurons. The vagus nerve projects directly to the HSD2 neurons and this could be another route whereby these neurons receive information about systemic sodium/aldosterone status. The peripheral sites of origin that contribute to this vagal projection remain unknown, and in the present study, we injected the transganglionic tracer, cholera toxin beta-subunit-horseradish peroxidase (CTb-HRP), into wall of various gastrointestinal organs (stomach, small and large intestine) or liver of rats. Confocal microscopy of brainstem sections stained by a double immunohistochemical procedure was used to analyze whether the HSD2 neurons received axonal contacts from specific gastrointestinal structures. The major source of afferents arose from the stomach, mainly from its pyloric antrum, but a weaker input originated from the fundus region. A trace amount originated from the duodenum. The terminal part of the small intestine and large intestine did not to contribute to this projection. Similarly, no afferent inputs from the liver or portal vein were found. In conclusion, HSD2 neurons receive an input mainly from the stomach and these results are considered as potential sites affecting sodium intake.

The transcription factor Phox2b is necessary for the development of the nucleus of the solitary tract (NTS). In this brainstem nucleus, Phox2b is expressed exclusively within a subpopulation of glutamatergic neurons. The present experiments in the adult rat were designed to test whether this subpopulation includes the aldosterone-sensitive NTS neurons, which express the enzyme 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2). Nuclear Phox2b was found in virtually all the HSD2 neurons (95-99%, n = 6 cases). Unlike the activity-related transcription factor c-Fos, Phox2b expression in the HSD2 neurons was not influenced by dietary sodium deprivation. The ubiquitous expression of Phox2b by the HSD2 neurons suggests that they are developmentally related to other Phox2b-dependent neurons of the NTS and that they release the excitatory neurotransmitter glutamate. This finding also suggests that human Phox2b mutations, which cause the central congenital hypoventilation syndrome (CCHS, also known as Ondine's curse), may also produce deficits in central aldosterone signaling and appetitive or autonomic responses to sodium deficiency.

The ventrolateral bed nucleus of the stria terminalis (BSTvl) receives direct input from two specific subpopulations of neurons in the nucleus tractus solitarius (NTS). It is heavily innervated by aldosterone-sensitive NTS neurons, which are selectively activated by sodium depletion, and by the A2 noradrenergic neurons, which are activated by visceral and immune- and stress-related stimuli. Here, we used a retrograde neuronal tracer to identify other brain sites that innervate the BSTvl. Five general brain regions contained retrogradely labeled neurons: cerebral cortex (infralimbic and insular regions), rostral forebrain structures (subfornical organ, organum vasculosum of the lamina terminalis, taenia tecta, nucleus accumbens, lateral septum, endopiriform nucleus, dorsal BST, substantia innominata, and, most prominently the amygdala--primarily its basomedial and central subnuclei), thalamus (central medial, intermediodorsal, reuniens, and, most prominently the paraventricular thalamic nucleus), hypothalamus (medial preoptic area, perifornical, arcuate, dorsomedial, parasubthalamic, and posterior hypothalamic nuclei), and brainstem (periaqueductal gray matter, dorsal and central superior raphe nuclei, parabrachial nucleus, pre-locus coeruleus region, NTS, and A1 noradrenergic neurons in the caudal ventrolateral medulla). In the arcuate hypothalamic nucleus, some retrogradely labeled neurons contained either agouti-related peptide or cocaine/amphetamine-regulated transcript. Of the numerous retrogradely labeled neurons in the perifornical hypothalamic area, few contained melanin-concentrating hormone or orexin. In the brainstem, many retrogradely labeled neurons were either serotoninergic or catecholaminergic. In summary, the BSTvl receives inputs from a variety of brain sites implicated in hunger, salt and water intake, stress, arousal, and reward.

Two specific groups of neurons in the dorsolateral pons are activated by dietary sodium deprivation. These two groups are the pre-locus coeruleus (pre-LC) and the inner subdivision of the external lateral parabrachial nucleus (PBel-inner). In each site, after rats are fed an extremely low-sodium diet for over a week, neurons increase their expression of an activity-induced transcription factor, c-Fos. Here, we confirm this observation and extend it by demonstrating that these two groups of neurons express a common marker gene, the constitutively-expressed transcription factor Forkhead box protein 2 (FoxP2). That is, virtually all of the c-Fos activated neurons in both regions also express FoxP2. The expression of FoxP2 by both these groups of neurons suggests that they are developmentally-related subsets derived from the same basic population. Given that FoxP2, unlike c-Fos, is expressed independent of sodium deprivation, this marker may be useful in future studies of the pre-LC and PBel-inner. The molecular definition of these neurons, which project to circuits in the forebrain that influence visceral, appetitive, and hedonic functions, may allow direct experimental exploration of the functional role of these circuits using genetic tools.

Cardiovascular changes occur during mental stress and in certain types of mood disorders. The neural basis for this phenomenon is unknown but it may be dependent on CNS neurons that provide branched projections to affective processing regions of the brain, such as the medial prefrontal cortex, and to the sympathetic outflow system. Because these putative neurons may be connected to these two target sites by chains of neurons, we performed double virus transneuronal tracing experiments and show here that a select subset of neurons in the medial preoptic nucleus (MPN), lateral hypothalamic area (LHA), and nucleus tractus solitarius (NTS) are co-linked to these two sites. Neurotensin MPN, orexin-containing LHA, and catecholamine NTS neurons were the major phenotypes involved in these projections. This novel class of neurons may coordinate cardiovascular changes seen in different emotional states.