Rapid and localized alterations of neuropeptide Y in discrete hypothalamic nuclei with feeding status

Neuropeptide Y (NPY) is believed to regulate the normal eating behavior and body weight in rats via central mechanisms. We have investigated whether NPY, which stimulates food intake, may in turn be modified by the nutritional state of the animals. Thus the impact of food deprivation (FD) (48 h) and subsequent refeeding on the levels of NPY in discrete hypothalamic areas was examined in this study. The results showed site specific change in only 3 of 7 hypothalamic sites. A 5-fold increment in NPY was reported in the paraventricular nucleus (PVN) and a 10-fold increase was observed in the arcuate nucleus-median eminence (ARC-ME). While subsequent refeeding for 6 h reversed the effect of FD in the ARC-ME, the levels of NPY in the PVN remained high in the refed rats. The perifornical lateral hypothalamus displayed a different pattern, namely, a significant increase in NPY content in refed as compared to satiated and deprived rats. The NPY levels in 4 other hypothalamic sites, namely, the dorsomedian, ventromedian, supraoptic and suprachiasmatic nuclei, and two extrahypothalamic sites, namely caudate nucleus and nucleus accumbens, showed total resistance to any change following deprivation and refeeding. These data emphasize the important and specific role of the paraventricular and arcuate nuclei in NPY's regulation of food intake and provide support for the idea that the variations of hypothalamic NPY after food deprivation reflect a specific physiological response of feeding regulatory system to alterations in the animal nutritional state and body weight.     

c-fos expression in the rat brain following central administration of neuropeptide Y and effects of food consumption

Administration of neuropeptide Y (NPY) intracerebroventricularly (i.c.v.) results in the release of a number of hypothalamic and pituitary hormones and stimulation of feeding and suppression of sexual behavior. In this study, we sought to identify cellular sites of NPY action by evaluating perikaryal Fos-like immunoreactivity (FLI), a marker of cellular activation, in those hypothalamic and extrahypothalamic sites previously implicated in the control of neuroendocrine function and feeding behavior. Additionally, we compared the topography of FLI in these brain sites when food was either available ad libitum or withheld after NPY injection (1 nmol/3 μl, i.c.v.). The results showed that one hour after NPY injection a large number of cells in the parvocellular region of the paraventricular nucleus (PVN) were FLI-positive in the absence of food consumption. However, in association with food intake, a significant number of cells were intensely stained in the magnocellular region of the PVN. An analogous increase in FLI in association with feeding was apparent in the supraotic nucleus (SON), the dorsomedial nucleus and the bed nucleus of the stria terminalis in the hypothalamus. Anong the extrahypothalamic sites, feeding facilitated FLI in a large number of cells located in the lateral subdivision of the central amygdaloid nucleus and the lateral subdivision of the solitary tract. FLI was observed in a moderate number of cells in the hypothalamic arcuate nucleus (ARC) and ventromedial nucleus, and this response was not changed by feeding. Cumulatively, these results show that neurons in a number of discrete hypothalamic and extrahypothalamic sites, previously implicated in the control of neuroendocrine function and feeding behavior, are activated by NPY and further, a divergent pattern of c-fos expression emerged in some of these sites if feeding occurs after NPY injection. Stimulation of FLI in cells of the PVN, SON and ARC by NPY imply the presence of NPY target cells that play a role in the neuroendocrine control of pituitary function. The finding that NPY induced FLI in cells located in the parvocellular subdivision of the PVN even in the absence of feeding, imply that cells involved in initiation of food intake by NPY may reside in this subdivision of the PVN. On the other hand, the appearance of Fos-cells in the magnocellular subdivision of the PVN in response to feeding, suggests neural mechanisms that operate during the post-ingestion period, including those associated with termination of NPY-induced feeding, may impinge upon this subdivision of the PVN.     

Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion

Neuropeptide Y (NPY), which is synthesized in neurons of the arcuate nucleus (ARC) that project to different hypothalamic nuclei, is known to have potent effects on eating behavior and hormone secretion after hypothalamic administration. To test the hypothesis that endogenous NPY is essential for the normal expression of these responses, the present study used two unmodified antisense oligodeoxynucleotides (ODNs) to disrupt the synthesis of NPY in the ARC and to examine the impact of this disturbance on nutrient intake, as well as on circulating levels of insulin and the adrenal steroids, corticosterone and aldosterone. Brain-cannulated rats maintained on macronutrient diets were given daily, bilateral injections, over a 4-day period, of NPY antisense ODNs, sense ODNs or saline into the ARC. The NPY antisense ODNs produced a significant decline (−33% relative to sense ODNs and −40% relative to saline, P < 0.05) in NPY levels in this nucleus, without causing any direct neural damage. Peptide levels in other hypothalamic areas, namely, the paraventricular nucleus and medial preoptic nucleus, were not significantly affected. In association with this reduction in ARC NPY, the antisense-treated animals exhibited a significant decrease in feeding behavior measured during the first 90 min of the natural feeding cycle, as well as over the 24-h period. In the 90-min interval, both carbohydrate and fat intake were suppressed by 65–70% (P < 0.05, relative to both saline and sense ODNs control scores). In addition, circulating insulin levels, in blood samples taken before the initiation of feeding, were significantly reduced by 50–55% (P < 0.05 relative to both saline and sense ODNs groups), while levels of corticosterone, aldosterone or glucose were unaltered. These findings provide the first evidence for physiological disturbances that may result from an inhibition of endogenous NPY production within neurons of the ARC.     

Neuropeptide Y in the arcuato-paraventricular pathway and diet selection in the vasopressin-deficient Brattleboro rat

Neuropeptide Y (NPY) is one of the most important brain peptides involved in feeding behavior. It influences both food choice and fluid homeostasis. The paraventricular and arcuate nuclei belong to the main pathway through which NPY stimulates carbohydrate intake. In this study, we measured NPY in various hypothalamic microdissected areas in Brattleboro di/di rats, a rat model of diabetes insipidus with specific dietary preferences. We confirmed that this rat is characterized by an increased fat intake (+10%; p < 0.001) and a decreased carbohydrate intake (−10%; p < 0.001) leading to a completely different dietary profile than that of di/+ controls. This profile was associated with a decrease in NPY in the paraventricular nucleus (−33%; p < 0.005) and in the ventromedial nucleus (−24%; p < 0.002). Intake of carbohydrate was negatively correlated with the gradient of NPY concentration between the arcuate and paraventricular nuclei. NPY could therefore contribute to the qualitative changes of feeding behavior in the Brattleboro rat through altered transport/release of the peptide and participate in the balance of neuropeptides that determines food choice in this strain of rat.     

Effects of monosodiuml-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus

Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodiuml-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.     

Effects of monosodium L-glutamate administration on neuropeptide Y-containing neurons in the rat hypothalamus

Immunocytochemical localization of neuropeptide Y (NPY) was performed in the hypothalamus of rats of which the arcuate nucleus had been destroyed with monosodium L-glutamate in the neonatal period. The treatment produced a disappearance of most of the NPY cell bodies normally found in the arcuate nucleus. The concentration of fibers was decreased in the paraventricular nucleus, but not in the other hypothalamic nuclei. The treatment also induced the appearance of a large number of immunoreactive cell bodies in the paraventricular nucleus. These results strongly suggest that arcuate NPY neurons are projecting to the paraventricular nucleus and that the arcuate nucleus probably exerts some inhibitory tonic influence on NPY paraventricular neurons.     

Decreased NPY innervation of the hypothalamic nuclei in rats with cancer anorexia

Whether the decrease in food intake that occurs at the onset of anorexia in tumor bearing (TB) rats is related to a change in the hypothalamic neuropeptide Y (NPY) system was tested by comparing NPY expression in sham operated Fischer Control and anorectic TB rats. Coronal cryocut sections of their fixed brain were processed by the peroxidase-antiperoxidase method with NPY polyclonal antibodies. NPY-immunoreactive fibers were widely distributed throughout the forebrain, but were most prominent in the hypothalamic paraventricular, suprachiasmatic, arcuate and periventricular nuclei. NPY-immunoreactive neurons were visualized in Control and anorectic TB rats in the preoptic region, the arcuate nucleus, and occasionally in the lateral hypothalamus. Semiquantitative image analysis showed a significant decrease in the NPY immunostaining in some hypothalamic nuclei of the anorectic TB rats, most prominently in the supraoptic nucleus, the parvocellular portion of the paraventricular nucleus, and, to a lesser extent, the suprachiasmatic and arcuate nuclei. No changes in NPY innervation were seen in the ventromedial nucleus and the lateral hypothalamus. The data support the hypothesis of an altered hypothalamic NPY system at the onset of anorexia in TB rats and also reveal the hypothalamic nuclei through which NPY influences food intake.     

Effect of d-fenfluramine on neuropeptide Y concentration and release in the paraventricular nucleus of food-deprived rats.

Recent evidence indicates that Neuropeptide Y (NPY) is an important signal in the hypothalamic neural circuitry that stimulates feeding in the rat. Administration of d-fenfluramine (FEN) has been shown to rapidly inhibit feeding in the rat. Because food deprivation increases the levels and release of NPY in the paraventricular nucleus (PVN) of the hypothalamus, the aim of this study was to investigate whether the rapid anorectic effects of FEN in food-deprived (FD) rats are associated with alterations in the hypothalamic NPYergic system. In the first experiment, the effect of FEN (10 mg/kg) on NPY concentrations in nine microdissected hypothalamic sites was assessed by radioimmunoassay (RIA) in rats either food deprived for 3 days or fed ad lib during the experimental period. In response to food deprivation, NPY concentrations increased significantly in the PVN and arcuate nucleus, but NPY levels remained unchanged in the remaining seven hypothalamic sites. In control rats maintained on ad lib food supply, FEN injection produced little effect on NPY concentration in hypothalamic sites. However, FEN suppressed NPY levels selectively in the PVN of FD rats, so that NPY concentrations measured in the nucleus were within the range found in satiated control rats. In the second experiment, the effect of FEN on NPY release in the PVN was examined in FD rats by the push-pull cannula (PPC) technique. NPY levels in the PPC perfusate were unchanged in FD rats during the period 30-120 min after saline or FEN injection. Also, the mean rate of NPY release was similar in vehicle- and FEN-treated FD rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

NPY mediates the feeding elicited by muscimol injections into the nucleus accumbens shell

Injections of muscimol into the nucleus accumbens shell (AcbSh) induce large increases in food intake in satiated rats and also activate neurons in a number of feeding-related brain regions, including NPY-containing neurons in the arcuate hypothalamic nucleus and cells in the paraventricular hypothalamic nucleus. This suggests that the NPY system may participate in the expression of AcbSh-mediated feeding behavior. Therefore, we examined the effects of intraventricular administration of the Y1 receptor antagonist 1229U91 or the Y5 receptor antagonist L-152,804 on AcbSh-mediated food intake. Intra-AcbSh muscimol elicited a large increase in food intake which was potently suppressed by blocking either central Y1 or Y5 receptors. Our results suggest that the AcbSh influences food intake, in part, through the release of NPY.     

Projection target‐specific action of nicotine in the caudal nucleus of the solitary tract

The brainstem nucleus of the solitary tract (NTS) is the key integrating relay in the central processing of sensory information from the thoracic and from most subdiaphragmatic viscera. Modulation of neuronal excitability and synaptic activity in the NTS by nicotinic agents can have potent effects on vital physiological functions, such as feeding, digestion, respiration, and blood circulation. Caudal NTS neurons demonstrate considerable heterogeneity in projection targets, synaptic properties, and expression of nicotinic acetylcholine receptors (nAChRs). However, despite its heterogeneity, the caudal NTS may contain discrete subsets of neurons with unique projection target‐specific properties. To test this hypothesis, we used in vivo fluorescent tracing and ex vivo patch‐clamp electrophysiology to evaluate responsiveness to nicotine of anatomically identified caudal NTS neurons that project to the hypothalamic paraventricular nucleus (PVN) and the brainstem caudal ventrolateral medulla (CVLM). The results of this study demonstrate that responsiveness to nicotine correlates with where the neurons project. Specifically, PVN‐projecting caudal NTS neurons respond to nicotine only presynaptically (i.e., via activation of presynaptic nAChRs and potentiation of synaptic release of glutamate), suggesting indirect, glutamate‐dependent effects of nicotine on the PVN‐projecting NTS circuitry. By contrast, CVLM‐projecting caudal NTS neurons exhibit only limited presynaptic, but dominant somatodendritic, responsiveness to nicotine, suggesting that the effects of nicotine on the CVLM‐projecting NTS circuitry are direct and largely glutamate independent. Understanding the relationships among function‐specific brainstem/hypothalamic neuronal networks, nuclei, and individual neurons could help develop therapies targeting identifiable neuronal circuits to offset impaired autonomic homeostasis. © 2014 Wiley Periodicals, Inc.     

Diurnal rhythm of neuropeptide Y-like immunoreactivity in the suprachiasmatic, arcuate and paraventricular nuclei and other hypothalamic sites

The diurnal rhythm of neuropeptide Y (NPY)-like immunoreactivity was examined in 9 discrete hypothalamic sites of rats maintained on a 12:12 h light/dark cycle. Significant bimodal rhythms of NPY concentration were detected in the suprachiasmatic and arcuate nuclei, with significant peaks just prior to onset of the nocturnal period and also at onset of the light period. In the parvocellular division of the paraventricular nucleus, a unimodal NPY peak was observed prior to dark onset. No diurnal rhythm was seen in the magnocellular division of the paraventricular nucleus, nor in 5 other hypothalamic areas examined.     

Elucidation of the anatomy of a satiety network: Focus on connectivity of the parabrachial nucleus in the adult rat

We hypothesized that brain regions showing neuronal activation after refeeding comprise major nodes in a satiety network, and tested this hypothesis with two sets of experiments. Detailed c‐Fos mapping comparing fasted and refed rats was performed to identify candidate nodes of the satiety network. In addition to well‐known feeding‐related brain regions such as the arcuate, dorsomedial, and paraventricular hypothalamic nuclei, lateral hypothalamic area, parabrachial nucleus (PB), nucleus of the solitary tract and central amygdalar nucleus, other refeeding activated regions were also identified, such as the parastrial and parasubthalamic nuclei. To begin to understand the connectivity of the satiety network, the interconnectivity of PB with other refeeding‐activated neuronal groups was studied following administration of anterograde or retrograde tracers into the PB. After allowing for tracer transport time, the animals were fasted and then refed before sacrifice. Refeeding‐activated neurons that project to the PB were found in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamic area; arcuate, paraventricular, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; parasubthalamic nucleus; central amygdalar nucleus; area postrema; and nucleus of the solitary tract. Axons originating from the PB were observed to closely associate with refeeding‐activated neurons in the agranular insular area; bed nuclei of terminal stria; anterior hypothalamus; paraventricular, arcuate, and dorsomedial hypothalamic nuclei; lateral hypothalamic area; central amygdalar nucleus; parasubthalamic nucleus; ventral posterior thalamic nucleus; area postrema; and nucleus of the solitary tract. These data indicate that the PB has bidirectional connections with most refeeding‐activated neuronal groups, suggesting that short‐loop feedback circuits exist in this satiety network. J. Comp. Neurol. 524:2803–2827, 2016. © 2016 Wiley Periodicals, Inc.     

Convergent influence of the central nucleus of the amygdala and the paraventricular hypothalamic nucleus upon brainstem autonomic neurons as revealed by c-fos expression and anatomical tracing

Combinations of anatomical tracing with detection of Fos (the protein product of the immediate early gene c-fos) consequent to the stimulation of the central nucleus of the amygdala were used to explore the possibility that the hypothalamic paraventricular nucleus participates in the activation of brainstem neurons in the nucleus of the solitary tract and ventrolateral medulla. After injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the paraventricular nucleus, labeled fibers and varicosities were found to impinge on catecholaminergic and non-catecholaminergic Fos-positive neurons in the brainstem. After injections of a retrograde tracer in the nucleus of the solitary tract or ventrolateral medulla, we observed that some of the Fos-positive neurons within the parvocellular paraventricular nucleus that project to the brainstem were catecholaminergic or oxytocinergic. The results indicate that direct and indirect inputs from the amygdala may influence the activity of autonomic neurons in the brainstem. The paraventricular nucleus, via its direct projections onto catecholaminergic and non-catecholaminergic neurons, may participate in activation of brainstem neurons. Activated catecholaminergic and oxytocinergic parvocellular neurons in the paraventricular nucleus may be involved in the transmission of autonomic signals from the amygdala toward the brainstem. ©1995 Wiley-Liss, Inc.     

Afferent neuropeptide Y projections to the ventral tegmental area in normal-weight male Wistar rats

The hypothalamic neuropeptide Y (NPY) circuitry is a key regulator of feeding behavior. NPY also acts in the mesolimbic dopaminergic circuitry, where it can increase motivational aspects of feeding behavior through effects on dopamine output in the nucleus accumbens (NAc) and on neurotransmission in the ventral tegmental area (VTA). Endogenous NPY in the NAc originates from local interneurons and afferent projections from the hypothalamic arcuate nucleus (Arc). However, the origin of endogenous NPY in the VTA is unknown. We determined, in normal-weight male Wistar rats, if the source of VTA NPY is local, and/or whether it is derived from VTA-projecting neurons. Immunocytochemistry, in situ hybridization and RT-qPCR were utilized, when appropriate in combination with colchicine treatment or 24 hr fasting, to assess NPY/Npy expression locally in the VTA. Retrograde tracing using cholera toxin beta (CTB) in the VTA, fluorescent immunocytochemistry and confocal microscopy were used to determine NPY-immunoreactive afferents to the VTA. NPY in the VTA was observed in fibers, but not following colchicine pretreatment. No NPY- or Npy-expressing cell bodies were observed in the VTA. Fasting for 24 hr, which increased Npy expression in the Arc, failed to induce Npy expression in the VTA. Double-labeling with CTB and NPY was observed in the Arc and in the ventrolateral medulla. Thus, VTA NPY originates from the hypothalamic Arc and the ventrolateral medulla of the brainstem in normal-weight male Wistar rats. These afferent connections link hypothalamic and brainstem processing of physiologic state to VTA-driven motivational behavior.     

Evidence that hypothalamic neuropeptide Y gene expression and NPY levels in the paraventricular nucleus increase before the onset of hyperphagia in experimental diabetes

Neuropeptide Y (NPY) is the most potent endogenous orexigenic signal. Several lines of evidence indicate that the site of NPY action in transducing feeding signal may reside in the paraventricular nucleus (PVN) and neighboring sites in the hypothalamus. To test the hypothesis that an increase in NPY activity in the ARC-PVN pathway precedes the onset of diabetic hyperphagia, we evaluated NPY levels in seven hypothalamic nuclei and NPY gene expression in the hypothalamus at 48, 72 or 96 h after streptozotocin (STZ) treatment in rat. In STZ-treated diabetic rats, NPY gene expression in the hypothalamus and NPY levels only in the PVN significantly elevated at 48 h, while hyperphagia occurred sometimes after 48 h post-injection. These results show that augmentation in NPY neuronal activity in the ARC-PVN axis precedes the onset of increased food intake produced by STZ-induced insulinopenia. These findings affirm the hypothesis that increased NPY neurosecretion in the PVN may underlie the diabetes-induced hyperphagia.     

Increased feeding and neuropeptide Y (NPY) but not NPY mRNA levels in the hypothalamus of the rat following central administration of the serotonin synthesis inhibitorp-chlorophenylalanine

Neurons containing serotonin (5-HT), a potent anorexic agent, come into contact with neuropeptide Y-ergic neurons, that project from the arcuate nucleus (ARC) to the paraventricular nucleus (PVN). NPY powerfully stimulates feeding and induces obesity when injected repeatedly into the PVN. We hypothesize that 5-HT tonically inhibits the ARC-PVN neurons and that balance between the two systems determines feeding and energy homeostasis. This study aimed to determine whether central injection of the 5-HT synthesis inhibitor p-chlorophenylalanine (pCPA), which increases feeding, increased hypothalamic NPY and NPY mRNA levels. pCPA (10 mg/kg in 3 μl) was administered into the third ventricle either as a single injection (n = 8) or daily for 7 days (n = 8). Control rats received a similar injection of saline. pCPA significantly increased food intake compared with controls after both single and repeated injections (P < 0.05). NPY levels were measured by radioimmunoassay in microdissected hypothalamic extracts. NPY levels in the acutely treated group were significantly increased in the paraventricular nucleus (PVN; by 41%,P = 0.01), anterior hypothalamic area (AHA; by 34%,P < 0.01) and lateral hypothalamic area (LHA; by 41%,P < 0.02). In the 7-day-treated group, NPY levels were also increased in the same areas, i.e. PVN (by 24%,P < 0.01), AHA (by 30%,P < 0.01) and LHA (by 38%,P = 0.01). There were no significant changes in the ARC or any other region or in hypothalamic NPY mRNA levels. pCPA administration increased NPY levels in several regions notably the PVN. This is a major site of NPY release, where NPY injection induces feeding. We suggest that the hyperphagia induced by pCPA is mediated by increased NPY levels and secretion in the PVN. This is further evidence for interactions between NPY and 5-HT in the control of energy homeostasis.     

Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus

Recent studies suggest that glucocorticoids may increase NPY and NPY mRNA levels. To determine if endogenous corticosterone affects the level of NPY mRNA in areas that control NPY levels in the paraventricular nucleus, we examined the effects of adrenalectomy and corticosterone replacement on NPY mRNA levels in the arcuate nucleus and brainstem. Rats were either adrenalectomized, adrenalectomized and corticosterone replaced, or sham-operated. The arcuate nucleus, hypothalamus (excluding arcuate nucleus), and brainstem were collected and the RNA isolated. Dot blots were made of each tissue and the NPY mRNA quantitated by densitometry. Adrenalectomy significantly reduced NPY mRNA levels in the arcuate nucleus, while corticosterone replacement restored the NPY mRNA levels. NPY mRNA levels in the remainder of the hypothalamus were not affected by adrenalectomy. Adrenalectomy also had no affect on NPY mRNA levels in the brainstem. These data suggest that the paraventricular nucleus may be affected by glucocorticoids via an NPY pathway and that the two major afferent pathways of NPY-containing neurons to the paraventricular nucleus may be regulated by different mechanisms.     

Intracerebroventricular injection of dibutyryl cyclic adenosine 3′,5′-monophosphate increases hypothalamic levels of neuropeptide Y

This investigation examined in vivo the relationship between the nucleotide cAMP and hypothalamic levels of two peptides, neuropeptide Y (NPY) and galanin (GAL), which are known to potentiate feeding behavior. In brain-cannulated rats, third ventricular injections of N⁶,2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate ((Bu)₂cAMP, 25 μg), compared to saline, caused a significant increase in NPY levels in the arcuate nucleus (ARC) and medial parvocellular portion of the paraventricular nucleus (mPVN), while having no impact in other hypothalamic areas. These site-specific changes in NPY occurred in the absence of any alteration in circulating levels of insulin, corticosterone, aldosterone or glucose, or of changes in hypothalamic levels of GAL. These findings implicate cAMP as having regulatory functions within specific hypothalamic NPY-synthesizing neurons, projecting from the ARC to the mPVN, that are believed to be involved in energy homeostasis.     

Aldosterone-sensitive neurons of the nucleus of the solitary tract: multisynaptic pathway to the nucleus accumbens

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.