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Arcuate NPY is involved in salt‐induced hypertension via modulation of paraventricular vasopressin and brain‐derived neurotrophic factor

Abstract Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt‐induced hypertension. Here, we demonstrate that wild‐type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY‐GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain‐derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV‐Cre recombinase into the Arc only of the NPY‐targeted mutant mice carrying a loxP‐flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt‐dependent blood pressure. In summary, our study uncovers an important Arc NPY‐originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.


| INTRODUCTION
Hypertension is a key contributor to major human health problems throughout the world and is associated with an increased risk of stroke, myocardial infarction, kidney failure, and mortality (Kong et al., 2016;McLaren et al., 2016). It is known that high levels of dietary salt (NaCl) intake are closely related to the prevalence of hypertension (Kim et al., 2011;Simmonds et al., 2014), and high salt intake is considered to be one of the major risk factors for the development of hypertension (He & MacGregor, 2012;F. J. He et al., 2013;Schmidlin et al., 2007). In recent years, the role of central regulation in salt-sensitive hypertension has increasingly received attention, particularly the hypothalamus. For example, one recent study showed that chronic high salt consumption in rats excites the neurohypophysis to release more vasopressin via BDNF-dependent pathways in the hypothalamic PVN and supraoptic nucleus (SON) of the rat, thereby blunting baroreflex-induced reduction of blood pressure (Choe et al., 2015). This study highlights the important involvement of different hypothalamic nuclei in the regulation of saltinduced hypertension; however, it remains elusive whether other hypothalamic regions, in particular, the hypothalamic arcuate nucleus (Arc) also participates in this critical fluid homeostasis regulation.
The Arc is located at the base of the hypothalamus with a notably semipermeable membrane that allows the neurons within the Arc to directly access peripheral nutritional signals (Loh et al., 2015). There are two populations of neurons within the Arc: neuropeptide Y (NPY)/agouti-related peptide (AgRP)-ergic neurons and proopiomelanocortin (POMC)/cocaine-and amphetamine-regulated transcript (CART)-ergic neurons that have opposing effects on food intake and energy homeostasis (Shi et al., 2013). NPY-ergic neurons are orexigenic and activation of NPY neurons in the Arc under a negative energy balance (e.g., fasting) project to the PVN and promote the feeding via the Y1 receptors in the PVN (Loh et al., 2015). NPY is widely distributed in the central nervous system with the highest concentration found in the Arc (Lin et al., 2004). Overexpression of Arc NPY leads to obesity and a reduction in energy expenditure in mice (Nguyen et al., 2012;Shi et al., 2013;Vähätalo et al., 2015). In addition to feeding, NPY is also indicated in other important functions including the regulation of bone mass, stress responses, neuroendocrine secretion (Ip et al., 2019;Shi et al., 2010;Tatemoto et al., 1982), and blood pressure (Zhu, Sun, Zhang, Song, & Lin, 2016).
Injection of NPY into the PVN or lateral ventricle decreases the activity of peripheral sympathetic nerves, heart rate, and blood pressure in rats (Morris et al., 2004) via an action mediated by vasopressin (Larsen et al., 1993;Urban et al., 2006). These data indicate a potential hypotensive effect of central NPY signaling.
However, it is unclear as to whether NPY in the Arc, which has direct access to peripheral nutritional signals, is involved in this process, and if so, what the underlying mechanisms are.
In this study, we demonstrate that salt intake induces hypertension and blunts expression of NPY in the hypothalamic Arc of WT mice. The suppression of NPY expression occurs in concert with the upregulation of mRNA expression of vasopressin and BDNF in the hypothalamic PVN, leading to elevated serum vasopressin levels.
Moreover, replenishment of Arc only-NPY in NPY-targeted mutant mice attenuated salt-dependent hypertension via downregulation of vasopressin and BDNF expression in the PVN. Thus, our study provides the first demonstration that chronic salt intake decreases NPY expression in the hypothalamic Arc, leading to vasopressindependent hypertension presumably via hypothalamic BDNF pathways, and emphasizes a novel and previously unexplored contribution of Arc NPY in the control of blood pressure independent of its traditional orexigenic effects.

| Animals
All experimental procedures were carried out in accordance with international ethical guidelines and the National Institutes of Health

| Body weight and water intake measurements
Forty 8-week-old C57BL/6J and eight 8-week-old NPY-GFP mice were administrated with either normal drinking water (NW) with <0.1% NaCl, or salt drinking water (SW) with 2% NaCl solution for 8 weeks. Body weight and water intake were measured weekly from 8 weeks of age onward. Urine production was measured. Body weight was always measured at the same time of the day to ensure consistency. Actual water intake was calculated as the water volume given minus the water volume that remained after 24 h.

| Generation of NPY stop cassette knock-in mice
The loxP-stop-loxP-NPY knock-in mouse line (NPY stop cassette knock-in line, referred to as NPY LSL/LSL ), in which a targeted allele of the stop cassette was cloned to upstream NPY coding sequences in all tissues, was obtained from Cyagen Biosciences Inc. In basal conditions, the presence of the stop codon blocks NPY expression in transgenic mice. However, the injection of adeno-associated virus ZHANG ET AL. | 2575 (AAV)-Cre recombinase (AAV-Cre) into specific tissues or regions can excise the loxP-flanked stop cassette and restore the expression of NPY. Therefore, AAV-Cre-injected mice can conditionally activate the expression of NPY, whereas the vehicle-injected mice, injected with vehicle solution carrying empty construct cannot modulate the expression of NPY.

| Stereotaxic virus injection
Twenty NPY LSL/LSL mice were injected with either AAV-Cre or AAVvehicle (n = 10 per group). After recovery from the surgery, each group of mice was divided into two groups. One group was given normal drinking water, and the other group was given 2% salt water for 8 weeks.
The NPY LSL/LSL mice were anesthetized with pentobarbital (80 mg/kg, IP) and placed in a temperature-controlled stereotaxic holder. A small hole was made in the skull. To selectively re-express NPY in the Arc, a total volume of 0.8 µl (0.4 µl each side) of AAV2-CAG-EGFP-T2A-Cre (OBiO Technology) was injected bilaterally into the Arc (coordinates relative to bregma were anteroposterior (AP)/ dorsoventral (DV)/mediolateral (ML): -1.2/-5.8/ ± 0.2 mm, (Franklin & Paxinos, 2017)) at a rate of 0.1 µl/min using a 1-µl Hamilton Syringe and syringe infusion pump (KDS 310 Plus, Kd Scientific). The needle was left in place for 10 min to allow sufficient diffusion.
Animals were kept on a heating pad during surgery until the recovery period. Mice were housed individually and subsequently monitored for 2 weeks before any studies were performed.

| Blood pressure measurement
The arterial pressure of the mice was measured weekly using the noninvasive Volume Pressure Recording System (CODA, Kent Scientific Corporation). To minimize the effects of stress, animals were trained using the tail-cuff method for at least 7 days before the first measurement. Blood pressure was measured by the same person at the same time of the day until the euthanization of the mouse. A conscious mouse was held in an individual plastic container, and a cuff with a pneumatic pulse sensor was wrapped around its tail. After warming animals on a heating plate for 10 min at 35°C, the measurements of arterial systolic blood pressure (SBP) and diastolic blood pressure (DBP) were performed for at least 20 consecutive cycles for each animal, and the mean values of several accurate measurements were used for the analysis of systolic and DBP.

| Determination of serum electrolytes
The concentration of serum Na + , Cl − , and K + was determined at the

| Immunohistochemistry
For c-fos detection in the brain 24 h after the start of treatment conditions, the animals were perfused with phosphate-buffered solution (PBS) at a pH of 7.4 by a cannula inserted in the left ventricle after anesthesia, followed by 4% paraformaldehyde. After perfusion, the brains were immediately removed, kept in 4% paraformaldehyde, immersed in 30% sucrose, and then stored at −80°C until use. Immunohistochemistry (IHC) staining was performed on cryostat sections. Adjacent serial 30-µm thick coronal brain sections were obtained with cryostat (Leica, CM 3000). The IHC staining was performed according to standard ABC protocol with rabbit polyclonal primary antibodies (anti-c-fos, 1:1,000, Cell Signaling Technology) and the signals were detected with a commercial ABC kit (ZsBio, Ltd.). The slides were photographed using an Olympus Imaging System (Olympus Corporation). The number of cfos-positive cells was quantified using ImageJ software (NIH). To maintain consistency in the analysis of cell counts among groups, sections of hypothalamic PVN regions from the salt-treated and control animals were matched based on the mouse brain atlas (Franklin & Paxinos, 2017).

| NPY-GFP positive cell counting
To assess the number of GFP positive cells in the Arc, NPY-GFP mice were fed with NW or SW for 8 weeks and were then perfused after treatment with 4% formaldehyde in PBS. The perfused brains were cut at 35 µm covering the hypothalamic Arc relative to Bregma anteroposterior, −1.46 mm to −1.94 mm (Franklin & Paxinos, 2017). Every third section was used for counting NPY-GFP positive neurons.
Fifteen sections from each brain corresponding to rostral-, medial-, and caudal-regions, were counted unilaterally from three different mice under a blinded condition. Two-tailed t tests were used.
Statistical significance was defined as p < 0.05.

| RNA extraction and qPCR analysis
At the end of each experiment, a microdissection procedure was used to isolate PVN and Arc areas as previously described (Kang et al., 2011). For mRNA expression analysis, total RNA was extracted from brain tissue using Trizol reagent (Aidlab) and cDNA was synthesized using a TRUEscript First Strand cDNA Synthesis Kit  Table 1. After PCR, a dissociation curve was added to confirm that no secondary products were formed. Standard curves were also performed to check the amplification efficiency. Relative quantification of mRNA was conducted using the 2 -ΔΔCt method and normalized respectively to the expression of β-actin. Every sample was performed in triplicate.

| In situ hybridization (ISH) by RNAscope technology
The brain samples were prepared as described above in IHC. The fixed frozen tissues were cryosectioned at 8 µm and stored at −80°C.
Commercially available RNAscope brown reagent kit and RNAscope probes (Advanced Cell Diagnostics) were used for transcript detection. ISH was performed according to the manufacturer's instructions for fixed-frozen tissue. The detection was operated in a hybridization oven (HybEZ, ACD) with RNAscope Probe-mm-NPY (ACD 313321), RNAscope Probe-mm-AgRP (ACD 400711), and RNAscope Probe-mm-vasopressin (ACD 401391). Ppib probe (ACD 313911), a mouse housekeeping gene, was used as the positive control probe, and bacterial dapB probe (ACD 310043) was used as the negative control probe. Each set of probes contained a tag that enabled target transcription to be visualized in a brown color. To compare the expression differences between the two groups, we quantified the integral optical density of positive NPY or vasopressin staining using ImageJ and normalized it by stained area. The mean intensities from three random areas of the same size in target areas were measured for each probe.

| Tissue collection
Upon the completion of the experiments, animals were anesthetized by IP administration of pentobarbital and then euthanized to collect blood and brain tissue for enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), in situ hybridization (ISH), immunohistochemical (IHC), and molecular studies. The collected tissues were snap-frozen and stored at −80°C until assayed.

| Statistical analyses
The data were expressed as mean ± SEM. Statistical analyses were performed using GraphPad Prism software. The significance of the differences between the two groups was analyzed by Student's t test.
To compare multiple time points, repeated measures ANOVA was used. Multiple comparisons were analyzed with a one-way analysis of variance (ANOVA). A value of p < 0.05 was considered significant.
As expected, serum Na + level in SW-treated mice was significantly elevated at the end of the experimental period (p < 0.001, Figure 1a). Cl − level was unaltered by NaCl consumption (Figure 1b). On the other hand, serum K + concentration was markedly lower in SWtreated mice compared to control mice ( Figure 1c). Consistent with elevated Na + levels in the blood, water consumption in SW-treated mice was significantly increased from the 2nd week onward throughout the rest of the experimental period compared to the water consumption of the control mice (p < 0.001, Figure 1d). Urine output measured at Week 4 was also concomitantly increased ( Figure 1e), suggesting the increased sense of thirst was due to drinking SW. Despite increased water consumption, there was no difference with regard to body weight ( Figure 1f ) and food intake measured at Weeks 1 and 4 ( Figure 1g) between SW and NW mice over the entire experimental period. In line with increased blood Na + concentration and water consumption in SW mice, from the third to the eighth week except for the fourth week, the systolic blood pressure (SBP) of SW-treated mice was significantly increased, compared to the SBPs of NW-treated mice in the same time period, and reached an average blood pressure of 143 ± 4 mmHg at the seventh and eighth week (Figure 1h). In contrast, NW mice displayed a stable SBP with an average of 127 ± 4 mmHg over the 8-week period. Similarly, the DBP in the SW-treated group was also markedly increased from the sixth week onwards over the same period of time F I G U R E 1 Salt water-induced hypertension in C57BL/6 wild-type (WT) mice. (a) Serum Na + was significantly increased in salt water-treated mice relative to normal water-given mice at the end of the experiment. (b) Serum Cldid not differ between the two groups at the end of the experiment. (c) Serum K + was decreased in salt water-treated mice. (d) Salt water-treated mice showed a marked increase in water consumption compared to control mice which was significant after 3 weeks and every week from the 4th week to the end of the study (n = 5). (e) Daily urine output measured after 1 and 4 weeks. (f) There was no significant difference in body weight between the two groups at any point over the experimental period. (g) Daily food intake measured at the 1st week and the 4th week. (h) Weekly systolic blood pressure profile and (i) diastolic blood pressure of normal water-and salt water-treated mice. The salt water-treated mice had significantly higher systolic blood pressure after 3 weeks of stimulation through to the end of the experimental period. Values are expressed as means ± SEM (n = 5). (*p < 0.05, **p < 0.01, ***p < 0.001, salt water vs. normal water) ( Figure 1i). Heart rate was measured and there was no difference between the two treatment groups ( Figure S1). Together, these data demonstrate that high salt water intake induces hypertension.

| Salt intake suppressed the expression and activity of hypothalamic Arc NPY neurons
The

| Enhanced c-fos immunoreactivity in the hypothalamic PVN
Having demonstrated a link between peripheral elevation of NaCl levels and Arc NPY, we next attempted to identify the critical brain regions and the downstream targets of Arc NPY signaling to characterize the neuronal pathways and circuits initiated by Arc NPY signaling. To capture the early responses to high salt treatment and avoid the potential secondary effects associated with prolonged salt treatment, we investigated the expression of early neuronal activation marker c-fos (Dragunow & Faull, 1989)

| Salt intake increased circulating vasopressin levels and vasopressin mRNA expression in the hypothalamic PVN
Vasopressin, also called arginine vasopressin or antidiuretic hormone (ADH), is a hormone that is produced in the hypothalamus of the brain and is stored in the neurohypophysis of the pituitary gland (Boone & Deen, 2008). It is released in response to increased blood osmolarity, including an increased Na + level (Choe et al., 2015; Prager-Khoutorsky et al., 2017). We, therefore, determined vasopressin concentration in the serum. However, due to its short circulatory half-life, it is inaccessible for clinical routine determination at an accurate level. We used serum copeptin (CPP), a 39-amino acid glycosylated peptide which is part of the original C-terminal of arginine vasopressin, as a surrogate in determining vasopressin level due to its in vivo stability and readiness to be detected (Masajtis-Zagajewska et al., 2015). As shown in Figure 4a, circulating CPP levels were significantly increased in SW mice (p < 0.0001), supporting the notion that high salt intake increases circulating vasopressin levels.
Since vasopressin is primarily produced in the hypothalamic PVN, we collected and processed this brain section from SW-and NW-treated mice and performed in-situ hybridization for vasopressin using the

| Salt treatment altered BDNF and KCC2 mRNA expression in the PVN
Having confirmed the activation of key brain regions in the hypothalamic PVN by c-fos, we wanted to further identify which proteins in the PVN were altered by salt intake. To achieve this, we used a candidate gene approach. Previous work has shown that brain-derived neurotrophic factor (BDNF) neurons in the PVN are involved in high salt-induced hypertension (Choe et al., 2015).  Figure S2C) in the NPY LSL/LSL mice were not different from those in the WT mice described above.
After AAV-Cre was introduced into the Arc, 2 weeks postinjection, brains from NPY LSL/LSL mice were collected to assess the mRNA expression of NPY using RNAscope in situ hybridization analysis. A significantly higher NPY mRNA expression was observed in the hypothalamus of AAV-Cre injected NPY LSL/LSL mice compared to AAV-Empty-injected mice (Figure 5d). This result has confirmed the successful reintroduction of NPY specifically into the Arc.
For functional evaluation, NPY LSL/LSL mice injected with the AAV-Cre vector or AAV-Empty vector were allowed to recover from surgery for 10 days before being given either SW or NW for 8 weeks.
Compared to normal-water-treated AAV-Cre-injected NPY LSL/LSL mice, mice treated with salt water for 24 h displayed a significant reduction in Arc NPY mRNA expression (Figure 6a), but no alteration such as in the AAV-Cre-injected NPY LSL/LSL mice, the SBP of SW mice was dramatically blunted to a level similar to that of the AAV-Empty-injected NPY LSL/LSL mice with NW intake (Figure 6c). On the other hand, the SBP in NW-given AAV-Cre injected mice was not different from that in NW-given AAV-Empty injected mice ( Figure 6b). These data reveal that the replenishment of NPY in the Arc could alleviate, at least partially, salt water-induced hypertension.
We also determined the mRNA levels of BDNF and vasopressin expression in the hypothalamic PVN of the four groups. We found that the mRNA expressions of both BDNF and vasopressin in SW groups were higher in comparison to the corresponding NW groups (Figure 6d,e). In addition, when AAV-Cre was injected into the Arc under SW condition, we found that the mRNA levels of BDNF and vasopressin in PVN were significantly decreased (Figure 6d,e).

| DISCUSSION
In this study, we demonstrated for the first time a critical involvement of hypothalamic Arc NPY in the regulation of fluid homeostasis and salt water-induced hypertension. In the salt water-induced Values are expressed as means ± SEM (n = 4) (*p < 0.05, ***p < 0.001, salt water vs. normal water). 3V, third ventricle hypertensive mouse model, Arc NPY mRNA expression was significantly reduced as evidenced by in situ hybridization and qPCR analysis as well as in NPY-GFP reporter mice. By using c-fos, a marker of neuronal activity response to external stimuli, we identified brain regions in the PVN that were activated by high Na + treatment, where we found that vasopressin and BDNF mRNA were also upregulated.
Importantly, by using unique NPY LSL/LSL mice where NPY expression is prevented by a loxP-flanked STOP cassette, we demonstrated that specific reintroduction of NPY using AAV-Cre into the Arc only in these NPY deficient mice reversed salt-induced upregulation of vasopressin and BDNF mRNA expression, thereby blunting the increases in blood pressure in salt-treated mice. This study has uncovered a crucial role of traditionally known orexigenic Arc-NPY in the control of a NaCl-triggered neuronal pathway.
It is well established that under the normal condition, elevated NPY in the hypothalamic Arc by a negative energy balance (e.g.,  (2017) fasting) stimulates feeding and inhibits energy expenditure to maintain energy homeostasis (Loh et al., 2015;Nguyen et al., 2012).
This study unravels an important and previously uncharacterized role of Arc NPY in salt-induced hypertension in addition to its effects on feeding behavior and energy balance. We found that peripheral rising NaCl levels markedly reduced Arc NPY expression, which triggered the changes in expression of several neurotransmitters in the PVN, thereby leading to hypertension. Vasopressin is critical for the regulation of blood osmolarity and blood volume (Choe et al., 2015;Prager-Khoutorsky et al., 2017), and high plasma Na + can induce hypothalamic magnocellular neurons to release vasopressin, consequently promoting vasoconstriction and water retention (Bourque, 2008;Danziger & Zeidel, 2015;Henderson & Byron, 2007).
We found that salt intake suppresses the Arc NPY expression leading to augmentation of vasopressin in the PVN, and this is consistent with a previous report.  (Graebner et al., 2015;Lee et al., 2020). We also observed that SBP in WT mice was similar to that in AAV-empty NPY LSL/LSL mice. While the exact F I G U R E 6 Effects of Arc NPY on the control of salt water drinking-induced hypertension in NPY LSL/LSL mice. (a) The mRNA levels of NPY and AgRP in AAV-Cre-injected NPY LSL/LSL mice administered with normal water or salt water were determined by RNAscope in situ hybridization analysis, n = 3. (b) Systolic blood pressure (SBP) of Empty-injected and Cre-injected NPY LSL/LSL mice in response to normal water or 2% salt water administration. SBPs of the four groups were similar in the first week. (c) AAV-Empty-treated mice on salt water showed a higher blood pressure compared to the other three groups, while AAV-Cre-induced restoration of Arc NPY significantly attenuated salt water-induced hypertension at the eighth week of the experiment. (d) qPCR results showed that the restoration of Arc NPY in AAV-Cre-injected mice blunted salt water drinking-induced vasopressin mRNA expression in the hypothalamic paraventricular nucleus (PVN) compared to AAV-Empty-injected mice. (e) qPCR results showed that the restoration of Arc NPY in AAV-Cre-injected mice blunted salt water-induced mRNA expression of brain-derived neurotrophic factor (BDNF) in the hypothalamic paraventricular nucleus (PVN) compared to AAV-Empty-injected mice. Values are expressed as means ± SEM (n = 4-5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to AAV-Empty normal water or comparisons indicated mechanism underlying this phenomenon is unclear, we speculate that similar to germline NPY knockout mice that display normal metabolic phenotypes (Erickson et al., 1996), inactivated NPY in NPY LSL/LSL mice may lead to developmental compensation due to the functional redundancy of homeostatic control. More studies will be warranted to determine the alteration of other neurotransmitters (e.g., POMC/ CART or GABA) secondary to the lack of NPY in the hypothalamus and other brain regions which are known to be important in the regulation of blood pressure. In addition, we have demonstrated that the hypothalamic PVN, the endocrine integration center that receives Arc NPY projections and is critical in relaying peripheral nutritional signals to maintain energy balance, also responds to changes in extracellular osmolality, thereby regulating fluid balance (Chu et al., 2010;Loh et al., 2015).
In this study, we also identified BDNF and KCC2, the two critical neurotransmitters in the PVN, could be potential mediators of the  (Gelfo et al., 2012), while administration of BDNF into the hypothalamic PVN blunts NPY expression in the Arc in response to a fasting state and decreased NPY-induced feeding (Wang et al., 2007). Our data suggest that a reciprocal relationship between PVN BDNF and Arc NPY in the context of food intake is also present in the context of salt-induced fluid balance and the development of hypertension, underlining the key involvement of Arc NPY-BDNF neuronal pathway in integrating nutritional and electrolyte signals, thereby regulating both energy homeostasis as well as fluid balance.
An important finding of our study is that Arc NPY modulates saltinduced hypertension independent of its traditional role in controlling food intake and energy expenditure. We found that salt treatment causes a decrease in Arc NPY expression, which is expected to lower food intake, however, reduced NPY expression in the Arc did not cause any change in body weight and feeding under current chow diet feeding conditions. Our results are supported by a previous study in rats where elevated central NPY reduced blood pressure without body weight changes (Michalkiewicz et al., 2003). The exact mechanism for this phenomenon is not clear, but it is possible that salt triggers other mechanisms or neurons in the hypothalamus that counteract the feeding effects of Arc NPY. For example, anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons have also been implicated in the salt loadinginvolved energy homeostatic regulation (Elkabes & Loh, 1988;Zhang et al., 2018). In addition, vasopressin is synthesized in the SON of the hypothalamus (Vandesande & Dierickx, 1975), a nucleus that has also been explored very recently (Choe et al., 2015) and receives projections from the Arc to regulate vasopressin level (Pineda et al., 2016). The SON also expresses abundant melanocortin 4 (MC4) receptors that mediate Arc POMC effects on feeding (Mountjoy et al., 1994). It is possible that the NPY in the hypothalamic Arc projects to the hypothalamic nuclei including the PVN and the SON to collectively modulate and fine tune the response to salt. Nevertheless, together these data highlight the complexity of the central nervous system and the coordination of multiple neuronal pathways in the control of salt-induced fluid balance.
Although we have identified a pathway by which NPY in the Arc affects vasopressin secretion and hence blood pressure, the precise mechanism(s) by which NPY expression is reduced in the Arc in response to salt treatment remains to be elucidated. One possibility is that high sodium levels can directly influence Arc NPY neurons considering the proximity of Arc to the third ventricle. The Na + /K +pump is essential for the transport of Na + into neurons and has been shown to modulate the activity of neurotransmitter receptors as previously reviewed (Pivovarov et al., 2018), and thus may modulate electrolyte sensing of Arc NPY neurons. Another possibility is that circulating vasopressin could directly interact with Arc NPY neurons.
While vasopressin receptors appear only to be expressed in the PVN and the SON (Dayanithi et al., 2018), some earlier studies in rat brains do suggest that vasopressin 1a receptor mRNA is present in the Arc (Ostrowski et al., 1994;Szot et al., 1994). It is unclear which population of neurons in the Arc express the vasopressin receptor. Likewise, there could be upstream signaling from the PVN to Arc controlling NPY expression, which has previously been demonstrated with PVN oxytocinergic neurons (Maejima et al., 2014). Further studies will be required to explore these possibilities.